Patent Application
20060234208
1. Field of the invention
This invention relates to the finding of a new mechanism of HIV-1 infection. This invention further relates to the development of novel drugs, e.g. synthetic peptide drugs blocking this mechanism, therefore containing HIV-1 infection.
2. Background of the invention
Several drugs have been developed to manage Human Immunodeficiency Virus-1 (HIV-1) infection and the consequent onset of AIDS. The advent of highly active antiretroviral therapy (HAART)—combinations of protease and reverse transcriptase inhibitors—provided a potent and clinically effective method of suppressing viral load in HIV-1—infected individuals. However, although initially successful, a broader clinical experience has revealed limitations in this therapeutic regimen. Consistent and long-term clinical benefit from triple therapy remains elusive owing to intolerance, non-compliance with drug dosing schedules and, most ominously, the development and transmission of resistant viruses. As a consequence, the search for anti-HIV agents continues, with renewed emphasis placed on novel targets in an attempt to increase the repertoire and efficacy of clinically available drugs.
Although the HIV-1 entry process was one of the earliest mechanisms examined as a target for therapeutic intervention, progress was difficult and success elusive. However, the elucidation of co-receptors that facilitate virus entry by triggering fusion, and the recently solved X-ray crystal structure of elements of the envelope glycoprotein gp120, have provided crucial new insights into the mechanism of HIV-1 entry and afforded significant new opportunities for HIV-1 drug discovery.
Virion attachment is mediated by the specific binding of gp120 to the receptor CD4, although it is also facilitated by non-specific interactions with other cell-surface molecules. The interaction of gp120 with CD4 results in the exposure of domains of gp120 that subsequently interact with one of several cell type-specific co-receptors, leading to destabilization of the gp120-gp41 protein complex. As a consequence, the gp41 subunit undergoes a conformational rearrangement, exposing the hydrophobic fusion peptide that inserts into the host cell membrane and initiates the fusion process (Chan and Kim, Cell 93 (1998), 681-684; Wyatt and Sodroski, Science 280 (1998), 1884-1888). This process occurs not only at the first interaction between the host cells and the virus, but characterizes the succeeding waves of spreading from lymphonodes, where HIV-1 replicates after the first infection, to the circulating lymphocytes. HIV-1 entry has been validated as a clinically relevant target, as demonstrated by several compounds that have been advanced into clinical trials (Table 1) (Blair et al., Drug Discov. Today 5 (2000), 183-194).
These reports confirm that discovering new targets is a crucial step for the development of innovative drugs to block virus entry. To be more efficient, these new drugs must be specific, but of broad-range application, that is, they have to block infection regardless of the viral strain. This characteristic is mandatory to fully circumvent drug resistance.
Recent attention has been placed in investigating the multifaceted activities of HIV-1 Trans Activator of Transcription (TAT) (Jeang et al., J. Biol. Chem. 274 (1999), 28837-28840). TAT is a nuclear factor with the main function of enhancing the transcription of viral RNAs, thus allowing the production of new viral particles and the consequent spreading of the virus.
Several other TAT functions have been described, and somehow related to AIDS pathogenesis. In particular, TAT is secreted by infected cells and is therefore detectable in the extracellular milieu, where acts as a growth factor binding trans-membrane receptors and inducing cellular pathways (Albini et al., Nat. Med. 2 (1996), 1371-1375); Mitolo et al., Blood 90 (1997), 1365-1372), or enters surrounding cells interfering with their gene expression. Interestingly, after release a portion of the protein binds the extracellular matrix (ECM)-associated heparan sulphate proteoglycans (HSPG) (Chang et al., Aids 11 (1997), 1421-1431). So, TAT is partially sequestered at the cell surface, which is, as previously described, the site for HIV-1 recognition and entry.
In the past few years, some peculiarities of TAT have been applied to biotechnological uses. In particular, a basic peptide derived from TAT (positions 48-60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, and has been employed for the delivery of exogenous proteins into the cells (Morris et al., Nat. Biotechnol. 19 (2001), 1173-1176). Moreover, TAT peptides facilitate intracellular delivery also of small colloidal particles, and of relatively large drug carriers, such as 200-nm liposomes (Torchilin et al., PNAS USA 98 (2001), 8786-8791). The mature HIV-1 particle has a diameter of about 100 nm, suggesting that such a mechanism of internalisation could be possible also in vivo.
The contribution of extracellular TAT to the progression of viral infection has been underlined by the ability of neutralizing anti TAT antibodies to reduce the viral load both in vitro and in vivo (Cafaro et al., Nat. Med. 5 (1999), 643-650; Goldstein, Nat. Med. 2 (1996), 960-964). These reports suggest that extracellular TAT can play a functional role in HIV-1 infection. However, until now the presence of TAT on the cell membranes and the ability of TAT basic region to deliver big particles into the cells have never been related, nor consequently investigated.
In accordance with one aspect of the present invention, the inventors have found a novel mechanism for HIV-1 entry into host cells. In particular, they have shown that HIV-1 TAT is sequestered at the cell surface, where it acts as a novel, specific receptor for gp120. This interaction enhances virus entry into permissive cells. This mechanism is independent of the viral strain, having similar outcomes on the infection by M-tropic (Ba-L) and L-tropic (III B) viruses. The inventors have further suggested a simple model of pathogenesis: HIV-1-infected cells release TAT into their environment; as a consequence, TAT binds the surface of surrounding, still uninfected cells, thus rendering those cells more permissive to HIV-1 infection.
Consequently, inhibitors of the interaction between HIV-1 TAT protein and HIV-1 gp120 are capable of inhibiting the entry of HIV-1 into a host cell. These inhibitors may be used for diagnostic or therapeutic applications, particularly for the treatment of HIV-1 infections. Further, methods and systems, e.g. kits, for identifying novel inhibitors are provided.
Second, the inventors have mapped the region of gp120 involved in TAT binding, by screening Phage Display libraries on TAT-expressing cells. In particular, two peptides were selected as specific TAT ligands, identifying a portion of the gp120 V1/V2 loop. The inventors have shown that these peptides, and they derivatives, interfere with TAT/gp120 interaction and revert TAT-mediated enhancement of virus entry. Finally, the inventors have shown that the peptides are equally efficient in inhibiting the infection of both M-tropic (Ba-L) and L-tropic (III B) HIV-1 strains.
Thus, the invention further relates to novel specific TAT ligands, particularly peptides selected from the gp120 V1/V2 loop or peptides homologous thereto.
A first aspect of the present invention relates to the use of inhibitors of the interaction between the HIV-1 TAT protein and HIV-1 gp120 for inhibiting the entry of HIV-1 into a host cell, particularly a human host cell.
In a first embodiment of this aspect, the inhibitor is selected from compounds which are capable of binding to TAT, more particularly from compounds which are capable of competing with gp120 for the binding to TAT. In a further embodiment, the inhibitor is selected from compounds which are capable of binding to gp 120, particularly from compounds which compete with TAT for the binding to gp120.
The inhibitors may be a peptide, e.g. a peptide having a length of from 4 to 25, particularly from 5 to 20 and more particularly from 6 to 15 amino acids including cyclic peptides, peptides containing non-naturally occuring amino acids, e.g. D-amino acids or peptide mimetics. In an especially preferred embodiment the peptide is homologous to the gp120 V1/V2 region. On the other hand, the inhibitor may be a non-peptidic compound, e.g. a low-molecular weight organic compound, particularly having a molecular weight up to 2000 Da. In an other embodiment, the peptide mimics the region of TAT involved in the interaction with gp120.
Specific examples of preferred peptidic inhibitors are selected from:
(d) a peptide comprising at least 5 contiguous amino acids from a peptide, selected from the group consisting of peptides (a)-(c),
(e) a peptide which has a sequence identity of at least 80% to the amino acid sequence of a peptide selected from the group consisting of peptides (a)-(d).
Further specific examples of preferred peptidic inhibitors are selected from:
(l) a peptide comprising at least 5 contiguous amino acids from a peptide selected from the group consisiting of peptides (a)-(k), (m) a peptide which has a squence identity of at least 80% to the amino acid sequence of peptide selected from the group consisting of peptides (a)-(I).
It is also possible to combine one or more of the peptidic inhibitors of the invention. Thus, a further aspect of the present invention is a peptide combination comprising at least two peptides with the sequences shown in SEQ ID NO: 2-127, especially in SEQ ID NO: 4-120.
Since many of the peptidic inhibitors contain one or more cysteine residue (e.g. peptides with the sequences shown in SEQ ID NO: 2-22 and 127), such peptides are able to form disulfide bridges.
Peptides derived from the cysteine containing peptides of the invention by formation of disulfide bridges are also within the scope of the present invention. Peptides, which are derived by Cys-Cys dimerization of two cysteine containing peptidic inhibitors are especially suitable. Thus, a further aspect of the present invention is a peptide combination comprising two peptides with the sequences shown in SEQ ID NO: 2-20 and 127, wherein one disulfide bridge is present.
However, it is also possible to use peptides of the invention, which contain two or more cysteine residues and are able to form more than one disulfide bridge. Thus, another aspect of the present invention are peptide combinations comprising at least two peptides with the sequences shown in SEQ ID NO: 2-127, wherein at least one disulfide bridge is present.
The inhibitors of the invention may be used for research purposes, e.g. in order to study the mechanism of HIV-1 infection. Further, the inhibitors are suitable for diagnostic and therapeutic applications.
In a preferred embodiment, the inhibitors are used for the manufacture of medicament for the treatment of HIV-1 infection, particularly for the containment of HIV-1 infection. Surprisingly, the inhibitors are capable for the treatment of infection caused by different HIV-1 strains, particularly M-tropic and L-tropic HIV-1 strains.
Thus a further aspect of the present invention relates to a pharmaceutical composition comprising as an active ingredient at least one inhibitor of the interaction between HIV-1 TAT protein and HIV gp120 and optionally pharmaceutically acceptable carriers, diluents and adjuvants. The composition may be in the form of a solution, suspension, emulsion, tablet, dragee, capsule, cream, ointment etc. The manufacture of the composition usually comprises a therapeutically effective amount of the active ingredient with pharmaceutically acceptable carriers, diluents and/or adjuvants according to known formulation methods. The composition may be administered by any suitable route, e.g. by parenteral, oral, topical, mucosal, or nasal administration.
The therapeutic effective dose of the ingredient may be determined by a skilled person without undue burden. Generally, e.g. for peptidic compounds, their therapeutically effective doses are in the range of 10 to 200 mg daily.
Still a further aspect of the present invention relates to a method for identifying and/or characterizing a compound which inhibits the entry of HIV-1 into a host cell comprising
In one embodiment the method is a screening method, wherein a plurality of compounds is tested in parallel or sequencial. For example, a compound library, e.g. a phage display library of peptidic compounds, a chemical library etc. may be tested.
Alternatively, the method is suitable for characterizing the property of an already known inhibitor, which may be used as a lead structure from which derivatives may be obtained, e.g. by molecular modelling and/or random variations.
The method may be carried out as a cellular-based assay, e.g. an assay wherein the inhibition of the infection of host cells by HIV viruses or HIV analogues is determined in the presence of the compound to be tested, optionally in comparision to a suitable control. On the other hand, the method may comprise a molecular-based assay, wherein the influence of a test compound on the interaction between purified e.g. recombinant TAT and gp120 is determined.
Finally, the method encompasses the formulation of a compound which has been identified as an inhibitor by the method as described above or a compound derived therefrom as a pharmaceutical composition.
The inhibitor of the present invention may be administered alone. Preferably, however, the inhibitor is administered as a component in a combination of different anti HIV agents, e.g. protease and/or reverse transcriptase inhibitors. On the basis of the mechanism of action of this type of inhibitors and on the proposed model of pathogenesis, this class of molecules can be characterized by anti-spreading activity.
Experimental Section
1. The first part of this section relates to the finding that TAT is released by producing cells and binds the cell surfaces, where it acts as an additional co-receptor for virus entry. In particular, in this part we will describe how the experimental model was designed and which are its properties, how TAT interacts with gp120 and enhances virus entry. Finally, we will propose a model of TAT-driven pathogenesis for the spreading of HIV-1.
1.1 Description of the Experimental Model
We have set up a model aimed to mimic in vitro the production of TAT by infected cells, observed in vivo in patients with AIDS (Westendorp et al., Nature 375 (1995), 497-500). This model consists of U937 pro-monocyte cells expressing TAT86 of HIV-1 strain III B (SEQ ID NO.1). In this paragraph we describe how the cells were prepared and characterized.
To characterize the U937/TAT cells, we have first evaluated the presence of TAT on their membranes.
We have then demonstrated that TAT is present in the conditioned media of U937/TAT cells. Since anti-TAT antibodies are poorly efficient, we could not detect TAT protein by Western Blot, suggesting that it is present in low amounts. To circumvent this problem we have tested the presence of TAT trans-activating activity in the culture media by a conventional CAT assay. The graph in
To complete the model, we have finally evaluated if TAT can bind the surface of close, untransfected cells. This simplified model should reproduce the way in which infected cells interact via TAT with surrounding, still uninfected cells. We have set up a co-culture system, in which U937/TAT cells are cultured in the lower compartment and C8166 T-lymphocyte cells are placed in the upper compartment of a transwell with a pore size of 0,4 μM (Falcon). The two compartments are divided by a membrane that allows the exchange of secreted proteins but not of the cells themselves.
1.2 Role of cell membrane-bound TAT in HIV-1 entry into the cells.
To assess the role of surface-bound TAT in HIV-1 infection, we have performed entry experiments using HIV-1 -derived lentiviral vectors (Dull et al., J. Virol 72 (1998), 8463-8471; Naldini et al., PNAS USA 93 (1996), 11382-11388). These vectors are a modification of the wild type virus, in order to reach higher safety for the operator, and easier detection of the infected cells. A complete viral particle is assembled by transfecting packaging cells (the human renal cancer cell line 293T) with 3 vectors. The first one carries all the genes of HIV-1 but the env gene. The second one carries the gene for the envelope protein (thus allowing the construction of viruses pseudotyped with different envelopes). The third one is a reporter vector for the expression of GFP. The mechanism of entry is the same as for the wild type virus, with the difference that transduced cells express GFP and can be readily detected by FACS analysis after 72 h from the infection. Moreover, since transduced cells are unable to produce new virions and to propagate the infection, by using these constructs it is possible to dissect the infection process, and to evaluate exclusively the phase of virus entry.
1.3 Discovering of the portion of TAT that binds to gp120
To determine the region of TAT required to bind gp120 we have produced different TAT from HIV-1 HXB2 as GST-fusion proteins, including full-length TAT (TAT86) and mutants lacking the C-terminal (TAT72) or containing mutated basic (TATBaSMut) or cystein-rich (TATCysMut) domains, and assayed the ability of these fusion proteins to bind gp120. All variants except for TAT72 could bind rgp 120-coated microwells, suggesting that the interaction with gp120 is mediated by TAT aminoacids 72-86 (
1.4 Proposed Model of Pathogenesis
We propose the following mechanism (see drawing in
HIV-1-infected cells release TAT into their environment; as a consequence, TAT binds the surface of surrounding, still uninfected cell, thus rendering those cells more permissive to HIV-1 infection. This amplification possibly causes an acceleration in the spreading of the virus. Until now the knowledge of TAT functions was not sufficient to confirm its direct involvement in HIV-1 infection. We demonstrate for the first time that these events depend on an enhanced TAT-driven virus entry into the cells, due to a specific interaction among gp120 and TAT at the cell surface.
This mechanism suggest a new target for the development of anti-HIV-1 drugs. In fact, blocking the TAT/gp120 interaction should inhibit the virus infection due to membrane-bound TAT. In the following part the study of such drugs is described in details.
2. The second part of this section relates to the discovery of peptides blocking the entry mechanism described in the first part. The described peptides have been selected from a random Phage Display library, and share homology with a region of gp120 whose function was unknown until now. We will describe how the peptide sequences have been selected and characterized. Moreover, applications of the cognate synthetic peptides as anti-HIV-1 drugs are disclosed.
2.1 Development of Targeted Drugs by Phage Display Screening
In a Phage Display library, a random oligonucleotide is inserted immediately before the gene coding for the pill coat protein so that, when the phage particle is assembled, a random peptide appears on its surface as a fusion with the pill protein itself. In a library, up to 109 different peptides are displayed on the phage surface, from which it is possible to select one or more peptides specific for binding a substrate of interest (Scott and Smith, Science 249 (1990), 386-390). The peptide drugs described in this invention have been developed by screening Phage Display libraries on U937/TAT cells. Accordingly to the model described in Part 1, one of the most representative proteins on the surface of U937/TAT cells should be TAT itself. Two different libraries, CX10C and CX3CX3CX3C, where C is Cys and X is any amino acid, have been used. After the second (CX3CX3CX3C library) or third round (CX10C library) of selection, we have obtained a significant enrichment in phage binding to U937/TAT cells compared to U937/PINCO cells. Sequencing the selected phage has revealed an enrichment in two peptides (phage inserts CTVECYFNCTPTC [SEQ ID NO.2] and CPDRKKKVVMVC [SEQ ID NO.3]) sharing high homology with gp120 . In particular, these peptides identify a portion of gp120 V1/V2 loop, where they are separated by a spacer of three amino acids only. Despite the high global variability of this loop, the portion mapped by the Phage Display-selected peptides appears to be conserved among different viral strains, particularly the (C-S/T/E-F-basic aa-apolar aa-S/T) motif (from SEQ ID NO.2) and the (R/K-D/N-basic stretch) (from SEQ ID NO.3), suggesting a functional role for this region. The peptide sequences of the described region of gp120 (from most of the known HIV-1 strains) have been deduced from Kuiken et al. (HIV Sequence Compendium 2000 (2000), Theoretical Biology and Biophysics Group, Los Alamos National Labroatory) and are shown in Tab. 1 (SEQ ID NO.4-22 are the homologues of SEQ ID NO.2; SEQ ID NO. 23-39 are the homologues of the 3 aa-spacer; SEQ ID NO. 40-120 are the homologues of SEQ ID NO.3).
A physiological role of the region mapped by the selected peptides has never been described in literature. Since we have based the rationale of the screening on the presence of surface-bound TAT, the selected regions of gp120 can be involved in binding TAT. A TAT-gp120 interaction has never been described before, so it is reasonable that new domains of the gp120 protein have been selected by the screening.
2.2 Properties of the Peptides
In the following Example Section the peptide properties and the ability of inhibiting HIV-1 infection are shown.
Example I
This example is illustrated in
Example II
This example is illustrated in
Example Ill
This example is illustrated in
Example IV
This example is illustrated in
Example V
This example is illustrated in
This example is illustrated in
METHODS
Methods that are not explicitly described in this disclosure and in other sections are amply reported in the scientific literature and are well within the scope of those skilled in the art.
Cell cultures. U937 and C8166 cells were cultured in RPMI -1640 medium supplemented with 10% heat inactivated FCS; Phoenix cells were cultured in DMEM supplemented with 10% FCS; 293T cells were cultured in Iscove's medium supplemented with 10% FCS. PBMCs from healthy donors were separated on a Ficoll gradient, and cultured in RPMI-1640 supplemented with 20% FCS and 200 U/ml Interleukin-2. Before infection, PBMCs were stimulated with 5 μg/ml PHA for 48 h.
Preparation of U937/TAT cells. The coding region of TAT86 IlI B was amplified by Polymerase Chain Reaction (PCR) and subcloned into the retroviral vector PINCO (Grignani et al. (1 998) supra) in the BamHI/EcoRI cloning site (
CAT assay. COS-7 cells were transiently transfected with a vector carrying the gene for the enzyme chloramphenicol acetyl transferase (CAT) under a LTR promoter responsive to TAT. The cells were incubated with conditioned media from U937/TAT cells for 48 h. The cell lysates were incubated with the substrate of CAT enzyme, 14C-labeled chloramphenicol, in the presence of Acetyl CoA. The products of the reaction, i.e. the acetylated forms of 14C-chloramphenicol, were resolved by thin layer chromatography and visualized by autoradiography.
Peptide libraries. The CX3CX3CX3C and CX10C (C, cysteine; X, any amino acid) libraries were prepared using synthetic oligonucleotides, containing respectively the core sequences TGT(NNK)3TGT(NNK)3TGT(NNK)3TGT and TGT(NNK)10TGT (N=equal molar mixture of A, C, G, T; K=G or T). The oligonucleotides were made double stranded by PCR amplification and ligated to the N-terminus of the pill gene of fUSE5 vector (Scott and Smith (1990, supra).
Cell panning. Cells were washed once in binding medium (DMEM supplemented with 20 mM HEPES and 2% FCS), and resuspended in the same medium at a concentration of 106 cells/mi. 1010 TU were added to 500 μl of the cell suspension, and the mixture was incubated overnight (first round) or for 2 h (successive rounds) at 4° C. with gentle rotation. Cells were washed five times in binding medium at room temperature and resuspended in 100 μl of the same medium. Phage were rescued by adding 1 ml of exponentially growing K91Kan Escherichia coli bacteria and incubating the mixture for 1 h at room temperature. Bacteria were diluted in 10 ml of LB medium supplemented with 0.2 μg/ml tetracycline and incubated for another 20 min at room temperature. Serial dilutions were plated on LB/agar plates containing 40 μg/ml tetracycline, and plates were incubated at 37° C. overnight before colonies were counted. When a significant increase in binding was observed, peptide sequences were inferred from the sequences of the nucleotide inserts derived from PCR analysis as described (Scott and Smith (1990), supra).
Co-cultures and incubations with rTAT. C8166 cells were co-cultured with U937/PINCO or U937/TAT cells in 6-well transwells for 4 h at 37° C., or incubated in culture medium containing rTAT (provided by The Centralized facility for AIDS Reagents, National Institute for Biological Standards and Controls, and the UK Medical Research Council, donor Immunodiagnostic) for 15 min at 37° C. After treatment, cells were extensively washed in PBS, and subjected to immunostaining or transduction with lentiviral vectors. PBMCs were incubated with 7 nM recombinant TAT86 III B for 15 min at 37° C., extensively washed and subjected to infection with HIV-1.
lmmunofluorescence. Cells were pre-fixed in 0,1 % paraformaldeyde in PBS for 15 min on ice, stained with the NT3 2 D1 anti-TAT mAb (provided by The Centralized facility for AIDS Reagents, National Institute for Biological Standards and Controls, and the UK Medical Research Council, donor Dr. J. Karn) diluted 1:20, and revealed with a TRITC-conjugated secondary antibody following a standard protocol. Cells were then fixed in 3% paraformaldeyde in PBS and photographed with an inverted microscope.
GST-fused TAT proteins
The following TATHXB2 variants were produced as GST-fusion proteins as described (Mitola, S. et al. Identification of specific molecular structures of human immunodeficiency virus type 1 TAT relevant for its biological effects on vascular endothelial cells. J Virol 74, 344-53 (2000): TAT86 (full-length), TAT72 (product of TAT first exon), TATBasMut (with the mutations R-49,52,53,55,56,57-A), TATCysMut (with the mutations C-22,25,27-A.
Surface plasmon resonance (SPR) analysis. Evaluation of interaction between TAT and gp120 III B recombinant proteins was performed using a SPR-based BlAcoreX biosensor (BIAcore AB). rTAT was diluted in 5mM maleate pH 6.0 and amine-conjugated to the dextran matrix on a CM5 sensor chip surface (BIAcore AB), in accordance with the manufacturer's protocol. Binding of recombinant gp120 III B was determined over a range of concentrations (1.25-50 nM) in 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% polysorbate 20 (HBS-EP buffer, BlAcore AB) with a flow rate of 5 μl/min at 25° C. Injection volume was 20 ml and flow rate was 5 μl/min. The surface was regenerated with 0.5% SDS. Kinetic analysis was performed using the BIAevaluation 3.0.2 software.
Transduction with HIV-1 -derived lentivirus. Vector stocks were prepared by transient co-transfection of 293T cells with the following combinations of plasmids: pRRL.hPGK.GFP.SIN-18+pCMV.DR8.2+pMD.G (Naldini et al. (1996), supra) (VSV-G-LV) or pEnvHXB (Huang et al., J. Virol. 72 (1998), 8952-8960) (TAT+/gp120-LV); pRRL.hPGK.GFP.SIN-18+pCMV.DR8.74 +pRSV-Rev ((Dull et al. (1998), supra)+pEnvHXB (TAT/gp120 -LV). Vector concentration was determined by HIV-1 p24 Core profile ELISA following the manufacturer's instructions. The entry experiments were conducted on 105 cells per well of a 48-well plate in the presence of 8 μg/ml polybrene. The number of GFP-positive cells was analysed by FACS.
Infection with wild type HIV-1. PHA-stimulated PBMCs (105 cells/well of a 48-well plate) were infected with HIV-1 IlI B or Ba-L for 2 h at 37° C. Samples were then processed as described, with slight modifications (Schmidtmayerova et al., J. Virol. 72 (1998), 4633-4642). Briefly, cells were lysed in 200 μl of 50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45% Nonidet P-40, 0.45% Tween 20, and 100 μg/ml proteinase K. After protein digestion (2 h at 56° C.) and inactivation of the proteinase (10 min at 95° C.), 0.1 μl of cell lysate was subjected to 35 cycles of PCR, with an annealing temperature of 58° C. Amplified DNA was analyzed by Southern blot hybridization with a 32P-labeled probe, and quantified by densitometry. The following primers and probe were used:
SEQUENCES MENTIONED IN THIS INVENTION
SEQ ID No. 4-120 Peptide sequences similar to gp120 (see Table 2); in particular, soluble peptides cited in the Examples:
SEQ ID No. 121-126 Synthetic peptides corresponding to SEQ ID No. 44 with single or double mutations in basic residues (K→A), in particular:
SEQ ID No. 127 Synthetic peptide corresponding to the selected region of gp120
Having now fully described the invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without due experimentation.
While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims. All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. o foreign patents, or any other references, are entirely incorporated by reference therein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.
Reference to known method steps, conventional method steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented therein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teaching and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.
| Number | Date | Country | Kind |
|---|---|---|---|
| 02020649.6 | Sep 2002 | EP | regional |
| 02028510.2 | Dec 2002 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/10162 | 9/12/2003 | WO | 6/7/2006 |
