Background of the Invention

The blood sucking ectoparasite Dermanyssus gallinae (De Geer 1 778), also known as the Poultry Red Mite (PRM), usually infests domestic birds but can also feed on cats, dogs, horses and humans, when the main host is absent (Brockis 1 980). PRM can bite through the skin of the host to feed on blood, causing itching, redness and stress to the host and may even cause death in poultry farms due to anemia (Schicht et al. 2013a). PRM are also vectors for several pathogens, as e.g., viruses and bacteria (Moro et al. 2009). 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). Other strategies focusing on biological methods were tested, such as insect growth regulators (C hauve 1998), infection of PRM with pathogenic bacteria (C hauve 1 998) or fungi (Steenberg and Kilpinen 2014), or the use of predatory mites (Lesna et al. 2012). However, neither of these strategies has proven largely successful in eradicating the pest from poultry farms. Therefore, there is a high demand for innovative strategies for the control of PRM.

An alternative to existing strategies to combat ectoparasites is based on vaccination (Sparagano 2009). The underlying principle is that antibodies are generated in the host, which are taken up by the parasite during the blood meal. If these antibodies are directed against an antigen present inside of the parasite (e.g. gut, intestine or germ tissues) they might have a negative effect onto their target tissue, thereby causing harm or even death of the parasite. Vaccines are non-toxic, do not cause problems with residual chemicals and resistance is unlikely to emerge. The first such animal vaccine against a tick (Boophilus microplus) was licensed in 2000 (Jonsson et al.) and consists of the protein Bm86. This antigen was identified via a laborious procedure involving multiple rounds of immunizations of cows with biochemical tick extract fractions (Willadsen and Kemp 1 988, Willadsen et al. 1989). The 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). On the other hand, 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). Thereby putative candidate antigens were identified in these extracts (Harrington et al. 2009b). In addition, histamine releasing factor and cathepsin D were investigated as potential vaccine candidates recently through gene-knockdown approaches (Kamau et al. 2013). However, none of the candidate antigens identified and/or studied so far was found positive for eliciting antibodies with anti-PRM activity.

There remains a need for an improved method to identify the candidate antigens which can positively elicit antibodies with anti-PRM activity.

Summary of the Invention

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.

Here, an innovative strategy is presented to identify potentially protective antigens in PRM extracts via combining biochemical fractionations, in vitro feeding of antibodies to PRM and proteomic techniques. By using this approach, a number of proteins are identified which, upon immunization into chicken, elicit antibodies with anti-PRM activity and can therefore be used as antigens for the development of a vaccine against

Dermanyssus 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.

Brief Description of the Figures Figure 1 : Detection of specific antibody production against total RVM extracts through ELISA using sera from 1 6 chickens immunized with PRM total extracts. Two different adjuvants (F = Freunds adjuvant; M = Montanide ISA 70 VG adjuvant) were tested.

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 : 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. Statistical analysis to evaluate the difference between the mortality was performed by using an unpaired t-test. Asterisk show a statistically significant difference (p < 0.03) between the mortality of mites fed with control IgY-M and mites fed with PRM IgY-M.

Figure 3: Dermanyssus gallinae 2 D SDS PAGE gels and Western blots. Analysis of 2 D SDS PAG E gels loaded with PRM total extracts stained with Coomassie blue (a and e) and Western blots (b-d). For Western blots IgY isolated from hens immunized with PRM total extracts and two different adjuvants (F = Freunds adjuvant and M = Montanide ISA 70 VG) were used as primary antibody. Control IgY-C (b) shows IgY isolated from chicken immunized without PRM extract. After analysis of the original Coomassie 2 D SDS gels (a) and Western blot films (b-d), spots recognized by IgY preparations displaying anti-mite activity were excised (U 1 -U 1 0 mark the respective positions of these spots).

Figure 4: 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).

Detailed Description of the Invention

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. 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. In one embodiment, 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 .

In one embodiment, 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 .

In one embodiment, 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 .

In one embodiment, 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 .

In one embodiment, 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 .

In one embodiment, 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 .

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 2.

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 3.

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 4.

In one embodiment, 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.

In one embodiment, 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.

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 8.

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 9.

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 0.

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 1 .

In table 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 0, and PRM-1 1 is defined as SEQ ID NO 1 1 . In another embodiment of the present invention, the polypeptides as described above can be used in the treatment of Poultry Red Mite (PRM).

In yet another embodiment the present invention also discloses the manufacture of a vaccine using the polypeptides as above described.

In one embodiment of the present invention, a vaccine against Poultry Red Mite can be produced comprising:

A polypeptide with the sequences SEQ IDs 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 or a mixture of these sequences as stated above, delivered as a recombinant protein or as its coding sequence (e.g. as part of a viral vector or a DNA vaccine plasmid or an isolated mRNA). After delivery into animals, these vaccine compositions elicit antibodies against said proteins.

In another embodiment the present invention provides isolated nucleic acid molecules comprising polynucleotides encoding the PRM polypeptides of the present invention. As used here in the term " isolated " refers to nucleic acid molecules purified to some degree from endogenous material. In one embodiment, 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. As used herein the term " specifically binds " refers to antibodies having a binding affinity (Ka) for PRM-polypeptides of 1 06M-1 or greater. As used herein, the term " antibody" refers to intact antibodies including polyclonal antibodies, and monoclonal antibodies. As used herein, 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. The term " 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.

2). 2D-Gels to identify proteins recognized by antibodies from step 1

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.

3). Immunization with proteins contained in the ten spots from step 2 The proteins in the gel spots from step 2 were recovered from the gel, purified and used to immunize chicken (this time Freund ^' s adjuvant was used). Subsequently, the antibodies elicited were again used in PRM feeding-assays. It was found that several of these proteins lead to antibodies with anti-PRM activity (Fig. 4).

4). 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.

Pharmaceutical compositions containing the PRM-polypeptides of the present invention are also provided. Such compositions 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 .

In one embodiment, 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.

In one embodiment 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 .

In one embodiment the pharmaceutical 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 .

In one embodiment of the present invention, the pharmaceutical composition comprising polypeptides having an amino acid sequence as set forth in SEQ ID N01 and 4.

In one embodiment of the present invention, the pharmaceutical composition comprising polypeptides having an amino acid sequence as set forth in SEQ ID N01 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 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 N01 and 3.

In one embodiment of the present invention, 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.

The pharmaceutical composition of the present invention further comprises a

pharmaceutical acceptable carrier or diluent.

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. Similarly, 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.

Examples

Materials and methods

Dermanyssus gallinae protein isolation

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 (eggs, nymphs, adult female and males) were subjected to homogenization and sonification on ice in different buffers (see below). For the isolation of PRM soluble proteins, mites were homogenized with phosphate buffered saline (PBS), pH 7.4, containing 1 x EDTA-free protease inhibitor (Roche Diagnostics). Then, 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).

The isolation of 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 μΙ PBS through 3 incubation steps for 5 min with gentle agitation.

2D SDS PAGE Gel and Western blots

To identify individual mite proteins 2D SDS PAGE gel analysis and Western blots were performed for PRM. First, PRM proteins were separated by 2 D SDS PAGE gel

electrophoresis using an Ettan IPGphor III IEF System (G E Healthcare) following the manufacturer ^' s instructions and based on a protocol described previously (Pohler et al., 2012). Briefly, at the beginning of the passive rehydration the thawing PRM extract was supplemented with rehydration buffer (7 M urea, 2 M thiourea, 2 % v/v C HAPS, 0.002 % v/v bromophenol blue) containing 1 .3 % v/v fresh added Ampholine (GE Healthcare). Thereafter, for the passive rehydration of 7 cm Immobiline DryStrip pH3-1 0 NL (GE Healthcare, Sweden) it was required 200 g PRM protein extract diluted in 100 μΙ of rehydration buffer. After 1 6 hours passive rehydration at RT, the isoelectric focusing (IEF) did run for 3.5 hours on an Ettan IPGphor III IEF System (GE Healthcare). The running conditions were 300 V for 60 min followed by linear gradients of 1 kV for 30 min, 5kV for 90 min, and 5kV for 30 min. Succeeding focusing, IPG strips were incubated with equilibration buffers as described before (Pohler et al., 2012). In the second equilibration buffer 2.5 % v/v iodoacetamide was added to prevent re-oxidation by atmospheric oxygen (Herbert et al. 2001 , Sechi et al. 1998). Finally, the second dimension was performed on 12 % SDS PAGE gels, which were stained with Coomassie Brilliant Blue (Fluka) or used for Western blotting. A protein size marker (PageRuler Prestained Protein Ladder Plus, Fermentas) was used.

Western blots: PRM proteins were separated through 2 D SDS PAGE gels before transfer onto nitrocellulose membranes overnight at 4°C . Antibodies were isolated from eggs collected from immunized hens following the manufacturer ^' s instructions of Pierce C hicken IgY Purification Kit (Thermo scientific). These eggs were from chicken immunized with PRM extracts and respective adjuvants (see below). IgY were diluted 1 : 50 with 5 % v/v milk (skimmed milk powder AppliChem) in PBS-Tween (0.1 v/v Tween 20) and 300 g IgY were incubated with the membrane for 2 hours. Next, 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).

The stained 2D SDS PAGE gels, but also the Western blot films were digitized using an Image Scanner III (GE Healthcare). Subsequent computer assisted image analysis of the PRM 2D gels and Western blot images were processed using the Delta2 D 3.6 software (DECODON, Germany) according to the manufacturer ^' s instructions. Immunization of hens and adjuvant tests

Twenty two Lohmann Brown hens at 1 8 weeks of age were housed together for at least 3 weeks before the beginning of the immunization studies (approval for animal experiments was obtained under Reg. -Nr. : TW 40/10 from Landesdirektion Leipzig). For the production of antibodies against mite proteins, hens were immunized

subcutaneously with total PRM extract and PRM proteins eluted from 2D gel spots (see above). C hicken were vaccinated 3 x during a period of 2 months (day 0, 28 and 56). In the first immunization study 2 different adjuvants (F = Freunds complete and incomplete adjuvant, Sigma; M = Montanide ISA 70 VG, Seppic) were used. In the second immunization study hens were immunized with Freunds adjuvant. Controls were immunized with PBS and adjuvant only. Freunds adjuvant was administrated in the ratio of 1 : 1 to provide a total volume of 300 μΙ vaccine per hen / immunization, and

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.

In the first immunization study 1 6 hens were randomly allocated to one of four treatment groups (4 hens / group): Two control groups (F and M adjuvants) and 2 groups immunized with PRM proteins (F and M adjuvants). In this first study each hen was immunized 3 times with 200 g per dose of total PRM extract in four-weeks intervals. In a second immunization study 6 hens were immunized with 20 g per dose of proteins eluted from 2 D SDS PAGE gel spots, using the same conditions and protocol as in the first experiment. 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).

In vitro feeding of PRM

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 μΙ) was spiked with antibodies (750 g) isolated from chicken eggs (see above) collected 2-4 weeks after the third immunization. In each glass chamber a filter paper (9 mm x 95mm) was placed and 10-20 PRM (adult females and males) were incubated in the dark with the blood reservoir 1 6 hours at 35°C ± 3°C and relative humidity of 70-80%. After mites were engorged, they were transferred from 35°C incubator to a RT incubator and were monitored daily for 1 -2 weeks after the feeding. During this period the mortality of 80-190 PRM was monitored per experiment, divided into triplicates for each test. Mites were defined as dead if they were immobile and unresponsive to stimulus as described before (McDevitt et al. 2006). Proteomics

After PRM protein separation through two-dimensional gel electrophoresis, spots were cut from the 2 D gel and the proteins were partially sequenced via MALDI TOF-TOF in a 4700 Proteomics Analyzer (AB Sciex). To obtain genomic protein sequences, total DNA and RNA were isolated from PRM (eggs, nymphs, adult female and males) according to the manufacturer ^' s instructions (TRI Reagent, Sigma), and 1 -2 g each (DNA and RNA) were applied for sequencing through lllumina HiSeq 2000.

Subsequently, the bioinformatics analysis was performed as follows: Sequencing adapters, indices and index adapters were removed with the software Cutadapt version 1 .3 (Martin 201 1 ). The revised RNA reads were then assembled to sets of contigs, representing partial or full transcripts, with Trinity version r20140413p1 (Grabherr et al. 201 1 ). In the next step, the proteins coded by this set of contigs were predicted in-silico with the software TransDecoder (Haas et al. 2013). The tandem of MS and MS/MS data was processed as follows: Peaks in 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).

Statistical analyses

The statistical analyses of ELISA and in vitro feeding studies were carried out using SigmaStat and SigmaPlot (Systat Software, San Jose, CA). The proteomics results were analyzed and clustered with statistics software R and the software EMBOSS stretcher version 6.6.0.0 (Rice et al. 2000). Results

Immunization with different Adjuvants and immune response against PRM fractions

The starting experiment for the investigation and detection of PRM proteins as potential vaccine candidate was the isolation of total mite protein. Hence, a complete PRM extract was generated and used for immunization of hens. Each hen was immunized three times with 200 g PRM extract per dose. In addition, two different adjuvants (F = Freunds adjuvant; M = Montanide ISA 70 VG) were tested to determine the most efficient combination for the production of antibodies against PRM (Fig. 1 ). To monitor the appearance of PRM antibodies, sera were collected from these hens before each immunization and 4 weeks after the third immunization. The results show specific humoral immune responses against PRM proteins upon immunization (Fig. 1 ). The IgY- titer was significantly higher in hens immunized with PRM-M extracts compared to hens from the control group.

In vitro feeding with PRM IgY-Fractions

Because antibodies are transferred from chicken to egg (Hamal et al. 2006, Harrington et al. 2009b), IgY were isolated from immunized hens. 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. In contrast, IgY derived from hens immunized with PRM extracts and Freund ^' s adjuvants only led to a marginal increase in PRM mortality. Therefore, this increase in mortality of 38.8% (Fig. 2) suggests the presence of anti-mite activity displayed by the antibodies isolated from hens immunized with PRM extracts and Montanide adjuvant.

2D SDS PAGE gel and Western blots To visualize individual protein antigens which are recognized by IgY preparations leading to PRM mortality, total PRM extracts were analyzed in 2D SDS PAGE gels and Western blots (Fig. 3). As can be seen in figure 3, the immune detection showed highly specific antibody production against PRM proteins by the hens immunized with PRM-M and PRM-F, whereas almost no proteins were stained when IgY from control-immunized animals were used. Mite mortality was significantly higher with IgY-M than with IgY from controls (Fig. 2) and from the IgY-F group. Hence, using computer assisted image processing, we compared the binding pattern of the different fractions and focused on spots recognized by the IgY-M preparation only or in a stronger way as compared to the other preparations. Ten of these spots were selected (Fig. 3e, spots U 1 -U 10) for further experiments.

Spots of interest: Immunization with PRM proteins

To analyze whether the proteins contained in these spots were able to elicit protective antibodies, proteins were eluted from the 2 D gel spots (Fig. 3e), before the material was injected into hens. To reach at least 20 g of protein for a single immunization (three immunizations in total), some spots were immunized together in the same hen: 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. Four weeks after the third immunization with these proteins, 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

After PRM in vitro feeding with IgY isolated from eggs of hens immunized with the proteins eluted from the 2D gel spots, mortality rates of PRM were determined. The results show that spots 1 , 2, 4, 5, 6, 7, 8 and 10 (Fig. 3e) are associated with increased mortality of PRM after feeding (Fig. 4). A statistically significant difference is observed between the mortality of mites fed with control IgY (immunization without PRM proteins) and mites fed with IgY isolated from hens immunized with spot-1 . The difference between the mortality of mites fed with control IgY and mites fed with IgY isolated from hens immunized with spot-2, spots-4, 5, 6 and spots-7, 8 was even greater. Consequently, 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. In addition, spot 1 0 (due to the marginally higher PRM mortalities) and spots 3 and 9 (eluted amount of proteins too low for immunizations) were analyzed.

Proteomics and vaccine candidate sequences

The above mentioned ten protein-spots were subjected to proteomic Mass Spec

(MS/MS). For the identification of PRM proteins, the MS/MS spectra were matched with PRM DNA and RNA sequences. PRM protein sequences were identified in all 10 spots

After determining the correlation between PRM 2D gel spots (Fig. 3e), PRM mortality rates (Fig. 4) and the results of the PRM sequencing data, eleven PRM proteins were identified as unique protein sequences and potential vaccine candidates (Table 1 ). These 1 1 vaccine candidate antigens were named PRM-1 to PRM-1 1 (Table 1 ) and were defined as 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).

candidate spots protein sequence type (2D gel)

PRM-1 111, 112 MRAFQVLSAALVATAAHGQLLSGYRTYGSSGGYG Complete

GAYNLGTYGLGGVSYGASYAPTSVRYSTGYTPSAV SYGSTYGAPVGVAYSSRRWSNAVPALTTTSVGAA PAI AVSTVPTAVNTISYG LG VN RG ISYG FG G G LS RG I SYG LG G G AG G LTTAG G VVTATGTG G G VRS H E VH TGGAGNWRVEEYKAGGQLIRVHDSAQPAAEWD VQGPAVAGNHVRLVSRTGGTQVERWHQDPSVQT F D VVN P PQ P AD RVVN I R R AP P P A ATV E LV AE H H QQ AVPEVWG G DE PNVQVQH VG G GAG VAVG G ASLS Y A A A P A V A H VQT V H A A PT VS V A A A P V AS Y AT R V H TVQAAPAISVAAAPVATYATRVHAAPAVSYSTVGV PAATYGTSYGTSYGTYG SG LG STLYGTG LG STVYG SGLGSTVYGSGLGNTLYGSYSSGLGRNVYGTSYGS YGSSLGGNSFGTTAVLSKKA*

PRM-2 U2 SSSAYRKMPRSPPPPPPPDHHRSRHRGDPYIDHPGY Internal

PSGHQSSSSSSHQPPRKSSYGSYRILLASNLNYKVND NLMRQALEDEFGRFGDLTVKLSQDAGDRVAYLYFR SYEEAKEARHAKSRMILFDKPIHIEPIVEEPPPRRKRTP SPPMDYRGMSPMTTRRRPPSMDRMPMYHHRGEH HH

PRM-3 U3 PTSNMGLRRSCWAQALLGLGLVALTNAQGQIGKE 5prime_

YRYSYTGHMWVSTTMHTQKPGMAFRSNVRVQRI partial VDNTYGLKIEDFEVATANERETDWSETEKMAFVKN ERLSSMVERPFLVHLERTENLSTYEFKSLETHSDDPV WSTNIKKGLVSLITLHVPFDNDEQIYSSVEPTVYGKC NVHWHRQSKPHWTRPDSKVLNISRAIDHENCETVA NRVYGSVMGTPCMDGSCQRHHTYPVSTSSQAKY SLVAASGDTNNWILETARDNSVFTHAPHTENGNVL KLKTAQRMRLIDTVDATEKLEIVGETQVDDQLMMH FTKRWRLERSVDLDKADDFVLDWDIRGCSNGTIALL

QKLRSLRNADDNQLGMFDNNVAETFSAAIEMMYL

LDREQLAWYDRIVKAAPEAELEQAQRLFVEVASAA

GSTTAMKFVMDKFQAGDISVRFMRPFLSGITRGLFD

RTSGALEIFENFCTSDQMKALPEERRQCLLAYTALVY

DTYRLEHFNVQKHERKDTRETIFKRIVPEYSVAVEEG

REAFKTYMLIAGNLKTASSVEFLARAVLDEKLDRLTA

MDAMRTLIMTADNSEVARKAALTVYADSSKPAELR

MLAAIVIMRSNPPLSVFYSMADRLLHEKSDQVRSYV

VSMFKELSQTTHPFFRHLANKAHYVAPKLEARLPSE

WKRKDYLTSHTKLASGYDPKYDHGGHSIISMIMSD

SYVPRDLYVNFGDFFAGFFFDNFGMSISQQGMERLI

DHFNNPRTFGNKFGRNLWNMAGRRRQTRDAASV

EHAMDEIDSTLNIHTKKYDPMRLDMTFSAFGENIHT

VSLNESFFLPLLSPDYKPGTLLNKILSTERDMHSFKNLR

DMTFMIPTAIGMPAWFDVQLPTVLSCRRKESSFDFG

NEGALKLKLDQRIVMDAQLEESLRFGVPGLQVSLGV

GFKRRLALNLPIKLDVDANVATGKVVMNQDFVLPR

DIMRYKFEPYTIEDNFKEPEKTTYLALFKDEELVEFKS

QPLKDLLGIDFFLEGKQLPKSVLSWDFSQWLKSDIRQ

KLYYAIVNPKWRPRSLSLSAAPAKEHPTTGRVLTFKH

KMTPPEATERPGSRFEEFEKDLPESFVHVIQVSNELTS

SKKRRADLEVRYSYTPNRVEHWIQLFYDRTPLSSQD

TDHTKLCMVAKVKQTTTDWEKLTKEQVMHVNDG

QRMDVLMNLQYGKSCKTGDEPDASSLQMLARAT

YEHSDEQKRWLSELTTGETRSRVHGLENPYMKFYTK

CIKYLEKGLLMPFACHKFIVHTSQLNNMTLDVEVND

RAHVLTNPCMSTLRAYLAAQYDANLRMFAPQRNV

SRPGHWHMNSWREPCINQRLADIAIVGPYHSSFW

ESVPVSWGAHWAPRTATHLGSTNMQSYSRKWFH

RYCDIQLNSVATFDNWYELPETNCWKVLAKDCSE

MKVFTLLG RANADKKKEIRLLVDKYEVHLKNVDGKI

VLTLNGAEEVLAERQPKLVKNENGFVTLVILNKGHG

LMQIDAPIYGIYMLAMDSAAFIQVAPYYRGKLCGL

CGNFNLDRQHEFHTTDGCHHRTPWSFARNFVIPSD QCAPVPEATDAGQPYC PAH*

PRM-4 U4, U6 MAGNFSYLTPELQAELRNTANKIVAPGKGILAADES Complete

TGTMGKRLASIGVENTEELRRKYRQMLFTSDKLLDS CISGVILFHETLYQKADDGTPFVKILQDRGIIPGIKVDT GVVPLMGTTDETTTQGLDDLSKRCAQYYKDGCRF AKWRCVLRIRKDAPTPLAVLENANVLARYATECQK NGIVPIVEPEILPDGDHDLVTCQKVTEKVLAAVYKAL SDHNVFLEGTLLKPNMCTPGQSCPVKSGPIDIAKAT VTALQRTVPAAVPGIVFLSGGQSEEEASVNLDAMN RLQGKKPWALSFSYGRALQASAIKAWGGKDENLK AGQDELLKRAKANSDASLGKYEGGVKGAAAEDTL FIKDHQY*

PRM-5 U5, U6, MVDQAVKDKLEAGFAKLQGAADCKSLLKKYLTRG Complete

U7, U8 VLDQLKDKKTAMGATLLDWQSGMENLDSGVGV

YAPDAESYKTFAPLFDPIIDDYHQGFKPTDKHPQSDF GDLNQLVNVDPEGKFVISTRVRCGRSLQGYPFNPCL TEAQYKEMEGKVSSTLKALEGELKGTYYPLTGMDK ATQQQLIDDHFLFKEGDRFLQAANAC RYWPAGRGI YHNDAKTFLVWCNEEDHLRIISMQKGGDLKQVFGR LTAAVKAIESKLPFSRDDRLGYLTFCPTNLGTTIRASV HIALPKLAADRAKLEEVASKYSLQVRGTRGEHTESE GGVYDISNKRRMGLTEFQAVKEMQDGILEMIKLEK AAA*

PRM-6 U5, U7 MDQETLKKAAIAVPALAVLCYVAYQGSNAIKNALA Complete

RKEVPQDVKDKLQAGFQKLQTSTQCKSLLKKYLTK EVLDKLQDQTTRMGATLLDVVQSGFANIDSGVGVY APDAESYTVFAALFDPIIDDYHQGFKSTDRHPATDF GDLSSFVNVDPEKKYVISTRVRCGRSLQGYPFNPCLT EAQYREMEDKVSSTLKNLSGELKGTYYPLTGMNKS TQQQLIDDHFLFKEGDRFLQTANACRYWPTGRGIF HNDAKSFLVWVNEEDHLRIISMQQGGDLKEVFTRL VEGVKAIESKLPFSRDNRLGYLTFCPTNLGTTIRASVH IKLPKLAANKARLEQVASKYNLQVRGTRGEHTESEG GVYDISNKRRLGLTEYQAVKEMQDGVLELIKLEAAA

Q*

PRM-7 U5, U7 MFFDDDDDDESSHPAPIPPSNEGTESEKTFSPSLLW Complete

RRACPRMFCLWEAVLVAETSNAIKNALARKEVPQD VKDKLQAGFQKLQTSTQCKSLLKKYLTKEVLDKLQ DQTTRMGATLLDVVQSGFANIDSGVGVYAPDAES YTVFAALFDPIIDDYHQGFKSTDRHPATDFGDLSSFV NVDPEKKYVISTRVRCGRSLQGYPFNPCLTEAQYRE MEDKVSSTLKNLSGELKGTYYPLTGMNKSTQQQLI DDHFLFKEGDRFLQTANAC RYWPTGRGIFHNDAKS FLVWVNEEDHLRIISMQQGGDLKEVFTRLVEGVKAI ESKLPFSRDNRLGYLTFCPTNLGTTIRASVHIKLPKLA ANKARLEQVASKYNLQVRGTRGEHTESEGGVYDIS NKRRLGLTEYQAVKEMQDGVLELIKLEAAAQ*

PRM-8 U5, U7 MSQRSNAIKNALARKEVPQDVKDKLQAGFQKLQT Complete

STQCKSLLKKYLTKEVLDKLQDQTTRMGATLLDVV QSGFANIDSGVGVYAPDAESYTVFAALFDPIIDDYH QGFKSTDRHPATDFGDLSSFVNVDPEKKYVISTRVR CGRSLQGYPFNPCLTEAQYREMEDKVSSTLKNLSGE LKGTYYPLTGMNKSTQQQLIDDHFLFKEGDRFLQTA NACRYWPTGRGIFHNDAKSFLVWVNEEDHLRIISM QQGGDLKEVFTRLVEGVKAIESKLPFSRDNRLGYLTF CPTNLGTTIRASVHIKLPKLAANKARLEQVASKYNLQ VRGTRGEHTESEGGVYDISNKRRLGLTEYQAVKEM QDGVLELIKLEAAAQ*

PRM-9 U7 MPNIKVFSGSSHCDLAQRIVDRLGINLGKWLKKFS Complete

NQETCVEIGESVRGEDVYIIQSGCGEVNDNLMELLI MINACKIASASRVSAVIPCFPYARQDKKDKSRAPIS AKLVANMLSVAGADHIITMDLHASQIQGFFDIPVDN LYAEPAVLKWIRENISDWRNAIIVSPDAGGAKRVTSI AD RLNVEFALI HKERKKAN EVASMVLVG DVRD Kl Al

LVDDMADTCGTVCHAADKLVEAGAQKVYAILTH GIFSGPAIQRINSAAFEAVWTNSIPQDQHMKDSPK VQCIDVSMILAEAIRRTHNGESVSYLFSHVPM*

PRM-10 U9 MRPFVGILRAGARYHRSAVGGQRRQLNHLARTTFS Complete

QTLFNMPETRVTTLANGVRVASEDNGAPTATVGIWI DAGSRYETEKNNGVAHFLEHMAFKGTGKRTQTQL ELEVENAGMHLNAYTSREQTVYYAKCLRKDLANA VDIVADITQNPKLGEQEIERERGVILREMEEVEGNLQ EWFDHLHAVAYQGTPLGLTILGPTKNIKSLQRQDLK DYIDTHYTGSRIVLACAGGVDHDELVKLAEQHFGK VGTGFDQQIIIPSPCRYTGSEVRVRDDDMPFAHVAI AIEGAGWTNPDNIPLMVANTMIGSWDRSHGGGA NASSRLAAVAATDEHRTMHSFQSFNTCYKDTGLW GVYFVANGDEHLDDCMSAVQNEWMRICTECVEA DVTRAKNLLKTNLLLQLDGTTPLCEDIGRQMLCYGR RIPLHELEARIDAVTANTVRDVALKYIYDRCPWAAV GPVSGLIDYVRIRSQMYRLRY*

P M-11 U10 ATTTTTSTTTTMTTTRSPHFAQALVPIKTLACRITGTIS 5prime_

RRRLRHYTRSRQQPPSTMRTLIVLAALVAAASAQIH partial ASSVAKAKKGCDFGVPINGDIGWVKVPCDKTNQA AKAIGLGEGAEFLGSSRLSGFVGAESYAIHGKIDGK CCSQR KIAGTATEHPFKAQEDELCC HETCPTWM EKC*