WO2020128496A1

WO2020128496A1 - Peste des petits ruminant virus (pprv) with chimeric n protein and corresponding vaccine

Info

Publication number: WO2020128496A1
Application number: PCT/GB2019/053641
Authority: WO
Inventors: Satya PARIDA; Mana MAHAPATRA; Maniswaran SELVARAJ
Original assignee: The Pirbright Institute
Priority date: 2018-12-21
Filing date: 2019-12-20
Publication date: 2020-06-25
Also published as: GB201820983D0

Abstract

The present invention provides a peste des petits ruminants virus (PPRV) comprising a chimeric N protein wherein the chimeric N protein comprises a variable C-terminus domain which comprises at least 50 amino acids from the variable C-terminus domain of a N protein from a second morbillivirus which is not PPRV.

Description

PESTE DES PETITS RUMINANT VIRUS (PPRV) WITH CHIMERIC N PROTEIN AND CORRESPONDING

VACCINE

FIELD OF THE INVENTION

The present invention relates to peste des petits ruminants virus (PPRV) and vaccines comprising said PPRV. In particular, the present invention relates to PPRV vaccines which provide an ability to differentiate infection in vaccinated animals (DIVA).

BACKGROUND TO THE INVENTION

Peste des petits ruminants (PPR) is an important infectious viral disease of domestic and wild small ruminants that threatens the food security and sustainable livelihood of farmers across Africa, the Middle East and Asia. Europe is free of the disease, except in Thrace (European part of Turkey) and Israel where outbreaks occur. An outbreak of PPR has recently been reported in Bulgaria in June, 2018. Following the successful eradication of Rinderpest (RP), PPR has been targeted by the OIE and FAO as the next viral pathogen to be eradicated by 2030.

PPR is currently controlled by vaccination mainly using two live attenuated PPRV vaccines (Nigeria 75/1 and Sungri 96). Similarly, two competitive ELISA (c-ELISA) kits are used to monitor the antibody response against the H and N proteins of PPRV. However, the critical drawback of these vaccines and associated c-ELISA kits is their inability to differentiate infection in vaccinated animals (DIVA) that slows down the implementation of PPR control through vaccination. The vaccinated animals develop a full range of immune responses to viral proteins and therefore cannot be distinguished serologically from those that have recovered from natural infection. This factor precludes meaningful assessment of vaccine coverage and epidemiological surveillance based on serology, in turn reducing the efficiency of control/eradication programme. Therefore, it is almost impossible to assess the quality and efficacy of existing PPR vaccines without knowing whether positive animals are vaccinated or infected. Unlike rinderpest, where cattle and buffalo are primary hosts, in PPR new crops (about 30-40%) of lambs and kids are produced every year and are the most susceptible population to bring back PPR outbreaks. This poses a serious problem for sero- surveillance programs. Furthermore, during the latter stages of any eradication programme use of a marker vaccine and associated DIVA diagnostics will enable the assessment of vaccine efficacy which is paramount for any successful vaccination campaign.

As an approach to achieve a DIVA target, an efficient reverse genetics system for PPRV Nigeria 75/1 vaccine strain was established and a version of PPRV Nigeria 75/1 vaccine strain with mutations in the haemagglutinin (H) gene was rescued. The rescued virus showed similar growth characteristics in vitro in comparison to parent vaccine strain and, following in vivo assessment the H mutant provided full protection in goats. However, although the C77 monoclonal antibody used in the c-ELISA was unable to bind to the mutated form of H in vitro , the mutation was not sufficient to enable DIVA in vivo (Muniraju, et al 2015; Vaccine 33, 465-471).

Accordingly, there remains a need for further PPRV vaccines and particularly PPRV vaccines which enable DIVA in vivo.

SUMMARY OF THE INVENTION

The present invention is based on the inventors’ surprising determination that a PPRV with a chimeric N protein retains the ability to be rescued using reverse genetic methods, and replicate and induce an immune response in a host. Further, the introduction of a chimeric N protein which comprises a variable C-terminus domain comprising a relatively large number of amino acids from a non-PPRV morbillivirus also enables in vivo DIVA. This is in contrast to prior art approaches in which minimal amino acid changes in the epitope of monoclonal antibodies used in commercial tests was not sufficient to enable DIVA in vivo.

Accordingly, in a first aspect the present invention provides a PPRV comprising a chimeric N protein wherein the chimeric N protein comprises a variable C-terminus domain which comprises at least 50 amino acids from the variable C-terminus domain of a N protein from a second morbillivirus which is not PPRV.

The present invention further provides a chimeric PPRV N protein according to the first aspect of the invention.

The invention also provides a nucleic acid sequence encoding a chimeric PPRV N protein according to the present invention.

The invention also provides a plasmid or a construct comprising a nucleic acid sequence according to the invention.

The present invention further provides a cell comprising a nucleic acid sequence, a plasmid or a construct according to the invention.

The invention also provides a vaccine comprising a PPRV, a chimeric PPRV N protein, a nucleic acid sequence or a plasmid according to the invention. In one embodiment, the present invention provides a vaccine comprising a PPRV according to the first aspect of the invention. The invention further relates to a method for treating and/or preventing a disease in a subject which comprises the step of administering a vaccine according to the invention to the subject. Preferably, the method is for preventing a disease in a subject.

The invention also provides a vaccine according to the invention for use in treating and/or preventing a disease in a subject.

The invention further relates to the use of a PPRV, a chimeric PPRV N protein, a nucleic acid sequence or a plasmid of the invention in the manufacture of a medicament for treating and/or preventing a disease in a subject.

The invention further relates to a method for making a PPRV of the invention which comprises the following steps: (i) introducing a plasmid according to the invention and one or more helper plasmids which between them encode the PPRV N, P and L proteins into a host cell; (ii) culturing the host cell under conditions in which virus is produced.

The invention further provides an antibody or antigen binding fragment thereof which (i) selectively binds a chimeric PPRV N protein according to the present invention; or (ii) selectively binds a native PPRV N protein.

The antibody or antigen binding fragment thereof may comprise complementary determining regions (CDRs) selected from: SEQ ID NOs: 15-17; SEQ ID NOs: 18-20; SEQ ID NOs: 21- 23; SEQ ID NOs: 24-26; SEQ ID NOs: 27-29; SEQ ID NOs: 30-32; SEQ ID NOs: 33-35; SEQ ID NOs: 36-38; SEQ ID NOs: 39-41 ; SEQ ID NOs: 42-44; SEQ ID NOs: 45-47; SEQ ID NOs: 48-50; SEQ ID NOs: 51-53; and/or SEQ ID NOs: 54-56 or derivatives thereof.

The antibody or antigen binding fragment thereof may comprise a combination variable heavy and variable light CDRs selected from the group consisting of:

(i) SEQ ID NOs: 15-17 and SEQ ID NOs: 18-20, or derivatives thereof;

(ii) SEQ ID NOs: 21-23 and SEQ ID NOs: 24-26, or derivatives thereof;

(iii) SEQ ID NOs: 27-29 and SEQ ID NOs: 30-32, or derivatives thereof;

(iv) SEQ ID NOs: 33-35 and SEQ ID NOs: 36-38, or derivatives thereof;

(v) SEQ ID NOs: 39-41 and SEQ ID NOs: 42-44, or derivatives thereof;

(vi) SEQ ID NOs: 45-47 and SEQ ID NOs: 48-50, or derivatives thereof; and

(vii) SEQ ID NOs: 51-53 and SEQ ID NOs: 54-56, or derivatives thereof.

The antibody or antigen binding fragment thereof may comprise a combination of a variable heavy domain and a variable light domain selected from the group consisting of: (i) an amino acid sequence which has at least 80% identity to SEQ ID NO: 57 and an amino acid sequence which has at least 80% identity to SEQ ID NO: 58;

(ii) an amino acid sequence which has at least 80% identity to SEQ ID NO: 59 and an amino acid sequence which has at least 80% identity to SEQ ID NO: 60;

(iii) an amino acid sequence which has at least 80% identity to SEQ ID NO: 61 and an amino acid sequence which has at least 80% identity to SEQ ID NO: 62;

(iv) an amino acid sequence which has at least 80% identity to SEQ ID NO: 63 and an amino acid sequence which has at least 80% identity to SEQ ID NO: 64;

(v) an amino acid sequence which has at least 80% identity to SEQ ID NO: 65 and an amino acid sequence which has at least 80% identity to SEQ ID NO: 66;

(vi) an amino acid sequence which has at least 80% identity to SEQ ID NO: 67 and an amino acid sequence which has at least 80% identity to SEQ ID NO: 68; or

(vii) an amino acid sequence which has at least 80% identity to SEQ ID NO: 69 and an amino acid sequence which has at least 80% identity to SEQ ID NO: 70.

The invention further provides a kit comprising:

(A) (i) a vaccine according to the invention; and (ii) (a) a chimeric PPRV N protein which is expressed by the PPRV of the vaccine of part (i), a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; or (b) a polypeptide comprising the variable C- terminus domain of the N protein of the chimeric PPRV N protein which is expressed by the PPRV in the vaccine of part (i), or a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; or

(B) (i) a vaccine according to the invention; and (ii) a reagent which is capable of specifically binding to either the chimeric PPRV N protein which is expressed by the PPRV of the vaccine of part (i) or a non-chimeric PPRV N protein;

(C) (i) a chimeric PPRV N protein according to the invention, a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; or a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein according to the invention, a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; and (ii) a detectable reagent which is (a) capable of binding to an anti-chimeric PPRV N protein antibody or (b) capable of specifically binding to the chimeric PPRV N protein; or

(D) (i) a chimeric PPRV N protein according to the invention, a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; or a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein according to the invention, a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; (ii) a capture reagent which is (a) capable of binding to an anti-chimeric PPRV N protein antibody or (b) capable of specifically binding to the chimeric PPRV N protein and (iii) a detectable reagent which is (a) capable of binding to an anti-chimeric PPRV N protein antibody or (b) capable of specifically binding to the chimeric PPRV N protein.

In an alternative embodiment, the kit described in part (D) may be directed to a native PPRV N protein. As such, the protein, polynucleotide, plasmid or construct and antibody(ies) may be directed to a native PPRV N protein instead of a chimeic PPRV N protein of the present invention.

The reagent which is capable of specifically binding to either the chimeric PPRV N protein of the present invention or a non-chimeric PPRV N protein may be an antibody according to the present invention.

The detectable reagent which is capable of specifically binding to either the chimeric PPRV N protein of the present invention or a non-chimeric PPRV N protein may be an antibody according to the present invention.

In another aspect the present invention relates to a method of distinguishing a subject that has been vaccinated with a vaccine according to the invention from a subject that has been infected with a non-chimeric PPRV; which method comprises contacting a chimeric PPRV N protein according to the invention or a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein according to the invention with (i) a sample from the subject; and (ii) a detectable reagent which is (a) capable of binding to an anti-chimeric PPRV N protein antibody present in the sample or (b) capable of specifically binding to the chimeric PPRV N protein or native PPRV N protein.

The detectable reagent of part (ii) which is capable of specifically binding to the chimeric PPRV N protein or native PPRV N protein may be an antibody according to the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : A) Mean antibody titre against H-protein following vaccination by c-ELISA. PI values above 50% are considered positive. B) Mean serum neutralising antibody titre of PPR-DIVA and PPRV vaccinated animals on 28 day post vaccination. C) Mean antibody titre against native PPRV N (Nv) and mutated N (mNv) in DIVA ELISA. D) Detection of PPRV genome in nasal excretions following challenge. Figure 2: A) Uninduced and induced cell lysates from PPRVNv and DMVNv protein were run on 12% SDS phage. M- Marker ; Lane 1 -uninduced PPRVNv protein; lane 2 -induced PPRVNv protein; Lane 3 -uninduced DMVNv protein; lane 4 -induced DMVNv protein. B) E. coli strain M15 was transformed with the expression plasmid encoding DMVNv protein and grown. Cell lysates were prepared and analysed on a 12% SDS-PAGE gel. The gel was stained with Coomassie blue and photographed. M, Marker; lane 1 , cell lysate from M15 control cells; lanes 2, cell lysate from clone expressing DMVN ₂o protein; lane 3 sonicated cell lysate; lane 4 flow through; lane 5 wash through; lane 6 Elutel ; lane 7 Elute 2; lane 8 Elute 3.

DETAILED DESCRIPTION OF THE INVENTION

Peste des petits ruminants virus (PPRV) and Peste des petits ruminants (PPR)

PPR is a viral disease of goats and sheep characterized by fever, sores in the mouth, diarrhoea and pneumonia. PPR can cause death of the subject. PPR is a disease listed in the Ol E Terrestrial Animal Health Code, and countries are obligated to report the disease to the OIE according to the criteria (OIE Terrestrial Animal Health Code). The disease primarily occurs in a band that spreads across Africa between the equator and the Sahara, through the Arabian Peninsula, the Middle East, south-west Asia, South Asia including India and recently China and Mongolia. PPR spread into North Africa for the first time in Morocco in 2008 and to Bulgaria in 2018.

PPR is caused by PPRV. The virus is secreted in tears, nasal discharge, secretions from coughing, and in the faeces of infected animals. Therefore, close contact between animals, especially through inhalation of fine droplets that are released into the air when affected animals cough and sneeze will spread the disease. Water, feed troughs, and bedding can also be contaminated with secretions and become additional sources of infection, however the virus does not survive for a long time outside the body of a host animal. Since animals excrete the virus before showing signs of the disease, it can spread by movement of infected animals.

PPRV belongs to the genus Morbillivirus of the family Paramyxoviridae and is an enveloped, negative-sense, single-stranded RNA virus with a single serotype. The PPRV genome is 15,948 nucleotides (nt) in length, but longer variants have been sequenced in outbreaks from China. The diameter of PPR virions ranges from 400 to 500 nm. The phosphoprotein (P) acts as a co-factor of large protein (L), which is the viral RNA dependent RNA polymerase (RdRp). There are three proteins associated with the host cell membrane- derived viral envelope. The matrix (M) protein acts as a link, which associates with the nucleocapsid and the two external viral proteins, the fusion (F) protein and the HN protein. The thickness of the PPRV envelope varies from 8 to 15 nm and the length of the surface glycoproteins ranges from 8.5 to 14.5 nm. Four distinct lineages of PPRV (I to IV) have been identified which correspond to the geographical distribution of the virus. Their classification is based on the nucleoprotein (N) or previously the fusion (F) protein gene. Lineages I and II are found mainly in West Africa. Lineage III is generally found in Middle East and East Africa. Lineage IV was long known as the Asian lineage, but has now spread to the African continent and become the most prevalent lineage of all.

The PPRV genome carries six transcriptional units; each encodes for a contiguous and non overlapping protein except the P gene, which also expresses C and V nonstructural proteins by an alternative open reading frame and RNA editing, respectively. All the genes in PPRV are arranged in an order of 3'-N-P/C/V-M-F-HN-L-5'.

Morbilliviruses such as PPRV replicate extensively within lymphoid and epithelial cells and the signaling lymphocyte activation molecule (SLAM) is a well-established receptor for morbillivirus infection.

PPR is currently controlled by vaccination using mainly two live attenuated PPRV vaccines (Nigeria 75/1 and Sungri 96).

Chimeric N Protein

The N protein surrounds the genomic RNA along with two other viral proteins, the L protein and the P protein to form the ribonucleoprotein (RNP). This RNP core encloses the entire genome of PPRV and protects from endonuclease digestion. Each molecule of N protein is associated with the six nucleotides of the genome, in accordance with the requirement of ‘the rule of six’ for paramyxoviruses including PPRV. Given that the PPRV genome contains 15,948 nucleotides (multiple of six bases), 2650 copies of N proteins are required to completely wrap up the genome. Electron microscopic analysis indicates that approximately 13 copies of the N protein constitute a single helix, and therefore a genome would involve around 200 turns of the nucleocapsid helix.

The N protein is 525 amino acids. The N protein of PPRV is likely to interact with other N proteins (N-N interaction), with the P protein (N-P interaction) and with polymerase units (P-L interaction) to take part in the replication complex. The N protein plays an essential role in the replication of PPRV.

It has been demonstrated that two domains, one at the N-terminus (1-120) and one in the central region (146-241), are responsible for the PPRV N-N self-assembly. Additionally, a short fragment in the N protein at amino acid 121-145 is essential for the stability of the resultant nucleocapsid structure.

Morbillivirus N proteins have defined regions with varying degrees of homology. The N- terminal part of the protein very well conserved, whilst the C-terminal region varies between related viruses. The present invention is based on the inventors’ determination that a PPRV comprising a chimeric N protein which comprises a variable C-terminus domain with at least 50 amino acids from the variable C-terminus domain of an N protein from a second morbillivirus which is not PPRV is capable of forming rescued virus, infecting and inducing an immune response in a host. Importantly, the present inventors have shown that infection by vaccination with the present PPRV can be distinguished from natural infection by a field PPRV. The present PPRV therefore enables DIVA in v/Vo when used in a vaccine.

Accordingly, in a first aspect, the present invention provides a PPRV comprising a chimeric N protein wherein the chimeric N protein comprises a variable C-terminus domain which comprises at least 50 amino acids from the variable C-terminus domain of an N protein from a second morbillivirus which is not PPRV.

Illustrative amino acid sequences for PPRV N proteins are provided by the Sungri 96 PPRV and Nigeria 75/1 N proteins as follows:

SEQ ID NO: 1 - Sunari 96 PPRV N protein

MATLLKSLALFKRNKDKAPTASGSGGAIRGIKNVI IVPIPGDSS I ITRSRLLDRLVRLAGDPDINGSK LTGVMI SMLSLFVESPGQLIQRITDDPDVS IRLVEVVQSTRSQSGLTFASRGADLDNEADMYFSTEGP SSGGKKRINWFENREI IDIEVQDPEEFNMLLASILAQVWILLAKAVTAPDTAADSELRRWVKYTQQRR VIGEFRLDKGWLDAVRNRIAEDLSLRRFMVSLILDIKRTPGNKPRIAEMICDIDNYIVEAGLASFILT IKFGIETMYPALGLHEFAGELSTIESLMNLYQQLGEVAPYMVILENSIQNKFSAGAYPLLWSYAMGVG VELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSVIAAELGITAEEAKLVSEIASQAGDERTAR GTGPRQAQVSFLQHKTGEGESSAPATREGVRAAIPNGSEERDRKQTRPGRPKGETPGQLLPEIMPEDE VPRESGQNPREAQRSAEALFRLQAMAKILEDQEEGEDNSQVYNDKDLLG

SEQ ID NO: 2 - Nigeria 75/1 N protein

MATLLKSLALFKRNKDKAPTASGSGGAIRGIKNVI IVPIPGDSS I ITRSRLLDRLVRLAGDPDINGSK LTGVMI SMLSLFVESPGQLIQRITDDPDVS IRLVEVVQSTRSQSGLTFASRGADLDNEADMYFSTEGP SSGSKKRINWFENREI IDIEVQDAEEFNMLLASILAQVWILLAKAVTAPDTAADSELRRWVKYTQQRR VIGEFRLDKGWLDAVRNRIAEDLSLRRFMVSLILDIKRTPGNKPRIAEMICDIDNYIVEAGLASFILT IKFGIETMYPALGLHEFAGELSTIESLMNLYQQLGEVAPYMVILENSIQNKFSAGAYPLLWSYAMGVG VELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSVIAAELGITAEEAKLVSEIASQTGDERTVR GTGPRQAQVSFLQHKTDEGESPTPATREEVKAAIPNGSEGRDTKRTRSGKPRGETPGPLLPEIMQEDE LSRESSQNPREAQRSAEALFRLQAMAKILEDQEEGEDNSQIYNDKDLLS

The variable C-terminus domain of the Sungri 96 PPRV and Nigeria 75/1 N proteins are shown as positions 406 to 525 of SEQ ID NO: 1 and SEQ ID NO: 2, respectively (shown as underlined text in SEQ ID NO: 1 and 2 above). The amino acid sequence for the Dolphin Morbillivirus (DMV) N protein is shown as SEQ ID NO: 3.

SEQ ID NO: 3 - Dolphin Morbillivirus (DMV) N protein

MATLRRSLALFKRNKDRTPLIAGSGGAIRGIKHVIVVPVPGDSS IVTRSRLLDRLVRLAGDPYI SGPK LTGVMI SILSLFVESPSQLIQRITDDPDVS IRLVEVIQSEKSLSGLTFASRGANMEDEADDYFS IQAG EEGDTRGTHWFENKEIVEIEVQDPEEFNILLASILAQIWILLAKAVTAPDTAADSETRRWIKYTQQRR WGEFRLDKGWLDAVRNRIAEDLSLRRFMVALILDIKRTPGNKPRIAEMICDIDTYIVEAGLASFILT IKFGIETMYPALGLHEFSGELTTVESLMNLYQQMGETAPYMVILENSIQNKFSAGSYPLLWSYAMGVG VELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSSLAAELGITAEDAKLVSEIAAQANDDRANR AIGPKQNQISFLHPDRGDASTPGNILANEGDGSTRMKRGGNIAQPKPTSIDQESTTQSKDTLDIEDQS DENTDDPI SIQKSAEALAKMRAMAKLLENQGPRDVTAHVYNDKDLLG

The variable C-ter inus domain of the DMV N protein is shown as positions 406 to 523 of SEQ ID NO: 3, respectively (shown as underlined text in SEQ ID NO: 3 above).

The variable C-terminus domain of the N protein corresponds to the positions shown as 406 to 525 of SEQ ID NO: 1 or 2. In other words, the variable C-terminus domain of the N protein of a morbillivirus is the amino acid sequence of the positions shown as 406 to 525 when the amino acid position numbering is identified by alignment of the N protein with the sequence of SEQ ID NO: 1 or 2. It will be appreciated that the actual number of the amino acid from the N-terminus of the protein may vary between N proteins of different morbilliviruses. However, it is clear from an alignment of the N proteins with the sequence of SEQ ID NO: 1 or 2 which is the "equivalent" amino acid position.

The present chimeric N protein comprises at least 50 amino acids from the variable C- terminus domain from a second morbillivirus which is not PPRV.

The chimeric N protein may comprise at least 60, at least 65, at least 70, at least 75, at least 80, at least 90, at least 100, or at least 115 amino acids from the variable C-terminus domain of a second morbillivirus which is not PPRV.

The chimeric N protein may comprise 55 to 115, 60 to 115, 70 to 115, 80 to 115, 90 to 115, or 100 to 115 amino acids from the variable C-terminus domain of a second morbillivirus which is not PPRV.

The chimeric N protein may comprise 55 to 80, 60 to 80, 65 to 80, 70 to 80 or 75 to 80 amino acids from the variable C-terminus domain of a second morbillivirus which is not PPRV.

Amino acids from the variable C-terminus domain of the PPRV N protein may be substituted with corresponding amino acids from a second morbillivirus which is not PPRV by any suitable method, for example, using site-directed mutagenesis. The variable C-terminus domain of the PPRV N protein may be substituted with the variable C-terminus domain from the N protein of a second morbillivirus which is not PPRV by genetic engineering and molecular biology techniques (e.g. restriction digestions, PCR and cloning).

Accordingly, the chimeric PPRV N protein is not a naturally occurring N protein. The polypeptide sequence of the N protein of the present invention has been modified to comprise an amino acid sequence which is not naturally present in a wild-type PPRV N protein.

The present PPRV is capable of forming rescued virus particles. The present PPRV is capable of replication in cell culture and in host (e.g. goats) and inducing an immune response in a host.

Methods for rescuing PPRV are known in the art and are described, for example, by Muniraju, et at. (2015; Vaccine 33, 465-471) and in the present Examples. Productiven PPRV replication may be determined by assessment of in vitro growth kinetics of the PPRVs, for example as described in the present Examples. The ability of the PPRV to induce an immune response in a host may be determined by assessing the anti-PPRV antibody titre in the host following administration of the PPRV (as described in the present Examples, for example). For example, the ability of a PPRV to induce an immune response may comprise determining the presence of anti-PPRV H protein antibodies in a sample from the subject.

Suitably, the second morbillivirus may be any morbillivirus provided that introducing the variable C-terminus domain of the N protein of that morbillivirus - or a variant thereof - provides a PPRV which is capable of forming rescued virus particles, and is capable of replication, infection of a host and inducing an immune response in a host.

The variable C-terminus domain of the N protein may be from dolphin morbillivirus (DMV).

The variable C-terminus domain may comprise the amino acid sequence shown as SEQ ID NO: 4 or a variant thereof. Suitably, the variant may have at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 4.

SEQ ID NO: 4 - Dolphin Morbillivirus (DMV) N protein variable C-terminus domain

ANRAIGPKQNQISFLHPDRGDASTPGNILANEGDGSTRMKRGGNIAQPKPTSIDQESTTQSKDTLDIE DQSDENTDDPISIQKSAEALAKMRAMAKLLENQGPRDVTAHVYNDKDLLG

The N-terminus domain of the N-protein may comprise the N-terminus domain of any PPRV.

The chimeric N protein of the present invention may comprise an N-terminal domain from a PPRV. For example, the chimeric N protein of the present invention may comprise an N- terminal domain from the Sungri 96 PPRV and Nigeria 75/1 N protein shown as SEQ ID NO: 1 and 2 or a variant thereof. The N-terminal domain of the Sungri 96 PPRV and Nigeria 75/1 N proteins is shown as positions 1 to 405 of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. Suitably, the variant may have at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to the amino acid sequence shown as positions 1 to 405 of SEQ ID NO: 1 or SEQ ID NO: 2.

Suitably, the N-terminus domain of the N-protein may comprise the N-terminus domain of the Sungri 96 PPRV or Nigeria 75/1 N proteins or a variant of the N-terminus domain of the Sungri 96 PPRV or Nigeria 75/1 N proteins. Accordingly, the N-terminus domain of the N- protein may comprise the amino acid sequence shown as SEQ ID NO: 5 or 6; or a variant of SEQ ID NO: 5 or 6. Suitably, the variant may have at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 5 or 6.

Other known PPRV strains include, but are not limited to, KJ867542.1_ isolate_Sungri_1996_MSD_(The_Netherlands),KM091959.1_isolate_China/XJYL/2013,X74 443.2_complete_genome, KY967608.1_SRMV/Lahore/UVAS/Pak/2015, KY967609.1_SRMV/ Faisalabad/UVAS/Pak/2015, KY967610.1_SRMV/Layyah/UVAS/Pak/2015, M F678816.1_100 8,KY628761.1_Small_ruminant_morbillivirus_strain_75/1 ,MG581412.1_PPRV/Bangladesh/B D2/2008, M F741712.1_PPRV/Sierra_Leone/048/2011 , MF737202.1_Georgia/Tbilisi/2016, KY888168.1_ PPRV/Mongolia/9/2016, KF727981.2_ isolate_Sungri/96, KY885100.1_ S15, KT860063.1_strain_IND/TN/VM/2014/02, KT860064.1_strain_IND/TN/VEL/2015/03, KT86006 5.1_strain_IND/TN/ED/2015/04,KX354359.1_isolate_PPRVFY,KX033350.1_strain_IND/Delh i/2016/05, KU236379.1_isolate_Lib/2015,KR828813.1_isolate_NGYO20132162,KR828814.1 _isolate_NGKW2012MSLN,KT633939.1_isolate_China/XJBZ/2015,KR781449.1_isolate_Be nin/10/2011 ,KR781450.1_isolate_Benin/B1/1969,KM816619.1_isolate_GZL14,KP868655.1_ isolate_CH/GDDG/2014,KR781451.1_isolate_CIV/01 P/2009, KT270355.1_strain_IND/TN/GI N/2014/01 , KR261605.1_isolate_lndia/TN/Gingee/2014, KR140086.1_ strain_lzatnagar/94, KP789375.1_isolate_E32/1969,KM089830.1_isolate_CH/HNNY/2014,KM089831.1_isolate_ CH/HNZK/2014,KM089832.1_ isolate_CH/HNZM/2014, KP260624.1_ strain_China/BJ/2014, KM463083.1_ isolate_KN5/201 1 , KJ867540.1_ isolate_Ethiopia_1994, KJ867541.1_ isolate_Ethiopia_2010, KJ867544.1_ isolate_Oman_1983, KJ867545.1_ isolate_UAE_1986, KJ867543.1_ isolate_Uganda_2012, KJ466104.1_ strain_Ghana/NK1/2010, KM212177.1_ isolate_SnDk1 1113, KC594074.1_isolate_Morocco_2008,JX217850.1_strain_Tibet/Bharal/20 08, JF939201.1_isolate_China/Tib/07,HQ197753.1_strain_Nigeria/75/1 ,FJ905304.1_strain_ China/Tibet/Geg/07-30, EU360596.1_ isolate_China/Tibet/0701_(N), EU267273.1_ strain_ICV89,EU267274.1_strain_Ng76/1 ,AJ849636.2_PPRV_complete_genome,NC_0063 83.2_PPRV_complete_genome, KX421384.1_China/2/2013, KX421385.1_China/3/2013, KX4 21386.1_China/4/2013.KX421387.1_China/5/2013, KX421388.1_China/33/2007,MF443335. 1_ChinaZJ2014,MF443336.1_ChinaYN2014,MF443337.1_ChinaSX2014,MF443338.1_Chin aSC2014, MF443339.1_ChinaSaX2014, MF443340.1_ ChinaNX2014, MF443341.1_

ChinaLN2014, MF443342.1_ChinaJX2014, MF443343.1_ ChinaJS2014, MF443344.1_ ChinaJL2014, MF443345.1_ChinaHN2014, MF443346.1_ ChinaHLJ2014, MF443347.1_ ChinaHeN2014,MF443348.1_ChinaHB2014,MF443349.1_ChinaGZ2014,MF443350.1_Chin aGX2014,MF443351.1_ChinaGS2014,MF443352.1_ChinaGD2014,MF443353.1_ChinaCQ2 014,MF443354.1_ ChinaAH2014.

The amino acid sequence of the N-protein of each of these strains is provided on the NCBI database. The N-terminus domain of the N-protein of each of the PPRV strains recited above is at least 97% identical to the N-terminus domain of the N-protein of each the Sungri 96 PPRV or Nigeria 75/1 N proteins.

SEQ ID NO: 5 - N-terminal domain of Sungri 96 PPRV N protein

MATLLKSLALFKRNKDKAPTASGSGGAIRGIKNVI IVPIPGDSS I ITRSRLLDRLVRLAGDPDINGSK LTGVMI SMLSLFVESPGQLIQRITDDPDVS IRLVEVVQSTRSQSGLTFASRGADLDNEADMYFSTEGP SSGGKKRINWFENREI IDIEVQDPEEFNMLLASILAQVWILLAKAVTAPDTAADSELRRWVKYTQQRR VIGEFRLDKGWLDAVRNRIAEDLSLRRFMVSLILDIKRTPGNKPRIAEMICDIDNYIVEAGLASFILT IKFGIETMYPALGLHEFAGELSTIESLMNLYQQLGEVAPYMVILENSIQNKFSAGAYPLLWSYAMGVG VELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSVIAAELGITAEEAKLVSEIASQAGDER

SEQ ID NO: 6 - N-terminal domain of Nigeria 75/1 N protein

MATLLKSLALFKRNKDKAPTASGSGGAIRGIKNVI IVPIPGDSS I ITRSRLLDRLVRLAGDPDINGSK LTGVMI SMLSLFVESPGQLIQRITDDPDVS IRLVEVVQSTRSQSGLTFASRGADLDNEADMYFSTEGP SSGSKKRINWFENREI IDIEVQDAEEFNMLLASILAQVWILLAKAVTAPDTAADSELRRWVKYTQQRR VIGEFRLDKGWLDAVRNRIAEDLSLRRFMVSLILDIKRTPGNKPRIAEMICDIDNYIVEAGLASFILT IKFGIETMYPALGLHEFAGELSTIESLMNLYQQLGEVAPYMVILENSIQNKFSAGAYPLLWSYAMGVG VELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSVIAAELGITAEEAKLVSEIASQTGDER

The present chimeric N protein may comprise the amino acid sequence shown as SEQ ID NO: 7 or 8; or a variant of SEQ ID NO: 7 or 8. Suitably, the variant may have at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 7 or 8.

SEQ ID NO: 7 - N-terminal domain of Sunori 96 PPRV N protein + C-terminal domain of DMV

MATLLKSLALFKRNKDKAPTASGSGGAIRGIKNVI IVPIPGDSS I ITRSRLLDRLVRLAGDPDINGSK LTGVMI SMLSLFVESPGQLIQRITDDPDVS IRLVEVVQSTRSQSGLTFASRGADLDNEADMYFSTEGP SSGGKKRINWFENREI IDIEVQDPEEFNMLLASILAQVWILLAKAVTAPDTAADSELRRWVKYTQQRR VIGEFRLDKGWLDAVRNRIAEDLSLRRFMVSLILDIKRTPGNKPRIAEMICDIDNYIVEAGLASFILT IKFGIETMYPALGLHEFAGELSTIESLMNLYQQLGEVAPYMVILENSIQNKFSAGAYPLLWSYAMGVG VELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSVIAAELGITAEEAKLVSEIASQAGDERANR AIGPKQNQISFLHPDRGDASTPGNILANEGDGSTRMKRGGNIAQPKPTSIDQESTTQSKDTLDIEDQS DENTDDPI SIQKSAEALAKMRAMAKLLENQGPRDVTAHVYNDKDLLG

SEQ ID NO: 8 - N-terminal domain of Nigeria 75/1 N protein + C-terminal domain of DMV

MATLLKSLALFKRNKDKAPTASGSGGAIRGIKNVI IVPIPGDSS I ITRSRLLDRLVRLAGDPDINGSK LTGVMI SMLSLFVESPGQLIQRITDDPDVS IRLVEVVQSTRSQSGLTFASRGADLDNEADMYFSTEGP SSGSKKRINWFENREI IDIEVQDAEEFNMLLASILAQVWILLAKAVTAPDTAADSELRRWVKYTQQRR VIGEFRLDKGWLDAVRNRIAEDLSLRRFMVSLILDIKRTPGNKPRIAEMICDIDNYIVEAGLASFILT IKFGIETMYPALGLHEFAGELSTIESLMNLYQQLGEVAPYMVILENSIQNKFSAGAYPLLWSYAMGVG VELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSVIAAELGITAEEAKLVSEIASQTGDERANR AIGPKQNQISFLHPDRGDASTPGNILANEGDGSTRMKRGGNIAQPKPTSIDQESTTQSKDTLDIEDQS DENTDDPI SIQKSAEALAKMRAMAKLLENQGPRDVTAHVYNDKDLLG

Percentage Identity

The percentage identity between two sequences (e.g. polypeptide or polynucleotide sequences) may be readily determined by programs such as BLAST, which is freely available at http://blast.ncbi.nlm.nih.gov. Suitably, the percentage identity is determined across the entirety of the reference and/or the query sequence.

Conservative amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to Table 1 below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.

Unless otherwise explicitly stated herein by way of reference to a specific, individual amino acid, amino acids may be substituted using conservative substitutions as recited below.

An aliphatic, non-polar amino acid may be a glycine, alanine, proline, isoleucine, leucine or valine residue. An aliphatic, polar uncharged amino may be a cysteine, serine, threonine, methionine, asparagine or glutamine residue.

An aliphatic, polar charged amino acid may be an aspartic acid, glutamic acid, lysine or arginine residue.

An aromatic amino acid may be a histidine, phenylalanine, tryptophan or tyrosine residue. Suitably, a conservative substitution may be made between amino acids in the same line in Table 1.

Nucleic Acid Sequence / Polynucleotide

The present invention also provides a nucleotide sequence capable of encoding a chimeric N protein of the present invention. As used herein, the terms“polynucleotide”,“nucleotide”, and“nucleic acid” are intended to be synonymous with each other. The nucleotide sequence may be natural, synthetic or recombinant. It may be double or single stranded, it may be DNA or RNA or combinations thereof. It may, for example, be cDNA, a PCR product, genomic sequence or mRNA.

The nucleotide sequence may be codon optimised for production in the host/host cell of choice.

It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Suitably, the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell.

Nucleic acids according to the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

The terms“variant”, “homologue” or“derivative” in relation to a nucleotide sequence or amino acid sequence includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequence.

It may be isolated, or as part of a plasmid, virus or host cell.

Plasmid

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. They are usually circular and double-stranded.

Plasmids, or vectors (as they are sometimes known), may be used to express a protein in a host cell. For example a bacterial host cell may be transfected with a plasmid capable of encoding a particular protein, in order to express that protein. The term also includes yeast artificial chromosomes and bacterial artificial chromosomes which are capable of accommodating longer portions of DNA.

The plasmid of the present invention comprises a nucleotide sequence encoding the chimeric N protein. It may also comprise one or more additional PPRV nucleotide sequence(s), or nucleotide sequence(s) capable of encoding one or more other PPRV proteins such as P, C, V, M, F, HN or L.

The plasmid may also comprise a resistance marker, such as the guanine xanthine phosphoribosyltransferase gene (gpt) from E. coli, which confers resistance to mycophenolic acid (MPA) in the presence of xanthine and hypoxanthine and is controlled by the vaccinia virus P7.5 early/late promoter.

Cell

The present invention further provides a cell comprising a nucleic acid sequence or a plasmid of the present invention. The cell may be, for example, a mammalian cell and suitably a Vero cell or a baby hamster kidney cell (e.g. BHK-21). The cell may be used to produce the PPRV of the invention.

The cell may be a Vero cell.

Suitably, the cell may express signaling lymphocyte activation molecule (SLAM) on its surface. SLAM is a well-established receptor for morbillivirus infection. The cell may be a VeroDogSLAMTag (VDS) cell (see Seki et at; J Virol; 2003; 77:9943-50 - which is incorporated herein by reference).

The present invention also provides a method for making a PPRV of the invention; which method comprises the following steps:

(i) introducing a plasmid of the invention and one or more plasmids which between them comprise the PPRV genome, and encode the N, P and L proteins into a host cell;

(ii) culturing the host cell under conditions in which virus is produced.

Suitably, the host cell is a cell according to the present invention.

The present PPRV may be produced by reverse genetic systems which are known in the art. For example the present PPRV may be produced using the method described by Muniraju, et al. (2015; Vaccine; 33, 465-471). The present invention also relates to a method for propagating a PPRV according to the first aspect of the invention; which method comprises the step of infecting cells, for example Vero cells of the invention, with a viral particle comprising a chimeric N protein as defined herein.

The cell may be from or part of a cell line. PPRV particles may be harvested, for example from the supernatant by methods known in the art, and optionally purified.

Vaccine

The present PPRV may be used to produce a vaccine.

The present PPRV may be a live, attenuated virus.

The present invention also relates to a method for producing such a vaccine which comprises the step of introducing polynucleotides encoding the present chimeric N protein along with polynucleotide(s) encoding further proteins required for PPRV production into cells, for example Vero cells.

The vaccine may further comprise a pharmaceutically acceptable carrier. As defined herein, “pharmaceutically acceptable carriers” suitable for use in the invention are well known to those of skill in the art. Such carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcohol/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusting agents may also be employed. Buffers include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid, e.g., citrates, ascorbic acid, gluconic acid, histidine-Hel, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, trimethanmine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EDTA), inert gases and the like may also be provided in the pharmaceutical carriers. The present invention is not limited by the selection of the carrier. The preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art. See, e.g., texts such as Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, pub!., 2000; and The Handbook of Pharmaceutical Excipients, 4.sup.th edit., eds. R. C. Rowe et al, APhA Publications, 2003.

The vaccine of the invention will be administered in a“therapeutically effective amount”, which refers to an amount of an active ingredient, e.g., a PPRV according to the invention, sufficient to effect beneficial or desired results when administered to a subject or patient. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a composition according to the invention may be readily determined by one of ordinary skill in the art. In the context of this invention, a "therapeutically effective amount" is one that produces an objectively measured change in one or more parameters associated with e.g. PPR condition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to reduce the incidence of PPR. As used herein, the term "therapeutic" encompasses the full spectrum of treatments for a disease, condition or disorder. A "therapeutic" agent of the invention may act in a manner that is prophylactic or preventive, including those that incorporate procedures designed to target animals that can be identified as being at risk (pharmacogenetics); or in a manner that is ameliorative or curative in nature; or may act to slow the rate or extent of the progression of at least one symptom of a disease or disorder being treated.

The present invention also relates to a method for producing such a vaccine which comprises the step of infecting cells, for example Vero cells, with a viral particle comprising a chimeric N protein as defined herein.

Vaccination method

The vaccine of the present invention may be used to treat and/or prevent a disease.

To "treat" means to administer the vaccine to a subject having an existing disease in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.

To "prevent" means to administer the vaccine to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease (e.g. infection) or to reduce or prevent development of at least one symptom associated with the disease.

The disease may be any disease caused by PPRV. Suitably, the disease may be PPR.

The vaccine of the invention may be used to treat an animal subject, in particular a ruminant. For example, the subject may be a small ruminant such as a goat or a sheep. The subject may be a juvenile, for example a kid or a lamb.

The vaccine of the invention may be administered by subcutaneous, intranasal or intravenous administration.

Typically, the amount of virus administered to a subject may be from about 10³ TCID₅₀ to about 10⁴ TCID_50. AS is well known in the art, TCID₅₀ refers to 50% Tissue culture Infective Dose.

The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the delivery or immunogenicity of the virus.

Antibody

In another aspect the present invention provides an antibody or antigen binding fragment thereof which selectively binds a chimeric PPRV N protein of the present invention. Suitably, selectively binding a chimeric PPRV N protein of the present invention may mean that the antibody is capable of selectively binding a variable C-terminus domain of an N protein from a morbillivirus which is not PPRV.

Descriptions of an antibody of the present invention provided herein are generally applicable to an antigen binding fragment thereof.

The variable C-terminus domain of an N protein from a morbillivirus which is not PPRV may be a variable C-terminus domain of a morbillivirus which is not PPRV as described herein. For example, the variable C-terminus domain of the N protein of the chimeric PPRV N protein may comprise the amino acid sequence shown as SEQ ID NO: 4 or a variant thereof. Suitably, the variant may have at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 4.

In a further aspect the present invention provides an antibody which selectively binds a native PPRV N protein. Suitably, selectively binding a native PPRV N protein may mean that the antibody is capable of selectively binding a variable C-terminus domain of a PPRV N protein. Suitably, the PPRV N protein (in particular the variable C-terminus domain of the PPRV N protein) may be an appropriate polypeptide sequence as described herein. For example, the variable C-terminus domain of the PPRV N protein may be the amino acid sequence shown as positions 1 to 405 of SEQ ID NO: 1 or SEQ ID NO: 2.

The variable C-terminus domain of the N protein of the native PPRV N protein may comprise the amino acid sequence shown as SEQ ID NO: 5 or 6 or a variant thereof. Suitably, the variant may have at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 5 or 6.

Suitably, an antibody which is capable of selectively binding a chimeric PPRV N protein of the present invention is not capable of binding a native PPRV N protein at a detectable level. Suitably, an antibody which is capable of selectively binding a native PPRV N protein is not capable of binding a chimeric PPRV N protein of the present invention at a detectable level.

Suitably, the chimeric PPRV N protein is any chimeric PPRV N protein of the present invention as described herein.

The terms“selectively binds/selecting binding” and“specifically binds/specifically binding” may be used interchangeably herein.

Methods for determining the binding specificity of an antibody include, but are not limited to, ELISA, western blot, immunohistochemistry, flow cytometry, Forster resonance energy transfer (FRET), phage display libraries, yeast two-hybrid screens, co-immunoprecipitation, bimolecular fluorescence complementation and tandem affinity purification.

To identify an antibody which is selective for either a chimeric PPRV N protein of the present invention, or a native PPRV N protein, the binding of the antibody to each of chimeric PPRV N protein and native PPRV N protein may be assessed. Typically, this is assessed by determining the binding of the antibody to each protein separately. An antibody which is selective binds to either a chimeric PPRV N protein of the present invention or a native PPRV N protein without significant binding to the other PPRV N protein.

The antibody may be a monoclonal antibody or a polyclonal antibody. Preferably, the antibody is a monoclonal antibody.

Examples of an antigen-binding fragment include, but are not limted to, a camelid antibody (VHH), an antigen-binding fragment (Fab), a variable region (Fv), a single chain antibody (scFv), a single-domain antibody (sdAb), a heavy chain variable region (VH), a light chain variable region (VL), and a complementarity determining region (CDR).

The antibody may be a full-length, classical antibody. For example the antibody may be an IgG, IgM or IgA molecule.

In preferred embodiments, the antibody is a full monoclonal antibody.

Antibodies may be obtained by techniques comprising immunizing an animal with a target antigen and isolating the antibody from serum. Monoclonal antibodies may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example. The antibody may also be a chimeric or humanized antibody.

“Heavy chain variable region” or“VH” refers to the fragment of the heavy chain of an antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs.“Light chain variable region” or“VL” refers to the fragment of the light chain of an antibody that contains three CDRs interposed between framework regions.

“Complementarity determining region” or“CDR” with regard to antibody or antigen-binding fragment thereof refers to a highly variable loop in the variable region of the heavy chain of the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs (heavy chain CDRs 1 , 2 and 3 and light chain CDRs 1 , 2 and 3, numbered from the amino to the carboxy terminus).

The CDRs of the variable regions of a heavy and light chain of an antibody can be predicted from the heavy and light chain variable region sequences of the antibody, using prediction software available in the art, e.g. using the Abysis algorithm, or using the IMGT/V-QUEST software, e.g. the IMGT algorithm (ImMunoGeneTics) which can be found at www.IMGT.org, (see for example Lefranc et al, 2009 NAR 37:D1006-D1012 and Lefranc 2003, Leukemia 17: 260-266). CDR regions identified by either algorithm are considered to be equally suitable for use in the invention. CDRs may vary in length, depending on the antibody from which they are predicted and between the heavy and light chains. Thus, the three heavy chain CDRs of an intact antibody may be of different lengths (or may be of the same length) and the three light chain CDRs of an intact antibody may be of different lengths (or may be of the same length). A CDR for example, may range from 2 or 3 amino acids in length to 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. Particularly, a CDR may be from 3-14 amino acids in length, e.g. at least 3 amino acids and less than 15 amino acids.

Examples of antibodies of the invention are further described herein.

In a particularly preferred embodiment, the antibody comprises three CDRs from the variable heavy chain sequence of an antibody which specifically binds to PPRVNv or DMVNv and/or three CDRs from the variable light chain sequence of an antibody which specifically binds to PPRVNv or DMVNv (preferably the same antibody or antibody fragment).

The antibody may comprises one or more CDR regions, selected from SEQ ID NOs: 15-56, or derivatives thereof (e.g. derivatives comprising 1 , 2 or 3 substitutions, preferably one substitution). In other words, in some embodiments the antibody comprises one or more CDR regions, selected from SEQ ID NOs: 15-56, or derivatives thereof (e.g. derivatives comprising 1 , 2 or 3 substitutions, preferably one substitution). Suitably, the antibody comprises three CDR regions selected from SEQ ID NOs: 15-56, or derivatives thereof.

Preferably, the antibody CDRs (CDR1 , CDR2, and CDR3), or derivatives thereof, are selected from the same variable chain. For example, the antibody may comprise SEQ ID NOs: 15-17; SEQ ID NOs: 18-20; SEQ ID NOs: 21-23; SEQ ID NOs:24-26; SEQ ID NOs: 27-29; SEQ ID NOs: 30-32; SEQ ID NOs: 33-35; SEQ ID NOs: 36-38; SEQ ID NOs: 39-41 ;

SEQ ID NOs: 42-44; SEQ ID NOs: 45-47; SEQ ID NOs: 48-50; SEQ ID NOs: 51-53; and/or SEQ ID NOs: 54-56 or derivatives thereof.

In preferred embodiments, the antibody or antigen-binding fragment thereof comprises a combination variable heavy and variable light CDRs as follows:

(i) SEQ ID NOs: 15-17 and SEQ ID NOs: 18-20, or derivatives thereof;

(ii) SEQ ID NOs: 21-23 and SEQ ID NOs: 24-26, or derivatives thereof;

(iii) SEQ ID NOs: 27-29 and SEQ ID NOs: 30-32, or derivatives thereof;

(iv) SEQ ID NOs: 33-35 and SEQ ID NOs: 36-38, or derivatives thereof;

(v) SEQ ID NOs: 39-41 and SEQ ID NOs: 42-44, or derivatives thereof;

(vi) SEQ ID NOs: 45-47 and SEQ ID NOs: 48-50, or derivatives thereof; or

(vii) SEQ ID NOs: 51-53 and SEQ ID NOs: 54-56, or derivatives thereof.

The antibody or antigen-binding fragment thereof may comprise or consist of a variable heavy domain variable heavy domain and variable light domain selected from SEQ ID NOs: 57-70 or a variant which is at least 80% identical to one or more of SEQ ID NOs: 57-70. The variant may be at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to one or more of SEQ ID NOs: 57-70.

Suitably, the antibody or antigen binding fragment thereof comprises a combination of a variable heavy domain and a variable light domain. Preferably, the antibody or antigen binding fragment thereof comprises a combination of a variable heavy domain and a variable light domain selected from:

(i) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 57 and an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 58; (ii) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 59 and an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 60;

(iii) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 61 and an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 62; (iv) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 63 and an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 64;

(v) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 65 and an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 66;

(vi) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 67 and an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 68; or

(vii) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 69 and an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 70.

The variants described herein retain antigen-binding ability. For example, the variants may be capable of selectively binding to PPRVNv or DMVNv.

Thus, in some embodiments the antibody comprises:

(i) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 57, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 15-17, respectively, and/or an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 58, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 18-20, respectively;

(ii) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 59, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 21-23, respectively, and/or an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 60, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 24-26, respectively; (iii) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 61 , wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 27-29, respectively, and/or an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 62, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 30-32, respectively;

(iv) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 63, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 33-35, respectively, and/or an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 64, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 36-38, respectively;

(v) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 65, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 39-41 , respectively, and/or an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 66, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 42-44, respectively;

(vi) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 67, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 45-47, respectively, and/or an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 68, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 48-50, respectively; or

(vii) an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 69, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 51-53, respectively, and/or an amino acid sequence which has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 70, wherein the amino acid sequence comprises CDR1 , CDR2 and CDR3 regions consisting of SEQ ID NOs: 54-56, respectively. Antibodies 93, 107, 162, 164, 241 , 249, 413 and 488 are capable of selectively binding to a chimeric PPRV N protein of the present invention. Antibodies 164, 247, 292, 315, 380, 383, 390, 394, 443, 455, 478, 54, 84, 182 and 486 are capable of selectively binding a native PPRV N protein.

DIVA

The present invention also relates to a method of distinguishing a subject that has been vaccinated with a vaccine of the present invention from a subject that has been infected with a non-chimeric PPRV; which method comprises contacting a chimeric PPRV N protein of the present invention or a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein of the present invention with (i) a sample from the subject; and (ii) a detectable reagent which is (a) capable of binding to an anti-chimeric PPRV N protein antibody present in the sample or (b) capable of specifically binding to the chimeric PPRV N protein or a native PPRV N protein.

The detectable reagent of part (b) may be an antibody of the present invention.

The present methods may further comprise identifying individuals in the population that have been infected with a native/field PPRV and/or have been vaccinated with a non-chimeric PPRV vaccine.

Suitably, the present invention may relate to a method of distinguishing a subject that has been vaccinated with a vaccine of the present invention from a subject that has been naturally infected with wild PPRV; which method comprises contacting a variable C-terminus domain of a native PPRV N protein (e.g. from an existing PPRV vaccine viruses known in the art) or a variable C-terminus domain of the N protein of a chimeric N protein of the present invention (or a virus comprising said protein) with (i) a sample from the subject; and (ii) a detectable reagent which is (a) capable of binding to an anti-chimeric PPRV N protein antibody present in the sample or capable of binding to an antibody specific for the variable C-terminus domain of the native PPRV N protein or (b) capable of specifically binding to the chimeric PPRV N protein / or a native PPRV N protein (in other words capable of specifically binding to the variable C-terminus domain of the present chimeric PPRV N protein or a native PPRV N protein).

The detectable reagent of part (b) may be an antibody of the present invention.

The non-chimeric/native PPRV is a field PPRV infection. As used herein the terms“chimeric N protein”,“mutated N protein” and“DIVA N protein” are intended to be synonymous and each refers to a PPRV N protein of the present invention.

As used herein the terms“non-chimeric N protein”, “native N protein” and“non-DIVA N protein” are intended to be synonymous and each refers to a wild-type or native PPRV N protein.

The polypeptide (e.g. the recombinant polypeptide) may comprise or consist of the variable C-terminus domain of the N protein of the chimeric PPRV N protein. The polypeptide (e.g. the recombinant polypeptide) may comprise or consist of the variable C-terminus domain of a native N protein.

The variable C-terminus domain of the N protein of the chimeric PPRV N protein may comprise the amino acid sequence shown as SEQ ID NO: 4 or a variant thereof. Suitably, the variant may have at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 4.

An anti-chimeric PPRV N protein antibody refers to an antibody which is capable of specifically binding to a chimeric PPRV N protein as defined herein. Suitably, the anti chimeric PPRV N protein antibody may be capable of specifically binding a chimeric PPRV N protein as defined herein but not capable of binding to a wild-type PPRV N protein. Examples of such antibodies include antibodies 162, 164 and 241 of the present invention. Further illustrative antibodies capable of specifically binding a chimeric PPRV N protein as defined herein but not capable of binding to a wild-type PPRV N protein are described in the present Examples.

Similarly an anti-native PPRV N protein antibody binds specifically to N protein of a field PPRV (including the N proteins of existing vaccine viruses), but not to the chimeric N protein of the present invention. Examples of such antibodies include antibodies 292, 54, 455 and 390 of the present invention. Further illustrative antibodies capable of specifically binding to the N protein of a field/native PPRV but not to the chimeric N protein of the present invention are described in the present Examples.

In certain embodiments, the present invention utilises a sample isolated from a subject. This sample may be referred to as a‘test sample’. Thus the present methods are typically practiced outside of the animal body, e.g. on a sample that was previously obtained from the subject to be tested.

Suitably, the method is an in vitro method.

The method of distinguishing a subject that has been vaccinated with a vaccine of the present invention from a subject that has been infected with a non-chimeric PPRV may be performed following administration of a vaccine to the subject, according to a method of the present invention.

The sample may be a blood, urine, or saliva sample.

In a preferred embodiment the sample is a blood sample. Suitably, the blood sample may be a whole blood sample. Suitably, the blood sample may be a blood fraction, for example a serum or plasma sample.

Techniques for collecting blood samples and separating blood fractions are well known in the art. For instance, vena blood samples can be collected using a needle and deposited into plastic tubes. The collection tubes may, for example, contain anticoagulants, spray- coated silica, or a polymer gel for separation. Plasma can be separated by centrifugation at 1300 RCF for 10 min at room temperature and stored in small plastic tubes at -80°C.

In a preferred embodiment the sample is a blood sample, in particular a plasma sample. In a preferred embodiment the blood sample is treated with anticoagulants (e.g. heparin) following collection.

The detectable reagent may be an antibody or a fragment thereof which is capable of specifically binding to the chimeric PPRV N protein or the variable C-terminus domain of the N protein of the chimeric PPRV N protein of the present invention. In other words, the detectable agent may be capable of specifically binding to the chimeric PPRV N protein of the invention but not capable of binding to a non-chimeric PPRV N protein.

The detectable reagent may be an antibody or a fragment thereof which is capable of specifically binding to a native PPRV N protein or the variable C-terminus domain of a native PPRV N protein. In other words, the detectable agent may be capable of specifically binding to a native PPRV N protein but not capable of binding to a chimeric PPRV N protein of the invention.

The method may be performed using a variety of suitable techniques known in the art. For example, distinguishing a subject that has been vaccinated with a vaccine of the present invention from a subject that has been infected with a non-chimeric PPRV may employ antibody-based arrays, enzyme linked immunosorbent assays (ELISA), non-antibody protein scaffolds, radioimmuno-assay (RIA), western blotting, or aptamers.

For example, the method may comprise the following steps: a) capturing an anti-chimeric PPRV N protein antibody present in a sample from a subject using a recombinant chimeric N protein or a recombinant polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein of the present invention; and b) detecting the anti chimeric PPRV N protein antibody using a detectable reagent which is capable of binding to an anti-chimeric PPRV N protein antibody present in the sample.

The method may further comprise c) capturing an antibody specific against the variable C- terminus domain of a native PPRV N protein present in a sample from a subject using a recombinant native PPRV N protein or a recombinant polypeptide comprising the variable C- terminus domain of the native PPRV N protein; and d) detecting the anti-native PPRV N protein antibody using a detectable reagent which is capable of specifically binding to the anti-native PPRV N protein antibody present in the sample.

The method may comprise the following steps: a) contacting a chimeric PPRV N protein of the present invention or a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein of the present invention with a sample from a subject; and a detectable reagent which is capable of specifically binding to the chimeric PPRV N protein. In this embodiment, the detectable reagent competes with anti-chimeric PPRV N protein antibody present in the sample from the subject for binding to the chimeric PPRV N protein or the polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein. Accordingly, if the sample comprises anti-chimeric PPRV N protein antibody the detectable signal from the detectable reagent is reduced compared to the level of signal from a corresponding assay where the sample does not comprise anti-chimeric PPRV N protein antibody.

The method may further comprise contacting a polypeptide comprising or consisting of the variable C-terminus domain of the native PPRV N protein with a sample from a subject; and a detectable reagent which is capable of specifically binding to the variable C-terminus domain of the native PPRV N protein. The method may comprise a competitive ELISA as described herein. In this embodiment, the detectable reagent competes with anti-native PPRV N protein antibody present in the sample from the subject for binding the polypeptide comprising or consisting of the variable C-terminus domain of the native PPRV N protein. Accordingly, if the sample comprises an anti-native PPRV N protein antibody which specifically binds the variable C-terminus domain the detectable signal from the detectable reagent is reduced compared to the level of signal from a corresponding assay where the sample does not comprise an anti-native PPRV N protein antibody which specifically binds the variable C-terminus domain of the native PPRV N protein.

An ELISA may be performed according to general methods which are known in the art. For example, the ELISA may be a competitive ELISA or indirect ELISA.

A competitive ELISA may comprise the following steps:

a surface (i.e. a microtitre plate well) is prepared to which a known quantity of capture agent is bound (e.g. recombinant chimeric PPRV N protein or recombinant polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein);

a labelled antibody which specifically binds chimeric PPRV N protein is incubated in the presence of a sample from a subject and the capture agent prepared in the first step to form a second sample;

the signal from the labelled antibody is detected.

The capture reagent may be bound to the surface using an antibody of the present invention. For example mAbs 292, 383 and 455 of the present Examples are suitable for use as a capture antibody for native PPRV N protein and mAbs 54, 84, 182 and 486 are suitable for use as a detecting antibody for native PPRV N protein. mAbs 249 and 488 are suitable for use a capture antibody against a chimeric PPRV N protein of the present invention and mAbs 93 and 162 are suitable for use as a detecting antibody for a chimeric PPRV N protein of the present invention.

Corresponding steps may be performed with a recombinant native PPRV variable C- terminus domain polypeptide. Said steps may allow individuals infected with field PPRV or previously vaccinated with non-chimeric PPRV vaccine to be identified.

Accordingly, a competitive ELISA enables the amount of anti-chimeric PPRV N protein antibody present in the sample from the subject to be determined.

An indirect ELISA may comprise the following steps:

a sample from a subject is applied to the surface; a detectable antibody (e.g. an enzyme-linked antibody) is applied as a detection antibody that binds specifically to any anti-chimeric PPRV N protein antibody retained on the plate;

the presence of the detectable antibody is determined by measuring e.g. the absorbance or fluorescence or electrochemical signal of the surface to determine the presence and quantity of antigen.

Accordingly, an indirect ELISA enables the amount of anti-chimeric PPRV N protein antibody present in a sample from the subject to be determined.

The detectable antibody may comprise a fluorescent moiety. The detectable antibody may comprise an enzyme conjugate. Various enzyme-substrate labels are available, e.g. as disclosed in US 4,275,149. The enzyme generally catalyses a chemical alteration of the chromogenic substrate that can be detected. For example, the enzyme may catalyse a colour change in a substrate, or may alter the fluorescence or chemiluminescence of the substrate. Examples of enzymatic labels include peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are well known.

Kit

The present invention further comprises a kit comprising: (i) a vaccine according to the invention; and (ii) (a) a chimeric PPRV N protein which is expressed by the PPRV of the vaccine of part (i), a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; or (b) a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein which is expressed by the PPRV in the vaccine of part (i), a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein.

The kit may further comprise a polypeptide which comprises or consist of a variable C- terminus domain of a native PPRV N protein, a polynucleotide, plasmid or construct encoding said variable C-terminus domain of a native PPRV N protein. The present invention further provides a kit comprising: (i) a vaccine according to the invention; and (ii) (a) a reagent which is capable of specifically binding to either the chimeric PPRV N protein which is expressed by the PPRV of the vaccine of part or a non-chimeric PPRV N protein.

The present invention further comprises a kit comprising: (i) a chimeric PPRV N protein according to the present invention, a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; or a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein according to the present invention, a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; and (ii) a detectable reagent which is (a) capable of binding to an anti-chimeric PPRV N protein antibody or (b) capable of specifically binding to the chimeric PPRV N protein.

The kit may further comprise a detectable reagent which is (a) capable of binding to an anti native PPRV N protein antibody or (b) capable of specifically binding to the variable C- terminus domain of a native PPRV N protein.

The reagent or detectable reagent may be an antibody or a fragment thereof.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. 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 also include the term "consisting of.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Results

Example 1 - Rescue and characterisation of PPRV DIVA vaccine strains from cDNA clones

Infectious recombinant viruses of PPRV Nigeria 75/1 , and rPPRV Sungri 96 containing the variable part of the N gene of DMV were rescued from respective synthetic cDNAs using reverse genetics (Muniraju, et ai\ 2015; Vaccine 33, 465-471). The CPE characteristic of PPRV infection was observed three days post transfection with 100% efficiency. The CPE observed for the two recombinant viruses appeared to be identical to that produced by the parental PPRV Sungri 96 and PPR Nigeria 75/1 vaccine strain. Total RNA isolated from the recovered PPRV recombinants at passage three were subjected to RT-PCR using PPRV genome specific primers using a -RT as a control for carry over DNA. The expected amplicon sizes were observed on an agarose gel and sequences were 100% identical to each respective plasmid. These DIVA vaccine viruses showed similar biological properties, replicative capacity, safety and stability on 9^th passage in cell culture. Multi-step growth curves were carried out to compare the growth of the recombinant vaccine viruses with that of the parental vaccine strains. The recombinant PPRVs grew to a similar titre and rate to that of the parental PPRV viruses.

Example 2 - In vivo evaluation of Sungri 96 PPR-DIVA and Nigeria 75/1 PPR-DIVA and comparison with parent conventional vaccines: These two DIVA vaccines along with parental conventional vaccines were tested in goats. No vaccination-associated side effects were observed in any of the animals vaccinated with PPR-DIVA or conventional vaccines. In addition, animals in both the vaccinated groups remained healthy during post challenge period, whereas all the five animals in the control group developed severe clinical symptoms (pyrexia, congested oro-nasal mucosa, mucopurulent nasal discharge, conjunctivitis, diarrhoea and anorexia) specific to PPR disease. Because of severe clinical signs three animals had to be humanely killed on 10 days post-challenge. The onset of disease in control animals were noticed from 4dpc, and showed a significant rise in rectal body temperatures (>40°C) until 8dpc in some animals with the peak temperatures observed on day 4 and 5 post-challenge. All the vaccinated animals maintained their rectal temperatures within the normal range throughout the study period, and showed no clinical symptoms.

Both the DIVA and parent vaccines provided full protection for vaccinated goats whereas the control animals were clinically infected indicating the DIVA vaccines are efficacious. No significant difference in antibody titre was detected in both the vaccinated groups on the day of challenge (Fig. 1A & B).

Goats in both the vaccinated groups did not excrete any virus in their body (nasal, ocular and saliva) excretions whereas very high copy number of viral genome was detected in control infected animals starting from 4 to 12 dpc (Fig. 1 D) indicating the virus is safe to use.

Two recombinant proteins (120 amino acid in length at the c-terminus variable part of the N gene), one native to PPR N gene (Nv) and the other one is mutated (mNv) PPR (replaced that of with DMV sequence) were expressed in E. coli and used in the DIVA ELISAs. PPR ELISA using Nv protein could detect the conventional vaccinated and control infected animals whereas mutated N protein (mNv) detected only DIVA vaccinated animals (Fig. 1C).

Example 3 - Generation of monoclonal antibodies for DIVA assays

Two rabbits each were immunised with recombinant proteins, PPRVNv and DMVNv and were boosted twice at 28 days interval. One week later blood samples were collected, serum separated and stored. These serum samples were tested in indirect ELISAs. The antibodies at a dilution of 1 : 1000 react only with the respective antigen giving an OD of ³1.5.

Similarly, five mice each were immunised with the recombinant proteins, PPRVNv and DMVNv and were boosted twice at 21 days interval. Two weeks later test bleed was collected and the serum samples were tested in ELISA to measure the antibody response to individual antigen. A third boost was given 28 days after the 2^nd boost. One week later blood samples were collected, serum separated and stored. The animals were sacrificed and the splenocytes from best responding mice were used for fusion.

Fusion supernatants were screened by antigen ELISA using both the antigens and the antibodies reacting only with their respective antigen without cross reaction were selected for further cloning to establish stable cell lines. A total of 40 supernatants were tested for PPRVNv out of which 15 that displayed strong reactivity with PPRVNv protein were selected (Table 2). Similarly, of 11 supernatants tested for DMVNv eight that displayed strong reactivity with DMVNv protein were selected (Table 3).

Table 2: Specificity results of fusion supernatants against PPRVNv protein.

Table 3: Specificity results of fusion supernatants against DMVNv protein.

Following cloning the mAbs were affinity purified, isotyped and an aliquot from the purified mAb was conjugated with HRP for further testing. A total of 15 Abs against PPRVNv protein and 8 mAbs against DMVNv protein were obtained (clones highlighted grey in Table 2 and 3). These mAbs were evaluated further for use as a capture antibody and/or conjugated detecting antibody in an ELISA.

At least three mAbs (292, 383 and 455) raised against PPRVNv protein were determined to be suitable for use as a capture antibody and at least four mAbs (54, 84, 182 and 486) were determined to be suitable for use as a detecting antibody.

At least two mAbs (249 and 488) raised against DMVNv protein were determined to be suitbable for use a capture antibody and at least two mAbs (93 and 162) were determined to be suitable for use as detecting antibody.

A representative selection of highly reactive mAbs raised against each protein (PPRVNv: 292, 390, 455, 54; DMVNv: 93, 162, 164, 241) were selected and sequenced.

Materials & Methods

Cell Culture

VeroDogSLAMtag (VDS) cells were used for virus rescue and propagation. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% (v/v) foetal calf serum (FCS, Gibco) and penicillin (100 U/ml, Sigma) at 37°C/5% C0₂. All viruses were rescued and grown in VDS cells until the detection of cytopathic effect (CPE) before being freeze-thawed, clarified and stored at -70°C. The Nigeria 75/1 vaccine was obtained commercially. Final virus titre was determined by 50% tissue culture infectious dose (TCID50).

Construction of recombinant PPRV cDNA clones Sunori 96

The full-length PPRV cDNA plasmids generated were based on the PPRV sugri 96 vaccine strain (GenBank accession number KJ867542). The plasmid containing the complete PPRV antigenome sequence (15948 nt) was designed and synthesised commercially. Novel restriction enzyme sites were inserted by nucleotide substitution into the untranslated regions (UTRs) of each gene and as such did not affect the total genome length or viral protein sequences. The full length clone was under the expression control of the T7 RNA polymerase promoter and cleaved at the antigenome promoter by the hepatitisdelta ribozyme. The synthesized plasmid, was sequenced in its entirety to ensure the sequence was 100% identical to the vaccine strain. The helper plasmids required for rescue of recombinant viruses, PPRV N (pN), P (pP)and L (pL), were cloned under the control of the T7 RNA polymerase promoter in the pGEM3z vector. The full length plasmids and helper plasmids were sequenced to ensure that no additional mutations had been incorporated through PCR and cloning.

Construction of recombinant PPRV-DMV cDNA clones Sunori 96 and Niqeri 75/1

Total RNA extracted from Dolphin Morbillivirus (DMV) grown on VDS cells was used to make cDNA using standard methods. The variable part of C terminus of the nucleocapsid (N) gene (gene encoding last 118 amino acids) of DMV was amplified from the cDNA using primer set PPR-DMV-F/DMV-PadR (GGCT GGGGACGAAAGAGCT AAT AGAGCAAT AGGT (SEQ ID NO: 9) /CGGCCTT AATT AAACGCTGCTCAGAGTGGATCC (SEQ ID NO: 10)). The N- terminus part of PPRV N-gene (~1.2kb) was amplified using primer set PPR-A cl IF/DMV- PPR-R (GCGCAAGATCTAACGTTATGGCGACTCTCC (SEQ ID NO: 11) /

ACCTATTGCTCTATTAGCTCTTTCGTCCCCAGCC (SEQ ID NO: 12)). These PCR products were gel purified and the DNA in the gels were used as the templates for overlapping PCR using primer set PPR-A cl IF/DMV-Pac-IR to generate a PCR product of expected size (2.1 kb) that encompasses the whole N-gene and the N-terminal 400 nucleotide of P-gene. The amplicon was cloned in to pT7Blue vector (pT7 Sungri-DMV ₂o plasmid) and sequenced on both the strands to ensure the there was no unwanted changes in the sequence. The chimeric N-gene was excised from the pT7 Sungri-DMV₁₀5 plasmid using Ac/I and Pad enzyme and cloned into the Ac/I and Pad digested ~19kb long product of pSungri plasmid to produce pSungri-DMV₁₂o plasmid. The pNig-DMV₁₂₀ plasmid was also generated using a similar approach. Transfection and recovery of recombinant PPRV from cDNA:

VDS cells (70% confluent) were grown in 6-well plates and infected with T7-polymerase expressing recombinant fowl poxvirus at a multiplicity of infection (MOI) of 0.2. Cells were washed and transfected with 1 pg of full-length PPRV cDNA plasmid and 1 pg pN, 1 pg pP and 0.05 pg pL using TransFast™ Transfection Reagent (Promega) at a ratio of 6: 1 (wt/wt) in a total volume of 0.75 ml of OPTI-MEM I reduced serummedium/well (Gibco). Media was changed on cells at 24 h posttransfection and observed for CPE for three days. Rescued viruses were harvested by freeze thawing and further passaged in VDS cells.

In vitro characterisation of recombinant viruses

To confirm the identity of rescued viruses, RT-PCR was performed using PPRV specific primers. Total RNA was extracted from rescued viruses at the third passage and analysed by RT-PCR. Immunofluorescence for the expression of N and H by the rescued viruses was carried out by labelling the N protein with an anti-PPRV-N C11 monoclonal antibody and the H protein with anti-PPRV-HC77 monoclonal antibody (BDSL, UK) and GFP autofluorescence. The in vitro growth kinetics of the recombinant PPRVs and the parental virus was assessed in a multiple-step growth cycle

Expression of recombinant proteins of the variable part of N gene of PPR and DMV

The 120 AA of variable part of DMV N gene (mNV) were amplified by PCR using primer sets DM V-BamH I F 1 /DM V-H /nd 111 R (GCGGAT CCGCT AAT AGAGCAAT AGGTCC (SEQ ID NO: 13) / GCGCAAGCTTGCCAAGT AGATCTTT ATC (SEQ ID NO: 14)), respectively using DMV cDNA as the template. The PCR products were then cloned into the pT7 Blue blunt end vector (pT7-DMV N₁₂o plasmid). The BamHI and H/ndlll restriction enzymes digested products of pT7Blue-DMV N₁₂o plasmid was cloned into the dephosphorylated pQE-30Xa vector to produce pQE-30Xa-DMVN₁₂₀ plasmid. mNV expression was induced using 1 mM IPTG that produced protein bands of 17kDa and 14kDa, respectively. The bacterial clones expressing mNv (120 amino acid) protein were cultured in large volume and the 6xHis tagged recombinant DMVNv protein was purified on a Ni-NTA agarose column and quantified using a protein assay kit (Bio-Rad). Using a similar approach the C terminus variable part of the nucleocapsid (N) gene (120 Amino Acids) of PPRV was expressed (Nv) in bacterial system.

Vaccine evaluation with challenge of virulent PPRV

A vaccination trial was carried out at the animal facility at the Pirbright Institute, UK to evaluate the DIVA capability of the vaccine viruses. Fifteen goats of either sex, aged 6 to 12 months were randomly divided into three groups (n=5/group). Goats were kept under observation for a week for acclimatization in an isolation unit. Animals in group 1 and 2 were vaccinated intranasally with 10⁴³ TCID50 of Sungri 96-DIVA, and Sungri 96 conventional vaccine (grown on Vero cells), respectively. Group 3 served as unvaccinated control. At four weeks post vaccination, all the animals were challenged with a virulent PPRV (10⁵ TCID₅₀) by the intranasal route (1 ml/nostril) using an Intranasal Mucosal Atomization Device. Animals were kept for 14 days post-challenge (dpc). A similar vaccine challenge experiment was carried out for Nigeria-DIVA vaccine.

For all animals, rectal temperatures and clinical assessments of animals were conducted twice daily. Blood, nasal, eye and mouth swabs were taken daily and analysed by real-time RT-PCR using PPRV N gene primers. RNA extraction was achieved using robotic extraction methods (MagNA Pure LC Total Nucleic Acid IsolationKit, Roche, UK) following the manufacturer’s protocols. Clotted blood samples were collected for the evaluation of the serum antibody response specific to the PPRV H protein using a PPR Antibody ELISA kit (BDSL, UK) and N ELISA kit (ID Vet France) and the development of PPRV neutralising antibodies evaluated using a virus neutralisation test (VNT).

Development of indirect ELISAs to detect PPR-DIVA N-specific antibodies using recombinant PPR NV and mNv proteins

Two indirect ELISAs were developed using E. coli-ex pressed mutated C-terminal variable region of N gene of PPRV (Nv) protein and C terminus variable DMV protein (mNv) as the antigen. Plates were coated with mNv protein (1 mg ml ¹) at a 1 : 2000 dilution in carbonate/bicarbonate buffer (pH 9.6) for 1 hour at 37°C. After washing with PBS, the serum sample was added at a 1 : 8 dilution in blocking buffer containing 0.1 % Tween 20 and 5 % Marvel in PBS and incubated for another hour. After a thorough wash, horseradish peroxidase-conjugated anti-bovine IgG (Sigma) was added at a dilution of 1 : 5000 in blocking buffer. After an hour of incubation at 37°C, the plates were washed and the OPD solution was added for development of colour. Colour development was stopped after 10 min by adding 1 M H₂S0₄. The plates were read in an ELISA plate reader using a 492 mm filter.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. A peste des petits ruminants virus (PPRV) comprising a chimeric N protein wherein the chimeric N protein comprises a variable C-terminus domain which comprises at least 50 amino acids from the variable C-terminus domain of a N protein from a second morbillivirus which is not PPRV.

2. A PPRV according to claim 1 wherein the variable C-terminus domain of the N protein corresponds to amino acid positions 406 to 525 when identified by alignment of the N protein with the sequence of SEQ ID NO: 1 or 2.

3. A PPRV according to claim 1 or 2 wherein the variable C-terminus domain of the N protein comprises at least 60, at least 65, at least 70, at least 75, at least 80, at least 90, at least 100, or at least 1 15 amino acids from the variable C-terminus domain of a N protein from a second morbillivirus which is not PPRV.

4. A PPRV according to any preceding claim wherein the variable C-terminus domain of the N protein is from a from dolphin morbillivirus (DMV).

5. A PPRV according to any preceding claim wherein the variable C-terminus domain of the N protein comprises SED ID NO: 4 or a variant which is at least 80% identical to SEQ ID NO: 4.

6. A PPRV according to claim 5 wherein the N terminal domain of the N protein comprises SEQ ID NO: 5 or 6; or a variant which is at least 80% identical to SEQ ID NO: 5 or 6.

7. A PPRV according to claim 5 wherein the N protein comprises SEQ ID NO: 7 or 8; or a variant which is at least 80% identical to SEQ ID NO: 7 or 8.

8. A chimeric PPRV N protein as defined in any of claims 1 to 7.

9. A nucleic acid sequence encoding a chimeric PPRV N protein according to claim 8.

10. A plasmid comprising a nucleic acid sequence according to claim 9.

1 1 . A cell comprising a nucleic acid sequence according to claim 9 or a plasmid according to claim 10.

12. A vaccine comprising a PPRV according to any of claims 1 to 7, a chimeric PPRV N protein according to claim 8, a nucleic acid sequence according to claim 9 or a plasmid according to claim 10.

13. A method for treating and/or preventing a disease in a subject which comprises the step of administering a vaccine according to claim 12 to the subject.

14. A vaccine according to claim 12 for use in treating and/or preventing a disease in a subject.

15. The use of a PPRV according to any of claims 1 to 7, a chimeric PPRV N protein according to claim 8, a nucleic acid sequence according to claim 9 or a plasmid or construct according to claim 10 in the manufacture of a vaccine for treating and/or preventing a disease in a subject.

16. A method, a vaccine for use, or a use according to claim 13, 14, or 15 wherein the disease is peste des petits ruminants (PPR).

17. An antibody or antigen binding fragment thereof which (i) selectively binds a chimeric PPRV N protein according to claim 8; or (ii) selectively binds a native PPRV N protein.

18. The antibody or antigen binding fragment according to claim 17 wherein the antibody comprises complementary determining regions (CDRs) selected from the group consisting of: SEQ ID NOs: 15-17; SEQ ID NOs: 18-20; SEQ ID NOs: 21 -23; SEQ ID NOs: 24-26; SEQ ID NOs: 27-29; SEQ ID NOs: 30-32; SEQ ID NOs: 33-35; SEQ ID NOs: 36-38; SEQ ID NOs: 39-41 ; SEQ ID NOs: 42-44; SEQ ID NOs: 45-47; SEQ ID NOs: 48-50; SEQ ID NOs: 51 -53; and/or SEQ ID NOs: 54-56 or derivatives thereof.

19. The antibody or antigen binding fragment according to claim 18 which comprises a combination variable heavy and variable light CDRs selected from the group consisting of:

(i) SEQ ID NOs: 15-17 and SEQ ID NOs: 18-20, or derivatives thereof;

(ii) SEQ ID NOs: 21 -23 and SEQ ID NOs: 24-26, or derivatives thereof;

(iii) SEQ ID NOs: 27-29 and SEQ ID NOs: 30-32, or derivatives thereof;

(iv) SEQ ID NOs: 33-35 and SEQ ID NOs: 36-38, or derivatives thereof;

(v) SEQ ID NOs: 39-41 and SEQ ID NOs: 42-44, or derivatives thereof; (vi) SEQ ID NOs: 45-47 and SEQ ID NOs: 48-50, or derivatives thereof; and

(vii) SEQ ID NOs: 51 -53 and SEQ ID NOs: 54-56, or derivatives thereof.

20. The antibody or antigen binding fragment according to claim 19, which comprises a combination of a variable heavy domain and a variable light domain selected from the group consisting of:

(i) an amino acid sequence which has at least 80% identity to SEQ ID NO: 57 and an amino acid sequence which has at least 80% identity to SEQ ID NO: 58;

21 . A method for making a PPRV according to any of claims 1 to 7 which comprises the following steps:

(i) introducing a plasmid according to claim 10 and one or more helper plasmids which between them encode the PPRV N, P and L proteins into a host cell;

(ii) culturing the host cell under conditions in which virus is produced; optionally further comprising the step:

(iii) recovering PPRV comprising the chimeric N protein from the virus from step (ii).

22. A method according to claim 21 wherein the host cell expresses signaling lymphocyte activation molecule (SLAM) on its cell surface.

23. A kit comprising:

(i) a vaccine according to claim 12; and

(ii) (a) a chimeric PPRV N protein which is expressed by the PPRV of the vaccine of part (i), a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; or (b) a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein which is expressed by the PPRV in the vaccine of part (i), a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; optionally further comprising:

(c) a polypeptide comprising the variable C-terminus domain of a native PPRV N protein, or polynucleotide, plasmid or construct encoding said variable C-terminus domain of a native PPRV N protein.

24. A kit comprising:

(i) a vaccine according to claim 12; and

(ii) a reagent which is capable of specifically binding to the chimeric PPRV N protein which is expressed by the PPRV of the vaccine of part (i) and/ora reagent which is capable of specifically binding to a non-chimeric PPRV N protein.

25. A kit according to claim 24 comprising:

(i) a vaccine according to claim 12;

(ii) (a) a chimeric PPRV N protein which is expressed by the PPRV of the vaccine of part (i), a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein;

(b) a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein which is expressed by the PPRV in the vaccine of part (i), a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; and/or

(c) a polypeptide comprising the variable C-terminus domain of a native PPRV N protein, or polynucleotide, plasmid or construct encoding said variable C-terminus domain of a native PPRV N protein; and

(iii) a capture reagent and/or a detectable reagent which is capable of selectively binding to the chimeric PPRV N protein; or

a capture reagent and/or a detectable regaent which is capable of selectively binding to the native PPRV N protein.

26. A kit comprising:

(i) a chimeric PPRV N protein as defined in claim 8, a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; or a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein as defined in claim 8, a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein; and

(ii) a detectable reagent which is (a) capable of binding to an anti-chimeric PPRV N protein antibody or (b) capable of specifically binding to the chimeric PPRV N protein; and optionally: (iii) a polypeptide comprising the variable C-terminus domain of a native PPRV N protein, a polynucleotide, plasmid or construct encoding said C-terminus domain of a native PPRV N protein; and/or

(iv) a detectable reagent which is (a) capable of binding to an antibody specific for the variable C-terminus domain of the native PPRV N protein or (b) capable of specifically binding to the variable C-terminus domain of the native PPRV N protein.

27. A kit according to claim 26 comprising:

(i) (a) a chimeric PPRV N protein as defined in claim 8, a polynucleotide, plasmid or construct encoding said chimeric PPRV N protein;

(ii) a capture reagent and/or a detectable reagent which is capable of selectively binding to the chimeric PPRV N protein; or

28. A kit according to any of claims 24 or 27 wherein the reagent and/or detectable reagent is an antibody or a fragment thereof.

29. A kit according to claim 28 wherein the reagent and/or detectable reagent is an antibody or a fragment thereof according to any of claims 17 to 20.

30. A method of distinguishing a subject that has been vaccinated with a vaccine according to claim 12 from a subject that has been infected with a non-chimeric PPRV; which method comprises contacting a chimeric PPRV N protein as defined in claim 8 or a polypeptide comprising the variable C-terminus domain of the N protein of the chimeric PPRV N protein as defined in claim 8 with (i) a sample from the subject; and (ii) a detectable reagent which is (a) capable of binding to an anti-chimeric PPRV N protein antibody present in the sample or (b) capable of specifically binding to the chimeric PPRV N protein.

31 . A method of distinguishing a subject that has been vaccinated with a vaccine according to claim 12 from a subject that has been infected with a non-chimeric PPRV; which method comprises contacting a native PPRV N protein or a polypeptide comprising the variable C-terminus domain of the native PPRV N protein with (i) a sample from the subject; and (ii) a detectable reagent which is (a) capable of binding to an anti-native PPRV N protein antibody present in the sample or (b) capable of specifically binding to the native PPRV N protein.

32. A method according to claim 30 wherein the detectable reagent is an antibody or a fragment thereof which is capable of specifically binding to the chimeric PPRV N protein.

33. A method according to claim 31 wherein the detectable reagent is an antibody or a fragment thereof which is capable of specifically binding to the native PPRV N protein.

34. A method according to claim 32 or 33 wherein the antibody or antigen binding fragment thereof is an antibody or a fragment thereof according to any of claims 17 to 20.