INTRODUCTION
Gnathostoma spinigerum is a nematode parasite, of which larvae cause gnathostomiasis in humans and certain animals, and is prevalent mainly in Asia [1,2]. Recently, human gnathostomiasis has become an emerging disease among travellers from western countries who visit endemic areas [3,4]. Human beings are accidental hosts, infected by consuming raw or semi-cooked foods that are contaminated with the infective larvae. The parasite rarely develops into a mature worm in humans but can survive for a long time in the body. It usually migrates into the subcutaneous tissue and causes intermittent migratory swellings [5]. Sometimes Gnathostoma larvae reach the central nervous system, resulting in various signs and symptoms that may be life-threatening [6-9]. The anthelmintic drug, albendazole, has been used for the treatment of gnathostomiasis [10]. However, the efficacy of this drug treatment is not very satisfactory and frequent failure was reported after a long term follow-up study [11]. Therefore, vaccine development is an alternative approach for prevention and control of this disease.
Although strong antibody responses are induced by infection with G. spinigerum in humans and mice [12-17], protective immunity against challenge infection with the same parasite species remains unclear. This suggests that although the Gnathostoma antigens are immunogenic, the level of antibody responses may be insufficient for protection or may be biased by antibody isotype switching. Therefore, the use of an appropriate immuno-modulating strategy is necessary to obtain protective immunity by vaccination.
Unmethylated CpG oligonucleotides (CpG ODNs) are known to modulate both innate and adaptive immune responses through initiating Toll-like receptor 9 [18,19]. Activation of dendritic cells by CpG ODN induces cell maturation and production of proinflammatory cytokines such as interleukin (IL)-1, IL-6, TNF-α, and type 1 interferon, as well as T-helper 1 (Th1)-promoting cytokine IL-12 [20,21]. Delivery of CpG ODN with various antigens can enhance antigen-specific cell-mediated and humoral immunity [22-24]. CpG ODNs have been used as an adjuvant for enhancing immunity against various parasitic infections, including malaria [25-29].
In this study, mice were immunized with crude antigens of G. spinigerum with the combined adjuvant of CpG ODN 1826 and Montanide ISA720. The antibody responses and protective effects against G. spinigerum challenge infection was investigated.
MATERIALS AND METHODS
Mice and parasites
Male Swiss albino mice, weighing 25-30 g, were obtained from the National Laboratory Animal Centre, Salaya, Nakhon Pathom, Thailand, and kept in the Animal Unit of the Faculty of Medicine, Khon Kaen University. Five mice were randomly placed into each cage containing wood shavings. Rodent's chow and water were given ad libitum. All animal experiments were performed according the Guidelines for Animal Experimentation of the National Research Council of Thailand and the study was approved by the Animal Ethics Committee of Khon Kaen University (reference no. 0501.04/0013). G. spinigerum advanced third-stage larvae (AL3) were maintained in our laboratory according to procedures described previously [30] and used for antigen preparation and challenge infection.
Crude G. spinigerum antigen, oil-based adjuvant, and oligonucleotides (ODNs)
Crude somatic antigen of G. spinigerum AL3 was produced as described previously [17]. The ODNs used in this study were CpG 1826 (TCCATGACGTTCCTGACGTT; the underlined nucleotides represent the immunostimulatory residues) and the control non-CpG ODN 2138 (TCCATGAGCTTCCTGAGCTT) (Coley Pharmaceutical Group, Wellesley Hills, Massachusetts, USA). Montanide ISA720 (Seppic, Paris, France), an oil-based adjuvant, was also used.
Immunization and challenge infection
The experimental design is summarized in Table 1. Ten mice of each group were immunized with either crude G. spinigerum antigen (GsAg; 350 µg/mouse) or normal saline solution (NSS) (control). Crude antigen or NSS incorporated in Montanide ISA720 only, or in Montanide ISA720 with CpG ODN 1826 or non-CpG ODN 2138, was administered on days 0 and 21 post-immunization (PI) via subcutaneous injections and then on days 42 and 56 PI via intraperitoneal injections. Mice were challenged orally with 4 live G. spinigerum AL3 28 days after the last immunization (i.e. day 84 after the start of the experiment). All mice were sacrificed at day 196 after the start of the experiment. The worm burdens were determined by counting the Gnathostoma larvae in all organs under a light stereomicroscope (×15-45).
Antibody assay
Sera were collected 1 day before and then every 1-4 weeks after the first immunization, and G. spinigerum-specific antibody levels were assessed by ELISA. The dilutions of serum, horseradish peroxidase (HRP)-conjugated anti-mouse antibody, biotin-conjugated anti-mouse antibody, and avidin-HRP conjugate in each assay are shown in Table 2. A high binding capacity ELISA plate (Corning Incorporated, New York, USA) was coated with 100 µl of 2.5 µg/ml of GsAg in a coating buffer overnight at 4℃. After 3 washes with 0.05% Tween 20 in PBS (PBST), the wells were blocked with 250 µl of 3% bovine serum albumin (BSA; fraction V; Sigma, St. Louis, Missouri, USA) at room temperature for 1 hr. After washing, 100 µl of optimum dilutions of serum were added. After incubation at 37℃ for 1 hr, the wells were washed. For IgG and IgG2b determination, HRP-conjugated anti-mouse IgG and IgG2b antibodies (Zymed Laboratories, South San Francisco, California, USA) were added, respectively. After incubation at 37℃ for 1 hr, the wells were washed and o-phenylenediamine (OPD; Sigma) substrate solution was added and incubated at room temperature for 30 min before adding 8 N H2SO4 to stop the reaction. The optical density (OD) was read at the wave-length of 490 nm by an ELISA reader. For IgM, IgE, IgA, IgG1, IgG2a, and IgG3 determination, biotin-conjugated anti-mouse IgM, IgE, IgA, IgG1, IgG2a, and IgG3 antibodies and avidin-HRP conjugate (BD Pharmingen, California, USA) were used, respectively.
RESULTS
Antibody response after immunization with crude G. spinigerum antigen
To examine the kinetics of the antibody responses after immunization, sera were collected from mice of all groups and assessed for IgG, IgM, IgE, IgA, and IgG isotypes (IgG1, IgG2a, IgG2b, and IgG3) specific for GsAg. Control mice in groups 4-7 (Table 1) did not produce antibodies to GsAg. Mice immunized with GsAg mixed with Montanide ISA720 and CpG ODN 1826 (group 1), or with Montanide ISA720 and non-CPG ODN 2138 (group 2), or with Montanide ISA720 alone (group 3) produced high levels of all antibody classes and IgG subclasses, except IgA antibody, specific to G. spinigerum antigen (Fig. 1). The IgG antibody responses in mice in groups 1-3 were not significantly different as determined immediately before the challenge infection (at week 4 after the last immunization) (mean OD±SE; 1.95±0.12, 2.04±0.10, and 1.94±0.10, respectively, P>0.05; Fig. 1C), as were IgM and IgE antibody responses (mean OD±SE: IgM; 0.98±0.13, 1.29±0.20, and 1.08±0.12, respectively, P>0.05; Fig. 1A; and mean OD±SE: IgE; 0.30±0.03, 0.34±0.05, and 0.30±0.05, respectively, P>0.05; Fig. 1B).
The IgG1 antibody responses in mice immunized with GsAg mixed with Montanide ISA720 and CpG ODN (group 1) were statistically significantly lower than those in mice immunized with the GsAg in Montanide ISA720 and non-CpG ODN, or with GsAg in Montanide ISA720 at 4 weeks after the last immunization (mean OD±SE; 1.66±0.02 versus 1.82±0.03, or 1.77±0.04, respectively, P<0.05; Fig. 1D). By contrast, IgG2a, IgG2b, and IgG3 antibody responses of mice immunized with GsAg mixed with Montanide ISA720 and CpG ODN were significantly higher than those of mice immunized with GsAg in Montanide ISA720 and non-CpG ODN or with GsAg in Montanide ISA720 at week 12 after the first immunization (mean OD±SE of IgG2a; 1.12±0.05, 0.56±0.12, 0.48±0.09, respectively, P<0.05; Fig. 1E mean OD±SE of IgG2b; 2.07±0.17, 1.45±0.15, 1.60±0.14, respectively, P<0.05; Fig. 1F mean O.D.±SE of IgG3; 2.05±0.19, 1.04±0.27, 0.35±0.12, respectively, P<0.05 Fig. 1G). In addition, the ratio of IgG2a/IgG1 antibody levels of mice immunized with GsAg in Montanide ISA720 and CpG ODN was higher than those of the other groups (Fig. 2A).
Antibody responses of immunized mice after challenge infection with G. spinigerum
Following a G. spinigerum challenge, antibodies in all immunized mice were monitored (Fig. 3). Control mice immunized with adjuvant only produced some IgG and IgM, but little IgE, and no IgA antibodies. The IgG and IgM antibody levels in control mice reached a peak between week 3 and 6 after the challenge but the levels were far less than those of immunized mice. In contrast, the IgG antibody levels of immunized mice after challenge infection remained high for a few weeks then gradually decreased regardless of the types of adjuvant used. In contrast, the IgM levels of immunized mice increased during the first week after challenge infection then gradually decreased. In terms of IgG subclass responses, IgG1 antibody levels remained high, whereas those of IgG2a, IgG2b, and IgG3 decreased continuously. Actually the IgG3 antibody levels started to drop after the 4th week post challenge (Fig. 3G), except for the group immunized with GsAg in Montanide ISA720 and CpG ODN, which increased for 2 weeks before dropping. The IgG2a/IgG1 ratios decreased over time after challenge infection (Fig. 2B).
Parasite burdens
We investigated the protective effect of the immunization by determining the worm recovery at week 16 post challenge. No significant differences of worm recovery were observed among groups of immunized and non-immunized mice (mean worm recovery±SE of groups 1-7; 3. 6±0.7, 3.9±0.3, 3.8±0.7, 3.9±0.4, 3.1±0.8, 3.1±0.8, and 3.3±0.7, respectively; P>0.05).
DISCUSSION
In an attempt to overcome a round worm infection, we used an immunostimulatory oligodeoxynucleotide containing unmethylated CpG motif (CpG ODN 1826) to combine with oil-based Montanide ISA720, which has been proven to be safe in human immunization trials [31]. After challenging with Gnathostoma larvae, immunized mice were not protected as demonstrated by the worm recovery. Our study showed that mice immunized with G. spinigerum crude antigen produced all antibody classes and subclasses to G. spinigerum protein except IgA antibodies (Fig. 1). Neither the presence of CpG ODN 1826 nor non-CpG ODN in the immunization, increased the levels of the IgG1 antibody responses, relative to Montanide ISA alone (Fig. 1D), suggesting that CpG ODN did not enhance an IgG1 response. By contrast, IgG2a, IgG2b, and IgG3 antibody levels were consistently higher after immunization with CpG ODN, compared to the other adjuvant regimes (Fig. 1E-G), suggesting a role of CpG ODN in the induction of a Th1-type biased response.
After challenge infection with live G. spinigerum AL3, antibody responses were observed in all groups of mice. In the control groups treated with adjuvants only, IgM and IgG1 were detected 1 week after infection and sustained at high levels for up to 16 weeks of study (Fig. 3A, D). In contrast, the IgG2a, Ig2b, and IgG3 antibody responses to the infection were negligible (Fig. 3E-G). These findings may suggest that infection with live G. spinigerum larvae results in a strong Th2-type biased response [32]. This suggestion was also supported by the data of the groups of mice immunized with G. spinigerum crude antigen prior to infection. These groups showed sustained levels of IgG1 but continuously decreasing levels of IgG2a, IgG2b, and IgG3 (Fig. 3D-G), and opposite IgG2a/IgG1 ratios before and after infection (Fig. 2). In addition, it is possible that the failure of mice to develop protective immunity and the survival of parasite larvae after challenge infection observed in this experiment may be due to the escape mechanisms of this parasite by a biased regulation of the type of antibody responses, through which h2-producing cells [33]. This needs to be further studied.
We conclude that the crude Gnathostoma larval antigen with CpG ODN can induce high immune responses with a shift to a Th1-type response after immunization but no protection is achieved together with the shift to Th2-response after infection. The development of vaccine for this disease may require more understanding of the mechanisms of immune regulation by the parasite.
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