DE4426453C1 - New fusion protein contg. fragments of two cytomegalovirus antigens - Google Patents

Abstract

Recombinantly produced fusion protein (I) contains at least (a) a fragment from the C-terminal region of the tegument protein pp150 of cytomegalovirus (CMV) and (b) a fragment from another antigenic protein of human CMV. Also new are test kits for detecting antibodies (esp. IgM) against CMV that contain (I).

Description

Human infections with the human cytomegalovirus (HCMV) are widespread and usually go away asymptomatic during childhood.

Let cytomegalovirus (HCMV) -specific IgG antibodies itself as a marker for an expired or latent infection depending on the tested population in 50 to 90% of adults determine. Congenital infections, however, as well as acute Infections in immunosuppressed patients, in particular Transplant patients and those infected with HIV, too lead to a life-threatening illness.

For early and effective antiviral therapy ensure it is of great interest to the clinic determine whether there is an acute infection.

Various can be used to diagnose an acute HCMV infection Methods are used. Especially at Transplant patients in whom a constant Accompanying diagnostics is possible in the past Years of the pp65-specific antigenemia test as well as the  Polymerase chain reaction (PCR) due to its superior Sensitivity largely enforced. The previously known molecular diagnostic techniques for the detection of HCMV infections are described, for example, by S. Chou in Multidisciplinary Approach to Understanding Cytomegalovirus Disease, Proc. 4th Intl. CMV Workshop, Paris, editor S. Michelson and S. Plotkin, Amsterdam 1993, pp. 183-194, described.

An acute infection is diagnosed in the routine laboratory, e.g. B. in viral differential diagnosis or in the context of Pregnancy care, usually by proof of Pathogen in cell culture and serologically by the Detection of specific antibodies, especially in the ELISA. Such methods are described, for example, by Drew in "Diagnosis of Cytomegalovirus Infection", Reviews of Infectious Diseases, Vol. 10, Supplement 3, 1988, pp. 468-476, described.

As specific antigens for antibody detection currently usually more or less poorly cleaned or poorly characterized lysates from HCMV-infected Cell cultures used. This leads in particular to IgM detection on specificity or sensitivity problems.

EPA 87.111726.3 describes a structural phosphor protein (pp150) of the human cytomegalovirus opted for the detection of specific antibodies has proven significant.

IgG antibodies directed against HCMV show that an infection tion with the human cytomegalovirus at some point has that. By determining the titer of the IgM antibodies against HCMV, on the other hand, one can determine whether a fresh In infection with HCMV viruses is present or not. The answer This question is particularly relevant for the risk patients mentioned of great importance for pregnant women. At In such a test it is of particular importance that  the evidence is highly specific and highly sensitive. If at too many positive, but not the test the test has obtained confirmable results only low informative value. On the other hand, however, the test fresh HCMV infections with sufficient certainty show, but the number of false positives Results should be reduced as much as possible.

The DNA sequence of the cytomegalovirus is already known. Chee et al. disclose in their publication in Current Topics in Microbiology and Immunobiology, Vol. 154 (1990), Pp. 125-169, the locations of the DNA sequence and an analysis the coding sequences of the cytomegalovirus. These Literature is hereby incorporated by reference into the Disclosure content of the present application locked in.

The present invention was able to determine that the C-terminal region of the pp150 tegument protein a very sensitive antigen for the detection of IgM antibodies. Within the scope of the present invention this is a range from 40 to 60 Amino acids at the C-terminus of the pp150 protein. Since the Amino acid sequence of the pp150 protein is already known this is the area of amino acids 988 to 1048. Particularly preferred in the context of the present Invention becomes the area of the pp150 tegument protein that comprises amino acids 994 to 1048 and this range will In the present case referred to as ppl150 / 7/2.

This C-terminal region of the pp150 tegument protein will fused according to the invention to a region with high IgM reactivity, that of another antigen of the HCMV virus comes from. Other antigens are, for example, those as pp65, designated antigens pp71, pp28 or gp116 / 58. As in the frame antigen particularly suitable for the present invention the antigen p52 and especially the C-terminal region of the p52 antigen exposed.  

In the context of the present invention, preference is therefore given to other component of the fusion protein an area from the C-terminus of the p52 used, which is about 100 to 150 amino acids of the p52 antigen. Very particularly preferred in the frame of the present invention is the region from p52 that the Contains amino acids 297 to 433.

In the context of the present invention, it was found that the Tegument protein pp150 with sera from healthy blood donors showed a very high IgM reactivity, which made the value this antigen clearly for the detection of acute infections is reduced. A share of about 35% in positive Results in the IgM test in healthy, seropositive Blood donation is not realistic and shows that the whole pp150 antigen not for the detection of IgM antibodies suitable is.

It was also found that the C-terminal region of the p52 antigen only a positive reaction with a serum shows. If only one were to use p52 as an antigen the sensitivity of this antigen would not be sufficient.

The present invention therefore relates to recombinant produced fusion proteins that comprise at least one fragment the C-terminal region of the pp150 tegument protein Cytomegalovirus (HCMV) and at least one more Fragment from another antigenic protein derived from human Cytomegalovirus.

In a preferred embodiment, the other is antigenic protein of the fusion protein of which as p52 designated protein, it being particularly preferred embodiment around the C-terminal region of the p52 antigen. This area extends in one very particularly preferred embodiment of about Amino acid at position 283 to amino acid No. 433.  

The amino acid sequence of a particularly preferred fusion protein is shown in Fig. 2. For immunological tests, the amino acids can of course be replaced by amino acids which have the same function, without the effect of the fusion protein being changed. It is well known to the person skilled in the art that amino acid exchanges do not affect the immunological properties of a protein as long as the essential structure of the epitopes is not changed. The invention therefore also relates to those fusion proteins which have a homology of at least 80% and in a particularly preferred form of at least 90% to the fusion protein shown in FIG. 2. A homology of 80% means that 80 out of 100 amino acids are identical at the corresponding sites, whereas 20% of the amino acids can be exchanged.

The fusion proteins used according to the invention thus exist on the one hand from the C-terminal part of the pp150 and on the other hand from an area with high IgM reactivity of another Cytomegalovirus antigen. In addition, the Fusion proteins according to the invention still a short proportion have by the recombinant production of Fusion protein is conditioned. That share, in particular comes from the expression vector, but according to the invention shows in preferred embodiment not more than 25 amino acids on.

The present invention also relates to test kits which for the detection of antibodies against human cytomegalovirus are suitable and the at least one according to the invention Have fusion protein. There are several in diagnostics various test methods, such as Western blot, radio immunoassay and others known. As part of the present Invention are particularly preferred test kits that are to carry out the ELISA test (Enzyme Linked Immuno Sorbent Assay) are suitable. A coupling takes place in these tests to a fixed phase. The antigen, in  in this case the fusion protein, on a solid phase, especially bound to microtiter plates and the unspecific bonds are saturated. In these The sample to be examined is then usually microtiter plates Serum, introduced, incubated and washed. The to the firm Antibodies are then against the Antigen to be detected. These antibodies are used in usually detected with anti-human antibodies to which a display component is bound. As particularly suitable the horseradish peroxidase has proven to be the catalyzes a color reaction based on the test result can be read.

The fusion proteins according to the invention and the latter Test kits containing are used to detect antibodies used against HCMV, the sample to be examined with is brought together with a fusion protein. The antibody Fusion protein complex can then be used with a detection component can be detected. By the invention Fusion proteins are provided antigens that at least show part of the "additional sensitivity" of the pp150, but at the same time have a high specificity. With the Fusion proteins according to the invention will therefore only be one low IgM reactivity with sera from healthy blood donors receive. So it is with the help of the invention Fusion proteins possible, a test for IgM antibodies perform with a sufficient sensitivity proves whether there is an acute infection and who at the same time the appearance of false positive results significantly reduced, if not excluded.

Fig. 1 shows the primers used for the cloning of the autologous fusion protein with the designation 52/3 | 105/7/2 for the polymerase chain reaction.

Fig. 2 shows the entire DNA sequence of the construct described in Example 1 and the corresponding amino acid sequence of the resulting fusion protein.

Fig. 3 shows the photograph of an SDS-polyacrylamide gel, which demonstrates the expression and purification of the recombinant fusion protein 52/3 | 150/7/2.

Fig. 4 shows the IgM courses of two patients (upper and lower figure), in whom in the IgM ELISA one C-terminal fragment of pp150 (amino acid 862-1048) and one the C-terminal fragment of p52 (amino acid 297-433) was used. The fusion protein 52/3 | 150/7/2 according to the invention was also used. The hatched columns labeled AG represent the antigenemia values determined by other methods. An acute infection could be observed at these times. Fig. 4 clearly shows that a clear IgM detection is possible with the fusion protein according to the invention, which, for example, is not possible with fragment 52/3 alone.

Fig. 5 shows Table 1, in which the IgM reactivity of different recombinant antigens is compared on the one hand with the sera of healthy seronegative blood donors and on the other hand with the sera of healthy seropositive blood donors. The fragment portion derived from the tegument protein pp150 (amino acid 862-1048 ≅ 150/7) has a high, obviously not disease-related IgM reactivity. When using the fusion protein according to the invention (52/3 | 105/7/2) the number of IgM-positive sera was considerably reduced.

Fig. 6 shows in Table 2 the OD values in the IgM ELISA with sera from various immunocompetent patients with acute HCMV infection. In comparison to the recombinant antigen p52 / 3 , the fusion protein according to the invention shows a significantly improved sensitivity.

example 1 Cloning of the autologous fusion protein 52/3 | 150/7/2

It is an autologous fusion protein that the complete range of 52/3 and the 54 C-terminal Combined amino acids of 150/7.

• 1. Starting from the clone puc8 / PCC150 / 7, which contains the partial sequence of pp150, which codes for the AS 862-1048, the partial fragment PCC150 / 7/2 was PCR amplified using the primers PCC15012.seq and PCC15013 .seq ( Fig. 1). In addition to the region complementary to pp150, both primers have overhangs which contain certain unique restriction sites, ie PCC15012.seq: EcoRI, PCC15013.seq: BamHI. These interfaces enable directional cloning of the amplificate.

The PCR amplification was carried out in a total volume of 100 µl consisting of 10 µl reaction buffer (Perkin-Elmer Cetus), 200 µM of the 4 deoxynucleotides, 0.5 µM of both PCR primers, 50-100 ng of the starting DNA and 2.5 units of the AmpliTaq DNA polymerase (Perkin-Elmer Cetus). The Amplification was done in the Perkin-Elmer Cetus DNA Thermal Cycler carried out in 25 cycles under the following conditions: 1 min - 55 ° C, 1 min - 72 ° C, 1 min - 94 ° C. The PCR reaction approach was in a submarine agarose gel chamber in a 1.2% Agarose gel (Ultra Pure Agarose, BRL) containing 0.5 µg / ml Ethidium bromide contains using a TBE running buffer (0.089 M Tris / Borate, 0.002 M EDTA) electrophoretic separated. The amplified DNA fragment was then used a UV lamp at 364 nm and the Gel area containing the corresponding band with a Cut out the scalpel. Elution of the DNA from the Gel fragment was carried out using a Biotrap chamber (Schleicher & Schüll) according to the manufacturer's instructions. That  added an additional purification of the DNA with a Elutip D-pillar (Schleicher & Schüll) according to the Instructions for use from the manufacturer.

The DNA fragment thus purified and dried was in Dissolved 80 µl distilled water. After adding 10 µl NEBuffer 4 (New England Biolabs) BamHI / EcoRI digestion was carried out by adding of 100 U each of both enzymes. After 2 hours there was one Phenol extraction followed by ethanol precipitation. 100 ng of the DNA fragment prepared in this way were included 200 ng BamHI / EcoRI-treated DNA of the standard vector puc8 ligated for 16h at 4 ° C and then E.coli JM109 transformed. The plating of the The transformation approach was carried out with ampicillin (50 µg / ml) and X-Gal (30 µg / ml) added agar plates. white Colonies were inoculated with ampicillin in 3 ml LB medium and incubated for 12-16h at 37 ° C with shaking. To The plasmid DNA was isolated using an EcoRI / BamHI Restriction digestion and electrophoresis in an agarose gel. A Clone, in which an additional DNA fragment of the expected size, was named puc8 / PCC150 / 7/2 and was used for further cloning.

• 2. Starting from the clone puc8 / PCC52 / 3 - the cloned fragment encodes AS 297-433 from p52 - PCR amplification carried out using the primers PCC525.seq and PCC526.seq and the later cloning of the amplified fragment as above described. Because of the overhangs of the two here used primer has the amplified fragment at the 5 'end a BamHI and at the 3'-end each a BglII and EcoRI interface. The corresponding clone was named puc8 / PCC52 / 3F designated.
• 3. The DNA of the clone puc8 / PCC150 / 7/2 was used for restriction digestion with BamHI and EcoRI. The released fragment of approx. 160 bp was isolated with the aid of agarose electrophoresis and eluted from the cut-out gel piece as described above. Approx. 50 ng of this DNA fragment were then ligated with 200 ng of the vector puc8 / PCC52 / 3F, which had been opened with BglII and EcoRI and also purified by electrophoresis. This was followed by transformation with E.coli JM109 and plating on agar plates containing ampicillin. Isolated colonies were inoculated with ampicillin in 3 ml LB medium and incubated at 37 ° C. for 12-16 h while shaking. After isolation of the plasmid DNA, a BamHI / EcoRI restriction digest was carried out with subsequent electrophoresis in an agarose gel. A clone with an insert of the expected size was designated puc8 / 52/3 150/7/2 and was used for the further experiments. This procedure takes advantage of the fact that the recognition sequences of BamHI and BglII have the same overhangs, which enables ligation. However, both interfaces are lost after ligation. The transition from 52 / 3F to 150/7/2 is chosen so that translation is possible in the same reading frame. Fig. 2 shows the entire DNA sequence of the construct described above and the corresponding AS sequence of the resulting autologous fusion protein.
• 4. To increase the expression of the autologous fusion protein ensure, the corresponding was recloned DNA fragment in the vector pET5c, which is the strong T7 promoter of the T7 bacteriophage gene 10. Behind it this vector has a ribosome binding site and a Start codon at an appropriate distance. Behind the start codon is a reading frame of 11 amino acids of the 10 des gene Bacteriophage T7 and a BamHI and an EcoRI interface. The reading frame of the BamHI interface is correct with that of the autologous fusion protein described above match. 100 ng of the EcoRI and BamHI restriction digest from puc8 / 52/3 | 150/7/2 released DNA fragment were with 200 ng of the same Restriction enzymes opened vector for 16h at 4 ° C ligated. This was followed by a transformation with E.coli JM109 and plating on ampicillin-containing plates. Colonies were isolated in 3 ml LB medium with ampicillin  inoculated and incubated for 16 hours with shaking at 37 ° C. To The BamHI / EcoRI restriction digest was used to isolate the plasmid DNA. A clone with an insert of the expected size was designated as pET5c / 52/3 | 150/7/2. Around to ensure the expression of the recombinant protein, was expression DNA in the chloramphenicol-resistant expression strain BL21 (DE3) pLysS transformed. One of the resulting clones was created for the used further work.

Example 2 Expression of recombinant fusion protein 52/3 | 150/7/2

• 1. Starting from a plate colony of the clone pET5c / 52/3 | 150/7/2 in BL21 (DE3) pLysS became a 15 ml Liquid culture - LB medium with ampicillin (Amp) and Chloramphenicol (CA) - at 37 ° C in a rotary shaker up to one optical density (600 nm) attracted from 1.8-2.0. The culture was then with glycerol (87%) to a final conc. of 15% (v / v) added, aliquoted in 0.1 ml portions and at -60 stored at -80 ° C until further use.
• 2. A frozen aliquot of the glycerol culture quickly became thawed and pipetted into 150 ml LB / CA, AMP medium. The This overnight culture was cultivated in a 1 liter Erlenmeyer flask (EMK) in a rotary shaker at 28 ° C and 100 rpm for 16h.
• 3. The 6 l main culture was grown in 12 Parallel batches of 0.5 l in 2 l EMF with baffles. The Medium (LB / CA, AMP) was preheated to 37 ° C. To Innoculation of the flasks with 10 ml of the preculture (1:51) the incubation took place at 37 ° C and 160 rpm Rotary shaker. The growth was continuous through Measurement of the OD tracked at 600 nm. With an OD of 0.6 expression of the recombinant antigen was added induced by IPTG to a final concentration of 1 mM.  
• 4. The harvest took place 3 hours after induction by centrifugation (6 × 1 L beakers, 4000 g, 0-4 ° C, 30 min). The well drained ones Bacterial pellets were resuspended in 200 ml ice-cold PBS and centrifuged again (2 × 250 ml beakers, 5000 g, 0-4 ° C, 10 min). The pellets were drained well again frozen and at -20 to -30 ° C until further Processing stored.
• 5. Before induction and before harvesting the bacteria were 1.5 ml aliquots of the bacterial suspension from selected Removed the flask, transferred to an Eppendorf tube and the Bacteria pelleted by centrifugation. The bacteria were then treated with SDS electrophoresis buffer and an aliquot of analysis in SDS electrophoresis Subjected to use of a 17.5% polyacrylamide gel. The samples taken before harvest showed compared to the an additional, strong sample taken before induction pronounced protein double band in the range of 25k.

Example 3 Disruption of the bacteria

The frozen bacterial pellets were thawed in 160 ml base buffer (Tris-HCl / 20mM / pH7.5) resuspended and then with a Teflon / Glass Potter homogenizer homogenized. The following additives were then added with stirring added: NP-40 (0.05%), PMSF (0.2 mM), Pefabloc (0.2 mM), EDTA (50 mM) and lysozyme (50 mg) to a total volume of 200 ml. The mixture was stirred vigorously for 60 min incubated at room temperature and then immediately on ice posed. All further steps were carried out on ice or chilled. After the incubation, glycerin (10%) and 2-mercaptoethanol (14 mM) added and the volume with basic buffer set to 280 ml. The lysis approach was then sonified (20 kHz, pulsating, 5 min,  Titanium trunk 3/4 ′ ′). Unsolubilized material has been removed Centrifugation removed (2 × 250 ml beakers, 27000 g, 30 min, 0-4 ° C). The pellets were discarded.

Example 4 Purification of the recombinant protein 52/3 | 150/7/2

• 1. The supernatant from Example 3 was stirred in an ice bath solid, finely ground ammonium sulfate up to a Concentration of 25% saturation slowly added. It closed incubation for 15 min with stirring. To Centrifugation (2 × 250 ml beakers, 27000 g, 30 min, 0-4 ° C) the pellets were discarded. The supernatant was again with Ammonium sulfate up to a concentration of 45% saturation offset and centrifuged as before. The supernatant was discarded. The pellets were placed in 25 ml base buffer, the additionally 2-mercaptoethanol (14 mM) and Pefabloc (0.1 mM) contains, resuspended, frozen and stored overnight at -20 to -30 ° C.
• 2. The protein fractionated with ammonium sulfate (25-45%) Solution was thawed and protein precipitated through Centrifugation (1 × 50 ml beaker, 40,000 g, 30 min, 0-4 ° C) away. The supernatant was over a Sephadex G-25 (coarse) column (volume at least 200 ml) chromatographed, where A (280 nm) and the conductivity measured in the flow become. Base buffer with 2-mercaptoethanol was used as the column buffer (1.4 mM) and Pefabloc (0.02 mM) were used. The protein in the exclusion volume was completely collected and immediately chromatographed on SP-Sepharose (Fast Flow). A column measuring 2.6 × 12 cm (60 ml) used. A (280 nm) and conductivity became continuous registered. The flow was 5 ml / min when column buffer was used Base buffer with 2-mercaptoethanol (1.4 mM) and Pefabloc (0.02 mM) was used. After sample order followed by 100 ml column buffer, became a linear NaCl gradient (dC / dV = 1 mM / ml, up to 300 mM) in column buffer. The  Eluate was collected in 10 ml fractions and frozen. The antigen-containing fractions were in the range between 100-200 mM NaCl. The detection was carried out by SDS-PA disc electrophoresis with subsequent Coomassie staining.
• 3. The fractions which were found to contain pure antigen (Coomassie gel) were thawed, combined, mixed with glycerol (20%) and concentrated by means of ultrafiltration (stirred cell, ice bath, membrane = Omega 30) to a final concentration of at least 2 mg / ml. The retentate was removed from the chamber and aliquoted, frozen and stored at -60 to -80 ° C. The course of cleaning is documented in Fig. 3.

Example 5 Reactivity of the purified recombinant antigen 52/3 | 150/7/2 in ELISA 1. Test execution

100 ng each of the purified recombinant antigen dissolved in 100 µl 0.01 M carbonate buffer pH 9.5 were in the Wells of microtiter plates (Nunc) are given and 16h in incubated in a grounded humid chamber. After adding 100 µl of a post-coating solution containing calf serum the incubation was continued for 2 more hours. The plates were then emptied and carefully knocked out. Then the plates for the actual test execution used or after drying in Vacuum cabinet and subsequent welding in Foil tube stored at -20 ° C until later use. The test cups of the ELISA plates filled with 100 µl 1:21 diluted serum, with taped with a plastic film and swimming for 1h at 40 ° C Incubated water bath. After washing three times in a bio-test ELISA Washer II was incubated with 100 µl of a anti-IgM, peroxidase-labeled, mouse monoclonal antibody (Janssen) for 30 min  40 ° C. After washing again, the bound ones Antibodies through a color reaction with 1,2-phenyldiamine as Chromogen made visible. The optical density of each Samples were taken at 495 nm (reference: 620 nm) on the Anthos HTII ELISA reader determined. All OD values were greater than 0.3 rated as a positive result.

2 results

The reactivity was checked with:

• - unselected sera from healthy blood donors with no evidence an acute HCMV infection. The division of these sera into HCMV positive / negative was done with an approved anti CMV IgG ELISA (Biotest)
• - selected sera of immunocompetent individuals with acute HCMV infection
• - selected courses of kidney transplant patients with acute HCMV infection.

Table 1 in Fig. 5 shows the IgM reactivity of the autologous fusion protein 52/3 | 150/7/2 with sera HCMV-seropositive (n = 54) or seronegative, healthy blood donor compared to the recombinant antigens 52/3 and 150/7, which were also expressed in pET5c and purified to homogeneity. While 150/7 showed a high, obviously not disease-related IgM reactivity (<32%) with HCMV-positive sera, the autologous fusion protein had the same low reactivity as 52/3 and thus a significantly better specificity than 150/7.

Table 2 in Fig. 6 shows the OD values in the IgM ELISA described above with 15 sera from immunocompetent people with acute HCMV infection. All sera were positive in the conventional IgM ELISA. The autologous fusion protein 52/3 | 150/7/2 showed a significantly improved sensitivity compared to the recombinant antigen 52/3, ie many of the sera with 52/3 negative sera showed OD values above the cut-off value with the autologous fusion protein from 0.3.

Fig. 4 shows the results of the recombinant proteins 52/3 | 150/7/2, 52/3 and 150/7 in the IgM ELISA described above with serum profiles of two transplant patients with acute primary HCMV infection. Both patients were HCMV seronegative before the transplant and received an organ (kidney) from a seropositive donor. The course of the acute infection was followed with the pp65-specific antigenemia test and the PCR. In both courses 52/3 showed no or only weak IgM reactivity, while 150/7 showed strong reactivity. The autologous fusion protein 52/3 | 150/7/2 showed a high IgM-specific seroreactivity in both patients, which correlated with the acute course of the disease.

Claims (8)

1. Recombinantly produced fusion protein, characterized in that it has at least one fragment from the C-terminal region of the tegument protein pp150 of the cytomegalovirus and at least one further fragment from another antigenic protein which is derived from the human cytomegalovirus. 2. Fusion protein according to claim 1, characterized in that that the other antigen, from the human cytomegalovirus derived protein is the p52 protein. 3. Fusion protein according to claim 2, characterized in that that the other antigen, from the human cytomegalovirus protein originating from the C-terminal region of the p52 protein comes from. 4. Fusion protein according to one of the preceding claims, characterized in that the fusion protein has an amino acid sequence which has a homology of at least 80% to the amino acid sequence shown in Fig. 2, Fig. 2 being part of this claim. 5. Fusion protein according to claim 1, characterized in that the fusion protein has the amino acid sequence shown in Fig. 2, Fig. 2 being part of this claim. 6. Fusion protein according to one of claims 1 to 5, characterized characterized in that the fusion protein is no more than Has 25 amino acids derived from a bacterial protein or the vector with the help of which the fusion protein is expressed. 7. test kit for the detection of antibodies, in particular IgM antibodies, against the cytomegalovirus, thereby characterized in that there is at least one fusion protein one of claims 1 to 6. 8. Test kit according to claim 7, characterized in that it is an ELISA test.