Vectors expressing attenuated RNA virus of the family arteriviridae and uses thereof

Abstract

47 ABSTRACT VECTORS EXPRESSING ATTENUATED RNA VIRUS OF THE FAMILY ARTERIVIRIDAE AND USES THEREOF Provided herein is a vector for use as a medicament, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein 5 the RNA virus is a virus of the family Arteriviridae. Also provided herein is a pharmaceutical composition comprising such vector and a method for preparing a vaccine against an RNA virus of the family Arteriviridae.

Description

[0001] VECTORS EXPRESSING ATTENUATED RNA VIRUS OF THE FAMILY ARTERIVIRIDAE AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods for treating and / or preventing an infection with a virus of the family Arteriviridae, preferably porcine reproductive and respiratory syndrome virus (PRRSV), and products for use therefor.

[0004] BACKGROUND OF THE INVENTION

[0005] Forty percent of the world's meat consumption is pork, with Europe being the second largest pork producer in the world with approximately 150 million pigs. However, as industrial systems are characterized by large numbers of animals being raised, predominantly in confinement, with rapid population turnover at a single site, the meat industry is vulnerable to viral disease outbreaks, which can lead to huge financial losses, threats to food security and animal suffering.

[0006] Porcine reproductive and respiratory syndrome (PRRS) is considered to be one of the most costly diseases affecting intensive pig production worldwide and is characterized by clinical signs varying from reproductive failure to respiratory disease. The causative agent is the PRRS virus (PRRSV), an enveloped, positive-sense, single-stranded RNA virus in the family Arteriviridae (order Nidovirales). Two different species of PRRSV have been described: Betaarterivirus suid 1 (PRRSV-1) and Betaarterivirus suid 2 (PRRSV- 2). PRRSV preferably infects subsets of differentiated macrophages in lung, placenta and lymphoid tissue. Control of PRRS is hard to manage and involves a combination of measures including diagnosis, monitoring, herd management, biosecurity, and sow and / or piglet vaccination for prevention and control of the disease. Unfortunately, despite the availability of different techniques, the effectiveness of PRRSV vaccination has proven to be unpredictable and suboptimal, with PRRS outbreaks occurring despite routine vaccination being practiced. For example, Eck et al., Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs, Vet Res, 2016, 47:33 describes recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) expressing PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N. However, while the VSV replicon vector can induce immune responses to heterologous proteins in pigs, the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic and fail to protect pigs against PRRSV infection. Thus, there is a need for the development of effective means for preventing PRRSV infection. European patent application EP2369001B1 concerns infectious clones of PRRSV VR-2332. EP2369001B1 describes the transfection of MARC-145 cells with VR-2332 purified viral RNA and the inoculation of pigs or young swine with cloned virus.

[0007] SUMMARY OF THE INVENTION

[0008] Present inventors are to their knowledge the first to demonstrate the in vivo efficacy of a vector comprising a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae, and more preferably a Porcine reproductive and respiratory syndrome virus (PRRSV), in the treatment and / or prevention of an infection with said RNA virus. More particularly, present inventors have demonstrated that vaccination of pigs with a vector according to the present invention comprising a viral expression cassette comprising a cDNA of an attenuated PRRSV genome operably linked to a promoter leads to the development of anti- PRRSV antibodies and the production of IFN-gamma by peripheral blood mononuclear cells (PBMCs) and isolated splenocytes. Furthermore, vaccination with such vector allows to protect against porcine reproductive and respiratory syndrome (PRRS) disease, as supported by the absence of PRRS-associated clinical symptoms as well as a significant decrease in PRRSV genome copies / ml of serum post challenge, when compared to challenge controls. The PRRS clinical symptoms and viremia observed after vaccination with the vector according to the present invention comprising the cDNA of an attenuated PRRSV genome were also lower than those observed after vaccination with a known anti-PRRSV vaccine, Progressis®. Moreover, unlike the known PRRSV vaccine Progressis®, the vector of the present invention comprising a cDNA of an attenuated PRRSV genome did not lead to an increase in the pigs' rectal temperature post challenge with a non-homologous PRRSV strain.

[0009] Accordingly, a first aspect provides a vector for use as a medicament, preferably wherein the medicament is a vaccine, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae.

[0010] In particular embodiments, the virus of the family Arteriviridae is a porcine reproductive and respiratory syndrome virus (PRRSV).

[0011] In particular embodiments, said vector is a bacterial artificial chromosome (BAC).

[0012] In particular embodiments, said vector further comprises an inducible bacterial origin of replication (ori) for amplifying said vector to a high copy number (i.e. at least 5 copies, such as at least 10 copies) per bacterial host cell in the presence of one or more inducers of said ori. A further aspect provides a vector for use in the treatment and / or prevention of a PRRSV infection in a subject, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated PRRSV virus genome operably linked to a promoter.

[0013] In particular embodiments, the PRRSV is a type I PRRSV or a type II PRRSV.

[0014] In particular embodiments of the invention, the PRRSV is a Lelystad strain, a P129 strain, a VR-2332 strain, or a derivative thereof.

[0015] In particular embodiments of the invention, said viral expression cassette comprises: a cDNA of an attenuated RNA virus genome, wherein the RNA virus is a virus of the family Arteriviridae, and a RNA polymerase driven promoter at the 5' end of said cDNA, preferably an RNA polymerase II promoter. In particular embodiments of the embodiments described above, said vector is administered to a subject and wherein said subject is member of the family Suidae, preferably a member of the genus Sus, more preferably a wild or domestic pig or a swine.

[0016] Irrespective of the above, in particular embodiments, said vector is administered intradermally, intramuscular, transdermally, intranasally, orally or subcutaneous to a subject.

[0017] In particular embodiments, the vector as described above is administered to a subject in a dose of from 100 to 2000 pg, preferably from 300 to 900 pg, of said vector.

[0018] In particular embodiments, the vector as described above is administered to a subject in a single-dose or double-dose regime.

[0019] A further aspect provides a pharmaceutical composition comprising a vector and a pharmaceutically acceptable carrier, wherein said vector comprises ; a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae, and preferably wherein said pharmaceutical composition is a vaccine.

[0020] A further aspect provides a method of preparing a vaccine against an RNA virus of the family Arteriviridae comprising the steps of: a) providing a host cell transfected with a vector as described herein, b) amplifying the vector, c) isolating the amplified vector, and d) formulating the isolated vector into a vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0021] Fig. 1. Percentage sequence identity between different PRRSV strains of type I and type II. Genbank sequences: Porcilis (formerly annotated under Genbank accession number KF991509; SEQ ID NO: 1 (Fig. 8)), Unistrain (annotated under Genbank accession number GU067771.1), ReproCyc (annotated under Genbank accession number KT988004.1), Suvaxyn (annotated under Genbank accession number MK876228.1), Ingelvac (annotated under Genbank accession number AF066183.4), VR2332 (annotated under Genbank accession number U87392.3), P129 (annotated under Genbank accession number AF494042.1), Fostera (annotated under Genbank accession number MK820650.1), PrimePac (annotated under Genbank accession number DQ779791.1) and Prevacent (annotated under Genbank accession number KU131568.1). Porcilis #13 is derived from the Porcilis® PRRS (MSD) (SEQ ID NO: 2), Ingelvac #7 (SEQ ID NO: 3) and #34 (SEQ ID NO: 4) are derived from Ingelvac PRRS (Boehringer Ingelheim).

[0022] Fig. 2. Schematic representation of an exemplary approach to clone the Porcilis PRRSV genome into the inducible BAC plasmid PLLAV-pVV6. The RNA extracted from the Porcilis PRRS vaccine was used as a template for 4 PCR reactions giving rise to fragments overlapping internally and with the pVV6 plasmid. Image generated with Biorender. F= fragment

[0023] Fig. 3. Schematic study design of the PLLAV-Porcilis vaccine dose-escalation and challenge study in pigs, i.m. = intramuscular; t.d. = transdermal

[0024] Fig. 4. The number of animals (expressed as percentage of 8 pigs) having detectable anti-PRRSV antibodies as measured by commercial ELISA at different timepoints post vaccination, i.m. = intramuscular; t.d. = transdermal

[0025] Fig. 5. The number of animals (expressed as percentage of 5 pigs) having detectable anti-PRRSV antibodies as measured by commercial ELISA at different timepoints post vaccination, i.m. = intramuscular; t.d. = transdermal

[0026] Fig. 6. The number of animals (expressed as percentage of 13 or 8 pigs depending on the respective group) having detectable anti-PRRSV antibodies as measured by commercial ELISA at different timepoints post vaccination. For the PROGRESSIS® and the PLLAV-Porcilis (300 pg dose level administered transdermally) groups the results of two studies were combined increasing the number of pigs to 13. i.m. = intramuscular; t.d. = transdermal

[0027] Fig. 7. Group mean PRRSV genome copies per mL serum (as measured by RT-qPCR) at different timepoints post challenge with PRRSV-1 strain Flandersl3. UUC stands for the group of pigs mock vaccinated and mock challenged; IUC is the group of pigs mock vaccinated but challenged; PROGRESSIS is the group of pigs vaccinated according to the prime-boost vaccination scheme with the Progressis® vaccine and challenged. PLLAV-Porcilis 300, PLLAV-Porcilis 600 and PLLAV-Porcilis 900 stands for the groups of pigs vaccinated with the PLLAV-Porcilis vaccine at dose levels of 300 pg DNA, 600 pg DNA and 900 pg DNA, respectively. All groups were (mock) vaccinated with their respective vaccine at the respective dose level on study day 0 and study day 21, and were (mock) challenged on study day 41 with 105TCID50 of PRRSV- 1 strain Flandersl3.

[0028] Fig. 8. Nucleotide sequences for PRRSV with Genbank entry no KF991509.1, PRRSV Porcilis #13, PRRSV Ingelvac #7, PRRSV Ingelvac #34, PRRSV P129 #3 and PRRSV Fostera #11 as referred to in the present application.

[0029] Fig. 9. Schematic representation of an exemplary approach to clone the P129 / Fostera PRRSV genome into the inducible BAC plasmid PLLAV-pVV6. Using a stepwise approach with 3 assembly steps, 6 synthetic DNA fragments covering the entire PRRSV genome was introduced in the PLLAV vector pVV6. Image generated with Biorender.

[0030] Fig. 10. Schematic study design of the PLLAV-Fostera immunogenicity study in pigs. i.m. = intramuscular; t.d. = transdermal

[0031] Fig. 11. The number of animals (expressed as percentage of 5 pigs) having detectable anti-PRRSV antibodies as measured by commercial ELISA at different timepoints post vaccination.

[0032] Fig. 12. The number of animals (expressed as percentage of 5 pigs) having detectable PRRSV type II targeted cellular immune response at different timepoints post vaccination as measured by a commercial enzyme-linked immune absorbent spot (ELISpot) assay (Pig IFN-y Single-Color ELISPOT, ImmunoSpot) performed on peripheral blood mononuclear cells (PBMCs) isolated from whole blood samples.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] The term "about", when used in relation to a numerical value, has the meaning generally understood in the relevant art. In certain embodiments the term "about" may be left out or may be interpreted to mean the numerical value +10%; or +5%; or +2%; or +1%.

[0035] Whenever used herein in relation to a percentage, w / w means weight / weight and w / v means weight / volume.

[0036] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0037] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of" when referring to recited members, elements or method steps also include embodiments which "consist of" said recited members, elements or method steps.

[0038] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements or steps and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

[0039] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment envisaged herein. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0040] Any references cited herein are hereby incorporated by reference.

[0041] Present inventors are to their knowledge the first to demonstrate that plasmid-based live-attenuated virus vaccine technology and more particularly Plasmid Launched Live Attenuated Virus (PLLAV) vaccine technology, could successfully be applied to RNA viruses of the family Arteriviridae, which belong to the order of Nidovirales and have a replication cycle that is significantly different from other, for example, viruses of the family Flaviviridae, which belong to the order of Amarillovirales. While the use of PLLAV vaccine technology is an attractive technology, there was no reasonable expectation of success given the higher complexity of the genome of the Arteriviridae, such as PRRSV.

[0042] A preferred RNA virus of the family of Arteri viridae is the PRRSV. More particularly, present inventors have demonstrated that vaccination of pigs with a vector according to the present invention comprising a viral expression cassette comprising a cDNA of an attenuated PRRSV genome operably linked to a promoter leads to a sustained and increasing anti-PRRSV antibody response and allows to protect against porcine reproductive and respiratory syndrome (PRRS) disease, as supported by the absence of PRRS-associated clinical symptoms and a significant decrease in PRRSV genome copies / ml of serum post challenge, when compared to challenge controls. Accordingly, in contrast to the prior art, such as European patent application EP2369001B1, the present application concerns the in vivo use of a vector comprising cDNA of an attenuated PRRSV genome for treatment and / or prevention of disease / infection.

[0043] In addition, the PRRS clinical symptoms and viremia observed post challenge in the groups vaccination with a vector according to the present invention comprising a cDNA of an attenuated PRRSV genome were also lower than those observed after vaccination with a known anti-PRRSV vaccine, Progressis®. Moreover, unlike the known PRRSV vaccine Progressis®, the vector of the present invention comprising a cDNA of an attenuated PRRSV genome did not lead to an increase in the pigs' rectal temperature post challenge.

[0044] Accordingly, the present invention provides a vector for use in therapy, more particularly for use as a medicament, preferably wherein the medicament is a vaccine, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteri viridae.

[0045] A further aspect provides a vector for use in the treatment and / or prevention of an infection with an RNA virus in a subject, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae, preferably PRRSV.

[0046] In particular embodiments, said vector is a BAC, preferably a BAC comprising an inducible bacterial origin of replication (ori) for amplifying said BAC to a high copy number (i.e. at least 5 copies, such as at least 10 copies) per bacterial host cell in the presence of one or more inducers of said ori, and a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteri viridae.

[0047] The present invention also provides a method of treating and / or preventing an infection with an RNA virus of the family Arteriviridae in a subject comprising administering to said subject a therapeutically and / or prophylactically effective amount of a vector comprising a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter. Furthermore, the present invention provides the use of a vector comprising a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, in the preparation of a medicament, preferably a medicament for the treatment and / or prevention of an infection with an RNA virus of the family Arteriviridae in a subject.

[0048] Also provided herein is a vector for use in reducing viraemia of an RNA virus of the family Arteriviridae, preferably PRRS viraemia, in a subject, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae. In other words, also provided herein is a method for reducing viraemia of an RNA virus of the family Arteriviridae, preferably PRRS viraemia, in a subject comprising administering to said subject a therapeutically and / or prophylactically effective amount of a vector comprising a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae. Also provided herein is a vector for use in inducing specific immune responses (such as humoral and cell-mediated immune responses) in a subject, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae. In other words, also provided herein is a method for inducing specific immune responses (such as humoral and cell-mediated immune responses) in a subject comprising administering to said subject a therapeutically and / or prophylactically effective amount of a vector comprising a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae.

[0049] Also provided herein is a vector for use in protecting a subject from PRRS clinical disease and / or from PRRSV-associated fever in a subject, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is PRRSV. In other words, also provided herein is a method for protecting a subject from PRRS clinical disease and / or from PRRSV-associated fever in a subject comprising administering to said subject a therapeutically and / or prophylactically effective amount of a vector comprising a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is PRRSV.

[0050] The family of Arteriviridae belongs to the order of Nidovirales. Nidoviruses are enveloped viruses having genomes consisting of a single strand of positive polarity RNA. The genomic RNA of a positive-stranded RNA virus fulfills the dual role in both storage and expression of genetic information. No DNA is involved in replication or transcription in Nidoviruses. The non-structural proteins are translated directly from the genomic RNA of nidoviruses as large polyproteins and subsequently cleaved by viral proteases into discrete functional proteins. A 3'- coterminal nested set of subgenomic RNAs (sgRNAs) is synthesized from the genome and are used as messenger RNAs for translation of the structural proteins. The reproduction of nidoviral genomic RNA is thus a combined process of genome replication and sgRNA synthesis.

[0051] Non-limiting examples of viruses belonging to the family Arteriviridae include PRRSV, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), wobbly possum disease virus and lactate dehydrogenase elevating virus (LDV). In particular embodiments, the virus of the family Arteri viridae is a PRRSV.

[0052] The term "porcine reproductive and respiratory syndrome" or "PRRS" as used herein refers to a viral disease caused by an infection with an PRRS virus (PRRSV). The PRRSV is a small, single-stranded, positivesense, enveloped RNA virus. It comprises a 15 kilobase (kb) genome consisting of one linear, single stranded RNA molecule consisting of a 5' untranslated region (UTR), 10 open reading frames, and a 3' UTR followed by a polyadenylation tail. ORFs include: the replicase complex (0RF1, comprised of ORFla and ORFlb) and ORFs that code for structural proteins consisting of nucleocapsid (N (ORF 7)), major structural proteins (GP5 (ORF 5); M (ORF 6)), minor structural proteins (GP2a (ORF 2a); E (ORF 2b); GP3 ( 0RF3); GP4 (ORF 4)), and other minor protein ORF5a. The PRRSV genome and replication cycle is as follows: 0RF1 codes the PRRSV non-structural replicase and transcription complex (RTC). The genomic replication cycle starts with the translation of polyproteins, PPla and PPlab. These polyproteins are cleaved into the 14 nonstructural proteins (nsps), which form the RTC. The RTC transcribes the RNA minus strand and subgenomic mRNA (sgRNA). Then, RTC switches to a discontinuous transcription, which allows the leader TRS (transcription regulatory sequence) to interact with the body TRS for subgenomic RNA synthesis. This may favor recombination and variation in the structural proteins that are translated from the sgRNA. These structural proteins are subsequently assembled into the new viral particle.

[0053] A large number of different PRRSV strains exist. PRRSV strains are classified in two different genotypes, based on genomic heterogeneity: type 1 (i.e. type I), which comprises commonly known European type strains, and type 2 (i.e. type II), which includes commonly known North American type strains. Although genetic similarity between type 1 and type 2 strains ranges from about 55% to about 63% for nonstructural proteins and from about 61% to about 81% for structural proteins, both genotypes are considered as the same virus and cause the same disease. However, as a consequence of their high genomic variability, type 1 and type 2 strains are biologically distinct. Interestingly, genetic similarity between type 1 strains may also range from about 85% (or even lower, such as from about 70%, from about 75% or from about 80%) to up to about 100% and between type 2 strains from about 82% (or even lower, such as from about 70%, from about 75% or from about 80%) to up to about 100% (as shown for exemplary PRRSV strains in Fig. 1). In particular embodiments, the PRRSV is a type I PRRSV or a type II PRRSV. Non-limiting examples of type

[0054] I PRRSV include Lelystad virus (for example, with a sequence as annotated under NCBI Genbank accession number M96262), SD03-15, SD02-11, SD01-07 and SD01-08, or any derivative thereof. Non-limiting examples of type II PRRSV include VR2332 (for example, with a sequence as annotated under NCBI Genbank accession number EF536003.1), SD 95-10, SD11-21, SD 95-47, ND 99-14, SD 04-89, P129, SDSU73, MN184, NADC30, and NADC31, or any derivative thereof.

[0055] In particular embodiments, the PRRSV is a Lelystad strain, a P129 strain, a VR-2332 strain, or a derivative thereof.

[0056] In particular embodiments, the PRRSV is a commercial vaccine strain, such as a type I PRRSV strain as used in Porcilis® PRRS (for example, with a sequence as annotated under NCBI Genbank accession number MT311646.1 or MW674755.1), Unistrain® PRRS (for example, with a sequence as annotated under NCBI Genbank accession number GU067771.1), ReproCyc® PRRS EU (for example, with a sequence as annotated under NCBI Genbank accession number KT988004.1), Suvaxyn® PRRS MLV (for example with a sequence as annotated under CBI Genbank accession number MK876228.1) , or such as a type II PRRSV strain as used in Fostera® PRRS (for example, with a sequence as as annotated under NCBI Genbank accession number MK820650.1), Fostera® PRRS modified live virus (MLV), Prime Pac® PRRS RR (for example, with a sequence as annotated under NCBI Genbank accession number DQ779791.1), Prevacent® PRRS (for example, SDll-21_P100 with a sequence as annotated under NCBI Genbank accession number KU131568.1), Ingelvac PRRSFLEX® EU (for example, with a sequence as annotated under NCBI Genbank accession number KT988004.1), Ingelvac PRRS® MLV (for example, with a sequence as annotated under NCBI Genbank accession number AF066183.4), or a derivative thereof. Preferably, the PRRSV is a commercial vaccine strain, such as a type I PRRSV strain as used in Porcilis® PRRS (for example, with a sequence as annotated under NCBI Genbank accession number MT311646.1 or MW674755.1), or a type

[0057] II PRRSV strain such as the strain used in Fostera® PRRS (for example, with a sequence as as annotated under NCBI Genbank accession number MK820650.1) or a P129 strain, or a derivative thereof.

[0058] In particular embodiments, the genome of the PRRSV comprises, consists essentially of or consists of a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, preferably at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, preferably 100%, sequence identity with one or more sequence of a type I PRRSV strain as annotated under Genbank accession number MT311646.1, Genbank accession number MW674755.1, Genbank accession number GU067771.1, Genbank accession number KT988004.1, Genbank accession number MK876228.1, or a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, preferably 100%, sequence identity with one or more sequence of a type II PRRSV strain as annotated under Genbank accession number AF066183.4, Genbank accession number U87392.3, Genbank accession number AF494042.1, Genbank accession number MK820650.1, Genbank accession number DQ779791.1 or Genbank accession number KU131568.1.

[0059] In particular embodiments, the genome of the PRRSV comprises, consists essentially of or consists of a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, preferably 100%, sequence identity with the sequence as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, preferably SEQ ID NO: 2.

[0060] In particular embodiments, the genome of the PRRSV differs by at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5, such as at most 4, at most 3, at most 2 or at most 1, nucleotides from the sequence as defined by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, preferably SEQ ID NO: 2.

[0061] A person skilled in the art is well aware of methods and tools to verify sequence homology, sequence similarity or sequence identity between different sequences of amino acids or nucleic acids. Non-limiting examples of such methods and tools are Protein BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), ClustalW2 (https: / / www.ebi.ac.uk / Tools / msa / clustalw2 / ), SIM alignment tool (https: / / web.expasy.org / sim / ), TranslatorX (http: / / translatorx.co.uk / ) and T-COFFEE (https: / / www.ebi.ac.uk / Tools / msa / tcoffee / ). The percentage of identity between two sequences may show minor differences depending on the algorithm choice and parameters.

[0062] The term "sequence identity" as used herein refers to the relationship between sequences at the nucleotide or amino acid level. The expression "% identical" is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions or deletions as compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not comprise insertions or deletions. A reference window is chosen and the "% identity" is then calculated by determining the number of nucleotides (or amino acids) that are identical between the sequences in the window, dividing the number of identical nucleotides (or amino acids) by the number of nucleotides (or amino acids) in the window and multiplying by 100. Unless indicated otherwise, the sequence identity is calculated over the whole length of the reference sequence. A skilled person is aware of the related, yet different interpretations in the art of the terms "similarity", "homology", and "identity" (explain in detail in e.g. Pearson, Current protocols in bioinformatics, 2014). In particular embodiments, the vector is capable of producing infectious PRRSV, such as in MARC-145 cells. In further particular embodiment, the vector is capable of infecting MARC-145 cells in vitro and causing at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of cytopathic effect (CPE) in said cells, preferably when measured from 1 day to 7 days post infection, such as 1 day post infection, 2 days post infection, 3 days post infection, 4 days post infection, 5 days post infection, 6 days post infection or 7 days post infection. CPE may be determined by any method known in the art, such as using microscopy.

[0063] The term "preventing" or "prevention" as used herein refers to a reduction in risk of acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). In the present case, the term prevention also encompasses reducing or preventing transmission of the causative agent and / or reducing or preventing infection with the causative agent, thereby actively preventing infection and / or disease development and progression.

[0064] The term "treating" or "treatment1of any disease or disorder includes, in one embodiment, to improve the disease or disorder (i.e., arresting or reducing the development of the disease or at least reducing one of the clinical symptoms of the disease). In another embodiment "treating" or "treatment" refers to improve at least one physical parameter, which may or may not be discernible by the subject, but which is based on or associated with the disease or disorder to be treated. In yet another embodiment, "treating" or "treatment" refers to modulating or alleviating the disease or disorder, either physically (e. g. stabilization of a discernible on non-discernible symptom), physiologically (e. g. stabilization of a physiological parameter), or both. In yet another embodiment, "treating" or "treatment" refers to delaying the onset or progression of the disease or disorder. Accordingly, "treating" or "treatment' includes any causal treatment of the underlying disease or disorder (i.e., disease modification), as well as any treatment of signs and symptoms of the disease or disorder (whether with or without disease modification), as well as any alleviation or amelioration of the disease or disorder, or its signs and symptoms. The terms "disease(s)" and "disorders)" are used largely interchangeably herein.

[0065] The term "effective amount" as used herein may refer to a prophylactically effective amount, which is an amount of an active compound or pharmaceutical agent, more particularly a prophylactic agent, that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician, or may refer to a therapeutically effective amount, which is an amount of active compound or pharmaceutical agent, more particularly a therapeutic agent, that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the signs and symptoms of the disease or condition being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses for the agents as taught herein. The effective amount can vary depending on the compound, the disease and its severity, and the condition, age, weight, gender etc. of the subject to be treated. More particularly, "therapeutically effective amount" or "therapeutically effective dose" indicates an amount of vector (e.g. BAC vector) or composition as described herein that when administered brings about a clinical positive response with respect to treatment of a subject afflicted by an infectious disease. Similarly, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of vector (e.g. BAC vector) or composition as described herein that inhibits or delays the onset of clinical manifestation of an infectious disease as being sought by a researcher, veterinarian, medical doctor or other clinician. A skilled person is aware that the terms "quantity", "amount" and "level", such as when used in the context of a dose, are synonyms and have a well-defined meaning in the art and appreciates that these may particularly refer to an absolute quantification of a vector or composition as described herein which is considered an effective amount for the applications described herein, or to a relative quantification of a vector or composition as described herein, such as for example a concentration of a vector or composition as described herein in function of the subject's bodyweight. Suitable values or ranges of values may be obtained from one single subject or from a group of subjects (i.e. at least two subjects). Except when noted, the terms "animal", "subject" or "patient" can be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably a member of the family Suidae, still more preferably a member of the genus Sus. Preferred subjects are wild or domestic pigs or swines. More preferred subjects are domestic pigs (Sus domesticus). Non-limiting examples of members of the family Suidae include Golden babirusa, Sulawesi babirusa, Togian babirusa, Giant forest hog, Desert warthog, common warthog, pygmy hog, bushpig, red river hog, Palawan bearded pig, bearded pig, Vietnamese warty pig, Visayan warty pig, Celebes warty pig, Flores warty pig, Mindoro warty pig, Philippine warty, Javan warty pig, wild boar and domestic pig.

[0066] In particular embodiments, the animal is a member of the family Suidae, a member of the genus Sus, preferably a domestic pig, wild pig, domestic swine or wild swine (i.e. wild boar).

[0067] The vector as described herein comprises a a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter. In particular embodiments, the vector is a mammalian expression vector. For example, the vector may be a vector comprising an inducible origin of replication such as described in published International patent application WO 2021 / 058722 Al on p. 20 to 27.

[0068] The term "vector" as used herein refers to a circular, double-stranded DNA molecule, to which nucleic acid fragments, preferably the recombinant nucleic acid molecule as defined herein, may be inserted and cloned, i.e., propagated. Hence, a vector will typically contain one or more unique restriction sites, and may be capable of autonomous replication in a defined cell or vehicle organism such that the cloned sequence is reproducible. In particular embodiments, the vector is a vector capable of replicating in bacterial cells. A vector may also contain a selection marker, such as, e.g., an antibiotic resistance gene, to allow selection of recipient cells that contain the vector. Vectors may include, without limitation, plasmids, cosmids, phagemids, bacteriophage-derived vectors, PAC, BAC, etc., as appropriate. The plasmids may be plasmids intended for viral vector construction such as inter alia retroviral vectors, lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors. Expression vectors are generally configured to allow for and / or effect the expression of nucleic acids or open reading frames introduced thereto in a desired expression system, e.g., in vitro, in a cell, organ and / or organism. For example, expression vectors may advantageously comprise suitable regulatory sequences. Throughout the present description, the term "vector" may refer to an empty vector or to the vector comprising the cDNA of the attenuated RNA virus genome as taught hereon, unless otherwise specified.

[0069] In particular embodiments, when reference is made to an empty vector (e.g. not comprising the viral expression cassette as taught herein), the vector may have a size of less than 10 kb, less than 9 kb, less than 8 kb, less than 7 kb, less than 6 kb, preferably less than 6 kb.

[0070] In particular embodiments, when reference is made to the vector comprising the cDNA of the attenuated RNA virus genome, the vector is a large vector, such as a vector with a size of at least 15.5 kb, at least 16 kb, at least 16.5 kb, at least 17 kb, at least 17.5 kb, at least 18 kb, at least 18.5 kb, at least 19 kb, at least 19.5 kb, at least 20 kb, at least 21 kb, or at least 22 kb, preferably at least 15.5 kb. "Kb", "kbp" or "kilo base pairs" as used herein refers to 1000 base pairs (bp) of DNA.

[0071] In particular embodiments, the vector is a bacterial artificial chromosome (BAC).

[0072] The term "bacterial artificial chromosome" or "BAC" refers to a plasmid DNA construct, based on the fertility or F-plasmid, used to clone large DNA sequences in bacterial cells, such as E. coli. BAC allows the insertion of a DNA sequence with a length of up to 300 000 base pairs. The BAC, with the inserted DNA, can be taken up by bacterial cells. The replication of a BAC is initiated at an origin of replication (ori) sequence, typically the oriS sequence. This replication is stringently regulated by gene products, generally the repE and / or repF, encoded by the BAC. The BAC further encodes for proteins, such as parA, B and protein binding sites like parC, directing the partitioning of the BAC copies to the daughter cells during division. BAC vectors may further comprise selectable markers, such as antibiotic resistance or an antibiotic-free selection marker, such as the complementation of an essential gene. Non-limiting examples of BACs include the pBeloBacI I (Shizuya et al. (1992) Proc. Natl. Acad. Sci. USA 89, 8794-8797), with GenBank Accession Number U51113. 1.

[0073] The term "attenuation" or "attenuated" as used herein refers to an alteration in the virulence of a diseasecausing parent virus by which the harmful nature of said virus is weakened (or attenuated). Attenuated vaccines can be derived in several ways from living, preferablyreplicating, viruses that have been weakened, such as by cultivation under sub-optimal conditions (also called attenuation), or from genetic modification, which has the effect of reducing their ability to cause disease. The term "parent virus" is used herein to refer to viral genomes from which new sequences, which may be more or less attenuated, are derived. Parent viruses are usually "wild type" or "naturally occurring" isolates for which it is desired to obtain a more highly attenuated virus. However, parent viruses also include mutants specifically created or selected in vitro on the basis of real or perceived desirable properties. Accordingly, parent viruses that are candidates for attenuation include mutants of wild type or naturally occurring viruses that have deletions, insertions, amino acid substitutions and the like, and also include mutants which have codon substitutions. In one embodiment, such a parent sequence differs from a natural isolate or strain by 50 amino acids or less, 40 amino acids or less, 35 amino acids or less, 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 10 amino acids or less or 5 amino acids or less. The subject being administered the vector as disclosed herein is envisaged to develop an immunological response to the strain used in the vector as well as to a PRRSV strain that is homologous or even heterologous (i.e. non-homologous) to the PRRSV strain used in the vector as disclosed herein. In accordance therewith, the RNA virus of the family Arteriviridae of which the attenuated genome is incorporated into the vector as disclosed herein, may be from the same or from a different genotype as the RNA virus of the family Arteriviridae of which infection is to be treated and / or prevented in a subject. In particular preferred embodiments, the RNA virus of the family Arteriviridae of which infection is to be treated and / or prevented in a subject, has the same genotype as the RNA virus of the family Arteriviridae of which the attenuated genome is incorporated into the vector. For example, if the RNA virus of the family Arter / v / r / doe of which infection is to be treated and / or prevented in a subject is a PRRSV type I virus, the RNA virus of the family Arteriviridae of which the attenuated genome is incorporated into the vector is also a PRRSV type I virus. For example, if the RNA virus of the family Arteriviridae of which infection is to be treated and / or prevented in a subject is a PRRSV type 11 virus, the RNA virus of the family Arteriviridae of which the attenuated genome is incorporated into the vector is also a PRRSV type II virus.

[0074] In particular embodiments, the vector as described herein allows to treat pan-genotype PRRSV.

[0075] The term "expression cassette" as used herein refers to nucleic acid molecules, typically DNA, to which nucleic acid fragments, preferably the cDNA of an RNA virus genome, may be inserted to be expressed, wherein said nucleic acid molecules comprise one or more nucleic acid sequences controlling the expression of the nucleic acid fragments. Non-limiting examples of such more nucleic acid sequences controlling the expression of the nucleic acid fragments include promoter sequences and transcription terminators. Preferably, the nucleic acid sequences controlling the expression of the nucleic acid fragments promote the transcription of the viral cDNA upon introduction of the vector as described herein in a mammalian cell and allow for processing of the transcribed RNA into infectious viral RNA. The expression "infectious viral RNA" as used herein refers to viral RNA which, upon introduction in its mammalian host, is sufficient to provide all viral functions required for viral replication and production of infectious viral progeny. This includes (i) serving as a transcriptional template for viral RNA synthesis and genome amplification, and (ii) serving as translational template for synthesis of viral proteins that are required for viral replication.

[0076] An "operable linkage" is a linkage in which regulatory sequences and sequences sought to be expressed are connected in such a way as to permit said expression. For example, sequences, such as, e.g., a promoter, may be said to be operably linked if the nature of the linkage between said sequences does not: (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter to direct the transcription of the ORF, (3) interfere with the ability of the ORF to be transcribed from the promoter sequence. Hence, "operably linked" may mean incorporated into a genetic construct so that expression control sequences, such as a promoter, effectively control transcription / expression of a sequence of interest.

[0077] As used herein, the term "promoter" refers to a DNA sequence that enables a gene to be transcribed. A promoter is recognized by a RNA polymerase, which then initiates transcription. Thus, a promoter contains a DNA sequence that is either bound directly by, or is involved in the recruitment, of RNA polymerase. A promoter sequence can also include "enhancer regions", which are one or more regions of DNA that can be bound with proteins (namely the trans-acting factors) to enhance transcription levels of genes in a gene-cluster. The enhancer, while typically at the 5' end of a coding region, can also be separate from a promoter sequence, e.g., can be within an intronic region of a gene or 3' to the coding region of the gene. When reference is made herein to an "inducible promoter", an inducible promoter / repressor system is intended comprising a promoter region and an operator region to which one or more regulatory proteins (repressor and / or activator) can bind (i.e. the operator). Binding of the one or more regulatory proteins to the operator region can increase or reduce the affinity of the promoter to the RNA polymerase. When reference is made herein to a "promoter", a promoter perse is intended, i.e. without the operator region.

[0078] Preferably, the promoter initiates the transcription of the viral cDNA upon introduction of the vector as described herein in a mammalian cell. Accordingly, particular embodiments, the promoter is an RNA polymerase driven promoter, preferably an RNA polymerase II promoter. Non-limiting examples of RNA polymerase II promoter include the Cytomegalovirus Immediate Early (CMV-IE) promoter, the Simian virus 40 promoter or functionally homologous derivatives thereof such as the CMV-IE chicken beta-actin chimeric (CAG) promoter or inducible versions of said RNA polymerase II operated promoters such as the tetracyline-operator minimal CMV-IE promoter. Alternatively, said RNA polymerase driven promoter is an RNA polymerase I (Russel and Zomerdijk (2006) Biochem. Soc. Symp. 73, 203-216) or a RNA polymerase III promoter, such as the U6 or Hl promoter.

[0079] In particular embodiments, the promoter is located upstream (i.e. at the 5' end) of the cDNA of the attenuated RNA virus genome.

[0080] Accordingly, in particular embodiments, said viral expression cassette comprises: a cDNA of an attenuated RNA virus genome, wherein the RNA virus is a virus of the family Arteriviridae, and an RNA polymerase driven promoter at the 5' end of said cDNA, preferably an RNA polymerase II promoter.

[0081] In particular embodiments, the viral expression cassette comprises an element for RNA self-cleaving for cleaving the RNA transcript of said viral cDNA at a set position, such as the cDNA of the genomic ribozyme of hepatitis delta virus or the cDNA of the functionally homologous hepatitis delta virus-like self-cleaving ribozymes RNA elements.

[0082] In particular embodiments, the vector comprises an inducible origin of replication, such as an inducible bacterial origin of replication. In particular embodiments, the vector comprises an inducible bacterial origin of replication (ori) for amplifying said vector to a high copy number (e.g. at least 5 copies, such as at least 10 copies) per bacterial host cell in the presence of one or more inducers of said ori. Preferably, the vector is a BAC comprising an inducible bacterial origin of replication (ori) for amplifying said BAC to a high copy number (e.g. at least 5 copies, such as at least 10 copies) per bacterial host cell in the presence of one or more inducers of said ori. The BAC as described herein may comprise an inducible bacterial ori for amplifying said BAC to a high copy number per bacterial host cell (or allowing to induce the amplification of said BAC to a high copy number) in the presence of one or more inducers of said ori.

[0083] The term "inducible origin of replication", "inducible ori", "iori", "conditional origin of replication" or "conditional ori" as used herein refers to a vector ori sequence that functions in a bacterial host cell and is responsive to one or more inducers of the inducible origin of replication foreign to the host cell (i.e. the bacterial cell). Preferably, the replication function of the inducible ori is severely suppressed or nonexisting in absence of said one or more inducers of the inducible origin of replication. In particular embodiments, the inducible ori amplifies the vector to a high copy number in the presence of said one or more inducers of the inducible origin of replication, preferably to at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 200, or at least 300 copies per cell, more preferably to more than 10 copies per cell, even more preferably more than 50 copies per cell. Preferably, the inducible ori responds to a single inducer of the inducible origin of replication.

[0084] The term "inducer of the inducible origin of replication", "inducer of vector replication" or "replicationinitiating agent" refers to an agent capable of inactivating (e.g. by removal) a repressor. A repressor typically inhibits or decreases the expression of one or more transcripts by binding to an operator sequence in the DNA. Binding of the repressor to the operator interferes with the binding of the RNA- polymerase to the promoter and thereby prevents transcription. An inducer of the inducible origin of replication can be capable of interfering with the binding of the repressor to the operator, thereby allowing transcription. The skilled person will understand that the inducible ori is chosen for compatibility with a known inducer of vector replication, for its normally tight down regulation in the selected host cells in the absence of the compatible inducer of vector replication, and for its strong inducible operability in the presence of the inducer of vector replication.

[0085] As a non-limiting example, the oriV may be used as an inducible bacterial ori in the present invention because of its broad host range, its known capacity to replicate DNA fragments of 100-kb or larger, its high copy number and its requirement for only one inducing protein. Further non-limiting examples of inducible origins of replication comprise the oriV / TrfA amplification system tightly controlled by the L- arabinose-inducible Para promoter (araC-PBAD)-which can be induced by the addition of L-arabinose, for example as disclosed for the amplification of a shuttle BAC vector in Wild J. et al., Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones. Genome Res. 2002.12(9):1434-1444. In a further example, the oriV / TrfA may be tightly controlled by a rhamnose- inducible Prha promoter (rhaS-Prha) as described in Wild J. et al., Copy-control tightly regulated expression vectors based on pBAC / oriV. 2004.267:155-167.

[0086] In particular embodiments, the vector as described herein is comprised in a pharmaceutical composition, preferably wherein said pharmaceutical composition is a vaccine.

[0087] Accordingly, in line therewith, the present invention also provides a pharmaceutical composition comprising a vector and a pharmaceutically acceptable carrier, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae.

[0088] Preferably, said pharmaceutical composition is a vaccine, more preferably a plasmid DNA vaccine. Such pharmaceutical formulations or compositions may be comprised in a kit of parts.

[0089] In particular embodiments, the vector is a BAC comprising an inducible bacterial ori for amplifying said BAC to at least 5 copies per bacterial host cell in the presence of one or more inducers of said ori, and a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae.

[0090] The term "pharmaceutically acceptable" as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof. As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active substance, its use in the therapeutic compositions may be contemplated.

[0091] The formulation of DNA into a vaccine preparation is known in the art. For example, details on acceptable carrier, diluents, excipient and adjuvant suitable in the preparation of DNA vaccines can be found in International patent application W02005042014. In certain embodiments, vaccines may include an adjuvant, i.e. one or more substances that enhances the immunogenicity and / or efficacy of a vaccine composition The vector as described herein may be administered to the subject by any safe route of administration. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intradermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Preferably, said vector is administered intradermally, intramuscular, transdermally, intranasally, orally or subcutaneously to a subject.

[0092] In particular embodiments, a single dose of the pharmaceutical composition comprises from 100 to 2000 pg, from 100 to 1500 pg, from 200 to 1250 pg, from 200 to 1000 pg, from 300 to 1000 pg, preferably from 300 to 900 pg, of said vector. The person skilled in the art will understand that the amount of vector in a single dose of the pharmaceutical composition can be adjusted depending on the type of animal, the age of the animal and / or the type of animal farm (e.g. breeding / nursery facility, weaner farm, fattening farm). In particular embodiments, a dose of from 100 to 2000 pg, from 100 to 1500 pg, from 200 to 1250 pg, from 200 to 1000 pg, from 300 to 1000 pg, preferably from 300 to 900 pg, of said vector is administered to a subject.

[0093] In particular embodiments, the vector as described herein is administered to a subject in a single-dose or double-dose regime. For example, the subject may be administered two doses of the vector as described herein, with an interval of at least 7 days, at least 14 days, at least 21 days or at least 28days. In particular embodiments, the subject is administered two doses of the vector as described herein, with an interval of from 7 to 35 days, from 14 to 35 days, from 14 to 30 days, from 7 to 28 days, from 14 to 28 days, or from 7 to 21 days, preferably from 14 to 28 days.

[0094] In particular embodiments, if the vector as described herein is administered to a subject in a double-dose regime, each dose of said vector as described herein comprises from 100 to 2000 pg, from 100 to 1500 pg, from 200 to 1250 pg, from 200 to 1000 pg, from 300 to 1000 pg, preferably from 300 to 900 pg, of said vector.

[0095] In particular embodiments, each dose of the double-dose regime is administered via the same delivery route.

[0096] In particular embodiments, the vector as described herein is administered to the animal when the animal is infected with PRRSV or when infection with PRRSV is to be prevented in said animal.

[0097] The vector of the invention may be employed in combination with other therapeutic agents for the treatment or prophylaxis of the infections or conditions indicated above.

[0098] The invention further provides the use of the vector as described herein in the preparation of a vaccine. In line therewith, also provided herein is a method of preparing a vaccine against an RNA virus of the family Arteriviridae comprising the steps of: a) providing a host cell (e.g. bacterial host cell) transfected with a vector comprising a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae, b) amplifying the vector, c) isolating the amplified vector, and d) formulating the isolated vector into a vaccine.

[0099] In particular embodiments, such as if the vector comprises an inducible origin of replication, the method of preparing a vaccine against an RNA virus of the family Arteriviridae comprises the steps of: a) providing a host cell (e.g. bacterial host cell) transfected with a vector comprising an inducible bacterial origin of replication (ori) and a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae, b) amplifying the vector by adding one or more inducers of said inducible ori, c) isolating the amplified vector, and d) formulating the isolated vector into a vaccine.

[0100] In particular embodiments, such as if the vector is a BAC, the method of preparing a vaccine against an RNA virus of the family Arteriviridae comprises the steps of: a) providing a bacterial host cell transfected with a BAC comprising: an inducible bacterial origin of replication (ori) for amplifying said BAC to at least 5 copies per bacterial host cell in the presence of one or more inducers of said ori, and a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteri viridae, b) amplifying the BAC by adding one or more inducers of said inducible ori, c) isolating the amplified BAC, and d) formulating the isolated BAC into a vaccine.

[0101] The term "bacterial cells", "bacterial host cells" or "bacteria" refers to bacteria suitable as a host cell for vectors. The advantages of producing vectors in bacteria are amongst other the relatively safe and straightforward handling of bacterial cells and the rapid replication cycles of the bacterial cells.

[0102] When increasing the yield of large vectors (e.g. bacterial artificial chromosomes (BACs) or vectors having a size of at least 15.5 kb) in bacteria, there is a legitimate concern that the strongly increased activity of the replication system will increase the mutation frequency in the vector DNA. The production of large vectors thus requires a manufacturing process wherein a high yield of vector is obtained, but wherein replication of the vectors occurs without intolerable introduction of mutations.

[0103] In particular embodiments, the method comprises limiting the copy number of the vector per cell unit during the growth phase (e.g. at an optical density at 600 nm (OD600) below 20) and inducing replication at a high biomass (e.g. OD600 of at least 20), such as described in published International patent application WO 2021 / 058722 Al. As a result thereof high-quality, pharmaceutical grade vector (i.e. with low mutation frequency) can be obtained at high yield.

[0104] In particular embodiments, if the vector (i.e. vector comprising the cDNA of the attenuated RNA virus genome) has a size of at least 15.5 kb and comprises an inducible origin of replication, the method comprises the consecutive steps of a) obtaining bacterial cells comprising a vector with a size of at least 15.5 kb, comprising an inducible origin of replication, b) inoculating culture medium with the bacterial cells comprising the vector, c) adding one or more inducers of said inducible origin of replication to the culture medium when the bacterial culture has reached an optical density at 600 nm (ODgoo) of at least 20, such as at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or at least 30, d) further culturing the bacterial cells in the culture medium, e) optionally separating the bacterial cells from the culture medium, and f) recovering the plasmid from the bacterial cells. In particular embodiments, such as if the vector is a BAC, the method comprises the consecutive steps of a) obtaining bacterial cells comprising the BAC as described herein, b) inoculating culture medium with the bacterial cells comprising the BAC, c) culturing the bacterial cells in the culture medium, d) adding one or more inducers of said inducible origin of replication to the culture medium when the bacterial culture has reached an optical density at 600 nm (ODgoo) of at least 20, such as at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or at least 30, e) further culturing the bacterial cells in the culture medium, f) optionally separating the bacterial cells from the culture medium, and g) recovering the plasmid from the bacterial cells. Inoculation of culture medium with the bacterial cells comprising the vector can be performed by any method known in the art to inoculate culture medium with bacterial cells. For example, a single colony of bacterial cells comprising the vector can be selected from an agar plate using a sterile pipette tip or toothpick, after which the tip or toothpick is dropped into liquid culture medium and the liquid culture medium comprising the pipette tip or toothpick is swirled. Alternatively, culture medium can be inoculated with a glycerol stock of the bacterial cells comprising the vector.

[0105] The culture medium can be any culture medium known in the art to culture bacterial cells. Non-limiting examples include Luria-Bertani Broth Medium (LB) (ATCC medium formulation 1065) or minimal medium, such as M9 minimal medium. The person skilled in the art will understand that if a minimal medium is used, supplementation with amino acids, salts and / or nutrients might be required. Use of a minimal medium offers the advantage of being able to tightly control the nutrients supplied to the bacterial cells. In particular embodiments, the method further comprises adding an inhibitor of bacterial protein synthesis to the culture medium after step d) and before step f). The term "inhibitor of bacterial protein synthesis" or "bacterial protein synthesis inhibitor" as used herein refers to mechanical (e.g. temperature shift) or chemical means (e.g. chemical agent) that inhibit or slow down the growth or proliferation of bacterial cells by disrupting the processes that lead directly to the generation of polypeptides or proteins. The inhibitor of bacterial protein synthesis may suppress the bacterial internal machinery and allows the bacteria to fully focus on plasmid production. Furthermore, the inhibitor of bacterial protein synthesis may stabilize the oxygen need of the bacteria. Non-limiting examples of such inhibitors include chloramphenicol, spectinomycin, streptomycin, aminoglycosides, tetracyclines, macrolides, lincosamides, or oxazolidinones.

[0106] Biologic oxidation of carbohydrates, such as glucose and / or glycerol, by bacteria results in synthesis of ATP as the chemical energy source and permits generation of simpler organic compounds needed by the bacteria for biosynthetic or assimilatory reactions.

[0107] Accordingly, in particular embodiments, glucose, glycerol and / or yeast extract are added to the culture medium during culturing the bacterial cells in the culture medium.

[0108] In particular embodiments, the culture medium comprising glucose is exchanged by culture medium comprising from 0.50% (v / v) to 2.0% (v / v) glycerol at least 30 minutes before adding the one or more inducers of plasmid replication to the culture medium.

[0109] In particular embodiments, step c) of culturing the bacterial cells in the culture medium is performed at a temperature of about 37°C. In other particular embodiments, step c) of culturing the bacterial cells in the culture medium is performed at a temperature of about 30°C and wherein the temperature is increased from a temperature of about 30°C to a temperature from 36.0°C to 38.0°C at least two hours before adding the one or more inducers of plasmid replication to the culture medium.

[0110] In particular embodiments, the inhibitor of bacterial protein synthesis is added to the culture medium at least one hour after the addition of the one or more inducers of the inducible origin of replication.

[0111] In particular embodiments, the bacterial cells are separated from the culture medium at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours or at least 12 hours, preferably at least 6 hours, after addition of the one or more inducers of the inducible origin of replication.

[0112] In particular embodiments, the inducible origin of replication comprises at least one LacOl operator and the inducer of said inducible origin of replication is isopropyl p-D-l-thiogalactopyranoside (IPTG) and / or alpha-D-lactose.

[0113] The inducible origin of replication may be an origin of replication such as described in published International patent application WO 2021 / 058722 Al on p. 23 to 27. In particular embodiments, said inducible origin of replication comprises a pMBl origin of replication:

[0114] - in which the RNAI promoter is deleted or mutated such that the function of the RNAI promoter is abolished or significantly reduced, thereby obtaining a truncated RNAII pre-primer; and

[0115] - in which the nucleic acid sequence encoding for RNAI is deleted or mutated such that the function of RNAI is abolished or significantly reduced, thereby obtaining a truncated RNAII pre-primer; and

[0116] - in which at least one LacOl operator is introduced upstream of the nucleic acid sequence encoding the truncated RNAII pre-primer, wherein the at least one LacOl operator is operably linked to the nucleic acid sequence encoding the truncated RNAII pre-primer.

[0117] In particular embodiments, the endogenous RNAII promoter of the pMBl origin of replication is replaced by a promoter which is not the endogenous promoter of the RNAII pre-primer of the pMBl origin of replication and wherein said promoter which is not the endogenous promoter of the RNAII pre-primer of the pMBl origin of replication is operably linked to the at least one LacOl operator. In further particular embodiments, the endogenous RNAII promoter of the pMBl ori is replaced by a promoter selected from the group consisting of the promoter of RNA I, the lac promoter, the trp promoter, the trc promoter or the T7 promoter, preferably the lac promoter, more preferably the L8 / UV5 lac promoter.

[0118] In particular embodiments, the nucleic acid corresponding to the nucleic acid at position 322 of the wildtype pMBl origin of replication is mutated from an A to a G and / or the nucleic acid corresponding to the nucleic acid at position 543 of the wild-type pMBl origin of replication is muted from a C to a T. In particular embodiments, the inducible origin of replication comprises a nucleic acid sequence having at least 95%, preferably 100%, sequence identity with the nucleic sequence as set forth in SEQ ID NO: 7.

[0119] The following examples are meant to illustrate the present invention and should not be construed as a limitation of its scope.

[0120] EXAMPLES

[0121] Example 1. Preparation of PLLAV-Porcilis (Fig. 2)

[0122] 1.1 Materials and methods

[0123] 1.1.1 Cells, vaccine and plasmid

[0124] Porcilis® PRRS Lyophilisate and solvent for suspension for injection for pigs (MSD) is commercially available (registration number(s) BE-V221182 and BE-V278433, batch number G121A01). The vaccine was resuspended according to manufacturer's protocol. The sequence for the Porcilis PRRSV strain comprises a sequence as set forth in SEQ ID NO: 1.

[0125] HEK293 cells were maintained in Gibco™ DMEM, high glucose, GlutaMAX™ Supplement (Thermo) supplemented with 10% fetal calf serum (Thermo) and 1% Gibco™ Penicillin-Streptomycin (Thermo).

[0126] MARC-145 cells were maintained in Gibco™ DMEM 10% fetal calf serum (Thermo) and 1% Gibco™ Penicillin-Streptomycin (Thermo).

[0127] A plasmid (named pVV6 ) was made comprising an inducible BAC vector containing the CMV promoter to drive gene expression, a hepatitis delta virus ribozyme sequence (HDr) and a SV40 polyA signal.

[0128] 1.1.2 RNA isolation and RT-PCR

[0129] Viral RNA was extracted from the Porcilis vaccine using the QIAamp Viral RNA Mini Kit (Qiagen). To obtain viral cDNA that could be cloned into the inducible BAC plasmid, the viral RNA genome was amplified in 4 RT-PCR reactions leading to overlapping PCR products that span the entire genome. Viral cDNA synthesis was performed using Superscript™ III One-Step RT-PCR System with Platinum™ Taq High Fidelity DNA Polymerase (ThermoFisher Scientific). The primers used to generate the PCR fragments are listed in Table 1. The forward primer Porcil is_FlF and the reverse primer Porcil is_F4R were designed to overlap with the linearized BAC plasmid. PCR fragments were purified with NucleoSpin Gel and PCR Clean-up (Macherey- Nagel). Table 1: list of primers for the generation of cDNA fragments

[0130] 1.1.3 Assembly of the PCR fragments in the linearized BAC plasmid

[0131] The pVV6 plasmid was cut with a unique restriction enzyme after the CMV promoter to linearize the vector. Equimolar amounts of plasmid and each PCR fragments were mixed with NEBuilder HiFi DNA Assembly Master Mix (NEB) according to manufacturer's protocol. The reaction was incubated for lh at 50°C, after which 2 pl was used to transform NEB 10-beta chemicompetent E. coli bacteria (NEB), according to manufacturer's protocol. The bacteria were plated on Lennox L agar (Thermo) plates containing kanamycine (50 pg / ml) as selection marker, and left overnight at 37°C. The transformed bacteria were screened for the presence of the Porcilis genome in a colony PCR with GoTaq® G2 Green master mix (Promega), using primers that bind on Nsp2 and Nsp3. Positive clones were used to inoculate 100 ml of Lennox L Broth Base (LB broth, Thermo) containing 50 pg / ml kanamycine. The inoculated cultures were incubated overnight in a shaking incubator at 37°C, after which the plasmids were harvested with the NucleoBond Xtra Midi EF kit (Macherey-Nagel) according to manufacturer's protocol. 1

[0132] 1.1.4 Virus reco very

[0133] One day prior transfection, HEK293 cells were seeded in a 24-well plate at a density of 80 000 cells / 500 pl. The next day 500 ng of plasmid was used to transfect the HEK293 cells using jetPEI® (Polyplus) as transfection agent, following manufacturer's guidelines. Three days post transfection, the transfected plates were stored at -80°C.

[0134] One day prior infection, MARC-145 cells were seeded in a 24-well plate at a density of 80,000 cells / 500 pl. On the day of infection, the plates containing the transfected HEK293 cells were defrosted, and the supernatant and transfected cells were transferred to a centrifuge tube. Cell debris was pelleted for 5 minutes at 1100 rpm, and the supernatant was added to the MARC-145 cells. After an incubation of 3-4h at 37°C, the supernatant was removed and fresh assay medium was added to the MARC-145 cells. After 3-4 days, the first signs of cytopathic effect (CPE) could be observed, after which the plates were stored at -80°C.

[0135] To confirm that the observed CPE was caused by a viral (Porcilis PRRSV) infection, present inventors performed an RNA extraction on the infected MARC-145 cells using the QIAamp Viral RNA Mini Kit, followed by an RT-PCR to investigate whether subgenomic RNA was present in the samples. As subgenomic RNA can only be produced when all nonstructural proteins of PRRSV are present, its presence can be used as a marker for viral replication. The RT-PCR was done using Superscript™ III One-Step RT- PCR System with Platinum™ Taq High Fidelity DNA Polymerase.

[0136] 1.1.5 Full Porcilis genome sequencing

[0137] The PLLAV-Porcilis clones that caused CPE in MARC-145 cell culture and that were positive for the production of subgenomic RNA, were fully sequenced with Sanger sequencing (LGC Genomics).

[0138] 1.2 Results

[0139] 1.2.1 Design of pVV6-Porcilis

[0140] In order to clone the Porcilis genome in the inducible BAC plasmid pVV6, present inventors designed 4 primer sets to generate overlapping PCR fragments covering the entire Porcilis genome. The range of overlap was between 46 and 67 bp. In addition, the forward primer for the 1stfragment contained a 30- bp tail to overlap with the CMV promoter in the pVV6 plasmid, while the reverse primer of the 4thfragment contained a 25-nt polyA tail and a 26-bp overlap with the HDr in pVV6. The length of the A-tail was chosen based on previous experiments described in literature. Fig. 2 shows the design of the construct. 1.2.2 Recovery of PLLAV-derived Porcilis PRRSV

[0141] Porcilis PRRS vaccine is a modified live vaccine. Given the high genetic variability of PRRSV (estimated at about 1 mutation per replication cycle (Risser J et al., Porcine reproductive and respiratory syndrome virus genetic variability a management and diagnostic dilemma, Virology Journal, 2021, 18:206), it can be expected that Porcilis PRRS vaccine contains many quasispecies of PRRSV. Indeed, Genbank entries MT311646.1 and MW674755.1 both correspond to sequenced Porcilis PRRS vaccine, but show only 99.07% sequence identity. In addition, mistakes made during RT-PCR cannot be excluded, leading to cDNA fragments that contain mutations compared to the theoretical sequence of Porcilis PRRS. Nineteen clones were selected for testing in cell culture. The transfection was done in HEK293 cells as this cell line is known to be easily transfected with a variety of transfection agents, and to give high yields of the recombinant product. However, this cell line is not permissive for PRRSV infection, and therefore cannot be used to assess the viability and infectibility of the different PRRSV clones present inventors have selected. Therefore, the supernatant and cell extracts of transfected HEK293 cells was used to infect the permissive cell line MARC-145. Three to 4 days post infection of the MARC-145 cells with the cell extracts of HEK293 cells, present inventors could observe CPE in 3 out of the 19 conditions. However, when present inventors extracted RNA from the infected MARC-145 cells and used this as a template in an RT-PCR to see whether subgenomic RNA was present in the sample, 7 out of 19 conditions showed multiple bands corresponding to the different lengths of the subgenomic RNAs that are produced during PRRSV replication. Of the 3 clones that gave CPE, clone #13 appeared to be the strongest clone as it gave 100% CPE 4 days post infection, while less CPE was observed for the other clones. Present inventors selected this clone for further characterization.

[0142] 1.2.3 Sequencing analysis of PLLAV-Porcilis #13

[0143] PLLAV-Porcilis #13 was fully sequenced (of which the PRRSV sequence is as defined in SEQ ID NO: 2), and the sequence compared to the Porcilis sequence (as previously annotated under Genbank no KF991509.1 and comprising the PRRSV as defined in SEQ ID NO: 1 (Fig. 8)). Present inventors observed a total of 25 mutations compared to the former Genbank sequence, of which 14 were silent mutations. Of the non- silent mutations, 1 was found in Nspip, 6 in Nsp2, 2 in 0RF2, 1 in 0RF5 and 1 in 0RF6. Most notably, present inventors observed a 222 bp deletion in Nsp2, but this was reported previously in literature (Darwich L. et al., Genetic and immunobiological diversities of porcine reproductive and respiratory syndrome genotype I strains. Veterinary Microbiology (2011) 150; 49-62). However, as present inventors were aware that Genbank entry no KF991509.1 was retracted, they repeated the sequence comparison of PLLAV-Porcilis #13 with Porcilis PRRS Genbank entries MT311646.1 and MW674755.1. Focussing on the 11 non-silent mutations only, 5 positions remained mutated in PLLAV- Porcilis #13, while 2 positions were similar to one of the two entries, but mutated in comparison with the other entry. Remarkably, the 222 bp deletion that present inventors observed in Nsp2 and that also was reported in literature, was not present in the sequence of MW674755.1, while a smaller part of 135 bp was missing in MT311646.1.

[0144] Example 2. PRRSV challenge studies using PLLAV-Porcilis in pigs

[0145] 2.1 Study 1: Dose escalation vaccine study in pigs followed by viral challenge

[0146] 2.1.1 Materials and methods

[0147] A total of forty-four Hypor x German Pietrain pigs (n=22 male and n=22 female) were included onto the study. Prior to the start of the study, all animals were seronegative for antibodies to PRRSV and Mycoplasma hyopneumoniae.

[0148] Pigs at 4.5 weeks of age were immunised in the neck area on "day 0" with either the full 2mL vaccine dose of Progressis® vaccine (Registration number BE-V221435, Batch: 001LR1KB; expiry date: 21 / 12 / 2023) via intramuscular injection using a needle and syringe (positive control, n=8 pigs), or with 300 pg PLLAV- Porcilis (comprising PRRSV Porcilis #13 as defined by SEQ ID NO: 2) via transdermal and needle-free administration (n=8 pigs), or with 600 pg PLLAV-Porcilis (comprising PRRSV Porcilis #13 as defined by SEQ ID NO: 2) transdermally and needle-free (n=8 pigs) or with 900 pg PLLAV-Porcilis transdermally and needle)free (n=8 pigs).

[0149] Non-vaccinated and non-challenged pigs served as a negative control (UUC, n=4 pigs). Non-vaccinated and challenged pigs served as challenge control (IUC, n=8 pigs).

[0150] The pigs received a booster immunization at day 21 (i.e., 21 days post vaccination or dpv) at the same dose level and via the same route as on day 0 (0 dpv).

[0151] The animals were challenged on day 41 (i.e., 41 dpv) via intranasal inoculation of 105TCID50 (50% tissue culture infectious dose) of PRRSV-1 strain Flandersl3 (13V091), using an intranasal mucosal atomization device. A total volume of 5 mL, i.e. approximately 2.5 mL per nostril, was inoculated. The animals of the UUC group were mock-challenged via the same route with 5 mL of phosphate-buffered saline (PBS).

[0152] Half of the animals was sacrificed on day 51 (i.e, 10 days post challenge (dpc)); the other half was sacrified on day 52 (i.e; 11 dpc).

[0153] A schematic study design is depicted in Fig. 3. Measurement of anti-PRRSV antibodies post vaccination

[0154] A commercial enzyme-linked immunosorbent assay (ELISA) (pigtype PRRSV Ab; Cat. No.: PT272753; INDICAL) was performed to detect anti-PRRSV antibodies in serum samples collected at -8, 0, 14, 20, 28, 35 and 41 days post vaccination (dpv). Animals having detectable anti-PRRSV antibodies according to the manufacturer's instructions were considered to have seroconverted to PRRSV.

[0155] Measurement of IFN-gamma response post vaccination

[0156] A commercial enzyme-linked immune absorbent spot (ELISpot) assay (Pig IFN-y Single-Color ELISPOT, ImmunoSpot) was performed on peripheral blood mononuclear cells (PBMCs) isolated from whole blood samples collected on day (41 dpv) to evaluate a PRRSV targeted cellular immune response pre-challenge.

[0157] Measurement of PRRS viraemia post challenge

[0158] A real time RT-PCR (RT-qPCR) was performed according to the World Organisation for Animal Health described procedure (PRRS (woah.org)) on RNA extracted from serum samples to detect viral PRRSV RNA copies. RNA extractions and RTqPCR were performed on serum samples collected at 0, 3, 5, 7 and 10 days post challenge (dpc).

[0159] Observation of PRRS associated clinical signs post challenge

[0160] Starting one day prior to challenge until study end (between 40-51 dpv), PRRS-associated clinical observations were made daily which included the assessment of demeanour / behaviour and evaluation of respiratory pattern (coughing, breathing pattern) according to the below scoring system.

[0161] Demeanour (Soc - Social behaviour):

[0162] 0 = Normal: Pig is vigorous, alert, and responding to its surroundings.

[0163] 1 = Mild depression: lethargy and apathy. Pig is inactive, slow to respond when promoted.

[0164] 2 = Severe depression: the pig is noticeably slow to respond, reluctant to move.

[0165] 3 = Moribund or coma: unconsciousness, unresponsive to stimuli; unable to rise; no interest in surroundings.

[0166] Cough:

[0167] 0 = Absent

[0168] 1 = Mild (<2 in 3 minutes observation)

[0169] 2 = Severe (>2 in 3 minutes observation) Respiratory pattern (Dysp - Dyspnea):

[0170] 0 = Normal.

[0171] 1 = Slight polypnea: slightly increased breathing rate.

[0172] 2 = Dyspnoea: markedly increased breathing rate; increased expiratory effort.

[0173] 3 = Severe respiratory distress; marked dyspnoea: laboured respiration; open-mouthed breathing.

[0174] Measurement of rectal temperature post challenge

[0175] Rectal temperature was measured in all pigs from day 40 to day 51 (i.e., -1 to 10 dpc) to monitor the effect of PRRSV-1 challenge. To obtain a baseline, temperatures were recorded starting 1 day before challenge. Rectal temperatures were measured without restraining or catching the pigs.

[0176] 2.1.2 Results

[0177] PLLAV-Porcilis induces anti-PRRSV antibodies post vaccination

[0178] None of the IUC and UUC pigs had detectable anti-PRRSV antibodies pre-challenge with PRRSV-1 Flandersl3, whereas all pigs vaccinated with PLLAV-Porcilis 900 seroconverted to PRRSV. A total of 7 / 8 pigs for both PLLAV-Porcilis 300 and PLLAV-Porcilis 600 had measurable anti-PRRSV antibodies. Irrespective of the PLLAV vaccine dose administered, seroconversion in the PLLAV-Porcilis vaccinated groups started at 20 dpv, hence, before the administration of a booster vaccination. In contrast, none of the PROGRESSIS-vaccinated animals had detectable anti-PRRSV antibodies prior to boosting and following a booster shot only 5 / 8 pigs were found to have anti-PRRSV antibodies (Fig. 4 and Table 2). Moreover, higher antibody responses as evidenced by higher S / P values were observed in the PLLAV-Porcilis vaccinated groups compared to the animals receiving the PROGRESSIS vaccine (Table 2).

[0179] Table 2. Number of animals showing anti-PRRSV antibodies as measured by ELISA and group average S / P value.

[0180] Note: an S / P value above 0.4 is considered positive for anti-PRRSV antibodies.

[0181] PLLAV-Porcilis induces IFN-gamma responses in PBMCs at day 41 post immunization

[0182] To assess cellular immunity responses post-vaccination, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples collected on day 41 (i.e., pre-challenge). PBMCs were restimulated in vitro with PRRSV-1, followed by an evaluation of IFN-y production by these cells. The IFN-y response was considered as proxy for a PRRSV-targeted cellular immune response.

[0183] None of the UUC and IUC pigs had detectable IFN-y responses on day 41. In contrast, IFN-y responses were observed in 6 / 8 pigs for all PLLAV-Porcilis groups (irrespective of the dose) whereas only one of 8 pigs vaccinated with the PROGRESSIS vaccine mounted an IFN-y response on day 41 (data not shown).

[0184] PLLAV-Porcilis vaccination reduces PRRS viraemia post-challenge

[0185] In order to evaluate vaccine efficacy, reduction in PRRS viraemia post-challenge was measured in addition to the above evidence of induction of immune responses post vaccination. Indeed, PLLAV-Porcilis vaccination significantly reduced viraemia, as evidenced by significantly lower PRRSV RNA copies in serum starting from 3 days post challenge up until 10 days post challenge, when compared to pigs that were left unvaccinated or those vaccinated with the PROGRESSIS vaccine (Fig. 7). No significant difference is PRRSV RNA copies was observed between the 3 different vaccine doses tested for PLLAV-Porcilis. Moreover, no significant reduction in viral load was observed in animals vaccinated with the commercial PROGRESSIS vaccine compared to mock vaccinated pigs (IUC group).

[0186] PLLAV-Porcilis vaccination protects pigs from PRRS clinical disease and from PRRSV-associated fever

[0187] Not only was PLLAV-Porcilis vaccination shown to be efficacious in reducing PRRS viraemia, PLLAV-Porcilis vaccination also provided full protection against clinical signs associated with PRRS disease. Indeed, 6 of 8 unvaccinated pigs and 3 of 8 PROGRESSIS-vaccinated pigs showed PRRS-associated signs of depression, respiratory distress and / or coughing during the 10-day monitoring period post PRRSV challenge. However, all PLLAV-Porcilis vaccinated pigs were fully protected against clinical PRRS disease regardless of the vaccine dose administered, except for a single isolated event of coughing on day 7 post challenge in one animal vaccinated with PLLAV-Porcilis at the 900 pg dose level.

[0188] Moreover, where unvaccinated pigs as well as pigs vaccinated with PROGRESSIS had group mean rectal temperature above 40°C (i.e., fever threshold) on day 2 post challenge, the groups of pigs vaccinated with PLLAV-Porcilis on average did not show signs of fever.

[0189] 2.2 Study 2: Immunogenicity study of PLLAV-Porcilis to assess different vaccine delivery routes

[0190] 2.2.1 Materials and methods

[0191] A total of fifteen pigs were included in this study. The piglets were 31 days old and Norsvin Landrace crossbred with Z-line with an average bodyweight of 7.03 ± 0.86 kg. The pigs were randomized into 3 groups (n=5 / group). All pigs were subjected to a prime-boost vaccination: on day 0 all pigs received a prime vaccination, followed by a booster vaccination on day 21. Animals were either vaccinated with the commercial Progressis® vaccine (Batch: 001LR1KB; expiry date: 21 / 12 / 2023) (PROGRESSIS, positive control) or with the plasmid-launched live attenuated porcine reproductive and respiratory syndrome virus vaccine based on the Porcilis DV strain (abbreviated as PLLAV-Porcilis, i.e. PLLAV-Porcilis #13 of which the PRRSV sequence is as defined in SEQ ID NO: 2), although dose and administration route differed.

[0192] Animals of group 1 (n=5) were vaccinated intramuscularly in the neck area with the full 2mL vaccine dose of Progressis® using a needle and a syringe. Animals of group 2 (n=5) were administered transdermally (t.d.) 300 pg of PLLAV-Porcilis. Transdermal administration was done using a needle-free delivery device at the left side of the neck area. The animals of group 3 were injected 300 pg of PLLAV-Porcilis (using a needle and a syringe) intramuscularly (i.m.). Henceforth, these groups are referred to as PLLAV-Porcilis t.d. and i.m., respectively.

[0193] The induction of humoral (anti-PRRSV antibodies) immunity was assessed at different timepoints post PLLAV-Porcilis vaccination. Hereto, serum samples collected at-3, 7, 14, 21, 28, 35, 41 and 49 days post vaccination (dpv) were tested in a commercial enzyme-linked immunosorbent assay (ELISA), designed to detect anti-PRRSV antibodies according to the manufacturer's instructions (pigtype PRRSV Ab; Cat. No.: PT272753; INDICAL). A positive ELISA result is an indication for a successful vaccination. In addition, a commercial enzyme-linked immune absorbent spot (ELISpot) assay was performed on 49 dpv (Pig IFN-y Single-Color ELISPOT, ImmunoSpot) on post-necropsy isolated splenocytes to evaluate a PRRSV targeted cellular immune response.

[0194] 2.2.2 Results

[0195] General safety and tolerability observations

[0196] The body weights of the animals in all groups steadily increased over time.

[0197] There was no evidence of any local or systemic reaction in any of the animals throughout the entire in-life phase of the study. Based on clinical observations, vaccine administration site assessment and body weight measurements no safety concerns were identified following administration of PLLAV-Porcilis.

[0198] PLLAV-Porcilis vaccination induces anti-PRRSV antibodies after transdermal as well as intramuscular delivery

[0199] The results of the ELISA are summarised in Fig. 5. Overall, 4 out of 5 and 3 of 5 pigs of the PLLAV-Porcilis t.d. and i.m. group respectively tested positive for anti-PRRSV binding antibodies at one or multiple consecutive timepoints. Seroconversion in these two groups was observed as early as 21 days post vaccination (hence prior to booster vaccination). In contrast, anti-PRRSV antibodies in the PROGRESSIS group were detected post booster vaccination only. Moreover, at best only 3 out of 5 animals seroconverted with a transient humoral immune response. Indeed, by day 49 none of the PROGRESSIS vaccinated animals tested positive for anti-PRRSV antibodies. In contrast, the antibody response following PLLAV-Porcilis vaccination was sustained or even increased over time.

[0200] PLLAV-Porilis vaccination induces an IFN-gamma response in post-necropsy isolated splenocytes on day 49 To assess cellular immunity responses post-vaccination, splenocytes were isolated on day 49 and restimulated in vitro with PRRSV-1, followed by an evaluation of IFN-y production by these cells. The IFN-y response was considered as proxy for a PRRSV-targeted cellular immune response.

[0201] None of the PROGRESSIS vaccinated pigs had detectable IFN-y responses on day 49. In contrast, IFN-y responses were observed on day 49 in 3 / 5 pigs for the PLLAV-Porcilis i.m. group and in all 5 of 5 pigs vaccinated with PLLAV-Porcilis via the needle-free transdermal route.

[0202] 2.3 Combination of studies 1 and 2

[0203] 2.3.2 Results

[0204] Fig. 6 combines the results of studies 1 and 2 and compares the seroconversion rates (i.e., percentage of pigs testing positive for anti-PRRSV antibodies at different timepoints post vaccination) of the animals vaccinated intramuscularly with the commercial Progressis® vaccine to those vaccinated with PLLAV- Porcilis vaccine (i.e. PLLAV-Porcilis #13, of which the PRRSV sequence is as defined in SEQ ID NO: 2) via the needle-free transdermal route at dose levels of 300 pg, 600 pg and 900 pg.

[0205] Clearly and irrespective of the dose level, PLLAV-Porcilis vaccination outperforms Progressis® vaccination by inducing an earlier, higher and more sustained anti-PRRSV antibody response.

[0206] Example 3. Preparation of PLLAV-lngelvac

[0207] 3.1 Materials and methods

[0208] 3.1.1. Cells, vaccine and plasmid

[0209] INGELVAC PRRS MLV (Boehringer Ingelheim) is commercially available (registration number BE-V179374, batch number 2451413AB). HEK293 cells, MARC-145 cells and the pVV6 plasmid were described in example 1.

[0210] 3.1.2 RNA isolation and RT-PCR

[0211] The procedure to clone the cDNA of Ingelvac PRR in the linearized plasmid pVV6 is similar to the procedure described in example 1 for Porcilis PRRS vaccine. Table 3 lists the primers used to generate the 4 overlapping cDNA fragments covering the entire Ingelvac genome.

[0212] Table 3: list of primers for the generation of cDNA fragments

[0213] 3.1.3 Assembly of the PCR fragments in the linearized PLLAV plasmid

[0214] The pVV6 plasmid was cut with a unique restriction enzyme after the CMV promoter to linearize the vector. Equimolar amounts of plasmid and each PCR fragments were mixed with NEBuilder HiFi DNA Assembly Master Mix (NEB) according to manufacturer's protocol. The reaction was incubated for lh at 50°C, after which 2 pl was used to transform NEB 10-beta chemicompetent E. coli bacteria (NEB), according to manufacturer's protocol. The bacteria were plated on Lennox L agar (Thermo) plates containing kanamycine (50 pg / ml) as selection marker, and left overnight at 37°C.

[0215] The transformed bacteria were screened for the presence of the Ingelvac genome in a colony PCR, using primers that bind on Nspip and Nsp2. Positive clones were used to inoculate 100 ml of Lennox L Broth

[0216] Base (LB broth, Thermo) containing 50 pg / ml kanamycine. The inoculated cultures were incubated overnight in a shaking incubator at 37°C, after which the plasmids were harvested with the NucleoBond Xtra Midi EF kit (Macherey-Nagel) according to manufacturer's protocol. 3.1.4. Virus recovery

[0217] One day prior transfection, HEK293 cells were seeded in a 24-well plate at a density of 80 000 cells / 500 pl. The next day 500 ng of plasmid was used to transfect the HEK293 cells using jetPEI® (Polyplus) as transfection agent, following manufacturer's guidelines. Three days post transfection, the transfected plates were stored at -80°C.

[0218] One day prior infection, MARC-145 cells were seeded in a 24-well plate at a density of 80 000 cells / 500 pl. On the day of infection, the plates containing the transfected HEK293 cells were defrosted, and the supernatant and transfected cells were transferred to a centrifuge tube. Cell debris was pelleted for 5 minutes at 1100 rpm, and the supernatant was added to the MARC-145 cells. After an incubation of 3-4h at 37°C, the supernatant was removed and fresh assay medium was added to the MARC-145 cells. After about 7 days, the first signs of cytopathic effect (CPE) could be observed, after which the plates were stored at -80°C.

[0219] To confirm that the observed CPE was caused by a viral (Ingelvac PRRSV) infection, present inventors performed an RNA extraction on the infected MARC-145 cells using the QIAamp Viral RNA Mini Kit, followed by an RT-PCR to investigate whether subgenomic RNA was present in the samples. As subgenomic RNA can only be produced when all nonstructural proteins of PRRSV are present, its presence can be used as a marker for viral replication. The RT-PCR was done using Superscript™ III One-Step RT- PCR System with Platinum™ Taq High Fidelity DNA Polymerase.

[0220] 3.1.5. Full Ingelvac genome seguencing

[0221] The PLLAV-lngelvac clones that caused CPE in MARC-145 cell culture and that were positive for the production of subgenomic RNA, were fully sequenced with Sanger sequencing (LGC Genomics).

[0222] 3.2 Results

[0223] 3.2.1 Design of PLLAV-lngelvac

[0224] Similar as the described in section 1.2.1 of example 1, present inventors designed 4 primer sets do generate overlapping cDNA fragments covering the entire Ingelvac genome. For cloning purposes, the forward primer of the 1stfragment contained a tail that overlapped with the CMV promoter of the PLLAV vector pVV6, while the reverse primer contained an Azs-tail and a 21 bp-region to overlap with the HDr in pVV6.

[0225] 3.2.2 Recovery of PLLAV-derived Ingelvac PRRSV

[0226] Present inventors have used a similar approach as described in section 1.2.2 for the recovery of Ingelvac PRRSV in cell culture, namely transfection of HEK293 using jetPEI, followed by infection of MARC-145 cells with cell extracts derived from the transfected HEK293. In an initial experiment, 5 constructs were tested in cell culture, of which only one (clone #7) gave signs of CPE 7 days post infection. In a second set of transfections, an additional 8 clones were tested. Two clones (#25 and #34) of pLLAV- Ingelvac gave signs of CPE in infected MARC-145 cell culture 3 days post infection. In a head-to-head comparison in MARC-145 cells, Ingelvac-PRRSV virus derived from clone #34 induced CPE in MARC-145 cells sooner than clone #7.

[0227] 3.2.3 Sequencing analysis of PLLAV-lngelvac #7 and #34

[0228] The sequence of PLLAV-lngelvac constructs #7 and #34 was analysed with Sanger sequencing and compared to the reference sequence of the MLV Ingelvac PRRS (Genbank no AF066183.4). Similar to Porcilis PRRS, this vaccine is expected to contain several quasi-species, hence present inventors expect to see mutations in the PLLAV-lngelvac constructs compared to the theoretical sequence.

[0229] For clone #7 (comprising a PRRSV sequence as defined by SEQ ID NO: 3), a total of 15 mutations were observed, of which 6 were silent mutation. The remaining 9 mutations were found in Nsp2 (S485G - 1642V - N707D), Nsp7P (L74P), NsplO (V37A), 0RF2 (A122S), 0RF3 (T64A - 194V) and 0RF5 (V94A).

[0230] Clone #34 (comprising a PRRSV sequence as defined by SEQ ID NO: 4) showed 22 mutations compared to the reference sequence of Ingelvac PRRS, of which 10 were silent mutations. Three mutations were found in Nsp2 (V142I - V699G - N707D) and 0RF3 (T64A - 194V - I214T) and 1 in Nsp5 (H21Y), Nsp7P (G76E), C- terminal part of Nsp9 (K153R), 0RF5 (L145I) and 0RF6 (189V).

[0231] When present inventors compared the mutations of clone #7 with the mutations of clone #34, they saw that 3 mutations are found in both clones: N707D in Nsp2, T64A in 0RF3 and 194V in 0RF3.

[0232] Example 4. Applicability of the technology to other strains

[0233] A. Preparation of PLLAV-P129 and PLLAV-Fostera (Fig. 9)

[0234] In order to demonstrate the wider applicability of the present invention, the experiments for the preparation were repeated using the well-established P129 and Fostera strains.

[0235] 4.1 Materials and methods for the preparation of the plasmids

[0236] 4.1.1. Preparation of synthetic DNA fragments

[0237] Based on the reference sequences of P129 (Genbank no AF494042.1) and Fostera (Genbank no MK820650.1), for each virus 6 synthetic DNA fragments, covering the entire genome, were designed in silico and supplied by the manufacturer (IDT and Thermo). A PCR using KAPA HiFi HotStart ReadyMix (Roche) was done on synthetic fragment 1 to create an overhang with CMV-promoter of the pLLAV pVV6 plasmid and on fragment 6 to create an A25-tail and an overhang with the HDr of the PLLAV vector. In addition, to allow for stepwise assembly, a PCR was done on fragment 3 and 5 to insert a unique restriction enzyme site (Notl) at the 3' end of the fragment, and to create an overhang with the HDr of the PLLAV vector. To this end, 10 ng of the synthetic DNA fragment was mixed with 12.5 pl of KAPA HiFi HotStart ReadyMix mastermix, and 0.5 pM of each primer (see Table 4). The PCR reactions were analysed on a 1% UltraPure agarose gel (Thermo) and purified with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel).

[0238] Table 4: list of primers for PCR on the synthetic fragments. The same primers were used to amplify the synthetic fragments of P129 and Fostera. 4.1.2. Assembly of the PCR fragments in the linearized PLLAV plasmid

[0239] The pVV6 plasmid was cut with a unique restriction enzyme after the CMV promoter to linearize the vector. The assembly of the 6 synthetic DNA fragments was done in a stepwise manner: in a first reaction, the PCR of fragment 1, fragment 2 and the PCR of fragment 3 were mixed equimolarly with an equimolar amount of linearized PLLAV and NEBuilder HiFi DNA Assembly Master Mix according to manufacturer's protocol. The mixture was incubated for lh at 50°C, after which 2 pl was used to transform NEB 10-beta chemicompetent E. coli bacteria (NEB), according to manufacturer's protocol. The bacteria were plated on Lennox L agar (Thermo) plates containing kanamycine (50 pg / ml) as selection marker, and incubated overnight at 37°C. The transformed bacteria were screened for the presence of both fragment 1 and fragment 3 using colony PCR. Positive clones were used to inoculate 100 ml of Lennox L Broth Base (LB broth, Thermo) containing 50 pg / ml kanamycine. The inoculated cultures were incubated overnight in a shaking incubator at 37°C, after which the plasmids were harvested with the NucleoBond Xtra Midi EF kit (Macherey-Nagel) according to manufacturer's protocol. The obtained PLLAV-P129_gbl23 or PLLAV-Fostera_gbl23 was sent for sequencing to confirm that no mutations were introduced during the assembly reaction. Once a correct clone was identified, the PLLAV-constructs were digested with the inserted unique restriction enzyme Notl-HF (NEB) in an overnight digest at 37°C, followed by a purification step with NucleoSpin Gel and PCR Clean-up (Macherey-Nagel).

[0240] Next, present inventors performed a second assembly reaction with fragment 4 and the PCR of fragment 5 and the linearized plasmid PLLAV-P129_gbl23 or PLLAV-Fostera_gbl23. Fragment 4 and the PCR of fragment 5 was added in a 2:1 ratio with the linearized plasmid and NEBuilder Hi Fi DNA Assembly Master Mix. The samples were again incubated for lh at 50°C, followed by bacteria transformation as described above.

[0241] Similar to the first assembly reaction, correct clones containing PLLAV-P129_gbl2345 and PLLAV- Fostera_gbl2345 were identified after colony PCR and Sanger sequencing. The correct clones were again digested with Notl-HF, and after an overnight incubation at 37°C purified using the NucleoSpin Gel and PCR Clean-up (Macherey-Nagel).

[0242] Finally, the last assembly reaction was performed with the linearized PLLAV-vectors and the PCR on fragment 6, where fragment 6 was added in a 2:1 ratio to the reaction mixture with NEBuilder HiFi DNA Assembly Master Mix. All following steps are similar to the first assembly reaction described above. In the end, a midiprep was performed on the PLLAV-P129 and PLLAV-Fostera plasmids.

[0243] 4.1.3. Re-cloning of the Fostera genome in PLLAV

[0244] After the assembly of the 6 synthetic DNA fragments covering the Fostera genome in the PLLAV, present inventors re-introduced the Fostera genome in the PLLAV, to remove any mutations in the PLLAV that had appeared during the assembly reaction and which might interfere with the launch of the Fostera genome after transfection in cell culture. To this end, present inventors PCR amplified the 5' half region of Fostera and the 3' half region using the primers listed in Table 5. To this end, 10 ng of PLLAV-Fostera was mixed with 12.5 pl of KAPA HiFi HotStart ReadyMix mastermix, and 0.5 pM of each primer. The PCR mixture was then incubated with 1 pl of Dpnl (Thermo) for lh at 37°C to digest the residual PLLAV-Fostera template, after which they were purified with the NucleoSpin Gel and PCR Clean-up (Macherey-Nagel). Table 5: primers used to the amplify the Fostera genome in 2 PCR reactions

[0245] The 2 PCR fragments were assembled in the linearized empty pVV6-plasmid in a 2:1 ratio, using NEBuilder HiFi DNA Assembly Master Mix according to manufacturer's protocol. The reaction was incubated for lh at 50°C, after which 2 pl was used to transform NEB 10-beta chemicompetent E. coli bacteria (NEB), according to manufacturer's protocol. The remainder of the protocol is similar as described above.

[0246] 4.1.4. Sequencing of PLLAV-P129 and PLLAV-Fostera

[0247] Initially, 4 clones of PLLAV-P129 and 8 clones of PLLAV-Fostera were sent for Sanger sequencing (LGC Genomics). An addition 3 clones of PLLAV-Fostera were sent for sequencing after recloning Fostera in pVV6.

[0248] 4.1.5. Virus recovery

[0249] The same cell culture assays as described in for PLLAV-Porcilis and PLLAV-lngelvac was performed for PLLAV-P129 and PLLAV-Fostera.

[0250] 4.2 Construction details and results

[0251] 4.2.1. Design of PLLAV-P129 and PLLAV-Fostera

[0252] For the construction of PLLAV-P129 and PLLAV-Fostera, present inventors divided the PRRSV's genome into 6 more or less equal parts, which were synthetically manufactured. Each fragment overlaps with the next for at least 30 bp. Present inventors would introduce the 6 fragments in the PLLAV in a stepwise manner, starting with fragments 1, 2 and 3. To this end, present inventors performed a PCR reaction on fragment 3 to introduce, at the 3' end, a unique restriction enzyme site, and a sequence that overlaps with the HDr region of the plasmid (see Fig. 9). Once the first 3 fragments were correctly inserted into the PLLAV, present inventors digested the plasmid with the unique restriction enzyme to linearize it, and start another round of assembly. For the second round, present inventors introduced fragments 4 and 5. This required them to PCR-amplify the 5thsynthetic DNA fragment to create, at its 3' end, a unique restriction enzyme site and an overlapping sequence. Correct clones were digested with the unique restriction enzyme Notl. As present inventors couldn't order a synthetic DNA fragment with the Azs-tail,, present inventors performed a PCR on the 6thsynthetic fragment, with a reverse primer that contained this A25- tail and an overlapping region with the vector.

[0253] 4.2.2. Confirmation of the sequence

[0254] Given the approach described in the previous section, each assembly step was accompanied by thorough sequencing of the intermediate constructs to avoid the introduction of mutations. In the end, present inventors obtained 7 clones of PLLAV-P129, of which 4 were fully sequenced. Clones #1, 2 and 4 were identical to the sequence found in Genbank no AF494042.1. Clone #3 (comprising a PRRSV sequence as defined by SEQ ID NO: 5) had a point mutation in 0RF3, leading to a G83E mutation.

[0255] For PLLAV-Fostera, after the introduction of the first 3 fragments, present inventors analysed 7 clones for the correct sequence. Six clones contained non-silent mutations, including deletions or insertions. The only clone that had incorporated the correct Fostera sequence contained a point mutation in the CMV promoter and a silent mutation in Nsp2.

[0256] Given the large size of the plasmid PLLAV-Fostera, present inventors preferred to redone Fostera into pVV6 to restore the mutation in CMV, rather than performing a site-directed mutagenesis. Clone #11 was chosen for further testing, as this clone contained only 2 silent mutations: one in Nsp2, and one in 0RF5.

[0257] 4.2.3. Recovery of PRRSV P129 or Fostera in cell culture

[0258] Present inventors tested the 7 clones of PLLAV-P129, even though only 4 were fully sequenced. Five days post infection of MARC-145 cells, clones #5, 6 and 7 all gave less than 50% CPE. Clones #1, 2 and 4, which all contained the correct sequence of P129, gave between 50- 70% CPE. Remarkably, clone #3 (comprising a PRRSV sequence as defined by SEQ ID NO: 5), which had the G83E mutation in 0RF3, showed the best results, with 90% of CPE after 5 days.

[0259] For PLLAV-Fostera, the initial clones contained a point mutation in the CMV promoter, of which present inventors did not know the effect on the launch of the Fostera genome. Quite to their surprise, 7 out of 8 clones of PLLAV-Fostera showed signs of CPE 6 days post infection of MARC-145 cells. The only clone that gave negative results, contained a mutation affecting both 0RF3 and 0RF4.

[0260] Despite the fact that the mutation in the CMV promoter did not appear to affect the launch of PRRSV Fostera after transfection / infection in cell culture, present inventors re-cloned the Fostera genome into the empty plasmid pVV6. The resulting constructs did not contain the mutation in the CMV promoter, but did contain silent mutations in the Fostera genome, namely in Nsp2 and 0RF5. This clone (#11) (comprising a PRRSV sequence as defined by SEQ ID NO: 6) was tested in cell culture and present inventors could observe CPE in infected MARC-145 cells 5-6 days post infection.

[0261] B. Immunogenicity study of PLLAV-Fostera in pigs

[0262] 4.3 Materials and methods

[0263] A total of ten pigs were included in this study. The piglets were approximately 4-5 weeks old and Norsvin Landrace crossbred with Z-line with an average bodyweight of 7.72 ± 0.90 kg. The pigs were randomized into 2 groups (n=5 / group). All pigs were subjected to a prime-boost vaccination: on day 0 all pigs received a prime vaccination, followed by a booster vaccination on day 21. Animals were either vaccinated with the live-attenuated PRRSV type II strain Fostera (in-house produced and abbreviated as "LAV-Fostera") or with the plasmid-launched live attenuated porcine reproductive and respiratory syndrome virus type II vaccine based on the Fostera strain (abbreviated as "PLLAV-Fostera", i.e. PLLAV-Fostera #11 of which the PRRSV sequence is as defined in SEQ ID NO: 6), although dose and administration route differed. The design of the study is visualized in Fig. 10.

[0264] Animals of the LAV-Fostera group (n=5) were vaccinated intramuscularly in the neck area with the full 2mL at 10A5 TCID50 (50% tissue culture infective dose) using a needle and a syringe. Animals of the PLLAV- Fostera group (n=5) were administered transdermally (t.d.) 900 pg of PLLAV-Fostera. Transdermal administration was done using a needle-free delivery device at the left side of the neck area.

[0265] The induction of humoral (anti-PRRSV antibodies) immunity was assessed at different timepoints post vaccination. Hereto, serum samples collected at -3, 7, 14, 20, 28, 34, 41, 49 and 56 days post vaccination (dpv) were tested in a commercial enzyme-linked immunosorbent assay (ELISA), designed to detect anti- PRRSV antibodies according to the manufacturer's instructions (pigtype PRRSV Ab; Cat. No.: PT272753; INDICAL).ln addition, a commercial enzyme-linked immune absorbent spot (ELISpot) assay was performed (Pig IFN-y Single-Color ELISPOT, ImmunoSpot) on peripheral blood mononuclear cells (PBMCs) isolated from whole blood samples collected on 7, 20, 34, 41 and 55 dpv to evaluate a PRRSV type II targeted cellular immune response.

[0266] 4.4. Results

[0267] 4.4.1. General safety and tolerability observations

[0268] The body weights of all animals in both groups steadily increased over time. There was no evidence of any local or systemic reaction in any of the animals throughout the entire in-life phase of the study. Group mean rectal temperatures remained below the threshold of 40.0°C (fever cutoff value) for all animals during the entire in-life phase of the study Based on clinical observations, administration site assessment and body weight measurements and rectal temperature recording, no safety concerns were identified following administration of PLLAV-Fostera.

[0269] 4.4.2. PLLAV-Fostera vaccination induces anti-PRRSV antibodies after transdermal needle-free delivery

[0270] The results of the ELISA are summarized in Fig. 11. Overall, 4 out of 5 pigs of the LAV-Fostera and PLLAV- Fostera groups tested positive for anti-PRRSV binding antibodies at multiple consecutive timepoints. Seroconversion in these two groups was observed as early as 14 days post vaccination (hence 7 days prior to booster vaccination on day 21).

[0271] 4.4.3. PLLAV-Fostera vaccination induces an IFN-qamma response post primo vaccination

[0272] To assess cellular immunity responses post-vaccination, PBMCs were isolated on 7, 20, 34, 41 and 56 days post vaccination and re-stimulated in vitro with PRRSV type II, followed by an evaluation of IFN-y production by these cells. The IFN-y response was considered as proxy for a PRRSV type Il-targeted cellular immune response. The results of the ELISpot are summarized in Fig. 12.

[0273] IFN-y responses were observed in all animals of both groups as early as on day 20 (hence prior to booster vaccination on day 21). PLLAV-Fostera vaccination via the needle-free transdermal route induces a consistent IFN-gamma response post primo vaccination.

Claims

CLAIMS1. A vector for use as a medicament wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae.

2. The vector for use according to claim 1, wherein the medicament is a vaccine.

3. The vector for use according to claim 1 or 2, wherein the virus of the family Arteriviridae is a porcine reproductive and respiratory syndrome virus (PRRSV).

4. The vector for use according to any one of claims 1 to 3, wherein said vector is a bacterial artificial chromosome (BAC).

5. The vector for use according to any one of claims 1 to 4, wherein said vector further comprises an inducible bacterial origin of replication (ori) for amplifying said vector to a high copy number per bacterial host cell in the presence of one or more inducers of said ori.

6. A vector for use in the treatment and / or prevention of a PRRSV infection in a subject, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated PRRSV virus genome operably linked to a promoter.

7. The vector for use according to any one of claims 2 to 6, wherein the PRRSV is a type I PRRSV or a type II PRRSV.

8. The vector for use according to any one of claims 2 to 7, wherein the PRRSV is a Lelystad strain, a P129 strain, a VR-2332 strain, or a derivative thereof.

9. The vector for use according to any one of claims 1 to 8, wherein said viral expression cassette comprises: a cDNA of an attenuated RNA virus genome, wherein the RNA virus is a virus of the family Arteriviridae, and a RNA polymerase driven promoter at the 5' end of said cDNA, preferably an RNA polymerase II promoter.

10. The vector for use according to any one of claims 1 to 9, wherein said vector is administered to a subject and wherein said subject is member of the family Suidae, preferably a member of the genus Sus, more preferably a wild or domestic pig or a swine.

11. The vector for use according to any one of claims 1 to 10, wherein said vector is administered intradermally, intramuscular, transdermally, intranasally, orally or subcutaneously to a subject.

12. The vector for use according to any one of claims 1 to 11, wherein a dose of from 100 to 2000 pg, preferably from 300 to 900 pg, of said vector is administered to a subject.

13. The vector for use according to any one of claims 1 to 12, wherein the vector is administered to a subject in a single-dose or double-dose regime.

14. A pharmaceutical composition comprising a vector and a pharmaceutically acceptable carrier, wherein said vector comprises a viral expression cassette comprising a cDNA of an attenuated RNA virus genome operably linked to a promoter, wherein the RNA virus is a virus of the family Arteriviridae, and preferably wherein said pharmaceutical composition is a vaccine.

15. A method of preparing a vaccine against an RNA virus of the family Arteriviridae comprising the steps of: a) providing a host cell transfected with a vector as defined in any one of claims 1 to 9, b) amplifying the vector, c) isolating the amplified vector, and d) formulating the isolated vector into a vaccine.

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