RECOMBINANT PROTEIN ANTIGENS WITH INCREASED IMMUNOGENICITY CONTAINING THE PEPTIDE REPRESENTED BY THE SEQUENCE VOGEESNDK

Description

IRI.3 NOTARBARTOLO & GERVASI 000071 '23PPP92 FIREl!ZE / MiNV£NZI0NI Description of the industrial invention entitled: ''Recombinant protein antigens with increased immunogenicity containing the peptide represented by the sequence VQGEESNDK IN THE NAME OF: Istituto Ricerche Immunobiologiche Siena Srl WITH HEADQUARTERS IN: Siena DESIGNATED INVENTORS: CHIARA Paolo, VILLA Luigi Deposit with the no. Field of invention The present invention relates to new recombinant protein antigens represented by the fusion of a recombinant protein antigen, or a fragment thereof, with the peptide of sequence VQGEESNDK (i.e.: Val-Gln-Gly-Glu-GluSer—Asn-Asp—Lys). State of the art It is well known that many recombinant protein antigens exhibit a poor immunogenic response, making them less effective for in vivo application. Therefore, numerous studies are underway to enhance the immune response of these antigens, especially for the preparation of vaccines based on genetically engineered proteins. Detailed description of the invention It has now been surprisingly found that by inserting the VQGEESNDK sequence into the proper sequence of recombinant protein antigens, or fragments thereof, using common genetic engineering methods, new TRI. 3 NOTARBARTOLO & GERVASI f Λ antigens whose in vivo immunogenicity is significantly increased and are therefore indicated for the preparation of vaccines useful for both oral and parenteral administration. In this way, for example, the immune response to the following classes of antigens can be increased: Bacterial toxins: pertussis toxin, tetanus toxin, diphtheria toxin, toxins from Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Shigella enterolytica, Bacillus anthracis, Clostridium botulinus, 130 kDa cytotoxin from Helicobacter pylori). Low immunogenic proteins: Ferritin, proteins of pathogenic viruses such as Hepatitis A, B, C, Polio, Papilloma, Influenzae. Protein carriers of glycoconjugates: CRM-197 conjugates with polysaccharides from meningococcus groups A, B, C (MenA, MenB, MenC respectively), with polysaccharides from Pneumococcus, from Haemophilus influenzae. Surface antigens of pathogenic or parasitic infectious agents · .* MOMP of Chlamydia, circumsporozyme surface proteins of malaria plasmodium. In particular, it should be noted that no adjuvants of any kind are currently available for oral vaccines, while recombinant antigens containing the sequence described demonstrate a significantly superior activity. These and other objects of the present invention will be more and better understood in light of the examples described below. IRI.3 NOTARBARTOLO & GERVASI and are reported here for illustrative but not limiting purposes. Antigone consists of the H chain of Ferritin into which the sequence VQGEESNDK has been inserted. A plasmid vector has been constructed that allows the synthesis of large quantities of the H chain of human ferritin. The plasmid was obtained by inserting the nucleotide sequence encoding ferritin into an expression vector characterized by the presence of the pL promoter of bacteriophage lambda and the ribosome attachment site (RBS) taken from the gene encoding the replicase of bacteriophage MS2. The coding sequence for the H chain of human ferritin, which is known to consist of 5 helices (A, B, G, D, E) linked together by loops, has been modified, by site-directed mutagenesis, to allow the introduction of a unique site for the restriction enzyme Smal in the region of the nucleotide sequence encoding the peptide that connects helix D and helix E in the three-dimensional structure of ferritin. The 5'-TGG GAG GGC CCG GGT CTG GCT TGG - 3' oligonucleotide used in the mutagenesis experiment replaces the codon coding for Jun 162 (GAA) with a codon coding for Gly (GGG) and at the same time introduces the aforementioned Smal site. This feature allows to easily introduce the desired oligonucleotide at this Smal site. IRI.3 NOTARBARTOLO & GERVASI (which is unique throughout the plasmid) and thus construct modified genes encoding hybrid proteins. Once expressed in a host bacterium, these genes drive the synthesis of ferritin molecules in which the oligopeptide encoded by the oligonucleotide is appropriately inserted covalently, as mentioned above, into the loop region connecting helix D to helix E. This particular region was preferred as it is certainly exposed on the outer surface of the Ferritin molecule. Using the technology described above, we inserted two oligonucleotides with complementary sequences into the Smal site of the plasmid as follows: GGG CCC CAA GTC CCA CTT CTT TCA TTG CTA TTT CCC GGG GTT CAG GGT GAA GAA AGT AAC GAT AAA, encoding the VQGEESNDK peptide. The structure of the obtained plasmid was verified by determining the nucleotide sequence using the Sanger method and it was possible to verify that, following the induction of the pL promoter activity, the expression of a protein with properties similar to those of human ferritin was obtained but with a slightly higher molecular weight (determined by electrophoresis on acrylamide gel under denaturing conditions). It has also been verified that the hybrid protein, IRI.3 NOTARBARTOLO & GERVASI, unlike natural ferritin, is able to interact with a monoclonal antibody specific for the oligopeptide of the sequence VQGEESNDK. Use of the Ferritin-VQGEESNDK hybrid protein It has been found that the immune response against Ferritin is increased when the above-described hybrid protein is used as an immunogen, administered either parenterally or orally. Parenteral immunization Groups of 3 8-week-old male C3H / Hej mice received ip on day 0 saline alone or containing 25 or 50 μg / kg of wild type ferritin (FerWT) or the hybrid protein containing the sequence VQGEESNDK (FerP20). After 4 days the spleens were tested in the Jerne test using FerWT covalently conjugated to SRBC by the Chromium Chloride method. Briefly, 350 µl of SRBC (sheep red blood cells) suspension is incubated for 5 minutes at room temperature with 200 µl of saline containing FerWT (125 µg / ml) in the presence of 50 µl of saline containing 6 mg / ml of CrClg. After washing with saline, the SRBC with FerWT conjugated are incubated between two slides for 45 minutes at 37°C with an appropriate dilution of a spleen cell suspension taken from the immunized mouse, in the presence of guinea pig serum. The FerWT-specific antibodies will bind to the antigen. IRI .3 NOTARBARTOLO & GERVASI conjugated to SRBC and the complement fixed by the secreted Ig will cause the lysis of the red blood cells. With an appropriate dilution of the spleen cells it will therefore be possible to observe the hemolysis plaques (PCF) well isolated from each other; each plaque corresponds to a plasma cell secreting specific Ig and it will therefore be possible to trace the number of PFC / spleen [see also Selected methods in cellular immunology B.Mishell and SMShig, Freeman and Co. USA (1980)], the data obtained are reported in TABLE I. TABLE I PFC anti-Fer / spleen immunization PFC anti-Fer / spleen total specific m ± sem (total - BkG) sol. physiol. (BkG) 716 ± 45 FerWT 25 / *g / Kg ip 750 + 30 34 50 833 + 34 117 FerP20 25 >*g / Kg ip 883 + 61 167 50 1400 + 105 684 Groups of 3 mice of the C3H / HeJ strain received ip daily either saline alone or containing 50 μg / kg of FerWT or FerP20. After 10 days, serum IgG levels against FerWT were determined using the standard ELISA technique with anti-mouse IgG conjugated to alkaline phosphatase. Data are shown in TABLE -II. IRI.3 NOTARBARTOLO & GERVASI TABLE II Optical density measured at 415 nm 1 / Dilution FerP20 treatment saline FerWT 50 0.184 0.256 250 0.042 0.058 1250 0.021 0.027 6250 0.017 0.013 Oral immunization Groups of 3 C3H / HeJ mice were immunized orally with saline alone or containing 50, 100 or 200 µg / Kg of ferWT or FerP20. After 4 days, the anti-ferWT PCF / spleen were determined as described above, the results were reported in TABLE III, TABLE III treatment PFC alone. physiol. (BkG) FerWT 50 / *g / Kg FerWT 100 FerWT 200 ” FerP20 50 >ug / Kg FerP20 100 FerP20 200 anti FerWT / spleen PFC anti FerWT / spleen total specifiche (m ± sem) (total -BkG) 1208 ± 1318 ± 3212 1450 ± 64 242 1525 + 29 317 2033 ± 157 825 2567 + 180 1359 2775 ± 53 1567

Claims

IRI.3 NOTARBARTOLO & GERVASI CLAIMS 1. Recombinant protein antigens with increased immunogenicity containing the peptide represented by the sequence VQGEESNDK 2. Antigens according to claim 1 wherein the peptide of sequence VQGEESNDK is fused to proteins selected from the group consisting of: bacterial toxins, poorly immunogenic proteins, protein carriers of glycoconjugates, surface antigens of pathogenic infectious agents or parasites, or fragments thereof.

3. Antigens according to claim 2 wherein the bacterial toxins are selected from the group consisting of: pertussis toxin, tetanus toxin, diphtheria toxin, toxins of Vibrio cholerae, Escherichia coli, Pseudomonas Aeruginosa, Shigella Enterolytica, Bacillus Anthracis, Clostridium Botulinus, 130 kDa cytotoxin of Helicobacter pylori.

4. Antigens according to claim 2 wherein the poorly immunogenic proteins are selected from the group consisting of Ferritin, proteins of pathogenic viruses such as Hepatitis A, B, C, Polio, Papilloma Influenza.

5. Antigens according to claim 2 wherein the glycoconjugate protein carriers are selected from the group consisting of: CRM-197 conjugates with meningococcal polysaccharides of groups A, B, C; polysaccharides of Pneumococcus, Haemophilus influenzae. IRI.3 NOTARBARTOLO & GERVASI 6. Antigens according to claim 2 wherein the surface antigens of pathogens or parasites are selected from the group consisting of: MOMP of Chlamydia, circumsporozyme surface proteins of malaria plasmodium.

7. Use of the antigens according to claim 2 for the preparation of vaccines or immunizing agents.

8. Use of the VQGEESNDK sequence peptide as an adjuvant for enhancing the immunogenic effect of recombinant protein antigens.

9. A method for enhancing the immunogenic effect of recombinant protein antigens, wherein the peptide of the sequence VQGEESNDK is inserted into the recombinant protein antigen by genetic engineering methods.

10. Vaccines containing antigens according to claim 1 suitable for parenteral administration to the patient.

11. Vaccines containing antigens according to claim 1, suitable for oral administration to the patient. Florence, 23 March 1992, p. Istituto Ricerche Immunobiologico Siena. The Representative Dr. Livio Brighenti of NOTARBARTOLO & GERVASI