Identification of B‐cell dominant epitopes in the recombinant protein P29 from Echinococcus granulosus

Abstract Introduction Echinococcus granulosus (E. granulosus) causes a hazardous zoonotic parasitic disease. This parasite can occupy the liver and several areas of the body, causing incurable damage. Our previous studies have provided evidence that the recombinant protein P29 (rEg.P29) exhibit immune protection in sheep and mice against pathological damage induced by E. granulosus, showing its potential as candidate for vaccine development. However, information on the B‐cell epitopes of rEg.P29 has not yet been reported. Methods Immunological model was established in mice with rEg.P29. SDS‐PAGE and Western blot were used to identify protein. Screening for B‐cell dominant epitope peptides of rEg.P29 by enzyme‐linked immunosorbent assay (ELISA) and immune serum. Dominant epitopes were validated using ELISA and flow cytometry. Multiple sequence alignment analysis was performed using BLAST and UniProt. Results Immunization with rEg.P29 induced intense and persistent antibody responses, and the epitope of the dominant antigen of B cells are identified as rEg.P29 166–185 (LKNAKTAEQKAKWEAEVRKD). Anti‐rEg.P29 166–185‐specific antibodies lack epitopes against IgA, IgE, and IgG3, compared to anti‐rEg.P29‐specific antibodies. However, anti‐rEg.P29 166–185 IgG showed comparatively higher titers, as determined among those peptides by endpoint titration. In addition, rEg.P29 and rEg.P29 166–185 promote B‐cell activation and proliferation in vitro. The dominant epitopes are relatively conserved in different subtypes of the rEg.P29 sequence. Conclusion rEg.P29 166–185 can act as a dominant B‐cell epitope for rEg.P29 and promote cell activation and proliferation in the same way as rEg.P29.


| INTRODUCTION
Echinococcus granulosus is a zoonotic parasitic disease with an extremely serious economic burden and poses great danger in human health. 1 Western China has a high incidence of hydatid disease. 2 E. granulosus grows slowly, and more than 80% of echinococcosis cysts occur in the host liver, causing incurable damage. 3,4 Currently, the main treatment methods for patients with cystic echinococcosis (CE) are surgery and drug treatment 5 ; however, drugs generally have side effects. Meanwhile, surgical treatment has a high recurrence rate and results in huge economic pressure and body damage to patients. [6][7][8] The massive use of anthelmintics has caused a series of problems, such as drug resistance, drug residues, and environmental pollution. 3 Vaccines are safe and residuefree and are important tools for disease prevention and control. 9,10 However, traditional vaccines are composed of attenuated or inactivated pathogenic microorganisms and may cause unwanted or harmful immune responses in the body. 11 Therefore, the development of an effective and safe vaccine against parasitic diseases is important in animal husbandry and public health.
Despite the wide range of preventative approaches explored, a human vaccine against E. granulosus is not yet available. In fact, the E. granulosus vaccine has been proposed for a long time, and many candidate proteins have been studied, such as Eg95, antigen B, and rEg.P29. [12][13][14][15][16] Our previous study found that rEg.P29 showed 94.5% and 96.6% protective efficacy in sheep and mouse models with secondary infection, respectively, and induced strong cellular and humoral immune responses against E. granulosus infection. 16,17 However, the anti-infective mechanism of rEg.P29 is still unclear.
In recent years, as our understanding of the immune response has improved and research on vaccine production has become more refined, the search for the most precise vaccine components, that is, antigenic epitopes, in recombinant vaccines has begun. These antigenic epitopes represent the smallest immunogenic regions of protein antigens and can induce a specific immune response with the desired effects in the body. 18 Considering the importance of rEg.P29, an epitope vaccine containing moderate antigenic peptides of the gene may serve as an efficient vaccine against E. granulosus infection.
Studies have shown that B-cell-mediated humoral immune responses play an important role against diseases. 18 Based on this study, we sought to screen for a dominant B-cell epitope of rEg.P29 and provide a basis for the construction of peptide-based Vaccines for rEg.P29.

| Animals and immunizations
This study was approved by the Experimental Animal Ethics Committee of the Ningxia Medical University. C57BL/6 female mice (SCXK2016-0006) aged 6-8 weeks were purchased from the Animal Center of Ningxia Medical University and kept for acclimatized feeding for 7 days before the experiment. Immunization protocols per mouse are summarized briefly as follows: 20 μg rEg.P29 was mixed with 20 μg CpG ODN 1826 (rEg.P29 + CpG), or 50 μl Freund's complete adjuvant (rEg.P29 + FCA), and phosphate-buffered saline (PBS) was used as control. Booster immunization was performed using Freund's incomplete adjuvant. For subcutaneous immunization, the mixture was suspended in PBS and injected in the lower quadrant of the abdomen (100 μl/mouse). Immunization was performed three times, 1 week apart ( Figure 1A).

| Amino acid sequence
The complete amino acid sequence of rEg.P29 was obtained from GenBank (accession number XP_024351425.1).

| Antigen and adjuvant
Protein purification and expression were performed as described previously. 15 Briefly, the positive strain was induced overnight with 0.05 mg/ml isopropyl-b-Dthiogalactoside (IPTG; Invitrogen) at 37°C to express the recombinant protein P29, which was then purified using an anti-His-tagged nickel purification column (Merck). Purified rEg.P29 was identified using Western blot analysis. A BCA Kit (KeyGEN Biotech Products) was used to detect the protein concentration. Next, an overlapping peptide library of rEg.P29 and CpG ODN 1826 (TCCATGACGTTCCTGACGTT) was synthesized with98% purity with the assistance of Shanghai Shenggong Biological Co., Ltd. Complete and incomplete Freund's adjuvants were purchased from Sigma-Aldrich.

| Sample collection and cell preparation
Blood samples were obtained from the orbit, and the serum was collected and purified via centrifugation at 400 × g at 4°C for 10 min. Splenocytes were isolated from F I G U R E 1 rEg.P29 immunization induced a strong humoral immune response. Mice were primed and boosted with PBS, rEg.P29+FCA, or rEg.P29+CpG, following the prime-boost protocol. (A) At the indicated time interval, mice were killed and serum was separated by centrifugation. (B) The expressed and purified recombinant P29 protein was detected by SDS-PAGE analysis. Lane M, protein marker with molecular mass indicated on the left; Lane 1, Escherichia coli containing pET28a before IPTG induction; Lane 2, E. coli containing pET28a-P29 6 h after induction; Lane 3, purified rEg.P29 using His-affinity chromatography, as indicated by the arrow. (C) Western blot identifies rEg.P29. Purified rEg.P29 was immunoblotted with anti-His tag antibody or postimmunized serum from mice. M, protein marker; Lane 1, anti-his antibody; Lane 2, serum in PBS group; Lane 3, immune serum in rEg.P29+CpG group; Lane 4, immune serum in rEg.P29+FCA group. Anti-rEg.P29 specific antibodies were detected by ELISA. (D) ELISA plate was coated with rEg.P29 (10 μg/ml). Then, serum levels of anti-rEg.P29 antibodies were measured by ELISA using HRP-labeled anti-mouse antibodies. The absorbance was read at 450 nm. ****p < .0001, ***p < .001, **p < .01, ns, not significant, p > .05 the tissue by filtering the tissue through a 70-μm strainer in a sample diluent, and the prepared cell suspension was transferred to a density gradient (Tianjin Haoyang biological products). Spleen lymphocytes were separated via centrifugation at 450×g for 20 min.
As for proliferation, the cells were washed two times with pre-warmed PBS supplemented with 0.1% BSA, carboxyfluorescein diacetate succinimidyl ester at a final concentration of 2.5 µmol/L (CFSE, Invitrogen) was added, and the resulting mixture was incubated for 15 min at 37°C in the dark. The reaction was terminated with precooled RPMI1640 containing 10% FBS, incubated at 4°C for 5 min, and washed two times with precooled RPMI1640 containing 10% FBS. CFSE-labeled cells were then cultured with or without 15 µg/ml rEg.P29 or peptide for 3 and 5 days at 37°C, 5% CO 2 . Cell samples were collected and stained with fluorochrome-conjugated mAbs for phenotyping at 4°C in the dark. The samples were subjected to FACSCeles-ta™, and data were analyzed using FlowJo software (TreeStar).

| Multiple sequence alignment
The amino acid sequences of P29 of different subtypes of E. granulosus were obtained using BLAST, and the conserved dominant epitopes were analyzed using UniProt (https:// www.uniprot.org/). Amino acid sequences obtained from E. granulosus were as follows: AHA85389.1 (E.

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc.) and SPSS 22.0. Unpaired Student's t-test was used for comparisons between two groups, and one-way or two-way analysis of variance was used for comparisons among more than two groups. The least significant difference test or Student-Newman-Keuls test was used to analyze data with normal distribution and homogeneous variance. Dunnett's T3 test or independent sample t-test was used to analyze data with normal distribution but had uneven variance. Data are represented as mean or mean ± SD. ****p < .001; ***p < .005; **p < .01; *p < .05 were considered statistically significant, whereas p > .05 was considered not significant (ns), as stated in figure legends.

| Expression, purification, and identification of rEg.P29
The rEg.P29 was successfully expressed and purified. SDS-PAGE analysis showed that rEg.P29 had a molecular weight of 31 kDa ( Figure 1B). The protein concentration was 1 mg/ml, and the protein was stored at −80°C). Western blot analysis showed that rEg.P29 could not be recognized by antibodies in the mouse serum samples from the PBS group but could be recognized by a mouse monoclonal His-tag antibody and mouse sera from the immunization group ( Figure 1C).

| rEg.P29 induced a sustained antibody response
To assess whether rEg.P29 induces specific immune responses in mice, we studied the antibody responses to rEg.P29 immunization in mice sera. Both immunization groups were compared with the control group. Immunization with rEg.P29 induced a high level of anti-rEg.P29specific antibodies. The antibody responses peaked at Week 2 after boost immunization and were maintained for more than 16 weeks, except for IgA and IgE. Specifically, IgA and IgE levels gradually decreased at Week 4, but IgG remained at high levels. Both immunization groups were compared with the control group ( Figure 1D).

| Immunization induced a polarized Th1 response
Characterization of IgG isotype responses in rEg.P29 + FCA and rEg.P29 + CpG group showed the induction of both IgG1 and IgG2a antibody responses, with the IgG2a isotype showing a comparatively higher level than IgG1 (Figure 2).

| Screening for the immunodominant linear B-cell epitope peptides
The sera of mice immunized with rEg.P29 and HRP-labeled goat anti-mouse IgM and IgG were used as antibodies to detect the interaction between the antigen peptide (Table S1) and the serum. The dominant epitopes were selected, as shown in Figure 3A,B. Compared to the control group, the synthesized antigen peptide (Table S1) could induce ELISAspecific reactions with the immune serum. ID34-ID36 IgG and IgM levels were significantly higher than those of the other antigenic peptides in both immune groups.

| rEg.P29 166-185 was relatively conserved in different subtypes of P29 sequences
Here, we analyzed the rEg.P29 sequences of different genotypes of E. granulosus and the corresponding regions of the dominant peptides. As shown in Figure S3, the dominant peptide (rEg.P29 166-185 ) was completely conserved in the P29 sequences of different subtypes.

| DISCUSSION
With the improvement in living standards and sanitary conditions, the incidence rate of E. granulosus has been declining annually. 4 However, it is still a serious public health problem in pastoral areas. New disciplines, such as bioinformatics, immunoinformatics, and rational vaccine design, have been applied in recent years. 19,20 Among them, the design of a new vaccine composed of multiple epitopes of a single antigen has become a new method to enhance the host's protective immune response at humoral and cellular level. The epitope, also known as the antigenic determinant, is a chemical group in the antigen molecule that determines antigen specificity. 21 Epitope vaccines are based on the characteristics of the amino acid sequence of antigenic epitopes. They have become a new direction invaccine research because of their highly effective immune protection, safety, and stability. 22,23 In 2013, Esmaelizad et al. 24 synthesized a multi-T-cell epitope antigen based on five proteins: EgGST, EgA31, Eg95, Eg14-3, and EgTrp. The antigen-stimulated mouse spleen cells to produce IFN-γ and obtained 99.6% protective efficacy in a mouse model. Simultaneously, multiepitope vaccines are also used in other fields. [25][26][27] The recombinant Bacillus Calmette-Guérin (BCG) vaccine (rBCG) constructed by Mohamud et al. 28 included two peptides, rBCG018 and rBCG032, that exhibited a stronger ability to induce cellular and humoral immune responses than BCG. Compared with traditional vaccines, synthetic peptide vaccines are easily prepared, have stable structure, and do not risk infection; thus, they are used for novel vaccine design at present.
Our previous studies have demonstrated that rEg.P29 has a favorable protective effect in sheep and mouse models. However, the B-cell epitopes of rEg.P29 remain unclear. Identification of the B-cell epitopes of rEg.P29 contributes not only in elucidating the mechanism underlying the B-cell immune process of E. granulosus but also to the development of more effective epitope vaccine candidates. Screening epitopes with high immunogenicity is used for epitope development. Here, after the primer and booster doses of rEg.P29, the specific antibody levels peaked at Week 2. Immunized mice expressed IgM, IgG, IgG2a, and IgG2b, and few expressed IgA and IgE. Antibody production was maintained for at least 16 weeks, except for IgA and IgE. Studies have shown that IgG, IgG2a, and IgG2b may participate in the protective immune response induced by the vaccine, and IgG1 may be related to susceptibility to hydatidosis. Meanwhile, IgG3 may be associated with the formation of a local inflammatory response early in the host. 29 Th2-related antibodies and cytokines mainly promote the escape of parasites and establish immune tolerance, while Th1 related antibodies and cytokines are closely related to anti-infection. 30,31 Serological antibody tests showed that the immunized mice had a predominantly IgG and IgG2a antibody response against rEg.P29, followed by IgG1. Moreover, rEg.P29 immunization induced a predominantly Th1 immune response. Our results showed that the dominant epitope lacks anti-rEg.P29 166-185 specific IgA, IgE, and IgG3 antibodies compared to anti-rEg.P29 specific antibodies. These results suggest that rEg.P29 166-185 may only induce protective immunity while eliminating allergic reactions. 32,33 However, this difference may be caused by the adjuvant or the weak immunogenicity of the peptide, suggesting that multiple epitopes or vectors should be combined to improve the immunogenicity of the peptide. Furthermore, lymphocyte activation and proliferation levels are indicators that reflect the body's immune function. Here, we screened rEg.P29 166-185 , for its ability to activate and proliferate B cells using flow cytometry. Results showed that rEg.P29 166-185 and rEg.P29 promote B-cell activation and proliferation, highlighting that specific B cells can rapidly activate and proliferate in response to infection.
In addition, sequence alignment of the dominant epitope with the P29 protein of different isolates of E. granulosus revealed that rEg.P29 166-185 was highly conserved, suggesting its application as a broad-spectrum vaccine. 34 Two adjuvants were used in the present study. CpG adjuvant is an oligodeoxynucleotide containing a CpG sequence with strong immune-activating properties; it was reported to show few side effects and can be used in humans. 35 Our results showed that both CpG and Freund's adjuvant enhanced the humoral immune response. However, Freund's adjuvant induces severe local necrotic ulcers and is considered to be toxic for human use. Considering the translational application of the vaccine, we used CpG adjuvant in subsequent experiments. Nonetheless, studies have shown that adjuvants alone do not induce specific antibody responses, 36 as shown in Figure S2.
This study has some limitations. Our study did not validate the immunoprotective effect after immunization of the dominant epitope. Further studies are warranted. In conclusion, our studies show that rEg.P29 immunization can induce a strong and sustained antibody response, and rEg.P29 166-185 can act as its dominant B-cell epitope. Furthermore, rEg.P29 and rEg.P29 166-185 stimulation in vitro can promote B-cell activation and proliferation.

ETHICS STATEMENT
All mouse experiments were given permission by the Ningxia Medical University Institutional Review Committee (Permit number: SCXK2016-0006) and carried out in strict accordance with national and institutional guidelines. The data obtained met the appropriate ethical requirements.

AUTHOR CONTRIBUTIONS
Wei Zhao and Changyou Wu were in charge of the project design; Yongxue Lv and Shasha Li were responsible for conducting the experiments, the data analysis, and paper writing; Tingrui Zhang and Jihui Yang were responsible for guiding Flow analysis in the experiment; Jia Tao was responsible for the design of relevant peptides in the experiment; Liangliang Chang and Yazhou Zhu were responsible for animal feeding; Yongxue Lv was in charge of the revision; Wei Zhao and Changyou Wu were responsible for the experimental guidance; all authors read and approved the final manuscript.

DATA AVAILABILITY STATEMENT
Contact the corresponding author for data and material requests.