Original article

Influence of DNA encoding cytokines on systemic and mucosal immunityfollowing genetic vaccination against herpes simplex virus

Sujin Lee a, Malgorzata Gierynska b, Seong Kug Eo c, Nelly Kuklin d, Barry T. Rouse a,*a Department of Microbiology, The University of Tennessee, M409 Walters Life Sciences Building, Knoxville, TN 37996-0845, USA

b Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Polandc Department of Microbiology, College of Veterinary Medicine, Chonbuk National University, Chonju 561-756, South Korea

d Merck Research Laboratories, WP 16-214C, West point, PA 19486, USA

Received 2 December 2002; accepted 21 March 2003

Abstract

The aim of our investigation was to improve the effectiveness of DNA vaccines against herpes simplex virus (HSV) infection. We chosecoimmunization with DNA encoding cytokines known to emphasize components of immune defense that best correlate with immuneprotection. These include interferon-producing T and NK cells and the IgG2a isotype immunoglobulin. Our results show that the coadminis-tration of plasmid DNA encoding IL-12 or IL-18 along with glycoprotein B (gB) DNA improves immune induction. Recipients of thecoimmunization procedure had elevated humoral as well as IFN-c-producing T cell responses and showed greater resistance to vaginalchallenge with a lethal dose of HSV-1. The adjuvant effects were observed when the vaccines were administered either systemically ormucosally. By most assays, the adjuvant effect of IL-18 was superior to IL-12, although gB DNA plus IL-18 failed to induce levels of immunityachieved by UV-inactivated HSV immunization. Mucosal immunization proved as an effective means of inducing systemic immunity, but wasless effective than the systemic route for inducing protection from vaginal challenge. Our results also demonstrated that protection from suchchallenges was mainly a property of IFN-c. Thus, immunized IFN-c–/– mice remained susceptible to challenges even while generating readilymeasurable immune responses. The approach of using DNA vaccines combined with DNA encoding cytokines holds promise and representsa potentially useful approach for vaccines.

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: DNA vaccine; Herpes simplex virus; IL-12; IL-18; IFN-c

1. Introduction

Herpes simplex virus (HSV) affects a majority of personsin most populations worldwide. The virus is a significantcause of morbidity and occasional mortality, yet, despitenotable efforts, it has not been successfully controlled byvaccines. Success might come from the use of novel ap-proaches based on a better understanding of viral pathogen-esis. Normally, the virus invades via the abraded skin ormucosal surfaces, so immunity at such entry sites representsan important component for successful defense [1].

We (and others) have evaluated several immunization ap-proaches for their efficacy at inducing mucosal immunity[2–6]. It appears that a variety of immune components may

mediate protection at mucosal sites. These include antibody,especially IgG2a, NK cells, as well as both CD4 and CD8 Tcells [7–9]. The cellular defenses appear to function prima-rily by producing interferon gamma (IFN-c), which eitheracts to inhibit viral replication or induces inflammatory ef-fects that are anti-viral [10]. Animals lacking the ability toproduce or respond to IFN-c are more susceptible to infec-tion [11], as are animals in which the IFN-c response isinhibited [12,13]. Vaccines such as plasmid DNA that areeffective inducers of the CD4+ Th1 subset and IFN-c-producing CD8+ T cells are expected to be especially valu-able. Such DNA vaccines have been investigated previouslyby our group as well as others [14–16], but their use has notbeen optimized. Plasmid DNA encoding a viral protein givenwith additional immune modulators could prove to be asuccessful approach for vaccination. This topic has alreadybeen investigated with variable results [3,4,17,18]. It would

* Corresponding author. Tel.: +1-865-974-4026; fax: +1-865-974-4007.E-mail address: btr@utk.edu (B.T. Rouse).

Microbes and Infection 5 (2003) 571–578

www.elsevier.com/locate/micinf

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seem that the vaccination route may also affect the outcome,as some studies show that enhanced levels of mucosal immu-nity may result from systemic rather than mucosal immuni-zation [3].

Since a major aim for mucosal defense against HSV is toinduce cells that produce IFN-c [13], we have further evalu-ated immunogen combinations that should achieve IFN-cinduction. For this purpose, we coadministered IFN-c-producing cytokines such as IL-12 or IL-18 plasmid DNAalong with glycoprotein B (gB) DNA, either intranasally orsystemically. Our results were evaluated by in vitro measure-ments of systemic and distal mucosal immunity as well as forresistance to vaginal challenge with HSV. We demonstratedthat the coadministration of either IL-18 or IL-12 enhancedthe systemic IFN-c-producing T cell immunity, with IL-18providing the superior adjuvant effect. Enhanced systemicresponses resulting from cytokine coadministration were ob-served when immunogens were administered either systemi-cally or mucosally. However, distal mucosal IgA responseswere present only in mucosally immunized mice; but, onceagain, the IL-18 plus gB DNA combination yielded the mostfavorable responses. Vaginal challenge experiments involv-ing systemic coadministration with either IL-18 or IL-12induced increased immunity compared to those that receivedgB DNA alone. Mucosal immunization, however, providedonly marginal protection. Again, recipients of either cytokinealong with gB DNA possessed increased resistance levels.Immunity to vaginal challenge was shown to primarily de-pend on the IFN-c response. Thus, IFN-c– /– mice systemi-cally immunized with IL-18 or IL-12 with gB DNA failed toresist challenge, even though they demonstrated gB-specificT cell immunity. Our results are discussed in terms of thevalue of cytokine coadministration for vaccine development.

2. Materials and methods

2.1. Mice

Female 3- to 4-week-old BALB/c mice were purchasedfrom Harlan Sprague-Dawley (Indianapolis, ID). Female 3-to 4-week-old GK/O mice were purchased from The JacksonLaboratory (Bar Harbor, ME). BALB/c mice were housed insterile microisolator cages in the animal facility. All manipu-lations were performed in a laminar flow hood. All investiga-tions followed the guidelines of the committee on the Care ofLaboratory Animals Resources, Commission of Life Sci-ences, National Research Council. The animal facilities ofthe University of Tennessee are fully accredited by theAmerican Association of Laboratory Animal Care.

2.2. Virus

HSV-1 strains KOS and McKrae were used. Viruses weregrown on Vero cell monolayers (ATCC cat no. CCL81),titrated, and stored in aliquots at –80 °C until used.

2.3. Plasmid DNA preparation

Plasmid DNA encoding gB (gB DNA) of HSV-1 KOSunder the cytomegalovirus promoter has been described else-where [19]. Plasmid DNA encoding murine IL-12 and IL-18were kindly provided by Kenji Okuda (Yokohama City Uni-versity School of Medicine, Yokoyama, Japan). All plasmidDNAs used in this work were inserted into the pCDNA 3.1expression vector (Invitrogen, Inc., San Diego, CA). Theplasmid DNAs were purified by polyethylene glycol precipi-tation by the method of Sambrook et al. [20] with somemodifications. Cellular proteins were precipitated with onevolume of 7.5 M ammonium acetate followed by isopropanolprecipitation. After polyethylene glycol precipitation, plas-mids were phenol–chloroform extracted (two times) andprecipitated with two volume of pure ethanol. The quality ofDNA was measured by electrophoresis on 1% agarose gel.The protein expression of the different plasmids was deter-mined by RT-PCR and dot blot after in vitro transfection intoChinese Hamster Ovary cells.

2.4. DNA immunization

Female 3- to 4-week-old BALB/c mice and GK/O micewere coimmunized intramuscularly and intranasally with100 µg of gB DNA plus 200 µg of cytokine DNA formulatedin phosphate-buffered saline (PBS) (pH 7.2). Immunizationwas performed three times at 5-d intervals. Preliminary ex-periments were performed to determine the most efficientconcentration of DNA for immunization. A dose-dependent(data not shown) immune response was observed, and wechose the optimal concentration for all experiments.

2.5. Delayed-type hypersensitivity (DTH)

DTH was measured as described elsewhere [21]. Theanimals received 20 µl of PBS containing UV-inactivatedHSV-1 (KOS). Ear thickness was recorded after 48 h with ascrew gauge micrometer (Oditest; H.C. Kroeplin GmbH,Schluechtern, Germany) and recorded as mean increase inthickness.

2.6. Sample collection

Serum samples from the mice were collected by retro-orbital bleeding. Vaginal lavage fluid was obtained by intro-ducing 100 µl of PBS (pH 7.2) into the vaginal canals andthen recovering it with a micropipette. Fecal samples wereweighed and suspended at 100 mg/ml in PBS containing0.1% sodium azide. Each sample was stored at –80 C untilused.

2.7. ELISA for gB-specific antibodies

For detection of antibodies (Abs) against gB, ELISAplates were coated with gB protein (kindly provided byChron Corporation, Emeryville, CA) at a concentration of

572 S. Lee et al. / Microbes and Infection 5 (2003) 571–578

2 µg/ml. A standard ELISA was performed to determine thegB-specific antibodies in the samples, and the concentrationwas determined. Briefly, after overnight incubation at 4 °Cwith gB protein, goat anti-mouse immunoglobulin G (IgG)(Southern Biotechnology Associate Inc, Birmingham, AL),goat anti-mouse IgG1 (Southern Biotechnology AssociateInc, Birmingham, AL), goat anti-mouse IgG2a (SouthernBiotechnology Associate Inc, Birmingham, AL), or rabbitanti-mouse IgA (Zymed, San Francisco, CA), the plates werewashed three times with PBS containing 0.05% Tween-20,and blocked by using PBS (pH 7.2) with 3% non-fat driedmilk for 2 h at 37 °C. A total of 200 µl of serum samples wereadded in duplicate and serially diluted twofold to a final100 µl final volume. Wells coated with goat anti-mouse IgG,IgG1, IgG2a, or IgA were treated with serially diluted stan-dard IgA (Zymed, San Francisco, CA), mouse IgG (SouthernBiotechnology Associate Inc, Birmingham, AL), mouseIgG1 (Southern Biotechnology Associate Inc, Birmingham,AL), or mouse IgG2a (Southern Biotechnology AssociateInc, Birmingham, AL). Plates were incubated for 2 h at37 °C. After being washed, 100 µl of goat anti-mouse IgGconjugated HRP (Southern Biotechnology Associate Inc,Birmingham, AL), goat anti-mouse IgG1-HRP (SouthernBiotechnology Associate Inc, Birmingham, AL), or goatanti-mouse IgG2a-HRP (Southern Biotechnology AssociateInc, Birmingham, AL) was added. To measure the IgA levelin vaginal lavage fluid and fecal samples, biotinylated goatanti-mouse IgA (Zymed, San Francisco, CA) was added for2 h at 37 °C, followed by peroxidase-conjugated streptavidin(Jackson Immuno Research Lab, West Grove, PA). The colorwas developed by adding the substrate solution (11 mg of2,2-azinobis-3-ethylbenxothiazoline-6-sufomic acid in 25ml of 0.1 M citric acid, 25 ml of 0.1 M sodium phosphate,10 µl of hydrogen peroxide). Ab concentrations were calcu-lated with an automated ELISA reader (Spectra MAX 340;Molecular Devices, Sunnyvale, CA).

2.8. HSV-specific lymphoproliferation

This assay has been described elsewhere [19]. Briefly, atday 14 following the final immunization, the splenocytes ofcoimmunized mice were enriched for T cells by a nylon woolcolumn and used as responder populations. These T cellswere restimulated in vitro with irradiated syngeneic enrichednaive dendritic cells (DCs) or DCs infected with UV-inactivated HSV (MOI of 1.5 before UV inactivation) andincubated for 5 d at 37 °C. ConA (5 µg/ml) was used as apolyclonal positive control and incubated for 3 d. Eighteenhours before harvesting, [3H] thymidine was added to thecultures.

2.9. Cytokine assay

For the cytokine (IFN-c and IL-4) assay, splenocytes frommice were suspended in 10% RPMI-1640, and 106 cells in1 ml were stimulated in vitro with irradiated syngeneic en-riched DCs pulsed with UV-inactivated HSV (MOI, 5.0 be-

fore UV inactivation). Similar numbers of cells were ConAstimulated (5 µg/106 cells/ml) in 96-well plates. Plates wereincubated at 37 °C for 72 h. These supernatants werescreened for the presence of IFN-c and IL-4 by ELISA, asdescribed previously [4].

2.10. Vaginal challenge

The mice were pretreated with progesterone to synchro-nize their estrous cycles, as described previously [22].BALB/c mice were injected subcutaneously with Depo-Provera (DP) (Upjohn Co., Kalamazoo, MI) at 2 mg permouse. Five days following the injection of DP, the micewere challenged intravaginally with 105 [50 LD50] PFU ofHSV-1 McKrae.

2.11. Virus recovery and titration

Vaginal washings were collected at various time pointspost-challenge, by micropipetting 100 µl of PBS into thevaginal cavity and then recovering it. The samples werestored at –80 °C until used. For detection and quantification,the samples (50 µl of each sample) were further diluted, andviral titers were determined by a plaque assay performed onVero cells as described elsewhere [23].

2.12. Determination of vaginal IFN-c secretion

Vaginal lavage fluid for IFN-c secretion was collecteddaily by introducing 100 µl of PBS (pH 7.2) into the vaginalcanals post-challenge and then recovering it with micropi-pette. The vaginal mucus was subsequently removed fromthe fluid by centrifugation at 10,000 rpm for 1 min. IFN-cconcentrations in vaginal lavages were determined by ELISAusing IFN-c anti-mouse Ab and biotinylated IFN-c Ab.

2.13. Statistical analysis

Significant differences among groups were evaluated us-ing Student’s t-test.

3. Results

3.1. Enhancement of humoral and cellular responsesfollowing intranasal or intramuscular coadministrationof plasmid DNA encoding IFN-c-inducing cytokines

Animals were given plasmid DNA encoding IFN-c-inducing cytokines (IL-18 or IL-12) together with gB DNAvaccine systemically or mucosally, and levels of serum andmucosal antibodies were measured by ELISA. As controls,additional groups of mice were immunized with UV-inactivated HSV or gB DNA mixed with control plasmid(pCDNA). As recorded in Table 1, systemic coadministrationwith IL-18, but not IL-12, led to enhanced serum antibodylevels. The response pattern emphasized that of IgG2a (Th1

573S. Lee et al. / Microbes and Infection 5 (2003) 571–578

pattern). Systemic immunization resulted in undetectabledistal IgA responses. Mucosal immunization, in contrast,resulted in IgA responses in both vaginal washings and feces.In this instance, both IL-12 and IL-18 coadministration pro-vided enhanced mucosal responses (serum antibody also),with IL-18 superior to the IL-12 enhancement.

The pattern of T cell responses in spleen revealed that bothIL-12 and IL-18 coadministration with gB DNA enhancedresponses measured by antigen-specific proliferation andIFN-c production (Table 2) as well as the DTH reaction(Table 3). The pattern was the same whether the immuniza-tion was systemic or mucosal, although the former inducedhigher responses.

3.2. Coadministration of IL-12 DNA or IL-18 DNAimproves the survival rate following intravaginal HSV-1challenge

Mice immunized as described in the section above werechallenged vaginally with 105 PFU (50 LD50) of HSV-1McKrae. The result shown in Table 4 indicates that systemicimmunization with gB DNA and gB DNA plus IL-12 orIL-18 induced protective immunity. All groups immunizedwith gB DNA were protected from HSV vaginal challenge.However, those given gB DNA plus either IL-12 or IL-18 hadhigher levels of protection compared to gB DNA (plus con-trol vector DNA)-immunized mice.

Table 1Serum and distal mucosal antibody responses following systemic and mucosal coadministration of gB DNA plus cytokines a

ImmunizationAnti-gB serum antibody Mucosal anti-gB antibody

IgG (ng/ml) IgG2a/IgG1 ratio Vaginal IgA (ng/ml) Fecal IgA (ng/ml)Systemic immunizationgB DNA + pCDNA 885 ± 162 12 <0.1 <10+ IL-12 DNA 369 ± 121 39 c <0.1 <10+ IL-18 DNA 1585 ± 789 b 47 d <0.1 <10HSV 58,731 ± 9875 195 <0.1 <10

Mucosal immunizationgB DNA + PC DNA 107 ± 36 4 0.7 ± 0.2 32.7 ± 18.8+ IL-12 DNA 399 ± 77 17 3.5 ± 1.2 107.6 ± 29.1+ IL-18 DNA 609 ± 173 26 6.4 ± 2.2 110.4 ± 31.6HSV 25,356 ± 3242 109 25 ± 7 232 ± 49

a Groups of animals were immunized either systemically or mucosally with 100 plus 200 mg of control vector DNA, IL-12 DNA, or IL-18 DNA. Theimmunization was repeated three times at 5-d intervals. Ten days following the last immunization, sera, feces and vaginal fluids were collected, and the level ofgB-specific antibodies was measured by ELISA. Vaginal IgA responses were measured from pooled samples, and fecal samples were resuspended at aconcentration of 100 mg/ml in PBS-0.1% sodium azide.

b Statistically significant between systemic gB DNA plus IL-18 DNA and mucosal gB DNA plus IL-18 DNA immunization (P < 0.01).c Statistically significant between systemic gB DNA plus IL-12 DNA and mucosal gB DNA plus IL-12 DNA immunization (P < 0.001).d Statistically significant between systemic gB DNA plus IL-18 DNA and mucosal gB DNA plus IL-18 DNA immunization (P < 0.001).

Table 2HSV-specific Th cell proliferation and cytokine productions of spleen in mice following systemic or mucosal coadministration of gB DNA plus cytokine DNAsa

ImmunizationResponses

Proliferation (cpm) Proliferation index IFN-g (ng/ml) IL-4 (ng/ml)Systemic immunizationgB DNA + pCDNA 8399 ± 1725 25.4 4.8 ± 1.6 <0.01+ IL-12 DNA 16,795 ± 3635 b 65.1 c 23.4 ± 2.3 c <0.01+ IL-18 DNA 17,241 ± 2981 d 66.7 e 25.9 ± 1.9 e <0.01

Mucosal immunizationgB DNA + pCDNA 1676 ± 216 3.9 1.8 ± 0.4 <0.01+ IL-12 DNA 5314 ± 319 12.5 5.4 ± 1.6 <0.01+ IL-18 DNA 5481 ± 520 12.7 6.2 ± 2.7 <0.01

a Groups of animals were immunized either systemically or mucosally with 100 plus 200 µg of control vector DNA, IL-12 DNA, or IL-18 DNA. Mice weresacrificed 12 d following the last immunization. Proliferation indices were calculated by cpm of HSV stimulation/cpm of unstimulation. For the measurementof IFN-c and IL-4, the T cells were restimulated in vitro with irradiated DCs infected with UV-inactivated HSV-1 KOS. Seventy-two hours later, culturesupernatants were collected and analyzed for IFN-c and IL-4 by ELISA.

b Statistically significant between systemic gB DNA plus IL-12 DNA and mucosal gB DNA plus IL-12 DNA immunization (P < 0.05).c Statistically significant between systemic gB DNA plus IL-12 DNA and mucosal gB DNA plus IL-12 DNA immunization (P < 0.001).d Statistically significant between systemic gB DNA plus IL-18 DNA and mucosal gB DNA plus IL-18 DNA immunization (P < 0.05).e Statistically significant between systemic gB DNA plus IL-18 DNA and mucosal gB DNA plus IL-18 DNA immunization (P < 0.001).

574 S. Lee et al. / Microbes and Infection 5 (2003) 571–578

In the mucosal immunized mice, whereas HSV immuni-zation resulted in excellent immunity, those immunized withgB DNA (with or without IL-12 or IL-18) had only partiallevels of resistance. Thus, all animals died from challenge,but some level of immunity was present, as indicated bydifferences in the average day of death measurements. Themost extended average day of death was observed in micemucosally immunized with gB DNA plus IL-18. As a furthermeasure of immunity, vaginal washings were collected frommice at various times, both to determine viral titers and levels

of IFN-c. As evident in Fig. 1, IFN-c was detectable invaginal washings by 1 d post-challenge in mice systemicallyimmunized with gB. Once again, the levels were higher inthose animals that received coimmunization with IL-12 orIL-18. By day 3–4 post-challenge, significant levels of IFN-chad disappeared, as had levels of detectable virus (Table 5).

In mucosally immunized mice, the pattern of both theIFN-c response and virus levels differed from that of thesystemic immune recipients. Here two peaks of IFN-c pro-duction were observed in all groups (including naïve andcontrols), with the first peak (elevated in gB plus IL-12 orIL-18 animals) occurring on day 2. This pattern was taken toindicate only partial levels of immunity.

Table 3Development of DTH in DNA- or HSV-immunized mice a

ImmunizationMean (±S.D.) increase in ear thickness Overprechallange thickness at 48 h (10–2 mm)UV HSV Vero extract

Systemic immunizationgB DNA + pCDNA 6.5 ± 2.1 2.0 ± 0.7+ IL-12 DNA 10.8 ± 2.2 b 2.7 ± 1.2+ IL-18 DNA 11.2 ± 1.8 c 1.8 ± 2.1HSV 13.5 ± 2.2 2.2 ± 0.4

Mucosal immunizationgB DNA + pCDNA 4.8 ± 1.4 2.3 ± 0.6+ IL-12 DNA 6.4 ± 1.1 2.5 ± 1.8+ IL-18 DNA 7.0 ± 1.4 2.0 ± 1.5HSV 10.6 ± 1.3 1.9 ± 2.4

a Groups of animals (n = 7) were immunized with one of the immuno-gens at day 0, 5 and 10. As a positive control, 106 PFU of HSV-1 KOS wasgiven intramuscularly or intranasally. For the DTH assay, each mouse wasinjected with either 105 PFU of UV HSV in the right ear pinna or Veroextract in the left ear pinna.

b Significantly different from values obtained for mice immunized intra-nasally with gB DNA plus IL-12 DNA (P < 0.05).

c Significantly different from values obtained for mice immunized intra-nasally with gB DNA plus IL-18 DNA (P < 0.05).

Table 4Resistance to vaginal challenge in DNA coimmunized mice a

ImmunizationNo. of mice survived/no. of mice challenged

Survivalrate (%)b

Average timeof death ± S.D. (d)

Naive 0/7 0 5.3 ± 1.2

SystemicimmunizationgB DNA +pCDNA

10/14 71.4 9

+ IL-12 DNA 14/14 100 ND c

+ IL-18 DNA 14/14 100 NDHSV 7/7 100 ND

MucosalimmunizationgB DNA +pCDNA

4/14 28.6 6.4 ± 1.0

+ IL-12 DNA 10/14 71.4 11.0 ± 1.1+ IL-18 DNA 11/14 78.6 12.2 ± 2.3HSV 7/7 100 ND

a Groups of animals (n = 14) were challenged with HSV-1 Mckrae 2weeks following the last immunization.

b In the case of mucosal immunization, the survival rate was counted 8 dfollowing vaginal challenge.

c ND, no death was observed until the end of the experiment.

Fig. 1. Vaginal IFN-c secretion following vaginal infection with HSV.Groups of mice (n = 12) coimmunized either systemically (A) or intranasally(B) with IL-12 or IL-18 plasmid DNA along with gB DNA were intravagi-nally challenged, and vaginal lavage fluid was collected every day. VaginalIFN-c concentrations were determined by ELISA. The results are expressedas a mean for 12 mice.

575S. Lee et al. / Microbes and Infection 5 (2003) 571–578

3.3. Role of IFN-c in protection from vaginal challenge

Since the above results and the work of others [24] indi-cate a role for IFN-c in protection from vaginal challenge,protective immunity was compared in IFN-c–/– (GKO) im-munized and wild-type mice. As evident in Table 6, the levelsof protective immunity against vaginal challenge induced inGKO mice were markedly less than those induced by sys-temic immunization of wild-type mice. Curiously the GKOmice responded to immunization, producing both Ab andantigen-specific T cell proliferative responses, with enhance-ment resulting from IL-12 and IL-18 coimmunization. How-ever, the in vitro serum IgG responses of GKO mice werearound 10-fold less than those observed in wild-type mice.Mucosal immunization of GKO mice generated minimalresponses. All mice died within 4–9 d regardless of the typeof immunization they received. These results show the neces-sity of IFN-c production for protection from HSV vaginalinfection and suggest that the enhanced protection affordedby the coadministration of IL-12 or IL-18 is due to their rolein the induction of IFN-c.

4. Discussion

The aim of our investigation was to improve the effective-ness of DNA vaccines against HSV infection. We chose

coimmunization with DNA encoding cytokines known toemphasize components of immune defense that best corre-late with immune protection. These include interferon-producing T and NK cells and the IgG2a isotype immunoglo-bulin [7,15,25]. Our results show that the coadministration ofplasmid DNA encoding IL-12 or IL-18 along with gB DNAimproves immune induction. Recipients of the coimmuniza-tion procedure had elevated humoral as well as IFN-c-producing T cell responses and showed greater resistance tovaginal challenge with a lethal dose of HSV-1. The adjuvanteffects were observed when the vaccines were administeredeither systemically or mucosally. By most assays, the adju-vant effect of IL-18 was superior to IL-12, although even gBDNA plus IL-18 failed to induce levels of immunity achievedby UV-inactivated HSV immunization. Mucosal immuniza-tion proved as an effective means of inducing systemic im-munity, but was less effective than the systemic route forinducing protection from vaginal challenge. Our results alsodemonstrated that protection from such challenges wasmainly a property of IFN-c. Thus, immunized IFN-c–/– miceremained susceptible to challenges even while generatingreadily measurable immune responses. The approach of us-ing DNA vaccines combined with DNA encoding cytokinesholds promise and represents a potentially useful approachfor vaccines.

Several previous investigations have explored the use ofcytokines as well as chemokines for adjuvant effects whenadministered with vaccines [26–28]. In some instances, theinfluence of multiple proteins was also studied [29]. A previ-ous report looked at the adjuvant effects of several cytokinesand chemokines on HSV vaccine efficacy in mice and dem-onstrated that systemic immunization with IL-2, IL-18 orMIP-1a enhanced IgG responses as well as T cell responses[4,30]. Our study confirms that IL-12 and IL-18 enhanceimmunity and extends the analysis to the mucosal immuni-zation route. Moreover, challenges were made at a mucosalsite distal to the site of immunization. Thus, in both theWeiner report and the present studies, the enhancement ofeffective immunity was achieved with IL-12 and IL-18 coad-ministration. Previous investigations had not evaluated theadjuvant effects of IL-18 given mucosally. Although thisim-munization route was effective at inducing systemic immu-nity, it proved disappointing with respect to functional immu-

Table 5Virus titration

Immunizationlog10 of viral titer at day post-challenge

1 2 3 4 5 6Systemic immunizationgB DNA + pCDNA 4.8 ± 0.2 4.4 ± 0.3 2.7 ± 0.3 1.6 ± 0.2 – –+ IL-12 DNA 4.9 ± 0.3 3.5 ± 0.2 1.9 ± 0.2 – – –+ IL-18 DNA 4.8 ± 0.4 3.2 ± 0.3 1.3 ± 0.2 – – –

Mucosal immunizationgB DNA + pCDNA 4.9 ± 0.3 4.7 ± 0.2 4.2 ± 0.5 3.6 ± 0.4 2.5 ± 0.4 1.3 ± 0.2+ IL-12 DNA 4.8 ± 0.5 4.3 ± 0.4 3.5 ± 0.5 2.7 ± 0.6 1.7 ± 0.4 –+ IL-18 DNA 4.9 ± 0.3 4.2 ± 0.5 3.3 ± 0.3 2.1 ± 0.2 1.4 ± 0.5 –

Table 6Challenge experiment in GKO mice

Immunization No. of mice survived/no. of mice challenged

Survivalrate (%)

Average timeof death ± S.D. (d)

Naive 0/3 0 5.2 ± 1.6

SystemicimmunizationgB DNA +pCDNA

0/6 0 6.3 ± 2.5

+ IL-12 DNA 0/6 0 6.8 ± 3.2+ IL-18 DNA 0/6 0 7.1 ± 2.4

MucosalimmunizationgB DNA +pCDNA

0/6 0 6.0 ± 0.8

+ IL-12 DNA 0/6 0 5.4 ± 1.2+ IL-18 DNA 0/6 0 5.8 ± 1.9

576 S. Lee et al. / Microbes and Infection 5 (2003) 571–578

nity at mucosal surfaces. Accordingly, although IgA-mediated immunity was detectable at mucosal sites, animalswere in fact less resistant to mucosal challenge when micewere immunized systemically (and which had undetectablemucosal IgA responses). The reasons for such susceptibilityremain to be established, but clearly the explanation was nota lack of antigen recognition, since systemic immunity oc-curred. We are currently evaluating whether the mucosalimmunization route results in suppressed Th1-mediated im-munity via IL-10 production. In this regard, Akbari et al. [31]observed that intranasal immunization with OVA proteinmay induce the CD4+ T cell tolerance mediated by pulmo-nary DCs that produce IL-10. In line with these studies, wehave observed, by RT-PCR, IL-10 production in mediastinallymph nodes following intranasal immunization with gBDNA, but not in popliteal lymph nodes following intramus-cular immunization (unpublished results). This issue is underfurther investigation.

We presume that the adjuvant effect of IL-18 coadminis-tration was explained by the well-known stimulatory effectof this cytokine on IFN-c production [32]. Thus, the bestcorrelate of resistance to mucosal challenge with HSV ap-peared to be IFN-c production by either NK cells or T cells,as was elegantly shown by the Milligan and Bernstein [33].We were able to show that IL-18 (and in most instancesIL-12) enhanced the levels of systemic IFN-c-producing Tcells, but could not demonstrate such cells in vaginal tissuesthemselves after immunization (data not shown).

This report extends the evidence that mucosal immunitydepends on IFN-c production by showing that systemicallyimmunized and responding GKO mice, unlike wild-typeanimals, failed to resist mucosal challenge. Such observa-tions are at variance with previous observations using sys-temic challenge, where immunized GKO did manifest immu-nity [34,35]. However, in support of the present investigators,Eriksson et al. demonstrated that protection could not berestored in vaccinated GKO mice in HSV-2 genital infection[24]. Three different knockout mice were used (IL-12–/–,IL-18–/– and GKO). Interestingly, in such studies, immunizedIL-12–/– and IL-18–/–, unlike the GKO animals, survived avaginal challenge.

In conclusion, our results serve to emphasize that DNAvaccines can achieve greater efficacy by using cytokines forcoimmunization, and IL-18 appeared to represent a valuablechoice for such purpose.

Acknowledgements

This work was supported by NIH (National Institutes ofHealth) grants AI 14981 and AI 46462.

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