Immune protection of three serine protease inhibitors vaccine in mice against Rhipicephalus sanguineus

Bioactive molecules in tick saliva are considered to be key to successful feeding and further the transmission of tick-borne pathogens. Problems such as pathogen transmission and animal weight loss result in tick infestation can cause tremendous economic losses to the livestock industry. Therefore, the development of a universal tick vaccine is urgently needed. In this paper, three serine protease inhibitor (serpin) proteins RMS-3, L7LRK7 and L7LTU1 were analyzed with bioinformatics methods. Subsequently the proteins were expressed and purified, and inoculated into Kunming mice for immune protection analysis. The amino acid sequence similarities between RMS-3, L7LRK7 and L7LTU1 were up to 90% in Rhipicephalus sanguineus. The recombinant RMS-3 + L7LRK7 + L7LTU1 showed anticoagulant reaction function and could inhibit the activity of CD4+ lymphocytes, when inoculated into Kunming mice. Additionally, After the immunized mice were challenged with Rhipicephalus sanguineus, the percentage of larvae and nymphs that were fully engorged dropped to 40.87% (P < 0.05) and 87.68% (P > 0.05) in the RmS-3 + L7LRK7 immune group, 49.57% (P < 0.01) and 52.06% (P < 0.05) in the RmS-3 + L7LTU1 group, and 45.22% (P < 0.05) and 60.28% (P < 0.05) in the RmS-3 + L7LRK7 + L7LTU1 immune group, in comparison with the control group. These data indicate that RmS-3 + L7LRK7 + L7LTU1 has good immune protection and has the potential to be developed into a vaccine against the larvae and nymphs of R. sanguineus.

The surface accessibility, flexibility and B cell epitope of RmS-3, L7LRK7 and L7LTU1 proteins was predicted and analyzed using DNAStar-protean software; T cell epitopes of RSM-3, L7LRK7 and L7LTU1 proteins were analyzed using AMPHI and Rothbard-Taylon algorithm in DNAstar-protean software.

Expression and purification of recombinant proteins
The codons of RmS-3, L7LRK7, and L7LTU1 gene were optimized using the codon optimization tool of the online program ExpOptimizer (https:// www.novop ro.cn/ tools/ codon-optim izati on.html) in order to more effectively express the aforementioned proteins.The recombinant expression vector was created using the pTYB12 plasmid, and an E. coli BL21 (DE3) competent strain (Sango Biotech, China) was transformed with the recombinant plasmid.
Briefly, monoclonal strains were inoculated in Luria-Bertani (LB) broth at 37 °C on a platform shaker until an optical density at 600 nm (OD 600 ) of 0.60 and subsequently induced for 24 h with 2 mM of Isopropyl b-d-1-thiogalactopyranoside (IPTG).The recombinant proteins were highly expressed and soluble.Bacteria pellets were resuspended in the lysis buffer (20 mM Tris-HCl (pH 8.5), 150 mM NaCl, 1 mM EDTA), ultrasonicated and centrifuged.The supernatant was purified with a Chitin (CBD-tag Affinity) Resin column (New England Biolabs, US).After purification, the fusion protein was cleaved at 16 °C or 23 °C for 24 h with a cleavage buffer (20 mM Tris-HCl (pH 8.5), 500 mM NaCl, 1 mM EDTA, 50 mM β-mercaptoethanol).Next, the target protein is eluted by the column buffer (20 mM Tris-HCl (pH 8.5), 500 mM NaCl, 1 mM EDTA).Finally, excess salt and β-mercaptoethanol are removed by desalting buffer.All proteins were determined by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).The protein was then lyophilized with the ALPHA 1-2 LD plus freeze dryer (Christ, Germany) and preserved in − 20 °C.

Vaccine formulation
Eight groups-RmS-3, L7LRK7, L7LTU1, RmS-3 + L7LRK7, RmS-3 + L7LTU1, L7LRK7 + L7LTU1, RmS-3 + L7LRK7 + L7LTU1 and control-were designed from 6-week-old female Kunming mice.Each group were vaccinated with corresponding antigens, while the control group were injected with PBS.Every group has 20 mice in it.The lyophilized protein was resuspended in sterile PBS to obtain a concentration of 1.0 mg/mL each.On days 0, 21 and 35, Kunming mice were subcutaneously injected, and the immunization dose of each mouse was 100 μL (25 μL antigen solution, 25 μL PBS solution and 50 μL adjuvant).50 μL PBS solution and 50 μL adjuvant was used for injection to each animal in the control group.In order to achieve better immunization effect, complete Freund's adjuvant (Sigma-Aldrich, Inc.) was used for the first immunization, and incomplete Freund's adjuvant (Sigma-Aldrich, Inc.) was used for the remaining two immunizations.On the fourth day after each inoculation, serum was collected and stored at − 20 °C.

Coagulation and routine analysis of blood
Orbital blood was collected from mice 7 days after the third immunization.The volume of blood collected from each mouse was 500 µL.Before detection, the blood was gently reversed and mixed for 10 times, and the blood routine was detected by automatic blood cell analyzer (BC-2800).The main detection indicators were as follows: white blood cell (WBC), lymphocyte (LYM), monocyte percentage (MON), neutrophil (NEU), red blood cell (RBC), hemoglobin (HGB) and platelet (PLT).
The clotting capability of the proteins RmS-3, L7LRK7, and L7LTU1 were measured by the following tests, prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT).On the 4th day after each immunization, 3.2% sodium citrate anticoagulant was added to collect mouse blood.The blood samples were centrifuged at 3000 rpm for 10-15 min within two hours to obtain plasma, stored at − 20 °C, transported on dry ice, and sent to Servicebio company for testing.

Antibody titer detection
A 96-well plate was coated with 0.1 μg/well recombinant protein in 100 μL carbonate/bicarbonate buffer (500 mM pH 9.6) and incubated overnight at 4 °C.The plate was washed three times (5 min each) with PBST (PBS containing 0.05% Tween-20) and then incubated with blocking solution (PBST containing 5% BSA) at 37 °C for 2 h.After washing with PBST three times, the plate was added with A series of 1:3 dilutions of mice serum and incubated for 1 h at 37 °C and washed as above.After that, 1 h incubation at 37 °C with 100 μL of HRPconjugated goat anti-mouse IgG (diluted with 1:5000).The plate was washed again, and incubated with 100 μL of 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB) for 15 min at room temperature in the dark. 2 M H 2 SO 4 (50 μL/well) was added for the end of the reaction, and the optical density (OD) of the product was measured at 490 nm by a microplate reader (Perlong Medical, Beijing).The OD450 nm (test group)/OD450 nm (negative control) ratio ≥ 2.1 was considered as a positive result.

Identification of salivary proteins in R. sanguineus
Immunized mice serum was utilized for the detection of proteins RmS-3, L7LRK7 and L7LTU1 in R. sanguineus salivary glands.The salivary glands of 10 partially engorged female ticks were collected in a centrifuge tube containing 200 μL of carbonate/bicarbonate buffer (500 mM, pH 9.6), crushed and diluted to 20 mL as an antigen coating solution for iELISA detection.The operation refers to the description in "Antibody titer detection" section.

Detection of CD4+, CD8+ T lymphocyte ratio
Two weeks after the last immunization, the mice spleen lymphocyte cells were harvested.In short, the mice were euthanized by cervical dislocation and soaked in 75% alcohol for 10 min for sterilization.Spleens were subsequently aseptically removed and a single cell suspension were prepared in PBS after lysis of red cells with adjustment of the cell concentration to 1 × 10 6 cells/100 μL.
The lymphocytes isolated above were incubated with fluorescently labeled antibodies (FITC anti-mouse CD3ε, PE anti-mouse CD4, APC anti-mouse CD8a) at 4 °C for 50 min in the dark.One blank control tube (only cells, without any dye staining) and 3 single-dye adjustment compensation tubes (1 tube is only dyed with FITC dye, 1 tube is only dyed with PE dye, and the other tube is stained with APC dye only) were used as controls.Cells were washed twice with PBS and then resuspended in 200 μL of PBS.Flow cytometer (Beckman Coulter, USA) was used for detecting levels of T lymphocyte subgroups (CD4 + and CD8 + ).

Challenge trials
On day 14 after the last immunization, tick challenge trial were performed on the immunized mice.The device for tick-infested mice was previously described 39 .For the tick challenge, there were 6 mice in each group, 3 mice were parasitized with 50 larval ticks per mouse; 3 mice were parasitized with 30 nymphs per mouse.The ticks were collected at 7 days after challenge, observe and count the changes in the tick weight, the number of engorged ticks and tick molting rate.

Approval for animal experiments
All animals used in the experiments were housed at the Vector Biology Laboratory, School of Life Sciences, Hainan University.The care and use of animals in this study were approved by the Hainan University Institutional Animal Use and Care Committee.The study is reported in accordance with ARRIVE guidelines.All the methods in this study were carried out in accordance with relevant guidelines and regulations.
The results of B cell epitope analysis were shown in Supplementary Fig. 1.There are 15 B cell antigen epitope fragments in RmS-3, 14 in L7LRK7, and 10 in L7LTU1.AMPHI algorithm and Rothbard-Taylor algorithm were used for predicting T-cell epitope of the three proteins.The comprehensive results of the two algorithms revealed that the T-cell epitope fragments were mainly residues 134-141, 158-165 in RmS-3, residues 38-45 in L7LRK7, and residues 134-141, 158-165 in L7LTU1.The presence of several epitopes suggested that these proteins might be immunogenic.The recombinant proteins RmS-3, L7LRK7 and L7LTU1 were expressed using the pTYB12 vector.The recombinant proteins of RmS-3, L7LRK7 and L7LTU1 with molecular weights of 43 kDa, 45 kDa and 44 kDa, respectively were obtained by Chitin Resin affinity chromatography (Fig. 2).The full length membranes of gel images were showed in Supplementary Figs.2-4.Eventually, the purified proteins were dried in a vacuum freeze dryer at − 45 °C and stored at − 20 °C.

Coagulation and immunity
Since serpins perform an important function in modifying the host immune response and coagulation cascade, they were validated by routine blood tests.In comparison with the control group, there was no significant difference in hematological indexes such as MON, NEU, RBC, HGB and PLT in the immune group.Nevertheless, except for the slight increase in RmS-3 immunized group, WBC and LYM in the other immunized groups showed a downward trend, and the WBC and LYM of L7LRK7 + L7LTU1 immunized group were significantly lower than those in the control group (Table 1).Prothrombin time (PT) and activated partial thromboplastin time (APTT) of each group were measured to confirm the function of the serpins in the coagulation cascade.PT index data revealed no significant difference between the vaccinated and control groups.Furthermore, in compared to the control group, APTT of L7LTU1 and RmS-3 + L7LRK7 + L7LTU1 in the inoculated group was considerably extended after the second immunization (Fig. 3).These findings from the immunological groups L7LTU1 and RmS-3 + L7LRK7 + L7LTU1 imply that serpins have a role in blood coagulation inhibition.
After the third vaccination of mice, the percentage of CD4+ and CD8+ spleen cells was determined by flow cytometry.Figure 4 illustrates how the proportion of CD4 + and CD8 + cells in the vaccinated group usually reduced when compared to the control group.The percentage of CD4 + cells in the vaccinated groups L7LTU1, www.nature.com/scientificreports/RmS-3 + L7LRK7, RmS-3 + L7LTU1, L7LRK7 + L7LTU1, and RmS-3 + L7LRK7 + L7LTU1 decreased to 59.98%, 15.3%, 57.32%, 19.14%, and 10.50 (P < 0.05) compared to the control group.There was no discernible difference between the vaccinated and control groups (P > 0.05), despite the fact that the proportion of CD8+ cells in the immunized groups was lower than that in the control group.Following the third mouse vaccination, the results of a routine blood test revealed that the WBC and LYM levels in the L7LRK7 + L7LTU1-immunized group were considerably lower than those in the control group.

Identification of RmS-3, L7LRK7 and L7LTU1 proteins in R. sanguineus
In order to identify whether RmS-3, L7LRK7 and L7LTU1 homologous proteins are contained in the salivary glands of R. sanguineus, the salivary glands of the partially engorged R. sanguineus were analysed by iELISA.The results (Fig. 6) showed that only the immunized groups L7LTU1 and RmS-3 + L7LTU1 showed positive in the iELISA, but RmS-3 and L7LRK7 were not detected.

Effect of vaccination on R. sanguineus infestation in Kunming mice
The immunized mice were challenged with R. sanguineus in order to compare the immunological effects of single-antigen vaccinations versus cocktail-antigen vaccines.In compared to the control group, the number of completely engorged ticks in both larval and nymphal ticks decreased.The fully engorged rate (%) of larvae and nymphs decreased to 40.87% (P < 0.05) and 87.68% (P > 0.05) in the RmS-3 + L7LRK7 immune group, 49.57% (P < 0.01) and 52.06% (P < 0.05) in the RmS-3 + L7LTU1 group, 45.22% (P < 0.05) and 60.28% (P < 0.05) in the RmS-3 + L7LRK7 + L7LTU1 immune group, as illustrated in Fig. 7.In the examined larvae and nymphs, however, there was no significant difference between groups in body weight or molting rate (P > 0.05).

Discussion
In order to reduce the problem of increasing acaricides resistance and acaricides residue caused by the abuse of acaricides, the development of tick vaccines is considered to be an effective way.Some tick serpins have been identified as candidate targets for effective anti-tick vaccines, and they have a certain protection as single antigen vaccine 33,[40][41][42] .nevertheless, due to the great number of tick species, the number of tick antigens with crossprotected epitopes is limited, resulting in poor broad-spectrum and generality of anti-tick vaccines [43][44][45][46] .The hunt for new antigens that are conserved among tick species can aid in the resolution of the aforementioned issues.In this study, three serpins, RmS-3 L7LRK7, and L7LTU1 protein were selected for their high identity in Rhipicephalus spp.RmS-3 protein was found in salivary glands of female R. sanguineus, while the other two proteins, L7LRK7, and L7LTU1, were deduced by sequencing 38 .Kunming mice were inoculated with recombinant  www.nature.com/scientificreports/proteins RmS-3, L7LRK7, and L7LTU1, and the efficacy of the three proteins as vaccinations against R. sanguineus was assessed.
The tick completes its blood meal by inflicting a wound on the host's skin and preventing healing and coagulation by secreting a number of bioactive substances in saliva [47][48][49] .As a consequence, avoiding anticoagulants helps to reduce tick blood feeding.The coagulation and immune protection roles of RmS-3, L7LRK7 and L7LTU1 were detected by routine blood test, coagulation reaction and T lymphocyte ratio.The APTT in the L7LTU1 and RmS-3 + L7LRK7 + L7LTU1 immunized groups was extended after the second immunization, according to the findings of the coagulation reaction.The intrinsic coagulation route is represented by APTT, while the extrinsic coagulation pathway is represented by PT.When the PT or APTT reaction time was extended, it showed that the immunological groups were more capable of inhibiting anticoagulation.As a result, the coagulation level was greater in the L7LTU1 and RmS-3 + L7LRK7 + L7LTU1 immune groups.Blood routine test results showed that there was no significant difference of RBC, HGB and PLT between the control group and the immunized after three immunizations; WBC and LYM in the L7LRK7 + L7LTU1 immunized group were significantly lower than in the control group.The percentages of CD4 + and CD8 + T lymphocytes in the RmS-3 vaccinated group decreased in the T lymphocyte subset level analysis experiment, which is consistent with the findings of Coutinho et al. which indicates that RmS-3 has a strong inhibitory effect on lymphocyte metabolic activity, IFN-production, and proliferation, but does not promote cell death 35 .Similarly, T lymphocyte subset identification findings in this experiment revealed that L7LRK7 and L7LTU1 had inhibitory effects on lymphocyte metabolic activity, which is consistent with the biological functions of RmS-3, L7LRK7, and L7LTU1 as serpins.The safety issue of immunosuppression caused by these antigens should be further evaluated in subsequent studies.
The iELISA results showed that all three proteins could stimulate high-affinity antibodies in the host, and the combination of RmS-3 + L7LRK7 and RmS-3 + L7LTU1 could produce higher affinity antibodies than the single RmS-3 and L7LTU1 proteins.Furthermore, the salivary glands of R. sanguineus were extracted and analyzed for the presence of these three proteins.The L7LTU1 protein can be detected by antibodies in serum.This means that the antibody can bind to the L7LTU1 protein in the salivary gland, thereby preventing its function.The other two proteins could not be detected, probably due to the absence or lower amounts in salivary glands.
Although it has been demonstrated that a cocktail of multiple antigens may be more effective 22,13 , the results of immunogenicity analysis in this study revealed that the immunogenicity of the cocktail vaccine of three antigens was lower, while the immunogenicity of the cocktail vaccine of two antigens was better.This may be explained by antigen competition and cross-immunity.Cross-immunity means that antibodies directed against a particular 14:7703 | https://doi.org/10.1038/s41598-024-58303-4www.nature.com/scientificreports/Production of recombinant RmS-3, L7LRK7 and L7LTU1

Figure 3 .
Figure 3. Coagulation reaction results.(a) Prothrombin time (PT) of blood in different groups.(b) Activated partial thromboplastin time (APTT) of blood in different groups; Abscissa: the immunization group that was the source of plasma.

Figure 4 .
Figure 4.The levels of CD4 + and CD8 + T lymphocyte subsets in different groups after immunization of mice.(a) The levels of CD4 + T lymphocyte subsets.(b) The levels of CD8 + .Ordinata: percentage of CD4 + and CD8 + T lymphocytes in total T lymphocytes; Abscissa: spleen lymphocytes of mice in each group after immunization.

Figure 6 .
Figure 6.Identification results of tick salivary gland proteins by iELISA.Ordinata: absorbance value of antibody in serum combined with protein in R. sanguineus salivary gland; Abscissa: serum of control group and immunized group.

Figure 7 .
Figure 7. Tick number, Tick weight, and Tick molting rate in different groups after challenge.(a) Molting rate of tick Larvae in different groups.(b) Molting rate of tick Nymphs in different groups.(c) Number of tick Larvae in different groups.(d) Number of tick Nymphs in different groups.(e) Weight of tick Larvae in different groups.(f) Weight of tick Nymphs in different groups; Ordinata: Tick number, number of ticks full of blood; Tick weight, weight of ticks after full blood; Tick molting rate, ratio of molting number to full blood of ticks after fully engorged; Abscissa: different mice immunized group.

Table 1 .
Blood test values after the third immunization of mice.WBC white blood cell, LYM lymphocyte, MON monocyte percentage, NEU neutrophil, RBC red blood cell, HGB hemoglobin (), PLT platelet.*(P < 0.05): significant difference between immune group and control group; ns no significant difference between immune group and control group, the same below.