Patent 6,737,066

The invention provides immunogenic compositions which enhance .beta.-chemokine levels in a mammal. The immunogenic compositions contain an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence (ISS) and an adjuvant. The HIV antigen can be a whole-killed HIV virus devoid of outer envelope protein gp120. Alternatively, the HIV antigen can be a whole-killed HIV virus, or a p24 antigen. Also provided are kits, the components of which, when combined, produce the immunogenic compositions of the invention. The invention also provides methods of making the immunogenic compositions, by combining an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence (ISS) and an adjuvant. The invention further provides a method of immunizing a mammal, by enhancing .beta.-chemokine production in the mammal by administering to the mammal an immunogenic composition containing an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence (ISS) and an adjuvant. Also provided is a method of inhibiting AIDS, by enhancing .beta.-chemokine production in the mammal by administering to the mammal an immunogenic composition containing an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence (ISS) and an adjuvant.

SUMMARY OF THE INVENTION

In the immunogenic compositions of the invention, the isolated nucleic acid molecule containing an ISS can be an oligodeoxynucleotide. The isolated nucleic acid molecule containing an ISS can contain two or more CpG sequences. Exemplary ISS-containing nucleic acid molecules contain the motif 5'-Cytosine, Guanine, Pyrimidine, Pyrimidine-3'. The isolated nucleic acid molecule can contain a phosphorothioate backbone. The isolated nucleic acid molecule can be conjugated to the HIV antigen.

In the immunogenic compositions of the invention, the adjuvant can be suitable for administration to a human. An exemplary adjuvant is Incomplete Freund's Adjuvant.

The immunogenic compositions of the invention can further enhance HIV-specific IgG2b antibody production in a mammal. The immunogenic compositions of the invention can also enhance an HIV-specific cytotoxic T lymphocyte response in a mammal.

Also provided are kits, which contain an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence (ISS) and an adjuvant. The components of the kits, when combined, produce the immunogenic compositions of the invention.

The invention also provides methods of making the immunogenic compositions, by combining an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence (ISS) and an adjuvant. The components can be combined ex vivo or in vivo to arrive at the immunogenic compositions.

The invention also provides a method of immunizing a mammal, by enhancing .beta.-chemokine production in the mammal by administering to the mammal an immunogenic composition containing an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence (ISS) and an adjuvant. Also provided is a method of inhibiting AIDS, by enhancing .beta.-chemokine production in the mammal by administering to the mammal an immunogenic composition containing an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence (ISS) and an adjuvant. In the methods of the invention, the mammal can be a primate, such as a human, or a rodent. In certain embodiments of the method, the primate is a pregnant mother or an infant. A human can be HIV seronegative or HIV seropositive. The immunogenic compositions can advantageously be administered to the mammal two or more times.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides immunogenic HIV compositions containing an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence, and an adjuvant. Also provided are kits containing the components of such compositions, for use together. The invention also provides methods of immunizing a mammal with such compositions, or with the components of such compositions, so as to enhance production of .beta.-chemokines in the immunized mammal. Advantageously, the compositions of the invention can also induce potent Th1 immune responses against a broad spectrum of HIV epitopes, and provide a strong HIV-specific cytotoxic T lymphocyte response. Thus, the immunogenic compositions of the invention are useful for preventing HIV infection and slowing progression to AIDS in infected individuals.

As used herein, the term "HIV" refers to all forms, subtypes and variations of the HIV virus, and is synonymous with the older terms HTLVIII and LAV. Various cell lines permanently infected with the HIV virus have *been developed and deposited with the ATCC, including those having accession numbers CCL 214, TIB 161, CRL 1552 and CRL 8543, all of which are described in U.S. Pat. No. 4,725,669 and Gallo, Scientific American 256:46 (1987).

As used he,rein, the term "whole-killed HIV virus" refers to an intact, inactivated HIV virus.

As used herein, the term "outer envelope protein" refers to that portion of the membrane glycoprotein of a retrovirus which protrudes beyond the membrane, as opposed to the transmembrane protein, gp41.

As used herein, the term "HIV virus devoid of outer envelope proteins" refers to a preparation of HIV particles or HIV gene products devoid of the outer envelope protein gp120, but: contains the more genetically conserved parts of the virus (eg. p24 and gp41).

As used herein, the term "HIV p24 antigen" refers to the gene product of the gag region of HIV, characterized as having an apparent relative molecular weight of about 24,000 daltons designated p24. The term "HIV p24 antigen" also refers to modifications and fragments of p24 having the immunological activity of p24. Those skilled in the art can determine appropriate modifications of p24, such as additions, deletions or substitutions of natural amino acids or amino acid analogs, that serve, for example, to increase its stability or bioavailability or facilitate its purification, without destroying its immunological activity. Likewise, those skilled in the art can determine appropriate fragments of p24 having the immunological activity of p24. An immunologically active fragment of p24 can have from 6 residues from the polypeptide up to the full length polypeptide minus one amino acid.

As used herein, the term "immunostimulatory sequence" or "ISS" refers to a nucleotide sequence containing an unmethylated CpG motif that is capable of enhancing the immune response in a mammal when administered in combination with an antigen. Immunostimulatory sequences are described, for example, in PCT publication WO 98/55495.

As ISS can contain, for example, at least one sequence consisting of 5'-Cytosine, Guanine, Pyrimidine, Pyrimidine-3'. For example, the sequence 5'-CGTT-3' is found in two copies in the sequence designated SEQ ID NO:1, described in Example I, and one copy each of the sequence 5'-CGTT-3' and the sequence 5'-CGCT-3' are found in the sequence designated SEQ ID NO:4, described in Example IV.

An ISS can contain the hexameric motif 5'-Purine, Purine, Cytosine, Guanine, Pyrimidine, Pyrimidine-3', such as the motif 5'-GACGTT-3', two copies of which are found in the nucleotide sequence designated SEQ ID NO:1. An ISS can also contain, for example, either the octameric motif 5'-Purine, Purine, Cytosine, Guanine, Pyrimidine, Pyrimidine, Cytosine, Cytosine-3' or 5'-Purine, Purine, Cytosine, Guanine, Pyrimidine, Pyrimidine, Cytosine, Guanine-3', such as the sequence 5'-AACGTTCG-3'. An exemplary isolated nucleic acid molecule containing the ISS motif 5'-AACGTTCG -3' has the nucleotide sequence designated SEQ ID NO:2, as described in Example I.

An ISS can contain more than one unmethylated CpG motif, such as two or more CpG motifs. An exemplary isolated nucleic acid molecule containing two CpG motifs has the nucleotide sequence designated SEQ ID NO:1 or the sequence designated SEQ ID NO:2, described in Example I, below. An exemplary isolated nucleic acid molecule containing three unmethylated CpG motifs has the nucleotide sequence designated SEQ ID NO:4, as described in Example IV. SEQ ID NO:4 also contains two copies of the hexameric motif 5'-Purine, Pyrimidine, Cytosine, Guanine, Pyrimidine, Pyrimidine-3', namely both the sequence 5'-GTCGCT-3' and the sequence 5'-GTCGTT-3'.

As used herein, the term "nucleic acid molecule containing an ISS" refers to a linear, circular or branched single- or double-stranded DNA or RNA nucleic acid that contains an immunostimulatory sequence. The term "isolated," with reference to a nucleic acid molecule containing an ISS, is intended to distinguish the ISS-containing nucleic acid molecule from an ISS that may naturally be present in a whole-killed HIV virus preparation. A nucleic acid molecule containing an ISS can contain multiple ISSs. The ISSs can be adjacent within the nucleic acid molecule, or they can be separated by additional nucleotide bases within the nucleic acid molecule. Such a nucleic acid molecule can be of any length greater than 6 bases or base pairs, and is preferably greater than about 15 bases or base pairs, such as greater than about 20 bases or base pairs, and can be several kb in length.

A nucleic acid molecule containing an ISS can be, for example, a synthetic oligonucleotide, a naturally occurring nucleic acid molecule of any species, or a vector. A nucleic acid molecule containing an ISS can contain either natural or modified nucleotides or natural or unnatural nucleotide linkages. Modifications known in the art, include, for example, modifications of the 3'OH or 5'OH group, modifications of the nucleotide base, modifications of the sugar component, and modifications of the phosphate group. An unnatural nucleotide linkage can be, for example, a phosphorothioate linkage in place of a phosphodiester linkage, which increases the resistance of the nucleic acid molecule to nuclease degradation. Various modifications and linkages are described, for example, in PCT publication WO 98/55495.

As used herein, the term "adjuvant" refers to a substance which, when added to an immunogenic agent, nonspecifically enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture. Adjuvants can include, for example, oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts), liposomes and microparticles, such as polysytrene, starch, polyphosphazene and polylactide/polyglycosides. Adjuvants can also include, for example, squalene mixtures (SAF-I), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873-875. For veterinary use and for production of antibodies in animals, mitogenic components of Freund's adjuvant (both complete and incomplete) can be used. In humans, Incomplete Freund's Adjuvant (IFA) is a preferred adjuvant. Various appropriate adjuvants are well known in the art and are reviewed, for example, by Warren and Chedid, CRC Critical Reviews in Immunology 8:83 (1988).

As used herein, "AIDS" refers to the symptomatic phase of HIV infection, and includes both Acquired Immune Deficiency Syndrome (commonly known as AIDS) and "ARC," or AIDS-Related Complex, as described by Adler, Brit. Med. J. 294: 1145 (1987). The immunological and clinical manifestations of AIDS are well known in the art and include, for example, opportunistic infections and cancers resulting from immune deficiency.

As used herein, the term "inhibiting AIDS" refers to a beneficial prophylactic or therapeutic effect of the immunogenic composition in relation to HIV infection or AIDS symptoms. Such beneficial effects include, for example, preventing initial infection of an individual exposed to HIV; reducing viral burden in an individual infected with HIV; prolonging the asymptomatic phase of HIV infection; increasing overall health or quality of life in an individual with AIDS; and prolonging life expectency of an individual with AIDS. A clinician can compare the effect of immunization with the patient's condition prior to treatment, or with the expected condition of an untreated patient, to determine whether the treatment is effective in inhibiting AIDS.

As used herein, the term ".beta.-chemokine" refers to a member of a class of small, chemoattractive polypeptides that includes RANTES, macrophage inflammatory protein-1.beta. (MIP-1.beta.) and macrophage inflammatory protein-1.alpha. (MIP-1.alpha.). The physical and functional properties of .beta.-chemokines are well known in the art.

As used herein, the term "enhances," with respect to an immune response such as .beta.-chemokine production, IgG2b production or cytotoxic T lymphocyte activity, is intended to mean that the immunogenic composition elicits a greater immune response than does a composition containing any two of the three components of the immunogenic composition, administered in the same amounts and following the same immunization schedule. As disclosed herein, the components of the immunogenic compositions of the invention can act in synergy. For example, the immunogenic compositions of the invention can enhance .beta.-chemokine production by eliciting production of a higher concentration of .beta.-chemokine than would be expected by adding the effects of pairwise combinations of components of the immunogenic composition.

The .beta.-chemokine production that is enhanced can be either "HIV-specific .beta.-chemokine production," which refers to production of a .beta.-chemokine in response to stimulation of T cells with an HIV antigen. Alternatively, or additionally, the .beta.-chemokine production that is enhanced can be "non-specific .beta.-chemokine production," which refers to production of a .beta.-chemokine in the absence of stimulation of T cells with an HIV antigen.

As used herein, the term "kit" refers to components packaged or marked for use together. For example, a kit can contain an HIV antigen, an ISS and an adjuvant in three separate containers. Alternatively, a kit can contain any two components in one container, and a third component and any additional components in one or more separate containers. Optionally, a kit further contains instructions for combining the components so as to formulate an immunogenic composition suitable for administration to a mammal.

The invention provides an immunogenic composition containing an HIV antigen, a nucleic acid molecule containing a immunostimulatory sequence (ISS), and an adjuvant. The immunogenic composition enhances .beta.-chemokine production in a mammal administered the composition.

In one embodiment, the HIV antigen in the immunogenic composition is a whole-killed HIV virus, which can be prepared by methods known in the art. For example, HIV virus can be prepared by culture from a specimen of peripheral blood of infected individuals. In an exemplary method of culturing HIV virus, mononuclear cells from peripheral blood (e.g. lymphocytes) can be obtained by layering a specimen of heparinized venous blood over a Ficoll-Hypaque density gradient and centrifuging the specimen. The mononuclear cells are then collected, activated, as with phytohemagglutinin for two to three days, and cultured in an appropriate medium, preferably supplemented with interleukin 2. The virus can be detected either by an assay for reverse transcriptase, by an antigen capture assay for p24, by immunofluorescence or by electron microscopy to detect the presence of viral particles in cells, all of which are methods well-known to those skilled in the art.

Methods for isolating whole-killed HIV particles are described, for example, in Richieri et al., Vaccine 16:119-129 (1998), and U.S. Pat. Nos. 5,661,023 and 5,256,767. In one embodiment, the HIV virus is an HZ321 isolate from an individual infected in Zaire in 1976, which is described in Choi et al., AIDS Res. Hum. Retroviruses 13:357-361 (1997).

Various methods are known in the art for rendering a virus non-infectious (see, for example Hanson, MEDICAL VIROLOGY II (1983), de la Maza and Peterson, eds., Elsevier,). For example, the virus can be inactivated by treatment with chemicals or by physical conditions such as heat or irradiation. Preferably, the virus is treated with an agent or agents that maintain the immunogenic properties of the virus. For example, the virus can be treated with beta-propiolactone or gamma radiation, or both beta-propiolactone and gamma radiation, at dosages and for times sufficient to inactivate the virus.

In another,embodiment, the HIV antigen in the immunogenic composition is a whole-killed HIV virus devoid of outer envelope proteins, which can be prepared by methods known in the art. In order to prepare whole-killed virus devoid of outer envelope proteins, the isolated virus is treated so as to remove the outer envelope proteins. Such removal is preferably accomplished by repeated freezing and thawing of the virus in conjunction with physical methods which cause the swelling and contraction of the viral particles, although other physical or non-physical methods, such as sonication, can also be employed alone or in combination.

In yet another embodiment, the HIV antigen in the immunogenic composition is a substantially purified gene product of HIV. Such gene products include those products encoded by the gag genes (p55, p39, p24, p17 and p15), the pol genes (p66/p51 and p31-34) and the transmembrane glycoprotein gp41. These gene products may be used alone or in combination with other HIV antigens.

The substantially purified gene product of HIV can be a substantially purified HIV p24 antigen. p24 can be substantially purified from the virus by biochemical methods known in the art, or can be produced by cloning and expressing the appropriate gene in a host organism such as bacterial, fungal or mammalian cells, by methods well known in the art. Alternatively, p24 antigen, or a modification or fragment thereof that retains the immunological activity of p24, can be synthesized, using methods well known in the art, such as automated peptide synthesis. Determination of whether a modification or fragment of p24 retains the immunological activity of p24 can be made, for example, by immunizing a mammal and comparing the immune responses so generated, or testing the ability of the modification or fragment to compete with p24 for binding to a p24 antibody.

The immunogenic compositions of the invention also contain an isolated nucleic acid molecule having at least one immunostimulatory sequence (ISS). The HIV antigen and the nucleic acid molecule can be mixed together, or can be conjugated by either a covalent or non-covalent linkage. Methods of conjugating antigens and nucleic acid molecules are known in the art, and exemplary methods are described in PCT publication WO 98/55495.

A nucleic acid molecule containing an ISS can be prepared using methods well known in the art including, for example, oligonucleotide synthesis, PCR, enzymatic or chemical degradation of larger nucleic acid molecules, and conventional polynucleotide isolation procedures. Methods of producing a nucleic acid molecule containing an ISS, including a nucleic acid molecule containing one or more modified bases or linkages, are described, for example, in PCT publication WO 98/55495.

Those skilled in the art can readily determine whether a particular nucleic acid molecule containing an ISS is effective in enhancing a desired immune response in a particular mammal by immunizing a mammal of the same species, or a species known in the art to exhibit similar immune responses, with a composition containing a particular ISS. For example, an optimal ISS to include in an immunogenic composition for administration to a human can be determined in either a human or a non-human primate, such as a baboon, chimpanzee, macaque or monkey.

The immunogenic compositions of the invention further contain an adjuvant, such as an adjuvant demonstrated to be safe in humans. An exemplary adjuvant is Incomplete Freund's Adjuvant (IFA). Another exemplary adjuvant contains mycobacterium cell wall components and monophosphoryl lipid A, such as the commercially available adjuvant DETOX.TM.. Another exemplary adjuvant is alum. The preparation and formulation of adjuvants in immunogenic compositions are well known in the art.

Optionally, the immunogenic compositions of the invention can contain or be formulated together with other pharmaceutically acceptable ingredients, including sterile water or physiologically buffered saline. A pharmaceutically acceptable ingredient can be any compound that acts, for example, to stabilize, solubilize, emulsify, buffer or maintain sterility of the immunogenic composition, which is compatible with administration to a mammal and does not render the immunogenic composition ineffective for its intended purpose. Such ingredients and their uses are well known in the art.

The invention also provides kits containing an HIV antigen, an isolated nucleic acid molecule containing an ISS, and an adjuvant. The components of the kit, when combined, produce an immunogenic composition which enhances .beta.-chemokine levels in a mammal.

The components of the kit can be combined ex vivo to produce an immunogenic composition containing an HIV antigen, a nucleic acid molecule containing an ISS and an adjuvant. Alternatively, any two components can be combined ex vivo, and administered with a third component, such that an immunogenic composition forms in vivo. For example, an HIV antigen can be emulsified in, dissolved in, mixed with, or adsorbed to an adjuvant and injected into a mammal, preceded or followed by injection of the nucleic acid molecule containing the ISS. Likewise, each component of the kit can be administered separately. Those skilled in the art understand that there are various methods of combining and administering an HIV antigen, an isolated nucleic acid molecule containing an ISS, and an adjuvant, so as to enhance .beta.-chemokine production in a mammal.

An immunogenic composition of the invention is effective in enhancing .beta.-chemokine production in a mammal administered the composition. As described in Examples I and III, below, production of the .beta.-chemokine RANTES can be detected and quantitated using an ELISA assay of supernatants of T cells (such as lymph nodes cells or peripheral blood cells) from mammals administered the composition. In order to determine antigen-specific .beta.-chemokine production, T cells from an immunized mammal can be stimulated with HIV antigen in combination with antigen-presenting thymocytes, and the .beta.-chemokine levels measured in the supernatant. In order to determine non-specific .beta.-chemokine production, either T cell supernatant or a blood or plasma sample from an immunized mammal can be assayed. Similarly, production of other .beta.-chemokines, such as MIP-1.alpha. and MIP-1.beta., can be detected and quantitated using commercially available ELISA assays, according to manufacturer's instructions.

An immunogenic composition of the invention can further be capable of enhancing HIV-specific IgG2b antibody production in a mammal administered the composition. As described in Examples II and III, below, HIV in combination with ISS, or with IFA, stimulate HIV-specific IgG1 antibody production, but not HIV-specific IgG2b antibody production. In contrast, the immunogenic compositions of the invention can stimulate potent HIV-specific IgG2b antibody production. High levels of IgG2b antibodies, which are associated with a Th1 type response, are correlated with protection against HIV infection and progression to AIDS.

An immunogenic, composition of the invention can further be capable of enhancing HIV-specific cytotoxic T lymphocyte (CTL) responses in a mammal administered the composition. As described in Example II, below, an HIV antigen in combination with an adjuvant elicited low levels of IFN-.gamma. production by either CD4+ T cells or CD8+ T cells. However, when an ISS was included in the composition together with an HIV and an adjuvant, there was a dose-dependent increase in IFN-.gamma. production by both CD4+ T cells and CD8+ T cells.

IFN-.gamma. production by CD4+ T cells is characterized as a classic Th1-type response. IFN-.gamma. production by CD8+ T cells, however, is considered to be a cytotoxic T lymphocyte (CTL) response, and is highly correlated with cytolytic activity. CTL activity is an important component of an effective prophylactic or therapeutic anti-HIV immune response. Methods of determining whether a CTL response is enhanced following administration of an immunogenic composition of the invention are well known in the art, and include cytolytic assays (described, for example, in Deml et al. supra (1999)), and ELISA and ELISPOT assays for CD8-specific IFN-.gamma. production (see Examples I and II, below).

The invention also provides a method of immunizing an individual. The method consists of enhancing .beta.-chemokine production in an individual by administering to a mammal an immunogenic composition containing an HIV antigen, an isolated nucleic acid molecule containing an ISS, and an adjuvant. The components of the immunogenic composition can be administered in any order or combination, such that the immunogenic composition is formed ex vivo or in vivo.

Preferably, the HIV antigen, ISS and adjuvant are administered simultaneously or at about the same time, in about the same site. However, administering the components within several minutes or several hours of each other can also be effective in providing an immunogenic composition that enhances .beta.-chemokine production. Additionally, administering the components at different sites in the mammal can also be effective in providing an immunogenic composition that enhances .beta.-chemokine production.

The immunogenic compositions of the invention can be administered to a human to inhibit AIDS, such as by preventing initial infection of an individual exposed to HIV, reducing viral burden in an individual infected with HIV, prolonging the asymptomatic phase of HIV infection, increasing overall health or quality of life in an individual with AIDS, or prolonging life expectency of an individual with AIDS. As described in Examples I-III, below, administration to a mammal of an immunogenic composition containing an HIV antigen, an isolated nucleic acid molecule containing an immunostimulatory sequence, and an adjuvant stimulates immune responses correlated with protection against HIV infection and progression to AIDS.

In particular, the immunogenic compositions enhance .beta.-chemokine production more effectively than would be expected by combination of any two components of the immunogenic compositions. Additionally, the immunogenic compositions promote strong Th1 type immune responses, including both Th1 type cytokines (e.g. IFN-.gamma.) and Th1 type antibody isotypes (e.g. IgG2b). Thus, the immunogenic compositions of the invention will be effective as vaccines to prevent HIV infection when administered to seronegative individuals, and to reduce viral burden, prolong the asymptomatic phase of infection, and positively affect the health or lifespan of a seropositive individual.

Individuals who have been exposed to the HIV virus usually express in their serum certain antibodies specific for HIV. Such individuals are termed "seropositive" for HIV, in contrast to individuals who are "seronegative." The presence of HIV specific antibodies can be determined by commercially available assay systems.

At the present time, serological tests to detect the presence of antibodies to the virus are the most widely used method of determining infection. Such methods can, however, result in both false negatives, as where an individual has contracted the virus but not yet mounted an immune response, and in false positives, as where a fetus may acquire the antibodies, but not the virus from the mother. Where serological tests provide an indication of infection, it may be necessary to consider all those who test seropositive as in fact, being infected. Further, certain of those individuals who are found to be seronegative may in fact be treated as being infected if certain other indications of infection, such as contact with a known carrier, are satisfied.

The immunogenic compositions of the invention can be administered to an individual who is HIV seronegative or seropositive. In a seropositive individual, it may be desirable to administer the composition as part of a treatment regimen that includes treatment with anti-viral agents, such as protease inhibitors. Anti-viral agents and their uses in treatment regimens are well known in the art, and an appropriate regimen for a particular individual can be determined by a skilled clinician.

As shown in Example IV, below, administration of the immunogenic compositions of the invention to a primate fetus or to a primate neonate resulted in the generation of a strong anti-HIV immune response, indicating that the immune systems of fetuses and infants are capable of mounting an immune response to such compositions which should protect the child from HIV infection or progression to AIDS. Accordingly, the immunogenic compositions of the invention can be administered to an HIV-infected pregnant mother to prevent HIV transmission to the fetus, or to a fetus, an infant, a child or an adult as either a prophylactic or therapeutic vaccine.

The dose of the immunogenic composition, or components thereof, to be administered in the methods of the invention is selected so as to be effective in stimulating the desired immune responses. Generally, an immunogenic composition formulated for a single administration contains between about 1 to 200 .mu.g of protein. Preferably, an immunogenic composition contains about 100 .mu.g of protein for administration to a primate, such as a human. As shown in Example IV, below, about 100 .mu.g of HIV antigen in an immunogenic composition elicits a strong immune response in a primate. As shown in Examples I-III, below, about 10 .mu.g of HIV antigen is suitable for administration to a rodent.

The immunogenic composition can further contain from about 0.1 .mu.g/ml to about 1 mg/ml of an isolated nucleic acid molecule containing an ISS sequence, such as about 1 .mu.g/ml, about 10 .mu.g/ml, or about 100 .mu.g/ml. As shown in Example I, below, a ratio of at least 5:1 by weight of nucleic acid molecule to HIV antigen was more effective than lower ratios for eliciting immune responses. In rodents, an effective amount of an oligonucleotide containing an ISS in an immunogenic composition is from 5 .mu.g to greater than 50 .mu.g, such as about 100 .mu.g. In primates, about 500 .mu.g of an oligonucleotide containing an ISS is suitable in an immunogenic composition. Those skilled in the art can readily determine an appropriate amount of ISS to elicit a desired immune response.

As with all immunogenic compositions, the immunologically effective amounts of the components must be determined empirically, but can be based, for example, on immunologically effective amounts in animal models, such as rodents and non-human primates. Factors to be considered include the antigenicity, the formulation (e.g. volume, type of adjuvant), the route of administration, the number of immunizing doses to be administered, the physical condition, weight and age of the individual, and the like. Such factors are well known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.

The immunogenic compositions of the invention can be administered locally or systemically by any method known in the art, including, but not limited to, intramuscular, intradermal, intravenous, subcutaneous, intraperitoneal, intranasal, oral or other mucosal routes. The immunogenic compositions can be administered in a suitable, nontoxic pharmaceutical carrier, or can be formulated in microcapsules or as a sustained release implant. The immunogenic compositions of the invention can be administered multiple times, if desired, in order to sustain the desired immune response. The appropriate route, formulation and immunization schedule can be determined by those skilled in the art.

Claim 1 of 24 Claims

What is claimed is:

1. An immunogenic composition, comprising:

(a) a whole-killed HIV virus devoid of outer envelope protein gp 120;

(b) an isolated nucleic acid molecule containing an immunostimulatory sequence (ISS); and

wherein said composition synergistically enhances .beta.-chemokine levels in a mammal.

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