**1. Introduction**

Ebola virus infections lead to severe hemorrhagic fevers in humans and nonhuman primates, with human fatality rates of up to 90% [1]. The first outbreak of Ebola virus disease occurred in the Republic of Zaire (now the Democratic Republic of the Congo, DRC) and southern Sudan in 1976. From these independent outbreaks, two distinct viruses were identified, Zaire Ebolavirus (EBOV) [2] and Sudan virus (SUDV) [3], which are members of the genus Ebolavirus and have been the cause of sporadic outbreaks in humans throughout the years [1]. Species of Ebolavirus include EBOV, SUDV, Tai Forest Ebolavirus (TAFV), Bundibugyo Ebolavirus (BDBV), Reston Ebolavirus (RESTV) [4]. Both EBOV and SUDV are pathogenic to humans and nonhuman primates, causing severe hemorrhagic fever with high mortality rates of 67–90% [5,6]. Past EBOV outbreaks have been sporadic in nature and confined to central Africa, and thus far, the biggest outbreak on record is the 2013–2016 epidemic in western Africa, with 28,464 cases and 11,323 fatalities [7]. Most of the cases were in Guinea, Liberia, and Sierra Leone, but some of them were imported into Europe and the United States (https://app.who.int/ebola/current-situation/ebola-situation-report-30-march-2016). Currently, the latest devastating outbreak of the Ebola virus disease in the DRC is ongoing. As of today, a total of 3250 cases were reported, including 3133 confirmed and 117 probable cases, of which there have been 2174 fatalities with a mortality rate of 67% (WHO situation report https: //www.who.int/csr/don/24-october-2019-ebola-drc/en/).

The Ebola virus is a non-segmented negative-strand RNA virus belonging to the family Filoviridae and the genus *Ebolavirus* [8]. The genome of filoviruses consists of a single-strand, negative-sense RNA genome of approximately 19 kb length, encoding the following genes in the following orientation: 3 -nucleoprotein (NP)-polymerase cofactor (VP35)-matrix protein (VP40)-glycoprotein (GP)-soluble GP (sGP)-small soluble GP (ssGP)-transcription activator (VP30)-minor matrix protein (VP24)-RNA dependent RNA polymerase (L)-5 [9]. GP, the critical target antigen that is expressed on the surface of mature virions, is responsible for mediating cell attachment and viral entry [10]. Several vaccine platforms have been reported for EVD vaccine research, including vesicular stomatitis virus (VSV) [11,12], DNA replication-defective adenovirus vectors (Adv) [13,14], human parainfluenza virus type 3 [15], rabies virus [16,17], cytomegalovirus [18], Venezuelan equine encephalitis virus (VEEV) replicons [19] and virus-like particles (VLPs) [20]. There are eight vaccine candidates currently in human clinical trials. The two most promising preclinical vaccine candidates, VSV-EBOV [21] and chAd-EBOV, are in phase 3 clinical trials. No effective treatment for EVD is commercially available; however, China and Russia were the first to license EBOV vaccines in 2018 [22]. At the same time, passive immunotherapy with sera of animal origin has been used for over 120 years to treat bacterial and viral infections and drug intoxications. Currently, there are many therapeutic antibody drugs for treating Ebola virus disease, such as ZMappTM [23] and immunoglobulin F(ab ) 2 fragment [24].

VLPs represent a promising vaccine platform for a diverse array of viruses that include influenza virus, rotoviruses, noroviruses, HIV, hepatitis B virus, parvoviruses, rift valley fever virus, human papillomavirus and filoviruses [25–27]. VLPs are assembled by one or several proteins, with the distinct advantage of being noninfectious because they lack the viral genome required for replication. VLPs are highly ordered compounds similar to an actual live virus in terms of structure and size. The granular structure of VLPs is beneficial for antigen presentation and cell uptake, which can stimulate powerful innate and adaptive immune responses [28]. VLPs have the advantages of rapid production in large quantities and can generate robust innate, humoral and cellular immunity in animals and

humans [29]. Furthermore, pre-existing immunity associated with live carrier vaccines is not hindered by VLP-based immunizations. Previous findings showed that SUDV VLPs could be readily assembled by the co-expression of insect cells with baculoviruses expressing GP, NP, and VP40 [30].

There are currently no approved specialized drugs or vaccines to protect against SUDV disease outbreaks, and thus there is an urgent need for the development of an efficacious, safe and economically viable vaccine or therapeutic antibody to control SUDV infections. Here, we report that production of SUDV VLPs has been accomplished in insect cells by the co-infection with recombinant baculoviruses rBV-GP-GP and rBV-VP40-VP40, and evaluate the ability of SUDV-VLPs to induce SUDV-specific humoral and cellular immune responses in vaccinated mice. Further, horses were immunized with SUDV VLPs, and horse serum was purified to prepare purified immunoglobulins and the purified immunoglobulins had neutralizing activity.
