Modified VP1-capsid protein of parvovirus B19

Abstract

The invention relates to a modified VP1-capsid protein of parvovirus B19 having a reduced phospholipase A2-like enzyme activity as compared to the wild type VP1-capsid protein of parvovirus B19 having the amino acid sequence of SeqID 1.

Claims

1. A pharmaceutical composition comprising a modified VP1-capsid protein of parvovirus B19, wherein a wild type sequence of the VP-1 capsid protein has been modified to contain amino acid substitutions at positions corresponding to histidine 153 of SEQ ID NO:1, tyrosine 157 of SEQ ID NO:1, lysine 162 of SEQ ID NO:1, and tyrosine 168 of SEQ ID NO:1, wherein said substitutions are histidine 153 to alanine, tyrosine 157 to phenylalanine, lysine 162 to leucine, and tyrosine 168 to phenylalanine, and wherein said modified VP1-capsid protein has a reduced phospholipase A2 enzyme activity as compared to the wild type VP1-capsid protein.

2. The pharmaceutical composition of claim 1, further comprising a carrier or adjuvant suitable for vaccination purposes.

3. The pharmaceutical composition of claim 1, further comprising an immunostimulatory substance selected from the group consisting of an immunostimulatory deoxynucleotide (ODN), a peptide containing at least two LysLeuLys motifs, a neuroactive compound, alum, Freund's complete adjuvant, and Freund's incomplete adjuvant.

4. The pharmaceutical composition of claim 1, further comprising a polycationic peptide.

5. The pharmaceutical composition of claim 1, further comprising a VP2-capsid protein.

6. The pharmaceutical composition of claim 5, wherein the modified VP1-capsid protein is fused to the VP2-capsid protein.

7. A pharmaceutical composition comprising (a) an adjuvant, and (b) a modified VP1-capsid protein of parvovirus B19, wherein a wild type sequence of the VP1-capsid protein of parvovirus B19 has been modified to contain an amino acid substitution at a position corresponding to tyrosine 157 of SEQ ID NO: 1, lysine 162 of SEQ ID NO: 1, or tyrosine 168 of SEQ ID NO: 1, wherein said substitution is selected from the group consisting of tyrosine 157 to phenylalanine, lysine 162 to leucine, and tyrosine 168 to phenylalanine; and wherein said modified VP1-capsid protein has a reduced phospholipase A enzyme activity as compared to the wild type VP1-capsid protein.

8. The pharmaceutical composition of claim 7, wherein the substitution is lysine 162 to leucine.

9. The pharmaceutical composition of claim 7, wherein the substitution is tyrosine 168 to phenylalanine.

10. The pharmaceutical composition of claim 7, wherein the substitution is tyrosine 157 to phenylalanine.

11. The pharmaceutical composition of claim 7, wherein said adjuvant is an immunostimulatory substance selected from the group consisting of an immunostimulatory deoxynucleotide (ODN), a peptide containing at least two LysLeuLys motifs, a neuroactive compound, alum, Freund's complete adjuvant, and Freund's incomplete adjuvant.

12. The pharmaceutical composition of claim 7, further comprising a polycationic peptide.

13. The pharmaceutical composition of claim 7, further comprising a VP2-capsid protein.

14. The pharmaceutical composition of claim 13, wherein the modified VP1-capsid protein is fused to the VP2-capsid protein.

15. The pharmaceutical composition of claim 1, wherein said wild type VP1-capsid protein has a sequence selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

16. The pharmaceutical composition of claim 7 where said wild type VP1-capsid protein has a sequence selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

Description

(1) The present invention is further illustrated by the following examples and the figures, from which further features, embodiments and advantages may be taken. It is to be understood that the present examples are given by way of illustration only and not by way of limitation of the disclosure.

(2) FIG. 1 shows an immunological comparison of wt and mutant protein

(3) FIG. 2 shows an alignment of Parvovirus VP1 protein. GENOTYP 1=SEQ ID NO: 1; GENOTYP 2=SEQ ID NO: 2; GENOTYP 3=SEQ ID NO: 3.

Example 1

(4) The production of VP1/VP2 antigens without enzymatic activity can be achieved by alteration of the residues that are part of the active centre by site-directed mutagenesis. Despite the fact that the overall size of cellular enzymes with Ca.sup.2+-dependent phospholipase A2 activity and the viral VP1-unique region are different, alignments comparing the amino acid sequences revealed a number of conserved residues in the region that represents the active centre of the enzyme. Conserved amino acids were observed at the following positions:

(5) Residue 153: histidine; residue 157: tyrosine; residue 162: lysine; residue 168: tyrosine; residue 195: aspartic acid. The respective amino acids have been described as parts of the catalytic triad of bovine pancreatic phospholipase A2 and to be involved in substrate orientation and specificity. Therefore the alteration of these residues in the VP1-unique region by site-directed mutagenesis was performed using polymerase chain reaction with mutated primer and overlap extension as initially described by Ho and coworkers (56). As shown in table 2 the phospholipase A2-like activity of the VP1-unique region could be reduced by exchanging both the tyrosine 157 and 168 to phenylalanine and by the alteration of lysine 162 to leucine. However the exchange of aspartic acid 195 to alanine led to an unexpected enhancement of the activity of the viral enzyme indicating distinct differences between viral and cellular phospholipase A2 enzymes. An almost total destruction of the enzymatic activity could only be achieved by the alteration of histidine 153 to alanine. It can be concluded that this amino acid residue is part of the active centre and most important for the enzymatic activity of the VP1-unique region. Its alteration is associated with the complete destruction of the viral phospholipase A2-like activity.

(6) TABLE-US-00001 TABLE 1 Autoimmune diseases that are reported in association to parvovirus B19 infection. Involved organs Disease Joints Arthralgias Arthritis Monoarthritis Oligoarthritis Polyarthritis Rheumatoid arthritis Juvenile idiopathic arthritis Connective tissue/vessels Systemic lupus erythematosus (SLE) Vasculitis Leukoclastic vasculitis Purpura Henlein-Schoenoch Papular-purpuric gloves-and-socks syndrome (PPGSS) Kawasaki disease? Giant cell arteritis (GCA) Polyarteritis nodosa Wegener's granulomatosis Dermatomyositis Blood cells Autoimmune neutropenia Autoimmune thrombocytpenia Idiopathic thrombocytopenic purpura (ITP) Autoimmune hemolytic anemia Virus-associated hemophagocytic syndrome (VAHS)

(7) TABLE-US-00002 TABLE 2 Enzmyatic phopholipase A2-like activity in the VP1- unique region of parvovirus B19 and variants constructed by site-directed mutagenesis. enzyme Position altered by acitivty site-directed mutagenesis (%) wildtype, genotype 1 100 active center mutants histidine 153 .fwdarw. alanine 0 tyrosine 157 .fwdarw. phenylalanine 10 lysine 162 .fwdarw. leucine 61 tyrosine 168 .fwdarw. phenylalanine 54 aspartic acid 195 .fwdarw. alanine 204 not-active center mutants leucine 76 .fwdarw. glutamine, phenylalanine 81 .fwdarw. alanine 80 isoleucine 66 .fwdarw. leucine, leucine 70 .fwdarw. glutamine 144 leucine 59 .fwdarw. glutamine, leucine 62 .fwdarw. glutamine 143 variations in non-conserved regions parvovirus B19, VP1-unique region genotype 2/strain Berlin 70 ala18.fwdarw.asp, gln21.fwdarw.lys, asn68.fwdarw.ser, asn72.fwdarw.asp, ser73.fwdarw.thr, ser96.fwdarw.pro, ala101.fwdarw.thr, val123.fwdarw.ile, val192.fwdarw.ala parvovirus B19, VP1-unique region, genotype 3/strain V9 59 lys4.fwdarw.thr, ser5.fwdarw.thr, gly6.fwdarw.asn, asp12.fwdarw.ser, lys17.fwdarw.gln, ala18.fwdarw.asp, gln21.fwdarw.lys, glu25.fwdarw.gln, val30.fwdarw.ala, asn68.fwdarw.ser, ser98.fwdarw.asn, his100.fwdarw.ser, val123.fwdarw.ile, ser144.fwdarw.asn, val192.fwdarw.ala

Example 2

Comparison Study for the Immunogenicity Between Wild Type and Mutant of VP1 Proteins

(8) Inoculation of Mice.

(9) Groups of 5 female Balc/C mice were inoculated with 50 ug of purified preparations of the VP1-unique region/wildtype and the VP1-unique region/H153A in emulsion with complete's Freund's Adjuvans. Retrobulbar blood samples were taken at days 0, 3, 7, 10, 14, 18 and 28 after inoculation. The sera were tested for the presence of IgG antibodies against the VP1-unique region/wildtype in ELISA assays.

(10) Protein Production.

(11) The sequences encoding the VP1-unique region/wildtype and the VP1-unique region/H153A were cloned into the T7-expression vector pET21a_int in fusion with an intein and a chitin-binding domain as described previously (Dorsch et al., 2001, Dorsch et al., 2002). The constructs were introduced into the E. coli strain BL21. Bacteria were inoculated with LB (luria broth) medium containing 100 g/ml ampicillin and incubated at 37 C. Expression of the recombinant protein was induced by addition of 1 mM IPTG for at least 3 h of culture. The bacteria were harvested by centrifugation, resuspended in 30 ml 20 mM HEPES, 1 mM EDTA, 100 mM NaCl, pH8.5 and lysed by the use of a French Press. The debris was pelleted at 10000 g. The supernatant was loaded on a chitin column (NEB) using FPLC-system (Pharmacia Biosystems, Freiburg). The column was washed with 2 volumes of 20 mM HEPES, 1 mM EDTA, 100 mM NaCl, pH8.5, 8 volumes of 20 mM HEPES, 1 mM EDTA, 2 mM NaCl, pH8.5 and 2 volumes of 20 mM HEPES, 1 mM EDTA, 100 mM NaCl, pH8.5. Afterwards the protein was eluted using 3 volumes of 50 mM DTT in buffer 20 mM HEPES, 1 mM EDTA, 100 mM NaCl, pH8.5. Fractions were tested for the recombinant proteins by SDS-PAGE and silver staining. Positive fractions were unified and concentrated by using a Centriplus concentrator (3 kD exclusion volume; Amicon, Beverly, USA). The protein concentration was determined after dialysis against PBS (0.9 mM KH.sub.2PO.sub.4, 8.0 mM Na.sub.2HPO.sub.212H.sub.2O, 2.7 mM KCl, 137 mM NaCl) using a Bradford assay (Bio Rad Laboratories, Hercules, USA).

(12) ELISA-Assay.

(13) Microtiter plates (Maxisorb, Nunc, Wiesbaden, FRG) were coated over night with purified protein (VP1-unique region/wildtype, 100 ng/well) in 0.2 M NaCO.sub.3 buffer, pH 9.2 containing 0.15 M NaCl. Sera were used in dilutions of 1:100 in PBS/0.5% Tween-20 and IgG-antibodies were detected using HRP-coupled polyclonal rabbit anti-mouse IgG as second antibodies (dilution 1:5000 in PBS/0.5% Tween-20, Dako, Hamburg FRG) and TMB as substrate, the optical density was determined at 450 nm.

(14) Results

(15) Starting from day 7 after inoculation IgG antibodies directed against the VP1-unique region/wildtype were detectable in mice that had been inoculated both with purified preparations of the VP1-unique region/wildtype and the VP1-unique region/H153A (FIG. 1). The amounts of antibodies continually increased until day 28 after inoculation. Differences in the reactivity of mice inoculated with either the VP1-unique region/wildtype or the VP1-unique region/H153A could not be observed. These results indicate that both proteins are highly antigenic. Antibodies induced against the variant VP1-unique region/H153A have the capacity to bind to the VP1-unique region/wildtype which had used as antigen in the ELISA indicating an high degree of cross reactivity. Mice that were inoculated with PBS in emulsion with complete Freund's adjuvans did not develop any significant amounts of VP1-specific antibodies.

(16) The wildtype VP1-unique region antigen and the mutant antigen (His153Ala) were inoculated in mice. VP1-specific antibody production was analysed by ELISA. No differences were observed in the capacity of both antigens to elicit VP1-specific antibody production. Antibodies against the mutant antigen His153Ala were similarly active to bind to the wildtype VP1-unique region and vice versa. This indicates that the mutant His153Ala variant of the VP1-unique region of parvovirus B19 has a comparable capacity to elicit the production of antibodies as the wildtype protein domain. Since the main neutralising epitopes are known to be located in protein parts different from the active centre of the viral phospholipase A2-like enzyme and were not affects by any of the introduced mutations effects on the protein's immunogenicity are unlikely (57).

(17) The combination of both approachesthe production VP1/VP2-capsids in recombinant S. cerevisiae and the destruction of the phospholipase A2-like activityhas the potential to produce a vaccine that allows the prevention of parvovirus B19 infection without a reduced number of side effects due to elevated leukotriene and prostaglandin production and without the dangerous potential to induce autoimmune reactions that may result in life-long rheumatic disease.

(18) Legend

(19) FIG. 1. The development of IgG-antibodies against the VP1-unique region/wildtype. Groups of 5 mice were inoculated with either the VP1-unique region/wildtype, the VP1-unique region/H153A or PBS. Serum samples taken at days after inoculation as indicated were tested in ELISA using the VP1-unique region as antigen. Average values obtained from testing the individual samples of 5 mice were determined in each case.

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