Evaluation of six novel antigens as potential biomarkers for the early immunodiagnosis of schistosomiasis
© Zhang et al. 2015
Received: 14 July 2015
Accepted: 17 August 2015
Published: 4 September 2015
Early diagnosis of schistosomiasis, prior to egg laying, would enable earlier treatment and help interrupt the transmission cycle of the parasite and the progress of the disease. Previously we identified six novel antigens with potential as diagnostic markers for human Schistosoma japonicum infections. In this study, we evaluated these antigens as candidate biomarkers for the early diagnosis of schistosomiasis in mice and rabbits.
The transcriptional profiles of the six antigens (SjSP-13, SjSP-23, SjSP-160, SjSP-164, SjSP-189 and SjSP-216) at different developmental stages were analyzed by quantitative PCR. The recombinant proteins were expressed in E. coli and purified with nickel chelate affinity chromatography. We then developed recombinant protein-based ELISA kits to analyze the kinetics of antigen-specific antibodies during the course of infection in mice and rabbits. The early diagnostic validity of the candidate SjSP-216 was further evaluated in mice and rabbits infected with S. japonicum.
Of the six antigens, SjSP-13, SjSP-160 and SjSP-216 were highly expressed in 21-day old young worms, while SjSP-23, SjSP-164 and SjSP-189 were highly expressed in eggs. In the mouse model, we detected a significant increase in antibodies against SjSP-13 and SjSP-216 at 3 weeks post-infection. However, in the rabbit model, only anti-SjSP-216 antibody showed a significant increase at this time point. We recorded 100 % diagnostic sensitivity and specificity of SjSP-216-based ELISA in both infected mice and rabbits, 3 weeks after infection.
This study strongly suggests that SjSP-216, a highly expressed gene in the young worms, could serve as a potential biomarker for the early immunodiagnosis of S. japonicum infections in vertebrate hosts.
KeywordsSchistosoma japonicum Schistosomiasis Biomarker Early diagnosis Animal model
Schistosomiasis is a chronic disease caused by parasitic trematodes of the genus Schistosoma that afflicts over 200 million people worldwide and kills >300,000 people annually [1–3]. Humans are infected by cercariae, which are released from infected snails when they come in contact with contaminated water . After the cercariae penetrate the skin, the parasites become schistosomula and over 4–6 weeks migrate and mature to adult male or female worms. Adult worms live as pairs in the portal and mesenteric veins (S. japonicum and S. mansoni) or in the veins of the bladder (S. haematobium). They are long-lived and produce hundreds of fertilized eggs per day . Although most eggs are retained within the host tissues, about one-third are excreted into the environment and hatch into free-living miracidia, which can infect susceptible snails in water, thus completing the cycle [6, 7]. The host’s immune response to antigens excreted from the trapped eggs can induce severe morbidity, including hepatic fibrosis, portal hypertension, urinary obstruction and bladder carcinoma [8, 9].
One factor contributing to high disease prevalence and severe morbidity is the absence of effective diagnostic methods for detecting schistosome infections, especially in the early phase . The traditional gold standard for the diagnosis of schistosomiasis involves detecting eggs in and hatching miracidium from the host’s feces or urine [11, 12]. However, in the majority of schistosome infections, the paired worms start to discharge eggs about 4 weeks after infection . Thus, these diagnostic methods are relatively insensitive and cannot detect early infections. If schistosome infections could be detected prior to egg deposition, the source of infection would be controlled and subsequent treatment with PraziquantelTM would effectively prevent the development of severe pathologic lesions.
Immunodiagnostic detection of antibodies against schistosome antigens is an attractive option for detecting early infections. However, currently available antibody detection assays are not suitable as they use antigens extracted from schistosome eggs or adult worms [14–16]. Previous studies have revealed that the reactivity of antibodies against crude worm antigens remains low until the infections become patent in the experimental hosts [17, 18]. Therefore, the ideal immunodiagnostic assay to detect early infections would use antigens from the schistosomula or cercariae . However, it is notoriously difficult to extract antigens from the schistosomula or cercariae.
In a previous study, we used a high-throughput glutathione S-transferase (GST) fusion protein array assay to identify several antigens (SjSP-13, SjSP-23, SjSP-160, SjSP-164, SjSP-189 and SjSP-216) with the potential to be immunodiagnostic markers for human schistosomiasis . In this study, we aim to verify the utility of these six antigens as candidates for the early diagnosis of S. japonicum infections in a murine and rabbit model.
Parasites and animals
A field-collected isolate of S. japonicum from Guichi County, Anhui Province, China was used in all the experiments. Parasites were maintained in Oncomelania hupensis snails and in rabbits. Female 12-week old New Zealand White rabbit and female 6–8 week old BALB/c mice were obtained from SLAC Laboratory Animal Co., Ltd. of the Chinese Academy of Sciences of Shanghai. All procedures performed on animals within this study were conducted in accordance with and by approval of the Internal Review Board of Tongji University School of Medicine.
Primers used for Real-Time PCR
Primer sequence (5′-3′)
Cloning, expression and purification of antigens
The gene fragments of antigens SjSP-13, SjSP-23, SjSP-160, SjSP-164, SjSP-189 and SjSP-216 (Genbank accession number: AY222880, AY814664, AY222887, AY814985, AY815838 and AY813624, respectively) were amplified from a mixture of cDNAs of cercariae, young worms, adult worms, and eggs by PCR with KOD polymerase (Toyobo, Japan). Specific primers with restriction enzyme sites are listed in Additional file 1. The PCR products were cloned into E.coli expression vector pGEX-4 T-1 (for SjSP-13, SjSP-160, SjSP-164, SjSP-189 and SjSP-216) or pET28a (for SjSP-23). The recombinant plasmids containing target DNA fragments were confirmed by DNA sequencing. Expression of recombinant proteins was induced with Isopropyl-D-1-thiogalactopyranoside (IPTG) at 1 mM. Recombinant proteins were purified from the insoluble inclusion body with a hexahistidine tag. The purified antigens were re-natured in refolding buffer C7 (1.0 mM TCEP, 250 mM NaCl, 12.5 mM β-cyclodextrin, 50 mM Tris–HCl pH 8.5) . Protein concentration was determined by the Bradford method . The predicted molecular weight of SjSP-23 and the GST fusion proteins of SjSP-13, SjSP-160, SjSP-164, SjSP-189, SjSP-216 were 13.0kD, 45.6kD, 51.9kD, 40.8kD, 41.1kD and 57.4kD, respectively.
Indirect enzyme-linked immunosorbent assay
The 96-well microliter plates (Corning, USA) were coated with 100 μL per well of 1 to 2 μg/ml antigens diluted in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) for 16 h at 4 °C. The plates were washed 3 times with washing buffer (0.15 M phosphate buffer saline containing 0.05 % of Tween 20, pH 7.4). The free sites were saturated with 200 μL per well of blocking buffer (5 % skim milk dissolved in washing buffer) at 37 °C for 1 h. After washing three times, 100 μL of individual mouse sera (diluted 1:100) in blocking buffer were added to the plates and were incubated at 37 °C for 1 h. The plates were submitted to 5 times of washing and incubated at 37 °C for 1 h with goat anti-mouse IgG or goat anti-rabbit IgG conjugated with peroxidase (Abcam, USA) diluted in blocking buffer at the dilution of 1:20,000. Plates were washed again and 100 μL of TMB substrate solution was added to each well. The enzymatic reaction was stopped after 10 min of incubation at 37 °C by adding 50 μL per well of 2 N H2SO4. The results were obtained as absorbance values at 450 nm by a microplate reader.
To analyze the dynamics of antigen specific antibodies during infection, three mice and three rabbits were infected with 30 ± 2 or 200 ± 10 cercariae by the subcutaneous route, respectively. Meanwhile, another three mice and three rabbits were used as non-infected controls. Serum samples were collected before infection and on week 1, 2, 3, 4, 5 and 6 after infection.
A separate experiment was performed to evaluate the early diagnostic validity of SjSP-216. Ten mice and six rabbits were infected the same as before; ten mice and six rabbits were used as a negative control of infection. Sera were collected at 3 weeks after infection.
Data of real-time PCR were expressed as the mean ± standard deviation. The absorbance values were expressed as the mean ± standard error. All data were analyzed by the Student’s t-test, with a p value < 0.05 considered significant. We used Prism 4.0 software for all statistical analyses.
Production of recombinant proteins
The gene sequences of the six proteins were verified as correct by DNA sequencing. The six recombinant proteins were successfully produced in E.coli (Additional file 2). The purified antigens were re-natured in refolding buffer C7 that was identified from the iFOLD Protein Refolding System 1 in our previous studies .
Analysis of transcriptional profiles of the six antigens at different developmental stages
Kinetics of specific antibodies in the sera of mice infected with S. japonicum
Kinetics of specific antibodies in the sera of rabbits infected with S. japonicum
Sensitivity and specificity of SjSP-216 as a candidate for early serological diagnosis of S. japonicum infections
Schistosomiasis remains a serious public health problem in developing countries. Morbidity and mortality from this disease are associated with the chronic infection stage, which occurs after egg deposition. Thus, an ideal diagnostic test for schistosomiasis should be capable of detecting parasites as early as possible after the onset of infection. An early diagnostic test would enable more rapid treatment and would interrupt the transmission cycle of the parasite and the progress of the disease. However, the current diagnostic standards for schistosomiasis all depend on the detection of eggs. In addition, as disease is asymptomatic in its early stages, clinical examinations can’t confirm infection. Recently, molecular techniques to detect schistosome infections have been developed to facilitate early diagnosis, but these are expensive and suffer from sampling limitations [22, 23]. Serologic assays to detect antibodies against schistosome antigens, however, have proven useful in the clinical diagnosis of schistosomiasis [24–26].
In definitive hosts, the immune systems must confront four life-cycle stages of the parasite: penetrating cercariae, schistosomula (young worms), adult worms and the eggs that are produced by adult worm pairs . As the host is exposed (internally) to cercariae and schistosomula earlier than they are exposed to eggs, antigens that are highly expressed or solely expressed in one of these two stages are likely be potential candidates for early diagnosis. For example, previous studies of the schistosomula (mechanically transformed cercariae) tegument antigen revealed this antigen had diagnostic value for early infections . In this study, we found that antigens SjSP-13, SjSP-160 and SjSP-216 were highly expressed in young worms, while antigens, SjSP-23, SjSP-164 and SjSP-189 were highly expressed in eggs. These distinct expression profiles correlate with the timing of host immune responses to specific stages. For example, seroconversion of young worm antigen SjSP-216 occurred 3 weeks post-infection, 3 weeks earlier than to the egg antigen SjSP-23.
The immunogenicity of antigens varied between the two host species. Previously, we found that SjSP-164 and SjSP-189 were recognized by the antibodies of human patients . In this study, we report that SjSP-164 and SjSP-189 stimulated humoral immune responses in infected mice but not in rabbits. We found that SjSP-13, which has been verified as a diagnostic marker for human schistosomiasis , is also recognized by IgG antibodies in both infected mice and rabbits. Nevertheless, the titers of antibodies in mice and rabbits were much lower than those reported for humans, indicating that the antigenicity of SjSP-13 was weaker in these species than in humans. Importantly, the antigenicity of SjSP-216 in mice and rabbits appeared very similar to that reported for this antigen in humans.
For these reasons, we selected the antigen, SjSP-216, as a candidate for early diagnosis of schistosomiasis. Our results confirmed that SjSP-216 was able to diagnose early schistosome infections in both mice and rabbits with 100 % sensitivity and specificity. Moreover, we observed that anti-SjSP-216 antibody levels increased throughout the period of the parasite exposure. Thus, SjSP-216 would also be useful for diagnosing chronic infections. Although early diagnosis may not be necessary in areas with high endemicity where most patients harbor chronic infections , it is vital for detecting new infections, and for ensuring effective disease surveillance in areas where the schistosomiasis has be controlled. In areas where schistosomiasis has been controlled, large-scale disease surveillance at the start of the transmission season could identify (and remove) sources of infection and prevent the recurrence of schistosomiasis. Moreover, early diagnosis will lead to earlier treatment, potentially preventing the development of severe pathologic lesions. This would be particularly relevant for susceptible individuals, such as travellers . Although PraziquantelTM is more effective on adult worms than young worms, high-dose (500 mg/kg) PraziquantelTM therapy or prolonging the course of treatment can damage the tegument or even kill young worms [29, 30].
We propose that SjSP-216 be further developed as an early serological diagnostic tool for human schistosomiasis, as this protein has strong antigenicity in different hosts, including humans. It is difficult to validate the early diagnostic efficacy of SjSP-216 in humans directly as current techniques can’t detect pre-patent human infections. In addition, it is not easy to regularly monitor antibody changes during the course of infection in human. However, it is possible to compare the positive rates of SjSP-216 and other antigens including SjSP-13 at the start of transmission season (i.e. in May) and at the end of epidemic season (i.e. in September). If the sensitivity of SjSP-216 was higher during the epidemic season, we can prove the early diagnostic efficacy of SjSP-216 indirectly.
This study strongly suggests that SjSP-216, a highly expressed gene in the young worm stage, could serve as a potential biomarker for the early immunodiagnosis of S. japonicum infections in vertebrate hosts.
This study was supported by grants from the National Natural Science Foundation of China (81430051) and the Shanghai Municipal Natural Science Foundation (15ZR1443500).
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