African swine fever DIVA immunoassay

Abstract

The present invention relates to the diagnostic use of African Swine Fever virus CD2v protein, methods, devices and kits for detecting the presence of ASFV antibodies in a test sample, in particular its use in a DIVA immunoassay.

Description

Technical Field

[0001] The present invention relates to the diagnostic use, method, apparatus and kit of African swine fever virus (ASFV) CD2v antigen for detecting the presence of African swine fever virus (ASFV) antibody in a test sample.

Background Art

[0002] The background of the disease African swine fever (African swine cholera) (ASF), its causative agent, the ASF virus, and attempts to control the virus have been the subject of numerous recent reviews (Arias et al., Vaccines 5 , 35, 2017; Galindo et al., Viruses 9 , 103, 2017; Revilla et al., Advances in Vir.Res. 100 , 2018; Sanchez et al., Vir.Res. 265 , 150 - 155, 2019; Blome et al., Vir.Res. 287 , 98099, 2020; Bosch - Camos et al., Porcine Health Management, 2020 6 : 17; Dixon et al., Annu.Rev.Anim.Biosci., 2020, 8(221-246). In the early 1900s, ASF was reported in East Africa as an acute hemorrhagic fever resulting in the death of almost all infected livestock pigs. The source of infection was identified as a virus that spread from an ancient forest cycle. Since then, ASFV has spread to most sub-Saharan African countries and Europe. Eradication of the disease was achieved in Europe by the mid-1990s. The outbreak in Georgia in the Caucasus region in 2007 harbingered a new era of infection, as ASFV subsequently spread to numerous countries, mainly in Eastern Europe. In 2018, the situation worsened significantly when ASFV was detected in China, which is thought to contain half of the world's pig population. The major socioeconomic impacts of ASF stem from the suffering of animals, business losses in the pig production chain, disease control costs, and commercial losses. Epidemics can lead to a dramatic reduction in the size of domestic pig populations, as well as soaring prices for pork and pork products. ASF is listed as a notifiable disease by the World Organisation for Animal Health (OIE).

[0003] The host range of ASFV is limited to suid animals and mites of the genus Ornithodoros. In its wild suid hosts in Africa, ASFV infection can cause mild clinical symptoms and lead to long-term persistent infection. In contrast, most ASFV isolates cause acute hemorrhagic fever with a near 100% mortality rate in domestic pigs and wild boars. The disease observed in domestic pigs and wild boars includes acute and hyperacute forms, which are caused by highly virulent isolates and result in death within 4–15 days after infection. Moderately virulent isolates result in a lower mortality rate (30–70%). Low-virulence isolates result in low mortality or no death at all and do not cause vascular lesions. However, signs of chronic disease such as arthritis may be observed. Clinical signs of acute ASF include high fever, loss of appetite, and lethargy and increased morbidity. Bloody diarrhea, vomiting, and miscarriage may also be observed.

[0004] ASFV is one of the largest and most complex cytoplasmic double-stranded DNA viruses. It replicates in mononuclear phagocyte cells, primarily monocytes and macrophages, but can infect other cell types. ASFV virions have an icosahedral structure of approximately 200 nm, consisting of an inner core, core-shell, inner membrane, and capsid, and in extracellular virions, an outer envelope formed by concentric layers. This virus is the sole member of the Asfaviridae family and is classified within the Asfivirus genus.

[0005] The ASFV genome exhibits varying lengths of 170–190 kbp across different ASFV strains. This is due to variability in the size of several open reading frames (ORFs), particularly in the multigene family (MGF) regions of the genome, and variability in short tandem repeats within genes and intergenetic regions. Depending on the strain, the genome contains 150–167 ORFs, which are involved in viral replication and morphogenesis, as well as the regulation of host cell function and immune evasion. Based on molecular genotyping analysis, 23 distinct ASFV genotypes have been described to date.

[0006] The roles of various structural and non-structural proteins of ASFV in viral infection, immunogenicity, and pathogenicity have been studied in the past, particularly by Jia et al., J.Vet.Res. 61 , 135-143, 2017; Blome et al., Virus Research 287 This has been reviewed in 98099, 2020; and Bosch-Camos et al., Porcine Health Management, 2020 6:17. More than 50 proteins are packaged within the viral particle, and more than 100 proteins are involved in infection. Currently studied ASFV proteins include, in particular, pp220, pp62, p54, p30, p72, p14.5, p17, CD2v, A238Lp, A179Lp, A238Lp, A224Lp, DP71Lp, and proteins encoded by MGF.

[0007] Despite several research groups developing novel vaccine technologies over the past few years, encompassing inactivated vaccines, recombinant protein / peptide vaccines, DNA vaccines, and attenuated live virus (LAV) vaccine candidates, there is currently no commercially viable, effective, and safe ASFV vaccine. Therefore, currently, only prevention, control, and eradication measures can be taken to combat ASF disease. These primarily rely on early detection through laboratory diagnosis, as well as the implementation of strict sanitary measures, restrictions on movement and trade, and culling of infected populations. These problems could theoretically be solved by the use of so-called marker vaccines. Such vaccines lack one or more immunogenic viral proteins, and as a result, animals immunized with marker vaccines do not produce antibodies against all immunogenic viral proteins. Detection of differences in the ASFV antibody palette between vaccinated and infected animals can be performed in diagnostic tests designed for this purpose. Thus, such tests enable "distinguishing infected animals from vaccinated animals" (DIVA).

[0008] The availability of an effective and safe ASF (marker) vaccine would improve ASF disease control and eradication programs, enhance animal welfare, and reduce economic losses. However, a lack of understanding of the complexity of the ASF virus itself, and the complexity of protective immunity against ASFV, has so far hindered the commercial availability of a safe and effective vaccine.

[0009] Inactivated ASFV vaccines are safe, but they do not provide protection, even in the presence of a potent adjuvant.

[0010] Several attempts to develop ASFV subunit vaccines have been reported (Bosch-Camos et al., 2020, op. cit.). Currently, more than 40 ASFV proteins are being studied. These include proteins such as p30, p12, p72, p54, p22, CD2v, and D117L. However, vaccines based on immunogenic subunit proteins have provided little to no homologous protection against pathogenic ASFV challenge.

[0011] Live attenuated virus (LAV) vaccines are considered the most promising type of vaccine for combating ASF. Recently, attempts have been made to develop recombinant LAVs based on live, replicating ASFV strains in which genes associated with blocking the host immune response and / or pathogenicity have been inactivated. Examples of ASFV genes targeted for deletion to improve the safety of ASFV strains include, among others, DP71L, several MGF360 and MGF505 genes, 9GL, DP96R, CD2v, A283L, A224L, EP153R, A276R, DP148R, B119L, and DP96R.

[0012] WO 2018 / 005358 (University of Connecticut) discloses a novel mutant ASFV-G Δ9GL / ΔUK virus resulting from extensive deletions in both the 9GL(B119L) and UK(DP96R) genes of the parent Georgia 2007 strain.

[0013] WO 2020 / 049194 (University of Madrid) discloses and characterizes a field isolate of ASFV named Lv17 / WB / Rie1. This ASFV strain was isolated from infected wild boars in Latvia. This novel ASFV strain was used as an attenuated live vaccine in wild boars administered orally and proved safe and effective.

[0014] US 2020 / 0129609 (Pirbright Institute) disclosed deletions in five MGF360 genes, namely 10L, 11L, 12L, 13L, and 14L, and three MGF505 genes, namely 1R, 2R, and 3R, as well as interruptions in additional genes (MGF360 9L, MGF505 4R, and DP148R). These mutations resulted in attenuation of the pathogenic virus, and vaccination with these novel mutant strains induced 100% protection against challenge by the parental ASFV strain.

[0015] It is generally accepted that, in order to successfully combat the current global ASFV epidemic, in addition to the availability of a safe and effective vaccine, additional requirements for a truly effective vaccination strategy must be met, namely, the availability of diagnostic assays that enable a reliable DIVA approach. Generally, DIVA diagnostic assays are diagnostic assays designed and adapted to be used in conjunction with a safe and effective DIVA vaccine. Such assays and accompanying vaccines together enable disease eradication based on immunological prevention and infection surveillance. Essentially, the active ingredient in a DIVA vaccine exhibits phenotypic / genotypic characteristics different from the pathogen (negative marker) prevalent in the field.

[0016] According to the European Union Reference Laboratory for ASF (eurl-asf), PCR is currently considered the “gold standard” test for the early detection of disease, for detecting the ASFV genome in any type of clinical sample from domestic pigs, swine, wild boar, and ticks, due to its superior sensitivity, specificity, robustness, and high-throughput application. Over the past 20 years, a variety of PCR tests, including both traditional and real-time PCR assays, have been developed and validated to detect a wide range of ASF isolates belonging to various known viral genotypes. All of these PCR assays are designed using the VP72 coding region, a highly conserved gene encoding the major viral protein, to ensure the (potential) detection of any ASFV isolate.

[0017] Detection of specific antibodies against ASFV by ELISA is, to date, the OIE-mandated test for international trade. Currently, numerous ASF ELISA modifications are available, as well as several OIE "in-house" forms of the test that use live virus as the antigen. Three commercially available ELISA kits (INGENASA, IDVET, and SVANOVIR) have been validated and are available for the detection of anti-ASFV antibodies. These ELISA assays are based on the most antigenic proteins described to date, such as p72, p32, pp62, and p54 (see https: / / asf-referencelab.info / asf / en / procedures-diagnosis / diagnostic-procedures).

[0018] Kollnberger et al. (J. Gen. Virol.) 83The researchers (1331-1342, 2002) identified the major serological immunodeterminants of ASFV by ELISA screening expressed ASFV proteins with convalescent antiserum, identifying 14 viral proteins that stimulate antibody responses recognized in ELISA. These include six proteins encoded by previously unassigned ORFs (B602L, C44L, CP312R, E184L, K145R, and K205R), as well as structural proteins (A104R, p10, p32, p54, and p73) and non-structural proteins [RNA reductases F334Lp, F778Rp, DNA ligase (NP419Lp), and thymidine kinase (K169Rp)] that have been studied in more detail.

[0019] In WO 2020 / 102370, ASFV diagnostic antigens were validated using convalescent serum. The chimeric antigen, designated KPI712, was recognized more strongly than p32, p54, p72, and pp62, which have been evaluated as diagnostic antigens to date.

[0020] However, none of the aforementioned literatures have identified an ASFV protein that can be used as an antigen in diagnostic assays on the one hand, and as an incidental marker immunogen in marker vaccines that enable DIVA on the other hand.

[0021] Therefore, the object of the present invention is to provide an in vitro diagnostic assay that can serologically distinguish between samples from animals vaccinated with an ASFV marker vaccine and samples from animals infected with ASFV that is prevalent in the field. [Brief explanation of the drawing]

[0022] [Figure 1] Schematic diagram of the full-length ASFV CD2v protein used in the examples, its domains, and fragments. Numbering is based on GenBank accession number CAD2068420. [Figure 2]Amino acid sequence alignment of ASFV CD2v proteins of various ASFV strains. Visualization of the alignment in MView (https: / / www.ebi.ac.uk / Tools / msa / mview / ). Numbers 1-8 are genotype II strains, serogroup 8 CD2v. Numbers 9-15 are genotype I strains, serogroup 4 CD2v. The correspondence with the sequence numbers is shown below. [Table 1] [Figure 3] Relative optical density measured at 450 nm in ELISA [CD2 "16-204" antigen (i.e., CD2 "16~204" antigen; the same applies hereinafter)] for various serum samples. [Figure 4] Relative optical density measured at 450 nm in ELISA (CD2 "132-204" antigen) for various serum samples. [Figure 5] Relative optical density measured at 450 nm in ELISA (CD2 "132-204" antigen) for various serum samples. [Figure 6] ELISA optical density measured at 450 nm (CD2 "132-204" antigen) at several serum sample dilutions in various sample diluents. [Figure 7] Effect of the size of the CD2v fragment on the performance of ELISA. ELISA optical density was measured at 450 nm using CD2v fragments of various lengths and various serum samples diluted 1:300. Note: CD2v fragment 132-204 was not tested with sera S13, S15, S19 or S21 due to the lack of peptide material. [Figure 8] Effect of surfactant concentration in the sample diluent on the P / N ratio of ELISA. The CD2v peptide fragment used was CD2 "132-204", and various serum samples at a series of dilutions were used. [Figure 9]Effect of salt concentration in sample diluent on the P / N ratio in ELISA. Several serum samples of different dilutions were used with the CD2 "132-204" peptide. Note: Data points for C-67 serum completely overlap with those for S3 serum. [Figure 10] Effect of PBS buffer in sample diluents on the P / N ratio in ELISA. Several serum samples at different dilutions were used with the CD2 "132-204" peptide. [Overview of the project]

[0023] Description of the Invention Surprisingly, it was found that this objective could be achieved by an in vitro diagnostic immunoassay for the detection of anti-ASFV antibodies, based on isolated ASFV CD2v antigen.

[0024] The observation that isolated ASFV CD2v antigens can be used to effectively distinguish between ASFV-infected animals and animals vaccinated with ASFV(CD2-) marker vaccines makes it possible for the first time to implement a DIVA strategy to combat the epidemic.

[0025] A key step leading to this favorable observation was the prior art report that the ASFV CD2v protein is a weak immunogen (Ruiz-Gonzalvo et al., Virology). 196 ,769-777,1993;Argilaguet et al.,PLoS ONE 7(9):e40942.doi:10.1371 / journal.pone.0040942;Gomez-Puertas et al., J.of Virol.Aug.1996,p.5689-5694;Lokhandwala et al.,Vet.Micr. 235Despite this, the inventors recognized that isolated ASFV CD2v antigens could be advantageously used in immunoassays for the purposes of the present invention (10-20, 2019 and PLoS ONE 12(5):e0177007.https: / / doi.org / 10.1371 / journal.pone.0177007, 2017).

[0026] Test samples obtained from animals vaccinated with the incidental LAV CD2v-marker vaccine can be serologically distinguished from test samples obtained from animals infected with the wild-type ASFV strain with the required specificity and sensitivity (Examples 1-3). This observation makes it possible for the first time to combat ASF epidemics with the DIVA strategy that the veterinary community has long awaited.

[0027] The examples also show that in antibody ELISA based on the CD2v antigen, convalescent ASFV pig antiserum could not be reliably identified from ASFV-negative control pig serum samples as a result of nonspecific binding between components in the anti-ASFV antiserum and the ASFV CD2v antigen in the immunoassay. Treatment of convalescent pig serum samples with sample diluents showed that: (i) the CD2v protein of ASFV can be used in immunoassays as an antigen to detect the presence or absence of anti-CD2v antibodies in pig test samples with sufficient specificity and sensitivity; (ii) the ASFV gene encoding the CD2v protein (EP402R) is a suitable target for genetic modification that gives LAV ASFV strains that can be used as DIVA vaccines; (iii) the CD2v protein in wild-type ASFV has sufficient immunogenicity to induce a detectable anti-CD2v antibody response in pigs; and (iv) modified LAV ASFV can be used in combination in immunoassays in advantageous diagnostic protocols that enable DIVA.

[0028] Accordingly, in a first embodiment, the present invention provides the use of an isolated African swine fever virus (ASFV) CD2v protein or its antigenic fragment conjugated to a solid support as an antigen in an immunoassay, characterized in that the CD2v protein or its antigenic fragment is used to detect the presence (including absence) of ASFV antibodies in a test sample obtained from a pig vaccinated with an accompanying ASFV attenuated live virus CD2v-marker vaccine (LAV CD2v-marker vaccine).

[0029] The ASFV CD2v protein is a well-known and established ASFV protein (Borca et al., Virology). 199 , 463-468, 1994; Rodriguez et al., J. Gen. Virol. 67(5312-5320, 1993). It is a glycoprotein with a relative molecular weight of approximately 105 kDa, encoded by the EP402R gene on the ASFV genome, which in vitro results in the erythrocyte adsorption phenotype of ASFV-infected cells. This ASFV protein is a viral homolog (CD2v) of the cell T lymphocyte surface adhesion receptor CD2 protein. Based on sequence data and hydropathy profiles, the ASFV CD2v protein is similar to a typical (CD2) class III transmembrane protein. Generally, the full-length ASFV CD2v protein contains four distinct parts: (i) a hydrophobic leader at the N-terminus of the protein, (ii) a hydrophilic extracellular domain containing numerous potential N-linked glycosylation sites, (iii) a hydrophobic extension of amino acids that functions as a transmembrane domain, and (iv) a C-terminal hydrophilic cytoplasmic domain containing numerous typical incomplete repeats of hexapeptides (PPPKPC) (Figure 1). Detailed information on the EP402 gene and ASFV CD2v protein of numerous ASFV strains [including genomic location of the ASFV gene, nucleotide / amino acid sequence information (and its alignment), identification of the four CD2v domains, and other annotations] can be found in Figure 2, as well as in various public nucleic acid and protein sequence databases such as the European Union Reference Laboratory for African Swine Fever (EURL-ASF) [e.g., the NCBI Genome Database, UniProt, EMBL / GenBank, and the European Union Reference Laboratory for African Swine Fever (EURL-ASF) at the Centro de investigacion en sanidad animal (CISA-INIA) (https: / / asf-referencelab.info / asf / en / sequence-data-base)]. In Zhu and Meng (Database, 1-9, 2020), the authors reported the establishment of an ASFV database in which collective, publicly available genomic and proteomic ASFV information is collected and made available.ASFVdb is freely accessible at http: / / asfvdb.popgenetics.net and the viruSITE genome browser; http: / / virusite.org / index.php, Stano, M., Beke, G., Klucar, L. (2016): viruSITE - integrated database for viral genomics.Database(Oxford).baw162.doi:10.1093 / database / baw162.

[0030] The sequences of the CD2v ASFV protein and its polypeptide fragment used in this invention may differ from the specific sequences disclosed herein. This is due to existing natural sequence variations between ASFV strains, as is evident from the sequences available in the aforementioned public sequence databases and Figure 2. The specific CD2v amino acid sequences and specific sequence number designations described herein relate to the ASFV reference strain Georgia 2007 / 1, which is also disclosed in GenBank accession number CAD2068420 (Sequence ID 1). Its complete genome nucleotide sequence and the amino acid sequence of the polypeptide encoded by the Georgia 2007 / 1 genome are also shown in GenBank accession number FR682468.

[0031] In particular, the ASFV CD2v protein as used herein is defined as a protein comprising an extracellular domain containing an amino acid sequence having at least 95% amino acid sequence identity with respect to SEQ ID NO: 2 (CD2 "16-204"), preferably at least 99% or 100% amino acid sequence identity with respect to SEQ ID NO: 2, in the region of duplication (alignment using the MUSCLE algorithm www.ebi.ac.uk / Tools / msa / muscle / ).

[0032] In the context of the present invention, antigenic fragments of the ASFV CD2v protein may also be used as antigens. Such antigenic fragments represent a truncated form of the CD2v protein and are polypeptides containing one or more epitopes that can be recognized by an anti-ASFV CD2v antibody in a test sample obtained from a pig infected with wild-type ASFV.

[0033] Preferably, the antigenic fragment is a polypeptide containing the extracellular domain of the CD2v protein or an antigenic fragment of the extracellular domain.

[0034] The extracellular domain of the ASFV CD2v protein is located at the N-terminus of the transmembrane domain.

[0035] The extracellular or transmembrane domain of the ASFV CD2v protein is, for example, Kyte and Doolittle (J.Mol.Biol.). 157 ,105-132) and Rodriguez et al. (J. Virol. 67 Such domains can be identified based on their typical amino acid sequence by methods known in the art, such as those described by (5312-5320, 1993). Alternatively, such domains can be identified by amino acid sequence alignment with one or more amino acid sequences of ASFV extracellular domains available from public sequence databases, or disclosed in public sequence databases. For example, the four domains of the Georgia 2007 / 1 CD2v protein extend to approximately the following amino acid regions: leader: aa(amino acids) 1-15; extracellular domain: aa 16-204; transmembrane domain: aa 205-229; and extracellular domain: aa 230-360 (where amino acid numbers are indicated with respect to the numbering of reference amino acid sequence SEQ ID NO: 1).

[0036] In a particularly preferred embodiment, the extracellular domain of the ASFV CD2v protein contains an amino acid sequence in the overlapping region that has at least 95% amino acid sequence identity with SEQ ID NO: 2, preferably at least 99% or 100% amino acid sequence identity with SEQ ID NO: 2.

[0037] In another preferred embodiment, the extracellular domain antigenic fragment for use in the present invention is a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 3 (CD2 "132-204"), preferably at least 99% or 100% amino acid sequence identity to SEQ ID NO: 3, in the overlapping region.

[0038] In a more preferred embodiment, the extracellular domain antigenic fragment for use in the present invention is a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24 in the overlapping region, more preferably at least 99% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24, and even more preferably 100% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24.

[0039] In the most preferred embodiment, the extracellular domain antigenic fragment for use in the present invention is a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 25 in the overlapping region, more preferably at least 99% amino acid sequence identity to SEQ ID NO: 25, and even more preferably 100% amino acid sequence identity to SEQ ID NO: 25.

[0040] In the present invention, sequence number 3 is CD2 "132-204", sequence number 23 is CD2 "132-194", sequence number 24 is CD2 "142-204", and sequence number 25 is CD2 "142-194".

[0041] In the examples, it has been shown that when the CD2 "132-204" fragment of the extracellular domain (of the genotype II strain) is used as an antigen in ELISA, genotype I-positive test samples also react with this antigen, while the complete extracellular polypeptide CD2 "16-204" is not recognized by antibodies in genotype I-positive samples. Therefore, the CD2 "132-204" fragment can be advantageously used in accordance with the present invention in a DIVA immunoassay for serologically differentiating between samples from pigs vaccinated with either genotype I or genotype II associated with the LAV strain and samples from pigs infected with wild-type ASFV containing the intact CD2v gene.

[0042] Therefore, in a more preferred embodiment, the extracellular domain antigenic fragment used herein is a polypeptide comprising an ASFV amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 3 (CD2 "132-204"), preferably at least 99% or 100% amino acid sequence identity with SEQ ID NO: 3, in the overlapping region.

[0043] In a more preferred embodiment, the extracellular domain antigenic fragment used herein is a polypeptide comprising an ASFV amino acid sequence having at least 95% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24 in the overlapping region, more preferably at least 99% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24, and even more preferably 100% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24.

[0044] In the most preferred embodiment, the extracellular domain antigenic fragment used herein is a polypeptide comprising an ASFV amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 25 in the overlapping region, more preferably at least 99% amino acid sequence identity with SEQ ID NO: 25, and even more preferably 100% amino acid sequence identity with SEQ ID NO: 25.

[0045] Alternatively, the extracellular domain antigenic fragments used herein are polypeptides comprising an ASFV amino acid sequence having at least 95%, at least 99%, or 100% amino acid sequence identity in the overlapping region with any of the fragments 132-194, 132-214, 122-194, 122-204, or 142-214 shown in SEQ ID NO: 1, and any of the fragments 132-194, 142-204, or 142-194 shown in SEQ ID NO: 1.

[0046] The aforementioned ASFV CD2v antigen may be from any serogroup known for the ASF virus, particularly serogroup 4 or 8, preferably serogroup 8.

[0047] Clustering of ASFV serogroups is based on testing for the suppression of the ASFV erythrocyte adsorption phenotype by serogroups belonging to the same group. Currently, the existence of serogroups 1-8 has been confirmed (Malogolovkin et al., J. Gen. Virol). 96 (866-873, 2015).

[0048] Furthermore, the aforementioned ASFV CD2v antigen may include a tag that enables the detection of protein expression or the purification of the antigen. Suitable tags include Chatterjee (Opin.Biotech). 17As disclosed in 353-358, 2006, the product includes a 6×His tag, a c-Myc domain: EQKLISEEDL (SEQ ID NO: 4), a hemagglutinin tag: YPYDVPDYA (SEQ ID NO: 5), a maltose-binding protein, a glutathione-S-transferase, a maltose-binding protein, a FLAG-tagged peptide, a biotin acceptor peptide, a streptavidin-binding peptide, or a calmodulin-binding peptide. The FLAG tag or His tag is preferred.

[0049] The production of the CD2v antigen used herein may utilize common and commercially available conventional peptide synthesis methods and recombinant DNA expression systems and methods, including bacterial, yeast, fungal, insect, and vertebrate cell expression systems. Detailed guidance on prokaryotic and eukaryotic expression systems can be found, in particular, in reviews and textbooks on recombinant DNA expression, such as Trepe, K., *Applied Microbiology and Biotechnology*. 72 ,Number 2(2006),211-222;Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, edited by Gellissen, G. Publisher: Wiley-VCR, ISBN: 3527310363 edition 2005, Expression systems, edited by Michael Dyson and Yves Durocher, Scion Publishing Ltd, ISBN. 9781904842439 edition 2007.

[0050] Advantageously, the CD2v antigen can be produced by using a baculovirus insect cell expression system. Examples of scientific papers, textbooks, and reviews illustrating this system include Luckow et al., 1988, Bio-technology, vol.6, p.47; Baculovirus Expression Vectors: A Laboratory Manual by David R. O'Reilly, Oxford University Press, 1993, ISBN:0716770172; The Baculovirus Expression System: A laboratory guide, King & Possee (eds.), 1992, ISBN:9401050473; and for a review, van Oers et al., 2015, J. of Gen. Virology, 96 Examples include 6-23. Expression and purification of ASFV polypeptides in E. coli and insect cell lines are described, for example, by Lokhandwala et al., PLOS ONE, May 2017, and Kollnberger et al. (cited above).

[0051] Means and kits for the efficient production of baculoviruses for use in the present invention are commercially available, including, for example, Bac-to-Bac® (Thermo Fisher Sci., Waltham, MA, USA); ProEasy® (AB Vector, San Diego, CA, USA); and flashBAC® (Oxford Expression Technologies, Oxford, UK).

[0052] "Marker vaccines" are a well-known concept in the field of veterinary vaccinology. Marker vaccines contain and / or express modified polypeptide immunogens that differ in immunogenicity from the wild-type polypeptide immunogen in that they lack at least one epitope or have a different form of epitope compared to the wild-type form. Typically, the polypeptide immunogen (or the gene encoding it) in, or expressed by, a marker vaccine is modified by biochemical or recombinant DNA technology, and as a result, it is possible to serologically detect infected animals unrelated to vaccination by taking advantage of the lack of an antibody response against the wild-type portion of the modified immunogen in the marker vaccine. This would enable serological DIVA. Typically, the modified immunogen is either a non-existent immunogen or a fragment of the wild-type polypeptide immunogen.

[0053] As used herein, the term "immunogen" refers to the ability of a molecule (e.g., a protein or polypeptide) to induce a specific antibody response by the immune system of an organism, while the term "antigen" refers to the ability of a molecule to specifically bind to antibodies produced by the immune system of an organism.

[0054] The epitopes used herein are typically 5-15 amino acid extensions within a protein or polypeptide that can induce an antibody response specific to that portion and / or bind to a specific antibody produced by such a response.

[0055] The LAV CD2v-marker vaccine used herein is a vaccine containing an attenuated bioreplicable ASFV marker vaccine strain capable of expressing a serologically distinguishable modified CD2v polypeptide immunogen from the CD2v polypeptide immunogen of a wild-type ASFV strain.

[0056] An "accompanying" LAV CD2v-marker vaccine means a vaccine containing the aforementioned CD2v-marker vaccine strain, where the modified CD2v polypeptide immunogen is aligned with the CD2v polypeptide antigen in the immunoassay and designed to be different from the CD2v polypeptide antigen in the immunoassay, so that the CD2v polypeptide antigen can serologically detect antibodies in the test sample that are specific to the wild-type portion of the CD2v polypeptide immunogen, but cannot recognize antibodies that are specific to the modified portion of the CD2v polypeptide immunogen.

[0057] Therefore, the incidental LAV CD2v-marker vaccine contains the aforementioned CD2v-marker vaccine strain, which induces an effective immune response in pigs, generating an antibody repertoire in vaccinated pig serum samples that lacks antibodies present in the antibody repertoire of wild-type ASFV-infected pig serum samples. Thus, the distinction between infected animals and vaccinated or negative animals is based on an immunoassay detecting antibodies specific to one or more ASFV CD2v epitopes that are missing from the marker vaccine.

[0058] In particular, the associated LAV CD2v-marker vaccine includes ASFV CD2v-marker vaccine strains that contain and / or can express truncated CD2v protein, or that do not contain and / or cannot express CD2v protein. Preferably, the truncated CD2v protein is a polypeptide fragment of the CD2v protein lacking an extracellular domain or a fragment thereof.

[0059] More preferably, the truncated CD2v protein is a polypeptide fragment of the CD2v protein lacking a fragment of the extracellular domain.

[0060] In a more preferred embodiment, the truncated CD2v protein is a polypeptide fragment of the CD2v protein lacking a fragment of the extracellular domain of the CD2v protein, and containing an amino acid sequence in the overlapping region that has at least 95% amino acid sequence identity with SEQ ID NO: 3 (CD2 "132-204"), preferably at least 99% or 100% amino acid sequence identity with SEQ ID NO: 3.

[0061] In a more even more preferred embodiment, the truncated CD2v protein is a polypeptide fragment of the CD2v protein lacking a fragment of the extracellular domain of the CD2v protein, and comprising an amino acid sequence in the overlapping region having at least 95% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24, more preferably at least 99% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24, and even more preferably 100% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24.

[0062] In the most preferred embodiment, the truncated CD2v protein is a polypeptide fragment of the CD2v protein lacking a fragment of the extracellular domain of the CD2v protein, and containing an amino acid sequence in the overlapping region that has at least 95% amino acid sequence identity to SEQ ID NO: 25, more preferably at least 99% amino acid sequence identity to SEQ ID NO: 25, and even more preferably 100% amino acid sequence identity to SEQ ID NO: 25.

[0063] In a more preferred embodiment, the truncated CD2v protein is a polypeptide fragment of the CD2v protein lacking a fragment of the extracellular domain of the CD2v protein, and comprising an ASFV amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 3 (CD2 "132-204"), preferably at least 99% or 100% amino acid sequence identity with SEQ ID NO: 3, in the overlapping region.

[0064] In a more even more preferred embodiment, the truncated CD2v protein is a polypeptide fragment of the CD2v protein lacking a fragment of the extracellular domain of the CD2v protein, comprising an ASFV amino acid sequence having at least 95% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24 in the overlapping region, more preferably at least 99% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24, and even more preferably 100% amino acid sequence identity with a sequence selected from SEQ ID NOs. 23 and 24.

[0065] In the most preferred embodiment, the truncated CD2v protein is a polypeptide fragment of the CD2v protein lacking a fragment of the extracellular domain of the CD2v protein, and comprising an ASFV amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 25, more preferably at least 99% amino acid sequence identity to SEQ ID NO: 25, and even more preferably 100% amino acid sequence identity to SEQ ID NO: 25, in the overlapping region.

[0066] In a particular embodiment of the use of the ASFV CD2v antigen in the immunoassay described above for detecting the presence (including absence) of ASFV antibodies in a test sample obtained from a pig vaccinated with an incidental LAV CD2v-marker vaccine, the ASFV CD2v antigen does not have a common epitope, in particular overlapping amino acid sequence, with the CD2v-marker vaccine strain or the modified CD2v polypeptide immunogen expressed by the CD2v-marker vaccine strain. The absence of overlapping amino acid sequences means that the ASFV CD2v antigen and the LAV CD2v-marker vaccine strain or the modified CD2v polypeptide immunogen expressed by the LAV CD2v-marker vaccine strain originate from different regions of the CD2v protein and do not exhibit overlap at their terminals.

[0067] More preferably, as described above, the ASFV CD2v antigen in the immunoassay and the modified CD2v polypeptide immunogen in the accompanying marker vaccine represent two distinct, non-overlapping fragments of the extracellular domain of the ASFV CD2v protein.

[0068] Suitable attenuated bio-ASFV CD2v-marker vaccine strains are known in the art, or can be produced by recombinant DNA technologies using standard methods such as CRISPR-Cas or homologous recombination, or can be isolated from the field.

[0069] Recently, the results of various research activities disclosing the (rational) design of ASFV LAV strains by genetically modifying ASFV strains have been published (see the aforementioned review of ASFV and the references cited therein). These prior art documents disclose various ASFV genes that can be mutated to obtain attenuated and effective ASFV vaccine strains.

[0070] Prior art also discloses the creation of various ASFV mutant strains containing or expressing modified CD2v protein: Gallardo et al. (Transbound.Emerg.Dis.66,1399-1404,2019) and Barasona et al. (Front.Vet.Sci.6;137,2019). The ASFV strain Lv17 / WB / Rie1 (WO 2020 / 049194) has been tested for its safety and efficacy profile after immunization in domestic pigs and wild boars. Lv17 / WB / Rie1 is a naturally attenuated strain possessing a truncated CD2v protein (encoded by the mutant EP402R gene) and exhibiting a non-erythrohemolytic phenotype in vitro. Another naturally occurring non-pathogenic ASFV isolate, OURT88 / 3, contains a frameshift mutation in the sequence encoding the cytoplasmic domain of CD2v, resulting in the last 215 amino acids not being translated. Borca et al. (J.Virol.72,2881-2889,1998 and Sci Rep.2020,10:494) and Monteagudo et al. (J.Virol.91,2017,91(21):e01058-17) have disclosed the creation of CD2v deletion mutants using recombinant DNA technology based on ASFV strains Malawi, Georgia 2007 / 1, and BA71, respectively. Chen et al. (Sci China Life Sci,63,2020) have disclosed the creation of an ASFV strain (HLJ / 18) with seven gene deletions that is effective and safe as an attenuated live virus vaccine in pigs. In particular, the gene encoding the CD2v protein is also deleted in HLJ / 18.

[0071] The ASFV CD2v antigen and ASFV CD2v-marker vaccine strains used in this invention may be derived from any ASFV genotype or any ASFV strain, such as one of the following strains: Georgia 2007 / 1, Benin 97 / 1, Kenya, and Malawi. The preferred ASFV genotype is I or II. ASFV genotyping is based on genetically characterizing the ASFV genome by sequencing the C-terminal portion of the p72 protein (encoded by the B646L gene), which corresponds to the major ASFV capsid protein. This method has determined 24 different genotypes to date (Bastos et al., Arch. Virol. 2003 Apr;). 148 :693-706.2003; Quembo et al., Transbound.Emerg.Dis.;65,420-431,2018).

[0072] In a preferred embodiment, the accompanying LAV CD2v-marker vaccine is based on the ASFV strain Lv17 / WB / Rie1 disclosed in WO 2020 / 049194, and the ASFV CD2v antigen is a polypeptide comprising an ASFV amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 3 (CD2 "132-204") in the overlapping region, preferably at least 99% or 100% amino acid sequence identity to SEQ ID NO: 3.

[0073] In a more preferred embodiment, the accompanying LAV CD2v-marker vaccine is based on the ASFV strain Lv17 / WB / Rie1 disclosed in WO 2020 / 049194, and the ASFV CD2v antigen is a polypeptide comprising an ASFV amino acid sequence having at least 95% amino acid sequence identity with sequences selected from SEQ ID NOs. 23 and 24 in the overlapping region, more preferably at least 99% amino acid sequence identity with sequences selected from SEQ ID NOs. 23 and 24, and even more preferably 100% amino acid sequence identity with sequences selected from SEQ ID NOs. 23 and 24.

[0074] In the most preferred embodiment, the accompanying LAV CD2v-marker vaccine is based on the ASFV strain Lv17 / WB / Rie1 disclosed in WO 2020 / 049194, and the ASFV CD2v antigen is a polypeptide comprising an ASFV amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO: 25 in the overlapping region, more preferably at least 99% amino acid sequence identity to SEQ ID NO: 25, and even more preferably 100% amino acid sequence identity to SEQ ID NO: 25.

[0075] The accompanying LAV CD2v-marker vaccine used in the present invention can be manufactured by conventional methods, such as those commonly used for commercially available attenuated live virus vaccines. Briefly, the attenuated live CD2v-marker vaccine strain is inoculated into a susceptible substrate, and it is grown until the virus replicates to a desired titer, after which the ASFV-containing material is recovered. The recovered material, which is then purified and / or concentrated as needed, is formulated with a pharmaceutically acceptable carrier or diluent into a pharmaceutical product having immunoassay properties. The carrier includes stabilizers, preservatives, and buffers. Suitable stabilizers are, for example, SPGA (sucrose, phosphate, glutamate, and albumin), carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, dextran, glutamate, or glucose), proteins (e.g., dried whey, albumin, or casein), or their degradation products. Suitable buffers are, for example, PBS buffer, Tris buffer, or HEPES buffer. Suitable preservatives are thimerosal, melthiolate, and gentamicin.

[0076] The vaccine can be administered intramuscularly, subcutaneously, intradermally, or intranasally by inoculation or injection in a dose effective in protecting pigs from ASF disease. This dose may vary depending on the animal being vaccinated, taking into account the animal's age and weight.

[0077] In the examples, it has been demonstrated that a successful ASFV DIVA approach was established for the first time by combining the DIVA diagnostic assay and the accompanying DIVA LAV CD2v-marker vaccine, both as defined above. The inventors confirmed that, on the one hand, ASFV CD2v corresponds to a suitable immunogen in wild-type ASFV, and on the other hand, ASFV CD2v corresponds to a suitable antigen that can be used in immunoassays with the necessary specificity and sensitivity to enable DIVA. The DIVA method enables vaccination while maintaining the possibility of serological surveillance for the presence of infection, thereby providing for the first time a powerful and practical means of combating ASF in animals that can be easily scaled. This is because, in particular, the method does not involve the use of live infectious ASFV, which would require such a method to be carried out in a highly containment facility.

[0078] Therefore, in certain embodiments, the ASFV CD2v antigen is used in an immunoassay characterized in that the immunoassay is a DIVA immunoassay.

[0079] In general, for infected animals to be ultimately distinguished from vaccinated animals, the test score must be interpreted as positive or negative. In practice, this means being above or below a certain threshold. This can be easily done by incorporating several reference samples tested in parallel with the test sample into the method, as described, for example, in the examples. Positive and negative reference samples can be prepared in pigs or are available from several institutions and (national) reference laboratories around the world (e.g., European Union Reference Laboratory for ASFV, Centro de investigacion en sanidad animal (CISA-INIA), Madrid, Spain).

[0080] The solid support used in the immunoassay described above can, in principle, be any solid support; however, it must enable the performance of use according to the present invention, in particular, enable the binding of the ASFV CD2v antigen to the solid support. It can be of various sizes, shapes, or forms. Binding may occur by conventional means, such as covalent or non-covalent interactions (i.e., adsorption or coating). Alternatively, binding may be achieved via biotinylated CD2v antigen bound to an avidin-coated solid support.

[0081] In particular, the solid support is a microtiter plate, vial, bead paper strip, membrane, gel, or lateral flow strip. Preferably, the solid support is a microtiter plate.

[0082] In another embodiment, the present invention provides a method for distinguishing between ASFV-infected animals (positive test result) and vaccinated animals (negative test result), wherein the method is an immunoassay, the isolated ASFV CD2v protein or its antigenic fragment conjugated to a solid support is used as the antigen, and the marker vaccine is an associated LAV CD2v-marker vaccine, and the method is characterized by comprising the step of testing a test sample obtained from an animal for the presence of an ASFV CD2v antibody conjugated to the antigen.

[0083] In this additional aspect and embodiment of the present invention, the definitions of certain terms referred to herein and in the various embodiments thereof are the same as those given with respect to the first aspect described above.

[0084] In one embodiment of this aspect, the present invention provides a method wherein the antigenic fragment is a polypeptide comprising the extracellular domain of the CD2v protein or an antigenic fragment of the extracellular domain, more particularly, the antigenic fragment of the extracellular domain is a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 3 (CD2 "132-204"), and even more particularly, the antigenic fragment of the extracellular domain comprises an ASFV amino acid sequence consisting of an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 3 (CD2 "132-204") in the overlapping region.

[0085] In a preferred embodiment of this design, the antigenic fragment of the extracellular domain is a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with a sequence selected from SEQ ID NOs: 23 and 24 in the overlapping region, more preferably at least 99% amino acid sequence identity with a sequence selected from SEQ ID NOs: 23 and 24, and even more preferably 100% amino acid sequence identity with a sequence selected from SEQ ID NOs: 23 and 24.

[0086] In the most preferred embodiment of this design, the antigenic fragment of the extracellular domain is a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 25 in the overlapping region, more preferably at least 99% amino acid sequence identity with SEQ ID NO: 25, and even more preferably 100% amino acid sequence identity with SEQ ID NO: 25.

[0087] In another embodiment of this aspect, the present invention provides a method wherein the accompanying LAV CD2v-marker vaccine comprises an ASFV CD2v-marker vaccine strain that includes and / or expresses a modified CD2v polypeptide immunogen, more specifically, the modified CD2v polypeptide immunogen lacks the extracellular domain or fragment of the CD2v protein, or the ASFV CD2v antigen and the modified CD2v polypeptide immunogen do not have overlapping amino acid sequences (all as described above).

[0088] The design of the immunoassays used in the various embodiments of the present invention described above is similar to commonly used immunoassays based on antigens bound to a solid support. In principle, immunoassays are based on the formation of antibody-antigen complexes and subsequent follow-up tests for the presence (including the absence) of such complexes. The following handbooks describe various diagnostic assays that may be used herein and their specific characteristics (Handbook of Immunoassay Technologies, Vashist, Sandeep K. and Luong, John HT, 2018; and Immunoassays: Development, Applications and Future Trends, R. O'Kennedy, C. Murphy 2017).

[0089] Detailed information regarding the preparation, protocol, standard operating procedures, reagents, etc., for the ASFV immunoassay used in this invention can be found, for example, in the European Union Reference Laboratory for ASFV (cited above), FAO (Beltran-Alcrudo et al., 2017, African swine fever: detection and diagnosis - A manual for veterinarians. FAO Animal Production and Health Manual No. 19, Rome), and Gallardo et al., Virus Research. 271 It was disclosed in 197676 and 2019.

[0090] In a more detailed embodiment of the method according to the present invention, the method is 1. A step of incubating the test sample with the antigen in the assay mixture. 2. A step to enable the formation of an ASFV CD2v antibody-antigen complex in the assay mixture, and 3. Step to detect the presence of antibody-antigen complexes in the assay mixture. Includes.

[0091] In this embodiment, the detection of the presence of an antibody-antigen complex may involve the use of a label-bound detection antibody. In particular, it may involve contacting the complex with an antibody-labeled conjugate.

[0092] The properties of the label are not important; it can be any label commonly used in immunoassays. A label is an entity that provides or can induce a detectable signal.

[0093] In particular, the labels are enzymes, fluorophores, chromophores, radioisotopes, enzyme substrates, chemiluminescent molecules, or gold colloids.

[0094] Preferably, the label is an enzyme that can be directly or indirectly bound to the detection antibody, particularly by biotin / avidin binding.

[0095] Typically, the enzyme used herein is horseradish peroxidase (HRP), and the enzyme substrate is TMB(3,3',5,5'-tetramethylbenzidine).

[0096] In the particularly preferred embodiments of the present invention described above, the immunoassay is an ELISA (enzyme-linked immunosorbent assay). The advantages of ELISA include its practicality, reliability, speed, and ease of scale-up. ELISA is well known in the art, and various types in terms of form and protocol may be applied herein.

[0097] The aforementioned immunoassays may be based on direct or indirect antigen-antibody reactions. A direct assay involves a one-step conjugation of a sample antibody to an antigen. An indirect assay involves a two-step conjugation process, including the use of a primary (sample) antibody and a labeled secondary (detection) antibody capable of binding to the primary antibody. An immunoassay can also be a competitive immunoassay, in which case the antibody in the sample competes with the labeled secondary antibody, which can bind to the antigen, for a limited number of antigen-binding sites.

[0098] In the preferred method according to the present invention described above, 1. A step of incubating the test sample with a solid support-bound antigen in the assay mixture. 2. Adding a labeled antibody capable of recognizing the anti-ASFV CD2v antibody to the assay mixture. 3. A step of adding an enzyme substrate to the assay mixture to generate a detectable signal, and 4. Steps to measure the signal An indirect ELISA including [specific component] is used.

[0099] When a chromogenic substrate is added to the assay mixture to induce color development, samples with higher antibody concentrations produce a higher signal than those with lower antibody concentrations.

[0100] In another preferred method according to the present invention, 1. A step of incubating the test sample and an antibody capable of binding to the antigen together with a solid support-bound antigen in an assay mixture. 2. A step of adding an enzyme substrate to the assay mixture to generate a detectable signal, and 3. Steps to measure the signal A competitive ELISA including [specific criteria] is used.

[0101] When a chromogenic substrate is added to the assay mixture to produce color, samples with high antibody concentrations produce a lower signal than samples with low antibody concentrations, resulting in an inverse correlation between antibody concentration in the sample and color development in the assay.

[0102] ELISA results are typically expressed in arbitrary absorbance units, usually optical density (OD) units ranging from 0.1 to 2.5, depending on the characteristics and settings of the technical instrument used for reading. Appropriate positive and negative control samples are usually included, and in most cases, the sample is tested multiple times. Standardization is achieved by including a defined reference sample (within its dilution range), which also allows for matching a certain score to a preset threshold for determining positive or negative, enabling correlation to biological significance, such as distinguishing between animals infected with wild-type virus and those vaccinated with a marker vaccine.

[0103] Particularly preferred ELISAs are shown in the examples.

[0104] In another method according to the present invention, the immunoassay is a lateral flow (immunochromatography) assay. Lateral flow immunoassays are commonly used in the art. In principle, a lateral flow immunoassay functions in the same way as the ELISA described above. In the lateral flow immunoassay used in the present invention, the antigen can be bound as a test line to a solid support having the ability to transport fluid as a result of capillary activity, such as porous paper or a (nitrocellulose) membrane, a microstructured polymer or a sintered polymer. Essentially, the solid support moves the sample solution of the test sample containing the antibody to be detected from an absorption zone along the surface of the support. An antibody-antigen complex is then formed on the test line and can be detected in the detection zone of the solid support to which the antigen is bound.

[0105] Therefore, in certain embodiments of the method of the present invention, the immunoassay used herein is a lateral flow immunoassay.

[0106] More specifically, lateral flow immunoassay is, 1. A step of incubating the test sample together with a solid support in the absorption zone. 2. A process that enables the formation of an antibody-antibody / labeled complex. 3. A step of enabling lateral movement of the composite via a solid support, 4. A step of capturing the complex with an antigen bound to a solid support in the test line, thereby enabling the formation of an antibody-antigen-antibody / labeled complex, and 5. Step of detecting the presence of the complex in the assay mixture in the detection zone. Includes.

[0107] The labels used in lateral flow immunoassays can be any labels commonly used in LF immunoassays, and in particular, they can be colored particles, such as latex particles, nanometer-sized particles or gold particles, fluorescently labeled, magnetically labeled or radio frequency identification (RFID) particles.

[0108] The LF immunoassays used herein may function as either competitive assays or sandwich assays.

[0109] The inventors initially observed that when ASFV CD2v antibody-positive serological samples were incubated with CD2v antigen in ELISA, the signal-to-noise ratio was below optimal, resulting in a negative impact on the specificity of the ELISA and preventing the acquisition of a reliable DIVA immunoassay. Subsequently, it was found that this limitation was due to short-term intermolecular interactions unrelated to specific antigen-antibody interactions. The examples demonstrate that this negative impact can be overcome by incorporating a dilution step (of porcine antiserum) in the immunoassay, which limits these nonspecific intermolecular interactions. Sample diluents that can be used in this step exhibit increased stringency.

[0110] The term stringency of the sample diluent is defined herein as a numerical value representing the ratio (P / N ratio) of the absorbance value (OD units) of the diluted positive serum control sample to the absorbance value (OD units) of the diluted negative serum control sample, as measured in ELISA, particularly as described in the examples.

[0111] Therefore, in the advantageous method of the present invention, the porcine test sample is diluted with a sample diluent of optimal stringency sufficient to limit undesirable nonspecific interactions without affecting specific antigen-antibody interactions to undesirable levels.

[0112] The sample diluent used in the present invention may have a stringency P / N ratio of 5 or higher, preferably 10 or higher, when measured by ELISA.

[0113] The examples demonstrate and provide further guidance on how incorporating a sample dilution step and increasing the stringency of the sample diluent together enable the CD2v antigen-based immunoassay to become a highly reliable DIVA immunoassay. The sample dilution step reduces nonspecific interactions between the anti-porcine ASFV antiserum and the CD2v antigen in Elisa, and therefore increases the P / N ratio. The sample dilution step can be designed by those skilled in the art to use an appropriate sample diluent with increased stringency, while simultaneously maintaining the Elisa(OD) signal for the positive control sample at an appropriate level.

[0114] Sample diluents that can be advantageously used in the method of the present invention may include conventional buffers, such as PBS or TRIS buffer, to which surfactants, such as Tween® 20 or Tween 80, Triton, sodium deoxycholate, sodium dodecyl sulfate, aminooxide, or CHAP surfactants have been added.

[0115] In a preferred embodiment of the identification method according to the present invention, or in a preferred embodiment of the determination method according to the present invention, the sample diluent comprises one or more surfactants selected from Tween® 20, Tween 80, and aminooxides. Preferably, the aminooxide is aminooxide WS35, also known as cocamidopropylamine oxide. More preferably, the aminooxide WS35 is a compound having CAS number 53988-60-6.

[0116] In preferred embodiments, the surfactant is present in the sample diluent at a concentration of 1-5% w / v, more preferably 2-4% w / v, and even more preferably 3% w / v.

[0117] Therefore, in a preferred embodiment of the present invention, the method according to the present invention includes the step of diluting a test sample with a sample diluent having a stringency that yields a P / N ratio of 5 or more, preferably 10 or more.

[0118] A favorable P / N ratio can also be obtained by diluting the porcine test sample with a sample diluent in a ratio of 1:100 to 1:2700, preferably 1:100 to 1:900, more preferably 1:100 to 1:300, and more specifically 1:300.

[0119] In a more preferred method according to the present invention, the porcine test sample is diluted with a sample diluent having a stringency that yields a P / N ratio of 5 or more, preferably 10 or more, at a dilution ratio of 1:100 to 1:2700, preferably 1:100 to 1:900, more preferably 1:100 to 1:300, and more specifically 1:300.

[0120] In a preferred embodiment of the identification method according to the present invention, or in a preferred embodiment of the determination method according to the present invention, the sample diluent contains 0.01 to 1 M, more preferably 0.05 to 0.5 M, and even more preferably 0.1 M of salt. In a preferred embodiment, the salt is magnesium chloride.

[0121] In the most preferred embodiment, the sample diluent comprises Tween, aminooxide, and magnesium chloride.

[0122] The various embodiments of the present invention described above can be advantageously applied by testing samples derived from pigs susceptible to ASFV infection. In particular, the pigs are pigs of the family Suidae, preferably pigs of the genus Sus, such as pigs or wild boars. Preferably, the pigs are domesticated pigs.

[0123] Therefore, in preferred embodiments of various aspects of the present invention, the test sample is derived from domestic pigs.

[0124] The test sample for use in various embodiments of the present invention may, in principle, be any type of sample from a pig that may contain anti-ASFV CD2v antibodies, such as a plasma sample or a serum sample. Preferably, the sample is a serum sample.

[0125] Another aspect of the present invention is an apparatus for use in a method for detecting the presence of ASFV CD2v antibodies in a test sample obtained from a pig vaccinated with the aforementioned incidental LAV CD2v-marker vaccine, the apparatus comprising isolated ASFV CD2v antigen conjugated to the aforementioned solid support.

[0126] Another aspect of the present invention is a diagnostic kit including the above-mentioned apparatus.

[0127] The diagnostic kit according to the present invention may include a single packaging unit containing additional components applied in the method according to the present invention.

[0128] In particular, diagnostic kits are • Sample diluent, • Antibody-labeled conjugate, • Positive control sample, and / or • Negative control sample Further includes one or more containers containing [the specified item].

[0129] In a more detailed embodiment, the diagnostic kit also includes instructions for using the kit with respect to test samples obtained from pigs vaccinated with the aforementioned supplemental LAV CD2v-marker vaccine.

[0130] In particular, the instructions for use state that the diagnostic kit can be used for DIVA, and that test samples from ASFV-infected pigs will be positive in the test, while test samples from vaccinated, uninfected pigs will be negative.

[0131] Examples Example 1 - ELISA based on ASFV CD2v To verify whether anti-CD2v antibodies in porcine serum can be detected by ELISA, we performed ELISA using a fragment of the ASFV CD2v protein called CD2 "16-204". CD2 "16-204" is elongated into the extracellular domain and lacks the leader sequence, transmembrane domain, and proline-rich intracellular portion of the full-length CD2v protein (Figure 1). It contains the GP64 signal peptide: MVSAIVLYVLLAAAAHSAFA (SEQ ID NO: 6) at its N-terminus and a 6×His tag at its C-terminus. It was produced by baculovirus expression followed by purification with GenScript.

[0132] Serum samples were obtained from the European Union Reference Laboratory for African Swine Fever (CISA-INIA, Spain).

[0133] For ELISA, 96-well microtiter plates were coated overnight at 2–8°C with a solution containing CD2v fragments at a concentration of 1 μg / ml. The plates were washed four times with washing buffer (0.04 M PBS + 0.15% Tween20) and then blocked with casein at 37°C for 1 hour. After washing the plates four times, 3-fold serial dilutions of serum samples in EIA buffer (0.2 M PBS + 0.1% BSA) were prepared in wells A–G of each row (well H contained only EIA buffer and was used as a control). Serum samples were pre-diluted 1:100 in EIA buffer. Plates containing serum dilutions were incubated at 37°C for 1 hour and then washed four times with washing buffer. A solution containing peroxidase-labeled goat anti-porcine IgG(H+L) antibody was added to each well, the plates were incubated at 37°C for 1 hour, and then washed four times with washing buffer. Next, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added to each well and incubated for at least 10 minutes. The coloring reaction was stopped by adding 4N H2SO4. Optical density was measured at 450 nm using a microtiter plate reader, and the data were analyzed.

[0134] The results are shown in Figure 3. Serum from pigs infected with genotype II ASFV, serotype 8 strains (S1, S2, and S3-ASFV strain Lv17 / WB / Rie1; WO 2020 / 049194) showed a clear positive signal above negative serum C-67 at a serum dilution of 1:900. Serum samples C+113 obtained from pigs that were twice infected with genotype I ASFV strains and then also infected with genotype II strains showed a clear positive signal set to 100%. Serum from pigs infected with genotype I, serotype 4 ASFV strains (S13, S15, S19, S21) could not be distinguished from the negative serum control. Therefore, the results indicate that CD2v is immunogenic and that ELISA based on the CD2 "16-204" fragment can be used to measure anti-CD2v antibodies induced by genotype II ASFV strains.

[0135] Example 2 - CD2v-based ELISA for detection of genotype I and II ASFV strains We designed a truncated form of the extracellular CD2v fragment CD2"16-204". This fragment, namely CD2"132-204", lacks the 131st amino acid at the N-terminus of the CD2v protein (Figure 1). It contains a 5×GlyGlyGlySer (SEQ ID NO: 7) linker and a subsequent Flag tag at its C-terminus and was produced by baculovirus expression followed by purification with GenScript.

[0136] An ELISA was performed as described in Example 1.

[0137] The results are shown in Figure 4. Serums from pigs infected with genotype II ASFV strains (S1 and S2) or genotype I ASFV strains (S13, S15, S19, S21) all showed a clear positive signal above negative serum C-67 at a serum dilution of 1:300. Serum sample C+113 also showed a clear positive signal set to 100%. Therefore, ELISA based on the CD2 "132-204" fragment can be used to measure anti-CD2v antibodies induced by genotype I / serotype 4 or genotype II / serotype 8 ASFV strains.

[0138] Example 3 - CD2v-based ELISA as a DIVA immunoassay The CD2v fragment used in Example 2, i.e., CD2 "132-204", was also used in this experiment. ELISA was performed as described in Example 1, but the serum was diluted in 0.04M PBS + 0.05% v / v Tween20 instead of EIA buffer (1:300). CD2v-positive serum samples C+113, S1, and S2, as well as the negative serum sample C-67, were included in the ELISA. Serum sample S3 was also included, which came from a pig immunized with the Lv17 / WB / Rie1 vaccine strain (which expresses only the first 131 amino acids of CD2v). The results are shown in Figure 5. The OD450 value obtained from the C+113 serum sample at a serum dilution of 1:300 was set to 100%. Serum from pigs infected with non-vaccine genotype II strains (C+113, S1, and S2) containing the intact EP402R gene showed a clear positive signal far exceeding that of the negative serum C-67. However, a serum sample (S3) derived from a pig immunized with Lv17 / WB / Rie1 produced a signal similar to that of the negative control serum. This is in contrast to the observation made in Example 1, which indicated the presence of anti-CD2v antibodies in sample S3. This can be explained by the fact that the anti-CD2v antibodies in sample S3 are against a portion of CD2 "16-204" that does not overlap with CD2 "132-204". Thus, the data demonstrates that the vaccine strain Lv17 / WB / Rie1 cannot induce antibodies against CD2 "132-204". Therefore, ELISA based on the CD2 "132-204" fragment can be used to distinguish ASFV-infected animals from ASFV-vaccinated animals when the ASF vaccine does not induce antibodies that react with the CD2 "132-204" fragment.

[0139] Example 4 - Effect of sample diluent In this embodiment, the effect of the sample diluent on the signal-to-noise ratio was investigated.

[0140] A In this experiment, the CD2v fragment CD2 "132-204" was used. ELISA was performed as described in Example 1, but serum was diluted with one of the following: EIA buffer (0.04M PBS + 0.2M NaCl + 0.1% w / v BSA), EIA / T (EIA + 0.05% v / v Tween), PBS / T (0.04M PBS + 0.15M NaCl + 0.05% v / v Tween20), or a low-metal salt / high-surfactant (LSHD) buffer containing 3% v / v Tween20, 3% v / v aminooxide WS35, and 0.1M magnesium chloride, without phosphate buffer. CD2v-positive serum sample C+113, negative serum sample C-67, and serum sample S3 (derived from an animal infected with the Lv17 / WB / Rie1 vaccine strain) were included in the ELISA. For a reliable DIVA immunoassay, the OD450 value of the S3 sample should be similar to that of the negative serum sample C-67.

[0141] Figure 6A shows that EIA, a buffer with a relatively high salt concentration and no surfactant, results in poor separation of the sample dilution curve. The OD450 signal of S3 is clearly higher than that of C-67, indicating that EIA is not a suitable buffer for the CD2-based DIVA ELISA. The assay can be improved by using a low-salt buffer PBS / T containing a low concentration of surfactant as the sample diluent (Figure 6B). The dilution curve of sample S3 overlaps with that of the negative serum sample, but the OD450 value of C+113 is slightly lower than that of EIA. The P / N ratio is 5.7. To further separate positive signals from negative signals, LSHD buffer was evaluated (Figure 6C). LSHD as a sample diluent exhibits the most optimal signal-to-noise ratio (P / N ratio 10.3) among the three buffers, allowing for clear separation of samples that should be negative in the assay from samples that should give a positive signal. Therefore, ELISA based on the CD2 "132-204" fragment can be used in combination with a low-salt, high-surfactant buffer to distinguish ASFV-infected animals from ASFV-vaccinated animals.

[0142] The addition of urea to the EIA / T sample diluent reduced the P / N ratio, primarily due to a decrease in the signal for the positive control.

[0143] B In this experiment, the CD2v fragment CD2 "16-204" was used. ELISA was performed as described in Example 1. The stringency of the sample diluents was altered by increasing the surfactant and metal salt content. The stringencies of the three sample diluents used were 2.9, 5.7, and 9.9, respectively. Table 1 shows that only sample diluents with a stringency of 5.7 or higher resulted in a clear difference between the OD values ​​of the negative control sample and the OD values ​​of the vaccine test sample. [Table 2] C In this experiment, the CD2v fragment CD2 "132-204" was used. ELISA was performed as described in Example 1. The same sample diluents and EIA sample diluents as in Experiment B were used. Serum samples were diluted as shown in Table 2. When serum at a 1 / 100 dilution was tested using a high-stringency sample diluent, the P / N ratio was 5.8, indicating that serum from vaccinated animals (S3) was as negative as the negative control, while the noise (0.4) was relatively high.

[0144] When serum at a 1 / 300 dilution was tested using this sample diluent, the P / N ratio was 10.0, indicating that serum from vaccinated animals (S3) was as negative as the negative control, with low noise (<0.2). The P / N results obtained with other sample diluents were not as satisfactory (<5) for all dilutions. [Table 3] Example 5 - Further characterization of CD2v fragment sizes used in ELISA Several short and long morphologies of the extracellular CD2v fragment "132-204" were designed. These fragments contained amino acids "122-204", "142-204", "132-194", "132-214", or "122-194" from CD2v. These fragments also had the linker of SEQ ID NO: 7 and a subsequent Flag tag at their C-terminus. The peptide fragments were prepared by baculovirus expression and subsequent purification with GenScript as described in Example 2. ELISA was performed as described in Example 1.

[0145] The results are shown in Figure 7. For all peptides tested, serum from pigs infected with genotype II ASFV strains (S1 and S2) or genotype I ASFV strains (S13, S15, S19, S21) showed a clear positive signal at a serum dilution of 1:300, outperforming the signal of negative serum C-67. Serum sample C+113 also showed a clear positive signal set to 100%. As before, serum from animals vaccinated with genotype II strain (S3) did not react in the ELISA.

[0146] This analysis clearly shows that CD2v peptide fragments 132-194 (SEQ ID NO: 23) and 142-204 (SEQ ID NO: 24) both showed positive signals in ELISA. Therefore, it can be confidently concluded that the relevant epitope on these peptides is located between amino acids 142 and 194 of the CD2v protein. Thus, peptides 142-194 (SEQ ID NO: 25) can be effectively used in the ASFV DIVA of the present invention.

[0147] Example 6 - Variation of sample diluent This example investigated the effect of variations in sample diluent composition on the signal-to-noise ratio of the ELISA for the present invention.

[0148] A: Effect of surfactant concentration in sample diluent ELISA was performed using CD2v fragment CD2 "132-204" as described in Example 1. However, in this case, the test serum was diluted in the following various forms of sample diluents: 0.01M PBS + 0.33% v / v Tween20; 0.01M PBS + 3% v / v Tween20; 0.01M PBS + 0.33% v / v Tween80; 0.01M PBS + 3% v / v Tween80; 0.01M PBS; or LSHD buffer.

[0149] The ELISA included CD2v-positive serum sample C+113 and CD2v-negative serum sample C-67, as well as serum sample S3 (derived from an animal infected with the Lv17 / WB / Rie1 vaccine strain). For a reliable DIVA immunoassay, the OD of the S3 sample was used. 450 The values ​​should be similar to those of the negative control serum.

[0150] Figure 8 shows the ELISA results for various sample diluents, and Table 3 shows the corresponding P / N scores.

[0151] Panels A-E in Figure 8 show that buffers containing Tween (Panels 8A-D) resulted in better separation of positive and negative samples than diluents containing only PBS without Tween (Panel 8E). This indicates that the presence of Tween in the sample diluent is important.

[0152] As indicated by the corresponding P / N ratios shown in Table 3, LSHD buffer (Panel 8F) yielded the best results, followed by a diluent containing 0.01M PBS + 0.33% v / v Tween20. This also shows which sample diluents meet the stringency requirement of 5 or higher or 10 or higher. [Table 4] B: Effect of salt concentration in sample diluent Again, the CD2v fragment CD2 "132-204" was used. ELISA was performed as described in Example 1, but the serum was diluted with the following various sample diluents: 0.01M PBS + 3% v / v Tween20 + 0.1M MgCl2; 0.01M PBS + 3% v / v Tween20 + 0.33M MgCl2; 0.01M PBS + 3% v / v Tween20 + 1M MgCl2; or LSHD buffer. CD2v-positive serum sample C+113 and negative serum sample C-67, as well as serum sample S3, were included in the ELISA.

[0153] The ELISA results are shown in Figure 9, and the corresponding P / N ratios are shown in Table 4.

[0154] Panels A–C of Figure 9 show that the lowest salt concentration in the sample diluent (i.e., 0.1 M MgCl2) yielded the best separation of the sample curves. In other words, increasing salt concentration reduces the ability to distinguish between positive and negative samples (Panels 9B and C). Seemingly, lower salt concentrations have a beneficial effect on the signal intensity of positive samples, which in turn has a positive effect on the P / N ratio (Table 4). LSHD as a sample diluent yielded the best signal-to-noise ratio among the three diluents tested (Figure 9D; Table 4). Note: In Panel 9D, the data point for C+113 serum at a dilution of 1:24300 is clearly an outlier and almost certainly an experimental error. [Table 5] C: Effect of buffer on sample diluent The CD2v fragment CD2 "132-204" was used. ELISA was performed as described in Example 1, thereby diluting the serum with sample diluents containing: 0.01M PBS + 3% v / v Tween20 + 0.1M MgCl2; 3% v / v Tween20 + 0.1M MgCl2; 0.01M PBS + 3% v / v Tween20; or LSHD diluent. CD2v-positive serum sample C+113 and negative serum sample C-67, as well as serum sample S3 from an animal inoculated with the Lv17 / WB / Rie1 vaccine strain, were included in the ELISA.

[0155] The ELISA results are shown in Figure 10, and the corresponding P / N ratios are shown in Table 5.

[0156] Figure 10 shows that all tested buffers resulted in clear separation of the sample dilution curves. Sample diluents without PBS (containing 3% v / v Tween20 + 0.1M MgCl2) performed almost identically to LSHD. Compare panels 10C and D, as well as the P / N ratios in Table 5. Therefore, it is preferable not to use PBS, or at least not to use phosphate buffer, in the sample diluents for the present invention. [Table 6]

Claims

1. The use of an isolated African swine fever virus (ASFV) CD2v protein or its antigenic fragment conjugated to a solid support as an antigen in an immunoassay, wherein the CD2v protein or its antigenic fragment is used to detect the presence of ASFV antibodies in a test sample obtained from a pig vaccinated with an adjunct ASFV attenuated live virus CD2v-marker vaccine (LAV CD2v-marker vaccine), and the antigen is a polypeptide comprising an antigenic fragment of an extracellular domain, wherein the antigenic fragment of the extracellular domain is a polypeptide comprising an ASFV amino acid sequence consisting of the amino acid sequence of SEQ ID NO:

25.

2. The use according to claim 1, characterized in that the accompanying LAV CD2v-marker vaccine comprises an ASFV CD2v-marker vaccine strain capable of expressing a modified CD2v protein, wherein the modified CD2v protein lacks a fragment of the extracellular domain containing the amino acid sequence of SEQ ID NO:

25.

3. The use according to claim 1 or 2, characterized in that the immunoassay is a DIVA (Diverted Immune-Assessed) immunoassay that distinguishes between vaccinated animals and infected animals.

4. The use according to any one of claims 1 to 3, characterized in that the CD2v protein or its antigenic fragment is also used to detect the presence of ASFV antibodies in test samples obtained from pigs infected with wild-type ASFV and / or pigs not infected with ASFV.

5. The use according to any one of claims 1 to 4, characterized in that the solid support is a microtiter plate, a vial, a bead paper strip, a membrane, a gel, or a lateral flow strip.

6. A method for determining whether a pig is infected with wild-type ASFV or has been vaccinated with a marker vaccine, wherein the method is an immunoassay using an isolated African swine fever virus (ASFV) CD2v protein or an antigenic fragment thereof conjugated to a solid support as an antigen in an immunoassay, the method comprising the step of examining a test sample obtained from a pig for the presence of an ASFV CD2v antibody that binds to the antigen, wherein the antigen is a polypeptide comprising an antigenic fragment of an extracellular domain, and the antigenic fragment of the extracellular domain is a polypeptide comprising an ASFV amino acid sequence consisting of the amino acid sequence of SEQ ID NO:

25.

7. The method is 1. A step of incubating the test sample with the antigen in the assay mixture.

2. A step to enable the formation of an ASFV CD2v antibody-antigen complex in the assay mixture, and 3. Step to detect the presence of antibody-antigen complexes in the assay mixture. The method according to claim 6, characterized by including the following:

8. The method according to claim 6 or 7, characterized in that the method includes detecting the presence of an antibody-antigen complex by contacting the complex with an antibody containing a label.

9. The method according to any one of claims 6 to 8, characterized in that the immunoassay is ELISA (enzyme-linked immunosorbent assay).

10. The method according to any one of claims 6 to 9, characterized in that the test sample is diluted with a sample diluent having a stringency of at least 5.

11. The method according to claim 10, characterized in that the sample diluent has a stringency of at least 10.

12. The method according to any one of claims 6 to 11, characterized in that the immunoassay is an immunoassay for "identifying infected animals from vaccinated animals" (DIVA).

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