KR20070019635A - Immunogenic hiv compositions and related methods - Google Patents

Abstract

The present invention provides immunogenic compositions that enhance the duration and intensity of an immune response in a mammal. The immunogenic composition contains an HIV antigen, an immunomer, and an adjuvant. The HIV antigen can be a killed whole HIV virus without the envelope protein gp120. Alternatively, the HIV antigen can be killed whole HIV virus or p24 antigen. Also provided are kits that produce the immunogenic compositions of the invention when the components are combined. The present invention also provides a method of making an immunogenic composition by combining an HIV antigen, an immunomer, and optionally an immunoadjuvant. The present invention also provides a method of immunizing a mammal by administering to the mammal an immunogenic composition containing an HIV antigen, an immunomer and optionally an immunoadjuvant, thereby enhancing the immune response in the mammal. Also provided is a method of inhibiting AIDS in a mammal by administering to the mammal an immunogenic composition containing an HIV antigen, an immunomer, and optionally an immunoadjuvant.

Description

Immunogenic HIV composition and related methods {IMMUNOGENIC HIV COMPOSITIONS AND RELATED METHODS}

The present invention relates to an immunogenic composition for use in the prevention and treatment of Acquired Immunodeficiency Syndrome (AIDS), more specifically AIDS.

More than 30 million people worldwide are now infected with human immunodeficiency virus (HIV), the virus responsible for AIDS. Although the HIV epidemic is spreading rapidly around the world, about 90% of HIV-infected individuals live in developing countries, including parts of sub-Saharan Africa and parts of Southeast Asia. Antiviral therapies that reduce viral burden and slow the progression to AIDS have recently become available. However, these drugs are ridiculously expensive to use in developing countries. Thus, there remains a urgent need to develop effective preventive and therapeutic vaccines to reduce the global AIDS pandemic.

To date, HIV has proved to be a difficult target in developing effective vaccines. Because of the rapid propensity for mutation of HIV, there are now a number of strains that dominate in different parts of the world and differ in epitopes. In addition, in certain infected individuals, the HIV virus may be out of control of the host immune system by the development of mutations in the epitope. There remains a need to develop improved HIV vaccines that stimulate the immune system to recognize a broad spectrum of conserved epitopes, including epitopes derived from the p24 core antigen.

In the 1990s, more than 30 candidate HIV-1 vaccines entered human clinical trials. The vaccine elicits a variety of humoral and cellular immune responses, which vary in type and strength depending on the particular vaccine component. There remains a need to develop HIV vaccine compositions that strongly elicit specific immune responses that correlate with protection against HIV infection.

The nature of the protective HIV immune response has been reviewed through studies of individuals infected with HIV for many years without remaining infected or suffering from AIDS despite repeated exposure to HIV. The study revealed that CD4 + T helper cells correlated significantly with defense against HIV infection and subsequent disease progression. In addition to antigen-specific CD4 + helper T cell responses, CD8 + cytotoxic T cell responses are considered important for early HIV infection and disease progression. During an effective anti-viral immune response, activated CD8 + T cells kill the virus infected cells directly and secrete cytokines with antiviral activity.

The β-chemokine system has also been shown to be important in defense against early HIV infection and disease progression. Infection of immune cells by most primary isolates of HIV requires interaction of the virus with CCR5, a common biological role of CCR5 as a major receptor for β-chemokines RANTES, MIP-1α and MIP-β. . Genetic polymorphisms leading to reduced expression of the CCR5 receptor have been shown to provide resistance to HIV infection. In addition, a significant correlation between β-chemokine levels and resistance to HIV infection was demonstrated in both exposed individuals and cultured cells. It has been suggested that β-chemokines can block HIV infectivity by several mechanisms including competing or interfering with the binding of HIV to CCR5 and down-regulating surface CCR5.

Because of the importance of β-chemokines in the prevention of early HIV infection and disease progression, effective HIV immunogenic compositions should induce high levels of β-chemokine production prior to infection and in response to infectious viruses. HIV immunogenic compositions capable of inducing [beta] -chemokine production are disclosed. However, immunogenic compositions that promote high levels of β-chemokine production, induce strong and durable HIV-specific Th1 cellular and humoral immune responses and have HIV-specific cytotoxic activity have not been disclosed. .

Compositions that elicit certain types of HIV-specific immune responses may not elicit other important protective responses. See, eg, Deml et al., Clin. Chem. Lab. Med. 37: 199-204 (1999) describe a vaccine comprising HIV-1 gp160 envelope antigen, immune stimulating DNA sequence and alum adjuvants, which induce antigen-specific Th1 cytokine responses. Nevertheless, antigen-specific cytotoxic T lymphocyte responses cannot be induced. In addition, vaccines containing only envelope antigens are not expected to elicit an immune response against HIV's more highly conserved core proteins.

Thus, there is a need for immunogenic compositions and immune methods that help prevent HIV infection or slow the progression of at least to AIDS in infected individuals. The present invention fulfills this need and provides related advantages as well.

Summary of the Invention

The present invention provides immunogenic compositions that can be used for enhancing the immune response potential in mammals. The immunogenic compositions of the invention can enhance the breadth, type, intensity and duration of the immune response induced. The immunogenic composition contains an optimized HIV antigen, an isolated nucleic acid molecule comprising an immunoomer and optionally an immune adjuvant. The HIV antigen can be a killed whole HIV virus without the envelope protein gp120. The HIV antigen can also be protease defective HIV particles, eg L2 particles. Alternatively, the HIV antigen may be a killed whole HIV virus, including p24 antigen, nef, gp41 and the like, or a combination of selected HIV antigens or peptides.

In immunogenic compositions of the invention in which an adjuvant is present, the adjuvant may be suitable for administration to a human. Exemplary immune adjuvant is Incomplete Freund's Adjuvant.

The immunogenic compositions of the invention also produce β-chemokine levels, interferon-γ (IFNγ), interleukin 2 (IL2), tumor necrosis factor α (TNFα), and interleukin 15 (IL15) production and / or HIV-specific in mammals. May enhance potent IgG2b antibody production. The immunogenic compositions of the invention can also enhance HIV specific helper CD4 + T cells, HIV-specific cytotoxic T lymphocyte responses, and non-cytotoxic inhibitory T lymphocyte responses in mammals.

Also provided are kits comprising an HIV antigen, an immunomer, and optionally an adjuvant. The components of the kit, when combined, produce the immunogenic compositions of the invention.

The invention also provides a method of making an immunogenic composition by combining an HIV antigen, an immunomer and optionally an immunoadjuvant. The components can be combined ex vivo or in vivo to obtain an immunogenic composition.

The present invention also provides a method of immunizing a mammal by administering to the mammal an immunogenic composition containing an HIV antigen, an isolated nucleic acid molecule comprising an immunomer and optionally an immunoadjuvant. Also provided is a method of inhibiting AIDS in a mammal by enhancing the immune response by administering to the mammal an immunogenic composition comprising an HIV antigen, an isolated nucleic acid molecule comprising an immunomer, and optionally an immunoadjuvant. In the method of the invention the mammal may be a primate, for example a human, or a rodent. In certain embodiments of the method, the primate is a pregnant woman or an infant. Humans may be HIV seropositive or HIV seropositive positive. The present immunogenic compositions may be advantageously administered to mammals by two or more times and by various routes of administration, including subcutaneous, intramuscular and intramucosal routes.

1 shows the chemical structure of an exemplary linker for binding oligonucleotides for the formation of immunomers (Yu et al., J. Med . Chem . 45: 4540-4548 (2002)); Yu et al., Nucl.Acids Res. 30: 4460-4469 (2002)).

2 shows a schematic of the immunomer HYB2055, also known as Amplivax®.

3 shows the induction of HIV-specific cytokines RANTES, MIP1α, MIP1β, Interleukin-10 (IL-10) and IL-5 by HIV-1 immunogens. The immunogen was administered subcutaneously. "*" Indicates significance in saline.

4 shows that the production induced by the HIV-1 immunogen of HIV-specific interferon-γ (IFNγ) is augmented in a dose dependent manner by Amplivax®. "*" Indicates significance for HIV-1 immunogen alone.

5 shows the effect of Amplivax® on RANTES, MIP1α, MIP1β, IL-10 and IL-5. The immunogen was administered subcutaneously. "*" Indicates significance for saline.

6 shows HIV-specific IFNγ production enhancement by Amplivax®. Similar results were found for RANTES, MIP1α, MIP1β and IL-10. The immunogen was administered subcutaneously. "*" Indicates significance for saline.

FIG. 7 shows the enhancement of the effects of Amplivax® on HIV-specific IFNγ-secreting T cells in Elispot assays. The immunogen was administered subcutaneously. "*" Indicates significance for HIV-1 immunogen alone.

8 shows that HIV-specific INFγ production is enhanced by Amplivax® in a dose dependent manner. The immunogen was administered subcutaneously. "*" Indicates significance for HIV-1 immunogen alone.

FIG. 9 shows that HIV-specific RANTES production is enhanced by Amplivax® in a dose dependent manner. The immunogen was administered subcutaneously. "*" Indicates significance for HIV-1 immunogen alone.

FIG. 10 shows that HIV-specific MIP-1α production is enhanced by Amplivax® in a dose dependent manner. The immunogen was administered subcutaneously. "*" Indicates significance for HIV-1 immunogen alone.

FIG. 11 shows that HIV-specific MIP-1β production is enhanced by Amplivax® in a dose dependent manner. The immunogen was administered subcutaneously. "*" Indicates significance for HIV-1 immunogen alone.

12 shows that HIV-specific IL-10 production is enhanced by Amplivax® in a dose dependent manner. The immunogen was administered subcutaneously. "*" Indicates significance for HIV-1 immunogen alone.

13 shows that HIV-specific IL-5 production is reduced by Amplivax® given subcutaneous administration (SC). "*" Indicates significance for HIV-1 immunogen alone.

14 shows the effect of Amplivax® on the titer of HIV-1 immunogen-induced p24 antibody in mice. The immunogen was administered subcutaneously.

FIG. 15 shows that whole HIV-1 vaccine (HIV-1 immunogen) in IFA induces HIV specific cytokine production upon subcutaneous (SC) and intramuscular (IM) administration.

16 shows that Amplivax® can be added before or after emulsification with IFA to enhance IFNγ production.

17 shows that Amplivax® may be added before or after emulsification with IFA to enhance RANTES production.

FIG. 18 shows that the whole HIV-1 vaccine vaccine including Amplivax®, which does not contain IFA, in mice immunized by subcutaneous administration triggers HIV-specific IFNγ production. "*" Indicates significance for HIV-1 immunogen (IM). The HIV antigen is a whole HIV vaccine containing no IFA.

FIG. 19 shows that the pre-HIV-1 vaccine vaccine containing Amplivax®, which does not contain IFA, in mice immunized by subcutaneous administration triggers HIV-specific IFNγ-secreting CD8 + T cell activity. "*" Indicates significance for HIV-1 immunogen (IM administration). The HIV antigen is a whole HIV vaccine containing no IFA.

FIG. 20 shows that the pre-HIV-1 vaccine vaccine including Amplivax®, including IFA, triggers HIV-specific RNATES production in mice immunized by subcutaneous administration. "*" Indicates significance for HIV-1 immunogen (IM administration). The HIV antigen is a whole HIV vaccine containing no IFA.

FIG. 21 shows that the percentage of α-defensin producing CD8 + T cells is increased by Amplivax® added in vitro.

FIG. 22 shows HIV-specific IFNγ-producing CD8 + T cell numbers in REMUNE® treated patients and HIV positive controls (0 μg / ml of Amplivax®).

FIG. 23 shows HIV-specific IFNγ-producing CD8 + T cell numbers in the presence of 0.1 μg / ml Amplivax® added ex vivo.

FIG. 24 depicts HIV-specific IFNγ-producing CD8 + T cell numbers in the presence of 1 μg / ml Amplivax® added ex vivo.

25 shows the number of HIV-specific IFNγ-producing CD8 + T cells in the presence of 10 μg / ml Amplivax® added ex vivo.

FIG. 26 shows IFN-γ ELIspot analysis in peripheral blood mononuclear cells (PBMC). HYB2055 was used at 1 μg / ml.

FIG. 27 shows phenotypic changes in CD4 T cells after the first injection of REMUNE® in patients with antiretroviral therapy (ART) naive.

FIG. 28 shows phenotypic changes in CD8 T cells after the first injection of REMUNE® in ART naïve patients.

The present invention provides immunogenic HIV compositions containing an HIV antigen, an isolated nucleic acid molecule comprising an immunoomer, and optionally an immunoadjuvant. Also provided is a kit comprising the components of such a composition for use together. The present invention also provides a method of immunizing a mammal with such a composition, or a component of such a composition, in order to enhance an immune response in an immunized mammal compared to an HIV antigen alone. Advantageously, the compositions of the present invention also induce HIV specific CD4 helper cells and CD8 + T cells to produce a potent Th1 immune response against a broad spectrum of HIV epitopes, resulting in a strong HIV-specific cytotoxic T lymphocyte response. to provide. Thus, the immunogenic compositions of the invention are useful for preventing HIV infection and / or slowing the progression to AIDS in infected individuals. The compositions and methods induce a potent Th1 cellular and humoral immune response specific for conserved HIV epitopes, elicit HIV-specific CD4 T helper cells, HIV-specific cytotoxic T lymphocyte activity, chemokines and It is used to promote the production of cytokines such as β-chemokine, interferon-γ, interleukin 2 (IL2), interleukin 7 (IL7), interleukin 15 (IL15), α-defensin, and to increase memory cells. Can be. Such vaccines can be administered through various routes of administration. Such vaccines can be used for the prevention of maternal transmission of HIV in vaccination of newborns, children and high risk individuals, and in vaccination of infected individuals. Such vaccines may also be used in combination with other HIV therapies, including antiretroviral therapy (ART) using various combinations of nuclease and protease inhibitors and agents for the blocking of viral entry, such as T20 ( See Baldwin et al., Curr . Med . Chem . 10: 1633-1642 (2003).

As used herein, the term "HIV" refers to all forms, subtypes and variants of the HIV virus and is synonymous with the older terms HTLVIII and LAV. Various cell lines capable of propagating HIV or permanently infected with the HIV virus have been developed and deposited with the ATCC, including HuT 78 cells and HuT 78 derivative H9, with accession numbers CCL 214, TIB 161, CRL 1552 and CRL 8543. And US Patent Nos. 4,725,669 and Gallo, Scientific. American 256: 46 (1987).

As used herein, “killed whole HIV virus” refers to an inactivated HIV virus intact. Inactivated HIV refers to a virus that cannot be infected and / or replicated.

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

As used herein, the term “HIV virus without envelope protein” refers to preparations of HIV particles or HIV gene products that lack the envelope protein gp120, but contain genetically more conserved portions of the virus (eg p24 and gp41). Indicates. HIV without the coat protein gp120 is also referred to herein as REMUNE ™.

As used herein, the term "HIV p24 antigen" refers to the gene product of the gag region of HIV called p24, characterized by an apparent relative molecular weight of about 24,000 daltons. The term "HIV p24 antigen" also refers to variants and fragments having the immunological activity of p24. Those skilled in the art can determine appropriate modifications of p24, such as, for example, additions, deletions or substitutions of natural amino acids or amino acid analogs that function to increase stability or bioavailability or to aid purification without disrupting immune activity. . Likewise, one of skill in the art can determine appropriate p24 fragments having the immunological activity of p24. A p24 fragment with immune activity may have up to six residues from this polypeptide up to the amino acid of full length polypeptide-1. Other HIV antigens encoded by other HIV gene products may include fragments or variants similar to those described above for the HIV p24 antigen. Other exemplary HIV antigens include, for example, gp41, nef, and the like.

As used herein, “immunomer” refers to an oligonucleotide comprising two smaller oligonucleotides linked at the 3 ′ end, which results in an oligonucleotide having two 5 ′ ends. Two smaller oligonucleotides of an immunomer may be of the same or unequal sequence and / or length but are generally the same. Immunomers, in addition to their immunostimulatory activity, contain 3'-3 'bonds and thus have no free 3' termini, thus increasing resistance to nuclease cleavage. The smaller oligonucleotides of the immunomers are generally at least about 5 or 6 nucleotides that are joined together to form two 5 'ends, but are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, It may be an immunomer of longer, smaller oligonucleotides, such as 17, 18, 19, 20 or even longer. One skilled in the art can readily determine the length and / or sequence of an immunomer sufficient to stimulate a larger immune response than seen with the antigen alone. Thus, in one embodiment the immunomer comprises two identical oligonucleotides linked through its 3 'end. Immunomers may also include modified bases. Immunomers are described, for example, in Kandimalla et al., Bioorg . Med . Chem . 9: 807-813 (2001); Yu et al., Nucl . Acids Res . 30: 4460-4469 (2002); Yu et al., Bioorg . Med. Chem . 11: 459-464 (2003); Bhagat et al., Biochem . Biophys . Res . Comm. 300: 853-861 (2003); And Yu et al., Biochem . Biophys . Res . Comm . 297: 83-90 (2002); Yu et al., Nucl . Acids Res . 30: 1613-1619 (2002); Yu et al., J. Med . Chem . 45: 4540-4548 (2002); Kandimalla et al., Bioconjugate Chem . 13: 966-974 (2002); Yu et al., Bioorganic Med . Chem. Lett . 10: 2585-2588 (2000); Agrawal and Kandimalla, Trends Mol . Med. 8: 114-121 (2002), each of which is incorporated herein by reference. Such immunomers may have stronger immune stimulating activity than immune stimulating sequences comprising CpG. Immunomers enhance immune responses upon administration in combination with antigens in mammals. Immunomers can be CpG immunomers or CpG immunofree as discussed below. Exemplary immunomers are described in Examples X and XI.

As used herein, “CpG immunomer” refers to an immunomer that specifically includes a CpG motif as described above. Thus CpG immunomers are oligonucleotides comprising two identical or non-identical smaller oligonucleotides, where one or more of the smaller oligonucleotides comprise one or more CpG motifs.

As used herein, “free CpG immunoomer” refers to an immunomer with the CpG motif specifically excluded. Thus, a CpG immunomer free is an oligonucleotide comprising two identical or unequal smaller oligonucleotides, wherein none of the smaller oligonucleotides contain a CpG motif.

Immunomers can include modified bases (see Kandimalla et al., Supra, 2001). For example, an immunomer may comprise an analog of CpG. For example, an immunomer may comprise a pyrimidine analog of deoxycytosine represented by Y. Particularly useful deoxycytidine analogs for use in immunomers are deoxy-5-hydroxycytidine or deoxy-N4-ethylcytidine. In other embodiments, the immunomers may comprise purine analogs of guanine represented by R. Particularly useful deoxyguanine analogs are 7-deazaguanine. Thus an immunomer may comprise a YpG motif, a CpR motif or a YpR motif, where Y and R are cytosine analogs and guanine analogs, respectively.

A method for combining two smaller oligonucleotides to form an immunomer is described above (Kandimalla et al., Bioorg . Med . Chem . 9: 807-813 (2001); Yu et al., Nucl. Acids Res . 30: 4460-4469 (2002); Yu et al., Bioorg . Med . Chem . 11: 459-464 (2003); Bhagat et al., Biochem . Biophys. Res . Comm . 300: 853-861 (2003); And Yu et al., Biochem . Biophys. Res . Comm . 297: 83-90 (2002); Yu et al., Nucl . Acids Res . 30: 1613-1619 (2002); Yu et al., J. Med . Chem . 45: 4540-4548 (2002); Kandimalla et al., Bioconjugate Chem . 13: 966-974 (2002); Yu et al., Bioorganic Med . Chem . Lett . 10: 2585-2588 (2000); Agrawal and Kandimalla, Trends Mol . Med . 8: 114-121 (2002)). Exemplary linkers include, for example, 3′-3 ′ conjugates via glyceryl linkers (Yu et al., Biochem . Biophys. Res . Comm . 297: 83-90 (2002)). Linkers are described in Yu et al., J. Med . Chem. 45: 4540-4548 (2002), which may be alkyl, branched alkyl or ethylene-glycol linkers (see FIG. 1). Those skilled in the art will readily recognize that these and other methods can be used to combine oligonucleotides through the 3 'end to produce two free 5' ends.

As used herein, the term "immunostimulatory sequence" or "ISS (immunostimulatory sequence)" refers to a nucleotide sequence comprising an unmethylated CpG motif capable of enhancing an immune response when administered in combination with an antigen in a mammal. Indicates. Immune stimulating sequences are described, for example, in PCT Publication No. WO98 / 55495.

As used herein, the term “nucleic acid molecule comprising an immunomer” refers to a linear, circular or branched single or double stranded DNA or RNA nucleic acid comprising an immunoomer. Nucleic acid molecules comprising an immunoomer may comprise one immunoomer. Nucleic acid molecules may also contain more than one immunomer if one or both free 5 'ends are joined to the 5' end of another nucleic acid sequence to produce another potential 3 'end for binding additional immunomers. have. Such nucleic acid molecules may be of any length of more than 6 bases or base pairs, in addition to immunomers, and are generally more than about 15 bases or base pairs, for example more than about 20 bases or base pairs, may be kb. If such nucleic acid comprising an immunomer is circular, or if it comprises a large number of immunomers requiring 5'-5 'binding, as found in short immunomers (Kandimalla et al., Supra, 2002, and Yu et al., Supra, 2000) As long as the 5'-5 'bonds embedded in the nucleic acid do not interfere with the immunostimulatory activity of the immunomer, for example, Kandamalla et al., Bioconjugate Chem . 13: 966-974 (2002), and Yu et al., Bioorgan . Med . Chem . 10: 2585-2588 (2000), it can be seen that the free 5 'end is bound. The nucleic acid comprising an immunomer may further comprise a nucleic acid sequence encoding one or more HIV antigens and for use as a DNA vaccine.

Immunomers or nucleic acids comprising immunomers, as disclosed herein, can be produced, for example, by chemically synthesizing oligonucleotides and chemically binding the oligonucleotides through their 3 'ends. Also, nucleic acid molecules comprising an immunomer or immunomer can be produced by synthesizing two halves of an immunonomer or nucleic acid comprising an immunomer in a recombinant manner and combining these two halves chemically through their 3 'terminus. have.

Immunomers can include natural or modified nucleotides or natural or unnatural nucleotide bonds. Modifications known in the art include, for example, modification of 3'OH or 5'OH groups, modification of nucleotide bases, modification of sugar components and modification of phosphate groups. Non-natural nucleotide bonds may be, for example, phosphorothioate bonds instead of phosphodiester bonds, which increase the resistance of the nucleic acid molecule to nuclease degradation. Various modifications and combinations are described, for example, in PCT Publication No. WO 98/55495.

As used herein, the term “immune adjuvant” when added to an immunogenic agent refers to a substance that, upon exposure to this mixture, nonspecifically enhances or enhances the immune response to that agent in the recipient host. . Immunoadjuvant may include, for example, oil-in-water emulsions, water-in-oil emulsions, alum (aluminum salts), liposomes and particulates such as polystyrene, starch, polyphosphazenes and polyacetide / polyglycosides. Immunoadjuvant also contains, for example, squalene mixture (SAF-I), muramyl peptides, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazenes and derivatives, and immunostimulating complexes (ISCOMs), for example Takahashi et al. (1990) Nature 344: 873-875. In veterinary use and in the production of antibodies in animals, the mitogenic component of Freund's immune adjuvant (both complete and incomplete) can be used. In humans, incomplete Freund's adjuvant (IFA) is a particularly useful immunoadjuvant. Various suitable immune adjuvants are well known in the art and are described, for example, in Warren and Chedid, CRC. Critical Reviews in Immunology 8: 83 (1988).

As used herein, "AIDS" refers to the symptomatic phase of HIV infection, see Adler, Brit . Med . J. 294: 1145 (1987), both acquired immune deficiency syndrome (commonly known as AIDS) and "ARC" or AIDS related complications. Immunological and clinical signs of AIDS are well known in the art and include, for example, cancer and opportunistic infections resulting from immunodeficiency.

As used herein, the term “inhibiting AIDS” refers to the beneficial prophylactic or therapeutic effect of an immunogenic composition in connection with HIV infection or AIDS symptoms. Such beneficial effects include, for example, the prevention or delay of the initial infection of individuals exposed to HIV; Reduction in viral burden in individuals infected with HIV; Prolonged asymptomatic phase of HIV infection; Maintenance of low viral load in HIV infected patients where the level of virus is lowered through anti-retroviral therapy (ART); Overall health or life expectancy in individuals with AIDS due to decreased CD4 T cell levels or decreased CD4 T cells, which can be both HIV-1 specific and nonspecific in patients treated with drug naïve and ART Increase in quality; And prolonging the life expectancy of the individual suffering from AIDS. The clinician can compare the immunization effect with the state of the patient prior to treatment, or the expected state of untreated patients, to determine if this treatment is effective in the inhibition of AIDS.

As used herein, the term "enhancing" with respect to an immune response is intended to mean that the immunogenic composition causes a larger immune response than a composition containing an HIV antigen alone produces an immune response. If the immunogenic composition contains three components, an HIV antigen, an immunomer, and an adjuvant, the immunogenic composition contains the same immunization schedule administered in the same amount, containing any two of the three components of the immunogenic composition. The later composition elicits an immune response that is greater than that which elicits an immune response. The components of the immunogenic compositions of the present invention may act synergistically. Enhancing the immune response can be, for example, increased production of chemokines and / or cytokines that promote memory cells, increased memory cells, increased IgG2b production, increased cytotoxic T lymphocyte activity, increased β-chemokine or IL15 production, and the like. have. As an example of enhancing immune response, the immunogenic compositions of the present invention can increase the production of γ-interferon by both CD4 cells (helper function) and CD8 cells (cytotoxic T lymphocytes; CTL).

As used herein, the term "β-chemokine" refers to a small amount of chemoattractive chemicals, including RANTES, macrophage inflammatory protein-1β (MIP-1β) and macrophage inflammatory protein-1α (MIP-1α). Represents members of polypeptides. The physical and functional properties of β-chemokines are well known in the art.

For β-chemokine production enhancement, β-chemokine production may be “HIV-specific β-chemokine production” indicating the production of β-chemokine in response to stimulation of T cells by HIV antigens. Alternatively, or in addition, enhanced β-chemokine production may be “non-specific β-chemokine production” indicating production of β-chemokine without stimulation of T cells by the HIV antigen.

As used herein, the term "kit" refers to packaging or commercial ingredients for use together. For example, the kit may include HIV antigens, immunomers and immune adjuvant in three separate containers. Alternatively, the kit may include any two components in one container and the third component and any additional components in one or more separate containers. Optionally, the kit further includes instructions for combining the components for formulation of an immunogenic composition suitable for administration to a mammal.

The present invention provides immunogenic compositions containing HIV antigens, immunomers and optionally immune adjuvant. The immunogenic composition enhances the immune response in the mammal to which the composition is administered. In one embodiment, the present immunogenic compositions enhance the HIV-specific cytotoxic T lymphocyte (CTL) response in mammals. In another embodiment, the immunogenic composition enhances HIV-specific CD4 + helper T cells.

In one embodiment the HIV antigen in the immunogenic composition is killed whole HIV virus, which can be prepared by methods known in the art. For example, the HIV virus can be produced by culturing from a peripheral blood sample of an infected individual. In an exemplary method of culturing HIV virus, mononuclear cells (e.g., lymphocytes) from peripheral blood are placed in a heparinized venous blood sample in a Ficoll-Hypaque density gradient and centrifuged. Can be obtained. Mononuclear cells are then collected and activated for 2-3 days, as in phytohemagglutinin, and cultured in a medium supplemented with a suitable medium, preferably interleukin 2 (IL2). Viruses can be detected by reverse transcriptase, antigen capture assays for p24, immunofluorescence, or electron microscopy to detect the presence of viral particles in cells, all of which are well known to those skilled in the art.

Methods of isolating pre-killed HIV particles are described, for example, in Richie et al., Vaccine 16: 119-129 (1998) and US Pat. No. 5,661,023 and US Pat. No. 5,256,767. In one embodiment the HIV virus is an HZ321 isolate derived from an individual infected in Zaire in 1976, which is described by Choi et al., AIDS Res . Hum . Retroviruses 13: 357-361 (1997).

Various methods of making a virus non-communicable are known in the art (see, eg, Hanson , MEDICAL). VIROLOGY II (1983), de la Maza and Peterson, eds., Elsevier,]. For example, viruses can be inactivated by treatment with chemicals or by physical conditions such as heating or irradiation. Preferably the virus is treated with agent (s) that maintains the immunogenicity of the virus. For example, the virus can be treated with β-propiolactone or gamma radiation, or both β-propiolactone and gamma radiation, at a dose sufficient to inactivate the virus and for a time sufficient to do so.

In another embodiment the HIV antigen in the immunogenic composition is a killed whole HIV virus devoid of envelope proteins, which can be prepared by methods known in the art. For the production of pre-killed viruses without envelope proteins, the isolated viruses are treated to remove the envelope proteins. Such removal is achieved by repeatedly freezing and thawing the virus along with other physical or non-physical methods, such as sonication, alone or in combination, but with physical methods that cause swelling and shrinkage of the virus particles. It is desirable to.

In another embodiment, the HIV antigen in the immunogenic composition is one or more HIV gene products that are substantially purified. Such gene products include the 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 and the nef protein. Such gene products may be used alone or in combination with other HIV antigens. The HIV antigen can also be a peptide fragment of an HIV gene product that elicits an immune response.

The substantially purified HIV gene product may be a substantially purified HIV p24 antigen or other HIV antigens and gene products. p24 and other HIV antigens may be substantially purified from viruses by biochemical methods known in the art, or by appropriate methods in the host organisms, such as bacteria, fungi or mammalian cells. By cloning and expression. Alternatively, the p24 antigen, or variants or fragments thereof retaining the immune activity of p24, and other HIV antigens or variants or fragments thereof can be synthesized using methods well known in the art, such as automated peptide synthesis. have. Whether the variant or fragment of p24 retains the immune activity of p24, or whether other viral antigens retain their respective immune activity, is, for example, the conventional lymphocyte proliferation assay (LPA) known in the art By comparison of mammalian immunization and the resulting immune response, or by testing the ability of a variant or fragment to compete with p24 for binding to p24 antibodies or binding other HIV antigens to their respective antibodies. As analyzed, it can be determined by the ability to stimulate the in vitro proliferation of previously immunized PBMCs.

In another embodiment the HIV antigen in the immunogenic composition is a substantially purified gene product of protease deficient HIV (see US Pat. No. 6,328,976 and US Pat. No. 6,557,296).

The replication process for HIV-1 has an error rate of about 1 per 5-10 base pairs. Since the entire viral genome is less than 10,000 base pairs, this leads to an error rate of about one base pair per replication cycle. This high mutation rate contributes to the broad variability of the virus in one person and even wider variability across the population.

Three HIV-1 variants described by this mutant and approximately 10 viral subspecies called "clades" were produced. This distinction is based on the structure of the envelope protein, which is particularly variable. The M (major) variant is by far the most dominant in the world. Within the M variants are clades A, B, C, D, E, F, G H, I, J and K, clades A through E represent the vast majority of infections worldwide. Clades A, C and D dominate in Africa. Claude B is the most widespread in Europe, North and South America, and Southeast Asia. Clades E and C dominate in Asia. The clades differ from each other by as much as 35%.

There are two important consequences from the very high mutation rate of HIV-1, which has profound consequences for the pandemic. First, high mutation rates are one of the mechanisms by which viruses are released from control by drug therapy. These new viruses represent resistant strains. High mutation rates also leave the patient's immune system by altering the structure in which the virus is recognized by immune components. The added consequence of this wide range of variability is that viruses can escape from control by vaccines, and vaccines based on envelope proteins are unlikely to be effective.

The largest variation in structure is seen in the envelope proteins gp120 and gp41. Less variation is seen in various internal proteins. As disclosed herein, REMUNE is an immunogen that is made from whole virus that does not contain the gp120 protein but contains most of the highly conserved epitopes of HIV-1 virus. Both the number of these epitopes and the low incidence of mutations of epitopes mean that the HIV virus without the envelope protein, eg REMUNE, stimulates an immune response with greater chance of success in the individual. In addition, the HuT 78 cell line was deliberately infected with a very early HIV virus mainly comprising both clades A and G in the conserved antigen retained across most of the variants in clades observed worldwide. 78 HIV infected cell lines provide the virus used as the HIV antigen. Thus in the immunization the use of HIV viruses comprising a large number of initial clades that are also free of envelope proteins can be effective across clades by providing conserved antigens that can be recognized by most proteins.

HIV antigens and immunomers may be mixed together or may be conjugated by covalent or non-covalent bonds. Methods for conjugation of antigens and nucleic acid molecules are known in the art, and exemplary methods are described in PCT Publication WO 988/55495.

Oligonucleotide components of an immunomer 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 preparing oligonucleotide components of immunomers comprising oligonucleotides comprising one or more modified bases or linkages are described, for example, in PCT Publication No. WO 98/55495.

One of ordinary skill in the art would be effective in enhancing the desired immune response of a particular immunogen in a particular mammal by immunizing mammals of the same species, or species known in the art as having a similar immune response with a composition containing a specific immunomer. Can be easily determined. Various assays known in the art can then be used to characterize and compare the characteristics of the elicited immune response. Optimized immunomers, for example, contained in immunogenic compositions for human administration, are human or non-human primates such as baboons, chimpanzees, macaques or monkeys, for example LPA, ELISPOT, and And / or by evaluating its immune activity by the ratio of generated IgG1 / G2 antibodies.

The immunogenic composition of the present invention may further contain an adjuvant, such as an adjuvant that has proven safe in humans. Exemplary immune adjuvants include incomplete Freund's immune adjuvants (IFA). Other exemplary immune adjuvants include the Mycobacterium cell wall component and monophosphoryl lipid A, such as the commercially available immune adjuvant DETOX ™. Another exemplary immune adjuvant is alum. The preparation and formulation of immune adjuvants in immunogenic compositions is well known in the art.

Optionally, the immunogenic compositions of the present invention may contain or be formulated with other pharmaceutically acceptable ingredients, including sterile water or physiologically buffered saline. Pharmaceutically acceptable components can be any compound that functions, for example, to stabilize, solubilize, emulsify, buffer, or maintain the bactericidal properties of an immunogenic composition, which compound is compatible with administration to a mammal. It is possible to ensure that the immunogenic composition is not invalidated for its intended purpose. Such ingredients and their use are well known in the art.

The present invention also provides kits containing HIV antigens, immunomers and optionally immune adjuvant. The components of the kit, when combined, produce an immunogenic composition that enhances an immune response in a mammal.

The components of the kit can be combined ex vivo to produce an immunogenic composition containing an HIV antigen, an immunomer, and optionally an immunoadjuvant. Alternatively, any two components can be combined in vitro and administered together with the third component such that the immunogenic composition is formed in vivo. For example, HIV antigens can be emulsified, lysed in adjuvant, admixed with adsorbent, adsorbed to adjuvant, and injected into mammals before or after injection of the immunomer. Likewise, each component of the kit can be administered separately. Those skilled in the art will recognize that there are a variety of ways to enhance the immune response in mammals by combining and administering HIV antigens, immunomers, and optionally immune adjuvant. As discussed in more detail below, the immunogenic compositions of the invention are well known in the art, including but not limited to intramuscular, intradermal, intravenous, subcutaneous, intraperitoneal, intranasal, oral or other mucosal routes. Local or systemic administration may be by known methods.

As disclosed herein, REMUNE has been found to be immunogenic in most patients, although with varying degrees of efficacy and duration (Example VIII). Thus immunogenic compositions containing HIV, eg, REMUNE, in combination with IFA immune adjuvant and without envelope proteins can be used to induce an immune response in most patients infected with HIV. The immunogenic compositions of the invention enhance the strength and potential of the immune response to HIV immunogens, eg REMUNE ™, thereby enhancing the therapeutic and / or prophylactic efficacy of the vaccine. Enhancing the immune response may include, for example, increased production of HIV-1 specific CD4 + helper T cells, chemokines and / or cytokines, increased memory cells, increased overall antibody production and more specifically an increase in the ratio of IgG2b production, cytotoxic T Increased lymphocyte activity, increased β-chemokine or IL15 production, and the like. As such, the immunogenic compositions of the present invention can be used for the enhancement of TH1 cytokine profiles (high IFNγ, high IgG2 / IgG1 ratios). As disclosed herein, the components of the immunogenic compositions of the invention may act synergistically. For example, the immunogenic compositions of the present invention can enhance β-chemokine production by inducing the production of higher concentrations of β-chemokine than would be expected by adding the effect of a pairwise combination of components of the immunogenic composition. .

Memory cells are required for long-term immunity after the initial acute state of infection. Significant amounts of immune cells induced against infectious agents during the systolic period after the initial acute state of infection are destroyed by apoptosis, leaving only viable cells to become memory cells . Thus the defense of HIV-specific CD4 and CD8 T cells from apoptosis promotes an increase in both HIV-specific CD4 helpers and CD8 CTL memory cells. The immunogenic compositions of the invention can be used to increase memory cells, thereby promoting long-term helper function and cell mediated immunity. The immunogenic compositions of the present invention can be used to increase the number of memory cells by reducing apoptosis or by stimulation of factors that promote memory cell survival.

The immunogenic compositions of the present invention can be used to convert TH2 into a TH1 response, thereby increasing cell mediated immune responses, including a stronger CD8 + response. As such, the immunogenic composition of the present invention can be used to enhance an immune response in a patient, which otherwise would only respond weakly, and the composition of the present invention could be used to convert the immune response to cell mediated immunity. have. Thus, the immunogenic compositions of the present invention can be used to increase the intensity and duration of an immune response in patients who respond weakly to HIV antigens similar to those used in immunogenic compositions.

Immunogenic compositions of the invention, in mammals to which the composition is administered, enhance immune responses, eg, β-chemokine and / or enhance IL15, IFN, IL2, TNFα production, HIV-specific CD4 helper cells, It is effective in IgG2b antibody production, HIV-specific cytotoxic T lymphocyte (CTL) production, increased IFNγ production by CD4 + cells and CD8 T cells, and the like. Β-, as described in U.S. Patent Application Nos. 09 / 565,906, filed May 5, 2000, and WO 00/67787, each of which is incorporated herein by reference, and Examples I and III below. The production of chemokine RANTES can be detected and quantified using ELISA assays of supernatants of mammalian derived T cells (eg lymph node cells or peripheral blood cells) to which the composition is administered. For the determination of antigen-specific β-chemokine production, T cells from immunized mammals can be stimulated with HIV antigens in combination with antigen presenting thymic cells and measure the level of β-chemokine in the supernatant. For measurement of nonspecific β-chemokine production, T cell supernatants or blood or plasma samples from immunized mammals can be analyzed. Similarly, the production of other β-chemokines such as MIP-1α and MIP-1β can be detected and quantified according to manufacturer's instructions using commercially available ELISA assays.

Methods of measuring the production of cytokines including interferon, IL15, IL2, TNFα, IL10 and IL7 by ELISPOT, ELISA, or intracellular cytokine staining are well known to those skilled in the art (see, eg, Robbins et al., AIDS 17: 1121-1126 (2003)).

The immunogenic compositions of the invention can also enhance the production of HIV-specific IgG2b antibodies in the mammal to which the composition is administered. High levels of IgG2b antibodies combined with Th1 type responses correlate with protection against HIV infection and progression to AIDS. As such, the present invention provides a composition that can increase the TH1 response.

The immunogenic compositions of the invention can also enhance HIV-specific cytotoxic T lymphocyte (CTL) responses in the mammal to which the composition is administered. The immunogenic compositions of the invention can increase IFN-γ production by both CD4 + T cells and CD8 + T cells.

IFN-γ production by CD4 + T cells is characterized as a traditional CD4 helper response important for cell mediated immunity. CD4 + T cells producing both IFN and IL2 may be most effective. IFN-γ production by CD8 + T cells is a representative cytotoxic T lymphocyte (CTL) response, which is highly correlated with cytolytic activity. Cells producing both IFN and TNFα may be most effective. CTL activity is an important component of an effective prophylactic or therapeutic anti-HIV immune response. Methods for determining whether a CTL response is enhanced after administration of an immunogenic composition of the present invention are well known in the art and are described in cell lysis assays and LPA assays (see, eg, Deml et al. Supra (1999)). Described; see Example III) and ELISA and ELISPOT assays for CD8-specific IFN- [gamma] production (see US Patent Application Nos. 09 / 565,906 and WO 00/67787 and Examples I and II below), FACS analysis using myriad antibodies to intracellular staining and cell surface markers.

The invention also provides a method for immunizing an individual. The method consists in augmenting an immune response in an individual by administering to the mammal an immunogenic composition containing an HIV antigen, an immunomer, and optionally an immunoadjuvant. The components of the present immunogenic compositions may be administered in any order or combination such that the immunogenic composition is formed ex vivo or in vivo.

In certain embodiments, the HIV antigens, immunomers and selective immune adjuvant are administered simultaneously or approximately simultaneously at approximately the same site. However, administration of components within minutes or hours with respect to each other may also be effective in providing immunogenic compositions that enhance an immune response. In addition, administering the components to different sites in a mammal can also be effective in providing an immunogenic composition that enhances an immune response. One of ordinary skill in the art would separately administer the components, ie HIV antigens, immunomers and selective immune adjuvant components, separately to provide a sufficient immune response by the administration of the separate components and measurement of the immune response at various times and places. Easily determine the right time and place. The present immunogenic compositions may also be desired multiple times, for example at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times. , Or any desired number of times to stimulate or enhance HIV-specific immune responses.

The immunogenic compositions of the present invention provide for the prevention of early infection in individuals exposed to HIV, reduction of viral burden in individuals infected with HIV, prolonged asymptomatic period of HIV infection, general health or life expectancy in individuals with AIDS. It can be administered to humans for suppression of AIDS, such as by increasing quality, or by prolonging the life expectancy of individuals with AIDS. As disclosed herein, administration of an immunogenic composition containing an HIV antigen, an isolated nucleic acid molecule comprising an immunomer, and optionally an immunoadjuvant to a mammal correlates with defense against HIV infection and progression to AIDS Immune response is stimulated.

In particular, the present immunogenic composition is expected by a combination of any of the individual components, or by a combination of any two components of the immunogenic composition, in a three component composition containing HIV antigens, immunomers and immunoadjuvant. More effectively enhances the immune response. In addition, the immunogenic compositions include a Th1 cytokine (eg IFN-γ) and a Th1 antibody isotype (eg IgG2b) to promote a strong Th1 immune response. As such, the immunogenic compositions of the present invention, when administered to a seropositive negative subject, prevent HIV infection, reduce viral burden, prolong the asymptomatic stage of infection, and positively affect the health or longevity of the seropositive subject. Effective as a vaccine.

Individuals exposed to the HIV virus generally express certain antibodies specific for HIV in their serum. Such individuals are termed "serum response positive" for HIV in contrast to individuals that are "serum response negative". The presence of HIV specific antibodies can be measured by commercially available assay systems.

At present, serological tests for detecting the presence of antibodies to viruses are the most widely used infection determination methods. In such methods, however, both false negatives in which the individual has reduced the virus but have not yet raised the immune response, and false positives in which the fetus acquires antibodies from the mother but no virus can be produced. If serological tests provide signs of infection, it may be necessary to consider all those tested positive for serologic reaction as actually infected. In addition, certain individuals found to be sero negative may be actually treated as infected if other predetermined signs of infection, such as contact with known carriers, are met.

The immunogenic compositions of the invention can be administered to a subject who is HIV seropositive or seropositive. In seropositive individuals it may be desirable to administer the composition as part of a treatment regimen comprising treatment with an anti-viral agent, eg, a protease inhibitor. Anti-viral agents and their use in therapeutic regimens are well known in the art, and the appropriate regimen for a particular individual can be determined by a skilled clinician.

As described in US Patent Application Nos. 09 / 565,906 and WO 00/67787 and as disclosed herein and in Example IV below, administration of an immunogenic composition of the invention to a primate fetus or primate newborn A strong anti-HIV immune response is generated, which indicates that the fetal and infant immune systems can boost the immune response to compositions that should not allow children to become infected with HIV or progress to AIDS. Thus, the immunogenic compositions of the present invention can be administered to HIV-infected pregnant women to prevent HIV transmission to the fetus, or can be administered to the fetus, infant, child or adult as a prophylactic or therapeutic vaccine.

The dose of immunogenic composition, or component thereof, to be administered in the method of the present invention is selected to be effective in stimulating the desired immune response. Generally, immunogenic compositions formulated for single administration contain between about 1 and 200 μg protein antigen. Immunogenic compositions generally contain about 100 μg of protein antigen in administration to primates, for example humans. As described in US Patent Application Nos. 09 / 565,906 and WO 00/67787 and disclosed herein by DP and illustrated in Example IV below, about 100 μg of HIV antigen in an immunogenic composition results in a strong immune response in primates. . About 10 μg of HIV antigen is suitable for administration to rodents. One skilled in the art can readily determine a suitable amount of HIV antigen for inclusion in an immunogenic composition of the invention sufficient to stimulate an immune response.

The immunogenic compositions of the present invention may further contain from about 5 μg to about 100 μg of immunomers, and if desired, may contain up to 10 mg of immunomers. For example, for doses for administration to humans, the dose is about 0.01 mg to about 5 mg, for example about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg , About 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.7 mg, or about 2 mg Can be. The amount of immunomer to be administered is generally from about 0.1 mg / kg to about 0.25 mg / kg up to about 5 mg / kg, for example about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.2, about 1.5, about 1.7, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, or about 5 mg / kg. The amount of immunomers is also about 0.2 μg / kg, about 0.5 μg / kg, about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 13 μg / kg, about 14 μg / kg , About 15 μg / kg, about 16 μg / kg, about 17 μg / kg, about 18 μg / kg, about 19 μg / kg, about 20 μg / kg, about 22 μg / kg, about 25 μg / kg, etc. have. As described above in US Patent Application Nos. 09 / 565,906 and WO 00/67787, nucleic acid molecules to HIV antigens in a weight ratio of at least 5: 1 were more effective than the lower ratios in inducing an immune response. One skilled in the art can readily determine the appropriate or optimized ratio of immunoomers to HIV antigens in inducing an immune response. For example, this ratio can vary and the immune response can be measured by the methods disclosed herein to determine a suitable or optimized ratio of immunomers to HIV antigens. In rodents, the effective amount of the immunomer in the immunogenic composition is from 5 μg to greater than 50 μg, for example up to about 100 μg. For primates about 500 μg of immunomers in immunogenic compositions are suitable. One skilled in the art can easily determine the appropriate amount of immunomer for eliciting the desired immune response.

As with all immunogenic compositions, an immunologically effective amount is determined empirically, but may be based on immunologically effective amounts in, for example, animal models such as rodents and extrahuman primates. Factors to be considered include antigenicity, formulation (e.g. type, volume of immune adjuvant), route of administration, number of immunization doses to be administered, physical condition, weight and age of the subject, and the like. Such factors are well known in the vaccine field and it is well within the skill of immunologists to make such determinations without undue experimentation.

The immunogenic compositions of the present invention may be administered topically 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. Can be. The present immunogenic composition may be administered in a suitable nontoxic pharmaceutical carrier, or the present immunogenic composition may be formulated in microcapsules or as a sustained release implant. The immunogenic compositions of the invention can be administered multiple times if desired to sustain the desired immune response. Appropriate routes, formulations, and immunization schedules can be determined by one skilled in the art.

It will be appreciated that modifications that do not substantially affect the activity of the various embodiments of the invention are included within the definition of the invention provided herein. Thus, the following examples are intended to illustrate the invention and are not intended to limit the invention.

Example  I

HIV  Cytokines, Antibodies and by Immunogenic Compositions Chemokines  Triggering of the reaction

This example demonstrates that immunogenic compositions containing HIV antigens, immunomers and immunosuppressive agents are potent stimulators of IFN-γ production (Th1 (CD8) and Th2 (CD4 helper) cytokines, antibody responses and β-chemokine production in mammals. Thus, immunogenic compositions containing HIV antigens, immunomers and immunoadjuvant mediate a powerful type of immune response that is important in the defense against HIV infection and disease progression, which is an effective prophylaxis. Or therapeutic vaccine.

Immunomers. Immunomers are synthesized as described above (Kandimalla et al., Bioorg. Med . Chem . 9: 807-813 (2001)); Yu et al., Nucl . Acids Res . 30: 4460-4469 (2002); Yu et al., Bioorg . Med . Chem . 11: 459-464 (2003); Bhagat et al., Biochem . Biophys . Res . Comm . 300: 853-861 (2003); And Yu et al., Biochem . Biophys . Res . Comm . 297: 83-90 (2002); Yu et al., Nucl . Acids Res . 30: 1613-1619 (2002); Yu et al., J. Med. Chem . 45: 4540-4548 (2002); Kandimalla et al., Bioconjugate Chem . 13: 966-974 (2002); Yu et al., Bioorganic Med . Chem . Lett . 10: 2585-2588 (2000); Agrawal and Kandimalla, Trends Mol . Med . 8: 114-121 (2002)).

Immunization. The HIV-1 antigen is prepared essentially as described above (WO 00/67787). Briefly, HIV-1 antigen is prepared from virus particles obtained from a culture of Hut 78 chronically infected with Zir virus isolate (HZ321) characterized as subtype "M", including env A / gag G recombinant virus. (Choi et al., AIDS Res . Hum . Retroviruses 13: 357-361 (1997)]. gp120 is removed during the two-stage purification process. Antigen is inactivated by addition of β-propiolactone and gamma irradiation of 50 kGγ. Western blots and HPLC analysis in the preparation of the antigens revealed that gp120 was undetectable. (Prior et al., Pharm Tech . 19: 30-52 (1995)]. In in vitro experiments, native p24 is preferentially dissolved from HIV-1 antigen purified using 2% Triton X-100 and then purified with Pharmacia Sepharose ™ Fast Flow S resin. Chromatography is performed at pH 5.0 and p24 is eluted with a linear salt gradient. The purity of the final product was found to be generally> 99% based on both SDS (sodium dodecyl sulfate) electrophoresis and reverse phase high pressure liquid chromatography. Immunomers are added to HIV-1 antigen diluted to at least 5% of the final volume.

Complete Freund's immune adjuvant (CFA) was resuspended at 10 mg / ml in mycobacterium tuberculosis H37RA (DIFCO, Detroit, MI) at IFA (DIFCO, Detroit, MI) Manufacture. IFA or ISA 51 is used to add 1 part of Montanide 80 (high purity Mannide Monooleate, Seppie, Paris, France) to 9 parts of Drakeol 6 VR light oil (Panreco, Carnes City, Pa.). Formulated by addition. The gp120 clearance HIV-1 antigen is diluted to 200 μg / ml in PBS and emulsified with the same volume of CFA or IFA with or without immunomers.

C57B1 mice maintained in a sterile facility are injected intravenously with 100 μl of emulsion. Each animal is given an immunomer of 1-10 μg of inactivated HIV-1 antigen, 10-100 μg of immunomer, or IFA + 10-100 μg in CFA or IFA. Two weeks later animals are primed with a subcutaneous route at the base of the tail using the same regimen, except that animals primed with HIV-1 antigen in CFA are instead boosted with HIV-1 antigen in IFA. Mice are primed and boosted with HIV-1 antigen in the presence of an immunoomer. Negative controls are administered as saline or IFA in saline. On day 28 animals are sacrificed for cytokine, chemokine and antibody analysis.

ELISA for antigen-specific antibodies. At the end of the study, whole blood is collected from immunized animals by cardiac puncture. Centrifuge the SST tube for 20 minutes at 800 rpm. Serum is aliquoted and stored at -20 ° C until analysis. PVC plates (polychlorinated biphenyl plates, Falcon, Oxnard, Calif.) Are coated with natural p24 diluted to 1 μg / ml in PBS and stored at 4 ° C. overnight. Plates are blocked by adding 200 μl of 4% BSA in PBS per well for 1 hour. Serum is diluted 1: 100 in 1% BSA in PBS and then serially diluted 4-fold. 100 μl of diluted serum is added in duplicate and incubated for 2 hours at room temperature. Plates are washed three times with 0.05% Tween 20 in PBS and blotted dry. Detection secondary antibodies (e.g. goat or rat anti-mouse IgG biotin, goat or rat anti-mouse IgG1 biotin, or goat or rat anti-mouse IgG2a biotin, Zymed, San Francisco, CA, USA) Dilute to. 100 μl of diluted secondary antibody is added to each well and incubated for an additional hour at room temperature. After washing of excess secondary antibody, Strept-Avidin-Biotin-HRP (Pierce, Rockford, Ill.) Is added at 50 μl per well and incubated for 30 minutes. Plates are washed three times with 0.05% Tween 20 in PBS. ABTS substrate (KPL, Gettersburg, Maryland) is added until blue green color develops. The reaction is stopped by adding 1% SDS and the plate is read with absorbance at 405 nm.

The antibody response reported with 50% antibody titer for all given samples is the inverse dilution rate equivalent to 50% of the maximum binding (highest optical reading). Absorbance values (OD at 405 nm) are plotted against log scale antibody dilution to generate sigmoidal dose response curves. 50% of the maximum binding is calculated by multiplying the highest OD by 0.5. This 50% value is located on the curve and the corresponding x-axis value is reported as the antibody dilution rate.

ELISA assay for cytokine and chemokine assays. Exhausted lymph nodes (inguinal and groin) are isolated from immunized animals two weeks after booster inoculation. Single cell suspensions from the lymph nodes are prepared by mechanical dissociation using a sterile 70 μm mesh screen. T cells are purified from lymph node cells by a panning method. Briefly, Petri dishes (100 × 15 mm) are precoated with 20 μg / ml rabbit anti-mouse IgG for 45 minutes at room temperature. Petri dishes are washed twice with ice cold PBS and once with ice cold 2% human AB serum in PBS. 1 x 10 ⁷ lymph node cells are added to the prewashed plates and incubated at 4 ° C. for 90 minutes. Non-adherent cells (enriched T cells) are then collected and transferred into sterile 50 ml conical tubes. Plates are washed twice and combined with non-adherent cells. The cells are then centrifuged and the cell pellet is resuspended at 4 × 10 ⁶ cells / ml in complete medium (25 mM hepes, 2 mM L-glutamine, 100 μg streptomycin and 5 × 10 ⁻⁶ M β-mer 5% human AB serum in RPMI 1640 with captoethanol).

Γ-irradiated thymic cells from C57BL mice are used as antigen presenting cells. 2 x 10 ⁵ enriched T cells and 5 x 10 ⁵ thymic cells are added to each well of a 96-round bottom plate. HIV-1 antigen and native p24 are diluted to 10 μg / ml in complete medium while con A is diluted to 5 μg / ml. 100 μl of each antigen or T cell mitogen is added in triplicate. Plates are incubated at 5 ° C., 37 ° C. for 72 hours. Supernatants are stored at -70 ° C until harvested and analyzed. Samples are analyzed for IL-4, IFN-γ and RNATES using commercially available kits specific to mouse cytokines and chemokines (eg Biosource, Camarillo, CA, USA).

Statistical method. Groups are compared using the Mann-Whitney U nonparametric statistical analysis. All p values are bilateral verification values.

Complete Freund's adjuvant (CFA) is currently the most potent immunoadjuvant known for stimulation of cell mediated immune response. However, CFA is not an appropriate adjuvant for use in humans due to safety issues. Thus the combination of IFA and immunomers for use in HIV immunogenic compositions provides a safe and effective vaccine for human treatment.

For investigation of dose related immune responses to IFN- [gamma], C58BL mice are immunized with inactivated gp120-removed HIV-1 antigen emulsified in IFA containing different concentrations of immunomers.

To investigate whether the antibody response to the HIV-1 antigen can also be augmented, the serum is analyzed for the total IgG and Th2 isotype (IgG1 and IgG2a) antibody responses to the p24 antigen.

As such, the immunogenic compositions of the present invention can be used to enhance β-chemokine production in a subject. Because of the strong correlation between β-chemokine levels and protection from HIV infection and disease progression, the compositions of the present invention are more effective than other disclosed compositions for inhibiting AIDS.

Example II

HIV  By immunogenic composition CD4  And CD8  Triggering an immune response

This example demonstrates the induction of potent CD4 helper function following immunization with immunogenic compositions containing HIV antigens, immunomers and immunosuppressive agents, CD8 HIV-specific Th1-type immune responses, and alterations to higher IgG2a / IgG1 antibody ratios. It is designed to show. Cytotoxic T lymphocytes, which are antigen-specific responses by CD8 +, are important factors in the prevention of early HIV infection and disease progression. Thus, this example provides additional evidence that the immunogenic compositions of the present invention are effective prophylactic and therapeutic vaccines.

HIV antigens, immunomers and IFAs are prepared essentially as described in Example I. C57BL mice are essentially immunized as described in Example I and sacrificed at 28 days for ELISPOT and p24 antibody analysis. p24 antibody analysis is performed essentially as described in Example I.

ELISPOT of γ-interferon from bulk and purified T cell populations. Single cell suspensions are prepared from spleens of immunized mice by compacting and compacting through sterile micromesh nylon screens in RPMI 1640 (Hyclone, Logan, Utah, USA). Spleen cells are purified by ficoll gradient centrifugation. CD4 and CD8 cells are isolated by magnetic bead removal. 2 × 10 ⁷ cells are stained with 5 μg rabbit or rat anti-mouse CD4 or rabbit or rat anti-mouse CD8. Cells are incubated for 30 minutes on ice and washed with 2% human AB serum in ice cold PBS. Pre-wash Dynabeads (DYNAL, Oslo, Norway) coated with goat anti-mouse IgG is added to the cell suspension and incubated with constant mixing at 4 ° C. for 20 minutes.

Purified CD4, CD8 and non-eliminated spleen cells were 5 × 10 ⁶ cells in complete medium (5% inactivated human serum, Pen-strep, L-glutamine and β-ME in RPMI 1640). Resuspend in / ml and use for ELISPOT analysis to count individual IFN-γ secreting cells. Briefly, 96 well nitrocellulose bottom microtiter plates (Millipore Co., Bedford, UK) are coated with 400 ng rabbit anti-mouse IFN-γ (Biosource, Camarillo, CA, USA) per well. After overnight incubation at 4 ° C., the plates are washed with sterile PBS and blocked with 5% human AB serum in RPMI 1640 containing pen-strep, L-glutamine and β-ME for 1 hour at room temperature. Plates are washed with sterile PBS and 5 × 10 ⁵ spleen cells (purified CD4, purified CD8 or non-removed) per well are added in triplicate and incubated overnight at 37 ° C., 5% CO ₂ . Cells are incubated with medium, OVA (egg of albumin, Sigma-Aldrich, St. Louis, MO), native p24 or gp120-removed HIV-1 antigen. CD4 Purification and CD8 Purification Spleen cells are analyzed in complete medium containing 20 units / ml of recombinant rat IL-2 (Pharmingen, San Diego, CA, USA).

After washing of unbound cells, add 400 ng of polyclonal rabbit anti-mouse IFN-γ per well and incubate for 2 hours at room temperature, followed by washing and goat anti-rabbit IgG biotin (Zymed, San Francisco, CA) Dye with After extensive washing with sterile PBS, avidin alkaline phosphatase complex (Sigma-Aldrich, St. Louis, MO) is added and incubated for an additional hour at room temperature. Spots were developed by adding a chromogenic alkaline phosphate substrate (Sigma, St. Louis, MO) and IFN- [gamma] cells were dissected with a highlight 3000 light source (Olympus, Lake Success, NY). Count using

Statistical method. Compare groups using the Mann-Whitney U nonparametric statistical analysis. Spearman rank correlation analysis is performed to investigate the relationship between CD4 and CD8 γ interferon production. All p values are bilateral verification values.

The production of IFN-γ by non-eliminated spleen cells and purified CD4 + or purified CD8 + populations is investigated. IFN- [gamma] production by CD4 + cells is a characteristic Th1 immune response, whereas IFN- [gamma] production by CD8 + cells correlates with the cytolytic activity of cytotoxic T lymphocytes (CTL). Total IgG, IgG1 and IgG2b specific for p24 are also examined.

In sum, this example shows that immunogenic compositions containing HIV antigens, immunomers, and immunoadjuvant can be used to generate potent HIV-specific CD4 and CD8 HIV-specific immune responses. Induction of CD4 T helper cells may be central to the production of CD8 effector cells. CD8 T cells may function as effectors for HIV by several mechanisms including direct cell lysis (CTL) activity and through the release of antiviral inhibitors such as β-chemokine and other less fully characterized factors. Can be. Thus the compositions described herein are superior to other disclosed compositions for use as HIV vaccines.

Example III

Comparison of immune responses elicited by different immunogenic compositions and immunization schedules

In this example, when the nucleic acid containing an immunomer is administered simultaneously with an HIV antigen and an adjuvant in inducing a protective immune response including RANTES production and HIV-specific IgG2b antibody production, the antigen and the immune It is designed to show that it is more effective than when used to prime a mammal one week before administration of an adjuvant. This example also shows that compositions containing HIV antigens, immunomers and immunoadjuvant promote antigen-dependent lymphocyte proliferation more effectively than compositions containing only HIV and IFA.

HIV antigens, immunomers and IFAs are prepared essentially as described in Example I. C57bBL mice (3 or more per group) are immunized at 7 days and primed at 0 days when indicated with the following compositions illustrated in Table 1.

group 0 days 7 days A Immunomer HIV-1 B HIV-1 C Immunomer HIV-1 / IFA D HIV-1 / IFA E HIV-1 / IFA / immunomer

Animals are sacrificed 21 days essentially for cytokine, chemokine and antibody assays and lymphocyte proliferation assays as described in Example I.

Lymphocyte proliferation assay. Single cell suspensions are prepared from the excretion of lymph nodes in immunized animals. B cells are removed from lymph node cells by panning. Briefly, lymph node cells are incubated for 90 minutes in anti-mouse IgG pre-coated Petri dishes. Non-adherent cells (enriched T cells) are collected and resuspended at 4 × 10 ⁶ cells / ml in complete tissue culture medium. Enriched T cells are incubated with p24 or HIV-1 antigen in the presence of γ-irradiated thymic cells for 40-48 hours at 37 ° C., 5% CO ₂ . Samples are pulsed with tritiated thymidine and incubated for an additional 16 hours. Cells are harvested and tritiated thymidine incorporation counted using a β-scintillation counter.

The production of cytokines in T cells, such as IFN-γ and β-chemokines such as RANTES, MIP-1β and MIP-1α, are measured using methods well known to those skilled in the art. Serum levels of total IgG, IgG1 and IgG2b specific for p24 are also examined. In addition, T cell proliferative responses to p24 antigen and pg120-depleted HIV are investigated.

As such, the immunogenic compositions of the present invention can effectively elicit HIV-specific Th1 cytokines (IFN-γ) and humoral responses (IgG2 antibodies) and enhance both nonspecific and HIV-specific β-chemokine production. Can be. This response to the present immunogenic composition correlates with a strong HIV-specific T lymphocyte proliferative response.

Example IV

HIV  Immunization of Primates Using Immunogenic Compositions

This example is designed to show that immunogenic compositions containing HIV antigens, immunomers and immunoadjuvant are effective for enhancing HIV-specific immune responses in primates.

Three babies are injected intrauterine with an immunogenic composition containing gp120-depleted HIV-1 (100 μg total protein, equivalent to 10 p24 units) in IFA containing 500 μg immunomer. Four weeks later, the fetus is boosted using the same regimen.

Peripheral blood mononuclear cells from neonatal babies are collected and analyzed for proliferative responses to p24 and HIV-1 antigens.

The production of HIV-specific antibodies, cytokines and β-chemokines is also measured at the same ratio. The results revealed that the type of immune response induced by the immunogenic composition described in Example I-III in rodents was also induced in primates.

These results demonstrate that the HIV immunogenic compositions and methods of the present invention are effective in stimulating HIV-specific immune responses in primates. The results also demonstrate that fetuses and infants can induce a strong HIV immune response against the immunogenic compositions of the present invention, suggesting that the compositions are useful for the prevention of maternal transmission of HIV and as a pediatric vaccine. Indicates.

Example  V

In the mouse model Immunomers and  Combined, inactivated gp120  remove HIV  Immune Response to Vaccination with Immunogen

This example describes the ability of immunogens to enhance the immunogenicity of HIV-1 antigens and HIV-1 immunogens (antigens emulsified in IFA) in a mouse model.

C57BL / 6 mice (6-8 weeks old) are injected as indicated below. The number per group is generally at least 8-10 mice.

1) PBS

2) 30 μg of immunomers per mouse = 1.5 mg / kg

3) Immunomer (highest 90 μg dose) = 4.5 mg / kg

4) HIV-1 immunogen (10 μg)

5) HIV-1 immunogen + immunomers (10 μg, 30 μg, 90 μg)

6) HIV-1 immunogen + immunomer 30 μg

gp120 Removal The HIV-1 antigen is diluted in phosphate buffered saline (PBS) to a concentration of 200 μg / ml and emulsified in the same volume of IFA with or without immunomers. Immunomers are added to diluted HIV-1 antigen and then emulsified to a volume of at least 5% of the final volume.

Initial single intradermal injections are performed on day 0 followed by intradermal injection two weeks later. Mice are sacrificed 2 weeks after booster injection. The HIV-1 immunogen used is an inactivated gp120 clearing HIV-1 antigen in IFA.

Immunological Analysis. New spleen mononuclear cells are isolated and stimulated for 4 days in vitro (Davis et al., J. Immunol . 160: 870-876 (1998)). Isolated cells are in medium alone; With native p24 antigen; Or stimulate with HIV-1 antigen.

Production of various cytokines is assessed using the ELISA method. Exemplary cytokines to be analyzed include, for example, IFNγ, IL-12, IL-4, IL-5, IL-10, MIP1α, MIP1β, RANTES, α-defensin as disclosed herein and as described above. It is analyzed by a method well known to those skilled in the art.

As described in Examples I and II, P24 antigen- and HIV-1 antigen-specific IFNγ production in CD4 and CD8 lymphocytes is assessed by ELISPOT assay.

P24 antigen-, HIV-1 antigen, and LPS-specific lymphocyte proliferation are assessed by standard proliferation assays using well known methods.

Example VI

HIV Previously immunized with -1 immunogen HIV -From infected patients PBMC Generated by HIV  For specific immune responses Immunomeric In vitro  effect

This Example assesses the ability of immunomers to increase HIV-specific immune responses in vitro in peripheral blood mononuclear cells (PBMCs) of patients treated with inactivated gp120 depleted HIV-1 antigen (REMUNE) in IFA. One is described.

The following groups of patients are examined: 15 HIV-infected HAART + REMUNE-treated patients; 15 HIV-infected HAART-treated patients. Patients are matched for disease duration, CD4 counts, HIV viremia, and the absence / presence of protease inhibitors (PI). Whole blood (530 ml) is drawn intravenously into EDTA containing tubes for later analysis. Immunomers are added to PBMC at the following concentrations: 0.1 μg / ml, 1.0 μg / ml, 10.0 μg / ml.

Various antigens such as HIV antigens, p24 antigens, HIV-1 antigens, env peptides, gag peptides; And specific response to flu (control antigen). Other HIV antigens can also be measured if desired. As described in Examples I and II, antigen-specific IFNγ-generation in CD4 and CD8 lymphocytes is assessed by ELISPOT assay. Antigen-specific lymphocyte proliferation is also assessed by standard proliferation assays.

The production of RANTES, α defensin is assessed by intracellular staining on CD8 + using fluorescence activated cell sorting (FACS) method. If desired, other cytokines or other cell types can be analyzed.

Example VII

Trimera ( Trimera ) Rat  In the model HIV  For specific immune responses Immunomeric In vivo  effect

This example describes the use of a trimera mouse model in determining the effect of an HIV immunogenic composition containing an immunomer.

The trimera mouse model is used to test the effects of combining immunomers with HIV antigens. Both induced and protective immunity can be monitored. Trimera mice are generated as described above (Reisner and Dagan, Trends Biotechnol. 16: 242-246 (1998); Ilan et al., Curr . Opin . Mol . Ther . 4: 102-109 ( 2002); US Pat. No. 6,254,867; WO 97/47654. Briefly, normal mouse hosts are immune-discharged by systemic irradiation of lethal split-dose. Mice are then radioprotected with T cell depleted murine SCID bone marrow and converted to trimera mice by intraperitoneal injection of human peripheral blood mononuclear leukocytes (PBMC). Engraftment of human cells in trimera mice is confirmed by fluorescence labeled cell separation (FACS) analysis of human T cell markers such as CD3 or the like.

Trimera mice are infected with HIV as a model of AIDS. Briefly, trimera mice are infected with one or more HIV-1 strains. Control animals are only trimera mice injected with media (not including HIV-1) and mice not injected with PBMC. Mice are evaluated at various time points for HIV-1 infection by measuring levels of HIV-1 RNA in blood, the presence of proviral DNA, and active viruses in co-culture experiments. The presence of proviral HIV-1 DNA is evidenced by PCR of the HIV-1 sequence, eg gag.

To test immunogenic compositions containing immunomers for stimulation of the immune response, trimera mice are injected with gp120-removed HIV-1 with or without one or more immunomers and with or without an adjuvant. For example, various ratios of antigens and immunomers can be used and described for optimized immune responses as described in Example V. Alternatively, the composition is pulsed into human autologous dendritic cells (DCs) and injected into DCs of the trimera mice. Optionally, mice can be further inoculated with similar compositions.

Blood and peritoneal lymphocytes are collected after immunization. Determine the presence of immunoglobulins specific for HIV antigens. Specific cellular anti-HIV responses are also measured in human lymphocytes isolated from mice. For example, IFNγ production in human lymphocytes recovered from trimera mice is measured after exposure to HIV-antigens. Enhancing immunogenic response to HIV antigens in the presence of immunomers is measured.

Protective immunity is monitored in a similar fashion, except that mice are immunized with various compositions and then infected with HIV. As described above, the ability of various compositions to affect the level of viral viremia developed is measured. The most effective vaccines are those that provide the most effective regulation of the circulation of the virus and / or prolong survival.

Example VIII

REMUNE (Trademark) HIV  Immunization of Infected Patients

This example describes the immunization of HIV-infected patients with REMUNE ™ (GP120-removed HIV-1 antigen in IFA), and this example demonstrates that most patients can boost immune responses, although varying in intensity and duration. To prove that. The purpose of this particular study was to treat with REMUNE in combination with HAART (indinavir / ZDV / 3TC) as compared to incomplete Freund's immune adjuvant (IFA) + highly active retroviral therapy (HAART). It was later assessed for HIV-1 specific immune responses.

Study protocols were randomized double blind, two arm, parallel group, immune adjuvant control, multicenter study. The number of subjects (total and number for each treatment) was 52 random patients and 43 trialable patients were tried to be analyzed for treatment (22: REMUNE + HAART; 21: IFA + HAART). Measures of diagnosis and diagnosis were HIV-1 infected patients with a CD4 count of> 350 cells / μL previously without using any HIV protease inhibitor or lamivudine (3TC). Test products, doses, and dosage forms include REMUNE (HIV-1 immunogen); 10 units (equivalent to a p24 content of 10 μg / ml) and a volume of 1.0 ml given by IM (Batch Numbers: 8155-015 and 8155-017). For duration of treatment, patients received HAART for 32 weeks. REMUNE or IFA placebo (control) were given at 4, 16 and 28 weeks. Reference therapy, dose, and mode of administration were in contrast to the adjuvant; IFA placebo was used (Batch No .: 8144-006 and 8160-005).

A measure of primary efficacy was lymphocyte proliferation (LP) response to HIV-1 antigen stimulation in peripheral blood mononuclear cells (PBMC). Measures for secondary efficacy include LP response to native p24 and BaL HIV-1 antigen stimulation in PBMC; Chemokine response to p24 and HIV antigen stimulation in PBMC; gag CTL activity (in patient subset); Change in CD4 cell count and CD4 percent; Changes in viral load as measured by blood RNA and PBMC DNA; And DTH skin test response to HIV-1 and p24 antigens. Statistical methods used were Fisher's Exact Test (two-sided) and Mann-Whitney two-sided in treatment intent analysis.

The primary analysis defined response rates as stimulation indices (SIs) for five-fold HIV-1 antigen compared to baseline at two time points. The results showed that there were 14/22 (64%) responders in the REMUNE + HAART group and 4/21 (19%) in the IFA + HAART group (p = 0.005). Secondary analysis defined response rates as stimulation indices (SI) for three times the BAL type HIV-1 antigen and / or p24 antigen compared to baseline at two time points. The results showed that there were 15/22 (68%) responders in the REMUNE + HAART group and 5/21 (24%) in the IFA + HAART group (p = 0.006). The magnitude of the LP response to HIV-1 (HZ321) antigen in subjects who received REMUNE + HAART, defined as the ratio of the geometric mean SI to the post-treatment value measured for each subject after the first injection, was determined by IFA + HAART. Greater than those received (p = 0.008).

Compared to the IFA + HAART group, the LP response rate to the native p24 (p = 0.0002) and HIV-1 BaL antigen (p = 0.007) was statistically significantly higher in REMUNE + HAART. There was no difference in LP responses to the recall antigens (candida, streptokinase, tetanus) between the two groups. The production of MIP-1β by PBMCs stimulated with HIV-1 antigen was not observed. It was significantly increased in the REMUNE + HAART group compared to the HAART group (p + 0.0007 at 32 weeks) in the IFA + HAART group for HIV-1 (53% vs. 9%) and native p24 antigen (47% vs. 0%). Compared to the REMUNE + HAART group, the DTH skin test response rate was higher.

Administration of REMUNE + ZDV / 3TC / indinavir significantly stimulated the lymphocyte proliferation (LP) response to the HIV-1 antigen in terms of both the number of responders and the magnitude of the response. REMUNE + ZDV / 3TC / Indie than the IFA + ZDV / 3TC / Indinavir group (4/21, 19%) in response rate, defined as the stimulation index for HIV-1 antigen, which was five times the baseline at two time points. The Navar group (14/22, 64%) had significantly higher responders (p = 0.005).

A high percentage of subjects who received REMUNE produced strong LP responses against native p24 antigens, demonstrating that REMUNE can specifically produce responses against more conserved HIV core antigens. Treatment with IFA did not stimulate the HIV-1 specific immune response to the antigen. REMUNE + ZDV / 3TC / indinavir produced significantly greater lymphocyte proliferation response to purified native p24 (p = 0.0002).

Administration of REMUNE stimulated the LP response to HIV-1 (HZ321) immunized antigen, clade B, HIV-1 antigen, and HIV-1 BaL antigen, which caused the immune response generated by REMUNE to cross the clade. and cross-clade and not limited to immunizing agents. REMUNE + ZDV / 3TC / indinavir produced significantly greater lymphocyte proliferation response to HIV-1 BaL antigen compared to IFA + ZDV / 3TC / indinavir (p = 0.007).

Antigen-stimulated MIP-1β production was significantly increased in the REMUNE + ZDV / 3TC / indinavir group throughout the study (p = 0.0007, 32 weeks) and changed in the IFA + ZDV / 3TC / indinavir group during the study. It wasn't. Subjects in both groups showed a significant increase in CD4 cell counts and a significant decrease in plasma HIV RNA and proviral HIV DNA copy numbers. In the analysis of the time when HIV RNA relapses, the REMUNE + ZDV / 3TC / indinavir group tends to have a lower risk of relapse, subjecting IFA + ZDV / 3TC / Indinavir between 16 and 32 weeks. Among 12/21 (57%), 6/22 (27%) of REMUNE + ZDV / 3TC / Indinavir patients recurred (p = 0.08, log rank test). Stronger and longer-lasting responses are expected by the administration of immunogenic compositions of the invention containing HIV antigens such as REMUNE and one or more immunomers.

These results demonstrate that REMUNE stimulates the immune response in most patients.

Example IX

REMUNE (Trade name) and Immunomer  Used HIV  Immunization of Infected Patients

In this example, immunization of HIV infected patients with REMUNE and immunomers is described.

HIV-1 following treatment with incomplete Freund's immune adjuvant (IFA) + immunomers and / or REMUNE in combination with immunomers and / or highly active retroviral therapy (HAART) (indinavir / ZDV / 3TC) compared to HAART The study is conducted for the purpose of evaluating specific immune responses.

The method utilizes a randomized double blind, two-cancer, parallel group, immunoadjuvant control study. The measure and diagnosis of inclusion of HIV-1 infected patients were patients with a CD4 count of> 350 cells / μL previously without any HIV protease inhibitor or lamivudine (3TC). Other measures of patient choice may also be used. Test products, doses, and dosage forms include REMUNE (HIV-1 immunogen); 10 units (equivalent to a p24 content of 10 μg / ml), volume of 1.0 ml given by IM. Doses of between about 1 and 5 mg / kg are administered. More or less other doses of immunomers can also be tested for effective enhancement of the immune response. In the duration of treatment in patients treated with HAART, the patient receives HAART for 32 weeks. REMUNE or IFA placebo (control) and immunomers are given at 4, 16 and 28 weeks. Reference therapy, dose, and mode of administration are immunoadjuvant controls in which IFA placebos are used.

The scale of evaluation in efficacy and safety is similar to that described in Example VIII. As also disclosed herein and as described in Examples I-III and V, assays for measuring immune responses can be performed, for example, in the production of interferon ELISPOT, IgG1 / IgG2 antibodies, in the production of cytokines. ELISA assay, lymphocyte proliferation assay, spleen cell stimulation, and the like.

Combination of immunomers with REMUNE or other HIV antigens is expected to enhance the immune response over HIV antigens that do not contain immunomers. Thus, the immune response in this example is expected to be stronger and / or longer than that observed in Example VIII.

This example describes the enhanced effects of administering an HIV antigen with an immunomer to stimulate an immune response in HIV infected patients.

Example  X

Immunomer  Containing HIV -1 antigen HIV Induce specific immunity

 This example describes the use of HIV antigens and immunomers to stimulate HIV-specific immunity.

HIV-1 immunogens show killed previral vaccines with gp120 removed formulated with incomplete Freund's immune adjuvant (IFA), previously reported to induce HIV-1 specific immune responses, and immunostimulatory cytosine-guanine ( Synthetic oligonucleotides comprising CpG) dinucleotide motifs are potent stimulators of cell mediated immune responses. The possibility of enhanced immunogenic production of HIV-1 immunogens in combination with immunomeric adjuvant (Amplivax®) was studied in a mouse model. In subsequent experiments, it was confirmed that the HIV-specific immune response could be caused by killing whole virus (HIV-1 antigen) + Amplivax® with gp120 removed without IFA.

In this study, the HIV-1 immunogen used was a killed previral vaccine with gp120 removed formulated with incomplete Freund's adjuvant (IFA). The experiment was performed essentially as described in Example V. This immunogen induces an HIV-specific immune response. Amplivax®, also referred to herein as an immunomer, is an immunomodulatory oligonucleotide comprising novel structural and synthetic immunomodulatory motifs. This immunomer induces a unique immune stimulation profile.

2 shows a schematic of Amplivax®, also referred to as HYB2055. HYB2055 is a second generation immunomodulatory oligonucleotide (IMO) consisting of a novel structure and a synthesized CpR immune stimulating motif. The immunomer stimulates the immune system by signaling through TLR9 and induces a Th1 immune response. This immunomer shows enhanced metabolic stability. Phase I clinical trials in healthy volunteers have been completed.

The mouse study was initiated in a mouse model to assess the ability of Amplivax® (HYB2055) to enhance the immunogenicity of the whole HIV-1 vaccine (HIV-1 immunogen) in IFA. The study also examined whether HIV-specific immune responses could be triggered by a pre-killed virus (HIV-1 antigen) that does not contain IFA, used in combination with Amplivax®.

Briefly, 10 μg of whole HIV vaccine (HIV-1 immunogen) plus three doses of Amplivax® (90, 30 or 10 μg / mouse) or whole HIV vaccine (IFA) in incomplete Freund's immune adjuvant (IFA) C57 / BL6 mice were immunized subcutaneously (SC) or intramuscular (IM) (day 0 and 14) with HIV-1 antigen, 10 μg / mouse) and Amplivax® (90 μg / mouse). Animals immunized with HIV-1 immunogen, Amplivax® alone, or PBS were used as controls (8-10 mice / group). Mouse immunomer, HYB 2048, was used as reference compound. Mice were sacrificed on day 28. HIV-1 antigen-stimulated or p24-stimulated cytokine production and IFNγ-secreting T cells in new spleen mononuclear cells were evaluated. P24 antibody production was assessed in serum.

As shown in FIG. 3, HIV-1 immunogens induce HIV-specific RANTES, MIP1α, MIP1β, IL-10 and IL-5 production. Table 2 shows that the combination of HIV-1 immunogen and Amplivax® alters the cytokine profile towards Th1 type response. The immunogen was administered subcutaneously. Exemplary values are mean values.

IFNγ pg / ml RANTES pg / ml MIP-1α pg / ml MIP-1β pg / ml IL-10 pg / ml IL-5 pg / ml Cytokine Profile Solo HIV-1 immunogen 12 101 28 317 67 644 Th2 HIV- 1 immunogen + Amplivax 1783 700 83 438 257 6 Th1 Solo Amplivax 0.06 107 27 91 3 - -

Similar assays are shown in Table 3, which also illustrates the ratio of IFN-γ to IL-5. Stimulation was with HIV-1 antigen. IFN-γ and IL-5 were measured by ELISA.

IFN-g pg / ml IL-5 pg / ml IFN-g / IL-5 pg / ml  Cytokine Profile HIV-1 immunogen 12 644 0.02 Th2 type HIV-1 immunogen + Amplivax 1828 5.7 321 Th1 type Amplivax 0.064 5.4 - -

4 shows that HIV-specific IFNγ production is enhanced by Amplivax® in a dose dependent manner. The amount of immunomer used is indicated in parentheses (μg / mouse). Similar results were observed for RANTES, MIP1α, MIP1β and IL-10. 5 shows the effect of Amplivax® on production levels of HIV-1 immunogen-induced RANTES, MIP1α, MIP1β, IL-10 and IL-5. FIG. 6 shows that HIV-specific IFNγ production is enhanced by Amplivax® (data shown for 90 μg / mouse of Amplivax®). Similar results were found in RANTES, MIP1α, MIP1β and IL-10. As shown in FIG. 7, Amplivax® has an enhancing effect on HIV-specific IFNγ-secreting T cells in Elispot assay. The immunogen was administered subcutaneously. The amount of immunomer used is indicated in parentheses (μg / mouse).

FIG. 8 shows that HIV-specific IFNγ production is enhanced by Amplivax® in a dose dependent manner. The amount of immunomer used is indicated in parentheses (μg / mouse). 9 shows that HIV-specific RANTES production is enhanced by Amplivax® in a dose dependent manner. FIG. 10 shows that HIV-specific MIP-1α production is enhanced by Amplivax® in a dose dependent manner. The amount of immunomer used is indicated in parentheses (μg / mouse). FIG. 11 shows that HIV-specific MIP-1β production is enhanced by Amplivax® in a dose dependent manner. The amount of immunomer used is indicated in parentheses (μg / mouse). 12 shows that HIV-specific IL-10 production is enhanced by Amplivax® in a dose dependent manner. The amount of immunomer used is indicated in parentheses (μg / mouse). 13 shows that HIV-specific IL-5 production is reduced by Amplivax® given subcutaneously. The amount of immunomer used is indicated in parentheses (μg / mouse).

14 shows the effect of Amplivax® on the titer of HIV-1 immunogen-induced p24 antibody in mice. The amount of immunomer used is indicated in parentheses (μg / mouse). FIG. 15 shows that whole HIV-1 vaccine (HIV-1 immunogen) in IFA induces HIV specific cytokine production upon subcutaneous (SC) and intramuscular (IM) administration.

16 shows that Amplivax® can be added before or after emulsification with IFA to enhance IFNγ production. 17 shows that Amplivax® may be added before or after emulsification with IFA to enhance RANTES production. As shown in Table 4, the combination of HIV-1 immunogen and Amplivax® alters the cytokine profile towards Th1 type response.

Ratio of IFN-γ / IL-10 Ratio of IFN-γ / IL-5  Cytokine Profile HIV-1 immunogen 1,42 0,02 Th2 type HIV -1 immunogen + Amplivax® 74,04 321 Th1 brother Amplivax® ------ ------ ------

FIG. 18 shows that whole HIV-1 vaccine containing Amplivax® without IFA triggers HIV-specific IFNγ production in mice immunized by subcutaneous administration. 19 shows that pre-HIV-1 vaccine containing Amplivax®, which does not include IFA, in mice immunized by subcutaneous administration triggers HIV-specific IFNγ-secreting CD8 + T cell activity. FIG. 20 shows that pre-HIV-1 vaccine containing Amplivax®, which does not include IFA, in mice immunized by subcutaneous administration triggers HIV-specific RNATES production.

C57 / BL6 mice subcutaneously immunized with a combination of an HIV-1 immunogen and Amplivax® produced significantly enhanced HIV-specific p24 antibody, HIV-specific IFN-γ, compared to HIV-1 immunogen or Amplivax® alone. The amount and number of CD4 and CD8 T cells), chemokines (RANTES, MIP-1α, MIP-1β), and IL-10. Importantly, enhancement by Amplivax® is still observed even when the HIV-1 antigen is not emulsified with IFA.

HIV- and p24-specific IFNγ, RANTES, MIP 1α, MIP compared to HIV-1 immunogen alone or Amplivax by immunization with both Amplivax® + HIV-1 immunogen and a combination of Amplivax® + HIV-1 antigen 1β and IL-10 production and the number of IFNγ-producing cells were significantly enhanced. The magnitude of the immune response observed in mice immunized with HIV-1 antigen or HIV-1 immunogen in combination with Amplivax was comparable.

The results show that immunization with HIV-1 immunogen + Amplivax® results in HIV-specific IFNγ, RANTES, MIP 1α, MIP 1β and IL-10 production and the number of IFNγ-producing cells compared to HIV-1 immunogen or Amplivax® alone. Shows significant enhancement. The magnitude of the immune response observed in mice immunized with HIV-1 antigen and Amplivax® (not including IFA) was comparable to the response obtained with HIV-1 immunogen (HIV-1 antigen + IFA) and Amplivax®. Amplivax®, combined with pre-killing viral vaccines, elicits a strong HIV-specific immune response independent of the use of IFA.

Amplivax, associated with either an HIV-1 immunogen or an HIV-1 antigen, elicits a strong virus-specific immune response independent of the use of IFA. Strong immunogenicity of the combination of HIV-1 + Amplivax warrants its use as a therapeutic vaccine for HIV infected patients.

Example XI

Human peripheral blood Mononuclear  Cell In vitro HIV In combination with immunomers for specific immune responses HIV  Immunogen Impact

This example describes the effect of Amplivax® on in vitro HIV-specific immune responses in human peripheral blood mononuclear cells (PBMC).

Initiating this study to evaluate whether Amplivax® can increase the in vitro HIV-specific immune response of human PBMCs from antiretrovirus-treated HIV infected patients, who are immunized or immunized with the entire HIV-1 vaccine. Did not do it.

Amplivax® was investigated in vitro for its ability to enhance HIV antigen stimulation of PBMCs isolated from HIV + patients treated with antiretroviral therapy (ART). The patient did not immunize or previously immunized with an HIV-1 immunogen. Both patient groups have comparable CD4 counts, HIV plasma viremia, duration of infection, and ART. The results indicate that Amplivax® is stronger HIV-in patients vaccinated with HIV-1 immunogens, as measured by the higher percentage of total spot and α defensin-producing cells generated in the IFN-γ ELISPOT assay. It has been found to induce specific cell mediated immune responses. Both effects were most evident using Amplivax® at 1 μg / ml.

Briefly, patients were vaccinated with all HIV four vaccines (REMUNE® = HIV-1 immunogen) at 6-24 doses. The last dose was given 6-8 months before blood collection. Unvaccinated patients were matched for CD4, HIV viremia and HAART exposure. Amplivax® was added to PBMC at four concentrations (0, 0.1, 1.0 and 10 μg / ml). Cells were stimulated with HIV-1, nP24, gag and flu antigens. Evaluation of CD8 +, an IFNγ-producing cell, was performed with ELIspot. The analysis of α defensin-producing S cells was performed by fluorescent labeling cell sorting (FACS) method.

FIG. 21 shows that the percentage of α-defensin producing CD8 + T cells is increased by Amplivax® added in vitro. FIG. 22 depicts HIV-specific IFNγ-producing CD8 + T cell numbers in REMUNE® treated patients and HIV positive controls (not including Amplivax®). FIG. 23 shows HIV-specific IFNγ-producing CD8 + T cell numbers in the presence of 0.1 μg / ml Amplivax® added ex vivo. FIG. 24 depicts HIV-specific IFNγ-producing CD8 + T cell numbers in the presence of 1 μg / ml Amplivax® added ex vivo. 25 shows the number of HIV-specific IFNγ-producing CD8 + T cells in the presence of 10 μg / ml Amplivax® added ex vivo.

FIG. 26 shows IFN-γ ELIspot analysis in peripheral blood mononuclear cells (PBMC). HYB2055 was used at 1 μg / ml.

Preliminary data were generated for REMUNE® (HIV-1 immunogen + IFA) in HAART naïve patients. Clinical trials monitor 50 HIV-1 positive subjects, including HIV-1 RNA in the range of 10,000-40,000 copies / mL and more than 350 cells / μL of CD4 cells at the completion of this trial. Patients were randomized into three groups: REMUNE® (HIV-1 immunogen in IFA); IFA immune adjuvant; Or saline solution.

Phenotypic changes in CD4 T cells (FIG. 27) and CD8 T cells (FIG. 28) were observed after the first injection of REMUNE® in antiretroviral therapy (ART) naïve patients. Preliminary data for the first few patients are illustrated. Further patients are analyzed similarly.

Preliminary data from ongoing clinical trials in drug-naive HIV + patients suggest that HIV-1 immunogens also have a positive effect on the generation of HIV-specific immune responses in the patient population. Potential potentiating effects of Amplivax® are investigated in the records of clinical trials in these same patients by adding Amplivax® to the HIV-1 immunogen as part of the vaccine.

Reference is made to various publications throughout this application. In order to more fully describe the state of the art to which this invention belongs, the entire disclosure of this publication is incorporated herein by reference.

Although the present invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments described in detail merely illustrate the invention. It should be understood that various modifications may be made without departing from the spirit of the invention.

Claims (34)

(a) killed whole HIV virus without envelope protein gp120; (b) immunomers; And (c) adjuvant Immunogenic composition comprising a. The immunogenic composition of claim 1, wherein the HIV virus is HIV-1. The immunogenic composition of claim 1, wherein the HIV virus is HZ321 strain virus. The immunogenic composition of claim 1, wherein the isolated nucleic acid molecule comprises a phosphorothioate backbone. The immunogenic composition of claim 1, wherein the HIV virus is conjugated to the nucleic acid molecule. The immunogenic composition of claim 1, wherein said immunomer is a CpG immunoomer. The immunogenic composition of claim 1, wherein said immunomer is a CpG-free immunomer. The immunogenic composition of claim 1, wherein the immunoadjuvant is suitable for use in humans. The immunogenic composition of claim 1, wherein the immunoadjuvant comprises an incomplete Freund's adjuvant (IFA). The immunogenic composition of claim 1, wherein the immunoadjuvant comprises a mycobacterium cell wall component and monophosphoryl lipid A. The immunogenic composition of claim 1, wherein the immunoadjuvant comprises alum. The immunogenic composition of claim 1 which enhances β-chemokine production. The immunogenic composition of claim 12, wherein the enhanced β-chemokine production is nonspecific β-chemokine production. The immunogenic composition of claim 12, wherein the enhanced β-chemokine production is HIV-specific β-chemokine production. The immunogenic composition of claim 1, wherein the β-chemokine is RANTES. The immunogenic composition of claim 1 which enhances HIV-specific IgG2b antibody production in a mammal. The immunogenic composition of claim 1 which enhances an HIV-specific cytotoxic T lymphocyte (CTL) response in a mammal. The immunogenic composition of claim 1, which enhances HIV-specific CD4 + helper T cells. (a) killed whole HIV virus without envelope protein gp120; (b) immunomers; And (c) immune adjuvant A kit comprising a, wherein the kit components are used in combination to produce the immunogenic composition of claim 1. (a) killed whole HIV virus without envelope protein gp120; (b) immunomers; And (c) immune adjuvant Comprising the step of combining, the method of producing an immunogenic composition of claim 1. The method of claim 20, wherein the combination is an ex vivo combination. The method of claim 20, wherein the combination is an in vivo combination. A method of immunizing a mammal, comprising administering the immunogenic composition of claim 1 to the mammal to enhance the immune response in the mammal. A method of inhibiting AIDS, the method comprising administering the immunogenic composition of claim 1 to a mammal to enhance an immune response in the mammal. The method of claim 23 or 24, wherein the mammal is a primate. The method of claim 25, wherein the primate is an infant. The method of claim 25, wherein the primate is pregnant. The method of claim 25, wherein the primate is a human. The method of claim 28, wherein said human is HIV sero negative. The method of claim 28, wherein the human is HIV seropositive. 31. The method of claim 30, wherein said mammal is a rodent. 32. The method of claim 30 or 31, wherein said composition is administered to said mammal two or more times. The method of claim 23 or 24, wherein said composition is administered to said mammal two or more times. The method of claim 23 or 24, wherein the composition is administered subcutaneously, intramuscularly or intramucosally.

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