Trichinellosis is a worldwide food-borne zoonosis. The parasitic nematode Trichinella spiralis, one of the most common etiologic agents of Trichinellosis, infects both wild and domestic animals. Human infection is mainly acquired by ingesting raw or undercooked meat infected with Trichinella larvae and contaminated pork is the major infective source in China (Cui and Wang, 2011; Ortega-Pierres et al., 2015). Trichinellosis clinical manifestations range from asymptomatic infection to fatal disease (Wilson N.O. et al., 2015). This zoonosis is not only a serious public health problem to human but also causes a big economic loss for the meat processing industry as China is now the largest producer and consumer of pork in the world (Dobrescu et al., 2014). Therefore, to develop a vaccine preventing swine infection would make a feasible contribution for the control of Trichinellosis.

During the past decades, many vaccine candidates against Trichinellosis have been investigated and reported, including those based on crude larval extracts (Deville et al., 2005), excretory-secretory (ES) products (Dea-Ayuela et al., 2006), DNA (Wang et al., 2016), recombinant proteins (Bi et al., 2015), peptides (Castillo et al., 2013) or combined DNA with protein (Gu et al., 2014), all of them induced different extents of partial protective immunity in animal models. However, due to the complexity of the life cycle, diversity of stage-specific antigens and immune-evasion strategies of T. spiralis (Zhang et al., 2011; Sun et al., 2015), researchers face the challenges in developing effective vaccines against Trichinellosis. Subunit vaccine based on multiple protective epitopes of several vaccine antigens possibly enables investigators to overcome these problems and provides a novel approach to develop vaccines against infectious diseases (Cao et al., 2015; Meza et al., 2017).

Paramyosin (Pmy) is a thick myofibrillar protein found only in invertebrates (Gobert and McManus, 2005). Pmy is not only a structural component of myofilament but also a promising vaccine candidate in some helminths such as Schistosoma mansoni (Ramirez et al., 1996), Brugia malayi (Li et al., 1993) and Taenia solium (Vazquez-Talavera et al., 2001). Our previous studies have identified that paramyosin of T. spiralis (Ts-Pmy) is a good vaccine candidate against Trichinella infection in term of its potential to induce protective immunity against the challenge of T. spiralis larvae (Yang et al., 2008; Yang J. et al., 2010). However, difficulty in expressing Ts-Pmy as a soluble rMEP due to its large molecular weight (102 kDa) prevents it from being scaled up for vaccine purpose. In addition, the identified complement-binding activities of recombinant Ts-Pmy protein raise a concern of possible interference of human normal complement function when Ts-Pmy is administered as a vaccine (Zhang et al., 2011; Sun et al., 2015). Compared to whole-protein antigens, an epitope-based vaccine may overcome these above-mentioned shortcomings and have many advantages such as increased safety and immunogenicity of vaccine for increased protective potency (Tang et al., 2015). The epitope-based vaccines are on the road to the market, especially in the treatment of cancer (Higashihara et al., 2014; Correale et al., 2016).

In our previous study, in an effort to understand immunoprotective mechanism of Ts-Pmy, a protective B cell epitope named YX1, including 20 amino acids within Ts-Pmy 88-107, has been identified to be recognized by a monoclonal antibody (mAb) 7E2 which protects passively transferred mice from being infected with T. spiralis (Wei et al., 2011). In addition, four potent CD4⁺ T cell epitopes of Ts-Pmy that could strongly induce both Th1 and Th2 immune responses were also identified (Gu et al., 2016). These results make it possible to design an effective epitope-based subunit vaccine against Trichinellosis. In this study a multi-epitope protein (MEP) vaccine was constructed based on previously identified B and T cell epitopes and its immunogenicity and vaccine efficacy was evaluated in a mouse model.

Immune-dominant epitopes of an antigen are very important in priming of the immune system efficiently as the immune response is always mounted against these regions. Therefore, immune-dominant epitopes, or collections of these epitopes, are considered as potential vaccine candidates (Rosa et al., 2010). Recombinant DNA technology has opens up a wide range of possibilities for designing a rational vaccine of chimeric proteins with several effective epitopes. Multi-epitope vaccines have the following advantages: several immune-protective epitopes can be included in a single immunogen, epitopes or regions without protective effect can be removed, and epitopes with adjuvant effects such as promiscuous T cell epitopes can be included to enhance protective efficacy (Fonseca et al., 2016).

Compared to vaccinating with the whole antigen, epitope-driven vaccine overcomes some potential safety concerns and appears to be capable of inducing more potent immune responses (Tang et al., 2015). To design a vaccine capable of inducing convenient humoral or cellular immune responses, it is essential to include the epitopes that could elicit the desired immune response (Soria-Guerra et al., 2015). The CD4⁺ T helper (Th) cells are activated when the peptide antigens are presented by MHC II molecules. Once activated, Th cells will divide rapidly and regulate the active immune response by secreting cytokines (Liu et al., 2011). As part of the adaptive immune response, CD4⁺ T cells play an important role in providing protective functions, including cytokine-mediated and contact-mediated signals to B cells, CD8⁺ T cells, and innate-immune cells, as well as direct attack on pathogens (Mettu et al., 2016). As CD4⁺ T cells are crucial in determining the functional status of both innate and adaptive immune responses, it is essential to comprise appropriate CD4⁺ T cell epitopes to improve the vaccine efficacy (Rosa et al., 2010). Many studies have shown that protective efficacy can be significantly improved by including an array of promiscuous T cell epitopes in vaccine constructs (Wen et al., 2014; Wu et al., 2016). Meanwhile, protective B-cell epitopes are also essential for developing epitope-based vaccines (Zhao et al., 2015; Sharma and Dixit, 2016). The multi-epitope vaccine is an attractive option and some epitope-based vaccines have been carried out in clinical trial against virus infection and carcinoma up to now (Lennerz et al., 2014; Nakamura et al., 2016).

Our previously identified B cell and T cell epitopes on a leading vaccine candidate Ts-Pmy laid a solid foundation for designing an effective epitope-based multivalent subunit vaccine against Trichinellosis. A novel multi-epitope vaccine called MEP was constructed in this study, which includes four T cell epitopes (P2, P3, P4, and P5) and one B cell epitope (YX1) of Ts-Pmy. The rationale for the design of MEP centers around two critical considerations. Firstly, a combination of T- and B-cell epitopes may enhance the induction of both cell-mediated and humoral immune responses. It has been proved that a mixed humoral and cellular immune response contributed to the protective immunity against T. spiralis infection (Bi et al., 2015; Liu et al., 2015). Secondly, the strategy involves positioning T cell epitopes on the N-terminal side and B-cell epitopes on the C-terminal side separated each epitope by a di-lysine linker. Some studies showed that the chimera with T cell epitope located in N-terminus and B cell epitope located in C-terminus induced higher affinity and specificity of antibody than did the reverse orientation (Partidos et al., 1992). The bi-lysine linker (KK) was inserted between different epitopes to preserve their independent immunological activities. KK is the target sequence of cathepsin B, which is one of the important proteases for antigen processing in the context of MHC-II antigen presentation (Sarobe et al., 1993). Splenocytes from mice immunized with rMEP showed increased T cell proliferations and cytokines secretion upon stimulation of individual T cell epitopes, P2, P3, and P4, but not by P5, indicating that most of the arranged epitopes were presented and processed correctly in immune response in vivo. B cell epitope constructed at the C-terminus of MEP vaccine could be recognized by the mAb 7E2 in Western Blot. It confirms that B cell epitope maintains its antigenicity in the MEP vaccine.

Mice immunized with E. coli-expressed soluble recombinant MEP protein elicited 55.4% muscle larval reduction against T. spiralis infection compared to mice administrated with PBS only, which is significant higher than 34.4% larval reduction induced by the parental rTs-Pmy and reaches the highest protection so far among all DNA or recombinant protein vaccine candidates developed in our lab. In our previous study, mice vaccinated with rTs-Pmy protein formulated with different adjuvants demonstrated a 33.7–36.7% ML burden reduction respectively against T. spiralis larvae challenge (Yang et al., 2008; Yang J. et al., 2010). Salmonella-delivered Ts-Pmy DNA vaccine also produced a significant 46.6% reduction in ML burden after challenge (Wang et al., 2016). Other vaccine candidates, for example, Ts-ES-1 and Ts87, were also investigated in our group. Recombinant Ts-ES-1 protein produced a 42.1% ML burden reduction (Bi et al., 2015) and Salmonella-delivered Ts87 DNA conferred a 34.2% ML burden reduction against challenge (Yang Y. et al., 2010). Two possible reasons might explain the higher protective efficacy of rMEP observed in the present study. Firstly, balanced cell-mediated and humoral immune responses induced by rMEP may contribute to the protective immunity. Our study showed that immunization with rMEP induced high levels of specific IgG and a mixed IgG1/IgG2a response (Figure 3), with an isotype ratio (IgG1/IgG2a) > 1.0, indicative of a Th2-dominated isotype response (Mosmann and Coffman, 1989). In addition, immunization with rMEP induced strong splenocyte proliferation and high levels of Th1 cytokine (IFN-γ) and Th2 cytokines (IL-4, IL-5) upon stimulation of rMEP (Figure 4). The stimulation with individual epitope peptide also showed increased T cell proliferation for all four T cell epitopes than the negative control, indicating these four T cell epitopes were presented effectively in vivo and stimulated cellular immune responses (Figure 4). Cytokines profiles results showed that three of four individual T cell epitopes (P2, P3, and P4) induced elevation of Th2 cytokines (IL-4 and IL-5). The possibility for impotency of P5 in cytokine induction presumably is that P5 was constructed at the end of T epitope array that may be folded inside of the structure and prevented its processing. The second reason for the better protective immunity induced by rMEP than its parental rTs-Pmy is that whole rTs-Pmy antigen may contain epitopes involved in host immune suppression. Trichinella and other nematode infection induce strong CD4⁺CD25⁺ regulatory T cell response characterized by high level of IL-10 and TGF-β (Beiting et al., 2007), possibly through secreting some proteins with immunomodulatory function to suppress host immune response as a survival strategy. Mice immunized with Ts-Pmy DNA vaccine induced a strong IL-10 response (Wang et al., 2016). Immunization of rTs-Pmy protein also induced CD4⁺CD25^-Foxp3⁺ T cell population associated with high levels of IL-10 and TGF-β, possibly through stimulating dendritic cells (Guo et al., 2016). Ts-Pmy also directly binds to human complement components C8/C9 (Zhang et al., 2011) and C1q (Sun et al., 2015) to inhibit the activation of human complement. These studies indicate that Ts-Pmy itself may play a direct role in the suppression of host immune response to Trichinella infection and to the immunization of vaccine antigen as well. Epitope-based subunit vaccine design should take the T cell subsets primed during immunization into consideration and circumvent potential regulatory T cells activation that can handicap efficacy (Moise et al., 2014). In this study, CD4⁺ T cell epitopes that could induce effective Th1/Th2 immune responses were selected to construct rMEP. Ts-Pmy did not induce comparable immunity as rMEP did, one possible reason is that only a few immunodominant epitopes in Ts-Pmy are sufficient to induce a protective response and the whole protein may contain some epitopes with adverse effect as a vaccine.

In conclusion, a novel multi-epitope vaccine (MEP) was constructed by using four CD4⁺ T cell epitopes and one B cell epitope from Ts-Pmy as an array separated with a bi-lysine spacer between each epitope. The MEP was expressed as a soluble rMEP in E. coli. Mice immunized with rMEP vaccine produced significant higher muscle larval reduction (55.4%) than that induced by the immunization of parental rTs-Pmy (34.4%) against T. spiralis infection. The higher protection is associated with rMEP induced high levels of anti-rMEP specific IgG and subclass IgG1/IgG2a, elevated splenocytes T cell proliferation and secretion of IFN-γ, IL-4 and IL-5. The cytokine profiles and T cell proliferation of splenocytes from mice immunized with rMEP upon stimulation of individual T cell epitope suggested that most of T cell epitopes are exposed and processed well during immunization that may contribute to the high protection induced by the immunization of rMEP. This study implies that epitope vaccine is a promising approach for the development of vaccines against Trichinellosis. In future studies, a multi-epitope vaccine containing more protective epitopes from other vaccine candidates will be designed in order to induce more comprehensive protective immunity. In addition, as different adjuvants may induce different immune responses (Wilson K.L. et al., 2015), other adjuvants inducing Th1 and/or Th2 responses will be attempted to produce better protective immunity of rMEP.

YG designed and performed the experiments; XS, BL, and JH helped with the animal experiments. YG wrote the paper; BZ and XZ gave critical reviews of the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.