# Patent

## Title
Bunyaviruses with segmented glycoprotein precursor genes and methods for generating these viruses

## Abstract

The invention relates to a bunyavirus, in which separated (NSm)Gn and Gc coding regions are functionally present on two different genome segments, preferably a bunyavirus that comprises a total of at least 4 genome segments. The invention further relates to methods for producing said bunyavirus, and to a composition comprising said bunyavirus and a suitable excipient.

## Background
RELATED APPLICATIONS
This application is the U.S. National Stage of International Application No. PCT/NL2014/050321, filed May 21, 2014, published in English, and claims the benefit of European Application Number 13168608.1, filed on May 21, 2013 and European Application Number 14150826.7, filed on Jan. 10, 2014, the entire teachings of the above applications are incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 11, 2017, is named P101141US00 seqlist_ST25.txt and is 142,777 bytes in size.
FIELD
The invention relates to the field of viruses, more specifically, to the field of molecular virology of bunyaviruses. The invention relates to methods for the generation of an avirulent bunyavirus, and to a virus that is produced by these methods. The resulting virus can be used as a vaccine against an infectious disease mediated by bunyaviruses.
INTRODUCTION
Bunyaviruses are negative-strand RNA viruses with three-segmented genomes (Elliott, R. M. 1996. The Bunyaviridae. Plenum Press, New York, N.Y.). The Bunyaviridae family comprises five genera: Phlebovirus, Orthobunyavirus, Nairovirus, Hantavirus and Tospovirus. With the exception of the plant-infecting Tospovirus genus, bunyaviruses are pathogens of animals and humans. The small (S) bunyavirus genome segment encodes a nucleocapsid (N) protein and, with the possible exception of the Nairovirus genus, a nonstructural protein named NSs. The NSs protein of animal-infecting bunyaviruses functions as an antagonist of host innate immune responses and is considered the major virulence factor. The medium-size (M) genome segment encodes the viral structural glycoproteins Gn and Gc and, depending on the genus, one or more nonstructural proteins referred to as NSm. The M segment of Rift Valley fever virus (RVFV, genus Phlebovirus) additionally encodes a 78-kDa protein believed to be a minor structural protein. The large (L) genome segment encodes the viral RNA-dependent RNA polymerase (RdRp). Bunyaviruses that cause serious disease in animals and/or humans are classified as biosafety level 3 (BSL-3) or 4 pathogens, complicating the development of vaccines.
RVFV is an example of a Phlebovirus that is classified as a BSL-3 pathogen. RVFV is endemic to the African continent, Madagascar, the Comoros Islands, Mayotte and the Arabian Peninsula, where it is transmitted among livestock by mosquito vectors. RVFV epizootics are characterized by near simultaneous abortions, particularly among sheep, and high mortality among young animals below the age of two weeks. Humans can be infected via mosquito bite, but more commonly by inhalation of aerosols released during slaughtering of viremic animals. Infection of humans generally results in transient febrile illness. However, a small percentage of individuals develop complications such as retinal lesions, hepatic disease with hemorrhagic fever or delayed-onset encephalitis.
No vaccine exists for the prevention of RVF in humans. Two classical vaccines are used for the prevention of RVF in domesticated ruminants. Both vaccines are produced by Onderstepoort Biologicals Products (OBP), South Africa. The first is based on inactivated-whole virus. Although this vaccine can safely be applied in animals during all physiological stages, optimal efficacy depends on a booster vaccination and yearly re-vaccinations. The alternative “Smithburn” vaccine is a live virus that was attenuated by passage of the virus in suckling mouse brain (Smithburn 1949. Br J Exp Pathol 30:1-16). A single injection of this vaccine can provide solid protection, but due to residual virulence, this vaccine is not safe for young or gestating animals. More recently, a RVFV with a large deletion in the NSs gene, named the Clone 13 virus, was marketed as a livestock vaccine in South Africa by OBP. Relevant publications regarding the Clone 13 vaccine virus include Muller et al., 1995. Am J Trop Med Hyg 53:405-411; Vialat et al., 2000. J Virol. 74:1538-1543; Bouloy et al., 2001. J Virol 75:1371-1377; Dungu et al., 2010. Vaccine 28:4581-4587; Von Teichman et al., 2011 Vaccine 29:5771-5777. Although the aforementioned studies suggest that the Clone 13 virus can safely be applied in animals during all physiological stages, the virus was described to cause low-level viremia in hamsters (Muller et al., 1995. Am J Trop Med Hyg 53:405-411) and was shown to be capable of causing lethal delayed-onset encephalitis in mice (Vialat et al., 2000. J Virol. 74:1538-1543). Furthermore, the Clone 13 vaccine virus was shown to replicate extremely well in insect cell cultures and in mosquito vectors (Moutailler et al., 2010. Vector Borne Zoonotic Dis 10:681-688). Feeding of a mosquito on a Clone 13-vaccinated animal could result in amplification of the Clone 13 virus in the mosquito vector and, potentially, reassortment of genome segments with field viruses that are already present in the mosquito vector.
The work that resulted in the present invention was aimed to develop a RVFV vaccine for application in livestock and humans that optimally combines the efficacy of live vaccines with the safety of inactivated vaccines by being avirulent and non-transmissible by arthropod vectors.
The present invention provides a bunyavirus, in which separated (NSm)Gn and Gc coding regions are functionally present on two separate genome segments. Said two separate genome segments, preferably minigenome segments, are either L-L segments, L-M segments, L-S segments, M-S segments, M-M segments or S-S segments. It was found that the separation of Gn and Gc coding regions on two different genome segments, for example two M genome segments, results in a virus that has reduced replication capabilities in mammalian cell culture and is unable to replicate in insect cell culture. A preferred bunyavirus of the invention comprises a total of at least 3 genome segments, such as 3 genome segments or 4 genome segments. A bunyavirus comprising 3 genome segments preferably comprises S-S-L or S-L-L genome segments. A most preferred bunyavirus of the invention comprises a total of 4 genome segments, whereby each of the four genome segments comprises an essential gene selected from RdRp, NSm, Gn, and Gc. A preferred bunyavirus of the invention additionally comprises a functional deletion of the NSs gene.
A preferred bunyavirus of the invention comprising 3 genome segments (LMS) in which either (NSm)Gn or Gc is functionally present only on the S-segment. A most preferred bunyavirus of the invention comprising 4 genome segments comprises separate M-segments (LMMS), in which (NSm)Gn and Gc are functionally present on the separate M-segments. Said most preferred bunyavirus preferably also comprises a functional deletion of the NSs gene. It is further preferred that the coding sequence of the (NSm)Gn and/or Gc is codon optimized for efficient expression in mammalian cells, for example to remove rare codons. Methods for codon optimization are known in the art and are publicly available, for example from www.entelechon.com and from www.genscript.com.
A bunyavirus according to the invention comprises, as sole genomic segments, a bunyavirus L genome segment; and a bunyavirus M genome segment from which the (NSm)Gn and/or Gc coding region has been functionally inactivated, whereby the (NSm)Gn and/or Gc coding region that is deleted from the M genome segment is functionally present on a second L genome segment, a second M genome segment, or a S genome segment; or a second and a third L genome segment, a second L genome segment and a S genome segment, or a S genome segment and a second S genome segment, whereby separated (NSm)Gn and Gc coding regions are functionally present either on said second and a third L genome segment, on said second L genome segment and said S genome segment, or on said S genome segment and said second S genome segment. Said bunyavirus optionally further comprises a S genome segment, a S genome segment comprising at least the N gene and the 3′ and 5′ untranslated regions (UTRs); or a S-minigenome segment. A marker gene, for example encoding green fluorescent protein (GFP) may be present on one of the genome segments, for example on said S (mini)genome segment. It will be clear to a skilled person that variants of the above indicated bunyavirus, such as a bunyavirus without a (substantially complete) L genome segment and in which the coding region of the RdRp gene, which normally resides on the L genome segment, is functionally present on a (second) M or (second) S genome segment, and in which separated Gn and Gc coding regions are functionally present on two different genome segments, are also covered by this invention.
The work that resulted in the present invention started with the hypothesis that bunyaviruses are either not capable of efficiently packaging more than three genome segments and/or not capable of efficiently packaging two of the same type of genome segments (S, M or L) in a single particle in cells, for example mammalian and/or insect cells. To test this hypothesis, a virus was produced by combining four genome segments, namely the S genome segment, the L genome segment and two M-type minigenome segments, one encoding the NSmGn sequence and one encoding the Gc sequence. In a mammalian cell system, this combination resulted in infectious particles able to spread autonomously. Growth of a four-segmented bunyavirus can be explained by packaging of all four genome segments into a single virus particle or, alternatively, by co-infection of a single cell with two or more complementing particles. The resulting virus replicates considerably slower in mammalian cell culture compared to a virus that contains the authentic M segment (FIG. 6), which can be attributed to one or more of the following features: 1. Impaired glycoprotein and/or particle assembly; 2. Impaired genome packaging; 3. Impaired genome replication due to the presence of a fourth genome segment in infected cells. These properties will attenuate the virus in vivo. Remarkably, low moi infection of insect cells did not result in virus growth (FIG. 6). This suggests that the aforementioned mechanism(s) is (are) particularly inefficient in insect cells.
The term (NSm)Gn sequence indicates that the coding sequence of NSm may be present or absent.
The term “L-genome segment” refers to a substantially complete L-genome segment. The term “substantially complete” is used to indicate that the L genome segment comprises cis-acting elements that mediate replication of the L genome segment and that mediate functional expression of the RdRp gene. The term “substantially complete” indicates that sequences that are not involved in replication of the L genome segment or in functional expression of the RdRp gene may be deleted or substituted. The term “functional expression” refers to expression of an RdRp protein, a viral RNA-dependent RNA polymerase, which is able to mediate replication and transcription of a bunyavirus genome segment or bunyavirus minigenome.
The term “S genome segment comprising the N gene” refers to an S genome segment, comprising the UTRs of both the 3′ and the 5′ end of the S genome segment and at least the nucleotide sequences for expression of the N protein, such as nucleotide sequences for transcription of the N-gene and translation of the N-gene-transcript. The term “S genome segment from which the NSs and N coding regions have been functionally inactivated” refers to an S genome segment, comprising the 3′ and 5′ UTRs and the untranslated intergenic region of the S genome segment.
The term “M genome segment”, as used in the present application, refers to an M genome segment comprising the UTRs of both the 3′ and the 5′ end of the M genome segment, of which at least one of the glycoproteins Gn and Gc has been functionally inactivated. In one embodiment, said M genome segment is a minigenome segment.
The term “minigenome” refers to an RNA molecule that comprises the 5′ and 3′ UTRs of a bunyavirus genome segment that functions in replication of the segment, but which lacks at least one bunyavirus coding region that is present on the wildtype genome segment. A minigenome may further comprise a foreign gene such as, but not limited to, a marker gene such as a fluorescent protein, beta-glucuronidase, beta-galactosidase, Gaussia luciferase, Renilla luciferase and secreted alkaline phosphatase.
The term “second L genome segment”, as used herein, refers to a second viral genomic segment that contains the 3′ and 5′ UTRs of the L segment. The UTRs serve as promoters for replication of the segment and for transcription of the encoded reading frame. In addition, each genome segment exhibits a pseudo-circular structure due to the presence of the partially complementary 5′ and 3′ UTRs, referred to as “panhandle” sequences. The term “second M genome segment”, as used herein, refers to a second viral genomic segment that contains the UTRs of the M segment on both the 3′ and the 5′ ends. The term “second S genome segment”, as used herein, refers to a second viral genome segment that contains the UTRs of the S segment on the 3′ and the 5′ ends.
Similarly, the terms “third L genome segment” and “third S genome segment” as used herein, refer to a third viral genome segment that contains the UTRs of the L segment or the S segment, respectively, on the 3′ and the 5′ ends. The term “UTR” is known in the art and refers to the untranslated regions at the terminal ends of the viral RNA. The terms “second and third genome segment” include a second and third minigenome segment.
The present invention is directed to bunyaviruses with segmented glycoprotein precursor (GPC) genes. The invention is based on the expression of the coding sequences for the structural glycoproteins (NSm)Gn and Gc from separate genome segments. By this method, viruses can be created that contain four, instead of three genome segments, which retain high replicating efficiencies in vitro but lack the ability to spread efficiently in vivo, especially in arthropods. The limited spread of these viruses in vivo holds promise as veterinary and human vaccines.
In a wildtype bunyavirus, transcription and subsequent translation of the M genome segment results in the synthesis of a precursor protein that is processed into the structural glycoproteins Gn and Gc as well as a 14-kDa nonstructural protein named NSm and a 78-kDa protein, which is believed to be a minor structural protein (De Boer et al., 2012. J Virol 86: 13767-13771). It is relevant to note that the M segment open reading frame (ORF) contains five in-frame start codons. Four of these AUG codons, AUG-1, 2, 4 and 5, were shown to be used as translation initiation sites (Suzich et al., 1990. J Virol 64: 1549-1555). Translation from the first AUG results in the production of a polyprotein that is co-translationally cleaved into the 78-kDa protein (also referred to as “NSm1”) and the Gc protein (Gerrard and Nichol, 2007. Virology 357: 124-133) (see FIG. 1). Translation initiation at the second AUG results in the 14-kDa NSm protein as well as the Gn and Gc glycoproteins, whereas translation from AUG-4 or -5 results only in Gn and Gc. The Gn and Gc proteins form heterodimers and subsequently capsomers, presumably already in the endoplasmic reticulum. In the mature virus particle, Gn and Gc are believed to function as the receptor binding protein and viral fusion protein respectively (Dessau et al., 2013. Proc Natl Acad Sci USA 110: 1696-1701; de Boer et al., 2012. J Virol 86: 13642-13652).
Expression of Gn and Gc from the GPC gene results in the assembly of Gn and Gc into particles morphologically resembling the authentic virus, even in the absence of genome segments. These particles are referred to as virus-like particles (VLPs) (De Boer et al., 2010. Vaccine 28: 2330-2339; Habjan et al., 2009. Virology 385: 400-408). We previously demonstrated that transfection of replicon cells containing L and S segments with a GPC expression plasmid results in the assembly and secretion of infectious replicon particles that where shown to resemble phenotypically an authentic virus (Kortekaas et al., 2011. J Virol 85: 12622-12630; de Boer et al., 2012. J Virol 86: 13642-52; De Boer et al., 2012. J Virol 2012 86: 13767-71).
To generate a bunyavirus with a segmented glycoprotein precursor (GPC) gene, the coding sequences for the structural glycoproteins NSmGn and Gc, or for Gn and Gc, are separated and expressed from different genome segments. The coding sequence of glycoprotein Gc is preceded by an artificial AUG start codon, preferably embedded in a Kozak consensus sequence (gccRccAUGG; whereby R indicates a purine). The independent glycoprotein expression cassettes are positioned on separate genomic segments, selected from a genomic L segment and M segment, an L segment and a S segment, an M segment and a S segment, two L segments, two M segments or two S segments. cDNAs of the bunyavirus L, S and M genome segments, including second and third genome segments, when present, are preferably present in a plasmid vector in the genomic-sense orientation or in the antigenomic-sense orientation, and are transfected into relevant cells for generation of a bunyavirus, as has been described (Kortekaas et al., 2011. J Virol 85: 12622-12630). When transcription plasmids encode the genome segments in genomic-sense orientation, a bunyavirus is created by co-transfection of expression plasmids providing the N protein, the L protein and, preferably, the GPC.
A preferred bunyavirus according to the invention comprises four genome segments of which each segment encodes an essential gene. The S segment encodes the N protein and the L segment encodes the RdRp protein. The (NSm)Gn and Gc genes are separately expressed from different L, preferably different S, more preferably different M-type minigenomes, or a combination thereof. The production of progeny virions depends on infection of a cell with a single virus particle containing all four genome segment, or a co-infection of a single cell with two or more replicon particles together delivering all four genome segments. Co-transfection with transcription plasmids encoding the L segment, the S segment (containing or lacking NSs) and two M-type minigenomes, one expressing NSmGn and one expressing Gc, results in infectious particles that can be produced to titers of 107 TCID50/ml.
We have found that a four-segmented bunyavirus is incapable of spreading among insect cells, particularly when infection is performed at low moi (FIG. 6). Thus, it is likely that a bunyavirus with a segmented GPC, preferably a four-segmented bunyavirus, is not amplified in arthropod vectors that come into contact with the vaccine virus when feeding on a vaccinated individual. Re assortment of genome segments of the vaccine virus and field virus in arthropod vectors is thereby also prevented.
The term moi refers to the ratio of virus particles, preferably infectious virus particles, to target cells. Using a moi of 5 or less, such as 2, 1, 0.5 and 0.1, a number of cells will receive only one virus particle. For example, at an MOI of 1, 37% of cells receive no virus particle, 37% of cells receive one virus particle, and 26% of cells are multiply infected, while at an moi of 0.1, 90% of cells receive no virus particle, 10% of cells receive one virus particle and almost no cells are multiply infected. The term “low moi” indicates that the ratio of infectious virus particles to target cells is below 1, more preferably below 0.5, more preferably below 0.1, more preferably below 0.01, more preferably below 0.001, more preferably below 0.0001.
By combining a segmented GPC gene with a complete deletion of the major virulence factor, NSs, an optimally safe vaccine virus will be generated. The major inventive step is the successful rescue of a (nonspreading) bunyavirus using a segmented glycoprotein precursor gene, where each of the glycoprotein coding regions is expressed from different (S, M or L) genome segments or minigenomes.
The (NSm)Gn and Gc coding regions may be divided over two S segments. Combining these segments (S-[NSm]Gn and S-Gc) with the L segment resulted in infectious particles. These particles were capable of autonomous spread in vitro, also at low moi, suggesting that in this case, virus is produced that contains two S-type genome segments and one L genome segment. Although the product is an autonomously replicating virus, virus replication was reduced in vitro, suggesting that this three-segment virus will be attenuated in vivo. Deletion of the NSs gene will result in an optimally safe virus with the added advantage that no M segment is present. The resulting virus can therefore not donate an M segment to a field virus by means of reassortment.
The term “functionally inactivated”, as used herein, refers to a genetic alteration that abolishes expression of (NSm)Gn and/or Gc from the M genome segment. Said alteration is either an insertion, a point mutation, or, preferably, two or more point mutations, or, more preferably, a deletion. The term “deletion” covers the replacement of nucleotide sequences in the coding part of (NSm)Gn and/or Gc for other nucleotide sequences. A preferred deletion covers more than 10 nucleotides. A preferred deletion is a single, large deletion or a combination of several deletions. Most preferred is a complete deletion of the (NSm)Gn and/or Gc gene.
The term “functional deletion”, as used herein, refers to a genetic alteration that abolishes expression of NSs. Said alteration is either an insertion, a point mutation, or, preferably, two or more point mutations, or, more preferably, a deletion. The term “deletion” covers the replacement of nucleotide sequences in the NSs-coding part of the S-segment. A preferred deletion covers more than 10 nucleotides. This helps to ensure that the virus does not revert back to a propagation-competent, pathogenic and/or transmittable phenotype known as ‘reverse to virulence’ or ‘genetic drifting’. A preferred deletion is a single, large deletion or a combination of several deletions. Again, this helps to ensure that the virus does not revert back to a propagation-competent, pathogenic and/or transmittable phenotype. Most preferred is a complete deletion of the NSs gene. Alternatively, the NSs gene can be replaced by the corresponding gene from a heterologous bunyavirus such as Punta Toro virus or Sandfly fever Sicilian virus (Lihoradova et al. 2013. PLoS Negl Trop Dis 7:e2181).
Separated expression of (NSm)Gn and Gc from two different genome segments results in the production of an optimally safe vaccine virus. The impaired growth and/or impaired genome replication in vivo will allow the vaccinated animal to mount an effective immune response, restricting dissemination of the vaccine virus in the vaccinated animal and providing protection from wildtype bunyavirus. A most preferred bunyavirus according to the invention comprises a bunyavirus L genome segment; a bunyavirus S genome segment or part of a S genome segment comprising at least the N gene and the 3′ and 5′ UTRs; and a bunyavirus M genome segment from which the NSmGn or Gc coding region has been functionally inactivated, whereby the NSmGn or Gc coding region that is deleted from the M genome segment is functionally present on a second M genome segment or on a second S segment.
The bunyavirus L genome segment and/or the S genome segment and/or, when present, the M genome segment, of a preferred bunyavirus according to the invention comprises a foreign gene. Said foreign gene is preferably selected from those encoding N, Gn or Gc, of another bunyavirus of the Phlebovirus, Nairovirus, Orthobunyavirus or Hantavirus genus.
In a preferred method according the invention, one or more of the bunyavirus L genome segment, the S genome segment, and/or, the M genome segment comprises a foreign gene. Said foreign gene is preferably derived from an organism that is a transmitter of an infectious disease. Said organism is preferably selected from adenovirus, African horsesickness virus, African swine fever, Bluetongue virus, Border disease virus, Borna virus, Bovine viral diarrhoe virus, Bovine respiratory syncytial virus, Cache Valley fever virus, Chikungunya virus, Chrysomya bezziana, Classical swine fever, Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus, Cochliomyia hominivorax, Coronavirus, Cytomegalovirus, Dengue virus, Eastern equine encephalitis virus, Ebola virus, Equine encephalomyelitis virus, Equine encephalosis virus, Foot and mouth disease virus, Goat pox virus, Hantaanvirus, Sin Nombre virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Herpes simplex virus, Highly pathogenic avian influenza virus, Human immunodeficiency virus, Human parainfluenza virus, Influenza virus, Japanese encephalitis virus, Kaposi's sarcoma-associated herpesvirus, Lassa virus, Lujo virus, Marburg virus, Marsilia virus, Measles virus, Monkeypox virus, Mumps virus, Nipah virus, Papillomavirus, Papova virus, Peste des petits ruminants, Polio virus, Polyomavirus, Rabies virus, Respiratory syncytial virus, Rhinovirus, Rinderpest virus, Rotavirus, Rubella virus, Sandfly fever Naples virus, Sandfly fever Sicilian virus, SARS coronavirus, Sheep pox virus, Simian immunodeficiency virus, Smallpox virus, St. Louis encephalitis virus, Toscana virus, Varicella-zoster virus, West Nile virus, Western equine encephalitis virus, Yellow fever virus, Bacillus anthracis, Bordetella pertussis, Brucella spp., Campylobacter jujuni, Chlamydia trachomatis, Clostridium botulinum, Coxiella burnettii, Francisella tularensis, Group B streptococcus, Streptococcus suis, Legionella pneumophila, Leptospira spp., Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Neisseria meningitidis, Salmonella, Shigella spp., Trypanosoma cruzi, Vibrio cholerae, Yersinia pestis, Mycoplasma mycoides, Plasmodium malariae, Plasmodium ovale, Plasmodium ssp., Plasmodium vivax, Taenia solium, Taenia spp., and Trypanosoma brucei. 
A bunyavirus according to the invention is preferably selected from the genera Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus, which include numerous virus species capable of causing severe disease in both animals and humans. Preferred viruses are Sin Nombre virus (SNV) and Dobrava-Belgrade virus (DOBV) (both of the Hantavirus genus), Crimean-Congo hemorrhagic fever virus (CCHFV) and Dugbe virus (both of the Nairovirus genus), Schmallenberg virus, Bunyamwera virus, Shuni virus and Oropouche virus (Orthobunyavirus), Rift Valley fever virus (RVFV, Phlebovirus genus) and further members of the Phlebovirus genus: Toscana virus, Sandfly fever Naples virus, Sandfly fever Sicilian virus, Punta Toro virus, Uukuniemi virus, Massilia virus and severe fever with thrombocytopenia syndrome (SFTS) virus. Further preferred bunyaviruses include, but are not limited to, viruses of the Dera Ghazi Khan virus Group, the Hughes virus Group, Nairobi sheep disease virus Group, Qalyub virus Group, Sakhalin virus Group, and the Thiafora virus Group.
A most preferred bunyavirus is selected from the group consisting of Crimean-Congo hemorrhagic fever virus, Shuni virus, SFTS virus, Schmallenberg virus, Bunyamwera virus and Rift Valley fever virus.
A bunyavirus according to the invention preferably is a chimeric virus in which the (NSm)Gn and/or the Gc coding region is derived from a bunyavirus that differs from the bunyavirus from which the L and S genome segments are derived. The resultant chimeric virus may then be used as a vaccine that protects against the virus from which the glycoprotein genes are derived and, preferably, as a bivalent vaccine. A preferred virus according to the invention comprises the (NSm)Gn and Gc coding region from SFTS virus, while the L and S genome segments are derived from a different bunyavirus, for example from Rift Valley fever virus or Uukuniemi virus.
A bunyavirus according to the invention preferably is a chimeric virus in which an additional N coding region is introduced, preferably by replacing the NSs gene on the S segment. The resulting S genome segment thus encodes two N proteins. The heterologous N gene is derived from a bunyavirus that differs from the bunyavirus from which the rest of the genetic material is derived. A preferred additional N coding region is from the CCHF virus. The sequence of a S genome segment encoding the RVFV N protein and the CCHFV N protein is depicted in FIG. 2H. The resultant chimeric virus may then be used as a vaccine that protects against the virus from which the heterologous N gene is derived and, preferably, as a bivalent vaccine. A preferred virus according to the invention comprises the N coding region of the CCHF virus, while the rest of the genetic material is derived from Rift Valley fever virus. The resulting vaccine can be used to vaccinate humans or animals against CCHFV and RVFV.
It is preferred that a NSs coding region on the S genome segment is functionally inactivated in a bunyavirus according to the invention. An important advantage of a bunyavirus according to the invention is that the virus is not capable of spreading in a vaccinated animal and/or arthropod vector. The inability to spread systemically, greatly adds to the safety of such virus. The inability to cause viremia in a vaccinated animal also prevents any concerns about possible transmission of this vaccine by insect vectors (see Moutailler et al. 2010. Vector Borne Zoonotic Dis 10:681-688). A vaccine comprising a bunyavirus according to the invention with a functional deletion of the NSs gene, preferably a complete deletion of the NSs coding region, is considered of optimal safety, due to the lack of this major virulence factor.
The invention further provides a method for producing a bunyavirus according to the invention, the method comprising A) providing a eukaryotic cell with growth medium; and B) infecting the eukaryotic cell with the recombinant bunyavirus according the invention, whereafter the bunyavirus is secreted into the growth medium and can be isolated therefrom.
Said eukaryotic cell is preferably a cell that can easily be infected using standard methods known to the skilled person. Said cell is preferably a eukaryotic cell, preferably a mammalian cell. Suitable cells comprise, for example, Baby Hamster Kidney cells such as BHK-21, Human Embryonic Kidney cells such as HEK293, VERO cells, VERO-E6, MDCK cells, CHO cells, Huh-7, Huh7.5 (Sumpter 2005. J Virol 79:2689-2699), HeLa, SW13 and PER.C6 cells (Fallaux, F. J. et al. 1998. Hum Gene Ther 9: 1909-1917). A preferred cell is BHK-21, or a derivative thereof such a BSR subclone (Sato M, et al. 1977. Arch Virol 54: 333-343) or BSR T7/5 (Buchholz et al., 1999 J Virol 73:251-259.
The invention further provides a composition comprising a bunyavirus according to the invention and a suitable excipient, preferably a suitable pharmaceutically and/or veterinary acceptable excipient such as a carrier, adjuvant or vehicle. Said excipient may comprise vitamins; sugars such as sucrose, lactose, D-mannose, D-fructose, and/or dextrose; amino acids such as, for example, glycerin and asparagine; inorganic salts such as, for example, sodium bicarbonate, aluminum hydroxide, benzethonium chloride, ammonium sulfate, magnesium sulfate, potassium phosphate, sodium phosphate, aluminum phosphate and aluminum potassium sulfate; micro crystalline cellulose, magnesium stearate, cellulose acetate phthalate, human serum albumin, fetal bovine serum, citric acid, iron ammonium citrate, peptone, bovine extract and/or gelatin.
A composition according to the invention preferably is an immunogenic composition, more preferably a composition that provides protection against a subsequent infection with a wildtype bunyavirus. Said protection against wildtype virus is characterized as a reduction of clinical disease, and/or a reduction of replication of wildtype virus in the host. Regarding control or eradication of a disease, protection is defined as reduction of onward spread of wildtype virus by any transmission route, via an arthropod vector, horizontally, vertically, and directly or indirectly. The time to onset of protection and long lasting protection are part of the efficacy of a vaccine. Further, broad protection in case of different virus species or serotypes, as will be provided by chimeric bunyaviruses according to the invention, is also part of efficacy.
The invention also provides a bunyavirus according to the invention, or a composition comprising a bunyavirus according to the invention, for use as a medicament. Said medicament is preferably used for amelioration of a bunyavirus infection in an animal, including human. A medicament comprising a recombinant bunyavirus according the invention may additionally comprise a pharmaceutical acceptable adjuvant, diluent or carrier. A medicament according to the invention is preferably combined with other therapeutic options.
The invention additionally provides a vaccine comprising a bunyavirus according to the invention, or a composition according to the invention. Said vaccine preferably comprises an adjuvant. Adjuvant substances are used to stimulate immunogenicity. Examples of commonly used immunological adjuvants are aluminum salts, immunostimulating complexes (ISCOMS), non-ionic block polymers or copolymers, cytokines (like IL-1, IL-2, IL-7, etc.), saponins, monophosphoryl lipid A (MPLA), muramyl dipeptides, vitamin E, polyacrylate resins, and oil emulsions. Preferably, the adjuvant is a sulfolipopolysaccharide, such as the SLP/S/W adjuvant described in Hilgers et al. Vaccine 1994 12:653-660. A further preferred adjuvant is provided by Montanide adjuvants, for example Montanide ISA 720 which contains a natural metabolizable oil and a highly-refined emulsifier from the mannide monooleate family, andby triterpene, such as squalene, and derivatives and modifications thereof.
The invention additionally provides a method for generating a bunyavirus, the method comprising A) providing a cell with a bunyavirus comprising at least one genome segment functionally encoding a RdRp gene and one genome segment encoding a N gene, and B) providing the cell with at least two further (mini)genome segments selected from L, M, and/or S genome segments comprising separated (NSm)Gn and Gc coding regions, meaning that separated (NSm)Gn and Gc coding regions are functionally present on two separate genome segments, whereby each of the two genome segments comprises either the (NSm)Gn coding region or the Gc coding region. A preferred method comprises A1) providing a cell with a bunyavirus L genome segment; A2) providing the cell with at least part of a bunyavirus S genome segment comprising the N-gene and the 3′ and 5′ UTRs; and B) providing the cell with at least two further (mini) genome segments selected from L, M, and/or S genome segments comprising separated (NSm)Gn and Gc coding regions, optionally providing the cell with a further M genome segment from which the (NSm)Gn and Gc coding region has been functionally inactivated; wherein the sequence of steps A1, A2, and B is random and all or part of these steps may be performed simultaneously.
Step B comprises steps B1, in which either the (NSm)Gn or the Gc coding region is retained on the M genome segment, and B2 in which both the (NSm)Gn and the Gc coding region are functionally deleted from the M genome segment. Step B1) comprises providing the cell with a M genome segment from which the (NSm)Gn or Gc coding region has been functionally inactivated, whereby the cell is further provided with a second L genome segment, a second M genome segment, or a second S genome segment on which the (NSm)Gn or Gc coding region that is deleted from the M genome segment is functionally present. Step B2) comprises providing the cell with a second and a third L genome segment, a second L genome segment and a second S genome, or a second S genome segment and a third S genome segment, whereby separated NSmGn and Gc coding regions are functionally present either on said second and a third L genome segment, on said second L genome segment and said second S genome segment, or said second S genome segment and said third S genome segment, and optionally a further M genome segment from which the (NSm)Gn and Gc coding region has been functionally inactivated; wherein the sequence of steps A1, A2, and B is random and all or part of these steps may be performed simultaneously.
It is preferred that a cell is provided with a genomic segment by providing the cell with a vector that comprises cDNA of said genomic segment which is flanked at the 5′ end by a promoter sequence for a DNA-dependent RNA polymerase. Said promoter sequence for a DNA-dependent RNA polymerase is preferably selected from known promoter sequences of DNA-dependent RNA polymerases such as, but not limited to, the promoter sequence of a eukaryotic RNA polymerase I such as, for example, a promoter for murine RNA polymerase I, the previously reported Pol-I/Pol-II-based reverse genetics system (Habjan et al., 2008. J Gen Virol 89: 2157-2166), and the bacteriophage polymerases SP6, T3 and T7. Although the description and the claims refer to T7polymerase, it is to be understood that the invention is not limited to T7 polymerase but includes other DNA-dependent RNA polymerase such as, for example, T3 polymerase and SP6 polymerase. A preferred promoter sequence for a T7 polymerase is TAATACGACTCACTATAG (SEQ ID NO: 1).
Copy DNA of bunyavirus genomic segments or fragments thereof are flanked at the 3′ ends by a cDNA encoding a ribozyme sequence that mediates 3′ end formation of the RNA by self-cleavage of the nascent RNA. A preferred ribozyme sequence is a hepatitis delta virus (HDV) ribozyme sequence. A termination sequence that mediates termination of the DNA-dependent RNA polymerase may further be present distal to the cDNA encoding the ribozyme sequence. In a preferred embodiment, the DNA-dependent RNA polymerase is T7 polymerase and the termination sequence is a T7 transcription termination sequence. Promoter sequences for DNA-dependent RNA polymerases, such as T7 polymerase, and termination sequences such as a T7 transcription termination sequence, are known to the skilled person.
In a preferred method according to the invention, a cell is provided with a genomic segment by providing the cell with a vector that comprises cDNA of said genomic segment which is flanked by a T7 promoter and cDNA of a ribozyme. Said method additionally comprises providing the cell with a T7 polymerase.
A cell is provided with sufficient T7 polymerase by freshly transfecting or infecting the eukaryotic cell with an expression vector that encodes the T7 polymerase. In one embodiment, the expression vector is a plasmid that encodes the T7 polymerase. Suitable plasmids are, for example, pCAGGS, and pcDNA. In a preferred embodiment, T7-expressing cells are infected with a non-recombinant poxvirus, preferably an avipoxvirus such as pigeonpox virus or fowlpox virus. Infection by the poxvirus will stimulate capping of T7 transcripts, which stimulates protein expression. In another preferred embodiment, the expression vector is a recombinant virus or viral vector that encodes the T7 polymerase. A suitable virus or viral vector is, for example, a replication defective retroviral vector such as a lentiviral vector, for example a HIV-based vector or an EIAV-based vector, or a replication defective MMLV-based vector. A further suitable virus or viral vector is provided by a replication defective adenoviral vector and a baculovirus vector. A preferred virus or viral vector is a replication defective poxvirus such as, for example, a vaccinia-based virus. In a most preferred method, the eukaryotic cell is provided with sufficient T7 polymerase by infecting the eukaryotic cell with a fowlpox virus (FPV)-based expression vector that encodes the T7 polymerase. The FPV may be replication competent or replication defective.
An advantage of a FPV is that it belongs to the genus Avipoxvirus and is capable of spreading in avian cells. In non-avian cells such as, for example, mammalian cells, FPV replication is abortive without any evidence of production of infectious virus. Therefore, when the eukaryotic cell is a non-avian eukaryotic cell, a replication competent FPV-based expression vector or a replication deficient FPV-based expression vector is preferably used for a method of the invention. When the eukaryotic cell is an avian eukaryotic cell, it is preferred that a replication deficient FPV-based expression vector is used for a method of the invention.
Said cell preferably is a eukaryotic cell. Said eukaryotic cell is preferably a cell that can easily be infected using standard methods known to the skilled person. Said cell is preferably a eukaryotic cell, preferably a mammalian cell. Suitable insect cells comprise, for example, Baby Hamster Kidney cells such as BHK-21, Human Embryonic Kidney cells such as HEK293, VERO cells, VERO-E6 cells, MDCK cells, CHO cells, HuH-7, Huh7.5 (Sumpter 2005. J Virol 79:2689-2699), HeLa, SW13 and PER.C6 cells (Fallaux, F. J. et al. 1998. Hum Gene Ther 9: 1909-1917). A preferred cell is BHK-21, or a derivative thereof such a BSR subclone (Sato M, et al. 1977. Arch Virol 54: 333-343) or BSR T7/5 (Buchholz et al., 1999 J Virol 73:251-259).



FIGURE LEGENDS
FIG. 1. Membrane topology and proteolytic processing of the RVFV glycoprotein precursor. Shown are the translation products starting from the in-frame AUG codons 1,2 and 4⁄5 of the open reading frame. The ‘Y’ symbols indicate the predicted N-linked glycosylation sites. The red ‘Y’ symbol is known not to be utilized. The 14-kDa, 78-kDa, Gn and Gc proteins are indicated, as well as the predicted transmembrane spanning regions.
FIG. 2. Schematic representations of relevant plasmid sequences of A) pCAGGSNSmGn, B) pCAGGS-Gn, C) pCAGGS-Gc, D) pUC57-S-NSmGn, E) pUC57-S-Gc, F) pUC57-S-de1NSs, G) pUC57-M-NSmGn, H) pUC57-M-Gc, I) pUC57-S-CCHFV-N, J) pUC57-S-NSmGn (non-optimized NSmGn sequence), and K) pUC57-S-Gc (non-optimized Gc sequence). Cartoons were produced using SnapGene® software (from GSL Biotech; available at snapgene.com). Sequences in bold indicate start and stop codons whereas underlined sequences represent restriction enzyme sites. Shaded sequences represent untranslated regions. The RVFV sequences in pUC57 plasmids are flanked by a T7 promoter and a ribozyme sequence.
FIG. 3. Replicon particle production by BHK-Rep2 cells. BHK-Rep2 cells stably maintaining the L and S-eGFP genome segments of RVFV were transfected with either pCAGGS-M, co-transfected with pCAGGS-Gn and pCAGGS-Gc, or co-transfected with pCAGGS-NSmGn and pCAGGS-Gc. One day post transfection supernatants were titrated on BHK-21 cells and titers of replicon particle progeny were determined.
FIG. 4. Visualization of RVFV containing two S genome segments. BHK-21 cells were infected with RVFV-SSL and after 2 days Gn and Gc expression was visualized using IPMA.
FIG. 5. Visualization of RVFV containing four genome segments. BSR T7/5 cells were infected with RVFV-SMML-NSs, RVFV-SMML-delNSs or RVFV-SMML-eGFP and after 2 days Gn and Gc expression was visualized using IPMA.
FIG. 6. Growth curves of RVFV-eGFP and RVFV-SMML-eGFP. BSR T7/5 and C6/36 cells were infected with the listed viruses with an moi of 0.001. Supernatants were collected at the indicated time points and titrated on BSR T7/5 cells.
FIG. 7. RVFV-SMML-eGFP-based virus neutralization test. Sera from experimentally infected sheep were analysed for RVFV neutralizing antibodies using a conventional VNT test and a RVFV-SMML-eGFP-based VNT.
FIG. 8. Creation of RVFV replicon particles using a segmented glycoprotein precursor gene. A) Schematic presentation of M-segment encoded proteins and protein processing. The localization of the segmentation site is indicated with an arrow. B) Schematic presentation of the NSR read-out system. BHK-Rep2 cells, stably maintaining replicating RVFV L and SeGFP genome segments were transfected with pCAGGS expression plasmids encoding Gn, (NSm)Gn, Gc or NSmGnGc. C) One day post transfection the titer of NSR progeny was determined in the supernatant. Bars represent means+standard error (SE) of three independent experiments.
FIG. 9. Rescue of RVFV-LMS-split and RVFV-LSS. A) Schematic presentation of the wild-type RVFV genome and the RVFV-LMS-split and RVFV-LSS variants. B) IFA (Gn antigen) of BSR cells infected with RVFV, RVFV-LMS-split or RVFV-LSS 48 h post infection. C) Northern blot analysis of RVFV, RVFV-LMS-split and RVFV-LSS RNA isolated of supernatants of infected cells. Used probes are indicated on the right. D) Growth curve of indicated viruses on BSR cells.
FIG. 10. Rescue of RVFV-4S A) Schematic presentation of the wildtype RVFV genome and of 4 segment variants. B) IFA (Gn antigen) of BSR cells infected with all the variants 48 h post infection. C) Northern blot analysis of RNA isolated from supernatants of RVFV-4S infected cells. Used probes are indicated at the right. D) Growth curve of indicated viruses on BSR cells.
FIG. 11. Localization of Gn and Gc in cells infected with RVFV-4S. BSR cells were infected at MOI 0.1 with RVFVeGFP or RVFV-LMMSeGFP. 16 h post infection cells were fixated and Gn and Gc antigen was detected.
FIG. 12. Growth of RVFV variants in insect cells. C6/36 cells and BSR cells were infected with the RVFV variants as indicated at an MOI of 0.01. Supernatants were collected at 4 days post infection and titrated on BSR cells. Bars represent means+SE of three independent experiments.
FIG. 13. Virulence of RVFV-4S. A) Survival curve of mice inoculated with a high or a low dose of RVFV-LMMSNSs via intraperitoneal route. As a control, mice were challenged with a low dose of authentic RVFV. Virus dissemination in the liver (B) and brain (C) of mice euthanized at different time points was determined by qRT-PCR.
FIG. 14. Vaccination-challenge experiment. A) Survival curve of mice inoculated with culture medium (mock), NSRGn, RVFV-LMMSeGFP or RVFV-LMMSdelNSs. Three weeks post vaccination mice were challenged with a lethal dose of wild-type RVFV. B) RVFV neutralization titers present in sera the day before challenge. Virus dissemination into the liver (C) and brain (D) was determined by qRT-PCR.
FIG. 15. Rectal temperatures of vaccinated (A) and mock-vaccinated (B) lambs before and after challenge with RVFV on day post vaccination (DPV) 21. Rectal body temperatures (° C.) were determined daily. Fever was defined as a body temperature above 40.5° C. (interrupted line). Rectal body temperatures of vaccinated lambs are depicted as averages (n=7) with SD. Rectal body temperatures of mock-vaccinated lambs determined after DPV 23, 24 and 27 are depicted as averages of 6, 5 and 4 measurements, respectively, since a lamb from this group died on each of these days.
FIG. 16. Monitoring of viremia in vaccinated (A) and mock-vaccinated (B) lambs by qRT-PCR. Viral RNA was detected by qRT-PCR in plasma samples obtained at different days post challenge with RVFV.
FIG. 17. Results from virus neutralization tests performed with sera obtained from vaccinated- or mock-vaccinated lambs at different time points after vaccination and challenge infection. Errors bars represent standard deviations. The detection limit of the assay is a VNT titer of 10. The arrow indicates the day of challenge.



EXAMPLES
Example 1
Materials and Methods
Cells and Growth Conditions.
All mammalian cell lines were routinely grown at 37° C. with 5% CO2. BHK cells were grown in Glasgow Minimum Essential Medium (GMEM; Invitrogen, Bleiswijk, The Netherlands) supplemented with 4% tryptose phosphate broth (TPH; Invitrogen), 1% non-essential amino acids (NEAA; Invitrogen), 5-10% fetal bovine serum (FBS; Bodinco, Alkmaar, The Netherlands) and 1% penicillin-streptomycin (Invitrogen). BSR-T7/5 cells, kindly provided by Prof. Dr. K. Conzelmann (Max von Pettenkofer-Institut, Munchen, Germany) and BHK-Rep2 cells (BHK-21 cells constitutively replicating L and S genome segments of RVFV isolate 35/74) were grown in the same medium as used for BHK cells, supplemented with 1 mg/ml geneticin. Cell culture in the BSL-3 laboratory was performed in closed containers and therefore required the use of CO2-independent medium (CIM; Invitrogen). For these experiments, the culture medium was replaced with CIM supplemented with 5% FBS and 1% penicillin-streptomycin. Aedes albopictus (C6/36) mosquito cells [9] were grown in L15 medium (Invitrogen) supplemented with 10% FBS, 1% NEAA, 2% TPH and 1% pencillin-streptomycin at 28° C. without CO2. QM5 cells were grown in QT35 medium (Invitrogen) supplemented with 5% FBS and 1% penicillin-streptomycin.
Viruses
RVFV strain 35/74 was isolated from the liver of a sheep that died during a RVFV outbreak in the Free State province of South Africa in 1974 (Barnard 1979. J S Mr Vet Assoc 50: 155) and was kindly provided by the Agricultural Research Council-Onderstepoort Veterinary Institute (Pretoria, South Africa). The virus was passaged four times in suckling mice by intra-cerebral injection and three times on BHK-21 cells. The virus was routinely grown on BHK-21 cells. Sequences of the L, M, and S genome segments can be found in GenBank under accession numbers JF784386, JF784387, and JF784388 respectively.
A fowlpox virus that expresses T7 polymerase, named fpEFLT7pol [11], from hereafter referred to as FP-T7, was kindly provided by the Institute for Animal Health (IAH, Compton, UK; Britton et al., J Gen Virol. 1996; 77:963-967.). The virus was grown and titrated on QM5 cells.
Titration
Virus titers were determined by serial dilution on cells of interest in 96 wells plates (10.000-40.000 cells/well). Two to five days post infection cytopathologic effect (CPE) was scored or virus growth was visualized using immunoperoxidase monolayer assay (IPMA, see below). Titers were determined as TCID50 as described (Kärber., 1931. Arch Exp Path Pharmak 162: 480-483; and Spearman, 19908. Br J Psychol 2: 227-242).
Plasmids
Expression Plasmids
pGAGGS expression plasmids, containing a CMV immediate enhancer/chicken β-actin (CAG) promoter (Niwa et al, 1991, Gene 108: 193-199) were used for transient expression of genes of interest (GOI). pCAGGS-M (Kortekaas et al., 2011. J Virol 85: 12622-12630) contains the ORF of the M-segment of RVFV isolate 35/74, starting at the first methionine codon. pCAGGS-NSmGn contains the NSmGn coding region of the M segment of RVFV strain 35/74 without the signal sequence of Gc (FIG. 2A). pCAGGS-Gn contains the Gn coding region of the M-segment of RVFV isolate 35/74 without the signal sequence of Gc (FIG. 2B). pCAGGS-Gc contains the Gc coding region of the M-segment of RVFV isolate 35/74 including its N-terminal signal sequence (FIG. 2C). Plasmids were designed to contain sequences optimized for expression in mammalian cells and Kozak consensus sequences were included to optimize expression.
Transcription Plasmids
RVFV transcription plasmids, pUC57-S, pUC57-M and pUC57-L (Kortekaas et al., 2011. J Virol 85: 12622-12630) encode the complete S, M or L segment of RVFV isolate 35/74, respectively, including the 3′ and 5′ UTRs. Transcription of the genome segments is controlled by a minimal T7 promoter. Transcription results in antigenomic-sense RNA genome segments. Plasmid pUC57-S-eGFP plasmid (Kortekaas et al., 2011. J Virol 85: 12622-12630) encodes an S segment RNA in which the NSs gene is replaced in its entirety by the gene encoding enhanced green fluorescent protein (eGFP). pUC57-S-NSmGn and pUC57-S-Gc encode the N protein in antigenomic orientation and the NSmGn protein and Gc protein, respectively, in genomic-sense orientation (FIGS. 2D and 2E). NSmGn and Gc-coding sequences were codon optimized for optimal expression in mammalian cells. The non-optimized pUC57-S-NSmGn and pUC57-S-Gc sequences are depicted in FIGS. 2J and 2K, respectively. The pUC57-S-delNSs plasmid encodes an S segment from which the NSs gene is deleted in its entirety (FIG. 2F). The pUC57-M-NSmGn and pUC57-M-Gc plasmids contain the authentic (non-codon optimized) sequences of NSmGn, respectively Gc, in antigenomic-sense orientation (FIGS. 2G and 2H). Kozak consensus sequences were included for optimal expression.
Immunoperoxidase monolayer assay (IPMA) and immunofluorescence assay (IFA). Monolayers of infected cells were fixed in 4% paraformaldehyde in PBS for 30 minutes and subsequently permeabilized with 100% cold methanol. After blocking the cells with 5% horse serum in PBS for 30 min, cells were incubated with primary antibody in blocking solution. Either a Gn-specific monoclonal antibody (mAb, provided by Dr. Connie Schmaljohn, USAMRIID, Keegan et al., 1986. J Virol 58: 263-270), a Gc rabbit polyclonal antibody (De Boer et al., 2012. J Virol 86: 13642-13652), or an N-specific mAb (F1D11; Kindly provided by Dr. Alejandro Brun, CISA-INIA, Spain; Martin-Folgar et al., MAbs. 2010; 2(3):275-284) was used. As secondary antibodies, HRP-conjugated rabbit anti-mouse IgG, goat anti-rabbit IgG (DAKO, Heverlee, Belgium) or a rabbit anti-mouse Texas Red conjugated antibody (DAKO) was used. All antibody incubations were performed for >1 hour at 37° C. and between antibody incubations cells were washed three times with PBS supplemented with 0.05% Tween 80. Antibody binding was visualized using an EVOS fluorescence microscope (Fisher Scientific) and a 3-amino-9-ethylcarbazole (AEC)-based substrate (DAKO).
Virus Rescue
BSR-T7/5 or BHK-21 cells were seeded on day 0 in 6-well plates (100.000-600.000 cells/well) in GMEM supplemented with 5% FBS. On day 1, cells were infected for one to two hours with FP-T7 (moi≈0.1) in Optimem (Invitrogen) containing 0.2% FBS and were subsequently transfected with 3 μg plasmid DNA (600-1000 ng/plasmid) using jetPEI transfection reagents according the manufactures description (Polyplus, Illkirch, France). Four hours post transfection, medium was replaced by complete CIM or GMEM medium. Three to five days post transfection, supernatants were collected and incubated with freshly seeded BSR-T7/5 or BHK-21 cells. Productive infection was visualized with the EVOS fluorescence microscope by detection of GFP expression or by staining RVFV-specific proteins by IPMA or IFA.
Virus Neutralization Test (VNT)
VNTs were performed with the 4S SLMM-eGFP virus expressing eGFP from the S segment. Sera were obtained from lambs that had previously been experimentally infected with the 35/74 isolate. To confirm the presence of RVFV-specific antibodies, the sera were analyzed with a recombinant N (recN) RVFV enzyme-linked immunosorbent assay (ELISA) (BDSL, Irvine, Ayrshire, Scotland, United Kingdom) prior to analysis by VNT. Serum dilutions were prepared in 96-well plates in 50 μl complete GMEM supplemented with 5% FBS. Culture medium containing ≈200 infectious particles in a 50 μl volume was added to the serum dilutions and the mixture was incubated for 1.5 h at room temperature. Next, 50 μl of CIM growth medium containing 40 000 BHK-21 cells was added to each well. Plates were incubated at 37° C. for 36 to 48 h. Neutralization titers were calculated by the Spearman-Kärber method.
Results
The Glycoprotein Precursor (GPC) is not Essential for Bunyavirus Assembly
The requirement for the (NSm)Gn and Gc glycoproteins of RVFV to be expressed from a polyprotein precursor was evaluated. The ORF encoding the GPC was divided into two non-overlapping ORFs encoding NSmGn and Gc, respectively. Specifically, the GPC gene was segmented at the tyrosine (Y)-675 codon, which is predicted to be the first amino acid of the signal sequence of Gc (Suzich et al., 1990. J Virol 64: 1549-1555; Gerrard and Nichol, 2007. Virology 357(2): 124-133).
To evaluate whether the (NSm)Gn and Gc proteins, expressed from dedicated expression plasmids, are able to package RVFV genome segments into infectious replicon particles, we co-transfected BHK-Rep2 cells (Kortekaas et al., 2011. J Virol 85: 12622-12630) with pCAGGS expression vectors encoding either Gn or NSmGn (pCAGGS-Gn or pCAGGS-NSmGn) and a pCAGGS plasmid encoding Gc (pCAGGS-Gc) and evaluated if replicon particles were produced. As a positive control, BHK-Rep2 cells were transfected with a pCAGGS plasmid encoding the complete GPC (pCAGGS-M) which is known to result in the production of replicon particles (Kortekaas et al., 2011. J Virol 85: 12622-12630). The results show that Gn and Gc, when expressed from two different expression plasmids, are able to package RVFV genome segments into infectious replicon particles (FIG. 3). These experiments resulted in average infectious particle titers of 10E4 TCID50/ml. The presence of the NSm coding region increased the yield of infectious particles to 10E6 TCID50/ml (FIG. 2). Although replicon particle yields when produced with pCAGGS-NSmGn and pCAGGS-Gc were approximately 10 times lower compared to the yields resulting from transfection with pCAGGS-M, the results clearly show that segmentation of the GPC ORF into two dedicated ORFs encoding the (NSm)Gn and Gc glycoproteins, respectively, does not abrogate particle assembly or glycoprotein function. From this, it is concluded that co-translational cleavage of the GPC is not essential for the generation of infectious replicon particles.
Rescue of a RVFV that Expresses NSmGn from the S-Segment
After demonstrating that RVFV L- and S genome segments can efficiently be packaged into infectious replicon particles using the pCAGGS-NSmGn and pCAGGS-Gc expression plasmids, we next investigated if virus can be generated expressing the NSmGn gene and Gc gene from separate genome segments. To this end, a transcription plasmid was created that encodes the Gc gene flanked by the M segment untranslated regions (UTRs, pUC57-M-Gc) and a transcription plasmid was created that encodes an S segment in which the NSs gene is substituted for the NSmGn gene (pUC57-S-NSmGn). BHK cells were infected with FP-T7 and transfected with transcription plasmids pUC57-L, pUC57-M-Gc and pUC57-S-NSmGn. Three days post transfection supernatants were collected and used to inoculated fresh BHK-21 cells. Three days later, CPE was observed, indicating the presence of virus. Titration of the virus showed a yield of 10E6 TCID50/ml. The results of this experiment demonstrate that co-translational cleavage of the GPC is not essential for RVFV particle assembly and glycoprotein function.
The virus based on expression of codon optimized NSmGn from the S-segment is referred to as RVFV-LMS-split-opt
Rescue of RVFV Containing Two S-Type Genome Segments
Our finding that the GPC is not essential for RVFV provides opportunities to study RVFV genome packaging. We investigated whether RVFV is able to package more than one genome segment of the same type. A plasmid was created that encodes an S segment in which the NSs gene is replaced with the Gc gene (yielding pUC57-S-Gc). FP-T7-infected BHK-21 cells were transfected with pUC57-L, pUC57-S-NSmGn and pUC57-S-Gc. Three days post transfection supernatants were analyzed for the presence of infectious virus by incubation with BHK-21 cells. Three days post incubation CPE was observed and rescue of virus was visualized with IPMA (FIG. 4). Titration of the virus revealed a yield of 10E6 TCID50/ml. The relatively efficient growth of a RVFV containing two S segments suggests that the virus can easily maintain two S-type genome segments.
The two S-segmented virus based on expression of codon optimized NSmGn and Gc from separate S-segments is referred to as RVFV-LSS-opt.
Rescue of Four Segmented RVFV
To further explore RVFV genome packaging, we evaluated if a virus can be rescued that contains 4 genome segments: one L, one S and two additional segments encoding the structural glycoproteins. We selected the M segment for expression of NSmGn and Gc. To this end, two transcription plasmids were created, pUC57-M-NSmGn and pUC57-M-Gc. BSR T7/5 cells were infected with FP-T7 and transfected with transcription plasmids pUC57-L, pUC57-M-NSmGn, pUC57-M-Gc and either pUC57-S, pUC57-S-eGFP or pUC57-S-delNSs. The pUC57-S-eGFP was used to facilitate detection of virus by fluorescence microscopy. The pUC57-delNSs was created by deleting the complete NSs gene from the pUC57-S plasmid. Three days post transfection, supernatants were collected and used to infect BHK cells. First experiments focused on the rescue of eGFP expressing virus, which was monitored using an EVOS fluorescence microscope. The increase in number of fluorescent cells demonstrated that virus was rescued and passage of the supernatant resulted in a virus titer of 10E7 TCID50/ml. Using similar methods, viruses either lacking or containing the NSs gene were rescued, which both yielded titers of 10E7 TCID50/ml. All three viruses were visualized using IPMA (FIG. 5). The successful rescue of a RVFV using four genome segments, named RVFV-SMML (also referred to as RVFV-LMMS), indicates that RVFV is able to maintain more than three genome segments in the virus population. The successful production of virus using this method can be explained by packaging of all four genome segments into a single virus particle or, alternatively, by co-infection of a single cell with two or more particles.
An interesting feature of the RVFV-SMML-eGFP virus is that the virus is not able to spread among insect cells in contrast to RVFV-eGFP (RVFV-eGFP contains the wildtype M genome segment, the wildtype L genome segment and a S genome segment in which the NSs gene is replaced for the gene encoding eGFP) (FIG. 6). RVFV-SMML-eGFP can thus be considered a non-spreading virus in C6/36 cells. In addition, the RVFV-SMML-eGFP virus grows slower in BSR-T7 cells compared to wild type RVFV-eGFP (FIG. 6). We propose that the RVFV-SMML viruses hold great promise as a vaccine that optimally combines the safety of inactivated vaccines with the efficacy of live vaccines. Compared to the previously developed replicon particles, RVFV-SMML viruses offer the advantage of easy production on a variety of mammalian cell types known to be suitable for RVFV production, as no cell line is required that expresses (NSm)Gn and Gc. To obtain optimal safety, the NSs gene in the S segment of RVFV-SMML viruses is either deleted in its entirety or replaced by the eGFP gene. Finally, it is interesting to note that the NSs gene can also be replaced by a gene of interest from another pathogen, offering possibilities to develop multivalent vaccines or vector vaccines.
Use of the RVFV-SMML-eGFP Virus in a Virus Neutralization Test (VNT)
The expression of eGFP by the RVFV-SMML-eGFP virus allows its use in VNTs. To evaluate whether a VNT based on the RVFV-SMML-eGFP virus is of similar specificity and sensitivity as the conventional VNT using the authentic, virulent RVFV virus or the VNT using replicon particles (Kortekaas et al., 2011. J Virol 85: 12622-12630) a panel of sera obtained from sheep experimentally infected with RVFV was tested. Two days post infection with RVFV-SMML-eGFP, reporter gene expression was used as a readout, while CPE was used as a readout in the conventional VNT. The results show that both tests have similar sensitivity (FIG. 7). After further validation, the RVFV-SMML-eGFP-based VNT could be used as an alternative for the conventional VNT. The major advantage of this novel VNT is that the test can be performed outside biosafety containment facilities. A second advantage is that the results of a RVFV-SMML-eGFP VNT are available after 48 hrs, whereas the conventional VNT, which depends on CPE, takes 5-7 days to completion.
Example 2
Materials and Methods
Cells and Viruses
BHK, BHK-Rep2 (Kortekaas et al., 2011. J Virol 85: 12622-12630), BSR-T7/5 (Buchholz et al., 1999. J Virol 73: 251-259) and C6/36 cells were maintained as described previously (Kortekaas et al., 2011. J Virol 85: 12622-12630). All RVFV variants described in this study contain the RVFV strain 35/74 genetic backbone (Kortekaas et al., 2011. J Virol 85: 12622-12630; Barnard 1979. J South African Vet Assoc 50: 155-157). Viral titers were determined as TCID50/ml using the Spearman-Kärber method.
Plasmids
All plasmids are described in Table 1. To transiently express genes of interest, pCAGGS plasmids were used. RVFV genome segments were transcribed from minimal T7 promoters on pUC57 plasmids. All plasmids were constructed using standard cloning techniques and gene synthesis (GenScript, New Jersey, USA). Plasmids containing half of the glycoprotein precursor (GPC) gene, either (NSm)Gn or Gc, were segmented at the tyrosine (Y)-675 codon of NSmGnGc (FIG. 8A), without any nucleotide overlap. (Y)-675 is predicted to be the first amino acid of the signal sequence of Gc (Gerrard and Nichol, 2007. Virology 357: 124-133; Suzich et al., 1990. J Virol 64: 1549-1555).
Production of RVFV Replicon Particles
BHK-Rep2 cells were seeded in 6 wells plates and after overnight incubation transfected with a total of 3 μg pCAGGS expression plasmid using JetPEI reagents (Polyplus-transfection SA, Illkirch, France) according the manufacturers' instructions. At 1 day post transfection supernatants were harvested and titrated on BHK cells.







TABLE 1







Plasmids used in this study












Plasmid
Type
Encodes
UTRs
Product (nt)
Reference















pCAGGS-M
expression
NSmGnGc


[*]


pCAGGS-NSmGn
expression
NSmGn


this study


pCAGGS-Gn
expression
Gn


this study


pCAGGS-Gc
expression
Gc


this study


puC57-S
transcription
N(+) + NSs(−)
S-type
1691
[*]


puC57-S-delNSs
transcription
N(+)
S-type
922
this study


pUC57-S-eGFP
transcription
N(+) + eGFP(−)
S-type
1621
[*]


puC57-S-NSmGn
transcription
N(+) + NSmGn(−)
S-type
2934
this study


pUC57-S-Gc
transcription
N(+) + Gc(−)
S-type
2484
this study


pUC57-M
transcription
NSmGnGc(+)
M-type
3885
[*]


pUC57-M-NSmGn
transcription
NSmGn(+)
M-type
2319
this study


pUC57-M-Gc
transcription
Gc(+)
M-type
1869
this study


puC57-M-N
transcription
N(+)
M-type
1032
this study


pUC57-L
transcription
polymerase(+)
L-type
6404
[*]





(NSm)Gn and Gc are segmented at the tyrosine (Y)-675 codon of NSmGnGc


(+)genomic sence orientation;


(−)anti-genomic sence orientation


All plasmids contain sequences with RVFV strain 35/74 background


(Accesssion numbers: JF784388.1, JF784387.1 and JF784386.1)


[*] Kortekaas et al., 2011. J Virol 85: 12622-12630






Rescue Experiments

BSR-T7/5 cells were seeded in 6 wells plates (500.000 cells/well) and after overnight incubation, infected for 2 h with a recombinant Fowlpox virus expressing T7 polymerase (FP-T7) (Britton et al., (1996). J Gen Virol 77: 963-967). As an alternative, BSR-T7/5 cells were infected for 2 h with a wildtype Fowlpox virus for rescue of four segmented RVFV. Subsequently, medium was refreshed and cells were transfected with a total of 3 μg pUC57 transcription plasmids per well using JetPEI transfection reagents according to the instructions from the manufacturer. Three to five days post transfection, supernatants were collected and used to infect freshly seeded BSR-T7/5 cells. Viral rescue was visualized using immunofluorescence assays (IFA).
Immunofluorescence
Immunofluorescence assays (IFA) were performed as previously described with some modifications (Oreshkova et al., 2013. PloS one 8(10):e77461). Briefly, infected cell monolayers were fixed with 4% (w/v) paraformaldehyde (15 min) and permeabilized with cold methanol (5 min). Blocking (30 min) and antibody incubations (1 h at 37° C.) were subsequently performed in PBS supplemented with 5% horse serum. To detect Gn expression, monoclonal antibody 4-39-cc was used (Keegan and Collett, 1986. J Virol 58: 263-270) in combination with a Texas Red-conjugated secondary antibody (Abcam, Cambridge, UK). To detect Gc expression, a polyclonal antibody (rabbit) was used (de Boer et al., 2012. J Virol 86: 13642-13652), in combination with an alexa fluor 350-conjugated secondary antibody (Life Technologies, Bleiswijk, The Netherlands). Between antibody incubations cells were washed 3 times with washing buffer (PBS, 0.05% v/v Tween-20). Antibody binding was visualized using an AMG EVOS-FL fluorescence microscope.
Northern Blotting
Northern blotting was performed using the DIG Northern starter kit (Roche, Woerden, The Netherlands) in combination with the Northern-Max-Gly kit (Ambion, Austin, Tex.) as previously described (Kortekaas et al., 2011. J Virol 85: 12622-12630). Primers used for the generation of the RNA probes are listed in Table 2. Viral RNA was isolated using Trizol LS (Sigma-Aldrich, Missouri, United States) in combination with the Direct-zolTM RNA Miniprep kit (Zymo research, California, United States) according the manufactures instructions.










Primers used for Northern blot probes









Primer 
Sequence
Application













JR597
TAATACGACTCACTATAGGGTCAGTGTTTCCTACTTGAAGGAGGCTT
(SEQ ID NO: 30)
Polymerase forward


 


JR598
AAGTCCACACAGGCCCCTTACATT
(SEQ ID NO: 31)
Polymerase reverse


 


JR599
TAATACGACTCACTATAGGGGGTCTGCGAAGTGGGGGTTCAAG
(SEQ ID NO: 32)
Gn forward (1)


 


JR600
GACAACCAATCCGTGAGGCTCA
(SEQ ID NO: 33)
Gn reverse (1)


 


JR601
TAATACGACTCACTATAGGGCGGACAACCAATCCGTGAGGCTCAC
(SEQ ID NO: 34)
Gn forward (2)


 


JR602
CGAAGTGGGGGTTCAAGCACTCAAA
(SEQ ID NO: 35)
Gn reverse (2)


 


JR603
TAATACGACTCACTATAGGGGTCTCAAGTGAGCTATCGTGCAGGG
(SEQ ID NO: 36)
Gc forward (1)


 


JR604
ATTGCATACCCTTTGCCTGGGCT
(SEQ ID NO: 37)
Gc reverse (1)


 


JR605
TAATACGACTCACTATAGGGAGACACGGCTGCTCCCACAAAGTC
(SEQ ID NO: 38)
Gc forward (2)


 


JR606
CAGTCAGTCAGAAAAGAGGCCCTTAG
(SEQ ID NO: 39)
Gc reverse (2)


 


JR607
TAATACGACTCACTATAGGGTCAAGCAGTGGACCGCAATGAGATTG
(SEQ ID NO: 40)
N forward


 


JR608
ATTCACTGCTGCATTCATTGGCTGC
(SEQ ID NO: 41)
N reverse


 


JR609
TAATACGACTCACTATAGGGATTCTATCTCAACATCTGGGATTGGAGGA
(SEQ ID NO: 42)
NSs forward


 


JR610
CACCTCCACCAGCAAAGCCTTTTCA
(SEQ ID NO: 43)
NSs reverse





_T7 polymerase recognition sequence


(1) Resulting probe recognizes genomic-sense RNA (in wild-type RVFV virus)


(2) Resulting probe recognizes antigenomic-sense RNA (in wild-type RVFV virus)






Animal Experiments

Viral Dissemination RVFV-LMMSNSs 

Nine-week-old female BALB/cAnCrl mice (Charles River Laboratories) were divided in two groups of 16 mice and one group of 10 mice, kept in type III filter top cages under BSL-3 conditions, and allowed to acclimatize for 6 days. At day 0 the two groups of 16 mice were inoculated via intraperitoneal route (1 ml) with either a low (10E3 TCID50) or high (5.10E5 TCID50) dose of RVFV-LMMSNSs. As a positive control, the group of 10 mice was infected with a low (10E3 TCID50) dose of authentic RVFV strain 35/74. Mice were observed daily and at day 1, 4, 8 and 11 post infection 4 mice were euthanized form the groups infected with RVFV-LMMSNSs. Viral dissemination in the liver and brain was evaluated by qRT-PCR as described (Kortekaas et al., 2012. Vaccine 30: 3423-3429).
Vaccination-Challenge Experiment
Six-week-old female BALB/cAnCrl mice (Charles River Laboratories) were divided in 4 groups of 10 mice, kept in type III filter top cages under BSL-3 conditions, and allowed to acclimatize for 6 days. At day 0, mice were vaccinated intramuscularly (thigh muscle) with either medium (Mock), NSR-Gn (Oreshkova et al., (2013). PloS one 8(10):e77461), 10E6 TCID50), RVFV-LMMSeGFP 10E6 TCID50 or RVFV-LMMSdelNSs in 50 μl. Mice were observed daily and three weeks post vaccination mice were challenged intraperitoneally with 10E3 TCID50 of RVFV strain 35/74 in 1 ml medium. One day prior challenge, RVFV specific neutralization titers in sera were determined as described (Kortekaas et al., 2011. J Virol 85: 12622-12630) using the 4S virus as antigen. Viral dissemination in the liver and brain was evaluated by qRT-PCR as described (Kortekaas et al., 2012. Vaccine 30: 3423-3429).
Results
Splitting of the RVFV GPC Gene does not Abrogate the Functionality of Gn and Gc
Bunyavirus M segments encode GPCs which are proteolytically cleaved into proteins that function in receptor binding and fusion. To evaluate whether proteolytic processing of the RVFV GPC is a prerequisite for the functionality of Gn and Gc, we constructed expression plasmids encoding either (NSm)Gn or Gc and evaluated their ability to facilitate production of RVFV replicon particles (also referred to as non-spreading RVFV (NSR) (Kortekaas et al., 2011. J Virol 85: 12622-12630). The GPC was split at the tyrosine (Y)-675 codon, because this codon is predicted to be the first amino acid of the signal sequence of Gc (Gerrard and Nichol, 2007. Virology 357: 124-133; Suzich et al., 1990. J Virol 64: 1549-1555) (FIG. 8A). BHK cells, stably maintaining replicating L and SeGFP genome segments
(BHK-Rep2) cells were co-transfected with pCAGGS-(NSm)Gn and pCAGGS-Gc (FIG. 8B). One day post transfection the level of progeny replicon particles was determined in the supernatant. As a positive control, BHK-Rep2 cells were transfected with pCAGGS-M, which encodes wild-type NSmGnGc (Kortekaas et al., 2011. J Virol 85: 12622-12630). Co-transfection of pCAGGS-Gn and pCAGGS-Gc resulted in average NSR particles of 10E4 TCID50/ml, whereas co-transfection of pCAGGS-NSmGn and pCAGGS-Gc resulted in average NSR particle production of 10E6 TCID50/ml, nearly reaching the production level of 10E7 TCID50/ml, generally obtained after transfection of BHK-Rep2 cells with pCAGGS-M (FIG. 8C). These results show that splitting of the GPC gene does not abrogate Gn and Gc functionality.
Rescue of RVFV with a Segmented GPC Gene
After demonstrating that RVFV L and S genome segments can efficiently be packaged into infectious replicon particles using the NSmGn and Gc expression plasmids, we investigated whether a virus expressing NSmGn and Gc from separate genome segments is viable. Transcription plasmids pUC57-L, pUC57-M-Gc and pUC57-S-NSmGn were used for the rescue of virus with NSmGn expressed form the NSs location of the S-segment and transcription plasmids pUC57-L, pUC57-M-NSmGn and pUC57-S-Gc were used for the rescue of virus with Gc expressed from the NSs location. Virus based on the expression of Gc from the S-segment and NSmGn from the M segment could be rescued, as evidenced by IFA and Northern blot (FIG. 9A-C). The virus, from hereafter referred to as RVFV-LMS-split, was able to grow up to 10E6 TCID50/ml in BSR cells (FIG. 9D). The successful rescue of the LMS-split virus indicates that Gn and Gc are fully functional when expressed from separate genome segments.
RVFV is Able to Maintain Two S-Type Genome Segments
The finding that Gn and Gc do not require processing as a GPC protein to produce progeny virus provided new opportunities to study the dynamics of RVFV genome packaging. In a first experiment, we investigated whether RVFV is able to package two S-type genome segments in the absence of an M-type genome segment. Rescue experiments were performed with transcription plasmids pUC57-L, pUC57-S-Gc and pUC57-S-NSmGn. In this situation, both NSmGn and Gc will be expressed from the NSs gene location of an S segment. In several attempts, the presence of infectious double S-segment virus, as evidenced by Northern blot and IFA, could be confirmed (FIG. 9A-C). The virus, from hereon referred to as RVFV-LSS, is able to grow up to titers of 10E5 TCID50/ml in BSR cells (FIG. 9D), which is about 10 times lower than observed with RVFV-LMS-split. The ability to rescue RVFV-LSS indicates that RVFV is able to package more than one S-segment into a single virion.
To further investigate RVFV genome packaging, we evaluated whether viruses could be constructed that comprise four instead of three genome segments (RVFV-4S); one L, one S and two M-type segments. Rescue experiments were performed with transcription plasmids pUC57-L, pUC57-S-eGFP, pUC57-M-NSmGn and pUC57-M-Gc. In this situation, the virus contains an authentic L segment, an S segment that encodes N and eGFP and two M-type segments that encode either NSmGn or Gc. In several attempts, as evidenced by Northern blot and IFA (FIG. 10A-C), the rescue of infectious four-segment RVFV was successful. The RVFV-4S eGFP variant, from hereon referred to as RVFV-LMMSeGFP is able to grow up to 10E7 TCID50/ml in BSR cells (FIG. 10D).
In addition to RVFV-LMMSeGFP, we tried to rescue RVFV-4S viruses with S-segments expressing the N protein and NSs or solely N. Rescue experiments were performed as described for RVFV-LMMSeGFP, but instead of pUC57-S-eGFP, pUC57-S(encoding N and NSs) and pUC57-S-delNSs were used. Both viruses, hereon after referred to as RVFV-LMMSNSs and RVFV-LMMSdelNSs were viable and able to grow in BSR cells up to 10E6 and 10E7 TCID50/ml, respectively (FIG. 10A-D).
RVFV is Able to Maintain 4 Genome Segments of which 3 are of the M-Type
The results so far strongly suggest that RVFV genome packaging is relatively flexible. To further study this flexibility, we tried to rescue a four segment virus with three instead of two M-type genome segments. In this situation NSmGn, Gc and also N are all encoded by genome segments with M-type UTRs. Rescue experiments were performed with transcription plasmids pUC57-L, pUC57-M-NSmGn, pUC57-M-Gc and pUC57-M-N. In several attempts, successful rescue of RVFV-LMMM could be confirmed by IFA and Northern blot analysis (FIG. 10A-D) and the virus was able to grow up to 10E6 TCID50/ml in BSR cells. The ability to rescue RVFV-LMMM virus emphasizes that RVFV genome packaging, at least in mammalian cells, is highly flexible.
Evidence for the Packaging of 4 Genome Segments into a Single Virion
To produce progeny virions, RVFV-4S should deliver all 4 genome segments into a single host cell. Theoretically, this can be achieved by infection with a single virion containing all four segments or, alternatively, by co-infection of complementing replicon particles, lacking at least one of the genome segments. To evaluate which of the two mechanisms is used by the RVFV-4S virus, we infected BSR cells with RVFV-LMMSeGFP and evaluated GFP, Gn and Gc expression 16 h post infection using IFA. RVFVeGFP was used as a reference. As expected, the fast majority (>99%) of RVFVeGFP virions contain at least one L, one M and one S segment as evidenced by the high percentage of infected cells that expressed eGFP, Gn and Gc (FIG. 11). Infrequently, cells were observed that expressed eGFP in the absence of Gn and Gc. Most likely these cells were infected by two segmented replicons lacking the M-segment.
Comparable to RVFVeGFP, almost all (>99%) eGFP-expressing cells showed expression of both Gn and Gc after infection with RVFV-LMMSeGFP. Once again, only a limited number of eGFP positive cells were observed that did not express Gn and Gc (FIG. 10). As expected, there were also some eGFP-positive cells that expressed Gn in the absence of Gc, or Gc in the absence of Gn, indicative for the presence of replicon particles lacking at least one of the four genome segments. These results, together with the observation that RVFV-4S is able to spread at low MOI (data not shown), indicate that RVFV-4S primarily produces progeny by infection with 4 segment virions rather than by infection with complementing replicon particles.
Growth of RVFV with Segmented Glycoprotein Precursor Genes in Insect Cell Culture
In the experiments described thus far, viruses with segmented glycoprotein precursor genes were grown in mammalian cells. Since RVFV is a mosquito-borne pathogen and able to grow efficiently in insect cells, we compared the growth of wildtype and mutant viruses in Aedes albopictus C6/36 insect cell culture. As a positive control, viruses were grown in BSR cells. As expected, authentic RVFV and RVFVeGFP were able to grow efficiently in the C6/36 cells. In sharp contrast, none of the viruses with a segmented glycoprotein precursor gene were able to spread efficiently in C6/36 cell culture (FIG. 12). This result suggests that RVFV GPC processing and/or genome packaging is less flexible in mosquito cells.
RVFV-4S Comprising all RVFV Genes is Innocuous in Mice
Since the growth of viruses with segmented glycoprotein precursor genes is somewhat impaired in mammalian cells and strongly impaired in insect cells, we hypothesized that these viruses might have reduced virulence. To study the effect of GPC gene segmentation on virulence we evaluated whether a four-segmented containing all RVFV genes, including the major virulence factor NSs (RVFV-LMMSNSs), is able to cause disease in a mouse model. Mice were infected with either a low (10E3 TCID50) or a high (5.10E5 TCID50) dose of RVFV-LMMSNSs and, after different time points, mice were sacrificed for the evaluation of virus dissemination to the organs. As a positive control, one group of mice was infected with a low dose of authentic RVFV. All mice infected with authentic RVFV died within four days post infection, whereas none of the mice infected with RVFV-LMMSNSs died or showed clinical symptoms, not even when inoculated with the 500-fold higher dose (FIG. 13A). Evaluation of virus dissemination to the livers and brains confirmed that RVFV-LMMSNSs was unable to spread systemically (FIG. 13B-C). Altogether, these results indicate that RVFV-4S is innocuous in mice.
RVFV-4S Induces a Protective Immune Response in Mice
Since RVFV-4S grows well in cell culture and is innocuous in mice, we considered this virus to be a highly promising vaccine candidate. To investigate whether RVFV-4S is able to induce a protective immune response in mice, we performed a vaccination-challenge experiment. Mice were intramuscularly vaccinated with 10E6 TCID50 of RVFV-LMMSeGFP or RVFV-LMMSdelNSs. As a positive control mice were vaccinated with 10E6 TCID50 NSR-Gn (Oreshkova et al., (2013) PloS one 8(10):e77461). At three weeks post vaccination mice were challenged with a lethal dose of authentic RVFV. Within 4 days post challenge all mock-vaccinated control mice succumbed to the infection (FIG. 14A). In contrast, mice vaccinated with RVFV-LMMSeGFP or RVFV-LMMSdelNSs remained healthy during the entire experiment. Analysis of sera and organs of vaccinated animals demonstrated the presence of a neutralizing antibody response (FIG. 14B) and the absence of systemic spread of challenge virus (FIG. 14C-D). Collectively, these results demonstrate that RVFV-4S can be used as a vaccine that optimally combines the safety of an inactivated vaccine with the efficacy of a live vaccine.
Example 3
Materials and Methods
Preparation Vaccine and Challenge Virus
RVFV-LMMSdelNSs was used as vaccine virus and was rescued and produced on BSR cells as described in Example 2. The virus was diluted in BSR growth medium consisting of CO2 independent medium (CIM, Invitrogen) supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin, hereafter referred to as complete CIM medium. The recombinant RVFV strain 35/74 (RVFV rec35/74) was used as challenge virus (Kortekaas et al., 2011. J Virol 85: 12622-12630). Titers were determined as 50% tissue culture infective dose (TCID50) using the Spearman-Kärber algorithm
Vaccination and Challenge of Lambs
Conventional 9-11 week-old lambs were divided into five groups of seven animals. After one week of acclimatization, lambs of groups 1-4 were vaccinated via either the subcutaneous or intramuscular (right thigh) route with a medium dose (MD, 105.1 TCID50) or high-dose (HD, 106.1 TCID50) of RVFV-LMMSdelNSs. Lambs of group 5 were mock-vaccinated. Three weeks post vaccination, all lambs were challenged via the intravenous route (jugular vein) with 105 TCID50 of RVFV rec35/74. Vaccine and challenge viruses were administered in 1 ml complete CIM medium. Prior to challenge, animals were sedated by intramuscular administration of medetomidine (40 μg/kg medetomidine hydrochloride, Sedator®, Eurovet, The Netherlands). Rectal temperatures were determined daily and serum blood samples were obtained weekly. EDTA blood samples were also obtained weekly. During the first 6 and 11 days post vaccination and challenge, respectively, additional EDTA blood samples were taken daily. At the end of the experiment (three weeks post challenge), or when humane endpoints were reached, animals were euthanized by exsanguination, after being anesthetized with 50 mg/kg sodium pentobarbital (Euthasol®, ASTfarma BV, The Netherlands) applied via the intravenous route. Plasma samples were analyzed for the presence of RVFV RNA with quantitative real-time PCR (qRT-PCR) as described previously (Kortekaas et al., 2012. Vaccine 30: 3423-3429). Virus neutralization titers were determined using a RVFV-LMMSeGFP-based virus neutralization test (VNT) as described in Example 1.
Results
Vaccination with RVFV-4S Protects Lambs from Viremia, Fever and Mortality
To evaluate the potential of RVFV-4S as a vaccine for sheep, we performed a vaccination-challenge experiment with lambs. These lambs were offspring from Texel-Swifter ewes and a Suffolk ram. Thirty-five lambs were divided into five groups of seven animals at day-7. At day 0, lambs of groups 1 and 2 were vaccinated subcutaneously with 105.1 TCID50 or 106.1 TCID50 RVFV-LMMSdelNSs. Lambs of group 3 and 4 were vaccinated intramuscularly with 105.1 TCID50 or 106.1 TCID50 RVFV-LMMSdelNSs. Lambs of group 5 were mock vaccinated and served as a challenge control group. No vaccine virus was detected in plasma samples by qRT-PCR before challenge, indicating that RVFV-LMMSdelNSs is unable to induce viremia and strongly suggesting RVFV-LMMSdelNSs is unable to spread efficiently in vivo. After challenge, mock-vaccinated lambs developed fever (>40.5° C.), starting within 2 days post challenge and lasting on average for four days (FIG. 15B). In the first week post challenge these animals also displayed a high viremia, as evidenced by qRT-PCR (up to ≈1010 RNA copies/ml plasma) (FIG. 16B). Two lambs in this control group succumbed to the RVFV infection 3 days after challenge infection and one lamb died 7 days after challenge infection. No fever was observed in any of the vaccinated lambs and no viral RNA or infectious virus could be detected in the plasma samples of the HD and MD intramuscularly and the HD subcutaneously vaccinated lambs (FIGS. 15A and 16A). Only two sheep vaccinated with the medium-dose and via the subcutaneous route displayed very low levels of systemic viral RNA at 4-7 days post challenge. Altogether these results indicate that sterile protection against RVFV challenge in sheep can be achieved by a single (preferably intramuscular) administration of 105 TCID50 RVFV-4S particles.
Intramuscularly Vaccinated Lambs Display Higher Neutralizing Antibody Responses Compared to Subcutaneously Vaccines Lambs
Using a previously developed highly sensitive VNT test (Example 1) sera were evaluated for the presence of RVFV-specific neutralizing antibodies. As expected, neutralizing antibodies were not detected in any of the sera collected on the day of vaccination or in sera of mock-vaccinated animals collected before challenge (FIG. 17). In contrast, high levels of neutralizing antibodies were detected in sera obtained one, two and three weeks post RVFV-4S vaccination (FIG. 17A). Remarkably, average titers of MD subcutaneously vaccinated animals were significantly lower at one and two weeks post vaccination compared to the MD intramuscular vaccinated group. In addition, only the MD subcutaneously vaccinated animals displayed a significant increase in VNT titer after challenge. Collectively, these results demonstrate that RVFV-4S is able to induce a substantial and effective systemic neutralizing immune response in sheep, especially when applied via the intramuscular route.

## Claims

1. A bunyavirus of the genus Phlebovirus, in which separated Gn and Gc coding regions, or separated NSmGn and Gc coding regions, are functionally present on two separate genome segments.

2. The bunyavirus of claim 1, comprising a total of 4 genome segments.

3. The bunyavirus according to claim 1, comprising
a bunyavirus L genome segment;
a bunyavirus S genome segment or part of a S genome segment comprising at least the N gene and the 3′ and 5′ UTRs; and
a bunyavirus M genome segment from which the Gn or Gc coding region, or the NSmGn or Gc coding region, has been functionally inactivated,
whereby the Gn or Gc coding region, or the NSmGn or Gc coding region, that is functionally inactivated on the M genome segment is functionally present on a second M genome segment.

4. The bunyavirus according to claim 1, wherein a bunyavirus L genome segment and/or a S genome segment comprises a foreign gene.

5. A bunyavirus according to claim 1, wherein the Gn and/or the Gc coding region, or the NSmGn and/or Gc coding region, is from a bunyavirus that differs from the bunyavirus from which the L and S genome segments were obtained.

6. A bunyavirus according to claim 1, further comprising a nucleocapsid (N) coding region from a bunyavirus that differs from the bunyavirus from which the genome segments and Gn and/or Gc coding region, or the NSmGn and/or Gc coding region, were obtained.

7. A bunyavirus according to claim 1, wherein an NSs coding region on a S genome segment is functionally inactivated.

8. A method for producing a bunyavirus, the method comprising
A) providing a eukaryotic cell with growth medium; and
B) infecting the eukaryotic cell with the bunyavirus according to claim 1.

9. A composition comprising a bunyavirus according to claim 1, and a suitable excipient.

10. The composition according to claim 9, which is an immunogenic composition.

11. The bunyavirus according to claim 1, for use as a medicament.

12. A vaccine comprising the bunyavirus according to claim 1.

13. A method for generating a bunyavirus of the genus Phlebovirus, the method comprising:
A) providing a cell with a bunyavirus of the genus Phlebovirus comprising at least one genome segment functionally encoding a RdRp gene and an N gene, and
B) providing the cell with at least two genome segments selected from L, M, and S genome segments in which separated Gn and Gc coding regions, or separated NSmGn and Gc coding regions, are functionally present on two separate genome segments.

14. The method according to claim 13, whereby the cell is provided with a genomic segment by providing the cell with a vector that comprises cDNA of said genomic segment which is flanked by a T7 promoter and cDNA of a ribozyme, further comprising providing the cell with a T7 polymerase.

15. The bunyavirus of claim 1, comprising a total of 3 genome segments.

16. The method according to claim 13, wherein the separated Gn and Gc coding regions are present on two separate minigenome segments, or on a genome segment and a minigenome segment.

17. The composition according to claim 9, for use as a medicament.

18. A vaccine comprising the composition according to claim 9.