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. 1998 Apr;112(1):69–73. doi: 10.1046/j.1365-2249.1998.00542.x

Differential antigen-stimulated proliferation of human mononuclear cells by recombinant Schistosoma japonicum antigens in a Chinese population

G J Waine *,†,‡, W Yang *,†, A G P Ross *,†, Y S LI *,†,§, A C Sleigh †, B H Kalinna *,†, J C Scott *,†, D Mazzer *,†, Y LI §, D P Mcmanus *,†,‡
PMCID: PMC1904938  PMID: 9566792

Abstract

Peripheral blood mononuclear cells (PBMC) from 117 individuals living on two islands in an area (Dongting Lake) endemic for schistosomiasis japonica in China, and 15 control individuals from a non-endemic area of China, were assessed for antigen-stimulated proliferation against five recombinant Schistosoma japonicum antigens of recognized interest in the development of immunity to schistosomiasis. Two recombinant antigens, paramyosin and 28-kD glutathione-S-transferase, stimulated cellular proliferation (stimulation index ≥ 3.0) in 38.5% and 42.5% of subjects, respectively, a level similar to that induced by a soluble whole parasite extract (51.3%). In contrast, three other recombinant antigens tested—a fatty acid binding protein, 22-kD tegumental membrane-associated antigen, and glyceraldehyde-3-phosphate dehydrogenase—stimulated PBMC proliferation in only 3–8% of subjects. Moreover, we also identified a positive association between the degree of exposure, and cellular proliferation following stimulation with recombinant paramyosin or whole parasite extract. Highly significant differences in antigen-stimulated proliferation were also observed between the two islands, Niangashan and Qingshan. The whole parasite extract stimulated proliferation in 90% of subjects from Niangashan island compared with only 42.1% of subjects from Qingshan island (χ2 = 16.88, P = 0.00004), while glutathione-S-transferase stimulated proliferation in 77.3% of subjects from Niangashan island compared with only 34.7% of subjects from Qingshan island (χ2 = 13.09, P = 0.003). A similar, but not significant, trend was observed for paramyosin and the fatty-acid binding protein. The identification of differential cellular proliferative responses to specific schistosome antigens within an infected human population may have important practical implications for vaccine development against schistosomiasis japonica.

Keywords: schistosomiasis japonica, human immune responses, cellular proliferation, recombinant antigens

INTRODUCTION

Current World Health Organization estimates indicate that over 600 million people are endangered by schistosomiasis (also known as Bilharziasis or snail fever), with about 200 million infected worldwide, mainly in rural agricultural and peri-urban areas. Of these, 20 million suffer major consequences from the disease and 120 million are symptomatic. Humans contract the infection by direct contact with infested surface water containing free-living forms of the schistosome parasite, known as cercariae, which can penetrate human skin. Chemotherapy remains the cornerstone of intervention, but reinfection necessitates frequent retreatment. There is thus a high priority need for the development of effective anti-schistosomiasis vaccines. Evidence that humans can acquire resistance to reinfection by schistosomes, together with the successful use of attenuated vaccines in animals under both laboratory and field conditions, indicate that development of a safe and effective human vaccine is an achievable goal (reviewed in [1]).

A number of immunoepidemiological studies have established associations between the presence of antibodies recognizing specific schistosome antigens and resistance to reinfection in humans. IgG antibodies against a 37-kD parasite antigen (subsequently shown to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) have been associated with resistance to reinfection in Schistosoma mansoni patients in Brazil [2], and in a study undertaken in Kenya an association was found between resistance and the presence of IgA antibodies against a schistosome 28-kD glutathione-S-transferase [3]. In another study in Kenya, IgE antibodies against a 22-kD tegumental membrane-associated antigen have been shown to be associated with the expression of resistance [4]. More recently, the 22-kD antigen was shown to be the dominant target of human IgE recognition for both S. japonicum in The Philippines and S. mansoni in Kenya [5].

Other schistosome antigens have been shown to confer protection against cercarial challenge in experimental animal models. One such protective antigen is paramyosin, a 97-kD myofibrillar protein found exclusively in invertebrates. Both the native molecule and a recombinant expression product encoding half of the full-length protein of S. mansoni paramyosin have been shown to confer significant levels of protection in mice [6,7], while even higher levels of protection have been reported for the Philippine strain of S. japonicum using purified native paramyosin [8]. Another molecule of importance is a 14-kD fatty acid-binding protein. A recombinant 14-kD fatty acid-binding protein of S. mansoni was shown to protect outbred Swiss mice by up to 67% against challenge with S. mansoni, and the same antigen also provided 100% protection against challenge with another important trematode parasite, Fasciola hepatica, in the same animal model [9].

Our laboratory has previously cloned and expressed the S. japonicum homologues of the GAPDH [10], the 22-kD tegumental membrane-associated antigen [11], paramyosin [12], the 14-kD fatty acid-binding protein [13], and 28-kD glutathione-S-transferase (unpublished). As a prelude to vaccine development and to dissect further the mechanisms involved in human immune responses during natural schistosome infection, we have investigated the cellular proliferative responses to these antigens of individuals living in an endemic area for S. japonicum in China, the results of which are reported here.

STUDY AREA AND METHODS

Study area and parasitological methods

Five villages situated on two islands (Qingshan and Niangashan) in the southeastern sector of Donting Lake, Hunan Province, China, were selected for this study. Qingshan island has four fishing villages with a total population of 2450 persons, while Niangashan island has one village and a total population of 540 individuals. The prevalence of schistosomiasis was estimated in 1996 to be 15% on Qingshan island and 24% on Niangashan island [14]. Diagnosis of schistosome infection was performed in 1996 before the current study, and was based on medical history, physical examination and parasitological examination of three stool samples by the Kato–Katz thick smear technique [14].

For the current study, we used a starting cohort of 249 individuals who had been extensively investigated as part of an ongoing longitudinal study of exposure and reinfection rates. The level of exposure to cercariae was determined primarily on the basis of activity diaries which individuals were asked to keep for a maximum of 105 days during the May–October transmission period. The diaries noted the duration and frequency of water contact, together with details regarding the time and location at which contact occurred, type of activity (swimming, bathing, playing, fishing, boating, cutting reeds, crop planting and herding), and which body parts were in contact with water (feet, legs, hands, arms or entire body). Assumptions were made that the relative risk of exposure was directly related to the type of activity undertaken (i.e. the proportion of body surface in contact with the water associated with various activities), the site of contact (sites were assessed for numbers of Oncomelania hupensis hupensis snails, the intermediate host), as well as the duration and frequency of water contact. The reliability of the diaries was validated by direct water contact observations undertaken at two different times during the transmission period. A strong positive linear relationship (r = 0.704) was identified between the activity diaries and the direct water observations.

From this cohort of 249 individuals (76% male; average age = 39.2 years), 180 were available at the time of the current study, and of these, 117 were analysed for cellular proliferative responsiveness. The latter reduction in cohort size was due mainly to the fact that some patients yielded insufficient numbers of cells for complete and valid proliferation assays to be performed. Other patients wished to terminate venepuncture before sufficient blood was collected. There was no bias observed in the smaller group analysed, in either the age or sex distribution (74% male; average age = 40.4 years). Fifteen control individuals were selected from another area of Hunan Province, China, where there was no schistosomiasis transmission.

Antigen preparation

The five recombinant S. japonicum antigens were expressed and purified essentially as previously described [10–13]. Briefly, cultures of recombinant clones in Escherichia coli were grown until an absorbance of A600 of 0.7–0.9 was reached, at which time expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) (final concentration 2 mm). Following a further 4–5 h incubation, cells were harvested by centrifugation and the pellet resuspended in 50 mm NaH2PO4/300 mm NaCl, pH 8.0, supplemented with 1 mg/ml lysozyme. The cells were lysed by a 1-h incubation at room temperature, two freeze thaw cycles and brief sonication. Following centifugation the cleared lysate was incubated for 1 h with TALON (Clontech, Palo Alto, CA) resin. The resin was loaded into a 1 cm diameter column and washed with 50 mm NaH2PO4/ 300 mm NaCl, pH 8.0, until the A280 was < 0.01. The recombinant protein was eluted with a linear gradient to 0.5 m imidazole in 50 mm NaH2PO4/300 mm NaCl, pH 8.0 (flow rate of 0.5 ml/min). Imidazole was removed using PD-10 desalting columns (Pharmacia Biotech, Uppsala, Sweden), according to the manufacturer's instructions. Glycerol (10% w/v as a protectant) was added and the recombinant protein aliquoted and stored at −80°C. Whole parasite extracts were prepared by harvesting the soluble fraction obtained from lysed S. japonicum adult worms.

Lymphoproliferation assays

Blood samples, collected into heparinized tubes at the study sites, were transported on ice to the Institute of Parasitic Diseases, Yueyang, for same day processing. Peripheral blood mononuclear cells (PBMC) were isolated from freshly collected human blood by density gradient centifugation using Ficoll–Hypaque (Pharmacia Biotech). After separation, the PBMC were washed in RPMI and adjusted to a concentration of 106 cells/ml in RPMI supplemented with 10% heat-inactivated human AB serum (Sigma-Aldrich, Castle Hill, NSW, Australia). Aliquots of 2 × 105 cells were then incubated in quadruplicate in 96-well flat-bottomed plates in the presence of antigen. The cells were stimulated with 10 μg/ml or 1 μg/ml of each of the five recombinant antigens or whole adult parasite extracts (SWAP), 10 μg/ml purified protein derivative (PPD; CSL, Melbourne, Australia), 1 μg/ml concanavalin A (Con A; Sigma-Aldrich), or control medium. Plates were incubated at 37°C in 5% CO2. Cell proliferation was measured by an 18-h incorporation of 1 μCi of 3H-thymidine added on day 5. On day 6 the cells were harvested onto filters and processed for scintillation counting. The stimulation index (SI) was calculated as the ratio of the mean ct/min of the antigen-stimulated wells to the mean ct/min of the cells cultured in medium alone. SIs ≥ 3.0 were counted as positive.

Statistical analysis

Epi-Info software version 6.01 (Centres for Disease Control and Prevention, Atlanta, GA, and World Health Organization, Geneva, Switzerland) was used for analysis. The statistical significance of differences in proliferative frequencies was determined using the Mantel–Haenszel χ2 test. Student's t-test or analysis of variance was used to detect differences between means. Linear regression analysis was used to evaluate the relationship between antigen-stimulated proliferation (SI) and the mean daily water contact (average hours/day). The minimum level considered significant was P < 0.05.

RESULTS

Figure 1 shows the percentage of proliferative responses in the 117 test subjects to each of the antigens tested. All of the control individuals from the non-endemic area gave SI values < 3. A high prevalence of antigen-stimulated proliferation was observed for two of the recombinant S. japonicum antigens, paramyosin (38.5%) and glutathione-S-transferase (42.7%), and for the whole parasite soluble extract (51.3%). Indeed, the frequency of cellular proliferation induced by paramyosin or GST28 was almost comparable to that induced when the cells were stimulated by the whole parasite extract. In contrast, the other three recombinant antigens stimulated PBMC proliferation in less than 8% of the study group. Specifically, the fatty acid-binding protein, tegumental membrane-associated antigen, and GAPDH stimulated PBMC proliferation in 7.7%, 3.4% and 4.3% of the study group, respectively. The positive control, Con A, induced proliferation in all subjects. The other antigen tested, PPD, induced stimulation in only 14% of subjects. While the reason for this is unknown, one likely possibility is that individuals in this area have had little exposure to bacille Calmette–Guérin (BCG) or related antigens.

Fig. 1.

Fig. 1

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The percentage of subjects tested from the schistosomiasis japonica-endemic area exhibiting a cellular proliferative response (SI > 3.0) following stimulation with recombinant or other antigens. Control individuals from an area non-endemic for schistosomiasis japonica gave SI values < 3.0. Recombinant antigens are paramyosin (PMY), 28-kD glutathione-S-transferase (GST28), 14-kD fatty acid-binding protein (FABP), 22-kD tegumental membrane-associated antigen (TegAg) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Other antigens are whole parasite extract (SWAP), purified protein derivative (PPD), and concanavalin A (Con A).

The study group was further analysed to determine if associations existed between cellular proliferation to any of the antigens and a range of other variables, including age, sex, island of residence, average hours of exposure, egg counts in the stool, and hepatomegaly over the midsternal or midclavicular lines. Testing associations for one variable at a time (t-test) linked cellular proliferative responses with average hours of exposure and island of residence (P < 0.05).

To determine if cellular proliferation was associated with exposure, we analysed the association between the average hours per day each individual spent in contact with water and the cellular proliferative response of each individual to each of the antigens. The results for the whole parasite extract, paramyosin and glutathione-S-transferase are shown in Fig. 2. The sample size for the remaining antigens was too small to give meaningful data. The results for both the whole parasite extract (r = 0.39) and paramyosin (r = 0.30) suggest a positive linear association between cellular responsiveness to these antigens and the degree of exposure. In contrast, a weak negative association was observed between cellular responsiveness to glutathione-S-transferase and the degree of exposure.

Fig. 2.

Fig. 2

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Relationship between exposure (average hours per day in contact with water) and cellular proliferation following stimulation with soluble adult worm extract (SWAP) (a), paramyosin (PMY) (b) and 28-kD glutathione-S-transferase (GST) (c). SI, Stimulation index.

To dissect the relationship between cellular proliferation and degree of exposure, individuals were stratified based on mean daily water contact (low water contact ≤ 1 h/day; high water contact > 1 h/day). A significant increase in cellular proliferation following stimulation with whole parasite extract (P = 0.0008) or paramyosin (P = 0.021) occurred in the group with higher water contact (Table 1). In contrast, there was no significant difference between water contact groups for cellular proliferation following stimulation with glutathione-S-transferase. Dividing the analyses further according to age did not alter these results.

Table 1.

Analysis of the relationship between high (> 1 h/day) or low (≤ 1 h/day) exposure and cellular responsiveness (mean proliferation) to whole parasite extract (SWAP), paramyosin, or glutathione-S-transferase (GST28)

graphic file with name cei0112-0069-t1.jpg

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Highly significant differences in antigen-induced cellular proliferation were also observed between the two islands. Proliferation was induced more frequently on Niangashan island, the smaller of the two islands, compared with Qingshan island (Fig. 3). Highly significant differences were observed for the whole parasite extract, which stimulated proliferation in 90% of subjects from the small island compared with only 42.1% of subjects from the large island (χ2 = 16.88, P = 0.00004), and for glutathione-S-transferase, which stimulated proliferation in 77.3% of subjects from the small island compared with only 34.7% of subjects from the large island (χ2 = 13.09, P = 0.003). A similar trend was observed with paramyosin and the fatty acid-binding protein, although these differences were not statistically significant.

Fig. 3.

Fig. 3

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Comparison of the relative frequency of proliferative responses to each antigen of subjects residing on either island. S1, Qingshan island; S2, Niangashan island. SI, Stimulation index.

DISCUSSION

This study is the first report describing the cellular proliferative response of a human population from a schistosomiasis-endemic area to a panel of defined, purified recombinant schistosomal antigens. Each of these antigens is of recognized interest in relation to the development of immunity to schistosomiasis. The homologous molecules of other schistosome species have been shown to confer a high degree of protection in immunization/challenge experiments in animal models, or to be recognized by specific antibodies, the presence of which has been correlated with resistance to schistosome infection in humans.

This study has identified a differential cellular proliferative responses to specific schistosomal antigens in a population endemic for schistosomiasis japonica. Two of the antigens in particular, paramyosin and GST28, stimulated PBMC proliferation in 38–42% of the population. The frequency of cellular proliferation induced by either of these two antigens was, perhaps surprisingly, comparable to that observed when a whole parasite extract, presumably containing all the schistosome antigens, was tested. In comparison, the three other recombinant antigens tested, fatty acid-binding protein, tegumental membrane-associated antigen, and GAPDH, stimulated PBMC proliferation in < 8% of the population.

Moreover, this study has identified positive associations between the levels of exposure to infested water, and human proliferative responsiveness not only to a whole parasite extract, but, more importantly, to paramyosin, an antigen of immense interest in anti-schistosomiasis vaccine research. These results may have potential practical implications for the development of immunoprophylactic therapies aimed at preventing schistosomiasis infection, as well as contributing to our general understanding of the mechanisms involved in cellular responsiveness to specific parasite antigens following natural infection of humans.

A noteworthy difference was observed between the proliferative responses of subjects that was related to their island of residence. Proliferative responses were more frequent in subjects residing on Niangashan island than in those residing on Qingshan island. This result parallels the known prevalence of schistosomiasis on each island, estimated in 1996 to be 24% on Niangashan island and 15% on Qingshan island [14], and therefore probably reflects differences in the level of exposure to infective cercariae. This difference may be quite important; although both islands are in close proximity to each other, transmission of schistosomiasis often varies substantially from one focus to another. Additionally, it should be noted that extensive control measures, including the annual treatment of a high proportion of known infected individuals, is carried out in the study area, and therefore current infection reflects recent (< 1 year) reinfections.

Although this is the first report utilizing purified recombinant antigens, other studies have examined human cellular responses to whole parasite extracts—soluble adult- or schistosomulum-stage antigens, soluble egg antigens (SEA), or fractions of membrane-associated adult worm antigens. Chronic infection with S. haematobium is associated with the reversible down-regulation of T cell proliferative responses and IL-4 release; cell depletion experiments indicate that CD4+ T cells are the target of this down-modulation [15]. Immuno-epidemiological investigations identified an association between cellular proliferative responses to parasite extracts and resistance to reinfection with S. mansoni [16,17]. A schistosome T cell-stimulating antigen (Sm10), associated with protective immunity in humans, has been described [18]. Another study has suggested that the response to adult worm antigens may be pivotal in determining not only the development of resistance but also the severity of disease, particularly hepatosplenic pathology [19].

We are monitoring the patients tested in the current study to determine if the capacity to respond to specific antigens relates to development of acquired resistance to reinfection after treatment. Future work will also determine cytokine profiles induced by the various antigens, as well as investigating whether there is any genetic influence on immune responsiveness and resistance or susceptibility to infection with S. japonicum.

Acknowledgments

We would like to thank Mary Duke for technical assistance. Our studies into the development of anti-schistosomiasis vaccines receive financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, and the National Health and Medical Research Council of Australia.

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