The present invention relates to a composition and method for enhancing the efficacy of a vaccine in a subject treated with the vaccine by administering to the subject an antigen in conjunction with a chemokine.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates generally to the use of chemokines, particularly chemokines which attract lymphocytes or antigen presenting cells, for enhancing an immune response to an antigen, particularly to a vaccine antigen.

In one aspect, the invention relates to a method for enhancing the efficacy of a vaccine in a subject. The method generally comprises administering to the subject: a first component selected from the group consisting of: (i) an antigen against which an immune response is desired in the subject; and (ii) a nucleic acid encoding the antigen of (i). In addition to the first component, the subject is also administered a second component selected from the group consisting of: (i) a chemokine selected from the group consisting of RANTES, MCP-1, MDC and BLC, or a functional equivalent (as defined herein) of said chemokine; and (ii) a nucleic acid encoding the chemokine of (i). The first component and second component are administered in an immunizingly effective amount (as defined herein).

In one aspect of the invention, the second component comprises a human chemokine or a functional equivalent thereof.

In another aspect of the invention, the second component is administered concurrently with the first component. The second component may also be administered within a time period before or after administration of the first component, which time period is sufficient to achieve enhancement of the efficacy of the vaccine.

The first component may be any of a wide variety of antigens known in the art. However, in a preferred embodiment of the invention, the first component is an HIV antigen. A preferred HIV antigen is a gp120 antigen, which in addition to a native gp120, also includes analogs, derivatives and fragments of gp120 which produce an antibody response (i.e., polypeptides which are functionally equivalent in that they produce an antibody response wherein the antibodies produced by such response will bind to native gp120), which antibodies also have specificity for a native HIV gp120.

In another aspect, the invention relates to a nucleic acid encoding both the first component and the second component.

In a method aspect of the invention, the first component and the second component are provided as nucleic acid sequences on the same or on separate nucleic acids and are administered directly to the subject. The first component and the second component may also be provided as nucleic acid sequences on the same or on separate nucleic acids and may be used to transform a cell, which cell is administered to the subject. The first component suitably comprises a nucleic acid encoding an HIV antigen, preferably a gp120 antigen. The subject is preferably a human and may be HIV positive or may exhibit behavioral patterns or occupational factors associated with risk of becoming HIV positive.

In another aspect, the invention relates to a method for improving the speed of an antibody response to a soluble antigen in a subject, comprising co-administering to the subject the soluble antigen with BLC. The soluble antigen is preferably an HIV antigen, more preferably a gp120 antigen. The subject is preferably a human.

The invention also provides a method for reducing the number of immunizations in an immunization regimen required to achieve an improvement in a subject's immune response to an antigen. This method generally comprises administering MDC and the antigen to the subject. The antigen may suitably be administered before, during or after the administration of MDC. However, the antigen is administered in sufficient temporal proximity to the administration of MDC to achieve an improvement in the subject's immune response to the antigen. The antigen is preferably an HIV antigen, and the subject is preferably a human.

In another aspect, the invention provides a method for suppressing an immune response in a subject in need thereof. This method generally comprises administering to the subject an amount of an MCP-1 antagonist which, in comparison to a corresponding immune response in the absence of the MCP-1, suppresses the immune response.

In yet another aspect, the invention provides a method for inducing or enhancing a humoral response in a subject in need thereof, the method comprising administering to the subject a humoral response-inducing amount of BLC.

The invention also relates to a method for inducing a subject to produce MIP-1.alpha.. This method aspect of the invention generally comprises administering to the subject an MIP-1.alpha.-inducing amount of RANTES. The subject is preferably HIV positive.

The invention also relates to compositions for achieving the various method aspects of the invention. For example, in one aspect, the invention relates to a composition comprising a first component selected from the group consisting of: (i) an antigen against which an immune response is desired in the subject, and (ii) a nucleic acid encoding the antigen of (i); along with a second component selected from the group consisting of: (i) a chemokine selected from the group consisting of RANTES, MCP-1, MDC and BLC, or a functional equivalent of said chemokine, and (ii) a nucleic acid encoding the chemokine of (i). This composition preferably also comprises one or more of each of the following pharmaceutically acceptable components: carriers; excipients; auxiliary substances; adjuvants; wetting agents; emulsifying agents; pH buffering agents; and other components known for use in vaccine or other pharmaceutical compositions.

The invention also relates to a nucleic acid comprising: a first nucleic acid sequence encoding an antigen against which an immune response is desired in the subject; and a second nucleic acid sequence encoding a chemokine selected from the group consisting of RANTES, MCP-1, MDC and BLC, or a functional equivalent of said chemokine. The first and second nucleic acid sequences are preferably expressed in a coordinated manner upon introduction into a subject to produce an amount of the first component that is immunogenic and an amount of the second component that is effective to enhance the efficacy of the vaccine. A related aspect of the invention involves the administration of this nucleic acid to a subject in need thereof to elicit an immune response to the antigen. The nucleic acid is suitably administered as a component of a pharmaceutical composition and may be administered directly to the subject and/or introduced into a suitable host cell and said suitable host cell is administered to the subject. The host cell may be obtained from the subject or from a cell culture originating from one or more cells obtained from the subject.

In a preferred embodiment, the invention provides a method for enhance the efficacy of an HIV vaccine. A preferred polypeptide for use with the HIV vaccine is gp120.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for enhancing the efficacy of a vaccine in a subject comprising administering to the subject (i) an antigen or a nucleic acid encoding an antigen and (ii) a chemokine and/or functional equivalent thereof or a nucleic acid encoding a chemokine and/or functional equivalent of the chemokine.

In empirical work supporting the present application, the following four chemokines were compared:

RANTES (regulated on activation, normally T cell expressed and secreted), which has a broad chemoattractant activity, for T cells and monocytes/macrophages, as well as basophils, eosinophils, natural killer cells, mast cells and dendritic cells, but is unable to attract B cells. It functions through CCR-1. CCR-3, CCR-5 and CCR-9 (Kim, C. H. et al., 1999).

MCP-1 (monocyte chemotactic protein-1), which is chemoattractant for monocytes and T cells, as well as monocytes/macrophages, basophils, eosinophils, natural killer cells, mast cells and hematopoietic progenitor cells (Yoshimura, T., 1989) via the receptors CCR2B and CCR-9 (Kim, C. H., et al., 1999).

MDC (macrophage-derived chemoattractant), which causes chemotaxis of monocytes/macrophages, activated natural killer cells and dendritic cells (Bochner, B. S., et al., 1999) by activating via CCR4 (Bochner, B. S., et al., 1999), although there are indications that MDC also functions via other still unknown receptors (Bochner, B. S., 1999; Struyf, S., 1998).

BLC (B lymphocyte chemoattractant) is involved mostly in chemotaxis of B lymphocytes (Legler, D. F., et al., 1998). It induces the formation of germinal centers by directing B cells to follicles of secondary lymphoid tissues (Foerster, R., 1996) and functions via CXCRS (BLR-1 or BCA-1) (Gunn, M. D., et al., 1998).

The receptor-specificity, cellular expression patterns and their chemoattractant are set forth in the following table -- see Original Patent.

Chemokines as Modulators of Immune Response

We hypothesized that RANTES, MCP-1, MDC, and BLC could modulate the elicitation of antigen-specific immune response. Such modulation would depend on the cell types responsive to each chemokine. Accordingly, we investigated these chemokines in immunization experiments using HIV-1.sub.Bal. Gp 120 and gp 160 as antigens. Modulation of immune response has been shown for several chemokines and cytokines, such as 11-12 (Sin, J. L. et al., 1999), 11-2, 114 and 11-10 (Chun, S., et al.,1998), RANTES (Xin, K. Q., et al., 1999), and MCP-1 (Ki,m J. J., et al., 1998). However, there is a critical lack of data investigating the role of chemokines in modulating immune response in a comparative way, and chemokines that are potentially important in recruiting antigen presenting cells (APCs) have not been investigated.

Possible mechanisms for modulation of the immune response by chemokines might involve augmented probability of assembling a target (antigen or antigen presenting cell (APC)) and effector (B or T cell) by chemoattraction. In addition, chemokine-mediated direct activation of the cells involved in the immune response may also take place in this system: locally increased chemokine concentration can stimulate the attracted cells in a paracrine manner (Behringer, D., 1997). Finally, the interaction between effector cells and APCs, upon recruition by chemokines, namely CD4-T cells (helper cells) and CD.delta. T cells or B cells, leads to mutual activation. In fact, the encounter of the antigen by its specific effector cells induces proliferation and/or differentiation.

In order to evaluate modulation of the immune response, we measured production of antigen specific antibodies (Abs) as an indicator of humoral immunity and the antigen-specific induction of interferon-.gamma.-producing (IFN-.gamma.) cells as marker for specific cytotoxic T lymphocytes (CTLS) in mixed lymphocyte assays. In addition, we evaluated the T helper (Th) cell profile, following the expression of the antigen and the reaction of the attracted cells in presence of the different chemokines, using different markers. The ratio of the immunoglobulin isotypes IgG1 and IgG2a and the production of the cytokines (Street, N. E. and Mosmann, T. R., 1991) in non-stimulated effector cell cultures: Interleukin-4 (IL-4), which is Th2-type associated, IFN-.gamma., which is Th1-type associated) and further macrophage inflammatory protein-1.alpha. (MIP-1.alpha.).

In the case of MDC we also explored the immuno-modulatory activities of this chemokine in a protein vaccination protocol, evaluating the dose dependency of the induced immune reaction. The results demonstrate the usefulness of tailoring immunization protocols by adding one or more chemokines at specific amounts to elicit an immune response of a desired profile regarded as most adequate to fight a pathogen or a neoplastic cell.

Methods and Compositions for Enhancing the Efficacy of a Vaccine

Where nucleic acids encoding the antigens and/or chemokines are employed, it will be understood that the respective coding sequences are operatively linked to gene regulatory sequences capable of directing the expression of the sequences upon administration to a subject and/or introduction into a suitable cell. For example, such coding sequences will preferably be expressable in a cell of a subject, preferably in a cell of a human subject.

The present invention provides methods for enhancing the efficacy of a vaccine in a subject, which methods comprise administering to a subject an immunizingly effective amount of one or more antigens against which an immune response is desired in the subject in conjunction with an amount of a chemokine or its functional equivalent effective to enhance the immune response against the antigen. In one aspect, the chemokine or its functional equivalent, is administered to the subject concurrently with (e.g., in the same composition with) the antigen or antigens against which an immune response is desired. In another, aspect, the chemokine or its functional equivalent, is administered either before or after the administration of one or more antigens against which immunity is desired in the subject, but is administered within such time that the chemokine or its functional equivalent enhances the immune response to the one or more antigens. For example, the chemokine or its functional equivalent is suitably administered during the time that the subject mounts an immune response against the administered one or more antigens. The chemokine or its functional equivalent is preferably administered within 30 minutes, 1 hour, 5 hours, 10 hours, 1 day, and/or 2 days of (preferably, after) administration of the one or more antigens against which immunity is desired. In a preferred embodiment, the site of administration of the chemokine is at or near the site of administration of the antigen.

The present invention further provides compositions comprising an immunizingly effective amount of one or more antigens and an amount of chemokine or its functional equivalent, effective to enhance the immune response to said antigen and, preferably, the composition further comprises a pharmaceutically acceptable carrier.

Any chemokine that is capable of enhancing the efficacy of a vaccine (for example, but not limited to, as determined by the assays described in Section 5.4, infra) can be used in the methods and compositions of the present invention.

In one specific embodiment the chemokine component is full length MDC, preferably full length MDC having the amino acid sequence of SEQ ID NO:2 (FIG. 1B, see Original Patent). In another embodiment, the chemokine component consists of amino acid residues 2-69 of SEQ ID NO:2 (FIG. 1B). In another specific embodiment chemokine component consists of amino acid residues 3-69 of SEQ ID NO:2 (FIG. 1B). In still another specific embodiment the N-terminal amino acid sequence of the chemokine component consists of the amino acid sequence Tyr-Gly-Ala-Asn-Met-Glu-Asp-Ser-Val-Cys-Cys-Arg-Asp-Tyr-Val-Arg-Tyr-Arg-L- eu (portion of SEQ ID NO:2). In yet another specific embodiment the N-terminal amino acid sequence of the chemokine protein consists of the amino acid sequence Pro-Tyr-Gly-Ala-Asn-Met-Glu-Asp-Ser-Val-Cys-Cys-Arg (portion of SEQ ID NO:2). In yet another specific embodiment the N-terminal amino acid sequence of the chemokine consists of the amino acid sequence Tyr-Gly-Ala-Asn-Met-Glu-Asp-Ser-Val-Cys-Cys-Arg-Asp-Tyr-Val-Arg-Tyr-Arg-L- eu (SEQ ID NO:2), which derivative has activity to enhance the efficacy of the vaccine. In yet another specific embodiment the N-terminal amino acid sequence of the chemokine consists of the amino acid sequence Pro-Tyr-Gly-Ala-Asn-Met-Glu-Asp-Ser-Val-Cys-Cys-Arg (SEQ ID NO:2), which derivative has activity to enhance the efficacy of the vaccine. In yet another specific embodiment, the chemokine component is a derivative of a corresponding native chemokine, which derivative has one or more insertions of or substitutions with one or more non-classical amino acids relative to the native chemokine, which derivative will enhance the efficacy of the vaccine.

In yet another specific embodiment, the chemokine is a derivative of a corresponding native chemokine that has only one or more conservative substitutions in sequence relative to the native chemokine, which derivative enhances the efficacy of the vaccine.

Chemokines useful in the present invention may be derived from any suitable source and obtained by any method known in the art.

Preferably, the chemokine is of the same species as the subject to which the vaccine is administered. In a preferred embodiment, a human chemokine is administered to a human subject. The present invention also provides a method to enhance the efficacy of a vaccine in a subject, which method comprises administering to a subject a first nucleic acid comprising a nucleotide sequence encoding an antigen against which an immune response is desired in a subject and a second nucleic acid comprising a nucleotide sequence encoding chemokine and/or its functional equivalent. The expression of the encoded antigen and the chemokine or its functional equivalent, is suitably controlled of one or more gene regulatory elements. Such regulatory elements are known in the art, and examples are described herein. Regulatory elements are selected and arranged so that upon introduction of the first and second nucleic acids into a suitable cell (e.g., a cell of the subject), the antigen and chemokine or its functional equivalent are coordinately expressed. Coordinate expression occurs where (i) the two components are expressed either at the same time or within an appropriate time period, i.e., the time period is sufficient for the chemokine to enhance the immune response against the antigen). Moreover, the antigen is preferably expressed in an immunizingly effective amount; and (iii) the chemokine or its functional equivalent is expressed in an amount sufficient to enhance the immune response against the antigen.

In a specific embodiment, the nucleotide sequences encoding (i) the chemokine or its functional equivalent and (ii) the antigen are present on separate nucleic acids. In another embodiment, the nucleotide sequences encoding (i) the chemokine or its functional equivalent and (ii) the antigen are present on the same nucleic acid.

The present invention also provides compositions to enhance the efficacy of a vaccine in a subject. The compositions generally comprise a first nucleic acid comprising a nucleotide sequence encoding an antigen and a second nucleic acid comprising a nucleotide sequence encoding a chemokine, wherein the nucleotide sequences encoding the antigen and the chemokine are operably linked to one or more gene regulatory elements such that, upon introduction of said first and second nucleic acids (or a single nucleic acid comprising both) into a suitable cell (e.g., a cell of the subject), the antigen and chemokine are expressed, preferably in a coordinated manner. The antigen and the chemokine are expressed in a manner which results in an immunizingly effective response.

The present invention also provides compositions to enhance the efficacy of a vaccine in a subject. These compositions generally comprise a nucleic acid comprising a first nucleotide sequence encoding an antigen and a second nucleotide sequence encoding a chemokine or its functional equivalent. The first and second nucleotide sequences may be joined to form a single nucleotide sequence encoding both the antigen component and the chemokine component. The first and second nucleotide sequences are each operably linked to one or more gene regulatory elements such that, upon introduction into a suitable cell, the antigen and the chemokine are expressed, preferably in a coordinated manner. The antigen and the chemokine are expressed in a manner which results in an immunizingly effective response.

Any nucleic acid comprising a nucleotide sequence encoding an chemokine or its functional equivalent capable of enhancing the immune response to the antigen can be used in the methods and compositions of the present invention.

Preferred chemokine components are RANTES, MDC, MCP-1 and BLC, and functional equivalents of these chemokines.

In another specific embodiment, the method or composition of the invention uses a nucleic acid encoding a chemokine derivative having one or more deletional, insertional or substitutional mutations, which derivative has activity to enhance an immune response against an antigen in a subject.

DNA vaccines according to the invention are suitably produced by any method known in the art for constructing an expression plasmid vector encoding for expression the nucleotide sequences of (i) an antigen; and/or (ii) a chemokine. Regulatory elements are selected to provide a vector suitable for expression of the encoded proteins in the subject or in cells recombinant for the expression vector, which cells are to be provided to the subject. Such expression vectors typically comprise promoters, terminators and polyadenylation coding regions to control the expression of the encoded protein.

The DNA vaccine can be administered by any method known in the art for administration of DNA. The DNA vaccine may be delivered either directly, in which case the subject is directly exposed to the DNA vaccine such that the DNA enters and is expressed in cells of the subject, or indirectly, in which case, the DNA vaccine is first introduced into suitable cells by any method known in the art in vitro, then the cells containing the DNA vaccine are transplanted into the subject.

n a specific embodiment, the DNA vaccine is directly administered in vivo, where it is expressed to produce the encoded antigen and chemokine. This can be accomplished by any of numerous methods known in the art. The chemokine-encoding nucleic acid and the antigen-encoding nucleic acid can be provided as components of one or more nucleic acid expression vector (i.e., the chemokine-encoding nucleic acid and the antigen-encoding nucleic acid can be components of the same or separate expression vectors) and administering the vector (or vectors) so that it becomes intracellular. Methods for administration of vectors are known in the art. Examples include infection using a defective or attenuated retroviral or other viral vector (see U.S. Pat. No. 4,980,286); direct injection of naked DNA; microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); coating with lipids or cell-surface receptors or transfecting agents; encapsulating the vector in liposomes, microparticles, or microcapsules; administering the vector linked to a peptide which is known to enter the nucleus; administering the peptide linked to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors). In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In a preferred embodiment, the nucleic acid of a DNA vaccine is injected into the muscle of the subject to be immunized.

Another approach is to introduce the nucleic acid of the DNA vaccine into a cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign nucleic acid into cells (see e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen, et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92 (1985)) and may be used in accordance with the present invention. Usually, the method of transfer includes the transfer of a selectable marker to the cells. Known techniques are then used to isolate those cells that have taken up and are expressing the transferred gene.

Cells into which a DNA vaccine can be introduced for purposes of immunization encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

The resulting recombinant cells can be delivered to a subject by various methods known in the art. In a preferred embodiment, the recombinant cells are injected, e.g., subcutaneously. In another embodiment, recombinant skin cells may be applied as a skin graft onto the patient. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously.

The cells can also be encapsulated in a suitable vehicle and then implanted in the subject (see, e.g., Dionne, et al. PCT Publication WO 92/19195, dated Nov. 12, 1992). The amount of cells envisioned for use depends on the desired effect, subject state, etc., and can be determined by one skilled in the art without undue experimentation.

By way of example, and not by way of limitation, a DNA vaccine may be generated as described by Lekutis, et al. for an HIV DNA vaccine (1997, J. Immunol. 158:4471-4477). Briefly, an expression vector is constructed with the promoter, enhancer and intron A of human cytomegalovirus (CMV) and the termination and polyadenylation sequences of bovine growth hormone in a plasmid backbone. Additionally, the nucleotide sequence for signal sequence of tissue plasminogen activator is either substituted for the signal sequence of the antigen, if the antigen has a signal sequence or is added onto the amino-terminus of the antigen, thereby eliminating the dependence on viral proteins for expression (e.g., in the case of gp120 expression, Rev and Env proteins are required unless the HIV-1 signal sequence is so substituted). The resulting formulation is then injected intra-muscularly.

Further examples of DNA vaccines are set forth in Boyer, et al. (1996, J. Med. Primatol., 25:242-250), which describes the construction of a plasmid encoding the HIV-1 gp160 envelope glycoprotein as well as the rev-tax region cloned into pMAMneoBlue vector (Clonetech, Inc., Palo Alto, Calif.), and a vector encoding the envelope glycoprotein and Rev from HIV-1 strain MN under the control of the CMV promoter. Another vector which can be used in the present invention is as described in Boyer, et al. (1997, Nature Medicine 3:526-532) and contains expression cassettes encoding the envelope and Rev proteins of HIV-1 strain MN, and encoding the Gag/Pol proteins of HIV-1 strain IIIB.

For the practice of the present invention, the nucleotide sequence for the chemokine or its functional equivalent, can either be incorporated into the same expression vector containing the nucleotide sequence encoding the antigen in such a manner that the chemokine is expressed. Alternatively, the nucleotide sequence encoding chemokine, or a functional equivalent thereof, can be cloned into a separate expression vector (e.g., as described above for the expression vector containing the sequences coding for antigen) and the expression vector that expresses the antigen mixed with the expression vector that expresses chemokine. The mixture of the two expression vectors can then be administered to the subject.

The methods and compositions of the present invention may be used as a vaccine in a subject in which immunity for the antigen(s) is desired. Such antigens can be any antigen known in the art to be useful in a vaccine formulation. The methods and compositions of the present invention can be used to enhance the efficacy of any vaccine known in the art. The vaccine of the present invention may be used to enhance an immune response to infectious agents and diseased or abnormal cells, such as but not limited to bacteria, parasites, fungi, viruses, tumors and cancers. The compositions of the invention may be used to either treat or prevent a disease or disorder amenable to treatment or prevention by generating an immune response to the antigen provided in the composition. In one preferred embodiment, the antigen(s) are proteins or fragments or derivatives thereof encoded by any genes of the HIV genome including the env, gag, pol, nef, vif, rev, and tat genes. In a more preferred embodiment, the antigen is an HIV associated gp120 protein.

The methods and compositions of the present invention may be used to elicit a humoral and/or a cell-mediated response against the antigen(s) of the vaccine in a subject. In one specific embodiment, the methods and compositions elicit a humoral response against the administered antigen in a subject. In another specific embodiment, the methods and compositions elicit a cell-mediated response against the administered antigen in a subject. In a preferred embodiment, the methods and compositions elicit both a humoral and a cell-mediated response.

The subjects to which the present invention is applicable may be any mammalian or vertebrate species, which include, but are not limited to, cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and ratsmice, monkeys, rabbits, chimpanzees, and humans. In a preferred embodiment, the subject is a human. The compositions and methods of the invention can be used to either prevent a disease or disorder, or to treat a particular disease or disorder, where an immune response against a particular antigen or antigens is effective to treat or prevent the disease or disorder. Such diseases and disorders include, but are not limited to, viral infections, such as HIV, CMV, hepatitis, herpes virus, measles, etc., bacterial infections, fungal and parasitic infections, cancers, and any other disease or disorder amenable to treatment or prevention by eliciting an immune response against a particular antigen or antigens. In another preferred embodiment, the subject is infected or at risk of being infected with HIV virus.

In another preferred embodiment, the invention provides methods and compositions to enhance the efficacy of an HIV vaccine, and such a vaccine can be administered to either prevent or treat HIV.

Chemokine Nucleic and Amino Acid Sequence and Functional Equivalents

The chemokine amino acid and nucleic acid sequences used in the methods and compositions of the invention can be obtained by any method known in the art. Chemokine nucleotide and amino acid sequences for humans and other animals are publicly available in public databases. For examples the Genbank Accession Nos. for MDC SEQ ID NO: 1; MCP-1 SEQ ID NO: 3; RANTES SEQ ID NO: 4; and BLC SEQ ID NO: 5 are set forth in the following table -- see Original Patent.

Chemokines useful in the methods and compositions of the present invention include, but are not limited to, chemokines from mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, and human. Preferred chemokines are of human origin. Nucleic acid sequences encoding suitable chemokines can be isolated from vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, as well as additional primate sources, etc.

DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library"), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions. Clones derived from cDNA will contain only exon sequences. Whatever the source, the chemokine-encoding nucleic acid sequence can be molecularly cloned into a suitable vector.

In the molecular cloning of the gene from cDNA, cDNA is generated from totally cellular RNA or mRNA by methods that are well known in the art. The gene may also be obtained from genomic DNA, where DNA fragments are generated (e.g. using restriction enzymes or by mechanical shearing), some of which will encode the desired gene. Linear DNA fragments can be separated according to size using standard techniques, e.g, agarose and polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNA fragment containing all or a portion of the chemokine gene may be accomplished in a number of ways.

A preferred method for isolating a chemokine gene is by the polymerase chain reaction (PCR), which can be used to amplify the desired chemokine sequence in a genomic or cDNA library or from genomic DNA or cDNA that has not been incorporated into a library. Oligonucleotide primers that hybridize to chemokine sequences can be used as primers in PCR.

Additionally, a portion of the chemokine (of any species) gene or its specific RNA, or a fragment thereof, can be purified (or an oligonucleotide synthesized) and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. and Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). DNA fragments with substantial homology to the probe will hybridize. Chemokine nucleic acids can be also identified and isolated by expression cloning using, for example, anti-chemokine antibodies for selection.

Alternatives to obtaining the chemokine DNA by cloning or amplification include, but are not limited to, chemically synthesizing the gene sequence itself from the known sequence or transcribing cDNA to mRNA which encodes the chemokine protein. Other methods are possible within the skill of the art and within the scope of the invention.

Once a clone has been obtained, its identity can be confirmed by nucleic acid sequencing (by any method well known in the art) and comparison to known chemokine sequences. DNA sequence analysis can be performed by any techniques known in the art, including, but not limited to, the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Pat. No. 4,795,699), use of an automated DNA sequenator (e.g., Applied Biosystems, Foster City, Calif.) or the method described in PCT Publication WO 97/15690. Nucleic acids which are hybridizable to a chemokine nucleic acid (e.g., having sequence SEQ ID NO: 1), or to a nucleic acid encoding a chemokine derivative can be isolated, by nucleic acid hybridization under conditions of low, high, or moderate stringency (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. USA 78:6789-6792).

Chemokines and their functional equivalents can be obtained by any method known in the art. Examples include recombinant expression methods, purification from natural sources, and chemical synthesis.

For example, chemokines can be obtained by recombinant protein expression techniques. For recombinant expression, a nucleic acid encoding the chemokine or its functional equivalent is inserted into an appropriate cloning vector for expression in a particular host cell. Many vector-host systems are known in the art. Examples include plasmids, cosmids, phagemids and modified viruses. The vector system should be compatible with the host cell used. Specific examples include bacteriophages, such as lambda derivatives; and plasmids, such as pBR322 or pUC plasmid derivatives or the BLUESCRIPT.TM. vector (Stratagene).

Insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and chemokine gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionation, can be accomplished prior to insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the isolated chemokine gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA.

A nucleotide sequence coding for a chemokine or its functional equivalent, can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. The necessary transcriptional and translational signals can also be supplied by the native chemokine gene and its flanking regions.

A variety of host-vector systems may be utilized to express the protein-coding sequence. These include, but are not limited to, mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).

Expression of a nucleic acid sequence encoding a chemokine or its functional equivalent may be regulated by a second nucleic acid sequence so that the chemokine polypeptide is expressed in a host transformed with the recombinant DNA molecule.

Expression of a chemokine or its functional equivalent may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the p-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-373 1), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift, et al., 1984, Cell 38:639-646; Ornitz, et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl, et al., 1984, Cell 38:647-658; Adames, et al., 1985, Nature 318:533-538; Alexander, et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder, et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert, et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf, et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer, et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey, et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram, et al., 1985, Nature 315:338-340; Kollias, et al., 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead, et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason, et al., 1986, Science 234:1372-1378).

For example, a vector can be used that comprises a promoter operably linked to a chemokine-encoding nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene).

In a specific embodiment, an expression construct is made by subcloning a chemokine coding sequence into the EcoRI restriction site of each of the three pGEX vectors (Glutathione S-Transferase expression vectors; Smith and Johnson, 1988, Gene 7:31-40). This allows for the expression of the chemokine product from the subclone in the correct reading frame.

The invention provides expression vectors comprising chemokine-encoding and/or antigen-encoding inserts. In a preferred aspect, the invention provides an expression vector comprising both chemokine-encoding and/or antigen-encoding inserts.

The expression vectors of the invention can be used to transform cells. Cells expressing the expression vectors can be identified by a variety of approaches known in the art. Examples include nucleic acid hybridization, identification of the presence or absence of "marker" gene functions, and expression of inserted sequences.

In the first approach, the presence of a target insert in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to the target insert.

In a second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a target insert. For example, if the target insert is inserted within the marker gene sequence of the vector, recombinants containing the target insert can be identified by the absence of the marker gene function.

In a third approach, recombinant expression vectors can be identified by assaying for the chemokine product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the chemokine in in vitro assay systems, e.g., binding with an antigen and/or anti-chemokine antibody or a corresponding chemokine receptor.

Appropriate cells expressing the chemokine-encoding and/or antigen-encoding inserts can be administered to a subject as a live bacterial vaccine. Moreover, the cells may be killed and administered to a subject as a killed bacterial vaccine.

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast can be used to produce a glycosylated product. Expression in mammalian cells can be used to ensure "native" glycosylation of a heterologous protein. Furthermore, different vector/host expression systems may affect processing reactions to different extents.

In other specific embodiments, the antigen and/or chemokine and/or functional equivalent can be expressed as a fusion, or chimeric protein product. A wide variety of combinations are possible, e.g., chemokine-antigen; chemokine-heterologous polypeptide; antigen-carrier protein; antigen-chemokine functional equivalent, etc. In a preferred aspect, the invention provides a fusion protein comprising an antigen and a chemokine. The fusion protein and the chemokine may suitably be separated by a spacer sequence, e.g., as described U.S. patent application Ser. No. 09/335,150. The fusion protein can be made by a variety of synthetic and recombinant methods known in the art. For example, nucleic acid sequences encoding the desired amino acid sequences can be ligated to each other in the proper coding frame, and the chimeric product can be expressed by methods known in the art. Alternatively, a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. In a specific embodiment, a chimeric protein containing all or a portion of a chemokine is joined via a peptide bond to all or a portion of an antigen against which immunity is desired.

The nucleic acids of the invention can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions. Any technique for mutagenesis known in the art can be used. Examples of suitable techniques include in vitro site-directed mutagenesis (Hutchinson, et al., 1978, J. Biol. Chem 253:6551), use of TAB linkers (Pharmacia), mutation-containing PCR primers, etc.

The experimentation involved in mutagenesis consists primarily of site-directed mutagenesis followed by phenotypic testing of the altered gene product. Some of the more commonly employed site-directed mutagenesis protocols take advantage of vectors that can provide single-stranded as well as double-stranded DNA, as needed. Generally, the mutagenesis protocol for such vectors is as follows. A mutagenic primer, i.e., a primer complementary to the sequence to be changed, but consisting of one or a small number of altered, added, or deleted bases, is synthesized. The primer is extended in vitro by a DNA polymerase and, after some additional manipulations, the now double-stranded DNA is transfected into bacterial cells. Next, by a variety of methods, the desired mutated DNA is identified, and the desired protein is purified from clones containing the mutated sequence. For longer sequences, additional cloning steps are often required because long inserts (longer than 2 kilobases) are unstable in those vectors. Protocols are known to one skilled in the art and kits for site-directed mutagenesis are widely available from biotechnology supply companies, for example from Amersham Life Science, Inc. (Arlington Heights, Ill.) and Stratagene Cloning Systems (La Jolla, Calif.).

Chemokine fragments can be obtained by proteolysis of the protein followed by purification using standard methods such as those described above (e.g., immunoaffinity purification).

Formulations and Methods of Administration

The formulations of the invention generally comprise, in association with a pharmaceutically acceptable carrier or excipient: (i) a chemokine or functional equivalent and an antigen; (ii) a nucleic acid sequence encoding a chemokine or functional equivalent and a separate nucleic acid sequence encoding an antigen; (iii) a nucleic acid sequence encoding a chemokine or functional equivalent and also encoding an antigen; or (iv) combinations of any of the foregoing components. A nucleic acid encoding any of the chemokine (or functional equivalent therof) and/or the antigen is configured to express the sequence encoded thereby. The invention also provides pharmaceutical compositions comprising live or killed bacterial vectors transformed using the expression vectors of the invention.

Pharmaceutically acceptable components of the formulations (e.g., carriers and excipients) are well known in the art. Examples include physiological saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

Where a live bacterial vector is used, an acceptable carrier is a physiologically balanced culture medium. The culture medium may, for example, comprise one or more stabilizing agents such as stabilized, hydrolyzed polypeptides, lactose, and the like.

In addition, if desired, the vaccine or composition preparation may also include minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine or composition.

Supplemental adjuvants may also be included in the compositions of the invention. Adjuvants include auxiliary agents which, when administered jointly with an antigen, increase the immunogenicity of the antigen or influence the quality of the immune response. Adjuvants are typically administered to improve the immunogenicity of an antigen, i.e., to increase antibody formation and/or to induce a stronger cell mediated immune response to the antigen.

Examples of suitable adjuvants include mineral gels (e.g., aluminum hydroxide); surface active substances (e.g., lysolecithin, pluronic polyols); polyanions; peptides; oil emulsions; and alum. See Derek T. O'Hagan, Vaccine Adjuvants: Preparation Methods and Research Protocols, Humana Press, 2000, for further examples. The effectiveness of an adjuvant may be determined by comparing the induction of antibodies directed against a composition of the invention composition in the presence and in the absence of the adjuvant. Specific methods are discussed in O'Hagan supra.

The composition can take any of a variety of forms known in the art, e.g., liquid solution, suspension, emulsion, microemulsion, tablet, pill, capsule, sustained release formulation, powder, etc.

Oral formulations suitably comprise standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

Polypeptide components of the compositions of the invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts and methods for making them are known in the art. Examples include acid addition salts (formed with free amino groups of the peptide). Acid addition salts may be prepared using inorganic acids (e.g., hydrochloric or phosphoric acids) or organic acids (e.g., acetic, oxalic, tartaric, maleic, and the like). Salts formed with free carboxyl groups may also be derived from inorganic bases, e.g., sodium potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Polypeptide components of the compositions of the invention may also be constructed as univalent, divalent or multivalent polypeptides. The polypeptides may contain multiple copies of the same antigen species or multiple (i.e., two or more) antigen species. Similarly, the polypeptides may contain multiple copies of the same chemokine species or multiple (i.e., two or more) chemokine species. The polypeptides may also comprise one or more chemokine species in a fusion polypeptide with one or more antigen species.

The compositions of the invention are suitably administered to a subject in an amount sufficient to elicit an enhanced immune response. The compositions of the invention are also suitably administered to a subject in an amount sufficient to enhance an ongoing immune response. The compositions of the invention are preferably administered to a subject in an amount sufficient to cause an immunizingly effective response.

The precise dose of the active components of the composition to be employed in the formulation will depend on a variety of factors known to those of skill in the art. For example, factors to be considered include the route of administration and the nature of the subject to be immunized. The effect of such factors, and other factors known in the art, is readily determined by one of skill in the art according to standard clinical techniques. Effective doses of the active components of the compositions of the present invention may also be extrapolated from dose-response curves derived from animal model test systems.

The invention also provides a pharmaceutical pack or kit comprising one or more containers comprising at least one antigen component (polypeptide and/or antigen-encoding nucleic acid) and at least one chemokine component (polypeptide and/or chemokine-encoding nucleic acid) for preparing a vaccine composition of the invention. A notice can be associated with such container(s). The notice is preferably in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products. Further, the notice may reflect approval by the agency for manufacture, use or sale for human administration.

Generally, the ingredients of the compositions of the invention are supplied either separately or mixed together in unit dosage form. An example of the former is a dry lyophilized powder or water free concentrate in a hermetically sealed container, such as an ampule or sachette indicating the quantity of active agent. Where the composition is administered by injection, an ampoule of sterile diluent can be provided so that the ingredients may be mixed prior to administration.

In a specific embodiment, at least one antigen and at least one chemokine of the invention is provided in a first container (or a fusion polypeptide comprising both antigen and chemokine or functional equivalent), and a second container comprises diluent consisting of an aqueous solution of 50% glycerin, 0.25% phenol, and an antiseptic (e.g., 0.005% brilliant green).

A variety of known methods for administering vaccines may be used to administer the compositions and formulations of the invention. These include but are not limited to: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intraosseous, intranasal routes, and via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle). The nucleic acid vaccines of the invention can be administered by any method known in the art for delivery of DNA to subject.

Assays

The activity of the compositions of the present invention can be validated by monitoring the immune response in test animals following immunization with a composition comprising a first component selected from the group consisting of: (i) an antigen against which an immune response is desired in the subject; and (ii) a nucleic acid encoding the antigen; and a second component selected from the group consisting of: (i) a chemokine selected from the group consisting of RANTES, MCP-1, MDC and BLC, or a functional equivalent of said chemokine; and (ii) a nucleic acid encoding the chemokine. As previously discussed, the chemokine and antigen components may also be administered as components of separate compositions, in any chronological order.

The response of the test animals can be compared to a response in control animals immunized with a corresponding antigen alone. Where the chemokine is provided as a component of a fusion polypeptide which also comprises the antigen, a suitable control will also include animals administered with a corresponding chemokine and a corresponding antigen not contained in a fusion polypeptide. Other controls will be apparent to persons of skill in the art.

An immune response is indicated, for example, by generation of a humoral (antibody) response and/or cell-mediated immunity. Test animals may include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and eventually human subjects. Assays for humoral and cell-mediated immunity are well known in the art. The immune response of the test subjects can be analyzed by various approaches well known in the art. Examples include testing the reactivity of the resultant immune serum to the antigen of the composition or fusion polypeptide, as assayed by known techniques (e.g., immunosorbant assay (ELISA), immunoblots, radioimmunoprecipitations, and the like).

Methods of introducing the composition into animals used in assays of the present invention may include oral, intracerebral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intraosseous, intranasal or any other standard routes of immunization.

As one example of suitable animal testing, compositions of the present invention may be tested in mice for the ability to enhance an antibody response to an antigen component of the composition and/or a delayed-type hypersensitivity (DTH) response, measured by an increase in footpad swelling after inoculation in the footpad of the test animal. These measurements can then be compared to corresponding measurements in control animals. As an example, BALB/c mice may be used as test animals.

Serum samples may be drawn from the mice after the final inoculation (for example every one or two weeks after inoculation). Serum can be analyzed for antibodies against the antigen using known methods in the art, e.g., using an ELISA. DTH responses to the antigen may be measured after the final inoculation (e.g. within 1-7 days). An increase in the serum titer of antibodies recognizing the antigen and/or an increase in footpad swelling in the animals receiving the composition of the invention as compared to the serum titer of the control animals, indicates that composition enhances the immune response to antigen.
 

Claim 1 of 3 Claims

1. A composition comprising (a) an isolated HIV antigen against an immune response is desired in a subject wherein the HIV antigen is selected from the group consisting of gp120 and gp160; (b) a second component comprising B lymphocyte chemoattractant (BLC); and (c) a pharmaceutically acceptable carrier.

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