Title:  Vaccines and immunotherapeutics derived from the human immunodeficiency virus (HIV) trans-activator of transcription protein for the treatment and prevention of HIV disease

United States Patent:  6,667,151

Issued:  December 23, 2003

Inventors:  Cohen; David I. (Pelham, NY)

Assignee:  InIst, Inc. (Rockville, MD)

Appl. No.:  636057

Filed:  August 10, 2000

Abstract

Anti-lentivirus vaccines and immunotherapeutics and methods for preparing and using same are disclosed. The vaccines and immunotherapeutics are produced using non-immunosuppressive lentivirus trans-activator of transcription (Tat) proteins. An associated in vitro ultra-sensitive macrophage Tat bioassay is disclosed for assessing the immunosuppressive qualities of the lentivirus Tat preparations of the present invention. Additionally, a related long-term T4 cell propagation system for characterizing lentivirus Tat is also disclosed. The present invention has additional utility in the treatment and prevention of AIDS.

SUMMARY OF THE INVENTION

For the purposes of clarification, and to avoid any possible confusion, the trans-activating (Tat) proteins of the present invention will be referred to hereinafter as either "C-Tat" when conventional immunosuppressive Tat protein is intended, "ox-C-Tat" for chemically oxidized C-Tat, "IS-Tat" when the immunostimulatory Tat protein found in long-term non-progressors is referred to, or "Tat" when both forms of Tat protein are included.

Therefore, it is an object of the present invention to provide lentivirus vaccines which are derived from non-immunosuppressive lentivirus Tat protein.

It is another object of the present invention to produce HIV vaccines derived from a non-immunosuppressive lentivirus C-Tat.

It is another object of the present invention to produce HIV vaccines derived from IS-Tat.

It is yet another object of the present invention to produce HIV vaccines from a non-immunosuppressive recombinant Tat using the nucleic acid sequence, or portions thereof, of the IS-Tat.

It is another object of the present invention to provide an in vitro ultra-sensitive macrophage Tat bioassay for determining the immunosuppressive activity of a lentivirus Tat protein.

It is another object of the present invention is to provide an in vitro ultra-sensitive macrophage Tat bioassay for determining the immunosuppressive activity of a lentivirus Tat protein.

It is yet another object of the present invention to provide a related long-term T4 cell propagation system for characterizing lentivirus Tat that is immunostimulatory rather than immunosuppressive.

It is yet another object of the present invention to provide an adjuvant component consisting of attenuated, or suitably modified, Tat protein, or immunostimulatory peptides derived from the Tat protein, useful in the formulation of other vaccines.

The Tat vaccines and Tat immunotherapeutics of the present invention are made from either inactivated (chemically or physically altered to render the Tat protein non-immunosuppressant) native C-Tat derived from Human Immunodeficiency Virus (HIV) infected individuals, or native non-immunosuppressive IS-Tat isolated from HIV infected individuals classified as long term non-progressors (LTNP) (collectively referred to herein as Tat vaccines). It is also envisioned that Tat vaccines and/or Tat immunotherapeutics of the present invention may be made using recombinant DNA techniques using either full or partial Tat sequences. The inactivated C-Tat, native IS-Tat and recombinant versions thereof, are tested for immunosuppressive capacity using the in vitro ultra-sensitive, macrophage Tat bioassay of the present invention.

In contrast to the prior art, the Tat vaccines and immunotherapeutics of the present invention are made using lentivirus Tat that is proven to be non-immunosuppressive using the in vitro ultra-sensitive, macrophage Tat bioassay of the present invention. Consequently, the Tat vaccines made in accordance with the methods of the present invention do not suppress the immune system of the recipient, resulting in a vaccine or immunotherapeutic that can be administered using vaccine protocols comparable with other commercial vaccines as opposed to prior art protocols which called for multiple vaccinations over a protracted time period.

The Tat vaccines and immunotherapeutics of the present invention induce the production of lentivirus Tat protein, neutralizing antibodies in the vaccine recipient, thus protecting them from HIV infection. Individuals that are already infected with a lentivirus, specifically HIV, benefit from the administration of the Tat immunotherapeutics of the present invention by producing a Tat neutralizing antibody that reduces the immunosuppressant capacity of the indigenously produced Tat.

In another embodiment of the present invention the IS-Tat made in accordance with the teachings of the present invention, and/or the chemically modified Tat proteins of the present invention, are used to provide adjuvants which stimulate T4 cell proliferation and heighten the immune response to the vaccine components.

In another embodiment of the present invention, the Tat vaccines and Tat immunotherapeutics are also compounded with known adjuvants to further increase the host's immune response to the Tat proteins. These previously-known adjuvants may include, but are not limited to, alum, mineral oil, mineral oil-detergent emulsions, Freund's complete adjuvant, incomplete Freund's adjuvant, liposomes, MF59 (Chiron, Inc., Emeryville, Calif.) IFA51 (Seppic, Inc., Fairfield, N.J.), MPL (Corixa, Seattle, Wash.) and others, just to name a few. In one embodiment of the present invention, oil-based adjuvants may be preferred due to their propensity to improve host immune response to small protein antigens such as Tat.

The in vitro ultra-sensitive, macrophage Tat bioassay of the present invention permits the direct assessment of the immunosuppressant qualities of any Tat protein. The in vitro ultra-sensitive, macrophage Tat bioassays of the present invention have been developed to detect a minimum of 500 pM of C-Tat. In accordance with the teachings of the present invention, macrophages exposed to C-Tat are stimulated to express Fas Ligand (FasL) on their surfaces, the expression of which is then detected. In one embodiment of the present invention FasL expression is detected using anti-FasL antibodies.

Additionally, a related long-term T4 cell propagation system useful for isolating and producing lentivirus IS-Tat is also disclosed.

Further objects and advantages of the Tat vaccines, Tat immunotherapeutics and the in vitro ultra-sensitive, macrophage Tat bioassays produced in accordance with the teachings of the present invention, as well as a better understanding thereof, will be afforded to those skilled in the art from a consideration of the following detailed description of exemplary embodiments thereof.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

For the purposes of clarification, and to avoid any possible confusion, the trans-activating (Tat) proteins of the present invention will be referred to hereinafter as either "C-Tat" when conventional immunosuppressive Tat protein is intended, "ox-C-Tat" for chemically oxidized Tat protein, "IS-Tat" when the immunostimulatory Tat protein found in long-term non-progressors is referred to, or "Tat" when both forms of Tat protein are included.

The present invention provides anti-lentivirus vaccines and immunotherapeutics (specially anti-Human Immunodeficiency Virus (HIV) vaccines and immunotherapeutics), for preventing and treating HIV infection, and a related in vitro ultra-sensitive, macrophage trans-activator of transcription (Tat) bioassay for assessing Tat immunosuppressant activity. The vaccines and immunotherapeutics of the present invention can be made from a variety of Tat sources. In one embodiment of the present invention the C-Tat is derived from cells infected, either naturally or experimentally, with a lentivirus, preferably a human lentivirus. In another embodiment of the present invention IS-Tat is derived from HIV strains associated with a class of patients known as long term non-progressors (LTNP) The IS-Tat is isolated from both naturally and experimentally infected cells. In yet another embodiment of the present invention both C-Tat and IS-Tat are produced using standard recombinant DNA techniques known to those skilled in the art.

The IS-Tat proteins can be isolated and characterized using a long-term T4 cell propagation system developed in accordance with teachings of this invention. In this embodiment, peripheral blood mononuclear cells (PBMC) are isolated and purified from non-infected individuals and infected with HIV isolated from LTNP using methods known to those skilled in the art. By way of example, and not intended as a limitation, approximately 3x10⁶ of freshly isolated uninfected PBMCs are co-cultured with and equal number of freshly isolated PBMCs from LTNPs. These freshly inoculated PBMC/HIV cultures are diluted to a very low density of approximately 100 cells per well in a 96 well tissue culture plate (approximately 100 to 200 .mu.L per well) and maintained in RPMI 1640 containing 10% fetal bovine sera (FBS) (media) at approximately 37^oC. with 5% CO₂. The cultures are monitored every 3-7 days for the appearance viable cell colonies, at which time fresh media is added. The presence of viable, free HIV is monitored using methods which include, but are not limited to, measuring either viral core p24 antigen or the enzyme reverse transcnptase.

Under conditions of very low cell density, cell culture conditions which normally kills primary T4 cells (the cells which host the HIV infection), cell lines which profoundly over-express IS-Tat protein are initiated (designated herein as Tat TcL). The Tat TcL cell lines are rapidly expanded by culture in medium supplemented with interleukin 2 (20 U/ml), or interleukin 4 (10 ng/ml), but most potently by co-culture with limiting numbers (<100) of allogeneic, PHA or anti-CD3+ anti-CD28-stimulated, peripheral blood cells. This IS-Tat differs from C-Tat insofar as it is growth promoting rather than apoptotic and the Tat TcL cell lines of the present invention produced IS-Tat to the virtual exclusion of other HIV viral proteins. When used in accordance with the teachings of the present invention, the long-term T4 cell propagation system described above permits the rapid characterization and robust production of Tat proteins, including IS-Tat. Moreover, the Tat TcL of the present invention may also serve as an in vivo source of IS-Tat by directly administering the Tat TcL cells to a patient.

Recombinant Tat can be made using methods known to those of ordinary skill in the art using expression systems which include, but are not limited to, eurkaryotic and prokaryotics cells. Non-limiting examples of eukaryotic cells suitable for the expression of Tat proteins include insect cells, yeast cells, and mammalian cells. Prokaryotic cell expression systems include bacteria. Suitable nucleic acid sequences include tat genes derived from lentivirus RNA and are available through public domain gene libraries such as, but not limited to, GenBank (National Center for Biotechnology Information). In another embodiment of the present invention, the tat gene is cloned from the Tat TcL cells discovered and sequenced by the present inventor.

Tat TcL genes of the present invention revel sequence variation in two regions of tat, at the amino terminus and within the first part of the second exon. Conventional Tat protein contains two prolines (P) within its first seven residues, a regulatory motif that is frequently observed in proteins designated for protolytic degradation via the proteosome. The second P at residue six is followed by a consensus charged residue at position seven for all clades (closely related taxonomic sub-groups of HIV viruses designated as clades A, B, C, D, etc.), the charged residue mimicking a constitutive phosphorylation that conventionally inhibits proteosomal degradation. For clade B C-Tat, the position seven charged residue is arginine (R). The growth promoting IS-Tats frequently have modified the R to serine (S), an event that would lead to rapid proteosomal degradation of IS-Tat in the absence of S phosphorylation. The second region of extensive variation in the growth-promoting IS-Tat is found in the first part of the second exon between residues 73 and 84. Indeed, some of these IYS-Tats have mutated to entirely lose second exons, while C-Tat proteins as short as 86 residues have been reported to be pro-apoptotic. Moreover, immunostimulatory peptides may ideally be derived from regions of the IS-Tat protein that are variant in LTNP, including but not limited to, the second exon and the amino terminus of the Tat protein.

Conventional-Tat and recombinant C-Tat are chemically or physically inactivated in accordance with the teachings of the present invention prior to being incorporated into the vaccines or immunotherapeutics of the present invention. These C-Tat proteins are inactivated to reduce or eliminate their immunosuppressant activity, which is verified using the in vitro ultra-sensitive, macrophage Tat bioassay of the present invention. The chemical and physical methods of the present invention used to inactivate the C-Tat include, but are not limited to, chemical oxidation and irradiation. In one embodiment of the present invention the C-Tat proteins are chemically oxidized using 3% hydrogen peroxide for one hour at approximately 25^oC. Other methods for chemical oxidation include: 1 mM to 1M sodium periodate for one hour at approximately 25^oC.; 1 mM to 1M peroxyacids for one hour at approximately 25^oC.; 1 mM to 1M m-chloroperbenzoic acid for one hour at approximately 25^oC.; and other chemical and physical oxidative processes known to those skilled in the art. Residual oxidants can be eliminated from the C-Tat preparation by adding a suitable biocompatible oxidizable substrate to the C-Tat preparation after the oxidation is complete. (Oxidized C-Tat preparations are referred to herein colleectively as ox-C-Tat). Samples of suitable biocompatible oxidative substrates include, but are not limited to, glycerol, carbohydrates and similar compounds known to those in the art. Immuno-stimulatory Tat proteins expressed by LTNP do not require inactivation prior to incorporation onto the vaccines and immunotherapeutics of the present invention. However, to verify that the IS-Tat proteins are naturally non-immunosuppressant, it is desired to test them using the in vitro ultra-sensitive, macrophage Tat bioassay of the present invention.

All forms of Tat proteins, C-Tat, ox-C-Tat, IS-Tat, and their corresponding recombinant proteins, are tested for immunogenicity in mice. The ox-C-Tat proteins produced in accordance with the teachings of the present invention were compared with identical vaccines composed of un-inactivated C-Tat. It was surprisingly found that the ox-C-Tat induced a stronger immune response in mice than non-inactivated C-Tat. Table 1 depicts the results of this study. Group I received the C-Tat, group II received ox-C-Tat. Anti-C-Tat titers were remarkably enhanced at 2 weeks and 6 weeks post immunization in the animals inoculated with ox-C-Tat as compared to animals inoculated with immunosuppressant C-Tat. Further studies (data not shown) demonstrated that immunosuppression continued for a minimum of 12 weeks post immunization, at which point the animals were sacrificed.

One possible, non-binding, theory offered to explain the surprising antigenic superiority of ox-C-Tat as compared to C-Tat is that the C-Tat may suppress antigen presenting cell (APC) activity in addition to T4 cell ftmction. To test this non-binding theory, a series of HIV viral antigens preparations were mixed with either ox-C-Tat or C-Tat and formulated into vaccines in accordance with the teachings of the present invention. The antigen preparations included C-Tat plus HIV p24 (a group associated antigen (gag) which makes up part of the viral core), ox-C-Tat plus HIV p24 and controls consisting of either HIV p24 alone and HIV p24 antigen oxidized using the same methods used to prepare the ox-C-Tat (ox-p24).

Vaccines were prepared using 5 .mu.g of C-Tat protein mixed with 5 .mu.g each C-Tat and recombinant p24 (Chiron Corp, San Jose, Calif.) in 100 .mu.l of complete Freund's adjuvant administered subcutaneously in the animals' flanks. Subsequently, sera were collected every other week for antibody response (up to ten weeks), or lymph nodes were harvested at 6 weeks for T cell proliferation assays. Immune response to the vaccine preparations were determined using T-cell proliferative assays described below and measurement of specific antibody responses using an enzyme-linked immunosorbant assay (ELISA). The ELISA assays were performed by methods known to persons of ordinary skill in the art. Briefly, viral antigens and various Tat preparations at 1 .mu.g/ml were applied to plastic, 96-microwell plates in carbonate/bicarbonate coating buffer pH 9.6 overnight at 4^oC., and blocked overnight at 4^oC. in phosphate buffered saline, pH 7.4 (PBS), with 0.05% Tween-20, 2.5% bovine serum albumin (Sigma), and 5% FBS (GIBCO, Grand Island, N.Y.) (blocking buffer). Sera, diluted 1:100, 1:1000, and 1:10,000 into assay buffer (PBS+0.05% Tween 9:1 blocking buffer), were incubated on the coated plates for 1 hr at 37^oC. Reactions were developed with affinity-purified, horseradish peroxidase conjugated anti-human IgG or IgM, or anti-mouse IgG (KPI, Gaithersburg, Md.) for 30 min at 37^oC., followed by tetra methyl benzidine (TMB) substrate, and stopped in 4N H₂ SO₄. Anti-p24 antibodies were measured by commercial ELISA. Plates were read (V Max Pro, Molecular Devices, Sunnyvale, Calif.) for the difference in optical density between 450 nM (signal) and 575 nM (background).

Animals which received the p24/C-Tat (non-inactivated C-Tat) vaccine mixture had a ten-fold weaker p24 immune response that those animals receiving either p24 alone, ox-p24 or p24/ox-C-Tat. Moreover, the animals which received just ox-C-Tat had a seven fold better response to the C-Tat vaccine component that those animals receiving non-inactivated C-Tat alone. These results support the non-binding theory of the present inventor that C-Tat vaccines which are administered without first inactivating C-Tat's immunosuppressant qualities results in a vaccine that acts as an overall immunosuppressant reducing the recipient's immune response to Tat and any other associated immunogens. Table 2 depicts the results of this study. Group I received p24 alone, group II received C-Tat plus p24, group III received ox-C-Tat plus p24 and group IV received ox-p24 alone. Anti-p24 and anti-Tat titers were remarkably enhanced in the animals co-inoculated with ox-C-Tat as compared to animals co-inoculated with immunosuppressant C-Tat.

In order to better understand which cells of the immune system were possibly effected by the immunosuppressant activity of C-Tat, T cell and macrophage cell populations were studied. The role of T cells in the immune response to Tat was assessed using T cells harvested from the animals vaccinated with the C-Tat plus HIV p24 and ox-C-Tat plus HIV p24 described immediately above. The T cells were then exposed to purified HIV p24 antigen at concentrations of 0.02, 0.12 and 2 .mu.g/ml. The T cells derived from the ox-C-Tat/p24 animals demonstrated a typical recall proliferative response (T cells populations previously sensitized to an active immunogen such as a vaccine will increase in numbers rapidly when exposed to the same immunogen post sensitization). However, when T cell populations taken from animals immunized with C-Tat/p24 were exposed to the same p24 preparations, the recall proliferative response was significantly reduced. The blunting, or reduction, in recall proliferative response seen in T cell populations taken from animals co-inoculated with C-Tat as compared the animals receiving ox-C-Tat vaccines demonstrates the immunosuppressive action of un-inactivated C-Tat on T cells. The significant increase in antibody response to antigens co-inoculated with inactivated C-Tat and the markedly superior T cell proliferative response in the same animals as compared to animals vaccinated with un-inactivated C-Tat, combine to support the utility of the vaccines and immunotherapeutics of the present invention.

As previously stated, the in vitro ultra-sensitive Tat bioassay of the present invention is used to assess the immunosuppressant activity of the Tat proteins used in vaccines and immunotherapeutics of the present invention. This assay utilizes fresh macrophage cells substantially purified from human peripheral blood using standard density gradient enrichment procedures, or other cell isolation protocols known in the art. The substantially purified macrophages are washed and then cultured in cell culture media using standard tissue culture techniques. In one embodiment of the present invention, the substantially pure macrophages are cultured in RPMI 1640 supplemented with 10% FBS at 37^oC.

The in vitro ultra-sensitive macrophage Tat bioassay of the present invention is performed using a positive control (FasL inducing compound) and a negative control (no active compound is added to the culture). Suitable positive controls include, but are not limited to, lipopolysaccharide (LPS) and/or tissue necrosing factor alpha (TNF .alpha.) at a final concentration of 100 ng/mL and at 50 ng/mL respectively. Test samples (Tat preparations) are run at final concentrations ranging from 50 pM to 50 nM and include C-Tat, ox-C-Tat, IS-Tat, Tat proteins which has been pre-reacted with antibodies and other combinations and Tat preparations.

The test samples and controls are individually mixed with the substantially pure macrophages seeded at a density of 10⁶ cells/mL in round bottom tubes (Falcon 2059) containing RPMI 1640 with 10% FBS (herein referred to collectively as assay cultures). The assay cultures are then incubated for a suitable period of time, preferably from five to six days, at 37^oC. An enriched carbon dioxide atmosphere may or may not be required depending on whether optional buffering systems (e.g. HEPES) have been added to the RPMI 1640 cell culture medium. In the absence of such optional buffering systems, a 5% carbon dioxide incubation environment is preferred.

At the end of the incubation period, cells are removed from each assay culture and the presence of any induced FasL expression is detected. In one embodiment of the present invention, the test and control cells are washed and stained using an anti-FasL antibody. Suitable antibodies include, but are not limited to, monoclonals such as NOK-1, NOK-2, G247-4 (Pharmingen, San Diego, Calif.), mab 33 (Transduction Laboratories, Lexington, Ky.), C20 (Santa Cruz Biotechnology, Santa Cruz, Calif.) or MIKE-2 (Alexis, San Diego, Calif.). The cells are then washed again and stained with an appropriate detection reagent such as, but not limited to, a fluorescein isothiocyanate (FITC) conjugate as known to those of skill in the art. If mouse monoclonals are used, an anti-mouse FITC conjugate is preferred (for suitable non-limiting examples see the Sigma Chemicals Biochemical and Reagents for Life Sciences Research Catalogue 1999 edition, pages 1367-1370, Sigma Chemicals, St. Louis, Mo.). For anti-FasL antibodies derived from other sources, an appropriate anti-species FITC conjugate is used. It is also within the scope of the present invention to use anti-FasL antibodies pre-conjugated to an appropriate detection reagent such as a fluorescent dye including FITC.

After the substantially pure macrophages have been stained with an anti-FasL antibody and the detection reagent, the cells are washed and the reaction between the anti-FasL and the surface of the substantially pure macrophage is determined using a detection system suitable for the detection reagent used. For example, if a fluorescence conjugate detection reagent is used, the macrophage-anti-FasL reaction is detected using a fluorescent activated cell sorter (FACS) system. Control staining is performed using the fluorescent detection reagent alone and subtracted from the FasL staining seen in the assay cultures. Using the data thus obtained the number of FasL positive macrophages and the intensity of the reaction is determined. FIGS. 1 and 2 depict scattergrams using such a FACS system. The greater the percentage of FasL positive cells in a given assay culture, the more immunosuppressant the compound in the assay culture is. Negative controls should always remain non-reactive with the anti-FasL antibody and the positive control should fall within predetermined ranges. The acceptable range for a given positive control will depend upon the control materials used, the detection reagent, and the detection system used.

The inventor of the present invention has developed vaccines, immunotherapeutics and related bioassays for the treatment and prevention of lenitvirus diseases including Acquired Immune Deficiency Syndrome (AIDS). Moreover, the present invention provides methods for treating lentivirus diseases by administering the immunostimulatory forms of Tat proteins of the present invention. These immunostimulatory Tat proteins include, but are not limited to, recombinant IS-Tat, natural and synthetic LTNP peptide, native IS-Tat and attenuated native Tat. Furthermore, the Tat TcL cell lines of the present invention may also be used as an in vivo source of immunostimulatory Tat when the Tat TcL cell line is directly administer to the mammal. In addition, co-infection of a lentivirus infected mammal using a viable virus isolated from a LTNP can also serve as an in vivo source of immunostimulatory IS-Tat.

While this invention has been described with respect to various examples and embodiments, it is to be understood that the invention is not limited thereto and that it can be variously practiced within the scope of the following claims

Claim 1 of 15 Claims

I claim:

1. An in vitro method for measuring the immunosuppressive activity of lentivirus Tat proteins comprising:

a) obtaining a substantially pure population of macrophage cells purified from mammalian peripheral blood, wherein said macrophage cells do not express FasL;

b) suspending said macrophage cells in cell culture media to form a population of in vitro cultured macrophage cells;

c) admixing said in vitro cultured macrophage cells with a lentivirus Tat protein containing sample;

d) incubating said in vitro cultured macrophage cells for a sufficient time and under sufficient conditions for the expression of FasL; and

e) detecting FasL expression by said in vitro cultured macrophage cells wherein said expression of FasL is indicative of said immunosuppressive activity of said lentivirus Tat protein.

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