EP3209324A2 - Développement d'un vaccin contre le poux rouge des volailles dermanyssus gallinae - Google Patents

Développement d'un vaccin contre le poux rouge des volailles dermanyssus gallinae

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Publication number
EP3209324A2
EP3209324A2 EP15784675.9A EP15784675A EP3209324A2 EP 3209324 A2 EP3209324 A2 EP 3209324A2 EP 15784675 A EP15784675 A EP 15784675A EP 3209324 A2 EP3209324 A2 EP 3209324A2
Authority
EP
European Patent Office
Prior art keywords
prm
polypeptide
seq
antibodies
igy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP15784675.9A
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German (de)
English (en)
Inventor
Sebastian Ulbert
Gustavo Rodrigues Makert dos Santos
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Original Assignee
Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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Publication date
Application filed by Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV filed Critical Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Publication of EP3209324A2 publication Critical patent/EP3209324A2/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0003—Invertebrate antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds

Definitions

  • Patent Application Vaccine development against the poultry red mite Dermanyssus gallinae
  • PRM Due to a significant impact on animal health and egg production, PRM cause high economic losses in poultry industry worldwide, and are recognized as a vast economic, welfare and epidemiological problem for both birds and humans (Sparagano et al. 2009). Only in Europe, the annual costs for the poultry industry correlated with PRM were estimated in about 1 30 million euro (Van Emous, 2005). Therefore, different methods are being used to try to control PRM infestations. Some strategies involve frequent cleaning of the poultry farms and application of desiccant powders (Carroll 1994), repellents derived from plants oils (Birkett et al. 201 1 ), or the treatment with acaricidal agents or traps (George et al. 2009, C hirico and Tauson 2002).
  • tick protein Bm86 was also tested for a potential protection of chicken from PRM, but results were negative, and a direct homologue of Bm86 could not be identified in Dermanyssus gallinae (Harrington et al. 2009a).
  • immunization of hens with total PRM protein extracts led to protection against PRM, as measured by applying in vitro PRM feeding systems for the analysis of specific antibody preparations (Wright et al. 2009).
  • the present invention relates to the identification of protein antigens of Dermanyssus gallinae which could be used for the development of an immunological control strategy based on mite proteins which are correlated with PRM mortality.
  • Immunization with mite extracts led to an increased mortality effect of the resulting IgY towards PRM in in vitro feeding assays.
  • An innovative combination of 2 D-protein-gels and the comparison of antibody binding patterns were used to identify protein spots which are recognized by antibodies correlated with anti-PRM activity.
  • the identification of proteins which are contained in these single spots led to a number of candidates for vaccine development against PRM. This is suggested by the observation that these proteins, after elution from the 2 D gel and injection into chicken, elicited IgY with anti-PRM activity. Therefore, when applied as an antigen, these proteins are associated with the generation of protective antibodies against PRM and therefore are able to form the basis of a vaccine against D. gallinae.
  • the present invention provides an isolated or synthesized polypeptide having an amino acid sequence at least 80% homologous to the SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 , preferably at least 85 % homologous, more preferably 90% homologous, even more preferably 95 % homologous, most preferably 98% homologous.
  • the present invention further provides a monoclonal antibody that is capable of specifically binding the polypeptide as described above, and its pharmaceutical use.
  • Controls were immunized with PBS and adjuvant only. Mean values of three independent experiments (performed in triplicate) are shown, and error bars represent the standard deviation. Asterisk show that there is a statistically difference (Mann-Whitney Rank Sum Test, p ⁇ 0.03) between the anti-mite antibody production in control mites and mites immunized with PRM-M.
  • Figure 2 shows PRM mortality (in %) after Dermanyssus gallinae in vitro feeding with fresh heparinized chicken blood spiked with 750 g antibodies. Mortality of 300 mites was monitored for 1 -2 weeks in three independent experiments (performed in triplicate). Controls are PRM fed with IgY isolated from hens immunized without PRM extract (PBS + adjuvant only). PRM IgY-F was isolated from hens immunized with PRM extract and Freunds adjuvant. PRM IgY-M was isolated from hens immunized with PRM extract and Montanide ISA 70 VG adjuvant. Error bars represent the standard deviation.
  • Figure 4 shows PRM mortality (in %) after in vitro feeding with different PRM antibodies (IgY) isolated from hens immunized with PRM proteins eluted from 2D gel spots (the numbers of the spots are indicated). Mortality of 400 mites was monitored for 1 -2 weeks in three independent experiments. Control (F) IgY show the mortality of PRM after feeding with antibodies isolated from hens immunized with PBS and Freunds Adjuvans. Statistical analysis to evaluate the difference between the mortality mean of mites fed with control IgY and PRM spot IgY was performed by using an unpaired t-test. Asterisks show statistical significant differences (*, p ⁇ 0.05 and * *, p ⁇ 0.01 respectively).
  • the aim of the present study was the identification of protein antigens of Dermanyssus gallinae which could be used for the development of an immunological control strategy based on mite proteins which are correlated with PRM mortality. Immunization with mite extracts led to an increased mortality effect of the resulting IgY towards PRM in in vitro feeding assays.
  • An innovative combination of 2 D-protein-gels and the comparison of antibody binding patterns were used to identify protein spots which are recognized by antibodies correlated with anti-PRM activity.
  • the identification of proteins which are contained in these single spots led to a number of candidates for vaccine development against PRM. This is suggested by the observation that these proteins, after elution from the 2 D gel and injection into chicken, elicited IgY with anti-PRM activity.
  • the present invention relates to an isolated or synthesized polypeptide having an amino acid sequence at least 80% homologous to the SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 .
  • the present invention relates to an isolated or synthesized polypeptide having an amino acid sequence at least 85 % homologous to the SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 .
  • the present invention relates to an isolated or synthesized polypeptide having an amino acid sequence at least 90% homologous to the SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 .
  • the present invention relates to an isolated or synthesized polypeptide having an amino acid sequence at least 95 % homologous to the SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 .
  • the present invention relates to an isolated or synthesized polypeptide having an amino acid sequence at least 98% homologous to the SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 .
  • the present invention relates to an isolated or synthesized polypeptide having an amino acid sequence selected from SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 . In one embodiment, the present invention relates to a polypeptide having an amino acid sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 1 .
  • the present invention relates to a polypeptide having an amino acid sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 2.
  • the present invention relates to a polypeptide having an amino acid sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 3.
  • the present invention relates to a polypeptide having an amino acid sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 4.
  • the present invention relates to a polypeptide having an amino aci sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 5.
  • the present invention relates to a polypeptide having an amino aci sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 6. In one embodiment, the present invention relates to a polypeptide having an amino acid sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 7.
  • the present invention relates to a polypeptide having an amino acid sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 8.
  • the present invention relates to a polypeptide having an amino acid sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 9.
  • the present invention relates to a polypeptide having an amino acid sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 1 0.
  • the present invention relates to a polypeptide having an amino acid sequence at least 85 %, preferably 90%, more preferably 95%, most preferably 98% homologous to the SEQ ID NO 1 1 .
  • PRM-1 is defined as SEQ ID NO 1
  • PRM-2 is defined as SEQ ID NO 2
  • PRM-3 is defined as SEQ ID NO 3
  • PRM-4 is defined as SEQ ID NO 4
  • PRM-5 is defined as SEQ ID NO 5
  • PRM-6 is defined as SEQ ID NO 6
  • PRM-7 is defined as SEQ ID NO 7
  • PRM-8 is defined as SEQ ID NO 8
  • PRM-9 is defined as SEQ ID NO 9
  • PRM-1 0 is defined as SEQ ID NO 1
  • PRM-1 1 is defined as SEQ ID NO 1 1 .
  • the polypeptides as described above can be used in the treatment of Poultry Red Mite (PRM).
  • the present invention also discloses the manufacture of a vaccine using the polypeptides as above described.
  • a vaccine against Poultry Red Mite comprising:
  • the present invention provides isolated nucleic acid molecules comprising polynucleotides encoding the PRM polypeptides of the present invention.
  • isolated refers to nucleic acid molecules purified to some degree from endogenous material.
  • the nucleic acid molecule of the present invention comprises a polynucleotide encoding the polypeptides of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0 and 1 1 . Due to the known degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from the standard. Such variant DNA sequences can result from silent mutations occurring during production, or can be product of deliberate mutagenesis of these sequences.
  • Nucleic acid molecules of the invention include DNA in both single-stranded and double- stranded form, as well as the RNA complement thereof. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combination thereof.
  • the present invention further includes antibodies which specifically bind to the PRM- polypeptides of the present invention.
  • specifically binds refers to antibodies having a binding affinity (Ka) for PRM-polypeptides of 1 06M-1 or greater.
  • the term " antibody” refers to intact antibodies including polyclonal antibodies, and monoclonal antibodies.
  • the term “ antibody " also refers to a fragment of an antibody such as F(ab), F(ab'), F(ab')2, FV, FC, and single chain antibodies which are produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • antibody also refers to bispecific or bifunctional antibodies, which are an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • the present invention further discloses a method for producing antibodies against PRM comprising: isolating the total PRM proteins extract; immunizing the chickens with isolated PRM protein extract and one adjuvant selected from Freund or Montanide, preferably Montanide; isolating the antibodies.
  • the present invention further provides methods of identifying the desired PRM- polypeptides, comprising: 1 ). Immunization with PRM extracts and measurement of anti-PRM activity
  • PRM were transformed in a protein extract and used to immunize chicken. Two different adjuvants were used; hence the groups were called extract + montanide (group IgY-M), extract + Freund ' s (IgY-F) and controls (Fig. 1 ). The antibodies generated by these chickens were used in PRM-feeding assays. It was found that only one of the Adjuvants (IgY-M) led to antibodies which killed the mites (Fig 2). In the present invention, group IgY-M is preferred.
  • Mite extracts were separated by 2 D gels and single protein spots were seen.
  • the proteins were transferred from the gels to membranes (Western blot) and incubated with the antibodies from step 1 .
  • Computer-assisted image processing using Delta2D 3.6 software DECODON, Germany) was used to compare the binding pattern of antibodies from chicken of group IgY-M, group IgY-F and controls (Fig. 3). As a result, 10 spots were identified which are correlated to enhanced or exclusive binding by antibodies from group IgY-M.
  • Protein identification The proteins used in step 3 were identified by mass spectrometry in combination with analysis of the PRM transcriptome, genome and proteome. Protein sequences were identified and are the key component of the present invention (Table 1 ). These proteins were shown to elicit, although to a different extend, antibodies with anti-PRM activity and therefore form the basis for a vaccine against PRM, D. gallinae. None of these proteins was described in the context of vaccine research against PRM before.
  • compositions containing the PRM-polypeptides of the present invention comprise a therapeutically or prophylactically effective amount of at least one polypeptide or protein of the present invention in admixture with pharmaceutically acceptable compounds, and physiologically acceptable formulation materials.
  • Said polypeptides having an amino acid sequence at least 85%, preferably 90%, more preferably 95 %, and most preferably 98% homologous to the SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0 and 1 1 .
  • said polypeptide of the above-mentioned pharmaceutical composition having an amino sequence as set forth in SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 .
  • a pharmaceutical composition of the present invention comprising two or three different polypeptides having an amino acid sequence at least 85 %, preferably 90%, more preferably 95 %, and most preferably 98% homologous to the SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 and 1 1 respectively.
  • the pharmaceutical composition of the present invention comprising at least one polypeptide selected from the polypeptides having an amino acid sequence as set forth in SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 . In one embodiment the pharmaceutical composition of the present invention comprising two different polypeptides selected from the polypeptides having an amino acid sequence as set forth in SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0 or 1 1 .
  • composition of the present invention comprising three different polypeptides selected from the polypeptides having an amino acid sequence as set forth in SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0 or 1 1 .
  • the pharmaceutical composition comprising polypeptides having an amino acid sequence as set forth in SEQ ID N01 and 4.
  • the pharmaceutical composition comprising polypeptides having an amino acid sequence as set forth in SEQ ID N01 and 5.
  • the pharmaceutical composition comprising polypeptides having an amino acid sequence as set forth in SEQ ID NO 4 and 5.
  • the pharmaceutical composition comprising polypeptides having an amino acid sequence as set forth in SEQ ID N01 and 3.
  • the pharmaceutical composition comprising polypeptides having an amino acid sequence as set forth in SEQ ID N01 , 4 and 5. In one embodiment of the present invention, the pharmaceutical composition comprising polypeptides having an amino acid sequence as set forth in SEQ ID NO 3, 4 and 5.
  • composition of the present invention further comprises a
  • the carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof.
  • the pharmaceutical carrier employed may include a solid, a gel, or a liquid.
  • Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil, water, emulsions, various types of wetting agents, and the like.
  • the carrier of diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
  • PRM were collected from an egg production poultry farm in Thuringia, Germany.
  • the storage conditions and protein isolation of PRM were modified from previously used protocols (McDevitt et al. 2006, Wright et al. 2009, Harrington et al. 2009b).
  • the mites were stored in a 75 cm2 CellStar tissue culture flask (Greiner Bio-One) with a filter cap. Before PRM protein isolation started, the mites had starved in the dark for 3-7 days at RT, to minimize the amount of ingested chicken blood. Then, these mites were transferred to a refrigerator and maintained at 5°C for 3-4 weeks.
  • Mites were subjected to homogenization and sonification on ice in different buffers (see below).
  • PBS phosphate buffered saline
  • EDTA-free protease inhibitor Roche Diagnostics
  • the homogenized mites were incubated for 20 min at 4°C before centrifugation at 24,000g for 20 min at 4°C.
  • the supernatant was retained and the pellet was homogenized as described before, but with a buffer containing 8 M urea (to retrieve insoluble proteins) in PBS.
  • the homogenate was incubated for 90 min at 37°C before centrifugation at 24,000g for 20 min at room temperature (RT).
  • PRM proteins for the analysis on 2 D SDS PAGE gel was similar to the protein isolation protocol used before, except the usage of an isolation buffer containing 7 M urea, 2 M thiourea, 2 % v/v CHAPS, and traces of bromophenol blue (0.002 % v/v). Succeeding the PRM separation through 2 D SDS PAGE gel (see below), spots were cut from the gel and PRM proteins were eluted from the gel in 250 ⁇ PBS through 3 incubation steps for 5 min with gentle agitation.
  • the membrane was incubated for another 2 hours with the peroxidase conjugated rabbitanti- chicken IgY antibody (Sigma) diluted 1 :20,000. After each incubation step the membrane was washed three times with PBS-Tween. The blots were developed using EC L Western Blotting Substrates (Thermo Scientific).
  • Montanide ISA 70 VG adjuvant was administrated in the ratio of 7:3 to provide a total volume of 1 ml vaccine per hen / immunization as recommended by the manufacturers.
  • Hen 1 was immunized with PBS and F adjuvant only (control), hen 2 was immunized with PRM-spot-1 proteins, hen 3 with PRM-spot-2 proteins, hen 4 with PRM- spots-4, 5, 6 proteins, hen 5 with PRM-spots-7, 8 proteins, and hen 6 was immunized with PRM-spot-10 proteins.
  • C hicken blood was collected before each immunization and also 4 weeks after the third immunization for the analysis of anti-PRM antibodies using an ELISA-test. Briefly, Nunc polysorb plates (Thermo Scientific) were coated overnight with 1 00 ng of total PRM extract before sera from immunized hens were diluted 1 : 100 and used as first antibody. Bound IgY were detected with a rabbit anti-chicken antibody (see above). The values of the optical density represent the mean of triplicate measurements detected at 450 nm (520 nm as reference wavelength) in an ELISA Reader (Infiniti M200, Tecan).
  • PRM were first incubated in the dark for 3-7 days at RT, followed by storage at 5°C ( ⁇ 1 °C) for 3-4 weeks, to minimize the amount of ingested chicken blood by digestion. Then, a PRM in vitro feeding system based on previously published protocols (McDevitt et al. 2006, Wright et Al. 2009, Harrington et al. 2009b) was performed : briefly, skins from one day old chicks (obtained from the Clinic for Birds and Reptiles, Leipzig University) were washed, plucked and stored at -20°C for 1 -2 weeks before use.
  • the blood reservoir was constructed from an inverted 10 ml pipette tip (Gilson) covered with a 2 cm2 strip of the chick skin (leaved at RT for at least 1 0 min).
  • the external surface of the skin was exposed to the mites present in a glass tube (DURAN), and the internal surface of the chick skin was in direct contact with chicken blood as previously described (McDevitt et al. 2006).
  • Fresh heparinized chicken blood 250 ⁇ was spiked with antibodies (750 g) isolated from chicken eggs (see above) collected 2-4 weeks after the third immunization.
  • MS/MS spectra were detected using the statistical software R version 3.1 (R Core Team 2014) and additional R packages: MALDIquant 1 .1 0 (Gibb and K. Strimmer 201 2), MALDIquantForeign 0.8 (Gibb 2014), and plyr 1 .81 (Wickham 201 1 ). Finally, MS/MS spectra peaks were matched with predicted proteins using the software X!Tandem version 13-09-1 (Craig and Beavis 2004).
  • IgY were isolated from immunized hens.
  • PRM in vitro feeding assay To detect antibodies which were produced after immunization of hens and which lead to mortality of mites upon ingestion, we performed a PRM in vitro feeding assay. Mites were monitored for 1 -2 weeks after the in vitro feeding and the mortality rates between the groups were analyzed. The results show (Fig. 2) that the mortality (61 .4%) of the PRM group fed with IgY isolated from hens immunized with total PRM extract an the Montanide adjuvant was significantly higher than the mortality (22.6%) of mites fed with IgY isolated from control hens.
  • proteins were eluted from the 2 D gel spots (Fig. 3e), before the material was injected into hens.
  • spots-4, 5, 6 and spots-7, 8 were pooled for single injections, respectively.
  • Spot-3 and spot-9 were not used for immunization, because the amount of PRM protein obtained was too low.
  • IgY was extracted from eggs for further PRM in vitro feeding assays. In vitro feeding: different anti-mite activity correlated with proteins eluted from 2 D-gels
  • the 2D gel spots 1 , 2, 4, 5, 6, 7 and 8 contain proteins which elicit antibodies with anti-PRM activity upon immunization and were further investigated in order to identify the corresponding PRM proteins.
  • spot 1 0 due to the marginally higher PRM mortalities
  • spots 3 and 9 eluted amount of proteins too low for immunizations
  • MS/MS MS/MS
  • PRM-1 to PRM-1 1 Table 1
  • SEQ ID NO 1 to 1 1 respectively.
  • Table 1 Dermanyssus gallinae protein sequences obtained from the 2 D gel spots analysed in this study. The first column shows 1 1 PRM proteins identified as potential vaccine candidates (8 candidates and possible 3 isoforms: candidates PRM-6-8). The second column shows the 2 D gel spots these sequences were obtained from. Protein sequences are given in the third column. The last column show which D. gallinae protein sequences were completely identified (according to Trinity analysis).
  • PRM-5 U5 U6, MVDQAVKDKLEAGFAKLQGAADCKSLLKKYLTRG Complete
  • PRM-6 U5 U7 MDQETLKKAAIAVPALAVLCYVAYQGSNAIKNALA Complete
  • PRM-8 U5 U7 MSQRSNAIKNALARKEVPQDVKDKLQAGFQKLQT Complete

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Abstract

Le poux rouge des volailles (PRV) Dermanyssus gallinae provoque d'importantes pertes économiques et représente l'un des parasites les plus destructeurs en élevage de volailles dans le monde entier. Différentes stratégies chimiques, physiques et biologiques tentent de lutter contre l'expansion du PRV. Cependant, la résolution de ce problème reste particulièrement prioritaire. La présente invention concerne un procédé innovant pour le développement d'une stratégie de lutte immunologique, basée sur l'identification d'antigènes protéiques d'acarien qui induisent des anticorps ayant une activité anti-acarien chez le poulet immunisé. Des poules ont été immunisées avec différents extraits de protéines de PRV et des anticorps IgY ont été extraits des œufs. Un test d'alimentation in vitro de PRV utilisant du sang de poulet additionné de ces IgY a permis la détection d'anticorps qui ont provoqué une mortalité du PRV. Une nouvelle combinaison d'analyse de gel 2D et analyse comparative de modèles de liaison d'anticorps suivie par des approches protéomiques ont conduit à l'identification d'antigènes candidats responsables de la production de ces anticorps protecteurs. Ces résultats indiquent le potentiel élevé de cette stratégie pour le développement d'un vaccin contre le poux des volailles Dermanyssus gallinae.
EP15784675.9A 2014-10-23 2015-10-22 Développement d'un vaccin contre le poux rouge des volailles dermanyssus gallinae Pending EP3209324A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP14189993 2014-10-23
PCT/EP2015/074542 WO2016062832A2 (fr) 2014-10-23 2015-10-22 Développement d'un vaccin contre le poux rouge des volailles dermanyssus gallinae

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EP3209324A2 true EP3209324A2 (fr) 2017-08-30

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CN108191975A (zh) * 2018-01-19 2018-06-22 广州益琪琳生物科技有限公司 螨变应原特异性卵黄免疫球蛋白抗体及其抗过敏制剂

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* Cited by examiner, † Cited by third party
Title
See references of WO2016062832A2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111683208A (zh) * 2020-06-09 2020-09-18 深圳拔越软件有限公司 一种电视墙的智能配置方法及装置

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