SYNTHETIC PEPTIDES SUITABLE TO ENHANCE HIV-1 VIRUS INFECTIVITY

Abstract: Synthetic peptides derived from V3 region of HTLV-III B strain and from MN strain of HIV-1 virus, suitable to enhance the infectivity of HIV-1 virus in cellular cultures in vitro and therefore useful for the determination for the same virus in blood and in other biological materials.

Description: SYNTHETIC PEPTIDES SUITABLE TO ENHANCE HIV-1 VIRUS INFECTIVITY. PRIOR ART A great deal of information is known regarding the characteristics of HIV-1 virus and the peptides derived from V3 region of gp 120. A characteristic feature of HIV-1 is its genetic variability, and naturally occurring viral variants show distinct biological properties which correlate with the severity of HIV-1 infection in vivo (Asjo, B., L. Morfeldt-Manson, J. Albert, G. Biberfield, A. Karlsson, K. Lidman, and E.M. Fenyo. 1986. Replicative capacity of human immunodeficiency virus from patients with varying severity of HIV infection. Lancet ii: 660-662; Fenyo, E.M., L. Morfeldt-Manson, F. Chiodi, B. Lind, A. Von Gegerfelt, J. Albert, E. Olausson, and B. Asjo. 1988. Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates. J. Virol. 62: . .l .- . l ; Fiore, J.R., M.L. Calabrό, G. Angarano, A. De Rossi, G. Fico, G. Pastore, and L. Chieco- Bianchi. 1990. HIV-1 variability and progression to AIDS: a longitudinal study. J.Med.Virol. In press). Aminoacid sequence mutations on the viral envelope protein (gp 120) may play a critical role in virus infectivity and antigenicity, since the envelope protein mediates virus attachment and penetration on the host cell and induces both humoral and cellular host immune responses (Cordonnier, A., L. Montagnier, and M. Emmeman. 1989- Single amino acid changes in the HIV envelope affect viral tropism and receptor binding. Nature 30: 571-57^; Mills, K.H.G., D.F.Nixon, and Andrew J. McMichael. 1989. T-cell strategies in AIDS vaccines: MHC- restricted T-cell responses to HIV proteins. AIDS 3: slOl-sllO) . The principal neutralizing domain (PND) of HIV-1 corresponds to a 24-aminoacid sequence arranged in a loop determined by a disulfide bridge in the third hypervariable region, V3, of the protein gp 120 (Goudsmit, J., C. Debouck, R.H. Meloen, L. Smit, M. Bakker, D.M. Asher, A.V. Wolff, C.J. Gibbs, and D.C. Gajdusek. 1988. Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees. Proc. Natl. Acad.). PND-derived peptides elicit antibodies that in vitro neutralize the infection and prevent fusion of virus-infected cells with uninfected CD^-bearing cells (Rusche, J.R. , K. Jvaherian, C. Mc Danal, J. Petro, D.L. Lynn, R. Grimaila, A. Langlois, R. Gallo, L.O. Arthur, J.P. Fischinger, D.P. Bolognesi, S.D. Putney, and T.J. Matthews. I988. Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid of the viral envelope gp 120. Proc. Natl. Acad. Sci. USA 85: 3198-3202). Moreover, PND-derived peptides evoke virus neutralizing responses in mammals, and they may represent good candidates for vaccine development against AIDS (Goudsmit, J. , C. Debouck, R.H. Meloen, L. Smit, M. Bakker, D.M. Asher; A.V. Wolff, C.J. Gibbs, and D.C. Gajdusek. 1988. Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees. Proc. Natl. Acad. Sci. USA 85: 4478-4482; Javaherian, K., A.J. Langlois, C. Mc Danal, K.L. Ross, L.I. Eckler, C.L. Jellis, A.T. Matthews. 1989- Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc. Natl. Acad. Sci. USA 86: 6768-6772; La Rosa G.J., J.P. Davide, K. Weinhold, J.A. Waterbury, A.T. Profy, J.A. Lewis, A.J. Langlois, G.R. Dreesman, R.N. Boswell, P. Shadduck, L.H. Holley, M. Karplus, D.P. Bolognesi, T.J. Matthews, E.A. Emini, S.D. Putney. 1990. Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science 29: 932-935)- The central portion of the V3-PND contains a sequence which is highly conserved in different HIV-1 isolated strains, whereas the aminoaclds flanking this sequence are variable, and the antibodies elicited by peptides designed from PND of different HIV-1 strains are viral-variant specific, and generally neutralize only the homologous virus. Nevertheless, in spite of all this knowledge, the biological properties of PND-derived peptides are still unknown and furthermore a still open problem is represented by the insufficient sensitivity of the methods known up to now for the determination of the virus in blood or in others biological materials in subjects whose infection is uncertain. SUMMARY The present invention concerns synthetic peptides derived from V3 region of HTLV-III B strain and from MN strain of HIV-1 virus, suitable to enhance the infectivity of HIV-1 virus in cellular culture in vitro. This enhancing effect occurs in the early steps of the viral infection and is not virus restricted. Said peptides have the following aminoacid sequences: NNTRKSIRIQRGPGRAFVTIGKIG (DB1) YNKRKRIHIGPGRAFYTTKNIIG (DB3) The addition of said peptides to CD4-positive cells cultures enhances the sensitivity of the determination of the virus in blood and in the other biological materials of subjects affected by HIV-1 infection. Therefore the invention concerns also the use of said peptides in cultures in vitro in order to enhance the HIV-1 infection in the uncertain cases in which the infection is limited and the current methods are not sufficiently sensitive. DETAILED DESCRIPTION OF THE INVENTION The characteristics and the advantages of the peptides according to the present invention will be better defined later-on in the following detailed description. Said peptides are derived from V3 region of HTLV-III B strain and from MN strain of virus HIV-1 and have the following aminoacid sequences: NNTRKSIRIQRGPGRAFVTIGKIG (DB1) YNKRKRIHIGPGRAFYTTKNIIG (DB3) Peptides synthesis Synthetic peptides DB1, DB2, DB3 were prepared according to aminoacid sequences of the V3-PND of HIV-1 strains, HTLV-III B, RF and MN respectively. A modified peptide consisting of 23 a inoacids (DB5) and a DB1 derivative formed by 10 aminoacids (DB6) were also prepared. Said peptides are reported in the following table 1. TABLE 1 Peptide HIV-1 strain Sequence DB1 HTLV-III B NNTRKSIRIQRGPGRAFVTIGK IG DB2 RF TK VIYAT-QI-- DB3 MN Y-K--R-H- Y-TKNI— DB5 —V-R-LS R-R--I-- DB6 - The abbreviations used for the aminoacid are those recommended by IUPAC-IUB Commission on Biochemical Nomenclature (cfr. J. Mol. Biol.52, 1-17 (1970) and J. Biol. Chem. 24j, 977-983 (1972)). The hatchings represent aminoacids identical to those of DB1. Said peptides may have the terminal or side-chain aminic and/or the carboxylic functions free, salified or protected with suitable alkyl, aryl, alkylaryl and arylalkyl groups. In particular the carboxylic function in terminal position may be represented by free carboxylic groups or salified with numerous bases, by primary or various by substituted amidic functions, or also by different estereal groups. The synthesis of the peptide chains was realized by using an automated synthetizer "Applied Biosystems 431 A" starting with 0.5 mmol of a resin previously functionalized with ter-butyloxy- carbonyl derivative of the aminoacid of the sequence ending with a carboxylic function. Hereinbelow, for example, the operating conditions of a peptide synthesis are reported. The following side-chain protection was used: acetamidoethyl for cystein, benzyl for serin and threonin; p-chlorocarbobenzoxy for lysine, benzylester for aspartic and glutamic acid; tosyl for arginine; 2,4 dinitrophenyl for histidine and 2- bromobenzyloxycarbonyl for tyrosine. Removal of BOC group was accomplished by a 3 minutes wash with an aqueous solution containing 25 % of trifluoroacetic acid, followed by a 16 minutes treatment with 50 % trifluoroacetic acid in CH 2 C1 2 . BOC protected aminoacids were activated by using dicyclohexylcarbodiimide and introduced as hydroxybenzotriazoles esters. Each residue incorporation was followed by using an acylation of the free aminic groups by acetic anhydride. At the end of the synthesis the peptide bonded to the resin was treated with suitable side chain protecting groups and with 10 ml of HF. The reaction mixture was stirred for 1 hour at a temperature ranging from -5 to 0 * C; HF was then removed by Nitrogen flow, and the residue dried under vacuum, was extracted with ethylether and with 30 ml of 30 % acetic acid. After lyophylization the peptides were purified by ion exchange chromatography using a Protein Pak SP5PW, 0.8x7.5 cm Waters column with different gradients of solution B (Na 2 HP0^ 10 mM pH6/NaCl 0.5 mM/10^CH 3 CN) mixed with a solution A (Na 2 HP0i j 10 mM, pH6) at a flow rate of 1 ml/min. Each peptide was then further purified by HPLC on a Delta Pak column 15μ, 30θA, 7.8x30 cm using H 2 0 0.1U trifluoroacetic acid with a CH CN gradient containing 0.1 trifluoroacetic acid. Each peptide purity was then confirmed by analytical chromatography on Delta Pak 5μ, lOOA, 3-9x15 cm and resulted higher than 90 %. The aminoacid composition was determined with an automated analyzer (Carlo Erba 280) . Viral infection tests M0LT-3 and H938 cells were employed for HIV-1 experiments. Both these cellular lines have a T-lymphocyte origin and express CD4 molecule. Cells were maintained in RPMI 1640 medium (Flow Laboratories, Irvine UK) supplemented with 10 % fetal calf serum, 2 % h- glutamine, and 50 μg/ml gentamycine, and cultured under standard conditions at 37°C in humidified air containing 5 % C0 2 . HTLV-III B, RF and MN HIV strain were grown in H9 cells, and the supernatant was collected, centrifugated at 2,000 x g for 15 minutes, to remove the cells, and filtered through a 0.22 μm filter. The virus content was determined by reverse transcriptase (RT) assay and the supernatants were stored at -80 °C. M0LT-3 cells resuspended at a concentration af 2.5x10 cells/ml were plated at 200 μl/well in a 24 wells plate (Costar, Cambridge, MA, USA) and incubated with 100 μl of a medium containing scalar doses of equimolar amounts of peptides. After 1 hour, cells were infected with 50 μl of an HIV-1 preparation containing 1x10-* cpm/ml of RT activity (0.01 cpm of RT activity cell). After 1 hour incubation with gentle shaking at 37 "C, 1 ml of complete medium was added. The cells were checked daily at the light microscope for syncytium formation. After 4 days, supernatants were collected for testing RT assay. H938 cells were plated, treated with peptides and infected as above described and 48 hours after HIV-1 infection, cellular extracts were prepared by 3 cycles of freeze-thawing and tested with CAT (chloramphenicol acetyl transferase) (Gorman, CM. , L.F. Moffat, and B.H. Howard. 1982. Recombinant genomes which express chloramphenicol acetyl transferase in mammalian cells. Mol.Cell.Biol. 2: 1044-1051). In the cultures treated with MN-derived peptide DB3 and infected with strain HTLV-III B of HV-1, large syncytia were observed within 48 hours from infection (A and B of figure 1 respectively after 2 and 4 days from infection) while untreated cells showed small syncytia only at 4 days post infection (C of figure 1) (D and E represent control trials). DB3 effect resulted dose dependent, and concentrations as low as 2.5 μM increased syncytium formation within 2 days after HIV-1 infection. Cultures treated with peptides DB1, derived from HTLV-III B strain, showed an increase in syncytium formation only at peptides doses of 10 and 20 μM. No such effect occurred in cultures treated with DB2, DB5 and DB6. RT values (expressed in cpm/ml) in surnatants collected 4 days after infection further reflected the enhancing effect of DB3, and to a lesser extent that of DB1 , on HTLV-III B strain infection , while no such increase was observed in cultures treated with peptides DB2, DB5 and DB6 also at maxima doses (fig. 2) . These findings demonstrate that the enhanced cytopathic effect observed is the result of a more intense viral replication and of the envelope gp 120 protein expression on the surface of infected CD4 positive cells. HIV-1 gene expression depends mainly on the sequences in the "Long Terminal Repeats" region (LTR) . LTR may be activated by viral factors as well by the transactivetor (tat) protein which interacts with the responsive elements (TAR) in LTR, and cellular factors recognizing target sequences in HIV-1-LTR. (Nabel, G., and D. Baltimore. 1987. An inducible transcription factor activates expression of human immunodeficiency virus in T cell. Nature 326: 711-713) and are induced by several T-cell mitogens (Greene, W.C., E. Bohnlein, and D. Ballard. 1 8 . HIV-1, HTLV-I and normal T-cell growth: transcriptional strategies and surprises. Immunol. Today 10: 272- 274). The effect of the peptides on HIV-1-LTR region were evaluated using H938 cells. These cells were permanently transfected with HIV-1-LTR bonded to CAT gene, thus LTR activation resulting in activation of CAT expression (Felber, B.K. , and G.N. Pavlakis. I988. A quantitative bioassay for HIV-1 based on trans-activation. Science 239: 184- 187) • Parallel cell cultures were incubated for 1 hour with scalar dilutions of peptides, and one culture for each dilution was then infected with HTLV-III B strain. After 48 hours both infected and non infected cells were processed for CAT assay. As shown in figure 3 t CAT expression, evaluated as the percentage of Cm (chloramphenicol) in AcCm (chloramphenicol in acetylated form), resulted increased in DB3 treated HIV-1 infected cultures. The effect of DB1 peptide on CAT expression is lower than that of DB3. DB2, DB5, DB6 have no effect on CAT expression, and the value obtained are very similar to those obtained with untreated HIV-1 infected cultures. The non infected cultures treated with peptides did not show CAT expression even at high doses. These results demonstrate that LTR region activation is due to viral tat protein overexpression by initial enhancement of HIV-1 infection, rather than a peptide mitogenic effect on the infected cells. Features of the activity of the peptides according to the invention A characteristic feature of the activity of the peptides according to the invention is that the enhancing effect exerted by the peptides occurs in the early steps of viral infection. To investigate this point, we treated the H938 cells with different doses of DB3 before, during and after HIV-1 infection. The enhancement effect in cells exposed for 1 hour to both virus and peptide simultaneously (fig. 4A) was quite similar to that obtained when cells were preexposed to peptide 1 hour before infection (see Fig. 3)- Furthermore viral enhancement also occurred when cells were treated with peptide for 1 hour, washed and then infected (Fig. 4B) . No enhancement was seen when cells were infected with HIV-1, and then washed to remove virtually all the virus particles still present in the medium, prior to exposure to peptide (Fig. 4C) . The observation that enhancement occurred only when peptide was present in cultures before or during HIV-1 infection, but not when it was added after entry into the cells, implies that the peptides acts during the early steps of viral infection. An other characteristic of the peptides of the present invention is that the infectivity enhancement is not virus restricted. The antigenic properties of V3 region are known to be virus- specific; antibodies elicited by MN-derived peptide do not neutralize HTLV-III B virus and vice-versa. Our observation that the peptide from the MN V3 neutralizing domain brought about a greater increase in HTLV-III B infection than that obtained with III B-derived specific peptide, demonstrates that the enhancement effect is not virus-restricted. To better evaluate this aspect, experiments were performed using MN and RF HIV-1 strains. Fig. 5 shows that the peptide effect in MN and RF infection was quite similar to that observed in HTLV-III B infection (fig. 3) - A final concentration of 20 μM DB3 increased RT values in M0LT-3 cells with MN and RF virus infected 20 and 12 times, respectively; and this effect was still evident with 0.62 μM and 5 yM in MN and RF infections, respectively (fig. 5B). CAT assay in H938 infected cells supported RT findings, and the increase in transactivation activity was peptide dose dependent (fig. 5A). As previously observed with HTLV-III B strain infection, DB1 was less effective than DB3 and brought about significant increases in both parameters only at maxima doses in both MN and RF strains infections. Interestingly, DB2 did not have any effect on heterologous MN and homologous RF virus infections. To determine if the enhancing effect of the peptides of the present invention depended on the viral infection dose, serial dilutions of stock virus, 10 times concentrated with respect to H9/HTLV-III B surnatants, were evaluated for infection on H938 cells in the presence and absence of peptide DB3 (10 μM final solution) by CAT assay. It was found that Cm conversion in untreated cells decreased linearly according to the viral dilution, and at 10 " ^ the signal was comparable to the negative control (fig 6A) ; the peptide treated cells showed almost 100 % Cm conversion up to 10 -2 viral dilution, and at 10 - Cm conversion was 13-9 % (fig- 6B) . Therefore by means of the peptides of the present invention the viral expression was amplified even if a low virus concentration was used for infecting. The results reported above find an important practical application since the addition of said peptides to culture of indicating cells CD4-positive increases the sensitivity in determining the virus in blood and in other biological materials of subjects infected by HIV-1. In addition said peptides can be used in all the cultures in vitro for enhancing HIV-1 infection, and therefore they find a very important use in diagnosis of uncertain case wherein the infection is limited and the methods used up to now are not enough sensitive. Mechanism of the enhancement of viral infectivity of the peptides according to the present invention Without undertaking to explain the mechanism by which the peptides of the present invention enhance HIV-1 infectivity, we report herewith the experimentation results which permit to hypothesize a possible mechanism. As already observed viral enhancement occurred in the early steps of virus entry, probably at the virus entry into the cells. Since the viral receptor on T lymphocytes is the CD4 molecule (Sattentau, Q.J., A.G. Dalgleish, R.A. Weiss, and P.C.L. Beverley 1986 epitopes of the CD4 antigen and HIV infection,Science 234: 1120), MOLT-3 cells were exposed for 1 hour to scalar doses of different peptides, and then analyzed by cytofluorimetry for CD4 expression using 0KT4 and 0KT4a monoclonal antibodies (MAbs) . As reported in figure 1 , a medium intensity fluorescence increase was recorded when cells were treated with the suitable doses of DB3. This increase was recorded within 3 minutes of DB3 treatment, and also observed in DB3 treated cells 1 hour after the peptide had been removed by washing. In order to better investigate the peptide effect on CD4 expression, experiments were also performed using the gangliosides GM1 and phorbol ester (Chieco-Bianchi L., M.L. Calabro, M. Panozzo, A. De Rossi, A. Amadori, L Callegaro and A. Siccardi 1989. CD4 modulation and inhibition of HIV-1 infectivity induced by monosiaganglioside GMl in vitro . AIDS 3: 501-507 ; Nakakuma H, T.Kawaguchi, Koito A, Hattori T. , Kagimoto T. , and Takatsuki K, 1989 Inhibition of Human immunodeficiency virus infection of human lymphocytes by gangliosides .JPN Cancer Res. 80 :702-705); Offner.H . , T. Thime , and A. A. Vanderbark 1987. Gangliosides induce selective modulation of CD4 from helper T lymphocytes . J Immunol. 139 :3295~ 3305; Acres,R.B., P.J. Conion, D.Y. Mochizuki and B. Ballis,1986. Rapid phosphorylation and modulation of the T4 antigen on cloned helper T-cells induced by phorbol myristate acetate or antigen.J.Biol. Chem.26l :16210- l62l4; Jaekyoon, S., C. Doyle, Z. Zang, D. Kappes, and J.L. Strominger .1990 Structural features of the cytoplasmatic region of CO . required for internalization. EMB0 J. 9: 25-434). The incubation of X10^ MOLT-3 cells for 1 hour with 50 μg/ml of GMl resulted in an almost complete block of CD4 expression (fig. 8). When GMl exposure was carried out 1 hour after the incubation with DB3 or DB1 peptides (20 μM final concentration) or was conducted simultaneously, cells expressed CD4 levels similar to those of GMl-untreated controls; on the contrary, 20 μM of DB2 peptide did not block the CD4 down-regulation exerted by GMl (fig. 8). DB3 and DB1 effects were dose dependent ; 10 μM of both peptides inhibited completely CD4 down-regulation induced by GMl; at lower doses, however, DB3 peptide was more active than DBl in blocking GMl activity (fig. 9)- When 5x10^ M0LT-3 cells were incubated for 4 hours with 100 ng/ml of TPA, less than 10 % of cells expressed CD4 on the membrane surface. However, if TPA exposure was preceded by 1 hour treatment with DB3 peptide (20 μM) , or carried out simultaneously, almost 50 % cells were still CD4 positive (fig. 8). DB3 was less effective on TPA than on GMl, and 5 μM of peptide did not inhibit TPA-induced CD4 down regulation (fig. 9). Unlike that observed with GMl treatment, DBl did not block TPA activity, even at its highest dose (fig. 8 and fig. 9). Said results permit to hypothesize a mechanism of peptides action involving the cellular receptor CD4.

Claims: CLAIMS 1. Synthetic peptides derived from V3 region of HTLV-III B strain and from MN strain of HIV-1 virus, suitable to enhance HIV-1 infectivity in cellular cultures in vitro. 2. The peptides as claimed in claim 1, having the following aminoacid sequence: NNTRKSIRIQRGPGRAFVTIGKIG 3 - The peptides as claimed in claim 1 having the following aminoacid sequence: YNKRKRIHIGPGRAFYTTKNIIG 4. Use of synthetic peptides derived from V3 region of HTLV-III B strain and from MN strain of HIV-1 virus enhancing HIV-1 virus infectivity in cellular cultures in vitro, in test suitable to determine HIV-1 virus in blood and in other biological materials. 5- The use as claimed in claim 4, wherein said peptides have the following aminoacid sequence: NNTRKSIRIQRGPGRAFVTIGKIG 6. The use as claimed in claim 4, wherein said peptides have the following aminoacid sequence: YNKRKRIHIGPGRAFYTTKNIIG 7- The use as claimed in claim 4, wherein said cellular cultures consist of CD4-positive cells.

Inventors: CHIECO-BIANCHI LUIGI, DE ROSSI ANITA, DETTIN MONICA, DI BELLO CARLO, MAMMANO FABRIZIO, PANOZZO MARINA, PASTI MARCELLA, CHIECO-BIANCHI, LUIGI, DE ROSSI, ANITA, DETTIN, MONICA, DI BELLO, CARLO, MAMMANO, FABRIZIO, PANOZZO, MARINA, PASTI, MARCELLA
Applicants: CONSIGLIO NAZIONALE RICERCHE, IST SUPERIORE SANITA, CONSIGLIO NAZIONALE DELLE RICERCHE, ISTITUTO SUPERIORE DI SANITA
Application year: 1992
Main classification: C07K7
IPC codes: C07K14, C12N7, G01N33