VIRAL CHIMERIC PARTICLE OF POTATO VIRUS X AND USE THEREOF FOR IN VITRO DIAGNOSIS OF SJÖGREN SYNDROME

Abstract

The present invention relates to a chimeric virus particle of potato virus X, said particle having, as a capsid protein, a fusion protein containing a capsid protein and an antigenic determinant of lipocalin, and the use thereof in the in vitro diagnosis of Sjgren's Syndrome using the ELISA method.

Claims

1) A fusion protein comprising or consisting of: an amino-terminal portion consisting of a peptide sequence comprising or consisting of an antigenic determinant of lipocalin; and a carboxy-terminal portion consisting of a peptide sequence comprising or consisting of the capsid protein of a PVX virus, said capsid protein being intact or deleted in the 5 portion of the gene which encodes the wild-type capsid protein, said amino-terminal portion being fused to said carboxy-terminal portion in such a way that said antigenic determinant is in frame with said capsid protein.

2) The fusion protein according to claim 1, wherein said antigenic determinant is selected in the group which consists of FEKAAGARGLST (SEQ ID NO:2), MSFEKAAGARGLST (SEQ ID NO:5).

3) The fusion protein according to claim 1, wherein the PVX virus is selected in the group consisting of PVX X3, BS, EX, NL4, HB, WS2, ROTH1, XS, UK3, OS, NL1, Taiwan, X4, preferably X3.

4) The fusion protein according to claim 1, wherein said capsid protein consists of the following sequence: TABLE-US-00007 (SEQIDNO:6) PGTPATASGLFTIPDGDFFSTARAIVASNAVATNEDLSKIEAIWKDMKV PTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRARLAAAIKEV CTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGVT NPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVT RGRITGTTTAEAVVTLPPP.

5) The fusion protein according to claim 1, said fusion protein having the following sequence: TABLE-US-00008 (SEQIDNO:7) MSFEKAAGARGLSTPGTPATASGLFTIPDGDFFSTARAIVASNAVATNE DLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSN GISRARLAAAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKP EHKFAAFDFFNGVTNPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKAR AQSNDFASLDAAVTRGRITGTTTAEAVVTLPPP

6) A polynucleotide which codes for the fusion protein as defined in claim 1.

7) The polynucleotide according to claim 6, said polynucleotide having the following sequence (SEQ ID NO:8): TABLE-US-00009 atgtcttttgaaaaggctgctggtgctagaggtttgtctactcccggga ctcctgccacagcttcaggcctgttcaccatcccggatggggatttctt tagtacagcccgtgccatagtagccagcaatgctgtcgcaacaaatgag gacctcagcaagattgaggctatttggaaggacatgaaggtgcccacag acactatggcacaggctgcttgggacttagtcagacactgtgctgatgt aggatcatccgctcaaacagaaatgatagatacaggtccctattccaac ggcatcagcagagctagactggcagcagcaattaaagaggtgtgcacac ttaggcaattttgcatgaagtatgctccagtggtatggaactggatgtt aactaacaacagtccacctgctaactggcaagcacaaggtttcaagcct gagcacaaattcgctgcattcgacttcttcaatggagtcaccaacccag ctgccatcatgcccaaagaggggctcatccggccaccgtctgaagctga aatgaatgctgcccaaactgctgcctttgtgaagattacaaaggccagg gcacaatccaacgactttgccagcctagatgcagctgtcactcgaggtc gtatcactggaacaacaaccgctgaggctgttgtcactctaccaccacc ataa

8) An expression vector comprising the polynucleotide as defined in claim 6.

9) A host cell comprising the expression vector as defined in claim 8.

10) A genome sequence of a PVX virus, said genome sequence comprising the polynucleotide as defined in claim 6.

11) A chimeric virus particle of potato virus X, said chimeric virus particle being characterized in that it comprises, as a capsid protein, the fusion protein as defined in claim 1, said viral particle having exposed, on the outside thereof, the amino-terminal portion comprising or consisting of an antigenic determinant of lipocalin.

12) The chimeric virus particle according to claim 11, wherein the viral genome has a polynucleotide having the following sequence (SEQ ID NO:8): TABLE-US-00010 atgtcttttgaaaaggctgctggtgctagaggtttgtctactcccggga ctcctgccacagcttcaggcctgttcaccatcccggatggggatttctt tagtacagcccgtgccatagtagccagcaatgctgtcgcaacaaatgag gacctcagcaagattgaggctatttggaaggacatgaaggtgcccacag acactatggcacaggctgcttgggacttagtcagacactgtgctgatga ggatcatccgctcaaacagaaatgatagatacaggtccctattccaacg gcatcagcagagctagactggcagcagcaattaaagaggtgtgcacact taggcaattttgcatgaagtatgctccagtggtatggaactggatgtta actaacaacagtccacctgctaactggcaagcacaaggtttcaagcctg agcacaaattcgctgcattcgacttcttcaatggagtcaccaacccagc tgccatcatgcccaaagaggggctcatccggccaccgtctgaagctgaa atgaatgctgcccaaactgctgcctttgtgaagattacaaaggccaggg cacaatccaacgactttgccagcctagatgcagctgtcactcgaggtcg tatcactggaacaacaaccgctgaggctgttgtcactctaccaccacca taa

13) A use of the chimeric virus particle according claim 11 for the in vitro diagnosis of Sjgren's Syndrome, for example using the ELISA, dipstick or microchip method.

14) A kit for the in vitro diagnosis of Sjgren's Syndrome, said kit comprising or consisting of the chimeric virus particle according claim 11, possibly in combination with suitable reagents for detection purposes.

15) A plant characterized in that it comprises within it the chimeric viral particle as defined in claim 11.

16) Plant cells, which express the chimeric viral particle as defined in claim 11.

17) A use of a peptide sequence comprising or consisting of an antigenic determinant of lipocalin for the preparation of the fusion protein as defined in claim 1.

18) A use of the plant according to claim 15, for the production of a chimeric viral particle.

19) A process for the preparation of the fusion protein according to claim 1, said process comprising or consisting of the following steps: a) cloning, in a vector, the peptide sequence comprising or consisting of the capsid protein of a PVX virus, said capsid protein being intact or deleted in the 5 portion of the gene which encodes the wild-type capsid protein; and b) cloning a peptide sequence comprising or consisting of an antigenic determinant of lipocalin, said peptide sequence being fused to said peptide sequence comprising or consisting of the capsid protein of a PVX virus in the vector resulting from step a).

20) The process according to claim 19, wherein said antigenic determinant of lipocalin is selected in the group which consists of FEKAAGARGLST (SEQ ID NO:2) and MSFEKAAGARGLST (SEQ ID NO:5).

21) The process according to claim 19, wherein the PVX virus is selected in the group consisting of PVX X3, BS, EX, NL4, HB, WS2, ROTH1, XS, UK3, OS, NL1, Taiwan, X4, preferably X3.

22) The process according to claim 19, wherein said capsid protein consists of the following sequence: TABLE-US-00011 (SEQIDNO:6) MPGTPATASGLFTIPDGDFFSTARAIVASNAVATNEDLSKIEAIWKDMK VPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGISRARLAAAIKE VCTLRQFCMKYAPWVVNWMLTNNSPPANWQAQGFKPEHKFAAFDFFNGV TNPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAV TRGRITGTTTAEAVVTLPPP.

23) A process for the preparation of a chimeric viral particle, said process comprising or consisting of the step of replacing the capsid protein of a PVX virus, in said PVX virus, with the fusion protein according to claim 1.

24) A process for the production of a viral particle; said process comprising or consisting of the following steps: a1) infecting a plant with the viral particle according to claim 11; b1) cultivating the plant obtained according to step a1); c) extracting said viral particle from said plant.

Description

[0059] The present invention will now be described, by way of illustration and not by way of limitation, according to a preferred embodiment thereof, with particular reference to the figures of the appended drawings, in which:

[0060] FIG. 1 shows a diagram representation of the genome of the potato virus X; RdRp, RNA-dependent RNA polymerase; MT, methyltransferase domain; HEL, helicase domain; POL, polymerase domain; 25K/12K/8K, movement proteins; CP, capsid protein; SGP, subgenomic promoter; sgRNA, subgenomic RNA; 7-methylguanine cap; terminal cap 5; Poly(A): 3 terminal poly(A) tail.

[0061] FIG. 2 shows a schematic representation of how the viral genome can be modified and how a chimeric virus particle (CVP) is constructed. A) modification by gene insertion: the nucleotide sequence encoding the protein of interest is added to the viral genome with a duplication of the subgenomic promoter of the CP. B) modification by gene fusion: the nucleotide sequence encoding the protein/peptide of interest is fused to the 5 portion of the CP gene (N-terminal portion of the protein).

R, viral replicase; M, movement proteins; CP, coat protein, capsid protein; arrow, viral subgenomic promoter.

[0062] FIG. 3 shows a schematic representation of the plasmid pPVX201 containing the genome of the PVX subform of cDNA, 35S, promoter derived from CaMV; 5UTR: 5 untranslated region; RdRp, RNA-dependent viral replicase; p8/p12/p25, viral movement proteins; prom sg CP, subgenomic promoter of the CP; prom sg2CP, duplicate subgenomic promoter of the CP; CP, coat protein; 3UTR: 3 untranslated region; NOS term, terminator derived from the nopaline synthase gene of A. tumefaciens; beta-lactamase: gene coding for the beta-lactamase. The main restriction sites and their corresponding nucleotide position on the vector are indicated.

[0063] FIG. 4 shows a schematic representation of the plasmid containing the PVX genome in the form of cDNA, both the wild-type and mutant variants with the deletion at 5 of the CP gene. The subsequent panels show the region undergoing modification in detail, with the main restriction sites, the two oligonucleotides (PVX Back and New) used in the PCR reactions and their respective nucleotide positions. A) pPVX201: the plasmid containing the wild genome of PVX (it originates wild-type viral particles, wt PVX). B) pPVXCC: the plasmid derived from pPVX201 containing the mutant with the N-terminal deletion of the CP. C) pPVX-Sma: the plasmid derived from pPVX-CC and further modified and used in this patent to construct the chimeric viral particles PVX-LIP.

[0064] FIG. 5 shows the nucleotide alignment between the CP gene of wt PVX (SEQ ID NO: 16) and that of the deleted CP of PVX-CC (SEQ ID NO: 15).

[0065] FIG. 6 shows the protein alignment between the CP of wt PVX (SEQ ID NO: 17), that of the deleted one of PVX-CC (SEQ ID NO: 18) and the deleted and mutated one of PVX-Sma (SEQ ID NO: 19).

[0066] FIG. 7 shows the nucleotide alignment between the deleted CP gene of PVX-CC (SEQ ID NO: 15) and that of the deleted and mutated CP of PVX-Sma (SEQ ID NO: 20).

EXAMPLE 1: PREPARATION OF THE CHIMERIC VIRAL PARTICLE PVX ACCORDING TO THE PRESENT INVENTION

[0067] Preparation of PVX.LIP

[0068] The pPVX-Sma plasmid was purified on a small scale and digested with the restriction enzymes NheI and XmaI to insert the sequence coding for the LIP peptide at the 5 end of the CP-Sma gene.

[0069] This sequence was obtained by in vitro pairing of two complementary synthetic oligonucleotides carrying suitable hemi-restriction sites at their ends, to enable cloning directly in the receiving viral vector, duly digested.

[0070] The protocol provides for the dilution in water of equimolar amounts of the two strands (sense and antisense) and boiling of the solution for 5 minutes; after cooling at room temperature the complementary strands will pair.

[0071] The designed oligonucleotides are the following:

TABLE-US-00005 senseLIP: (SEQIDNO:9) 5'-CTAGCCTCGAGATGTCTTTTGAAAAGGCTGCT GGTGCTAGAGGTTTGTCTACTC-3' antisenseLIP: (SEQIDNO:10) 5'-CCGGGAGTAGACAAACCTCTAGCACCAGC AGCCTTTTCAAAAGACATCTCGAGG-3'

[0072] The sense and antisense oligonucleotides coding for the LIP peptide were designed in such a way as to have the codon usage of Nicotiana benthamiana, the 5 end compatible with the NheI restriction hemisite and the 3 end compatible with the XmaI hemisite, so that once in vitro pairing had taken place they would be ready for direct cloning in the pPVX-Sma plasmid digested with NheI-XmaI (FIG. 4c). Moreover, a serine was inserted immediately after the initial methionine to improve the characteristics of stability of the chimera in vivo.

[0073] The doubly digested pPVX-Sma plasmid was purified from the agarose gel using a commercial kit and the quantity and quality were re-verified in agarose gel and by spectrophotometric analysis.

[0074] On completion of the ligation reaction between the digested and purified vector and the oligonucleotides paired in vitro, the recombinant plasmid obtained is inserted into bacterial cells of the XL1-Blue strain by electroporation and plated in selective LB-agar medium containing ampicillin. The plates are incubated at 37 C. and the resulting colonies analysed by PCR with oligonucleotides specific for the nucleotide region of the PVX vector straddling the cloned region (FIG. 4):

TABLE-US-00006 5' back (SEQIDNO:11) 5' AGCAGTCATTAGCACTTC3' 3' new (SEQIDNO:12) 5' CACCTTCATGTCCTTCCA3'

[0075] The expected band is 370 bases. The positive colonies were used for small-scale purification of the plasmid and analysis thereof via sequencing in order to verify the correct presence of the additional LIP sequence. The positive clone identified was purified on a large scale and used for a first cycle of infections of plants of N. benthamiama. The plants of N. benthamiana were grown in hothouses with a photoperiod of 16 hours of light and 8 hours of darkness, at a constant temperature of 24 C. The light intensity in the hothouses is about 16000 lux.

[0076] For every leaf of N. benthamiana to be infected (2 leaves per plant) a solution containing 20 g of plasmid diluted in 50 l of water is prepared for the primary infection, whilst for the subsequent infections 50 l of crude extract drawn from the infected tissue is used. The infection is carried out mechanically on the adaxial (upper) side of the leaf by lightly rubbing with the fingers in the presence both of the prepared solution and silicon carbide, or Carborundum (VWR International, Prolabo). The action of this powder causes microabrasions on the leaf surface, enabling the plasmid to enter effectively into the plant cells and express themselves.

[0077] After the onset of systemic symptoms in a time and manner similar to PVX-wt was verified, the tissue was used to:

[0078] 1. extract the viral RNA, carry out a reverse transcription to DNA (RT) and a PCR, sequence the PCR fragment and verify the correct presence of the LIP sequence at the nucleotide level;

[0079] 2. derive a crude protein extract with which to repeat a cycle of infection in vivo.

[0080] The entire process was then repeated another time to determine the stability of the chimeric PVX for LIP over three generations.

[0081] In order to determine the stability of the chimeric PVX, between 6 and 8 days after the infection, RNA was extracted from the systemically infected leaves by means of the RNeasy plant mini kit (Qiagen) (http://www1.qiagen.com/literature/render.aspx?id=352) and RT-PCR reactions were carried out using the GeneAmp RNA PCR Kit (Perkin Elmer). The cDNA was synthesized with oligo d(T) and PCR was performed using the 5 back and 3 new oligonucleotides. The cDNA thus obtained was sequenced and the presence of the correct sequence coding for the LIP peptide was verified.

[0082] Then followed a large-scale infection with purification of the chimeric PVX (PVX-LIP) and PVX devoid of the LIP peptide (PVX-wt) as a control.

[0083] For the purpose of protein extraction, the plant tissue, frozen at 80 C. and mechanically pulverized in a mortar chilled in liquid nitrogen, is mixed in an equivalent volume of phosphate-buffered saline 1 (1PBS) (10 mM Na2HPO4, 10 mM NaH2PO4, 150 mM NaCl, pH 7.2) and mechanically crushed using an Ultraturrax electric homogenizer brought up to maximum speed. The homogenate must then be centrifuged at about 20000 g for 3 minutes.

[0084] For the purification of the chimeric viral particles, the following protocol is used.

[0085] Homogenize 50 g of tissue stored at 80 C. and pulverized with a mortar and pestle in 2 volumes of cold boric acid 0.5 M pH 7.8 (about 200-300 ml); filter the mixture through 3 layers of sterile gauze and adjust the pH to 6.5 with HCl. Then add 0.2% (w/v) of ascorbic acid and 0.2% (w/v) of sodium sulphite and leave under stirring until completely dissolved. Remove the particulate by centrifuging at 5500 g for 20 minutes at 4 C. and leave the supernatant a room temperature for 3 hours after having added 0.15 volumes of a 0.5% silver nitrate solution. After repeating the centrifugation at 5500 g for 20 minutes at 4 C., allow the supernatant to settle overnight at 4 C. in 0.2 volumes of a solution containing 1M NaCl and 20% PEG in boric acid 0.5 M pH 7.8. The next day, precipitate the viral particles by centrifuging at 8000 g for 30 minutes at 4 C., and resuspend the pellet in 10 ml of a solution containing boric acid 0.5 M, urea 0.5 M, 0.1% 2-mercaptoethanol, pH 7.8; centrifuge the mixture at 8000 g for 30 minutes at 4 C. Load the supernatant containing the virus on a 30% sucrose cushion prepared in water and centrifuge at 72500 g for 2 hours and 30 minutes at 4 C. Finally, resuspend the pellet in 1 ml of boric acid 0.5 M pH 7.8 and, after two hours of settling, centrifuge at 6000 g for 15 minutes at 4 C. The supernatant containing the virus is then loaded onto a 10-45% sucrose gradient prepared in boric acid and centrifuged at 90000 g for 1 hour. The individual fractions of the gradient are then analysed to identify those with the highest content of viral particles. The protocol is drawn from Udhe et al. 2005. The purified virus was dialyzed and concentrated by filtration through a membrane using Vivaspin 2 HS columns (Vivascience, http://teachline.ls.huji.ac.il/72682/Booklets/VIVASPIN_Ultrafiltration_products_II.pdf).

[0086] The purified virus was then analysed through silver-stained polyacrylamide gel to verify the degree of purity of the preparation and the presence of a band with a higher weight than the wt CP due to the presence of the LIP peptide and was quantified by reading with a spectrophotometer at a wavelength of 280 nm considering the molar extinction coefficient E equal to 1.25 ml/(mg*cm).

[0087] An LAL assay was also performed (following the protocol provided by pbi International); it verified the presence of endotoxins in the preparations described, presumably due to an environmental contamination given by the laboratory instrumentation. An EndoTrap red1/1 column (Hyglos) was used to remove the endotoxins, following the manufacturer's instructions.

[0088] The PVX-LIP and wt PVX viral particles were purified from the plant with a yield of 0.12 mg per gram of fresh tissue.

[0089] Preparation of CPMV.LIP

[0090] For the production of chimeric particles derived from the CPMV (Cowpea Mosaic Virus) which expose the LIP peptide, use was made of the pEAQ-HT system, in which the precursor of the coat protein (VP60) and the 24K protease are separately introduced in two pEAQ constructs to produce particles similar to empty viruses (devoid of genetic material). The sequence encoding the LIP peptide was cloned in the vector by ligation of the vector itself digested with the enzymes NheI and AatII and the sequence encoding the peptide obtained by pairing the primer CPMVfor 5-CTAGC ACT CCT CCT GCT TTT GAA AAG GCT GCT GGT GCT AGA GGT TTG TCT ACT CCA TTT TCA GACGT-3 (SEQ ID NO:13) and the primer CPMV-rev 5-C TGA AAA TGG AGT AGA CAA ACC TCT AGC ACC AGC AGC CTT TTC AAA AGC AGG AGG AGT G-3(SEQ ID NO:14). The vector thus obtained, called pEAQ-HT-VP60-LIP, permits the exposure of the peptide in a BB-BC loop of the small subunit of the coat protein and in this manner the peptide is exposed on its outer surface. Subsequently, the vectors pEAQ-HT-VP60-LIP and pEAQ-HT-24K were electroporated in cells of Agrobacterium tumefaciens strain LBA4404 and both used to agroinfiltrate plants of N. benthamiana. In particular, the bacterial cultures transformed for the two vectors were made to grow separately in a liquid medium containing antibiotic agents, precipitated by centrifugation at 4000 g and resuspended in MMA (10 mM MES pH 5.6, 10 mM MgCl2 and 100 mM acetosyringone) until arriving at an O.D.600 equal to 0.8. Subsequently, after 4 h of incubation at room temperature, equal volumes of the two bacterial suspensions were mixed and used for infiltration, by means of a needleless syringe, of leaves of 4- to 5-week-old plants of N. benthamiana. Four leaves per plant were infiltrated. Samples were then taken of the infiltrated leaves 6 days after infiltration (dpi). For the expression of the empty CPMV the same procedure was carried out with the vectors pEAQ-HT-VP60 and pEAQ-HT-24K.

[0091] For the purification of the viral particles, 30 g of infiltrated leaves were processed. The plant material was homogenized with 4 volumes of sodium phosphate 0.1 M pH 7.0 containing 2% polyvinylpolypyrrolidone and a protease inhibitor (Complete EDTA-Free Cocktail, Roche, 04693132001).

[0092] The extract was clarified by filtration with two sheets of Miracloth paper (Merck Millipore, 475855-1R) and subsequent centrifugation (30000 g, 1 h, 4 C.). The supernatant was loaded onto an anion-exchange resin (DEAE Sephadex A-50, GE Healthcare, 17-0180-01), with a sample:resin ratio of 4:1. The flow-through was then concentrated at 4 ml, centrifuged (10000 g10 minutes) and loaded into a size-exclusion chromatography column (HiPrep 16/60 Sephacryl S-500 HR, GE Healthcare, 28-9356-06) in 0.1 M sodium phosphate pH 7.0 containing 0.15 M of NaCl at a flow velocity of 0.8 ml/min. The eluted fractions were analysed using 12% SDS-PAGE gel stained with silver nitrate. The fractions containing CPMV. LIP and CPMV were joined separately and filtered with a 100 kDa centrifugal filter system (Amicon Ultra-15, Merck Millipore, UFC910024). The purified particles were analysed using 12% SDS-PAGE gel stained with silver nitrate and subsequently quantified by reading the absorbance with a spectrophotometer at 280 nm.

EXAMPLE 2: ELISA DIAGNOSTIC TEST USING THE CHIMERIC VIRAL PARTICLE OF PVX ACCORDING TO THE PRESENT INVENTION

[0093] With the aim of optimizing the sensitivity and specificity of this diagnostic test and overcoming the methodological obstacles (type of plate), an assessment was made as to the advisability of performing an ELISA test that simultaneously exploited the synthetic LIP peptide and the LIP peptide expressed by chimeric particles of PVX (PVX-LIP), as well as by the control PVX, in order to be able to compare the result for the individual patient serum tested, by eliminating intra-experiment variability. We proceeded in an analogous manner, on a different plate, using the synthetic LIP peptide and the LIP peptide expressed by chimeric particles of CPMV, as well as by the control CPMV.

[0094] The following groups of subjects were enrolled in the present study: [0095] 1) 91 patients with the disease (5 men and 86 women) who met the AECG diagnostic criteria; [0096] 2) 60 healthy donors, comparable in terms of age and sex; [0097] 3) 60 patients with other autoimmune diseases (20 with systemic sclerosis, 20 with rheumatoid arthritis and 20 subjects with systemic lupus erythematosus).

[0098] In order to evaluate the bond of the antibodies present in the serum of the patients, ELISA assays (Immunolon II-Dynax and Maxisorp-NUNC) were set up with the LIP peptide produced by chemical synthesis and with PVX.LIP and wild-type CVP.

[0099] The plates were coated with 50 l of 40 g/ml of synthetic peptide in 1PBS (2 micrograms of peptide) and with 50 l of a 50 g/ml solution of wild-type PVX and purified PVX.LIP (2.5 micrograms of chimeric CP corresponding to about 131.6 ng of peptide), similarly diluted in 1PBS. The reference scale was obtained using known concentrations of immunoglobulin pools from donors (Sandoglobulin 50 and 100 g/mL). The plates were then blocked using 3% BSA in 1PBS and subsequently incubated with the serum of the subjects belonging to the three above-described experimental groups diluted 1:50 in 1PBS with 1% BSA for 4 hours at room temperature. After the incubation, a sequence of washes was carried out, once with a solution of 1PBS and Tween-20 and 3 successive washes in 1PBS. Subsequently, the plates were incubated at 4 C. overnight with a human anti-IgG conjugated with alkaline phosphatase. At the end of the incubation the plates were treated with a new sequence of washes (1PBS solution and Tween20 once and 3 successive washes in 1PBS) and the signal was detected using p-nitrophenyl phosphate and a TECAN SUNRISE III microplate reader (wavelength 405 nm).

[0100] The result of the ELISA test revealed several difficulties of a methodological type. First, despite using plates polystyrene plates that were highly specific for the bond to small peptides, we had to limit the analysis of the result to plates that allowed the comparability of the data obtained, for the same subject, with the use of the synthetic LIP peptide and of the LIP peptide expressed by the chimeric particles of PVX and CPMV. Second, we had to discard the use of the LIP peptide expressed by the chimeric particles of CPMV, since the result obtained was not analysable. In fact, the absorbance obtained from the reaction with the viral particles alone, for all subjects, irrespective of whether they were patients or control subjects or affected by another autoimmune pathology, demonstrated to be higher than the value obtained with the LIP peptide expressed by the chimeric particles of CPMV. The CPMV system was thus excluded from the analysis and considered unsuitable for performing a diagnostic test.

[0101] The results of the ELISA test obtained using the synthetic peptide and the peptide mounted on the PVX viral scaffold showed a sensitivity of 84.7% and 97.1% respectively, whereas the specificity in both systems was equal to 90.0%.

[0102] The sensitivity of the ELISA test conducted using the peptide of the invention on the subgroup of patients with SS characterized by an ANA-negative antibody profile is equal to 98.7%, as compared to a value of 75% obtained with the use of the synthetic peptide.

[0103] In order to further validate the diagnostic method just described, we decided to assess the stability and reproducibility of the ELISA test with the PVX.LIP system. We therefore prepared plates coated with the PVX.LIP system (i.e. the wild-type PVX and PVX.LIP) and stored the same at 4 C. for use 1, 15, 30 and 60 days after coating them. The plates were tested with the serum of 30 subjects with SS, 10 patients with rheumatoid arthritis, 10 with Lupus and 10 control individuals and the result was substantially consistent. In particular, the data remained unchanged over time, both when we analysed the result for individual subjects or groups of subjects, and assessed the sensitivity and specificity of the test.

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