Patent Application
20100310592
The present invention generally falls within the immunology field and in particular relates to a truncated form of the HIV p17 protein that is particularly suited to be used as a vaccine against the HIV-1 virus.
The development of an effective anti-HIV-1 vaccine represents one of the main objectives for the worldwide scientific community. Such a vaccine must primarily be able to evoke both a significant cell response and the development of a vigorous humoral activity, the latter characterized by the production of antibodies capable of neutralizing the infectivity of the virus.
In these last years, the attention of the scientific world has been directed to virus-derived polypeptides to be used as a basis for the design of antiviral vaccines.
The p17 protein, which composes the matrix of the HIV-1 virus, represents a biological target that is particularly interesting from such a point of view. In fact, even though it has been classified as a structural protein, it is now clear that p17 performs several functions in many phases of the viral replication cycle, from the entry of the virus into the cytoplasm to the integration of the viral genetic material into the cellular one up to the assembly of new virus particles, thus playing a role of primary importance not only in the virus architecture but also in the HIV-1 replication cycle.
Recent studies have enabled to point out that p17 has a structure very similar to that of gamma-interferon, a human pro-inflammatory cytokine. Moreover, cell biology studies have enabled to point out the ability of p17 to modulate the biological activity of several immune system cells, both from the native and adaptive compartments. In particular, the activity of p17 on the CD4+ T lymphocyte subpopulation, the preferential target for HIV-1, has showed that this viral protein is able to stimulate the lymphocyte cells by activating them and thus making them more susceptible to viral infection. Furthermore, p17 is able to induce the activated T lymphocytes to release pro-inflammatory cytokines, such as gamma-interferon and tumor necrosis factor-alpha, into the cell culture microenvironment, thus generating a favorable environment for the optimum HIV-1 replication.
The activity of p17 as a “virokine” depends on the direct interaction of the protein with a specific receptor expressed on the surface of the target cells.
The evidence that p17 is able to act, from the biological point of view, on HIV-1 target cells, by affecting their functionality and inducing an increase in the HIV-1 replication activity, causes such a protein to be considered as an excellent target for the establishment of vaccination strategies against AIDS. An essential prerequisite is that p17 should be released into the cell microenvironment from HIV-1-infected cells. Actually, in vitro HIV-1-infected H9 cells release high amounts of p17 into the cell culture supernatant. Furthermore, recently it has been proven that p17 is present as a protein deposit in the lymph nodes of HIV-1 seropositive patients, also in the anti-retroviral therapy (HAART). It is interesting to note that p17 was detectable in the lymph node tissue in the absence of viral particles and/or viral genomes. This suggests that p17, as already demonstrated for other HIV structural proteins, acts on certain target cells as an exogenous protein, independently from the presence of the virus. This explains the data obtained by some researchers which pointed out a connection between the presence of high levels of anti-p17 neutralizing antibodies and a significant delay of the acquired immunodeficiency syndrome progression. As p17 is inside the glycoprotein coat in the viral architecture and as such not accessible to antibodies, such data were inexplicable before finding out that p17 is released by the infected cells.
The International patent application WO03/016337 describes short peptides isolated from HIV p17, which represent the p17 neutralizing epitope (residues from 9 to 22) and which are capable of evoking a neutralizing immune response when administered as a vaccine.
However, the use of peptides that only represent limited epitopic regions as the basis for the formulation of vaccines or as molecules capable of stimulating and directing the humoral immune response shows some drawbacks, the major one depending on the fact that peptides usually exhibit a different conformation from that showed by the full-length protein. As a result, the antibodies produced following the immunization with such peptides bring about a poor recognition of the native viral protein and therefore generate a low-efficiency humoral immune response.
The International patent application WO03/082908 describes the use of the full-length p17 protein as a vaccine in order to evoke an immune response capable of neutralizing the immunostimulating activity exerted by p17 on human cells. However, the use of the full-length protein as a vaccine shows the drawback that the administered full-length protein is capable of having the same noxious biological effects as the native viral protein.
To overcome such drawbacks, the present invention provides a truncated form of the p17 protein, designated as “AT96”, which exhibits the same immunogenic features as the full-length p17 protein, but without showing the typical noxious biological activities of the full-length protein.
The truncated AT96 protein of the invention consists of the amino acids 1-96 of the p17 protein. Preferably, AT96 is encoded by the nucleotide sequence shown as SEQ ID NO:1 in the Sequence Listing. Still more preferably, AT96 has the amino acid sequence shown as SEQ ID NO:2 in the Sequence Listing.
The truncated AT96 protein of the invention is a very promising anti-HIV therapy molecule because, as demonstrated in the experimental section below, even though it is missing the p17 carboxy-terminal end (that is the amino acids 97-132), which is important for the biological activity of the native protein, still it retains the immunogenically important epitopes, namely the epitopes capable of promoting the neutralizing cell-mediated and humoral immune response.
The truncated AT96 protein of the invention has been produced by per se known recombinant DNA methods.
In short, the AT96 protein was produced by amplifying the nucleotide sequence encoding for the amino acids 1-96 of p17 and then cloning it into the prokaryotic pGEX-4T expression vector (GE Healthcare). Such a nucleotide sequence was obtained by mutational PCR starting from the sequence encoding for the full-length p17 derived from the BH10 virus strain (NCBI accession number M15654). The nucleotide sequence of the full-length p17 used is shown in the Sequence Listing as SEQ ID NO:3 and the corresponding encoded amino acid sequence is shown as SEQ ID NO:4.
By using the specially designed mutagenic AT96BamHI and AT96ECORI primers, the following were created:
The sequences of the AT96BamHI and AT96ECORI primers are shown in the Sequence Listing as SEQ ID NO:5 and SEQ ID NO:6, respectively.
The amplification reaction (final volume 200 μl) was carried out by using 20 ng of a plasmid containing the nucleotide sequence of the full-length p17 as a template. The conditions used for the PCR reaction were the following: 94° C., 30 sec; 50° C., 30 sec; 72° C., 60 sec for a total of 30 cycles. The amplified product was purified by using standard protocols (Qiaquick PCR Purification Kit, Qiagen), then digested with the restriction enzymes BamHI and EcoRI and finally cloned into the multiple cloning site of the pGEX-4T plasmid, previously digested with the same restriction enzymes. The obtained construct (pGEX-AT96) was sequenced by using the Big Dye Terminator labeling (Applied Biosystems) in conjunction with analysis by sequencer and capillary chromatography (ABI PRISM® 7700) following the standard protocol.
The recombinant DNA technology was also used for the production of the AT96 protein according to per se known methods.
The pGEX-AT96 construct (20 ng) was introduced into E. coli (BL21) by electroporation and the transformed bacteria were selected on plates containing ampicillin. The presence of the AT96 insert in the selected colonies was checked by extraction, purification and restriction of the plasmid DNA.
The positive colonies were used to set up high-volume (2-4 liters) production cultures in liquid LB medium with ampicillin. The bacteria were incubated at 37° C. until reaching an optical density value corresponding to the beginning of the plateau phase (0.8 AU) and then induced to produce the protein by the addition of IPTG (isopropyl-β-thiogalactopyranoside, Sigma Aldrich) up to a final concentration of 100 mM and at the temperature of 30° C. The IPTG, by removing the block on the lacZ operon, allows the recombinant protein to be expressed in high amounts.
The AT96 protein, produced as a fusion with the GST enzyme (Glutathione-S-Transferase, the sequence of which is already inserted into the vectors of the pGEX series), was extracted by sonication of the bacterial cells and purified by using an affinity column made up by Glutathione Sepharose 4B beads (GE Healthcare), which, as being extremely related to the GST enzyme, capture the fusion protein in a selective way.
AT96 was then separated from GST by proteolytic excision with the thrombin protease (GE Healthcare). Alternatively, it may be eluted from the glutathione sepharose matrix by using a buffer containing reduced glutathione and kept in solution with the aid of chaotropic substances. The protein was finally subjected to buffer exchange, by dialyzing it against a sodium chloride solution with a 3500 kDa cut-off cellulose membrane (Pierce), and was further purified by HPLC (GE Healthcare) with an ion exchange column.
The immunogenic features of the truncated protein were checked experimentally as described in the following experimental section, which refer to the attached figures, wherein:
The AT96 responsiveness to the anti-p17 antibodies capable of binding to the p17 neutralizing epitope was assessed by immunoenzymatic (ELISA) and Western blot assays (
In order to verify that the conformation of the functional epitopes at the interaction with the cell receptor is retained in the AT96 protein, a competition ELISA test was established to evaluate the ratio of the AT96 interaction in solution with the MBS-3 monoclonal antibody, which recognizes the functional portion of p17 and inhibits the p17/receptor interaction [1]. The activity of AT96 in liquid phase as a competitor on the binding between MBS-3 and p17 present in the well (solid phase) was then compared to that of p17 used as a self-competitor.
The purified antibody was incubated with increasing concentrations of the AT96 or p17 protein and then the immunocomplexes obtained were used for ELISA assessment of the MBS-3 binding activity to the wild-type p17 fixed to the solid phase (on the bottom of the plate well). Recombinant p17, derived from the BH10 viral strain, was added to each well of a polystyrene plate for immunoenzyme assays at the concentration of 1 μg/ml in 100 μl PBS, the plates were incubated overnight at room temperature. The antigen-coated plates are designated as detection plates; whereas the uncoated plates are designated as reaction plates. In order to minimize the non-specific absorbance of the proteins, 200 μl of PBS containing 2% BSA (assay buffer) were added to each well of the detection plate. Both the reaction and detection plates were incubated for 1 hour at 37° C. The plates were washed with PBS containing 0.1% tween 20 (wash buffer). To each well of the reaction plate, 100 μl of the appropriate dilution of MBS-3 anti-p17 monoclonal antibody were added, as well as 100 μl of AT96 at concentrations ranging from 0.1 to 25 μg/ml assay buffer, or 100 μl of assay buffer alone. After a 2 hour incubation at room temperature, 100 μl aliquots were transferred from the wells of the reaction plate to those of the detection plate. The detection plates were incubated for 2 hours at room temperature and then washed four times with the wash buffer. The MBS-3 monoclonal antibody binding in solid phase was detected by adding an HRP-conjugated anti-mouse antibody.
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Cells: The ability of AT96 to bind the p17 cellular receptor was assessed on cells from the Raji lineage, that is to say Burkitt's lymphoma cells that exhibit a high expression of the p17 receptor. The cells were cultured in RPMI 1640 medium (Gibco BRL) containing 10% FCS (fetal calf serum), 100 U/ml penicillin, 50 μg streptomycin and 1 mM L-glutamine.
Conjugation of AT96 to biotin: The AT96 protein was conjugated to biotin to allow for the detection thereof by cytofluorometry through covalent binding to fluorescent streptavidin. The protein was subjected to buffer exchange through a dialysis membrane (3500 kDa cut-off, Pierce) against a sodium chloride/sodium bicarbonate solution, pH 8.0. The protein was then reacted with biotin succinimide ester (BioSPA) for 3 hours at 4° C. and further dialyzed overnight against PBS (phosphate buffered saline) containing 0.2 M sodium chloride.
Binding and neutralization tests: The ability of the sera collected from animals immunized with a peptide that mimics the amino-terminal portion of p17, designated as AT20 (SEQ ID NO: 7 in the Sequence Listing), to block AT96 binding to the p17 receptor (p17R) was assessed by cytofluorometry. The sera of AT20-immunized animals, diluted in PBS (1:50), were incubated with biotinylated AT96 (50 ng) for 20 minutes at 4° C. Raji cells were treated for 15 minutes at 4° C. with FcR blocking reagent (Miltenyi Biotech), centrifuged and then re-suspended in the sera pre-incubated with AT96. Sera from animals immunized with a peptide that mimics the carboxy-terminal portion of p17, designated as CT18, were used as a control. The cells were then washed with PBS and incubated on ice with 100 ng of streptavidin conjugated to the APC or PE Cy5.5 fluorochrome (Becton Dickinson). The cytofluorometric analysis of the samples was performed with a FACSCalibur instrument and the data were analyzed with the CellQuest Pro program (Becton Dickinson).
Synthetic peptides: The AT20 synthetic peptide consists of the 20 amino-terminal amino acids of the p17 protein, included between the amino acid positions 9 and 28 (SEQ ID NO: 7). Whereas the CT18 synthetic peptide consists of the 18 carboxy-terminal amino acids of p17, included between the amino acid positions 115 and 132 (SEQ ID NO:8). These peptides were synthesized in the free form and then were conjugated to the KLH (Keyhole Limpet Hemocyanin) carrier from Primm.
Immunizing protocol: C57BL/6 mice were immunized by the intraperitoneal route with AT20-KLH or CT18-KLH emulsified with Freund's complete adjuvant at the doses of 1, 5 and 25 μg/mouse and boosted, for 2 consecutive times at a 15 day interval, with the same dose of immunogen in incomplete adjuvant. The detection of the anti-AT20 and anti-CT18 antibodies in the sera of the immunized animals was carried out by ELISA.
ELISA: The presence of anti-AT20 and anti-CT18 antibodies in the sera of the immunized mice was assessed by an ELISA test. The wells of an ELISA plate (Nunc) were covered with 100 ng of AT96 protein overnight at room temperature in PBS. After 2 washes with the wash buffer (PBS+0.1% tween), 100 μl of the assay buffer (PBS+2% BSA) were added and incubated at 37° C. for 1 hour. After 4 washes, 100 μl of stepwise dilutions of the sera to be tested (from 1:100 to 1:3200) were transferred into each well. After incubation for 1 hour at 37° C., 4 washes were performed and 100 μl/well of a 1:1000 dilution of HRP-conjugated anti-mouse antibody were added. After incubation for 1 hour at 37° C., 4 washes were performed and 100 μl of the tetramethylbenzidine substrate were added. The chromogenic reaction was stopped with 100 μl of 2N sulphuric acid per well.
The experiments so far illustrated have allowed to establish the integrity of the amino-terminal portion of the AT96 protein, hypothesized as the region that allows p17 to interact with its cellular receptor. As a result, AT96 should be able, just like the wild-type p17 protein, to interact with the receptor on the cell membrane.
Experimentally, this has been assessed by assaying the binding of AT96 to B cells from the Raji lineage which express the p17 receptor on their surface at a high density. The cells were incubated with the biotin-conjugated AT96 protein for 30 minutes. After several washes, the binding of biotinylated AT96 to the cell surface was detected by adding fluorescent streptavidin (conjugated with the APC or PE Cy5.5 fluorochrome).
Similarly, the ability of antibodies directed towards a specific amino-terminal neutralizing epitope, designated as AT20 [2], to block the in vitro interaction between p17 and the cellular receptor was also assessed. The AT96 protein was incubated with sera from animals immunized with the peptide that reproduces the sequence of the amino-terminal epitope of p17 (AT20) or with sera from animals immunized with a peptide capable of mimicking a carboxy-terminal sequence of p17 and not existing in AT96. The AT96/p17 receptor interaction was then detected by using fluorescent streptavidin as the fluorescent tracer. From the executed experiments, AT96, just like wild-type p17, resulted to be neutralized, with regard to its binding activity to the specific receptor, by antibodies directed towards the amino-terminal portion (
Recently, it has been demonstrated that p17, as an antigen, is able to induce neutralizing and cell-mediated humoral immune responses in animal models [2]. Furthermore, p17 is able to act as a booster antigen in vitro, inducing proliferation of T lymphocytes taken from animals previously immunized with the viral protein.
Immunizing protocol: The data obtained in the present study, which was carried out by using the full-length p17 and the truncated AT96 protein of the invention in parallel, point out that the latter is also capable of inducing neutralizing humoral responses. The immunization was performed as follows: 30 9-week-old mice were immunized with different doses of p17 and AT96 administered in the presence of Freund's incomplete adjuvant. Each group, consisting of 5 mice, was administered by the intra-peritoneal route with 1, 5 and 25 μg/mouse of p17 or AT96 protein. Subsequently, 2 consecutive booster injections were given at a 15 day interval with the same dose of immunogen. The negative control was prepared by inoculating PBS and Freund's incomplete adjuvant into the mice. The immunized mice were bled at 0, 10, 24, 35 days post-immunization.
The immunization schedule with p17 and AT96 for Balb/mice is given in the following Table 1.
ELISA: The sera were stored at −20° C. The serum reactivity against AT96 and p17 was tested by solid-phase ELISA, the results of which are shown in
Neutralization test: The sera obtained through the immunizing program were used to perform neutralizing experiments on the interaction with the p17 or AT96 protein receptor. Raji cells were incubated for 30 minutes on ice with different amounts of biotinylated p17 or AT96, from 10 to 400 ng/ml. Subsequently, the cells were labeled for 30 minutes on ice with PE-conjugated streptavidin. For the neutralizing experiments, the Raji cells were incubated with the immunocomplexes obtained by pre-incubating the biotinylated p17 with sera from immunized (Ab) or non-immunized (K) animals at a 1:100 final dilution. The inhibition ratio was calculated as follows: % of receptor-positive cells in K−% of receptor-positive cells in Ab/% of receptor-positive cells in K. The Table 2 below shows the neutralization of the AT96/p17R or p17/p17R interaction by antibodies generated in animals immunized with different doses of p17. The % inhibition was calculated as indicated above.
The AT96 protein resulted to be also able to induce, as the full-length p17, cell-mediated responses in animal models and to act in vitro as a booster antigen, inducing proliferation of T lymphocytes taken from animals immunized with the maximum dose of p17 or AT96 (
Proliferation experiments: For the execution of the proliferation experiments, mouse spleen cells were sown into 96-well U-bottom cell culture plates at 2×105 cells/well and grown in complete RPMI-1640 medium (containing penicillin, streptomycin and serum) in the presence or absence of p17 or AT96 (10 μg/ml). 5 Days later, 1 μCi of tritiated thymidine was added to the cells and these were then sacrificed after further 18 hours of culture. The data are presented as a stimulation index (SI), which is defined as the ratio of the amount of tritiated thymidine incorporated by the cells in the presence of the antigen (AT96 or p17) and the amount of tritiated thymidine incorporated by the cells in the absence of the antigen.
The cytofluorometric analyses of the spleen cell cultures extracted from animals immunized with 25 μg of AT96 and re-stimulated in vitro for seven days with the same protein, also show the specific expansion of both CD4+ and CD8+ T lymphocytes (
The biological functionality of p17 was assessed on Raji cells. First of all, the ability of p17, subsequent to its binding with the cellular receptor, to affect the activity of the cell kinases was estimated, the which cell kinases are enzymes activated through phosphorylation, which in turn are able to phosphorylate different cellular substrates, activating them. In particular, the MAPKs (ERK 1/2) and pAKT were assessed, the cell survival and the inhibition of the pro-apoptotic processes depending on the activation thereof.
The biological power of the p17 protein is demonstrated by its influence on the phosphorylation index of the kinases, also detected at low doses and after an exposition time of a few minutes. The phosphorylated kinases in the Raji cells were detected by the Western blot method using monoclonal antibodies as specific reagents (Santa Cruz). The Raji cells were stimulated for 5 minutes with different concentrations (from 25 ng/ml up to 2 μg/ml) of the p17 protein. In
Consequent to this important demonstration of the biological activity of p17 in Raji cells, we similarly proceeded to estimate the biological activity of the AT96 protein. Surprisingly, unlike what was revealed with the full-length p17, the AT96 protein proved not to be able to inhibit the phosphorylation of ERK 1/2 and pAKT, which were still active (phosphorylated) even at AT96 concentrations of 2 μg/ml (
Thus, it can be inferred from the experimental data obtained that AT96 exhibits the same immunogenic features as the wild-type p17 protein, evoking both humoral and cell-mediated immune responses that are qualitatively and quantitatively similar. Also with regard to its interaction with the cellular receptor, AT96, as the wild-type p17 does, results to be able to bind the cellular receptor. Nevertheless, AT96 and the wild-type p17 differ greatly as for the cellular signal that leads to the kinase phosphorylation-mediated cell activation. Such a difference implies that the truncated AT96 protein of the invention is substantially devoid of those cell kinase activating biological activities that contribute to the activation of pro-apoptotic processes clearly noxious for the cell. On the contrary, the full-length p17 protein has such cell-damaging biological activities, which prevents the effective use thereof in anti-HIV treatment strategies.
The use of the viral truncated AT96 protein of the invention represents an extremely valuable and innovatory approach to the development of therapeutic and vaccine strategies against AIDS. Given the great importance of the matrix p17 protein in the biology of the virus, this protein represents a biological target of great interest.
The removal of the carboxy-terminal portion of p17 caused no structural alterations in the immunogenic epitopes. Moreover, the AT96 protein was shown to maintain the ability to interact with the cellular receptor.
The experimental evidences gathered during the course of this study have also allowed to extrapolate extremely significant structure-function features related to the HIV matrix p17 protein:
On the basis of these findings, it is evident that the truncated AT96 protein of the invention may be extremely promising for the development of anti-HIV-1 vaccine strategies designed to evoke specific neutralizing, cell-mediated and humoral, immune responses without biological activities that are potentially detrimental to an HIV-1-infected organism.
Therefore, the use of the truncated AT96 protein according to the invention as a medicament, particularly as a medicament designed to evoke an immune response that neutralizes the biological activity of the HIV p17 protein, and thus suitable for use in the treatment or prevention of HIV infections, falls within the scope of the present invention.
To this end, the truncated AT96 protein of the invention may be manufactured in the form of a pharmaceutical composition, preferably an immunogenic or vaccine composition, comprising a pharmaceutically effective diluent or carrier and optionally an adjuvant. The immunogenic or vaccine pharmaceutical composition of the invention is administered through any suitable administration route including—without any limitation—the intravenous, subcutaneous, intramuscular, nasal, mucosal routes, and so on. The selection of the types and amounts of excipients, diluents, adjuvants, and carriers, which may be selected depending on the specific administration route chosen, falls within the abilities of the person of skill in the art. The immunogen dose in the pharmaceutical or vaccine composition of the invention also varies depending on several factors and the determination thereof falls within the abilities of the person of skill in the art, also taking into account the indications provided in the experimental part of the description. It is however possible to contemplate a dose comprised within the range of 1-200 μg, preferably 10-100 μg.
| Number | Date | Country | Kind |
|---|---|---|---|
| TO2008A000027 | Jan 2008 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB08/55341 | 12/16/2008 | WO | 00 | 7/13/2010 |
