Peptide-simulator epitope protein gp120 of hiv-1

 

(57) Abstract:

The invention relates to biotechnology, genetic and protein engineering. The essence of the invention is to obtain by using phage display of peptides imitators antigenic determinants of a protein gp120 of human immunodeficiency virus type 1, HIV-1, recognizable neutralizing monoclonal antibody 2G12. The method of affinity selection of phage peptide libraries derived peptides that mimic the full-length glycoprotein gp120 binding neutralizing antibody 2G12. Peptides exposed on the surface of filamentous bacteriophages in the composition of the minor envelope protein pIII, each phage particle contains 5 copies of the same peptide. 4 Il.

The invention relates to biotechnology, genetic and protein engineering, specifically to obtain using phage display of peptides imitators antigenic determinants of a protein gp120 of human immunodeficiency virus type 1 (HIV-1), recognizable neutralizing monoclonal antibody 2G12.

One of the main problems in creating an effective vaccine against AIDS is the high antigenic variability of HIV. Intensive destruction of T-lymphocytes bearing which contributes to the emergence and selection of genetic variants of HIV-1 even within individual infectious process [1]. When using inactivated televizionnyh HIV vaccines there is also the risk of autoimmune processes due to mimicry patterns of viral protein gp120 under the structure of immunoglobulins [2].

The problem may help to use when creating a vaccine peptides that differ in amino acid sequence from the antigenic determinants of the virus, but retains the ability to bind to anti-viral antibodies. Such peptides are imitators of antigenic determinants. Peptides-simulators can be quite degenerate and have cross-reactivity with antibodies to different isolates of the virus. Degenerate peptides can have the property to induce the immunization antibodies, cross-reactive antigenic variants of the same virus (the phenomenon of molecular mimicry). Peptides that mimic neutralizing epitopes of the virus, apparently, will cause the production of protective antibodies and can be consequently used to create vaccines against HIV and other vysokopribylnyj pathogens. For example, it was shown that simulators HBsAg, electrovanne with subtype-specific serum when injected experimental animals with"ptx2">

A unique opportunity to obtain peptides imitators natural antigens provide a phage library of peptides, which includes all possible peptides of a given length, the exposed part of one of the surface proteins of filamentous bacteriophages. In the genome of each phage of the library encoded sequence only one of the variants of the peptides. Selection of peptides is carried out using specific antibodies specificity (upon receipt of vaccines that should be protective antibodies). While it is an interesting fact that monoclonal antibodies (MAB) from ragovoy peptide libraries are usually selected a number of peptides with different amino acid composition from the original antigen, but mimicking the binding.

A good example of peptide mimics of HIV-1 is the work of Keller and co-authors [4] , which shows that the peptides electrovanne of ragovoy peptide libraries for binding to the MAB 447-52D to the V3-loop of gp120 of the human immunodeficiency virus, can induce specific humoral immune response to gp120.

One of the most promising neutralizing MCA are 2G12 [5]. It was shown that monoclonal antibodies 2G12 protiv, activate the complement system by the classical pathway and induce antibody-dependent cellular cytotoxicity. None of the known antibody does not compete with for 2G12 binding protein gpl20. In accordance with this µa 2G12 belong to a unique group of antibodies, which currently µa 2G12 is the only representative of [6].

The MAB 2G12 in complex with other antibodies used for passive immunotherapy of AIDS patients in clinics in the USA and Europe [7]. In this regard, is a promising use of the 2G12 epitope to be exhibited in the composition of recombinant vaccines against HIV-1 [8]. The structure of the epitope, recognized by the MAB 2G12, is still not defined, only shows that he is strongly dependent on the conformation of the surface glycoprotein gp120 [5]. Not currently known fragments of gp120, the ability to communicate with MCA 2G12.

An object of the invention is the search for peptide mimics of the epitope of the protein gp120 of HIV-1, recognized by the MAB 2G12, using phage peptide libraries.

This goal is achieved by affinity selection of phage peptide libraries containing tens of millions of different peptides of length 15. Oh, those peptides that are able to communicate with moneny on the surface of bacteriophages in the composition of the minor envelope protein R, each phage particle contains 5 copies of the same peptide [9].

Method affine selection is as follows. Biotinylated monoclonal antibodies are immobilized on a plastic surface treated with streptavidin, and thereto is added phage library. After the occurrence of kinetic equilibrium unbound phages are washed with buffer, and bound peroxidase antibody phage suiryudan and breed for a new cycle of selection. Three rounds of affinity selection is usually sufficient to obtain enriched populations of phages containing the target sequence [10, 11]. To select all of the options specific clones in the first round of selection using saturating with respect to rahovym the particle number of the ICA. To create competition between the phage for binding to MCA and selection of the most specific options in the second and third rounds of selection using smaller relative to the phage antibody concentrations. Enrichment ragovoy population is determined by the increase in the yield of phage after each round of selection. Rich thus ragovoy library after 1st, 2nd, 3rd rounds of affinity selection select individual phage clones and determine the specificity of their tie is necessaty composition is specific to the MCA peptides located on the surface of phage particles, determined by sequencing of phage DNA using a modified method of Sanger [12] encodes a peptide region.

The analysis of similarity of amino acid sequences of selected peptides with the sequence of gp120 was carried out based on eksponirovannoi amino acids on the surface of the protein by x-ray analysis [13], showed that the total structural motifs electrovanne peptides are located in the region of 380 to 420 amino acid residues numbering gp 120 of HIV-1. Apparently, amino acid residues Phe-383, Tyr-384, Ser-387, Ser-393, Thr-394, Trp-395, Leu-416, Arg-419, Leu-420 define the binding of the MAB 2G12 with protein gpl20. These amino acids are removed from each other in sequence but close in space and form on the surface of gpl20 the epitope recognized by the MAB 2G12 (Fig.4).

Practical use.

For practical use electrovanne us a peptide mimetics can be obtained by chemical synthesis or, more economically, using recombinant technology in various protein carriers. The use of carrier protein expression vector based on filamentous bacteriophages (M13,fd), allowing exonerates in E/Coli, released into the culture medium not lysira the host cell, which simplifies the process of cleaning it;

- phage accumulated in the culture medium at a concentration of 1012virions per milliliter, so it is easy to produce in large quantities;

the conformation of a peptide selected as part of the phage, is optimal when using other protein carriers may vary considerably;

- when embedding peptide phage surface protein (2700 copies per virion) is very effective multimeric system presentation, allowing to overcome the problem of weak immunogenicity of the peptides. After his performance this system is often superior systems such as HBC antigen.

The invention consists in the fact that electrovanne of ragovoy peptide library for peptides have the ability to contact neutralizing HIV-1 MAB 2G12.

Our results on the localization of the epitope, recognized by the MAB 2G12, consistent with the views of other researchers, as well as complement them, because for the first time we were predicted amino acid residues that are part of this epitope. Since these antibodies have significant immunotherapy lastmonday system for the induction of humoral response [8] , the peptides imitators of the 2G12 epitope can be used in the development of recombinant vaccines against HIV-1 diagnostic test-systems.

Thus, we first obtained peptides that mimic the full-length glycoprotein gp120 binding neutralizing antibody 2G12. Peptides exposed on the surface of filamentous bacteriophages in the composition of the minor envelope protein R, each phage particle contains 5 copies of the same peptide.

The invention is illustrated by the following figures.

Fig.1. The affine scheme selection.

Fig. 2. The results of the interaction of phage clones in ELISA. As a negative control taken phage fuse2, which does not exhibits a randomized peptide.

Fig.3. Amino acid sequences of peptides imitators neutralizing epitope of the protein gp120 of HIV-1, recognized by the MAB 2G12. Selected matching amino acid residues.

Fig. 4. Surface molecules gpl20 of HIV-1. Black color represents amino acid residues that are part of the epitope, recognized by the MAB 2G12.

For a better understanding of the essence of the present invention the following are examples of its implementation.

Example 1. The way proonline antibody 2G12.

The first round of affinity selection ragovoy the libraries according to the following scheme [10] . On a plastic Petri dish with a diameter of 35 mm put 900 μl of distilled water and 100 µl of 1 M NaHCO3pH of 8.6, add 10 ál of water streptavidin (1 mg/ml). The Petri dish is incubated overnight at 4oWith, merge streptavidin and pour 1 ml of blocking solution (0.1 M NaHCO3, 5 mg/ml BSA, 0.1 mg/ml streptavidin, 0,02% sodium azide), incubated Cup at 4oC for 1 h Washed the Cup 6 times the buffer TBS/Tween (50 mm Tris-HCl, rn,5, Tween-20 0,5% v/v) and add 400 μl of TBS/Tween containing BSA (1 mg/ml) and 0.02% sodium azide, biotinylated antibodies (final concentration in solution of 250 nm), incubated Cup for 2 h at 4oWith, then add 4 μl 10 mm Biotin, incubated at 4oC for 1H, washed the Cup 6 times with TBS/Tween, inflict 400 μl of TBS/Tween, 4 μl 10 mm Biotin and 5 ál ragovoy library (5x1011virions). Cup incubated at 4oC for 4 h, washed 10 times in TBS/Tween, inflict 400 ál of eluting buffer (with 0.1 N HCl, pH is brought glycine to 2.2, 1 mg/ml BSA), incubated 10 min, the eluate is transferred into plastic tubes containing 75 μl of 1M Tris-Hcl pH 9.1 to neutralize the acid, final pH (7-8.5) check the indicator paper. Cap, grown on the TV medium (1,2% lactotropes, a 2.4% yeast extract, 0.5% glycerol, 1.7 mm KH2PO4, 7.2 mm TO2NRA4). Phages grown in LB medium [10]. Phages titrated using cells COP grown on a TV medium, followed by plating on plates with agar containing kanamycin (100 μg/ml) and tetracycline (40 mg/ml) and calculating the grown colonies. The titer of the phages was determined by the number of colonies (transducers units (TU) by G. P. Smith [10]. The number of phage particles are calculated according to the formula 1 TU = 20 virions.

The second and third round of affinity selection carried out as follows. In a plastic test tube add 100 ál ragovoy suspension (1011TU), biotinylated MCA (final concentration of antibodies in the second round is 100 nm, the third - 0.1 nm), incubated overnight at 4oTo add 400 μl of TBS/Tween and immediately put on streptomycinresistant Cup (as described above), incubated 10 min at room temperature. A Cup of washed TBS/Tween, who contacted the phage elute, titrated and amplified as described above. After the third round of selection receive individual phage colony, which is used for producing single-stranded DNA phages for enzyme-linked immunosorbent assay (ELISA). The definition of nucleotide 5'-TGAATTTTCTGTATGAGG [10].

Example 2. The analysis of binding peptides that mimic the highly conserved epitope of the protein gpl20 of HIV-1 with monoclonal antibodies 2G12 by ELISA.

Confirmation of the binding specificity of the selected phage with the MCA carried out by indirect enzyme-linked immunosorbent assay [11].

96-well plate for ELISA (VNII MediaPlayer) put 50 ál into the hole phage preparation (1010virions) and incubated over night in the fridge. After 3 times washing PBS/Tween to each well add ml blocking buffer (1% solution of casein in PBS) to block nonspecific designated sorption on plastic 1 h at 37oC, washed 3 times with PBS/Tween and add 1 μg biotinylated MCA in a volume of 50 μl. After incubation over night in the fridge, washed 3 times with PBS/Tween and add 50 ál of diluted 500 times (in blocking buffer) conjugate streptavidin-horseradish peroxidase. The reaction is carried out for 30 min at 37oC, washed tablet 6 times with PBS/Tween and add 50 ál of the CPB (with OFD. Stand the tablet 20 min in the dark at room temperature and stop the reaction by adding 50 μl of 2N sulfuric acid. The optical density is measured on a Multiscan spectrophotometer (Labsystem).

Excess mn is serviceline effectively interact with MCA and mimic recognizable µa epitopes of the protein gp120 of HIV-1.

Thus, for the first time obtained peptides imitators neutralizing epitope of the protein gpl20 of HIV-1, recognizable neutralizing antibody 2G12.

Literature

1. Coffin J. M.// Science.-1995.-Vol.267.-P. 483-489.

2. Atlan H., Gerstenm M. J., Salk P. L, Salk J.// Immunol. Today., 1993. Vol. 14., p. 200-202.

3. Delmastro P., Meola, A., Monaci, P., Cortese R., Golfre G. //Vaccine., 1997., Aug;15(11), p. 1276-1285.

4. Keller P. M., Arnold B. A., Show A. R., Tolman, R. L., Van Middlesworth F. , Bondy , S., Rusiecki V. K, Koening, S., et al.// Karpova et al., 1993, V. 193, p. 709-716.

5. Trcola A. , Purtscher, M. , Muster, T. et al // J. Virol., 1996, P. 1100-1108.

6. Moore J., Sodroski J.//J. Virology, 1996., P. 1863-1872.

7. Trcola A. , Pomales A. B., H. Yuang et al. // J. Virology, 1995. P. 6609-6617.

8. Kunert R., F. Ruker, Katinger H. // AIDS Res. Hum. Retrovir. - V. 14. , 13. - P1115-1128.

9. Scott J. K., Smith G. P. Searching for peptide ligands with an an epitope library.//Science., 1990., V. 249, p. 386-390.

10. G. P. Smith Cloning in fuse vectors (laboratory manual)//1992.

11. Motti C. , Nuzzo, M., A. Meola, G. Galfre, F. Felici, R. Cortese, M. Nicosia And Monaci, P.//Gene, 1994, V. 46 I, p. 191-198.

12. Kretz, K., W. Callen, Hedden V, Cycle sequencing// Methods of Enzimology. V. 154. P. 527-536.

13. Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski J, Hendrickson W. A. //Nature.,1998., V. 393., P. 648.

Peptide-simulator epitope protein gp120 of HIV-1, recognizable neutralizing monoclonal antibody 2G12, having an amino acid sequence selected in the group serial is

 

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