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The vaccination of mice with rSmKI-1 resulted in reduced numbers of parasites and eggs.
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The rSmKI-1 vaccine produced a solid IgG response, with the IgG1 isotype being predominant.
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The rSmKI-1 vaccine down-regulated the Th2 immune response, reducing disease pathology.
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SmKI-1 has potential for development as part of a schistosomiasis cocktail vaccine.
Abstract
Objective
The aim of this study was to develop a vaccine against schistosomiasis, which is a major challenge due to the complex lifecycle of the causative schistosome parasite.
Methods
SmKI-1 is a 16-kDa Kunitz-type protease inhibitor present in the excretory–secretory products and tegument of adult worms and eggs of Schistosoma mansoni. Two independent vaccine trials were performed in mice to determine the efficacy of rSmKI-1 in developing protective immunity.
Results
The results obtained showed reductions of 23–33% in adult worms, 28–31% in intestinal eggs, 33–39% in faecal eggs, and 20–43% in liver eggs. Furthermore, rSmKI-1 significantly increased the production of interferon gamma, interleukin (IL)-10, and IL-6 in vaccinated mice, maintaining a Th1/Th2-type balanced protective response.
Conclusions
rSmKI-1 generated partial protection against schistosomiasis mansoni in the murine model of infection and could be developed as part of a combination vaccine with other vaccine candidates to provide an even more solid level of protection.
). Current treatment involves the drug praziquantel (PZQ). However, with increasing concerns about the possible development of PZQ-resistant parasites exacerbating local schistosomiasis transmission, the development and deployment of a vaccine represents an essential component of future efforts to effectively control schistosomiasis (
). The complex developmental cycle of the schistosome parasite and the expression of antigenic components by each life stage present a major challenge in the goal to identify vaccine targets that will prevent human infection (
). However, an effective molecule or combination of molecules as a vaccine could reduce the number of adult worms and eggs produced, thus slowing and reducing schistosomiasis pathology and transmission (
). In practice, an effective vaccine would likely be a crucial component in an integrated schistosomiasis control package, combined with chemotherapy and other interventions (
). Schistosomes reside in human blood, which is a hostile immunological environment. Subsequently, they have evolved highly effective mechanisms to evade the host immune response and to live for a considerable period in the bloodstream without being damaged or expelled. Overcoming these evolutionary adaptations to parasitism is the goal for identifying and subsequently developing suitable anti-schistosomal vaccine candidates; however, this process has been slow despite extensive research over several decades (
) have received particular attention as encouraging vaccine targets, since they are readily accessible to the host immune system. Protease inhibitors secreted by schistosomes play important roles in successful invasion and migration through inhibiting digestive hydrolysis by host proteases (
). SmKI-1 was the first Kunitz-type serine protease inhibitor identified from S. mansoni; it is a component of adult worm ES products and exhibits both anticoagulatory and anti-inflammatory properties (
), SmKI-1 was tested as a potential vaccine candidate.
Materials and methods
Ethics statement
All animal experimentation was conducted in strict accordance with protocols approved by the QIMRB Animal Ethics Committee (project number P242), which adheres to the Australian code of practice for the care and use of animals for scientific purposes, as well as the Queensland Animal Care and Protection Act 2001; Queensland Animal Care and Protection Regulation 2002.
Recombinant SmKI-1 production and vaccine trial
Recombinant SmKI-1 (rSmKI-1) was expressed in Escherichia coli and purified as described previously (
). Lipopolysaccharide (LPS) levels in the purified recombinant SmKI-1 were determined using a Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific Inc., IL, USA) with a sensitivity of 0.1 EU/ml (approximately 0.01 ng endotoxin per ml) and removed using Pierce high capacity endotoxin removal spin columns (Thermo Fisher Scientific Inc.). Recombinant human TFPI (RPA394Hu02) and a polyclonal antibody against human TFPI (PAA394Hu02) were purchased from USCN Life Science Inc. (Wuhan, Hubei, China) and used in Western blot analysis to determine whether there was any immunological cross-reactivity of rSmKI-1 with human TFPI.
Groups of 9–10 female CBA mice aged 11 weeks were used in two independent vaccine trials, each comprising three groups: control adjuvant group, control lipopolysaccharide (LPS) group, and rSmKI-1 vaccinated group. To comply with strict ethical guidelines, hunched and less active mice, likely due to a heavy infection, were euthanized prior to perfusion. Each mouse was injected with 50 μg of rSmKI-1 protein mixed with 10 μg Quil A adjuvant in phosphate-buffered saline (PBS) three times at two-weekly intervals. The control group of mice was given PBS and Quil A only. The LPS control group comprised LPS (L2880; Sigma Aldrich, USA) in PBS, an amount equivalent to the calculated LPS contamination of the bacterially produced rSmKI-1 protein. Two weeks after the last of the three injected doses, the mice were each challenged subcutaneously with 100 S. mansoni cercariae. After 7 weeks, the mice were sacrificed and parasites were perfused from the intestinal mesenteric veins. Blood samples were collected prior to vaccination and prior to cercarial challenge. Further details of the schedule for both vaccine trials are shown in Figure 1.
Figure 1Design of the two independent vaccine trials.
Separated, diluted sera were aliquoted and stored at −80 °C until required. Immediately prior to perfusion, faecal samples were collected from each mouse, weighed, and stored in 10 ml of 10% (v/v) phosphate-buffered formalin (PBF) until processed. Adult worm numbers in each mouse were counted following perfusion and stored in PBF for subsequent morphological analysis. The caudate lobe of each liver was fixed in 10% (v/v) PBF for histological granuloma analysis and the remainders of the livers from each mouse were weighed and stored in dry tubes at −20 °C for determining egg burdens. In addition, the small intestine of each mouse was dissected, cleaned, weighed, and stored in a dry tube at −20 °C for determining the number of eggs present.
Total egg counts per gram of liver, intestine, and faeces were individually determined for each mouse, as described previously (
). The lengths of male and female worms from 10 worm pairs from each mouse were also measured using an Olympus CKX41 inverted microscope. The total worm/egg reductions (as a percentage change) were calculated using the following published formula (
): % change = (mean number of worms or eggs in infected control mice−mean number of worms or eggs in the immunized group)/mean number in infected controls × 100.
Determination of different IgG subtypes in sera by ELISA
Sera collected from mice prior to vaccination with rSmKI-1 and prior to cercarial challenge were tested for different anti-rSmKI-1 immunoglobulin antibody subtypes (IgG, IgG1, IgG2a, IgG2b, and IgG3) using an indirect ELISA. MaxiSorp 96-well plates (Nunc, Roskilde, Denmark) were coated with 0.5 μg of rSmKI-1 protein per well at 4 °C overnight and ELISA was performed as described previously (
Splenocytes from vaccinated and control mice were cultured and stimulated in vitro with rSmKI-1 (25 μg/ml). After 72 h, supernatants were collected and splenocytes were analysed by flow cytometry for cytokines released by CD8+ and CD4+ cells (
Comparative pathogenesis of eosinophilic meningitis caused by Angiostrongylus mackerrasae and Angiostrongylus cantonensis in murine and guinea pig models of human infection.
). A mouse cytometric bead array (CBA) Th1/Th2/Th17 kit (BD Biosciences, San Jose, CA, USA) was used to determine the levels of cytokines (interleukin (IL)-10, IL-17A, tumour necrosis factor (TNF), interferon gamma (IFN-γ), IL-6, IL-4, and IL-2) present in splenocyte culture supernatants. The array was performed in accordance with the manufacturer’s instructions and data were acquired on a BD LSR Fortessa analyser (BD Biosciences).
Data analysis and statistics
The statistical analysis of data was performed using the Mann–Whitney test, unpaired two-tailed Student t-test, and one-way analysis of variance (ANOVA) with GraphPad Prism 7.01 software. p-Values of ≤0.05 were considered statistically significant.
Results
Analysis of rSmKI-1
According to the NCBI BLAST results, SmKI-1 shares 57% amino acid sequence identity with human tissue factor pathway inhibitor 2 (TFPI2) (GenBank number AAA20094), which is a classical anti-coagulation Kunitz-type protein (Figure 2). Western blot analysis showed that human TFPI did not bind anti-SmKI-1 antibodies, and the rSmKI-1 protein was not bound by the anti-TFPI antibody (Figure 3). There was clearly no cross-reactivity between anti-SmKI-1 antibodies and human TFPI, a feature that reinforces the suitability of rSmKI-1 as a potential vaccine candidate for use in humans.
Figure 2Sequence alignment of human TFPI2 (GenBank number AAA20094) and SmKI-1.
Figure 3(A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots (WB) of rSmKI-1 incubated with (B) anti-TFPI antibody and (C) anti-rSmKI-1 antibody. Lane 1, rSmKI-1 (3 μg for SDS-PAGE and 1.5 μg for WB); lane 2, TFPI (3 μg for SDS-PAGE and 1.5 μg for WB); M, BenchMark pre-stained protein ladder.
The results of the two independent vaccine trials in mice showed a statistically significant reduction in worm and egg counts in the rSmKI-1 vaccinated group compared with the adjuvant control group (Figure 4). The mean adult worm pair count reduction was 23–33% in the rSmKI-1 vaccinated group. The average lengths of male and female worms obtained from the rSmKI-1 vaccinated mice were not significantly different from those in the control group. The pathology of schistosomiasis is mainly linked to the parasite eggs trapped in different tissues. The mean egg count reductions per gram of small intestinal tissue and liver tissue were 28–31% and 20–43%, respectively (Table 1), in the vaccinated group compared with the adjuvant control group. There were no significant differences among any of the adult worms, liver, faecal, or intestinal egg counts in the LPS control group compared with the adjuvant control group. Parasitological data for mice in the LPS control group over the two independent trials are presented in the Supplementary Material (Table S1).
Figure 4Results of the two vaccine trials. Worm counts and mean egg counts per gram of liver tissue and intestinal tissue per individual mouse presented for the total number of mice in the two trials in the buffer control group and rSmKI-1 vaccinated group.
Table 1Antibody titres and parasitological data for mice in the rSmKI-1 vaccinated and control groups over two independent trials (trial 1, n = 8, 8, 9; trial 2, n = 9, 9, 9).
Antibody endpoint titres at 6 weeks post vaccination
Adult worm pairs, range
Adult worm pairs Mean ± SEM (% reduction)
Faecal eggs Mean ± SEM (% reduction)
Liver eggs Mean ± SEM (% reduction)
Intestinal eggs Mean ± SEM (% reduction)
Trial 1 Control (PBS) n = 8
IgG IgG1 IgG2a IgG2b IgG3
1:200 1:200 1:200 1:200 1:200
16–25
19.75 ± 1.112
2831 ± 352.4
21 390 ± 1209
23 521 ± 1405
rSmKI n = 8
IgG IgG1 IgG2a IgG2b IgG3
1:256 000 1:256 000 1:32 000 1:32 000 1:2000
11–20
12.56 ± 1.872 (33%) p = 0.0039
1737 ± 262.3 (39%) p = 0.0460
17 029 ± 1880 (20%) p = 0.0062
16 318 ± 2199 (31%) p = 0.0409
Trial 2 Control (PBS) n = 9
IgG IgG1 IgG2a IgG2b IgG3
1:200 1:200 1:200 1:200 1:200
16–23
18.11 ± 0.7158
3120 ± 383.2
24 889 ± 1406
14 375 ± 971.5
rSmKI n = 9
IgG IgG1 IgG2a IgG2b IgG3
1:512 000 1:256 000 1:32 000 1:32 000 1:2000
10–17
13.89 ± 0.7158 (23.3%) p = 0.0006
2096 ± 336.5 (32.8%) p = 0.0243
13 944 ± 1798 (43%) p = 0.0012
10 355 ± 962.5 (28%) p = 0.0142
PBS, phosphate-buffered saline. n = number of mice per group in the two trials that survived the trials and were necropsied.
An endpoint IgG titre of 1:512 000 was measured in mice vaccinated with rSmKI-1. The antibody response to individual IgG subtypes for the vaccinated group was 1:256 000 for IgG1, 1:32 000 for IgG2a and IgG2b, and 1:2000 for IgG3. In comparison, the endpoint titres of the control mice sera for all IgG subtypes were all 1:200 (Table 1). Antibody endpoint titres for mice in the LPS control group over the two independent trials are presented in the Supplementary Material (Table S1). It was clear that rSmKI-1 had induced a specific IgG response in the vaccinated mice compared with the controls.
Images of liver sections stained with haematoxylin and eosin were taken with an Aperio AT turbo (Leica Biosystems, Wetzlar, Germany), and the sizes of the granulomas were measured using ImageScope software (Leica Biosystems). Granuloma area was calculated as a percentage of the whole liver area. There was a significant reduction in granuloma area in the livers of SmKI-1-vaccinated mice compared with the controls (Figure 5).
Figure 5Liver granuloma size reduction by SmKI-1. (A) Percentage of the total granuloma area, and (B) representative images of granulomas detected in haematoxylin and eosin-stained liver sections from mice in the two trials; buffer control group (upper panels) and rSmKI-1 vaccinated group (lower panels).
When stimulated by rSmKI-1 in vitro, CD8+ splenocytes isolated from the rSmKI-1 vaccinated group had a significantly higher expression of IFN-γ compared with the rSmKI-1 stimulated splenocytes of control mice (Figure 6A). However culturing of splenocytes with rSmKI-1 caused an increase in CD4+ T-cell IFN-γ production, independent of whether the cells had been exposed to SmKI-1 in vitro (data not shown). Of the cytokines measured, significantly higher levels of IFN-γ, IL-10, and IL-6 were detected in the culture supernatants of in vitro rSmKI-1 stimulated splenocytes from vaccinated mice compared with in vitro rSmKI-1 stimulated splenocytes from control animals (Figure 6B).
Figure 6Cytokine profile of splenocytes recovered from SmKI-1 vaccinated and control mice. (A) Expression of IFN-γ by CD8+ cells was increased by stimulation with rSmKI-1 (25 μg/ml) in the vaccinated group, and (B) higher levels of IFN-γ, IL-6, and IL-10 were detected in the vaccinated mouse splenocyte culture supernatants compared with those of controls.
This is the first report of a vaccine trial against S. mansoni using a Kunitz-type protease inhibitor. The present study showed that despite the very low expression of SmKI-1 detected in the ES products of adult worms (
), rSmKI-1 was able to generate partial immune protection against schistosomiasis mansoni, thus appearing to play an important role in the functional biology of S. mansoni in vivo. One concern in vaccine development is the potential induction of autoimmunity, although there have only been very few documented cases of autoimmune diseases induced by vaccines (
). Sequence homology searches and the comparison of SmKI-1 and the human proteome revealed high sequence identity of SmKI-1 with human TFPI2, mainly in the first Kunitz domain of the three Kunitz domains of TFPI2. Accordingly potential cross-reactivity of SmKI-1 with human TFPI was assessed using Western blotting. This analysis indicated no immunological cross-reactivity between TFPI and SmKI-1, which provided the impetus to initiate vaccine trials with rSmKI-1.
According to the World Health Organization (WHO) benchmark for progression of a schistosome vaccine antigen into clinical assessment, a molecule that can consistently induce 40% protection or better is considered as an optimal anti-schistosome vaccine candidate (
). Furthermore, it is important to reduce parasite fecundity and egg viability following vaccination so as to reduce the rate of schistosomiasis transmission and pathology (
In this study, the vaccination of mice with rSmKI-1 resulted in a 23–33% reduction in total worm numbers. Most importantly, rSmKI-1 caused a reduction of 33–39% in faecal egg output. A reduction in faecal egg output not only reduces the parasite load in the host but also in the environment, reducing disease transmission. The reduction in egg burden in the liver and intestinal tissue was 20–43% and 28–31%, respectively, indicating that vaccination with rSmKI-1 would also have an impact in reducing disease pathology. Eggs trapped in the liver cause the formation of hepatic granulomas, whereas eggs trapped in the intestinal tissue result in severe lesions and colonic polyps (
). Furthermore, a decrease in the intestinal egg burden might reflect a consequent reduction in disease transmission by reducing the passage of schistosome eggs into the intestinal lumen from the mesenteric veins.
IgG is the dominant immunoglobulin evoked as the result of an immune response (
IgG antibody and TH1 immune responses to influenza vaccination negatively correlate with M-protein burden in monoclonal gammopathy of undetermined significance.
), and in this study, rSmKI-1 was shown to be immunogenic, producing a solid IgG response. Regarding the IgG subtypes generated, rSmKI-1 induced a much more predominant IgG1 than IgG2a, IgG2b, and IgG3 response. It is known that Th2-type responses during a helminth infection can promote B-cell class switching to IgG1 (
). The immune correlates of protection induced by other recognized schistosome vaccine candidates, such as the tetraspanins, have been shown in mice to be dominated by IgG1 antibody recognition (
), so that the high level of IgG1 response induced by rSmKI-1 is noteworthy and encouraging.
In the initial stages of a schistosome infection, parasites induce mainly a Th1-type immune response, and at around 6 weeks, at the commencement of female worm egg deposition, the immune response shifts dramatically to Th2 (
). According to the cytokine analysis undertaken here, rSmKI-1 induced the production of significantly higher IFN-γ levels, which would tend to down-regulate the Th2 immune response. Th2 immunity is necessary for granuloma formation and the development of hepatic fibrosis (
Cytokine production in acute versus chronic human schistosomiasis mansoni: the cross-regulatory role of interferon-γ and interleukin-10 in the responses of peripheral blood mononuclear cells and splenocytes to parasite antigens.
Gamma interferon responses of CD4 and CD8 T-cell subsets are quantitatively different and independent of each other during pulmonary Mycobacterium bovis BCG infection.
). This could possibly be the reason for the increased numbers of IFN-γ-expressing CD8+ cells among the splenocytes obtained from vaccinated mice after in vitro stimulation by rSmKI-1. According to previous studies, peripheral blood mononuclear cells of human subjects who are naturally resistant to schistosomiasis produce high levels of IFN-γ when stimulated with schistosome antigens (
). A number of antigens tested as promising S. mansoni vaccine candidates have shown an association between increased IFN-γ production and protection against schistosome challenge (
Prime-boost and recombinant protein vaccination strategies using Sm-p80 protects against Schistosoma mansoni infection in the mouse model to levels previously attainable only by the irradiated cercarial vaccine.
). Furthermore, significantly higher levels of the modulatory cytokine IL-10, as well as IL-6, were also observed in SmKI-1-vaccinated mice compared with controls. IL-6 is known to be protective against Schistosoma-induced pulmonary hypertension and intimal remodelling, reducing disease pathology (
). A Th1-type immune response is important for eliciting immune protection, whereas a Th2-type response is known to provide resistance to helminth infections (
). Overall, the protection induced by the rSmKI-1 vaccine was probably the result of a balanced Th1/Th2 response.
Schistosomes have been remarkably successful in colonizing mammalian hosts. These worms produce numerous molecules that are involved in the avoidance of host immune reactions so that they remain untouched in the mesenteric veins for a considerable period of time; the result of this is that the identification of key vaccine candidates is a major challenge (
). It is unlikely that a single vaccine candidate would be sufficient to evoke a high level of protective immunity. Thus, rSmKI-1 in combination with one or two additional molecules that are crucially important for parasite survival should in future be tested as a cocktail vaccine (
In summary, the data presented here demonstrate that immunization with rSmKI-1 generated marginal protection against murine schistosomiasis in two independent, blinded vaccine trials. SmKI-1 should in future be combined and tested as a cocktail vaccine with one or two additional molecules, as even a partially protective anti-schistosomiasis vaccine could be promising along with mass drug administration (
) and emphasizes the importance of SmKI-1 in the biology and survival of S. mansoni in its mammalian host.
Financial support
This project was funded by program (APP1037304 to DPM) and project (APP613671 to DPM) grants from the Australian National Health and Medical Research Council (NHMRC). DPM is a NHMRC Senior Principal Research Fellow and Senior Scientist at QIMR Berghofer.
Conflict of interest
The authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.
Appendix A. Supplementary data
The following is Supplementary data to this article:
Comparative pathogenesis of eosinophilic meningitis caused by Angiostrongylus mackerrasae and Angiostrongylus cantonensis in murine and guinea pig models of human infection.
Prime-boost and recombinant protein vaccination strategies using Sm-p80 protects against Schistosoma mansoni infection in the mouse model to levels previously attainable only by the irradiated cercarial vaccine.
Cytokine production in acute versus chronic human schistosomiasis mansoni: the cross-regulatory role of interferon-γ and interleukin-10 in the responses of peripheral blood mononuclear cells and splenocytes to parasite antigens.
Gamma interferon responses of CD4 and CD8 T-cell subsets are quantitatively different and independent of each other during pulmonary Mycobacterium bovis BCG infection.
IgG antibody and TH1 immune responses to influenza vaccination negatively correlate with M-protein burden in monoclonal gammopathy of undetermined significance.
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