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Enhancement of DNA vaccine potency against herpes simplex virus 1 by co-administration of an interleukin-18 expression plasmid as a genetic adjuvant

^1,3Department of Biophysics, Institute of Basic Medical Sciences¹ and Department of Ophthalmology, Peking Union Hospital³, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, PR China ²Department of Pharmacology, Beijing Institute of Ophthalmology, Beijing 100062, PR China

Correspondence Guoxing Song songgx{at}ms.imicams.ac.cn

Received 13 June 2002 Accepted 15 November 2002

	Abstract

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In this study, the immune-modulatory and vaccine effects of using an interleukin (IL)-18 expression plasmid as a genetic adjuvant to enhance DNA vaccine-induced immune responses were investigated in a mouse herpes simplex virus 1 (HSV-1) challenge model. BALB/c mice were immunized by three intramuscular inoculations of HSV-1 glycoprotein D (gD) DNA vaccine alone or in combination with a plasmid expressing mature IL-18 peptide. Both the serum IgG2a/IgG1 ratio and T helper 1-type (Th1) cytokines [IL-2 and interferon (IFN)-] were increased significantly by the co-injection of the IL-18 plasmid compared with the injection of gD DNA alone. However, the production of IL-10 was inhibited by IL-18 plasmid co-injection. Furthermore, IL-18 plasmid co-injection efficiently enhanced antigen-specific lymphocyte proliferation and the delayed-type hypersensitivity response. When mice were challenged with HSV-1 at the cornea, co-injection of IL-18 plasmid with gD DNA vaccine showed significantly better protection, manifested as lower corneal lesion scores and faster recovery. These experiments indicate that co-injection of an IL-18 plasmid with gD DNA vaccine efficiently induces Th1-dominant immune responses and improves the protective effect against HSV-1 infection.

	Introduction

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Nucleic acid immunization is an important vaccination strategy that has many characters desirable for an ideal vaccine, including induction of broad immune responses (humoral and cellular), long-lasting immunity and simple and cheap production. This technique is being explored as a vaccination strategy against a variety of infectious diseases, autoimmune diseases and cancers. The first generation of DNA-immunization experiments have shown that delivery of DNA constructs encoding a specific immunogen into the host could elicit effective immune responses in vivo in a safe and well-tolerated manner in various model systems. However, more efficacious and specific immune responses against the target pathogen are required in order to enhance its clinical utility. One strategy is the use of molecular adjuvants. Molecular or genetic adjuvants are different from the traditional adjuvants in that they consist of gene-expression constructs encoding immunologically important molecules, such as cytokines, chemokines and co-stimulatory molecules (Iwasaki et al., 1997; Kim et al., 1997, 2000; Sin et al., 1999). Previous reports have shown that co-administration of genetic adjuvant constructs with immunogen constructs can modulate antigen-specific immune responses (Kim et al., 1998, 1999a).

Interleukin (IL)-18, first designed as an interferon (IFN)--inducing factor, is a recently identified cytokine of the T helper 1 (Th1) type. It has been known to induce IFN- production by both CD4⁺ T cells and natural killer (NK) cells (Okamura et al., 1995; Ushio et al., 1996) and to stimulate naive T cells to promote the development of Th1 cells (Kohno et al., 1997). Since IFN- is one of the most important cytokines that contributes to host defence, a cytokine capable of up-regulating IFN- should also play a key role in host defence. Indeed, IL-18 plays a critical role in the eradication of various pathogens including Leishmania major (Ohkusu et al., 2000), Mycobacterium leprae (Garcia et al., 1999), encephalomyocarditis virus (Tovey et al., 1999), human immunodeficiency virus (Billaut-Mulot et al., 2001) and herpes simplex virus (HSV) (Fujioka et al., 1999).

HSV is the causative agent of a spectrum of human diseases including ocular infections, encephalitis and genital infection. Immunizing animals with recombinant glycoprotein D (gD) protein, one of 11 known HSV glycoproteins, provides effective protection against both HSV-1 and HSV-2 infection in mice (Keadle et al., 1997; Corey et al., 1999). Similarly, gD DNA vaccine also protects mice against challenge by HSV-1 or HSV-2 (Bourne et al., 1996; Inoue et al., 2000). Since it has the best immunogenicity among the 11 glycoproteins and is highly conserved and antigenically cross-reactive between HSV-1 and HSV-2, gD has become the most important candidate immunogen.

Several cytokine genetic adjuvants including IL-2, IL-12 and granulocyte-macrophage colony-stimulating factor have been used in combination with gD DNA vaccine in immunization protocols to induce more protective immune responses against HSV (Sin et al., 1998, 1999; Inoue et al., 2000). The use of a plasmid encoding the Th1-inducing cytokine IL-18 has also been reported to exert immunomodulatory properties in vivo (Kim et al., 1999b; Kremer et al., 1999). Based on these observations, in this study, we tested the immune-modulatory and vaccine effects of using an IL-18 expression plasmid as a genetic adjuvant to enhance gD DNA vaccine-induced preventive immune responses in a mouse HSV-1 challenge model.

	METHODS

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Virus.

HSV-1 KOS strain was propagated in 2BS cells. At maximum cytopathic effect, the viruses were harvested by three cycles of freezing and thawing. After centrifugation at 5000 r.p.m. for 5 min, the supernatant was aliquotted and stored at -80 °C before use.

DNA plasmids.

The two plasmids pgD (pcDNA3.1-gD), encoding HSV-1 gD protein, and pIL-18 (pcDNA3.1-IL-18), the IL-18 expression plasmid, were constructed and identified in our laboratory as described previously (Zhu et al., 2002). Briefly, the whole gD gene was amplified by PCR from the HSV-1 genome and then inserted into pcDNA3.1 to give the gD expression plasmid. pIL-18 was constructed by inserting the kappa leader sequence-fused mature human IL-18 cDNA, which was obtained by PCR from a human tonsil cDNA library, into the pcDNA3.1 backbone. For DNA immunization, plasmid DNA of pcDNA3.1, pgD and pIL-18 was prepared using the Endofree Plasmid Giga kit (Qiagen).

DNA inoculation of mice.

Six- to eight-week-old female BALB/c mice were used in this study. Two days before DNA inoculation, the quadriceps muscles were injected with 100 µl of a solution containing 0.25 % bupivacaine hydrochloride to enhance subsequent DNA absorption. For DNA inoculation, 100 µg of each DNA construct in PBS was injected into the same region of the muscle as the bupivacaine injection. Since our preliminary experiments showed no difference in immune responses when inoculating 100 µg pgD either alone or together with 100 µg empty pcDNA3.1, in our present study, the gD-immunization group was injected with pgD plasmid alone. Co-administration of pIL-18 involved mixing the chosen plasmids prior to injection. The mice were boosted with the same dose at weeks 2 and 4.

ELISA.

The induced antibody response was quantified by ELISA as described previously (Zhu et al., 2002). In particular, HSV-1 gD (ViroStat) at 2 µg ml^-1 in carbonate/bicarbonate buffer was used as a coating antigen. To determine ELISA titres, sera were serially diluted to 1 : 1000, 1 : 2000, 1 : 3000, 1 : 4000, 1 : 5000, 1 : 6000, 1 : 7000 and 1 : 8000. The end-point titres were expressed as the last reciprocal serum dilution that gave an absorbance greater than 0.1 and at least twofold higher than the absorbance of control mouse sera at the same dilution. For the determination of relative levels of gD-specific IgG subclasses, an ELISA was performed with biotinylated goat anti-mouse IgG1 and IgG2a (Pharmingen) and horseradish peroxidase-conjugated streptavidin.

Splenocyte-proliferation assay.

Lymphocytes were prepared from mouse spleens. The isolated cell suspensions were resuspended to a concentration of 4 x 10⁶ cells ml^-1. A 100 µl aliquot containing 4 x 10⁵ cells was added immediately to each well of a 96-well flat-bottomed microtitre plate. One-hundred microlitres of HSV-1 gD protein was added to wells in duplicate, to a final concentration of 10 µg ml^-1. The cells were incubated at 37 °C in 5 % CO₂ for 66 h. One microcurie of ³H-thymidine was added to each well and the cells were incubated continuously for 6–8 h at 37 °C. The plate was harvested and the amount of ³H-thymidine incorporated was measured in a Beta plate-reader. To ensure that cells were healthy, 10 µg ConA ml^-1 was used as a polyclonal stimulator-positive control. The stimulation index (SI) was calculated as experimental c.p.m./spontaneous c.p.m.

Th1 and Th2 cytokine assays.

Cytokines were assayed in supernatants of spleen cells (4 x 10⁶ cells ml^-1) cultured for 72 h in the presence of HSV-1 gD protein (10 µg ml^-1). IL-2, IL-10 and IFN- levels were determined by using commercial cytokine kits (Diaclone).

Delayed-type hypersensitivity (DTH) assay.

Two weeks after the final immunization, mice were injected in the dorsal side of each pinna. The right pinna was injected with 10 µl (1 x 10⁶ p.f.u. ml^-1) UV light-inactivated HSV antigen. The left pinna was injected with the same amount of supernatant of 2BS cell lysate as a control. Forty-eight h later, the thickness of each ear was measured with an engineer's micrometer. The DTH response in each mouse was expressed as the difference in thickness between the left and right pinnae.

Virus challenge of the cornea and evaluation of the results.

Two weeks after the last immunization, all corneas of the mice were scarified in a criss-cross pattern 10 times with a 27-gauge needle. Five microlitres of solution containing 1 x 10⁶ p.f.u. virus ml^-1 was instilled into the conjunctival sac of each eye.

Clinical evaluation of viral infection challenge was carried out as described previously (Inoue et al., 2000). Briefly, every day from day 1 to day 5 and on days 7 and 9 after the viral challenge, the same observer examined the eyes with a slit-lamp biomicroscope and scored the severity of epithelial lesions by the following criteria: 0, no epithelial lesion or punctate epithelial erosion; 1, stellate keratitis or residue of dendritic keratitis; 2, dendritic keratitis occupying less than a quarter of the cornea; 3, dendritic keratitis occupying a quarter to half of the cornea; 4, dendritic keratitis extending over more than half of the cornea.

Statistical analysis.

Statistical analysis was performed using Student's t test and the Kruskal–Wallis H test. Values were compared between different immunization groups. P values < 0.05 were considered statistically significant.

	RESULTS

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Antibody responses

To determine whether co-injection of IL-18 expression plasmid (pIL-18) with gD DNA vaccine (pgD) could influence the humoral immune response against gD, sera obtained 2 weeks after the final DNA inoculation were tested by ELISA. When pIL-18 was co-injected, the geometric mean titre was increased to about 6000, significantly higher than the group immunized with pgD alone, in which group the mean titre was only about 4400 (P < 0.05).

To characterize the immune response elicited against gD antigen, IgG isotypes were identified. The results showed that pIL-18 co-injection resulted in a significant increase in the IgG2a/IgG1 ratio, indicating the dominance of Th1 cell function in the humoral immune response (Table 1).

Splenocyte proliferation

Splenocyte proliferation is a standard parameter used to evaluate the potency of cell-mediated immunity. It was carried out 2 weeks after the last immunization. As shown in Table 2, a low background level of proliferation was observed in the negative control. However, gD DNA vaccine-stimulated cells had an enhanced proliferative response. When the mice were co-injected with pIL-18, the level of splenocyte proliferation was further increased.

Levels of Th1 and Th2 cytokines

Th1 and Th2 cytokines play different roles in the polarization of immune responses. Th1 cytokines are thought to drive induction of cellular immunity, whereas Th2 cytokines preferentially drive humoral immunity. To understand the role of IL-18 in the development of immune responses, we examined the effect of co-injection of pgD with and without pIL-18 on changes in Th1 and Th2 phenotypes. As shown in Fig. 1, co-injection of pIL-18 significantly increased the production of IFN- and IL-2 (Th1-type) compared with pgD alone. In contrast, the IL-10 (Th2-type) level was decreased by pIL-18 co-injection.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Levels of cytokine production from splenocytes after gD stimulation in vitro. Groups of mice (n = 2) were immunized with 100 µg pgD alone (shaded bars) or in combination with 100 µg pIL-18 (filled bars) at 0, 2 and 4 weeks; pcDNA3.1-immunized mice (open bars) were used as a negative control. Two weeks after the final immunization, mice were sacrificed and the splenocytes were isolated. Splenocytes were then stimulated with 10 µg gD protein ml-1 for 3 days. The experiment was repeated with similar results; data from the two experiments were included in the analysis. Data are the means ± SD of four mice. Statistically significant differences (P < 0.05 using Student's t test) are indicated by * (compared with negative control) or ** (compared with pgD alone).

DTH

As shown in Table 3, intradermal injection of HSV antigen in the pinna resulted in a significant DTH response in mice immunized with pgD compared with negative controls. The DTH response was further increased in the pgD+pIL-18 group to a level significantly higher than that in the group that received pgD alone (P < 0.05).

Results of viral challenge

In control mice, epithelial lesion scores peaked on day 2 after viral challenge and then declined gradually. About 7 days after the challenge, all the epithelial lesions had recovered. However, pgD limited the lesion score to a level significantly lower than that observed in the negative control. When the mice were co-injected with pIL-18, the epithelial lesions were further reduced and the recovery time was also shortened significantly (P < 0.05; Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Clinical scores for severity of epithelial keratitis. Groups of mice (n = 7) were immunized with 100 µg pgD alone () or in combination with 100 µg pIL-18 () at 0, 2 and 4 weeks; pcDNA3.1-immunized mice () were used as a negative control. Two weeks after the final immunization, virus was instilled into the conjunctival sac as described in Methods. Values are mean scores and bars reflect SD. Statistically significant differences (P < 0.05 using Kruskal–Wallis H test) are indicated by * (compared with negative control) or ** (compared with pgD alone).

	DISCUSSION

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Cytokines play important roles in the immune and inflammatory responses as indicators and regulators of the immune network (Cohen et al., 1998). Recombinant cytokines have been used clinically in the treatment of human diseases including cancers and infectious diseases (Nash et al., 1993; Opal et al., 1998; Bukowski, 2000). However, the short half-life of recombinant cytokines and the side effects due to repetitive administration are still insoluble problems (Hara et al., 2000). Previous reports have shown that direct injection of cytokine genes into muscle resulted in the characteristic biological actions of these cytokines in vivo and could modulate immune responses (Iwasaki et al., 1997; Sin et al., 1999). However, there has been no study on the modulatory effect of using the human IL-18 gene as a molecular adjuvant to enhance the potency of DNA vaccine in an HSV-1 challenge model.

In the present study, we observed a significant increase in gD-specific IgG production through vaccine modulation with an IL-18 expression plasmid. This is compatible with a previous report (Kim et al., 1998). Although both IgG2a and IgG1 were increased, the increase in IgG2a was more remarkable compared with that of IgG1 and resulted in a significantly increased IgG2a/IgG1 ratio. Since the IgG2a isotype is driven by Th1 cells, while the IgG1 isotype is driven by Th2 cells, this result suggested that Th1 immune responses were dominant when pIL-18 was co-injected. This was further verified by the cytokine production profile; we observed that co-injection with pIL-18 induced both IL-2 and IFN- secretion, but appeared to inhibit IL-10 production. Furthermore, significant increases of lymphocyte proliferation and the DTH response were achieved by co-injection of pIL-18. We also investigated the induction of a gD-specific cytotoxic T lymphocyte (CTL) response by gD DNA vaccination, but no CTL activity was observed (data not shown), which was also consistent with previous reports (Ghiasi et al., 1995; Scott & Trinchieri, 1997; Cruz et al., 1999). In our experiments, the adjuvant activity cannot be attributed to immunostimulatory sequences at the DNA level, as a preliminary experiment showed that mixing pgD with pcDNA3.1 vector did not demonstrate similar immune-modulatory function (data not shown). Thus, the use of an IL-18 expression plasmid in gD DNA vaccination may be an effective approach for inducing a serum antibody response as well as cell-mediated immune responses.

Previous reports have shown that humoral or cellular immune responses or both are responsible for protective immunity against HSV infection (Price et al., 1975; Rager-Zisman & Allison, 1976; Nash & Cambouropoulos, 1993). During primary infection, neutralizing antibodies can inactivate free virus particles (Notkins, 1974). On the other hand, HSV-specific antibodies, which are present at high levels in humans, are insufficient to prevent HSV latency in the central nervous system (McKendall, 1983). Furthermore, it has been suggested that HSV-specific cellular immunity, mediated particularly by CD4⁺ and not CD8⁺ cells, play a major role in eradicating HSV-infected cells and controlling recurrent HSV infection (Sethi et al., 1983; Sin et al., 1999).

In this study, co-injection of pIL-18 increased protection against HSV corneal challenge significantly, compared with pgD alone. By co-injecting pIL-18 with pgD, corneal lesion scores were decreased significantly and recovery from the herpetic lesion was speeded up. This protective immunity might be attributed to both humoral immunity, which is interpreted as increased production of gD-specific IgG, and cellular immunity, which is interpreted as increased splenocyte proliferation, DTH response and levels of cytokine (IL-2 and IFN-) production, when pgD was co-injected with pIL-18. IFN- might play a critical role in the protective immunity, since we observed that co-injection with pIL-18 induced significant IFN- production from splenocytes in vitro. Many studies have revealed effects of IFN- on HSV infection (Neumann-Haefelin et al., 1985; Geiger et al., 1995; Cantin et al., 1995). IFN- takes effect in host resistance directly or via induction of an antiviral state in lymphocytes and macrophages (Landolfo et al., 1995). Furthermore, IFN- is also able to enhance NK cell activity, which has been reported to suppress HSV infection in vivo and in vitro (Reiter, 1993; Tanigawa et al., 2000).

The experiments described here demonstrate an enhanced immune response and protection against HSV challenge as measured 2 weeks after the final immunization. Clearly, it will be interesting to know how long these effects last in this animal model. This is the subject of further experimentation. Another issue that deserves to be addressed in future is whether this enhanced immune response might be preventive against establishment of HSV latency in the trigeminal ganglion, since this is the most distinct feature of this kind of virus.

In conclusion, the data presented here suggest that co-injection of an IL-18 expression plasmid with gD DNA vaccine could induce Th1-dominant immune responses efficiently, manifested as increases in the IgG2a/IgG1 ratio, IL-2 and IFN- but a decrease in IL-10, and achieve better protection against HSV-1 challenge. This study also demonstrates the potential of the IL-18 gene as a molecular adjuvant, which appears promising for the prevention of infectious diseases.

	Acknowledgments

 
This work was supported by grants from the Natural Science Foundation of Beijing and the Bureau of Health, Beijing, PR China.

	Footnotes

 
Abbreviations: DTH, delayed-type hypersensitivity; gD, glycoprotein D; HSV-1, herpes simplex virus 1; IFN, interferon; IL, interleukin.

	REFERENCES

TOP Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 

Geiger, K. D., Lee, M. S., Baugh, C. & Sarvetnick, N. E. (1995). Protective effects of interferon- in intraocular herpes simplex type 1 infection do not depend on major histocompatibility complex class I or class II expression. J Neurovirol 1, 405–409.[Medline]

Kim, J. J., Nottingham, L. K., Tsai, A. & 9 other authors (1999b). Antigen-specific humoral and cellular immune responses can be modulated in rhesus macaques through the use of IFN-, IL-12, or IL-18 gene adjuvants. J Med Primatol 28, 214–223.[Medline]

Kohno, K., Kataoka, J., Ohtsuki, T., Suemoto, Y., Okamoto, I., Usui, M., Ikeda, M. & Kurimoto, M. (1997). IFN--inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J Immunol 158, 1541–1550.[Abstract]

Neumann-Haefelin, D., Sundmacher, R., Frey, H. & Merk, W. (1985). Recombinant HuIFN- prevents herpes simplex keratitis in African green monkeys: demonstration of synergism with recombinant HuIFN- 2. Med Microbiol Immunol (Berl) 174, 81–86.[CrossRef][Medline]

Ushio, S., Namba, M., Okura, T. & 13 other authors (1996). Cloning of the cDNA for human IFN--inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol 156, 4274–4279.[Abstract]