key: cord-0714847-s5zjde9j
authors: Calvo-Pinilla, Eva; Marín-López, Alejandro; Utrilla-Trigo, Sergio; Jiménez-Cabello, Luís; Ortego, Javier
title: Reverse genetics approaches: a novel strategy for African horse sickness virus vaccine design
date: 2020-07-10
journal: Curr Opin Virol
DOI: 10.1016/j.coviro.2020.06.003
sha: 6e4bef901b69bfc64406b940ed38253d69cf45ee
doc_id: 714847
cord_uid: s5zjde9j

African horse sickness (AHS) is a devastating disease caused by African horse sickness virus (AHSV) and transmitted by arthropods between its equine hosts. AHSV is endemic in sub-Saharan Africa, where polyvalent live attenuated vaccine is in use even though it is associated with safety risks. This review article summarizes and compares new strategies to generate safe and effective AHSV vaccines based on protein, virus like particles, viral vectors and reverse genetics technology. Manipulating the AHSV genome to generate synthetic viruses by means of reverse genetic systems has led to the generation of potential safe vaccine candidates that are under investigation.

Eva Calvo-Pinilla 1 , Alejandro Marín-Ló pez 2 , Sergio Utrilla-Trigo 1 , Luís Jimé nez-Cabello 1 and Javier Ortego 1 African horse sickness (AHS) is a devastating disease caused by African horse sickness virus (AHSV) and transmitted by arthropods between its equine hosts. AHSV is endemic in sub-Saharan Africa, where polyvalent live attenuated vaccine is in use even though it is associated with safety risks. This review article summarizes and compares new strategies to generate safe and effective AHSV vaccines based on protein, virus like particles, viral vectors and reverse genetics technology. Manipulating the AHSV genome to generate synthetic viruses by means of reverse genetic systems has led to the generation of potential safe vaccine candidates that are under investigation.

African horse sickness virus (AHSV) causes lethal disease in horses and is transmitted by hematophagous biting midges of the genus Culicoides [1, 2] . AHSV infects mainly equids, causing high mortality rates up to 90% in horses, while mules and donkeys are less susceptible [3] . The virus belongs to genus Orbivirus, family Reoviridae, and nine serotypes (AHSV-1 to AHSV-9) have been identified upon the specificity of their reactions with neutralizing antibodies (NAbs) [4, 5] . AHSV virion is a non-enveloped isometric particle composed of three concentric protein layers surrounding 10 lineal double-stranded RNA genome segments [6] . Together with the seven structural viral proteins (VP), the genome encodes other five nonstructural (NS) proteins [6] [7] [8] .

Despite endemicity of AHSV is constrained to Sub-Saharan Africa, the virus has caused devastating losses in indigenous horses outside of its current endemic zone during epidemics in Middle East, India, Pakistan, North Africa and Europe caused by multiple serotypes [9] . Recently (February the 24th, 2020), new outbreaks of AHSV-1 with an unknown origin have been documented in racehorses in Thailand, with 191 confirmed cases and 175 deaths, and a 91.62% of fatality rate, being the first AHSV outbreak described in this country (https://www. oie.int/wahis_2/public/wahid.php/Reviewreport/Review? page_refer=MapFullEventReport&reportid=33768). The increasing global trade and the climate changes may facilitate the spread of vector-borne diseases, as shown by recent outbreaks of Bluetongue and Smallenberg viruses and demonstrating the rising viral transmission by Culicoides in non-endemic areas [10, 11] . This suggests that AHSV can also emerge outside of Africa, causing huge direct and economic losses in horse industry as occurred in the past [12] . This scenario requires the development of an effective and safe vaccine capable to protect equids against all AHSV serotypes.

Currently, the control of AHSV in endemic African countries relies on a polyvalent live attenuated vaccine (LAV) administering seven serotypes in two doses; AHSV-5 and AHSV-9 are not included in the vaccine since cross-protection with serotypes 8 and 6 respectively has been documented [3, 13] . Of concern, LAVs are associated with reversion to virulence, vector's transmission, absence of DIVA (Differentiating Infected from Vaccinated Animals) capacity, teratogenicity, and gene reassortment that lead to the establishment of new genetic variants [3,14- [20, 23] . In horses, prime/boost with MVA expressing VP2 from serotype 9 provided sterilizing protection against a lethal dose of AHSV-9 without any adjuvant in the vaccine composition [22] . Interestingly, simultaneous vaccination with MVA-VP2 of serotypes 4 and 9 triggered NAbs against serotype 6 [43 ] . After four months, vaccination with MVA-VP2 (AHSV-5) of previously immunized horses induced an anamnestic response towards AHSV-5, 4, 6 and 9 as well as the cross-reactive AHSV-8.

As antigenic variability of AHSV is the main hurdle of cross-protective immunity, several studies have been focused on NS1 protein with a highly conserved amino acid sequence among all serotypes (97.26-99.82% sequence identity). Importantly, CD8 T-cell epitopes have been identified in NS1 in mice and they are conserved among AHSV serotypes [44] . As cross-reactive T-cell responses are critical for multiserotype protection, vaccines based on NS1 have been analyzed. Immunization with DNA/MVA expressing AHSV-4 NS1 or two doses of MVA-NS1 reduced viremia in mice after challenge with a heterologous serotype, AHSV-9 [24]. In a more recent work, NS1 from AHSV-4 was incorporated 50 Preventive and therapeutic vaccines Figure 2 ).

ECRA viruses are deficient in VP6 and cannot complete the whole replication cycle due to the lack of function of VP6 as part of the transcriptase and packaging complex. However, they still initiate the replication cycle and synthesize a single round of viral mRNAs following entry and express viral proteins in normal cells. In contrast, for vaccine production, the in trans expression of VP6 in a helper cell line is required, to allow viral growth [66] . For AHSV, ECRA-AHSV viruses have been generated for all the nine serotypes by introducing multiple stop codons in the coding region of segment S9, then disrupting the expression of VP6 and also NS4 protein (encoded in the same segment) [58 ] . Previous works reported that NS4 is not essential for BTV replication in vitro but antagonizes Interferon-I expression in vivo [67, 68] Representation of different developed plasmid-based RG strategies for AHSV. The T7 RNA polymerase promoter is represented in green, the ORF of AHSV segments are colored blue and the hepatitis delta virus (HDV) ribozyme is emphasized in red. The CAG promoter is showed in yellow. (a) Ten plasmids containing the AHSV cDNA segments are co-transfected into BSR cells that express constitutively the T7 polymerase (BSR-T7). The viral positive-sense mRNAs with native 5 0 and 3 0 ends are produced due to cytoplasmic transcription of transfected cloned cDNAs. (b) Only five plasmids encoding the viral genome via transcription cassettes containing two AHSV cDNA segments are co-transfected into BSR-T7 cells in an identical procedure to that for the previous ten-plasmids RG system. (c) T7 polymerase is encoded in the plasmid that includes the AHSV segments 2 and 6 (S2-S6) under control of a CMV promoter. Cotransfection of this set of 5 plasmids is conducted in cells that do not express constitutively the T7 polymerase (BSR or L929). (d) A double transfection procedure is performed using BSR cells. First, transfection of six expression plasmids containing AHSV cDNA segments 1, 3, 4, 5, 8 and 9 is performed. A second transfection event of a whole set of ten T7 transcripts representing all AHSV dsRNA segments results in virus rescue. Expression plasmids enhance virus recovery events as it optimizes the formation of the primary replication complex. In all previous cases, once the virus is recovered, BSR cells are used for viral amplification and isolation is conducted by plaque assay.

presence on virus in blood and allowing only local replication in infected cells; reducing the risk of propagation or transmission by midge vectors during feeding [59] . RG-generated DISA-AHSV-4, with a total deletion of NS3/3a, was used for horse immunization (n = 2) following a prime/boost regimen (4 Â 10 4 TCID50) [28 ] .

No adverse reactions were detected in vaccinated animals. Seroconversion was observed, showing the peak of VP7 antibodies after boost (35 dpv). After challenge with AHSV-4, a horse developed severe clinical signs and high fever and viremia, and finally was euthanized. The second horse developed mild edema of the supraorbital fossae, slightly elevated body temperature and viremia, becoming negative at 28 dpi and survived. In the same study, DISA-AHSV-5, with an in-frame deletion of amino acid codon 25-101 in the S10 (77aa deletion in NS3/3a), was used to test safety (2 Â 10 7.7 TCID50) and efficacy (2 Â 10 5 TCID50) in ponies. After confirming the absence of side effects, clinical signs and viremia, and the presence of AHSV VP7 specific antibodies, immunized ponies were challenged with AHSV-5. Three out of four immunized animals survived to the infection and showed a delay in viremia, with lower titers compared to control ponies. Thus, DISA vaccine partially protected against AHS although did not induce measurable NAbs titers. The better results obtained in the latter experiment compared to that performed in horses might be due to the differences in vaccines doses and strains, virulence between strains used for the challenge or susceptibility to AHSV between horses and ponies.

Several research groups have developed promising vaccine candidates against AHSV. These approaches show improvements compared to marketed vaccines such as safety and allow a DIVA strategy. AHSV vaccines based on poxvirus recombinant vectors, such as MVA and canarypox [22, 30] , have displayed high levels of protection with absent of clinical signs and viremia in immunized horses. Although further optimization of reverse genetics vaccines is needed to abolish viremia completely in vaccinated animals, reverse genetics technology to create ECRA and DISA AHSV vaccines looks promising. Further research will be necessary to determine the optimal dose requirement and to perform a deep characterization of immune responses elicited for these vaccines. As the activation of cytotoxic CD8 T cells and other subsets of immune cells have been shown to have a key role in the virus clearance, cell-mediated immunity by AHSV RG vaccines need to be elucidated in the future. In 

Repl ica tion-Abort ive)

Vir aem ia Inhibited

Antigenpresen ting Cell

Schematic representation of the modified live attenuated vaccines (MLVAs) based on RG systems against AHSV. ECRA (Entry-competent Replication-Abortive) vaccines, formerly known as DISC (Disabled Infectious Single Cycle) vaccines, are deficient in VP6 and NS4, both encoded in segment 9. As a consequence, the replication cycle cannot be completed, although expression of viral proteins leads to an immune response. DISA (Disabled Infectious Single Animal) vaccines are based on attenuated viruses lacking the non-structural protein NS3/NS3a, encoded by segment 10. Therefore, viral egress is interrupted, inhibiting viraemia and allowing only local replication. The delayed egress of new viral particle results into a more prolonged antigen exposure and induces a potent immune response.

any case, having reverse genetics systems that allow the rapid development of safe and effective vaccines against the different serotypes of the virus by single S2[VP2] exchange, makes these vaccine platforms promising AHSV vaccine candidates for all current AHSV serotypes.

Nothing declared. The authors tested an immunization approach comprises NS1 of AHSV-4 incorporated into avian reovirus muNS protein microspheres (MS-NS1) and/or expressed using recombinant modified vaccinia virus Ankara vector (MVA-NS1). The results indicated that immunization based on MS-NS1 and MVA-NS1 afforded complete protection against the infection with homologous AHSV-4. Moreover, priming with MS-NS1 and boost vaccination with MVA-NS1 triggered NS1 specific cytotoxic CD8 + T cells and prevented AHSV disease in IFNAR (À/À) mice after challenge with heterologous serotype AHSV-9. The induction of crossprotective immune responses is highly important since AHS can be caused by nine different serotypes. 

Manning NM, Bachanek-Bankowska K, Mertens PPC, Castillo-Olivares J: Vaccination with recombinant Modified Vaccinia Ankara (MVA) viruses expressing single African horse sickness virus VP2 antigens induced cross-reactive virus neutralising antibodies (VNAb) in horses when administered in combination. Vaccine 2017, 35:6024-6029. In this work, the administration of two different MVA expressing VP2 from serotypes 4 and 9 induced neutralising antibodies against the homologous AHSV serotypes. A booster with MVA-VP2 of AHSV-5, given four months later to ponies resulted in the induction of NAbs against serotypes 4, 5, 6, 8 and 9. The anamnestic antibody response following the MVA-VP2-AHSV-5 boost suggests that it is possible some shared epitopes exist between different serotypes. 

Conradie AM, Stassen L, Huismans H, Potgieter CA, Theron J: Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus. Virology 2016, 499:144-155. In this work, three plasmid only based reverse genetics systems for AHSV recovery were developed. Initially, ten plasmids containing AHSV-4 segments 1-10 were transfected in cells expressing the T7 polymerase, allowing for recovery of AHSV-4 from cDNA plasmids only. A more efficient viral recovery was achieved by reducing the number of plasmids used from 10 to 5. In addition, cloning of the T7 polymerase into one of these five plasmids enabled AHSV rescue in cell lines that have not been engineered to express T7 RNA polymerase. The authors showed that recovery of specific reassortant viruses can be attained by this method.

Du Toit R: The transmission of bluetongue and horse sickness by Culicoides

African horse sickness virus: history, transmission, and current status

African horse sickness

The isolation and identification of further antigenic types of African horsesickness virus

Immunological types of horse sickness and their significance in immunization

African horse sickness virus structure

Characterising nonstructural protein NS4 of African horse sickness virus

Molecular epidemiology of the African horse sickness virus S10 gene

African horse sickness outbreaks caused by multiple virus types in Ethiopia

Schmallenberg virus: a novel viral disease in northern Europe

Invasion of bluetongue and other orbivirus infections into Europe: the role of biological and climatic processes

African horse sickness in Portugal: a successful eradication programme

African horse sickness

African horse sickness in naturally infected, immunised horses

African horse sickness caused by genome reassortment and reversion to virulence of live, attenuated vaccine viruses

Reverse genetics for fusogenic bat-borne orthoreovirus associated with acute respiratory tract infections in humans: role of outer capsid protein sigmaC in viral replication and pathogenesis

An improved reverse genetics system for mammalian orthoreoviruses

Development of reverse genetics for Ibaraki virus to produce viable VP6-tagged IBAV

Establishment of an entirely plasmid-based reverse genetics system for bluetongue virus

Development of a reverse genetics system for African horse sickness vaccines generated by reverse genetics Calvo-Pinilla et al. 55 www.sciencedirect.com Current Opinion in Virology

VP2 exchange and NS3/NS3a deletion in African Horse Sickness Virus (AHSV) in development of disabled infectious single animal vaccine candidates for AHSV

Structural protein VP2 of African horse sickness virus is not essential for virus replication in vitro

African horse sickness virus NS4 is a nucleocytoplasmic protein that localizes to PML nuclear bodies

A lassa virus liveattenuated vaccine candidate based on rearrangement of the intergenic region

Influenza A virus attenuation by codon deoptimization of the NS gene for vaccine development

Identification of the mechanisms causing reversion to virulence in an attenuated SARS-CoV for the design of a genetically stable vaccine

Bluetongue virus without NS3/NS3a expression is not virulent and protects against virulent bluetongue virus challenge

Generation of replicationdefective virus-based vaccines that confer full protection in sheep against virulent bluetongue virus challenge

Identification and characterization of a novel non-structural protein of bluetongue virus

Bluetongue virus NS4 protein is an interferon antagonist and a determinant of virus virulence

The protective efficacy of a recombinant VP2-based African horsesickness subunit vaccine candidate is determined by adjuvant

This work was supported by the Spanish Ministry of Science (AGL2017-82570-R) and EU Horizon 2020 Program (NO.727393-PALE-Blu). Sergio Utrilla-Trigo was a recipient of a predoctoral fellowship from the Instituto Nacional de Investigació n y Tecnología Agraria y Alimentaria, Centro de Investigació n en Sanidad Animal (program FPI-SGIT-2018). 

Matsuo E, Celma CC, Roy P: A reverse genetics system of African horse sickness virus reveals existence of primary replication. FEBS Lett 2010, 584:3386-3391. The authors successfully implemented for the first time a reverse genetics system for AHSV based only on core transcripts. They also demonstrated that utilization of T7 transcripts derived from a cDNA clone leads to virus recovery. Rescue of AHSV-4 as well as reassortant viruses between AHSV-6 and AHSV-4 was achieved. Moreover, they determined that AHSV replication is a two-step process, in which the second set of transcripts transfected defines the serotype of the rescued viruses. 

Lulla V, Lulla A, Wernike K, Aebischer A, Beer M, Roy P: Assembly of replication-incompetent African horse sickness virus particles: rational design of vaccines for all serotypes. J Virol 2016, 90:7405-7414. In this article, they first established a highly efficient reverse genetics system for AHSV serotype 1 (AHSV-1) and, subsequently, a VP6-defective AHSV-1 strain in combination with in trans complementation of VP6. Then, it was used to generate defective particles of all nine AHSV serotypes, which required the exchange of two to five RNA segments to achieve equivalent titers of particles. Furthermore, these replicationincompetent AHSV particles were demonstrated to be highly protective against homologous virulent virus challenges in type I interferon receptor (IFNAR)-knockout mice.