LDHs enhanced the immune response of animals to foot-and-mouth disease virus

Background: Foot-and-mouth disease (FMD) is a highly transmissible disease that leads to vast economic losses in many countries. Prevention using inactivated vaccines is one effective measure used to control FMD. Unfortunately, inactivated FMD vaccines provide only short-term protection and require a cold-chain system. In recent years, many studies have shown that layered double metal hydroxides (LDHs) carrying antigens can be used to strongly induce immune responses. In this study, LDH nanoparticles (NPs) were prepared by hydrothermal synthesis. LDH particle size, electric potential, and morphology were measured and observed. The adsorption capacity of LDH NPs to FMDV was tested. The effects of LDH as an adjuvant on inactivated FMDV vaccines were further evaluated and compared with commercial FMDV ISA-206 in BALB/C female mice and Yorkshire pigs. Results: LDH NPs were successfully prepared with a uniform particle size of ~87.21 nm, regular edges, a loose hexagonal shape and positive zeta charge of 32 mV. The maximum absorption concentration was 0.16-0.31 µg FMDV/µg LDH. In the mouse experiment, antibody of immunized with LDH + FMDV were induced significantly higher from days 42-98 compared to saline + FMDV (P<0.01) and significantly higher compared to ISA-206+FMDV on day 56 post-immunization (P<0.05). After day14 post-immunization, IFN-γ content was significantly increased (P<0.05). In the pig experiment, antibody levels in both the ISA-206 + FMDV and LDH + FMDV were positive and were significantly higher compared with the PBS group on day 7 (P<0.005). Antibody levels in 90% pigs were positive on day 56 in the LDH group. The neutralizing antibody levels in the LDH and ISA-206 groups were significantly higher from days 7-28 compared to the PBS control group (P<0.05). Thus, LDH NPs were effective at inducing an immune response against FMDV. Conclusions: LDHs with a loose hexagonal shape and a positive charge were prepared. LDHs can effectively induce humoral- and by injection of All pigs were randomly divided into groups. Five pigs were raised together without bedding. The PBS control group (n = 5) was injected with 1 ml PBS/pigs. The ISA-206 group (n = 10) was injected with 0.5 ml inactivated virus O/MYA/BY/2010 + 0.5 ml ISA-206 adjuvant /pigs. The LDH group (n = 10) was injected with 0.5 ml inactivated virus O/MYA/BY/2010 + 0.5 ml LDH NPs /pigs. The final O/MYA/BY/2010 virus concentration in immunized groups was 6 µg/ml. All injections were administered intramuscularly. Blood was collected from anterior vena cava in pigs under abdominal anesthesia with sodium pentobarbital (40 mg/kg). Day 0 prior to immunization and subsequently at day 14, 21 and 28 for serum collection. The FMD antibody level was detected using the Lanzhou Veterinary Institute FMDV type O liquid phase blocking diagnostic kit. The virus micro-neutralization test (VNT) was performed by Tiankang Biotechnology Co., Ltd. in P3 laboratory.

LDH is a layered structure: the laminates have a structural positive charge, and the interlayers are composed of anions and water molecules. The interlayers are bound together by electrostatic interaction. Recent results have demonstrated that dispersion-stable LDH NPs are efficient vaccine carriers, stimulate higher levels of antibodies for a longer period, maturate dendritic cells (DCs) and promote stronger specific T cell immune responses [31]. For example, antigen BSA-Cy7 loaded LDH complexes generate loosely structured agglomerates either in solution or within nodules formed at the injection site and recruit immune cells into injection nodules and over a prolonged period [32].
LDH-adjuvanted multiple-antigen vaccine formulations can efficiently stimulate strong humoral, cellular and mucosal immune responses that are capable of preventing E. coli from adhering to mammalian cells more efficiently than the commercial adjuvant formulation [27,33]. A dispersionstable LDH-based vaccine induced stronger cytotoxic T-lymphocyte (CTL) responses and significantly inhibited tumor growth [34]. Therefore, LDH is a promising adjuvant vaccine due to the small particle size, stable dispersion, large specific surface area, positive charge, large cargo load, sustained release, easy absorption, low toxicity, low cost, and significantly improved the cellular immune response.
FMD vaccines not only focus on antigens but also focus on the adjuvant technology. Efficacy, source, cost and safety should be taken into account in adjuvant selection. In this study, LDH NPs were prepared by hydrothermal synthesis, and their properties analyzed. The mouse is a typical model animal and one of the representatives of typical mammals. The immune effects in mice were initially easy to observe. Pigs are susceptible to foot-and-mouth disease. It is practical to evaluate the effects of vaccines and adjuvants. The effects of LDH as adjuvant on inactivated FMDV vaccines were further 5 evaluated compared with the commercial ISA-206 adjuvant in mice and pigs. LDH may be an effective and safe adjuvant improve FMD vaccine efficacy.

Results
Physicochemical properties of LDH NPs LDH NPs (Mg 2 Al-Cl-LDH) were synthesized via rapid precipitation followed by hydrothermal treatment.
The particle size of LDH had only one peak at 87.21 nm, which showed homogeneously dispersed suspension (Fig. 1A). The equivalent mean hydrodynamic diameter was 70.96 nm, and LDH size was no greater than 200 nm. The test LDH NPs were positively charged (zeta potential 32 mV). The TEM image showed that the LDH crystallites were well crystallized with a typical hexagonally-shaped morphology (Fig. 1B). The final concentration of LDH was 48.62 mg/ml.

Effective adsorption of LDHs for FMDV
The adsorption capacity of LDH NPs to FMDV 146 s was determined by the amount of free virions in the supernatant after the binding of LDHs and FMDV. After 1536 µg inactivated FMDV was added to 100 µl (4862 µg) of LDHs particles, a large number of viruses began to appear in the supernatant as observed by OD260/OD280 (Fig. 1C). The LDH NPs were able to absorb the maximum FMDV 768-1536 ug, that is, the maximum absorption concentration was 0.16-0.31 µg FMDV/µg LDH.

Cytotoxicity of LDH NPs
LDH toxicity was evaluated using BHK-21, MDBK, and SKC cells. Incubation with different concentrations of LDH with 38 h had no effect on any of the three cell types, and there was no statistically significant difference when compared with the PBS control group (Fig. 2). The results showed that LDH NPs can be used as an adjuvant in organisms.

Evaluation of LDHs + inactivated virus immunized BALB/C mice
The LDH was formulated with inactivated FMDV to determine its potential adjuvant efficacy. IL-4 and IFN-γ secretions were measured 14d post-immunization. The IFN-γ level was significantly higher different in three groups vaccinated with FMDV + LDHs, FMDV + ISA-206 and FMDV + saline compared to pre-immunization (P < 0.01). There was no difference in IL-4 level among the groups (Fig. 3A, B).
Th1 and Th2 represent the two extremes of the adaptive immune response [35]. In general, IL-4 is secreted by activated Th2 cells, and IFN-γ is secreted by Th1 cells. The results showed that the 6 adjuvant LDH and ISA-206 caused strong cellular immunity, but the level of humoral immunity was still weak in the early stage. The average antibody titer in different stages was measured and results showed a significantly higher difference in antibodies induced by LDHs + FMDV compared to the saline group from day 42 to 98 (P < 0.01) and a significantly higher compared to the ISA-206 group on day 56 post-immunization (P < 0.05) (Fig. 3C). The humoral immune effect reached the highest on day 56 and remained at a high level on days 70, 84, and 98 (Fig. 3C). FMDV VP1 antibody levels on day 70 post-immunization showed that the VP1 antibody titer of the LDH group was significantly higher compared to the control group immunized virus alone (P < 0.05). In addition, the positive rate in LDH group was 1/2, the ISA-206 group was 1/3, and the virus only group was negative compared with the positive serum (Fig. 3D). The antibody titer of the liquid phase blocking ELISA was basically the same as that of FMDV VP1. The results showed that LDH as an adjuvant not only induced cellular and humoral immunity, but also had sustained release. were positive and were significantly higher (P < 0.005) compared to the PBS group, in which antibody level was negative (Fig. 4A). There was no significant difference between the LDH group and the ISA-206 group. In the LDH group, antibody levels of 9 heads (10 in total) were positive on day 7. The number of positive heads decreased on day 28, but returned to 9 on day 56 ( Fig. 4B), which may indicate that LDH was continuously releasing antigen. In conclusion, the FMDV antibody level detected by ELISA was not very high, which may have been due to the low immune dose.

Evaluation of LDHs + inactivated virus immunized pigs
Results of the virus micro-neutralization test showed that antibody levels of pigs immunized with LDH + FMDV and ISA-206 + FMDV ranged from 1:4 to 1:32, and were significantly higher from day 7 to day 28 compared to the PBS control group (P < 0.01) (Fig. 4C). The pig numbers of the LDH group with positive titers was higher than the ISA-206 adjuvant group on day 28 post-immunization. These results demonstrate that the efficacy of LDH NPs for inducing specific antibody responses against 7 FMDV approximated that of commercial ISA-206 adjuvant in pigs.

Discussion
LDHs have a very strong load capacity [36] and can carry proteins, DNA and other substances. The positive charge property can combine with the negative charge group of the cell membrane to enter the cell smoothly [37]. Compared with aluminum salt adjuvant, nano-adjuvant has a better effect on spleen cell activation and cytokine secretion [38]. When the nano-adjuvant is combined with virus, the carrier effect of the conventional adjuvant can be avoided and antigens can be protected. LDHs have the dual effects of immunostimulant and antigen carrier. This study showed that LDH had a loose hexagonally-shaped structure and a positive charge, and could effectively carry FMDV virus.
The current study also demonstrated that sustained antigen release can be accomplished using an LDH nano-adjuvant, which promotes antigen presentation and produces a long-lasting and efficient memory immune response, thus reducing the number of vaccinations and the amount of antigen [31][32][33][34]. In this study, antibody levels in mice immunized with LDH + FMDV were significantly higher from day 42 to day 98 compared to the saline group (P < 0.01) and were significantly higher compared with the ISA-206 group on day 56 (P < 0.05). The humoral immune effect remained at a high level on day 98, indicating that LDH had a slow-release effect and continuously produced antibodies. FMDV combined with LDH enhanced the immunogenicity. IFN-γ and IL-4 are important immunomodulators that have multiple biological functions. IFN-γ is a Th1 cytokine produced by activated T cells and NK cells, which has a variety of biological activities. IFN-γ is anti-viral, antiparasitic and inhibits cell proliferation, which can induce at Th1 type immune response [39]. IL-4 is a cytokine produced by activated Th2 cells, which can enhance the interaction between B cells and T cells, promote the humoral immune response, and induce mononuclear-macrophages to express MHC-II molecules [40]. Th1 cells induce cell-mediated immunity whereas Th2 cells induce strong antibody responses [36].After day14 post-immunizing mouse, IFN-γ content increased significantly, indicating that LDH induced cellular immune response at an early stage. In addition, in pigs, average antibody levels in both the ISA-206 and LDH group were positive and were significantly higher (P < 0.005) compared with the PBS group on day 7. Furthermore, the number of pigs with positive 8 antibody titers returned to 9 on day 56, indicating that LDH has sustained release behavior. The neutralizing antibody levels of LDH and ISA-206 groups were induced significant higher from day 7 to day 28 compared to the PBS control group (P < 0.01). Both the ELISA and cell suspension measurements showed that LDH NPs had effective induced specific antibody responses against FMDV in mice and pigs, which may be also applied to cattle and sheep.
The preparation process of LDH is relatively simple, and provides a basis for large-scale industrial production. Due to the low production cost of LDH, the use of it as an adjuvant will greatly reduce the breeding cost to farmers. As an inorganic substance, with stable dispersion properties, LDHs can be stored for a long time. Because of the production and preparation through high temperature and high pressure, the product is sterile, which is a necessary condition for the production of vaccines.

Conclusion
In this study, LDH NPs were synthesized using hydrothermal treatment. The LDHs had an average

LDHs and inactivated virus immunized pigs
All pig experiments were performed according to the guidelines of the Animal Ethics Committee of the Shihezi University. The pigs were CL grade and were farmed on the ground with sufficient light. The pigs were allowed free access to clean water and limit complete diet pellets. All treatments were aseptic and clean feeding. The breeding temperature was ~ 27 °C. After the experiment, the pigs were euthanized by intraperitoneal injection of excessive sodium pentobarbital (200 mg/kg weight).
All pigs were randomly divided into groups. Five pigs were raised together without bedding. The PBS control group (n = 5) was injected with 1 ml PBS/pigs. The ISA-206 group (n = 10) was injected with

Virus micro-neutralization test (VNT) procedure
The valence of serum neutralizing antibody was determined via the cell suspension method.

Ethical approval and consent to participate
All animal experiments were approved by the Animal Welfare Institute of Shihezi University, and all operations were performed in accordance with animal welfare requirements (the written document, reference number A2018-163-01).

Consent for publication
Not applicable.

Availability of data and material
All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
We are grateful for the financial support from the National Natural Science Foundation of China 13 (U1803236, 31572491), the Bingtuan Science and Technology Cooperation Program (2018BC011).
The fund (U1803236) mainly supports the feed, mice and pigs, and all expenses incurred in carrying out animal experiments; the fund (31572491) mainly supports the test reagents and consumables, sampling and labor expenses, and analysis and testing expenses; the fund (2018BC011) mainly supports the some kits for serum detection.

Authors' contributions
CC designed the study, led collection of samples. PW designed the study and drafting of the manuscript. YZ performed data analysis and wrote the manuscript. XY contributed to cell culture and animal test. YH and QZ performed data regarding the mice and pigs serum samples. All authors approved the final manuscript.

Supplementary Files
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