**4. Discussion**

The mechanism of natural immunity against EBOV remains unclear. The efficacy of different types of vaccines may vary depending on the vaccine platform: antibody is the major immune correlate factor of the rVSV-ZEBOV vaccine [33]. Meanwhile, CD8 T cell responses have been attributed to protection by the Ch-Ad5 vaccine [34]. Previous VLP vaccine studies showed it is efficacious against lethal Ebola challenge, however, with different adjuvants to save antigen and enhance vaccine-induced immune responses [35,36].

The impact of SUDV outbreaks in recent years, and the potential for viral spread to non-endemic regions or countries, makes the development of safe and efficacious vaccines urgent.

Previous findings revealed that SUDV VLPs could be readily assembled by the co-expression of baculoviruses expressing GP, NP and VP40 in insect cells [30]. Here, we demonstrated that co-infection of rBV-GP-GP and rBV-VP40-VP40 recombinant baculoviruses can result in the successful assembly of VLPs in insect cells, the morphology of SUDV VLPs is similar to a native virus, and the immunogenicity of VLPs was tested with an ISA 201 adjuvant as a candidate vaccine in mice.

In our study, we generated recombinant baculaviruses rBV-GP-GP and rBV-VP40-VP40 using the pFastBacDual vector to increase the protein production utilizing the dual promoter. To determine the optimal proportion of two recombinant baculaviruses rBV-GP-GP and rBV-VP40-VP40, we also tried to co-infect Sf9 cells with recombinant baculaviruses rBV-GP-GP and rBV-VP40-VP40 at ratios of 1:1, 1:2, 1:2.5, 1:3, 2:1, 2.5:1, and 3:1. At a ratio of 2.5:1, the morphology of VLPs was similar to the native virus, as assessed by TEM (Figure 3A). The Western blot result (Figure 3B) showed that SUDV GP and VP40 efficiently assembled SUDV VLPs.

The immunization results depicted the failure of the group vaccinated with 20 μg of SUDV VLPs-only to stimulate the production of humoral immune responses in mice, while the group vaccinated with 20 μg of VLPs mixed with an ISA 201 adjuvant could stimulate the production of humoral immunity in mice. Presumably, immunization with 20 μg of SUDV VLPs-only is an insufficiently low dose of an immunogen that cannot stimulate the mice to produce an immune response, and the use of an ISA 201 adjuvant with SUDV VLPs might cause low-dose immunogens to initiate a response, and using with adjuvant can save antigen amount (Figure 4C). The same SUDV VLPs antigen dose (20 μg) mix different adjuvants could stimulate mice to produce different degrees humoral immune responses. The ISA 201 adjuvant showed the most effective adjuvant than other adjuvants in mice. The detection of neutralizing antibody showed that sera from mice immunized with 20 μg SUDV VLPs mixed with ISA 201 adjuvant could neutralize approximately 50% of the pseudotyped viruses at an average 1:320 dilution, while individual samples were effective with as high as a 1:640 dilution (Figure 4D).

Both humoral and cellular responses are indispensable for providing protection. IFN-γ is a Th1-type cytokine involved in the antiviral action of cellular immune responses and IL-4 is mainly produced by Th2 cells and is associated with humoral immune responses. The SUDV VLPs could effectively stimulate Th1 and Th2 cytokine production in vaccinated mice (Figure 5A,B). The levels of cytokines secreted from splenocytes, such as IL-2, IFN-γ, and TNF-α secreted from Th1 cells (Figure 6A–C) and IL-4 and IL-10 were secreted from Th2 cells (Figure 6D,E), were significantly increased after vaccination. Moreover, our study revealed that the B cells of mice of the group vaccinated with SUDV VLPs mixed with an ISA 201 adjuvant were activated at one week after the primary immunization (Figure 7).

In addition, we have conducted research on therapeutic antibodies with the goal of preparing antibodies for the post-exposure treatment of SUDV. Horses were selected as immunized animals in our study because horse anti-immunoglobulins have been used in the treatment of various viral infections [37–39]. SUDV VLPs can effectively induce humoral immune responses in horses after immunization (Figure 8A). The horse anti-SUDV immunoglobulin was obtained from #392 horse serum through crude extraction and purification. The neutralizing antibody titer of purified #392 IgG was 1:5120, which was one time lower than that before purification (Figure 8B).

We tested the SUDV VLPs immunogenicity and neutralizing activity of hours purified IgG by using pseudo typed viruses, and efficacy studies still need to be performed. These SUDV VLPs vaccine and horse purified IgG provide ideas for the development of vaccines and therapeutic antibodies that could prevent and treat SUDV infections. The Ebola epidemic is still spreading, vaccination is an effective means of preventing and controlling the outbreaks, and effective antibodies represent key drugs for the treatment of Ebola patients. Therefore, further Ebola vaccine and therapeutic antibody research are needed.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4915/12/1/64/s1, Figure S1: Diagram of the plasmids encoding the pFastBacDual-GP-GP (A) and pFastBacDual-VP40-VP40 (B).

**Author Contributions:** Y.Z. and S.Y. designed the experiments. F.W., Y.Z., R.M., F.Y., H.C. performed the experiment. H.W., N.F., T.W. analyzed the data. S.Z. and F.W. wrote the manuscript. X.X., Y.Z., S.Y., G.W. and Y.G. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Project for Prevention and Control of Transboundary Animal Diseases (Grant No. 2017YFD0501804).

**Conflicts of Interest:** The authors declare no conflict of interest.
