RUBELLA VIRUS SPIKE CONSTRUCT

Abstract

The Rubella Virus Spike construct comprises at least one E1 component and one E2 component, which are linked together. The E1 component consists of the E1 envelope protein, whose C-terminal transmembrane region and intravirional domain are removed and whose N-terminus comprises the ectodomain of the E1 envelope protein. The E2 component consists of the E2 envelope protein whose transmembrane regions and intravirional domain removed and whose C-terminus comprising the ectodomain of the E2 envelope protein. The C-terminus of the E2 component is connected to the N-terminus of the E1 component by direct fusion or by means of a linker to form an E1-E2 fusion protein.

Claims

1. Rubella virus antigen, wherein it is a rubella virus E1-E2 envelope protein complex construct (synonym: rubella spike construct) comprising at least one E1 component and one E2 component which are linked, wherein the E1 component consists of the E1 envelope protein whose C-terminal transmembrane region and intravirional domain are removed and whose N-terminus comprises the ectodomain of the E1 envelope protein, and wherein the E2 component consists of the E2 envelope protein whose transmembrane regions and intravirional domain are removed and whose N-terminus comprises the ectodomain of the E2 envelope protein, and wherein the C-terminus of the E2 component is connected to the N-terminus of the E1 component directly or by means of linkers.

2. The rubella virus antigen according to claim 1, wherein the antigen is produced as a recombinant E1-E2 fusion protein.

3. The rubella virus antigen of claim 2, wherein the E1-E2 fusion protein is coupled to a signal sequence.

4. The rubella virus antigen according to claim 1, wherein the E1-E2 fusion protein is coupled to an affinity tag, preferably a streptavidin affinity tag, preferably arranged C-terminal.

5. The rubella virus antigen according to claim 1, wherein the ectodomain of the E1 envelope protein comprises the amino acids (of positions) 1-446 according to SEQ ID NO: 6 or the amino acids AA583-1028 of the reference sequence UniProtKB/SwissProt—P08563 (POLS_RUBVM), Sequence Update May 30, 2006 (version 2 of the sequence).

6. Rubella virus antigen according to claim 1, wherein the ectodomain of the E2 envelope protein comprises the amino acids (of positions) 1-234 according to SEQ ID NO: 8 or the amino acids AA301-534 of the reference sequence UniProtKB/SwissProt—P08563 (POLS_RUBVM), Sequence Update May 30, 2006 (version 2 of the sequence).

7. The rubella virus antigen according to claim 1, wherein the linker is a flexible linker comprising glycine and/or serine.

8. Rubella virus antigen according to claim 2, wherein the fusion protein comprises the amino acid sequence according to SEQ ID NO: 2, wherein the signal sequence at the N-terminal end and/or the linker sequence in the middle region of this amino acid sequence is optionally present.

9. Rubella virus antigen according to claim 2, wherein the fusion protein comprises the amino acid sequence according to SEQ ID NO: 4, wherein the signal sequence at the N-terminal end and/or the linker sequence in the middle region of this amino acid sequence is optionally present.

10. A recombinant DNA molecule encoding a rubella virus antigen and comprising a nucleotide sequence encoding a rubella E1-E2 fusion protein according to claim 2.

11. Recombinant DNA molecule according to claim 10, wherein it comprises the nucleotide sequences according to SEQ ID NO: 5 and SEQ ID NO: 7.

12. Recombinant DNA molecule according to claim 10, wherein it comprises the nucleotide sequence according to SEQ ID NO: 1, wherein the signal sequence at the N-terminal end and/or the linker sequence in the middle region of this nucleotide sequence is optionally present.

13. Recombinant DNA molecule according to claim 10, wherein it comprises the nucleotide sequence according to SEQ ID NO: 3, wherein the signal sequence at the N-terminal end and/or the linker sequence in the middle region of this nucleotide sequence is optionally present.

14. An expression vector comprising a recombinant DNA molecule according to claim 10 in operative linkage.

15. Host cell transformed with an expression vector according to claim 14.

16. The host cell of claim 15, wherein it is an insect cell, preferably a Drosophila Schneider 2 (S2) cell.

17. A process for preparing a rubella virus E1-E2 envelope protein complex construct (synonym: rubella spike construct) according to claim 1, comprising the steps: (a) Cultivation of host cells, preferably Drosophila Schneider 2 (S2) cells; (b) Transforming the host cells with an expression vector comprising a nucleotide sequence encoding the rubella E1-E2 fusion protein in operative linkage; (c) Cultivation of the transfected host cells, whereby they express the Rubella E1-E2 fusion protein and secrete the Rubella spike constructs from the host cell; (d) Purification of the fusion protein.

18. Use of a rubella virus E1-E2 envelope protein complex construct (synonym: rubella spike construct) according to claim 1 in a method for the qualitative and/or quantitative detection of anti-rubella antibodies in a liquid sample, as capture reagent and/or binding partner for the anti-rubella antibodies.

19. Reagent kit (test kit) for carrying out a method for the qualitative and/or quantitative detection of anti-rubella antibodies in a liquid sample, wherein this kit contains at least one rubella virus E1-E2 envelope protein complex construct (synonym: rubella spike construct) according to claim 1 as antigen.

20. A vaccine preparation comprising a rubella virus E1-E2 envelope protein complex construct (synonym: rubella spike construct) according to claim 1 as the antigenic active ingredient.

Description

[0061] The invention is explained in more detail in the following on the basis of design examples and illustrations.

[0062] Short description of the illustrations:

[0063] FIG. 1: Models of the glycoprotein E2/E1 spike configuration. [0064] (A) Configuration of the glycoproteins E2 and E1 on the surface of a native rubella virus. [0065] (B) Configuration of the soluble rubella spike constructs. In these constructs the transmembrane regions and the intravirional domains (light grey lines) are removed and the ectodomains of E1 and E2 (black lines) are connected by a flexible glycine-serine linker.

[0066] FIG. 2: amino acid sequence of the rubella spike construct; [0067] (A) rubella spike construct “long” (i.e. with long linker) [0068] (B) rubella spike construct “short” (i.e. with short linker) [0069] Smooth underline: BiP signal sequence [0070] Bright gray background: Amino acid sequence of the E2 fragment [0071] Punctured Underlined: Glycine-serine linker short (8 amino acids) [0072] White background: Amino acid sequence of the E1 fragment

[0073] FIG. 3: Characterization of rubella spike constructs on SDS-PAGE: [0074] Aliquots of the purified rubella spike constructs (“short” and “long”) after separation by SDS-PAGE (4-20% tris-glycine gels, article number TG 42010, Anamed Elektrophoresis GmbH) under reducing conditions and subsequent staining with Coomassie-Brillant-Blue. [0075] Lane 1: Molecular weight marker (kDa), [0076] Lane 2: Rubella spike construct “long”, [0077] Lane 3: Rubella spike construct “short”.

[0078] FIG. 4: Characterization of the rubella spike constructs in a Western blot: [0079] Reaction of the rubella spike protein constructs purified by SDS-PAGE and then transferred to PVDF membrane with monoclonal antibodies directed either against rubella glycoprotein E1 (A) or rubella glycoprotein E2 (B). [0080] (A): Antibody=Anti-rubella virus structural glycoprotein, monoclonal, mouse (MABR23-Ru6, ibt - immunological and biochemical testsystems GmbH). [0081] Lane 1: Molecular size marker (kDa), [0082] Lane 2: Rubella spike construct “long”, [0083] Lane 3: Rubella spike construct “short”. [0084] (B): Antibody =Anti-rubella Virus E2 monoclonal antibody (D92G) (MA5-18255, Thermo Fisher Scientific). [0085] Lane 1: Molecular weight marker (kDa), [0086] Lane 2: Rubella spike construct “long”, [0087] Lane 3: Rubella spike construct “short”.

[0088] FIG. 5: Immunological reactivity of different rubella spike constructs (as antigens) compared to whole virus—in particle test with different rubella IgM positive/negative human sera. [0089] x-axis: Human sera [0090] y axis: MFI=Mean fluorescence intensity

[0091] FIG. 6: Immunological reactivity of different rubella spike constructs (as antigens) compared to whole virus—titration behaviour in the particle test with a selected, for rubella IgM positive, high titre human serum.

[0092] FIG. 7: Immunological reactivity of different rubella spike constructs (as antigens) compared to whole virus—p/n (positive/negative) ratio in particle-based detection of rubella IgM antibodies in different human sera.

[0093] FIG. 8: Immunological reactivity of different rubella spike constructs as antigens in an immunodiagnostic test—detection of anti-rubella IgM antibodies in selected, pre-characterized anti-rubella IgM human sera compared to a whole virus based reference test (ELISA). [0094] (A) (Part 1 and Part 2): Table of values [0095] ID: Sample identification [0096] OD: Optical density at 405 nm [0097] MFI: Mean fluorescence intensity [0098] pos: Positive rating for anti-rubella IgM antibodies [0099] neg: Negative rating for anti-rubella IgM antibodies  FIG. 8 (A)—part 2 is the continuation of table FIG. 8 (A)—part 1 in vertical direction. [0100] (B): Diagnostic performance (diagnostic efficiency)  ROC (receiver operating characteristic) curve

[0101] FIG. 9: Immunological reactivity of different rubella spike constructs as antigens in an immunodiagnostic test—detection of anti-rubella IgG antibodies in selected, pre-characterized anti-rubella IgG human sera compared to a whole virus based reference test (ELISA). [0102] (A) (Part 1 and Part 2): Table of values [0103] ID: Sample identification [0104] OD: Optical density at 405 nm [0105] MFI: Mean fluorescence intensity [0106] pos: Positive rating for anti-rubella IgG antibodies [0107] neg: Negative rating for anti-rubella IgG antibodies  FIG. 9 (A)—part 2 is the continuation of table FIG. 9 (A)—part 1 in vertical direction. [0108] (B): Diagnostic performance  ROC (receiver operating characteristic) curve

EXAMPLE 1

Generation of the Soluble Recombinant Rubella Spike Constructs according to the Invention

[0109] The production of the rubella spike constructs according to the invention is principally carried out in such a way that according to the schematic illustration in FIG. 1 the transmembrane region and the intravirional domain of the E2 glycoprotein and the C-terminal transmembrane region of the E1 glycoprotein are removed and the ectodomains of E1 and E2 are connected (coupled) to each other by a flexible glycine-serine linker.

[0110] In a concrete exemplary design, in which the rubella spike constructs according to the invention are derived from the glycoproteins E1 and E2 from the rubella strain M33, the C-terminus of the ectodomain of E2 (amino acids 1-234 of E2 according to SEQ ID NO: 8) is connected to the N-terminus of the ectodomain of E1 (amino acids 1-446 of E1 according to SEQ ID NO: 6) via a short linker (rubella spike “short”) or a long linker (rubella spike “long”). (Reference sequence see: UniProtKB/SwissProt—P08563 (POLS_RUBVM), version 2 of the sequence, sequence update May 30, 2006). The amino acid sequence of these rubella spike constructs is shown in FIG. 2A and SEQ ID NO: 2 (rubella spike construct “long” i.e. with long linker) and in FIG. 2B and SEQ ID NO: 4 (rubella spike construct “short” i.e. with short linker).

[0111] When deriving the rubella spike constructs according to the invention from the glycoproteins E1 and E2 from the rubella strain rubella TO-336 (RUBV) UniProtKB/SwissProt—P08564 (POLS_RUBVV) Version 3 of the sequence, Sequence Update May 30, 2006, which has a 99% amino acid sequence homology to strain M33, the construct is basically build in the same way.

[0112] For expression of the rubella spike construct in Drosophila melanogaster Schneider 2 (S2) cells, the protein construct should preferably be provided with a BiP Drosophila signal sequence or a functionally analogous signal sequence at the N-terminus. The BiP Drosophila signal sequence promotes the secretion of the expressed rubella spikes from Drosophila S2 cells particularly well.

[0113] Production of rubella spikes is not restricted to stable or transient transfected Drosophila S2 cells. Other eukaryotic expression systems such as the baculovirus expression system may also be considered. When using other expression systems, other signal sequences may be required, e.g. the gp64 or HMS signal sequence for the baculovirus expression system.

[0114] In order to enable an effective purification of the expressed rubella spike constructs, the E1-E2 fusion protein C-terminal according to the invention is provided (coupled) with a streptavidin affinity tag in the present execution example.

[0115] Other affinity tags, e.g. a His tag, are also possible. The position of the affinity tag, too, is not restricted to the C-terminus of the E1-E2 fusion protein but can also be located at the N-terminus or in the linker.

[0116] (A) Production of the Nucleotide Sequence Encoding the Rubella Spike Constructs

[0117] The production of the nucleotide sequence coding for the invention's E1-E2 fusion protein of the rubella spike construct according to FIG. 1 (B) is preferably carried out using known rubella virus RNA sequences for the envelope proteins E1 and E2 deposited in sequence databases.

[0118] For example, for the above-mentioned rubella spike construct derived from rubella strain M33, a nucleotide sequence is used that is constructed based on the rubella virus 24S mRNA according to GenBank, Accession X05259, VersionX05259.1.

[0119] In detail, the following procedure is followed:

[0120] The production of the rubella spike constructs “short” (with short linker) and “long” (with long linker) is basically the same. Therefore, the following description of the manufacturing process, only refers to rubella spike construct.

[0121] The nucleotide sequence encoding the rubella spike construct (e.g. SEQ ID NO: 1 or SEQ ID NO: 3) is created synthetically, preferably supplemented with an affinity tag sequence, and cloned into a standard pMX cloning vector (Invitrogen/Geneart, Regensburg). It is codon-optimized for expression in Drosophila melanogaster and contains a Kozak sequence (gccaccATG) to ensure an efficient start of translation during protein biosynthesis in Drosophila S2 cells. In addition, the DNA construct contains an EcoRI and a Notl restriction site for the purpose of cloning the rubella spike DNA construct into the expression vector pExpres2.1 (see EP2307543B1; commercially available from Expres2ion Biotechnologies, Horsholm, Denmark).

[0122] (B) Production of the Transfection Vector for Recombinant Protein Production

[0123] Nucleotide sequences produced according to (A), which encode rubella spike constructs, —here for example rubella spike constructs derived from rubella strain M33 (e.g. SEQ ID NO: 1 or SEQ ID NO: 3)—, are cloned into a transfection vector suitable for the cells of the intended cell culture system.

[0124] As cell culture system especially Drosophila melanogaster Schneider 2 (S2) cells are considered. A suitable transfection vector for these cells is the Drosophila S2 expression vector pExpres2.1 (commercially available from Expres2ion Biotechnologies, Horsholm, Denmark; see also EP2307543B1). The pExpres2.1 expression vector contains the Zeocin resistance gene as selection marker.

[0125] For expression in Drosophila (S2) cells according to the present execution example, the nucleotide sequence produced according to example (A) (coding for the rubella spike constructs) was inserted into the expression vector pExpres2.1.

[0126] The necessary cloning steps were performed according to pExpres2.1 manufacturer's instruction and are generally known and familiar to the expert.

[0127] Drosophila S2 cells were transfected with the generated transfection vector.

[0128] (C) Expression of Rubella Spike Constructs in Drosophila S2 Cell cultures

[0129] Drosophila S2 cells are cells from the embryonic Schneider 2 cell line Drosophila melanogaster, which are deposited with and can be obtained from DSMZ—Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstraβe 7 B, 38124 Braunschweig, Germany, under depot number DSMZ ACC 130, and with the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108, USA, under depot number CRL-1963.

[0130] The Drosophila S2 cells used in the execution example described here originate from cell cultures of the company Expres2ion Biotechnologies, Horsholm, Denmark, they are commercially available as so-called “ExpreS.sup.2 cells”, and they are referred to as “S2 cells” in the following.

[0131] Used Materials: [0132] Fetal Calf Serum “FCS” [0133] Serum-free medium for insect cells, e.g. EX-CELL® 420 (article number 14420, Sigma) [0134] Zeocin (article number R25001, Thermo Fisher Scientific) [0135] Penicillin-Streptomycin, 10,000 U/ml Penicillin, 10 mg/ml Streptomycin (article number P06-07050, PAN Biotech) [0136] (Drosophila melanogaster) S2 cells (ExpreS.sup.2 cells, Expres2ion Biotechnologies) [0137] ExpreS.sup.2 Insect-TRx5 Transfection Reagent (article number S2-55A-001, Expres2ion Biotechnologies) [0138] Buffer W: 100 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 mM EDTA [0139] Buffer E: 100 mM Tris/NaCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM Desthiobiotin [0140] BioLock-Biotin Blocking Solution (article number 2-0205-050, IBA Lifesciences) [0141] Cell culture flasks with Filter Cap, 25 cm.sup.2 (T-25) (article number 156367, Thermo Fisher Scientific) [0142] Cell culture flasks with Filter Cap, 80 cm2 (T-80) (article number 178905, Thermo Fisher Scientific) [0143] Shaker bottle with filter cap, 250 mL (article number 431144, Corning) (working volume: 20-60 mL) [0144] Roller bottle, PET 850 cm.sup.2 (article number 680180, Greiner bio-one GmbH) with ventilation cap (article number 383382, Greiner bio-one GmbH) (working volume: 200-400 mL) [0145] Cryotubes with female thread (article number 114841, Brand GmbH) [0146] Cell counting chamber, Neubauer Improved (Brand GmbH) [0147] Shaking incubator (CERTOMAT BS-1, Sartorius Stedim Biotech) [0148] TFF membrane, Vivaflow 200, 10,000 MWCO Hydrosart Membrane (article number VF20H0, Sartorius Stedim Biotech) [0149] Filter (0.45 μm), Minisart (article number 16555-K, Sartorius Stedim Biotech) [0150] Äkta-pure chromatography system (GE Healthcare) [0151] Strep-Tactin® Superflow® high capacity cartridge, 5 mL (article number 2-1238-001, IBA Lifesciences)

[0152] (i) Transfection and Production of a Stably Transformed Drosophila S2 Cell Line

[0153] To generate the stably transformed S2 cell line, the pExpres2-1 expression vectors produced according to (B) are transfected into the S2 cells with the inserts coding for the rubella spike constructs (“short” or “long”). For this purpose, the S2 cells of the shaker bottle are counted and adjusted to 2×10.sup.6 cells/mL in EX-CELL420 medium. Per transfection 5 mL of this cell suspension are transferred into a T-25 cell culture flask. Then 50 μL of transfection reagent is added and the transfection reagent is evenly distributed in the medium by tilting the bottle. Plasmid DNA (preferably 5-15 μg) is then added and evenly distributed by tilting the bottle. The T-25 bottle is incubated at 23-27° C. After about 3-4 hours 1 mL FCS is added. After 2 days the selection phase is initiated by adding Zeocin up to a final concentration of about 1500-2000 μg/mL. The cells are counted every 3-4 days. As soon as their concentration is higher than 1×10.sup.6 cells/mL, the cells are diluted in fresh selection medium (EX-CELL420 +10% FCS and Zeocin (final concentration about 1500-2000 μg/mL)) to 1×10.sup.6 cells/mL in a final volume of 6 mL. This procedure is repeated several times at intervals of several days. After 2-4 weeks of selection in medium containing Zeocin, the cell line can be considered stable. Following the selection phase and as soon as the cells have reached a concentration of >6×10.sup.6 cells/mL, 6 mL of this cell suspension are transferred from the T-25 to a T75 bottle containing 4 mL fresh medium (EX-CELL420+10% FCS). Once these cells have recovered, 5 mL of fresh medium (EX-CELL420+10% FCS) is added again. The cells are counted after 3-4 days. As soon as the cells have reached a concentration of >6×10.sup.6 cells/mL, 15 mL of these cells are transferred to a 250 mL shaker bottle and 15 mL medium (EX-CELL420) is added. The cells are expanded as described in (ii).

[0154] (ii) Expansion of Drosophila S2 Cells in Shaking Flasks

[0155] The S2 cell culture prepared according to (i) is maintained and monitored in the expansion process. As soon as the viability of the cells is >90% and the cell concentration >8×10.sup.6 cells/mL, the cells are diluted in EX-CELL420 medium to 8×10.sup.6 cells/mL. If the total volume of the culture does not exceed the allowable working volume of the shaking flask, the fresh medium of the original flask is simply added for this purpose. If the total volume would exceed the allowable working volume of the original bottle, the cells are diluted to 8×10.sup.6 cells/mL and distributed to several shaking flasks. Typically, the cells are diluted every 3-4 days. After 3-4 days a cell concentration of 25-50×10.sup.6 cells/mL is typically reached.

[0156] (iii) Production of the Rubella Spike Constructs

[0157] For the production and recovery of the rubella spike constructs, the polyclonal cell lines obtained according to (i) or (ii) shall be cultivated in serum-free medium. After approximately four days of cultivation the cells are removed by centrifugation and the supernatant is harvested. Rubella spike constructs are isolated from the supernatant by affinity chromatography over Strep-Tactin columns using their C-terminal Streptavidin affinity tag labelling.

[0158] In detail, the following procedure is used for example:

[0159] The cell density of the cell suspension obtained in (i) or (ii) is adjusted to 8×10.sup.6 cells/mL and the cells are transferred to a roller bottle (with vent cap) with the addition of penicillin-streptomycin (1:000). The roller bottles are incubated upright in a shaking incubator at 27° C. and 120 rpm for 4 days. Then the cells are separated and removed by centrifugation (4,400×g, 20 min). The cell supernatant is first concentrated by tangential flow filtration (Vivaflow 200, 10,000 MWCO Hydrosart membrane) to 1/10 of the initial volume and then continuously diafiltered with the same membrane against ten times the volume of buffer W. PMSF (final concentration 1 mM) is added to inhibit proteases in the concentrated cell supernatant. To remove biotin from the concentrated cell supernatant, a BioLock biotin blocking solution (3 mL/L) is added, incubated for 15 min with stirring at room temperature and then centrifuged (10,000×g, 30 min, 4° C.). The supernatant is filtered with a 0.45 μm filter. For purification of the strep-tagged rubella spike constructs, the supernatant is loaded onto a Strep-Tactin Superflow column of an Äkta-pure chromatography system. After application, the supernatant is washed with 4 column volume buffer W and then eluted with 5 column volume buffer E. The obtained eluate is collected in several separate fractions. The fractions containing the rubella spike construct are combined and a protein concentration determination (e.g. according to Bradford) is performed.

EXAMPLE 2

Characterization of the Antigenic Properties of Rubella Spike Constructs using SDS-PAGE and Western Blot

[0160] Aliquots of the rubella spike constructs “short” and “long” purified according to example 1 are applied to SDS-PAGE (4-20% tris-glycine gels, article number TG 42010, Anamed Elektrophorese GmbH), separated under reducing conditions and then stained with Coomassie brilliant blue. The results are shown in FIG. 3. The comparison with the molecular weight marker bands (kDa) in lane 1 shows that rubella spike construct “long” (lane 2) and rubella spike construct “short” (lane 3) have molecular sizes of about 80 kDa. These values correspond to the molecule sizes that can be expected mathematically based on their amino acid sequence, namely 77 kDa for rubella spike construct “long” and 76 kDa for rubella spike construct “short”.

[0161] For the Western blot, the proteins from the SDS-PAGE are transferred to a PVDF membrane. After blocking free protein binding sites, the proteins on the membrane are incubated with a monoclonal antibody for rubella glycoprotein E1 and a monoclonal antibody for rubella glycoprotein E2. The antibodies used are for example (a) anti-rubella virus structural glycoprotein, monoclonal, mouse (MABR23-Ru6, ibt—immunological and biochemical testsystems GmbH), which reacts with glycoprotein E1, and (b) rubella virus E2 monoclonal antibody (D92G) (MA5-18255, Thermo Fisher Scientific), which reacts with glycoprotein E2. For visualization, incubation with an anti-mouse IgG antibody from goat (alkaline phosphatase conjugate) is subsequently carried out. The results of the Western blots are shown in FIG. 4 (A) and (B). The comparison with the molecular weight marker bands (kDa) in lane 1 shows that both the rubella spike construct “long” (lane 2) and the rubella spike construct “short” (lane 3) are recognized by the anti-rubella E1 and anti-rubella E2-antibodies. These results clearly show that epitopes of rubella glycoprotein E1 and rubella glycoprotein E2 are present in both the “long” and “short” constructs.

EXAMPLE 3

Investigation of Immunological Reactivity of Rubella Spike Constructs in Immunodiagnostic Test: Detection of Anti-Rubella IgM Antibodies in Human Sera

[0162] Two different test systems were used—a conventional ELISA system and a particle-based system based on flow cytometry and a fluorescence read-out of the conjugate signal. Both systems (ELISA and particle-based) are representative systems (antigen carrier—particle or plate) for detection methods currently used in rubella IgM and IgG serology.

[0163] For the investigation of immunological reactivity of rubella spike constructs with respect to patient IgM antibodies, different test runs were performed, each in comparison to the whole virus (as the antigen commonly used in state-of-the-art technology):

[0164] (a) A particle test with four different human sera (i.e. sera from four different patients) yielded the results shown graphically in FIG. 5. The mean fluorescence intensity (MFI) on the y-axis is a measure of the amount of detected patient IgM antibodies in each sample. The results demonstrate that the rubella spike constructs “short” and “long” according to the invention at least match the detection performance of the whole virus, in most cases they even exceed it.

[0165] (b) A particle test with different dilutions of a selected human serum positive for rubella IgM and with high titre gave the results shown graphically in FIG. 6. The mean fluorescence intensity (MFI) on the y-axis is a measure of the amount of detected patient IgM antibodies in the respective dilution of the sample (x-axis). The results demonstrate that with a human serum the rubella spike constructs “short” and “long” according to the invention show a very similar and even improved “titration behaviour” compared to the whole virus. This is an essential property of an antigen for a serological test.

[0166] (c) The positive-to-negative ratio (P/N) for the antigens rubella spike construct “long” and “short”, each in comparison to the whole virus, was determined by means of known methods using a particle-based in-house procedure and using different human sera positive and negative for rubella IgM. For each antigen or test setup the corresponding positive to negative ratio (P/N) was determined. The results are shown graphically in FIG. 7. They demonstrate that in a serological test for the detection of rubella IgM antibodies, the rubella spike constructs “short” and “long” according to the invention have a positive-to-negative ratio (P/N) that is very similar to that of the whole virus and in some cases superior to that of the whole virus. The positive-to-negative ratio (P/N) of a diagnostic test is, like the titration behaviour, an essential parameter of this test procedure and a measure of the so-called diagnostic performance: the higher the P/N ratio, the higher or better the diagnostic performance

[0167] (d) In an additional comparative test, the two different test systems ELISA and particle-based system were used to investigate the immunological reactivity of the rubella spike constructs “short” and “long” with respect to anti-rubella IgM antibodies in 36 pre-characterized (i.e. pre-analysed for anti-rubella IgM antibodies) human serum samples, 17 positive and 19 negative. A whole-virus coated ELISA was used as reference.

[0168] To compare and determine the cut-off values for the experimental setups, a ROC analysis was performed in Microsoft Excel using the software “Analyse-it” (Analyse-it Software, Ltd., UK). The test results are shown in tabular and graphical form in FIG. 8A and FIG. 8B.

[0169] From FIG. 8A and FIG. 8B it can be seen that the new rubella spike construct antigens “short” and “long” can be successfully used for the serological differentiation of rubella IgM positive and rubella IgM negative patient samples in the ELISA setup as well as in the particle-based test. With both rubella spike construct antigens (“short” and/or “long”) a clear differentiation of positive and negative samples from/in different donor collectives (panel) is possible. In comparison to the reference test (ELISA), sensitivities and specificities of 100% were achieved with the rubella spike construct “long” on both test platforms. With the rubella spike construct “short”, sensitivities and specificities of 100% were also achieved in the ELISA. In the particle-based test, the sensitivity was 94.1% and the specificities were again 100%.

EXAMPLE 4

Investigation of Immunological Reactivity of Rubella Spike Constructs in Immunodiagnostic Test: Detection of Anti-Rubella IgG Antibodies in Human Sera

[0170] As in example 3, two different test systems were used - a conventional ELISA system and a particle-based system based on flow cytometry and on a fluorescence read-out of the conjugate signal.

[0171] For the investigation of the immunological reactivity of the rubella spike constructs “short” and “long” with respect to anti-rubella IgG antibodies, 44 pre-characterized (i.e. pre-analysed for anti-rubella IgG antibodies) human serum samples, 21 positive and 23 negative, were tested on both platforms against a commercial reference ELISA. The reference ELISA was coated with whole virus.

[0172] To compare and determine the cut-off values for the experimental setups, a ROC analysis was performed in Microsoft Excel using the software “Analyse-it” (Analyse-it Software, Ltd., UK). The test results are shown in tabular and graphical form in FIG. 9A and FIG. 9B.

[0173] FIG. 9A and FIG. 9B show that the new rubella spike construct antigens “short” and “long” can be successfully used for the serological differentiation of Rubella IgG positive and Rubella IgG negative patient samples in both ELISA setup and particle-based assays. With both rubella spike construct antigens (“short” and/or “long”) a clear differentiation of positive and negative samples from/in different donor collectives (panel) is possible. In comparison to the reference test (ELISA), specificities of 100% were achieved with both rubella spike constructs “long” and “short” on both test platforms. The sensitivities of both constructs in the ELISA were also 100% in each case, in the particle-based test the sensitivity of both constructs was also 95.2% in each case.

Non-Patent Literature Cited

[0174] Battisti et al., 2012. Cryo-Electron Tomography of rubella Virus, J Virol. 2012 October; 86(20): 11078-11085.

[0175] DuBois et al., 2012. Functional and evolutionary insight from the crystal structure of rubella virus protein E1. Nature. 2013 Jan. 24; 493(7433): 552-6.

[0176] Hobman et al., 1993: The rubella virus E2 and E1 spike glycoproteins are targeted to the Golgi complex. J. Cell Biol. 121: 269-281 (1993).

[0177] Hobman et al., 2007. Fields Virology Vol. 1 (ed. D. M Knipe) 1069-1100 (Lippincott Williams & Wilkins, 2007).

[0178] Katow et al., 1988. Low pH-induced conformational change of rubella virus envelope proteins. J Gen Virol 1988; 69(pt 11): 2797-2807.

[0179] Perrenoud G. et al. 2004: A recombinant rubella virus E1 glycoprotein as a rubella vaccine candidate. Vaccine 2004; 23(4): 480-8.

[0180] Prasad et al., 2017. Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus. PLoS Pathog. 2017 Jun. 2; 13(6).

[0181] Schneider, I., 1972. Cell lines derived from late embryonic stages of Drosophila melanogaster. Journal of Embryology and Experimental Morphology 27 (2): 353-365. http://www.ncbi.nlm.nih.gov/pubmed/4625067.

[0182] Seppanen et al. 1991: Diagnostic potential of baculovirus-expressed rubella virus envelope proteins. Clin. Microbiol, 1991,1877-1882.

[0183] Waxham et al., 1985. Detailed immunologic analysis of the structural polypeptides of rubella virus using monoclonal antibodies. Virology 1985; 143:153-165.