Background
Schistosomiasis, caused by trematodes belonging to the
Schistosoma genus, is an important neglected tropical disease with a substantial impact on public health. Over 200 million people are infected with approximately half of these being school aged children [
1‐
3]. It has long been argued that the schistosomiasis disease burden is greatly underestimated, and it has been suggested that the number of infected individuals surpasses 400 million [
4]. This underestimation is likely due to the inability of current diagnostic methods to detect light infections. Schistosomiasis is a chronic illness that significantly diminishes quality of life, and, in affected children, interferes with growth and cognitive development [
2,
5,
6]. Treatment of schistosomiasis relies solely on the drug praziquantel which is distributed in mass drug administration control programs. However, program outcomes have been disappointing [
3]. In 2011, of the 112 million children in need of praziquantel, only 16 million received treatment [
7]. School-based mass drug administration programs do not take into account that as many as 40 % of children in sub-Saharan Africa are not enrolled in school [
8]. It has been shown that less than 5 % of the schistosomiasis affected population is treated with praziquantel, indicating that control programs are not reaching sufficient coverage [
9]. Furthermore, these mass drug administration programs are not sustainable because praziquantel does not prevent re-infection. Therefore continuous drug distribution at an optimal timing is necessary in order to maintain disease control and break the transmission cycle [
8‐
10]. The current amount of praziquantel donated by the various sources, such as the Schistosomiasis Control Initiative, MedPharm, and Merck KgaA, is not sufficient to meet the global need [
9]. Vaccines, and/or chemotherapy combined with vaccination, present the best strategy for long-term sustained control of schistosomiasis. An anti-schistosome vaccine could contribute to decreased disease spectrum and transmission by reducing worm and egg burdens. It has been stated that even a partially effective anti-schistosome vaccine could significantly help accelerate the elimination of schistosomiasis if incorporated with other control efforts such as mass drug administration, intermediate host control, and improved sanitation [
11,
12].
Our group has chosen to focus on
Schistosoma mansoni (Sm) cathepsin B as a vaccine candidate. This cysteine peptidase, originally cloned and identified by Klinkert et al., [
13,
14] is predominantly found in the adult worm and migratory larva. Sm-cathepsin B is involved in the digestion of blood macromolecules. It plays a key role in hemoglobin degradation and is involved in digestion of host serum albumin or immunoglobulin G [
15‐
19]. The physiological importance of Sm-cathepsin B has been highlighted by RNA interference (RNAi) technology which demonstrated that suppression of cathepsin B results in impeded parasite growth [
20]. The present study was designed to investigate the protective potential of a Sm-cathepsin B formulation with the adjuvant Montanide ISA 720 VG (SEPPIC Inc., Fairfield, NJ, USA) in a mouse model of schistosomiasis. Montanide is a squalene based adjuvant containing a mannide mono-oleate emulsifier. The adjuvant forms water-in-oil droplets that allow for slow antigen release at the injection site. Montanide was chosen as an adjuvant because it is acceptable for use in humans; it is both safe and well-tolerated. The different Montanide adjuvants have been used in over fifty clinical trials including malaria, cancer, and human immunodeficiency virus (HIV) vaccine trials [
21‐
23]. These adjuvants have also continuously shown to elicit robust antibody responses. In this communication, we report that an immunization regimen involving a formulation of recombinant Sm-cathepsin B and Montanide ISA 720 VG can significantly reduce worm and egg burdens.
Discussion
Due to its essential role in parasite growth and development and its continuous interplay with the host immune system, our group has focused on Sm-cathepsin B as a potential vaccine candidate. During the mid-1990s, the WHO Special Programme for Research and Training in Tropical Diseases (TDR/WHO) planned for the independent testing of various
Schistosoma mansoni antigens with the hopes of uncovering an optimal vaccine candidate. The committee’s goal was to find an antigen that could consistently induce 40 % protection or better [
29]. Recently, our collaborators have demonstrated the inbuilt adjuvant properties of Sm-cathepsin B. Immunizations with unadjuvanted Sm-cathepsin B could decrease both worm and hepatic egg burdens in a mouse model of schistosomiasis by 66 % and 51 %, respectively, when compared to saline control mice [
30]. However, the intestinal egg burden was not significantly reduced (24.7 %). The pathological importance of egg burden in the intestines cannot be neglected. The inflammation surrounding eggs trapped in intestinal tissues results in the formation of severe lesions and colonic polyps [
31‐
33]. Furthermore, a decrease in intestinal egg burden could reflect a consequent decrease in transmission by reducing the passage of parasite eggs from the mesenteric veins to the intestinal lumen. The development of an anti-schistosome vaccine needs to target all forms of parasitological burden: worm, hepatic egg, and intestinal egg burdens. The data presented in this manuscript for the formulation of Sm-cathepsin B plus Montanide ISA 720 VG shows comparable protective results for both worm burden; decreased by 60 % compared to adjuvant controls and 66 % compared to saline controls. The reduction in hepatic egg burden was greater in the animals immunized with antigen plus adjuvant (reduction of 62 % compared to adjuvant controls and 69 % compared to saline controls). In this study, we showed that animals immunized with Sm-cathepsin B plus Montanide ISA 720 VG had a significant decrease in intestinal egg burden (56 % compared to the adjuvant control animals and 59 % compared to saline controls). The Montanide-adjuvanted recombinant protein formulation was capable of achieving significant reductions of all parasitological burdens (worms, hepatic eggs, and intestinal eggs). Therefore, using Montanide ISA 720 VG as an adjuvant with the recombinant Sm-cathepsin B improves the protective potential of Sm-cathepsin B.
The
S. mansoni radiation attenuated (RA) cercariae vaccine has been essential to study immune effector mechanisms associated with protection as well as discovering potential parasite points of vulnerability. The RA vaccine is considered the gold standard for reproducible induced anti-worm immunity. Exposure to the attenuated cercariae can elicit significant protection levels against challenges with normal cercariae. Protection levels have been shown to range from 56 % all the way to 80 % when multiple vaccinations are performed in the mouse model [
34‐
37]. The protection levels attained in this study with Sm-cathepsin B plus Montanide ISA 720 VG, 56-69 %, are beyond the 40 % threshold established by the TDR/WHO, and are comparable to those generated by the RA vaccine.
Several studies have demonstrated that IgG antibodies play an important role in protection against schistosomiasis. The passive transfer of sera from chronically infected or protected animals results in significantly reduced parasite burdens in the recipient animals [
38‐
42]. Studies utilizing the olive baboon and rhesus macaque models have shown that high IgG titers at the time of cercarial challenge correlated with low worm burdens [
43,
44]. Furthermore, the worms collected from the macaques, upon perfusion, were physiologically crippled. The compromised structure of these worms is suggested to be a result of antibody-dependant mechanisms where IgG has a blocking or stimulatory effect leading to parasite starvation or exhaustion, respectively [
44,
45]. In vitro work using immune sera has also highlighted antibody dependant cell mediated cytotoxicity and antibody dependant complement mediated cytotoxicity as potential effector mechanisms involved in protection against schistosomiasis [
41,
46]. Immunizations with the recombinant Sm-cathepsin B in the presence of Montanide ISA 720 VG resulted in the production of elevated antigen-specific IgG titers in the experimental mice (mean endpoint titers at week 9 = 122,880). Immunization with Sm-cathepsin B alone yielded endpoint titers of approximately 3,500 [
30] (data not shown) thereby, demonstrating that the addition of the adjuvant Montanide ISA 720 VG increases the production of Sm-cathepsin B specific antibodies by a factor of 35. These values demonstrate this vaccine formulation’s substantial impact on humoral immunity. There were prominent differences when comparing endpoint titers of different IgG subclasses. The adjuvanted recombinant protein formulation elicited a significantly stronger production of the IgG1 subclass (mean endpoint titers at week 9 = 48,640) over IgG2c (mean endpoint titers at week 9 = 1,840). The IgG1 subclass is known to be representative of a T-helper cell 2 (Th2) phenotype as its production is stimulated by IL-4 whereas the IgG2c subclass is a marker for a Th1 [
47].
It has long been a consensus that Th1 responses play a key role in protection against schistosomiasis. For instance, peripheral blood mononuclear cells isolated from individuals, who are considered naturally resistant to schistosomiasis, produce high levels of interferon-gamma (IFN-ɣ) when stimulated with schistosome antigen [
29,
48]. Moreover, immunological studies involving the RA vaccine have shown that, in protected animals, the migration of the schistosomulae is terminated in the lungs due to the formation of inflammatory foci consisting of monocytes and CD4
+ T-cells with Th1 characteristics [
37,
45,
49]. In the RA model, IL-12 was shown to prime the Th1 response by inducing IFN-ɣ, which then acts in an autocrine manner to amplify the response [
37,
45,
49]. The RA vaccine model has also elucidated the important role of TNF-α in protection against schistosomiasis [
50]. Both Th1 cytokines IFN-ɣ and TNF-α are secreted by cells in the inflammatory foci, and they are both capable of activating macrophages. Other studies with promising
S. mansoni vaccine candidates have shown an association between protection against parasite challenge and an increased IFN-ɣ production [
51‐
53]. The formulation of Sm-cathepsin B plus Montanide ISA 720 VG was able to significantly increase the secretion levels of the three Th1 cytokines IFN-ɣ, TNF-α, and IL-12 compared to both the saline and adjuvant controls. However, our experimental formulation was also able to increase the secretion levels of Th2 cytokines; the secretion levels of IL-4 and IL-5 were significantly increased in the experimental animals compared to the controls. Recent studies have discussed the importance of inducing a Th2 response as well to achieve protection against schistosomiasis. Inducing such a response aims to involve eosinophils and basophils in the response to the parasite [
30,
54]. The formulation of Sm-cathepsin B in the presence of Montanide ISA 720 VG was able to stimulate both a Th1 (IFN-ɣ, TNF-α, and IL-12) and a Th2 response (IL-4, IL-5, and IgG1). This resulting mixed Th1/Th2 response may explain the higher levels of protection obtained in this study compared to our previous study using a CpG adjuvanted Sm-cathepsin B formulation [
24]. The CpG formulation generated a more Th1-biased response. Furthermore, a previous study showed that immunization with Sm-cathepsin B alone leads to increased secretion of Th2-associated cytokines such as IL-4, IL-5, and IL-13, but not Th1-associated cytokines such as IFN-γ thus, creating a Th2-biased response [
30]. The Sm-cathepsin B + Montanide ISA 720 VG also significantly increased the secretion levels of IL-17 in the experimental animals compared to the adjuvant control animals. This observation highlights the potential role of Th17 responses in protection against schistosomiasis. Increases in IL-17 levels have been correlated to lower worm burdens [
55]. IL-17 may contribute to protection by mediating the recruitment and activation of neutrophils which can impede larval migration via their extracellular traps [
54]. Furthermore, this implication of IL-17 was not observed in our previously tested formulation of Sm-cathepsin B + CpG [
24] thus, demonstrating the multi-faceted immune involvement mediated by this Montanide adjuvanted formulation.
Levels of both GM-CSF and CCL5 were also significantly increased in the experimental animals compared to the control animals. GM-CSF is involved in macrophage recruitment whereas CCL5 is involved in the recruitment of T cells and granulocytes. Furthermore, CCL5 mediates proliferation and activation of natural killer (NK) cells, which play a role in schistosomiasis protection in elderly populations [
56]. Increased secretion levels of CCL5 were also observed with our previous formulation of Sm-cathepsin B + CpG [
24]. However, elevated levels of GM-CSF were not observed in our previous formulation thus, representing another immunological difference generated by the formulation containing the Montanide ISA 720 VG adjuvant.
The results obtained from this study are promising and promote further testing of the vaccine candidate Sm-cathepsin B. In the future, the formulation can potentially be tested in a non-human primate model of schistosomiasis. These animals manifest a disease that is similar to that observed in humans, and the data from such a study may be more clinically applicable than data from a mouse study.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AR was involved in all aspects of the study including conceptualizing and conducting the experiments, analyzing the data, and writing the manuscript. KV contributed by performing all of the ELISA repeats for this study (total IgG, IgG1, and IgG2c). JPD provided the Sm-cathepsin B clone as well as invaluable intellectual input throughout the research. His vaccine expertise greatly enriched the study. MN supervised and coordinated the direction of this entire study as well as managed the data analysis and the interpretation of results. All of the authors have read and approved the final version of the manuscript.