WO2012018856A2 - Hiv vaccine therapy with concomitant antiviral monotherapy - Google Patents

Hiv vaccine therapy with concomitant antiviral monotherapy Download PDF

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Publication number
WO2012018856A2
WO2012018856A2 PCT/US2011/046324 US2011046324W WO2012018856A2 WO 2012018856 A2 WO2012018856 A2 WO 2012018856A2 US 2011046324 W US2011046324 W US 2011046324W WO 2012018856 A2 WO2012018856 A2 WO 2012018856A2
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hiv
vaccine
vector
dna
inhibitor
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PCT/US2011/046324
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French (fr)
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WO2012018856A3 (en
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Riku Rautsola
Tessio Rebello
Frank Lemiale
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Virxsys Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A61K31/24—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group having an amino or nitro group
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
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    • A61K31/65—Tetracyclines
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
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    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention describes an improved therapeutic option for treatment and management of human immunodeficiency virus (HIV) infection.
  • This invention more particularly relates to an NRTI-sparing regimen consisting of a therapeutic vaccine plus an HIV replication inhibitor-based monotherapy.
  • HAART highly active antiretroviral therapy
  • VL viral load
  • NRTIs nucleoside or nucleotide reverse transcriptase inhibitors
  • PI protease inhibitor
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • NRTIs have poor tolerability and significant toxicities which can compromise compliance and promote drug resistance [14-16].
  • mitochondrial dysfunction induced by NRTIs produces a spectrum of illnesses including peripheral neuropathy, myopathies, steatohepatitis, pancreatitis, lipoatrophy, tubular acidosis and lactacidosis [17-20].
  • regimen simplification is a strategy used to decrease toxicities, and avoid drug interactions or improve antiretroviral compliance and convenience.
  • regimen simplification using boosted protease inhibitor monotherapy ( ⁇ /r) is not
  • First generation HIV vaccines currently in development may offer some form of protection, but they are not entirely protective. Prophylaxis to disease is unlikely to occur with first- generation prototypes of AIDS vaccines. However, even a partially effective vaccine could be very promising in protecting some individuals against infection. In addition to reducing the rate of transmission of HIV, lowering viral load in infected individuals, would slow progression to AIDS and prolong life expectancy. More likely, these vaccines will affect the clinical course of the disease, not prevent infection, and will reduce the viral load and prolong symptom alleviation or symptom- free survival by slowing progression to AIDS.
  • a vaccine may affect person to person transmission, since high viral loads have been strongly correlated to increased rates of HIV transmission. Accordingly, a vaccine may reduce viral load to a level that results in decreased transmission. Regardless of the actual benefits to individual patients, then, use of a vaccine on an individual level may be extremely beneficial on an epidemiological level, at the scale of a whole population.
  • NRTI- sparing regimen that is able to control viral replication would be a very useful addition to the HIV treatment armamentarium.
  • Such additions could include treatment vaccines that are designed to induce potent cytotoxic T-cell responses.
  • the present invention addresses these and other unmet needs by providing a novel HIV therapeutic vaccine co-administered with an antiretroviral backbone monotherapy regimen for treatment of HIV infection.
  • the present invention provides improved methods for therapeutic treatment of human immunodeficiency virus (HIV) infection.
  • This invention more particularly relates to methods for treatment of HIV-infected host in need thereof comprising administration of an HIV vaccine in combination with an HIV inhibitor-based monotherapy.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in amounts sufficient to produce and maintain virologic suppressed status.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non- nucleoside reverse transcriptase inhibitors.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV virologically suppressed host in need thereof a therapeutically effective amount of an HIV vaccine and one or more HIV protease inhibitors in amounts sufficient to maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic HIV vaccine and an HIV protease inhibitor(s) regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV virologically suppressed
  • the HIV vaccine comprises a viral vector-based HIV vaccine, an HIV protein-based HIV vaccine, a plasmid DNA-based HIV vaccine, an anti-HIV antibody-based vaccine, or any combination thereof.
  • the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines, or a combination thereof.
  • the HIV monotherapy comprises a small molecule inhibitor, a protease inhibitor, an HIV CCR5 antagonist, a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor or a combination thereof.
  • the protease inhibitor further comprises a booster agent.
  • the administration of the NRTI-sparing regimen comprising administration of an HIV vaccine plus an HIV replication inhibitor-based monotherapy may be for either "prophylactic” or “therapeutic” purpose, or for both purposes.
  • the administration of the NRTI-sparing regimen comprising administration of an HIV vaccine plus an HIV replication inhibitor-based monotherapy is used in a therapeutic context.
  • the administration of the NRTI-sparing regimen comprising administration of an HIV vaccine plus an HIV replication inhibitor-based monotherapy is used in a prophylactic context in either in pre-exposure (PrEP) or post-exposure prophylaxis (PEP) settings.
  • PrEP pre-exposure
  • PEP post-exposure prophylaxis
  • the NRTI- sparing regimen comprising administration of an HIV vaccine plus an HIV replication inhibitor-based monotherapy in such PrEP or PEP settings would be performed, for example,
  • the HIV vaccine further comprises an adjuvant or immune modulator, or a combination thereof.
  • the pre-vaccination virological status of the host is achieved by administration of a combination of antiretrovirals (ARV) comprising a combination of drugs including two nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) with either a protease inhibitor (PI), or boosted protease inhibitor, or a non- nucleoside reverse transcriptase inhibitor (NNRTI).
  • ARV antiretrovirals
  • NRTIs nucleoside or nucleotide reverse transcriptase inhibitors
  • PI protease inhibitor
  • NRTI non- nucleoside reverse transcriptase inhibitor
  • the host is an HIV first line treatment patient, an HIV second line treatment patient, an HIV third line treatment patient, or those HIV patients that harbor multi-drug resistant viruses (HIV salvage patients), or those patients that have less than two viable treatment options left, or any combination thereof.
  • HIV salvage patients multi-drug resistant viruses
  • the plasma viral load in the patient so treated is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 copies per milliliter.
  • the CD4 + T lymphocyte count in the patient so treated is greater than about 500, 600, 700, 800, 900, 1000 cells per milliliter.
  • FIG. 1 depicts an illustrative HIV vaccine candidate and various analogs thereof (SEQ ID NOs:l-2).
  • This candidate is an HIV based lentiviral vector (LV) using the native HIV LTR as promoter. It expresses gag, pol and rev genes as immunogenic payloads.
  • LV HIV based lentiviral vector
  • An important feature of this LV resides in the engineering or placement of the RRE-containing sequence upstream of the full length Rev coding sequence.
  • this LV important feature of this LV is the presence of a splice acceptor (SA) site located between the RRE-containing sequence and the Rev coding sequence. Another important feature of this LV is the immediate proximity of the immunogenic payload(s) to the RRE-containing sequence.
  • SA splice acceptor
  • This LV optionally contains a non-protein coding genetic sequence (Gtag) to allow its identification and discrimination from wt-HIV in an HIV therapeutic vaccine setting. This sequence can be placed either upstream of downstream of the SA site.
  • Gtag non-protein coding genetic sequence
  • the five other schematic representations depicted in Figure 1 are non-limiting representative examples of possible LV vaccine vector analogs.
  • the second construct shows that the RRE element can be placed upstream or downstream of the immunogenic payload(s).
  • the third construct shows that the Gtag sequence can be disposable.
  • the fourth construct shows that downstream of the sequence encoding for the Rev protein another genetic payload(s) can be placed, payloads able to encode for genetic adjuvant, other immunogens, RNA antisense, ribozymes, etc.
  • the fifth construct is a combination of the schematic vector construct representations described supra.
  • the final and sixth construct contains an additional element which can be introduced for an additional regulation of transgene expression where SD/SA sites will be optional. An internal promoter/Rev combination could be used in all above listed constructs.
  • FIG. 2 depicts a generic vaccine HIV-based lentiviral vector construct and analogs thereof (SEQ ID NOs:3-4).
  • the generic vaccine vector is an HIV based lentiviral vector (LV) using the native HIV LTR as promoter. It contains the minimal elements for production, encapsidation and integration in transduced host cells (psi, cPPT/cts and ppt elements).
  • LV HIV based lentiviral vector
  • the important feature of this LV resides in the engineering or placement of the RRE-containing sequence upstream of the full length Rev coding sequence, the placement of the splice acceptor (SA) site between the RRE-containing sequence and the Rev coding sequence, and the immediate proximity of the immunogenic payload(s) to the RRE sequence.
  • SA splice acceptor
  • This LV contains a non-protein coding genetic sequence (Gtag) to allow its identification and discrimination from wt-HIV in an HIV therapeutic vaccine setting.
  • This sequence can be placed either upstream of downstream of the SA site.
  • the three other schematic representations are several non- limiting examples of possible LV generic vector analogs.
  • the second construct shows that the RRE element can be placed upstream or downstream of the genetic payload(s).
  • the third construct shows that the Gtag sequence can be disposable.
  • the fourth construct shows that downstream of the sequence encoding for the Rev protein another genetic payload(s) can be placed, payloads able to encode for genetic adjuvant, other immunogens, RNA antisense, ribozymes, etc.
  • the final and fifth construct shows a configuration with an internal promoter regulated Rev and
  • FIG. 3 depicts HIV vaccine candidate and analogs using a SIN configuration (SEQ ID NOs:5-6).
  • This figure present schematic representation of the HIV vaccine vector candidate (first from top) described in the application.
  • This candidate is an HIV based lentiviral vector (LV) with the native HIV LTR promoter activity disrupted either by deletion, mutation or insertion of elements such as Insulators.
  • This SIN based HIV vaccine LV expresses gag, pol and rev genes as immunogenic payloads.
  • the important feature of this LV resides in the engineering or placement of the RRE- containing sequence upstream of the full length Rev coding sequence, the placement of the splice acceptor (SA) site between the RRE-containing sequence and the Rev coding sequence, and the immediate proximity of the immunogenic payload(s) to the RRE sequence.
  • This LV contains a non-protein coding genetic sequence (Gtag) to allow its identification and discrimination from wt-HIV in HIV therapeutic vaccine setting. This sequence can be placed either upstream of downstream of the SA site.
  • Gtag non-protein coding genetic sequence
  • the four other schematic representations are non- limiting examples of possible SIN-based LV vaccine vector analogs.
  • the second construct shows that the RRE element can be placed upstream or downstream of the immunogenic payload(s).
  • the third construct shows that the Gtag sequence can be disposable.
  • the fourth construct from the top shows that downstream of the sequence encoding for the Rev protein another genetic payload(s) can be placed, payloads able to encode for genetic adjuvant, other immunogens, RNA antisense, ribozymes, etc.
  • the final and fifth construct is a combination of the schematic vector construct representations described supra.
  • SEQ ID NO: 1 is an example of an HIV vaccine vector of FIG. 1.
  • SEQ ID NO: 2 is an example of a vaccine vector of FIG. 1.
  • SEQ ID NO: 3 is an example of a DNA prime plasmid construct of
  • SEQ ID NO: 4 is an example of FIG. 2.
  • SEQ ID NO: 5 is an example of FIG. 3.
  • SEQ ID NO: 6 is an example of FIG. 3.
  • SEQ ID NO: 7 is an exemplary packaging signal sequence.
  • SEQ ID NO: 8 is an example of 5' LTR of HIV of vector pNL4-3.
  • SEQ ID NO: 9 is an example of 3' LTR of HIV of vector pNL4-3. sd-564347 [0046] SEQ ID NO: 10 is an example of
  • SEQ ID NO: 11 is an example of
  • SEQ ID NO: 12 is an example of
  • HIV vector pNL4-3 HIV vector pNL4-3.
  • SEQ ID NO: 13 is an example of
  • SEQ ID NO: 14 is an example of
  • SEQ ID NO: 15 is an example of
  • SEQ ID NO: 16 is an example of
  • SEQ ID NO: 17 is an example of
  • HIV vaccine includes both HIV-l-based vaccines and HIV-2-based vaccines.
  • HIV infection refers to indications of the presence of the HIV virus in an individual including asymptomatic seropositivity, aids-related complex (arc), and acquired immunodeficiency syndrome (AIDS).
  • arc aids-related complex
  • AIDS acquired immunodeficiency syndrome
  • HIV viral load refers to the number of viral particles in a sample of blood plasma HIV viral load is increasingly employed as a surrogate marker for disease progression. It is measured by PCR and cDNA tests and is expressed in number of HIV copies or equivalents per millilitre.
  • virologic suppressed status refers to the amount of HIV vaccine and/or HIV inhibitor administered either solely or in combination to achieve a suppressed viral load of less than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 copies per milliliter and a CD4 + T lymphocyte count of greater than about 500, 600, 700, 800, 900, 1000 cells per milliliter.
  • DNA polymerase refers to a DNA polymerase enzyme that transcribes single- stranded RNA into single-stranded DNA. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of normal transcription. Reverse transcriptases are ubiquitous to retroviruses. Common examples include HIV reverse transcriptase, M-MLV
  • nucleoside and nucleotide reverse transcriptase inhibitors refers to nucleosides, nucleotides, and analogues thereof which mimic natural nucleoside and nucleotide bases.
  • NRTI nucleoside and nucleotide reverse transcriptase inhibitors
  • non-nucleoside reverse transcriptase inhibitors refers to compounds which bind to a retrovirus reverse transcriptase, such as HIV type l's reverse transcriptase, and inhibits its enzymatic activity.
  • the binding by the "NNRTI” causes a conformational shift in the reverse transcriptase which prevents the enzyme from binding nucleoside and nucleotide bases, resulting in DNA chain termination.
  • protease inhibitor refers to compounds which bind to active site of a retroviral protease enzyme, such as HIV's protease enzyme.
  • the binding by the "PI” causes a conformational shift in the retroviral protease enzyme, making it no longer able to cleave large viral precursor proteins into smaller functional proteins. Viruses that are produced are defective and unable to infect other cells.
  • the term "entry/fusion inhibitor” or “entry or fusion inhibitor” refers to compounds which interfere with the binding, fusion and entry of an HIV virion to a human cell. By blocking this step in HIV's replication cycle, such agents slow the progression from HIV infection to AIDS.
  • Integrase inhibitor refers to compounds which interfere with the action of integrase, an enzyme that integrates genetic material from the virus into the host's DNA. Integrase inhibitors are also called strand transfer inhibitors. Strand transfer refers to the process by which the viral DNA strands are transferred from the viral genome to the host genome. Integrase inhibitors may be taken in combination with other types of retroviral drugs to minimize adaptation by the virus.
  • the term "maturation inhibitor” refers to compounds which interfere with the assembly and budding of virion particles, by binding to the viral gag polyprotein.
  • the bound gag polyprotein can no longer be processed to functional subunits by viral protease enzymes.
  • the resulting virus particles are structurally defective and are incapable of spreading infection.
  • inhibitor of CYP3A4 or "CYP 450 inhibitor” refer to any member of the class of pharmaceuticals and/or natural products which inhibit at least the CYP3A4 isoform of the cytochromes P450.
  • the class includes, but is not limited to, amprenavir, atazanavir, cobicistat, clarithromycin, cyclosporin, diltiazem, erythromycin, itraconazole, indinavir, ketoconazole, mibefradil, nefazodone, nelfinavir, ritonavir, vitamin E, bergamottin, dihydroxybergamottin and grapefruit juice. See GK Dresser et al. Clin. Pharmacokinetics 2000 January; 38(1): 41-57 for a review of clinically-relevant CYP3A4 inhibitors.
  • the preferred inhibitor of CYP3A4 is ritonavir.
  • treatment means the administration of the antivirally active compounds according to this invention in combination or alternation according to the present invention to alleviate or eliminate symptoms of the viral infection and/or to reduce viral load in a patient.
  • the invention relates to improved methods for therapeutic treatment of human immunodeficiency virus (HIV) infection, and more specifically to treatment of an HIV infection in a host comprising administration of an HIV vaccine in combination with an HIV inhibitor-based monotherapy.
  • HIV human immunodeficiency virus
  • HIV vaccines and HIV inhibitors that may be used in combination therapy to achieve and maintain an HIV virologic suppressed status.
  • the treatment regimen comprises administration of an HIV vaccine wherein the HIV vaccine comprises an LV-based HIV vaccine, an MLV-based HIV vaccine, an adenovirus-based HIV vaccine, an AAV-based HIV vaccine, a vaccinia virus-based HIV vaccine, a HIV protein- based HIV vaccine, a plasmid DNA-based HIV vaccine, an anti-HIV antibody-based vaccine, or any combination thereof.
  • the HIV vaccine comprises an LV-based HIV vaccine, an MLV-based HIV vaccine, an adenovirus-based HIV vaccine, an AAV-based HIV vaccine, a vaccinia virus-based HIV vaccine, a HIV protein- based HIV vaccine, a plasmid DNA-based HIV vaccine, an anti-HIV antibody-based vaccine, or any combination thereof.
  • the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines, or a combination thereof.
  • the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines comprising Vacc-4x (BioNor Pharma peptide vaccine), AGS 004
  • the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines comprising HIV AX (Genecure, replication-defective lentiviral based vaccine), GeoVax MVA boost (GeoVax, DNA prime expressing gag, pol, env plus boost with recombinant MVA plus GCSF adjuvant), VIR201 (Virax, Pty, LTD., expresses highly conserved regions of HIV and interferon-gamma), Vacc-5q (Bionor Immuno, HIV/ AIDS therapeutic vaccine), DP6-001 (Cytrx, polyvalent DNA prime vaccine followed by protein boost), LC-002 (Derma Vir) (Research Institute for Genetic & Human Therapy (RIGHT), NIAID/ACTG, Genetic Immunity), MRKAd5 (Merck & Co., adenovirus- type 5 (Ad5) intramuscular HIV vaccine expressing gag, pol, and nef gene
  • HIV AX Genecure, replication-defective lent
  • vCP1452 Sanofi-Aventis Pasteur, canarypox vector encoding env, gag, the protease- encoding portion of the pol gene and CTL epitopes from the nef and pol gene products
  • NYVAC-HIV Sanofi-Aventis Pasteur, attenuated vaccinia vector expressing multiple HIV genes
  • Lipopeptides Aventis Pasteur, peptides from Gag, Nef and Pol proteins
  • VRC- HIVDNA009-00-VP VRC/NIAID, DNA vaccine encoding gag, pol, nef, and multigene (A, B, and C) env genes together with an adjuvant gene encoding IL-2 fusion protein
  • ALVAC vCP1452
  • ACTG Aventis, canarypox vector encoding env, gag, the protease-encoding portion of the pol gene and CTL epitopes from the nef and pol gene products
  • HIV Autologous dendritic cell HIV vaccination with conserved HIV-derived peptides (University of Pittsburgh), or any combination thereof.
  • the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines comprising Multi-epitope DNA vaccine (Epimmune, 21 CTL epitopes and proprietary, non-HIV derived "universal" CD4 T cell epitpe), DNA/MVA
  • the vaccine based vectors of the present invention can comprise at least one, but can optionally comprise two or more nucleotide sequences of interest.
  • the IRES/2 A(s) may be of viral origin (such as EMCV IRES, PV IRES, or FMDV 2A-like sequences) or cellular origin (such as FGF2 IRES, NRF IRES, Notch 2 IRES or EIF4 IRES).
  • viral origin such as EMCV IRES, PV IRES, or FMDV 2A-like sequences
  • cellular origin such as FGF2 IRES, NRF IRES, Notch 2 IRES or EIF4 IRES.
  • Non-limiting examples of lentiviral vector constructs of the present invention that utilize an IRES sequence may be found in Figures 2 and 3 infra.
  • nucleotide sequences of interest or "payload" sequences can also be included in the HIV vaccine and comprise such nucleotide sequences encoding enzymes, cytokines, chemokines, growth factors, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin- like molecules, a single chain antibody, fusion proteins, immune co-stimulatory molecules, immunomodulatory molecules, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumor suppresser protein and growth factors, membrane proteins, pro- and anti- angiogenic proteins and peptides, vasoactive proteins and peptides, anti-viral proteins and derivatives thereof (such as with an associated reporter group).
  • the nucleotide sequences of interest may also encode pro-drug activating enzymes.
  • the nucleotide sequences of interest may also encode reporter genes such as, but not limited to, green fluorescent protein (GFP), luciferase, beta-galactosidase, or resistance genes to antibiotics such as, for example, ampicillin, neomycin, bleomycin, zeocin, chloramphenicol, hygromycin, kanamycin, among others.
  • the nucleotide sequences of interest may also include those which function as anti-sense RNA, small interfering RNA (siRNA), other non-coding RNAs or ribozymes, or any combination thereof.
  • the additional antigenic or immunogenic sequences contemplated for use in the HIV vaccines of the methods of the present invention may further comprise any tumor antigens as are known in the art.
  • a tumor antigen is a protein or protein or peptide fragment thereof produced in tumor cells that triggers an immune response in the host. Tumor antigens are useful in identifying tumor cells and are potential candidates for use in cancer therapy. Tumor antigens may include alphafetoprotein, carcinoembryonic antigen, CA-125, epithelial tumor antigen, tyrosinase, melanoma associated antigen, by way of example.
  • another aspect of the method of the present invention is antigenic compounds, proteins and protein fragments associated with Alzheimer's disease.
  • the level of payload sequence or antigen expression can be increased by using codon-optimized genes as well.
  • Synthetic genes created with the codon bias found in highly expressed human genes, have been shown to be much more efficiently expressed in human cells and to elicit higher and more reproducible levels of immune responses than did native coding sequences.
  • Another implication of over-expression is that the amount of a lentiviral vector or DNA prime that needs to be administered to elicit an immune response can be greatly reduced.
  • the HIV vaccine further comprises an adjuvant or immune modulator comprising lipid-A portion of gram negative bacteria endotoxin, trehalose dimycolate of mycobacteria, the phospholipid lysolecithin, dimethyldictadecyl ammonium bromide (DDA), certain linear
  • POP-POE polyoxypropylene-polyoxyethylene block polymers, aluminum hydroxide, and liposomes, immunostimulants (e.g., various interleukins and cytokines that are known to enhance the immune response including GM-CSF, IL-2, IL-12, TNF and IFN.gamma.), toll like receptors, CpG, immunomodulators and/or antibiotics (e.g., antibacterial, antifingal, anti- pneumocysitis agents), or any combination thereof.
  • immunostimulants e.g., various interleukins and cytokines that are known to enhance the immune response including GM-CSF, IL-2, IL-12, TNF and IFN.gamma.
  • toll like receptors including GM-CSF, IL-2, IL-12, TNF and IFN.gamma.
  • CpG immunomodulators and/or antibiotics (e.g., antibacterial, antifingal, anti- pneumocy
  • the lentiviral vectors contemplated for use as HIV vaccines in the methods of the present invention may include, without limitation, those lentiviruses that infect primates (HIV, HIV-2, simian
  • SIV immunodeficiency virus
  • FAV equine infectious anemia virus
  • BIV Bovine Immunodeficiency Virus
  • CAEV caprine arthritis encephalitis virus
  • VV visna maedi virus
  • JDV Jembrana disease virus
  • lentiviral vectors for use as vaccines in the methods of the present invention could be also modified by removing the transcriptional elements of HIV LTR; such as in a so-called self-inactivating (SIN) vector configuration.
  • the modalities of reverse transcription which generates both U3 regions of an integrated provirus from the 3 ' end of the viral genome, facilitate this task by allowing the creation of so-called self-inactivating (SIN) vectors.
  • Self-inactivation relies on the introduction of a disruption (employing for example, deletion, mutation and element insertion) in the U3 region of the 3 ' long terminal repeat (LTR) of the DNA used to produce the vector RNA.
  • LTR long terminal repeat
  • Non- limiting representative examples of SIN-based lentiviral vectors of the present invention may be generated from one or more of the constructs specifically shown in Figures 1, 2, and/or 3 described herein or any combination thereof.
  • the lentiviral vector vaccine constructs of the present invention further comprise those lentiviral vectors in which the lentiviral integrase function has been deleted and/or abrogated by site directed mutagenesis. Insertional mutagenesis has been observed in clinical trials with oncoretro viral vectors and this has prompted detailed study of genotoxicty of all integrating vectors. The most straightforward approach for several vaccine applications would be avoiding the possibility of integration. Non-integrating lentiviral vectors have been developed by mutating the integrase gene or by modifying the attachment sequences of the LTRs.
  • the D64V substitution in the catalytic domain has been frequently used because it shows the strong inhibition of the integrase without affecting proviral DNA synthesis. It has been reported that the mutation allows a transduction efficiency only slightly lower than integrative vectors but a residual integration that is about 1000-fold lower than an integrative vector at low vector doses. Another mutation described, D116N, resulted in residual integration about 2000 times lower than control vectors. In a couple of instances it has been shown that a single administration of an integrase (IN)- defective SIN LV elicits a significant immune response in the absence of vector integration and may be a safe and useful strategy for vaccine development.
  • integrase (IN)- defective SIN LV elicits a significant immune response in the absence of vector integration and may be a safe and useful strategy for vaccine development.
  • Non-limiting representative examples of Integrase-deficient SIN-based lentiviral vectors of the present invention may be generated from one or more of the constructs specifically shown in Figures 1, 2, and/or 3 described herein or any combination thereof.
  • the HIV vaccine for use in the methods of the present invention comprises the lentiviral vector described in Assignee's co-pending PCT application (PCT Application No. PCT/US09/038535) which was published as PCT
  • the HIV vaccine comprises a lentiviral vector comprising
  • LTR long terminal repeat
  • a second nucleic acid sequence operably linked to said 5' LTR comprising a functional REV coding sequence and a rev response element (RRE)-containing sequence wherein the RRE- containing sequence is located upstream of the REV coding sequence; and wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR.
  • RRE rev response element
  • the lentiviral vector employed in the methods of the present invention comprises one or more of the lentiviral vector constructs depicted in Figure 1, Figure 2 or Figure 3, or any combination thereof.
  • the lentiviral vector employed in the methods of the present invention comprises a lentiviral vector for vaccine delivery comprising a 5' long terminal repeat (LTR) (SEQ ID NO: 8) and a 3' LTR (SEQ ID NO: 9); a first nucleic acid sequence operably linked to said 5' LTR, also referred to herein as the "payload"; and a second nucleic acid sequence, that is operably linked to said 5' LTR comprising a functional REV coding sequence (SEQ ID NO: 10) and a rev response element (RRE)(SEQ ID NO: ll)-containing sequence wherein the RRE-containing sequence is located upstream of the REV coding sequence, and wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR.
  • LTR 5' long terminal repeat
  • RRE rev response element
  • the present invention is characterized in that expression of the payload is driven by the 5 'LTR and also where expression of the payload depends on the activity of Rev and the RRE-containing sequence.
  • the 5' LTR can be a powerful enough promoter to drive expression of the payload and REV.
  • the lentiviral vectors employed in the methods of the present invention may comprise a 5' LTR from a lenti virus; a 3' LTR from a lentivirus; a first nucleic acid sequence, i.e., a payload; and a second nucleic acid sequence, that is operably linked to said 5' LTR an RRE-containing sequence and a REV coding sequence wherein the RRE-containing sequence is located upstream of the REV coding sequence, wherein the first nucleic acid sequence expresses the full length wild-type sequence of either one or both of Gag and Pol; and wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR.
  • the lentiviral vectors employed in the methods of the present invention may comprise a 5' long terminal repeat (LTR) and a 3' LTR; a first nucleic acid sequence operably linked to said 5' LTR; and a second nucleic acid
  • the lentiviral vector construct may further comprise one or more of the known lentiviral regulatory elements such as Tat, Nef, Vif, Vpu, Vpr or a combination thereof.
  • the lentiviral vectors employed in the methods of the present invention specifically exclude all known lentiviral regulatory elements such as Tat, Nef, Vif, Vpu, and Vpr.
  • the nucleic acid sequence(s) encoding at least one of Vpu, Vpr, Vif, Tat, Nef, or analogous lentiviral proteins are disrupted such that the nucleic acid sequence(s) are incapable of encoding functional Vpu, Vpr, Vif, Tat, Nef, or the nucleic acid sequence(s) encoding Vpu, Vpr, Vif, Tat, Nef, or analogous auxiliary proteins are removed from the lentiviral vector system of the present invention.
  • the lentiviral vector further comprises a nucleic acid sequence encoding one or more functionally active lentiviral RNA packaging elements.
  • HIV and other lentiviruses have the unique property to replicate in non-dividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell.
  • a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap; the central DNA flap.
  • This central DNA flap is a region of triple- stranded DNA created by two discrete half-genomic fragments with a central strand displacement event controlled in cis by a central polypurine tract (cPPT) and a central termination sequence (CTS) during HIV reverse transcription.
  • cPPT central polypurine tract
  • CTS central termination sequence
  • the upstream plus strand segment initiated at the 3 ' PPT will, after a strand transfer, proceed until the center of the genome and terminate after a discrete strand displacement event.
  • This last event of HIV reverse transcription is controlled by the central termination sequence (CTS).
  • CTS central termination sequence
  • the lentiviral vector further comprises a nucleic acid sequence encoding functional central polypurine tract (cPPT), and central termination sequence (cTS) (SEQ ID NO: 13), and 3' LTR proximal polypurine tract (PPT) (SEQ ID NO: 9) elements.
  • cPPT functional central polypurine tract
  • cTS central termination sequence
  • PPT 3' LTR proximal polypurine tract
  • the vector further comprises expression of said one or more sequences of interest which is dependent upon REV-RRE activity. It is a feature of the invention that Rev and RRE as expressed by the same 5' LTR and are required for the transport of the payload to the cytoplasm after translation in the cell nucleus.
  • Rev is a small regulatory protein of HIV that is essential for virus replication. The biological role of Rev is to control the pattern of viral gene expression by promoting the transition from the early phase of infection, during which small regulatory proteins are expressed, to the late stage, when larger structural proteins are synthesized and assembled into viral particles.
  • Rev binds to a highly structured RNA region, the Rev response element (RRE), where it forms an oligomeric ribonucleoprotein complex with about 8 to 10 Rev proteins binding to the RRE.
  • the most important region of the RRE consists of five stem-loop domains radiating from a central junction, with the core element for Rev binding consisting of a stem loop- structure containing a purine rich bulge region (SEQ ID NO: 11).
  • expression of said one or more antigenic sequences of interest in the lentiviral vector depends on REV-RRE activity.
  • the first nucleic acid sequence comprises a Gag/Pol coding sequence or derivative thereof.
  • the first nucleic acid sequence of the lentiviral vector encodes one or more antigenic sequences of interest.
  • the first nucleic sequence is an unmodified sequence.
  • the Gag/Pol coding sequence comprises a modified Gag/Pol coding sequence.
  • the lentiviral vector also supports a high level of expression of gag-pol fusion protein not mediated by translational frameshifting.
  • Myristylation is a key component in the HIV particle assembly process. Gag p55 undergoes posttranslational myristylation at the N- terminus of matrix, which allows the Gag complex to attach to the cell membrane via the myristic acid moiety. Disruption of the myristylation site results in an accumulation of Gag proteins in the cytoplasm. The higher concentration of proteins within the cytoplasm most likely increases proteosomal degradation of these polypeptides into the endogenous antigen pathway, resulting in the loading of Gag peptides on MHC class I molecules leading to higher
  • the invention further comprises at least one nucleotide substitution which alters the myristylation receptor glycine residue of Gag (SEQ ID NO: 17).
  • the cPPT/cTS is a part of a Pol coding sequence of said Gag/Pol coding sequence.
  • the functionally active lentiviral RNA packaging elements comprise a Gag packaging sequence or derivative thereof.
  • the lentiviral vector may further comprise a heterologous promoter located 3' of said RRE, wherein said heterologous promoter comprises a viral promoter, a human promoter, or a synthetic promoter, or a synthetic promoter, or a combination thereof.
  • the lentiviral vector for use in the methods of the present invention further comprises a nucleic acid sequence encoding functionally active lentiviral RNA packaging elements (SEQ ID NO: 12).
  • SEQ ID NO: 12 The full-length lentiviral RNA is selectively incorporated into the viral particles as a non-covalent dimer.
  • RNA packaging into virus particles is dependent upon specific interactions between RNA and the nucleocapsid protein (NC) domain of the Gag protein.
  • NC nucleocapsid protein
  • incorporation of the HIV genomic RNA into the viral capsid involves the so-called Psi region located immediately upstream of the Gag start codon and folded into four stem- loop structures, is important for genome packaging; SL1 to SL4.
  • SL1 contains the dimerization initiation site (DIS), a GC-rich loop that mediates in vitro RNA dimerization through kissing - complex formation, presumably a prerequisite for virion packaging of RNA. Additional cis- acting sequences have also been shown to contribute to RNA packaging. Some of these elements are located in the first 50 nucleotides (nt) of the Gag gene, including SL4, whereas others are located upstream of the splice-donor site (SD1), and are actually mapped to a larger region covering the first 350 ⁇ 400 nt of the genome, including about 240 nt upstream of SL1.
  • SD1 splice-donor site
  • the SL1-4 region is an example of a simple sequence essential for RNA packaging. Other such sequences are known by those of skill in the art.
  • the transcription of the payload as well as the Rev coding sequence and RRE-containing sequence is driven by the same promoter, i.e., the 5' LTR (SEQ ID NO: 8).
  • the 5' LTR has sufficient basal activity to drive transcription of a payload comprising nucleic acids that encode full length antigenic sequences, as well as packaging sequences, and Rev coding sequence and RRE-containing sequence on the same transcription unit, with the proviso that the RRE-containing sequence is
  • the 5' LTR can be derived from various strains and clades of HIV, as are known in the art, and optimized for stronger basal promoterlike function.
  • the 5 ' LTR from HIV Clade E can exhibit strong basal promoter activity.
  • HIV groups M (for major)(A, B, C, D, E, F, G, H, I, and J), O (outlier or "outgroup"), which is a relatively rare group currently found in Cameroon, Gabon, and France, and a third group, designated N (new group), and any circulating recombinant forms thereof.
  • the 5' LTR further drives expression of the payload and Rev proteins.
  • the HIV Rev protein directs the export of unspliced or partially spliced viral transcripts from the nucleus to the cytoplasm in mammalian cells. Rev contains the RNA binding domain, which binds the RRE present on target transcripts. Export activity is mediated by a genetically defined effector domain, which has been identified as a nuclear export signal.
  • the heterologous promoter comprises viral, human, and/or synthetic promoters or a combination thereof.
  • heterologous viral promoters comprise Mouse Mammary Tumor Virus (MMTV) promoter, Moloney virus, avian leukosis virus (ALV), Cytomegalovirus (CMV) immediate early promoter/enhancer, Rous Sarcoma Virus (RSV), adeno-associated virus (AAV) promoters; adenoviral promoters, and Epstein Barr Virus (EBV) promoters, or any combination thereof.
  • heterologous human promoters comprise
  • Apolipoprotein E promoter Albumin promoter, Human ubiquitin C promoter, human tissue specific promoters such as liver specific promoter, prostate specific antigen (psa) promoter, Human phosphoglycerate kinase (PGK) promoter, Elongation factor-1 alpha (EF-la) promoter, dectin-2 promoter, HLA-DR promoter, Human CD4 (hCD4) promoter, or any combination thereof.
  • the synthetic promoters comprise those promoters described in US Patent 6,072,050, the contents of which are incorporated by reference in its entirety.
  • various elements of the lentiviral vector construct may include functional equivalent derivatives, fragments or modifications thereof that have been engineered into the nucleotide coding sequences and/or amino acid sequences of the first nucleic acid sequence or the second nucleic acid sequence of the lentiviral vectors of the present invention. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural genotypic, allelic variation, or that have been artificially engineered, and that
  • functional equivalent derivatives, fragments or modifications thereof of the first nucleic acid sequence or the second nucleic acid sequence of the lentiviral vectors of the present invention can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of marker nucleic acids, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidinea), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • the lentiviral vector for use in the methods of present invention comprises a packaging signal sequence as described in SEQ ID NO: 7, a 5' LTR of HIV vector pNL4-3 as described in SEQ ID NO: 8, a 3' LTR of HIV vector pNL4- 3 as described in SEQ ID NO: 9, a REV gene of HIV vector pNL4-3 as described in SEQ ID NO: 10, a sequence of functional RRE of HIV vector pNL4-3 as described in SEQ ID NO: 11, a minimum packaging sequence of HIV vector pNL4-3 as described in SEQ ID NO: 12, a cPPT/cTS of HIV vector pNL4-3 as described in SEQ ID NO: 13, PPT sequence of HIV vector pNL4-3 as described in SEQ ID NO: 14, a gag/pol coding sequence of HIV vector pNL4-3 as described in SEQ ID NO: 15 a frameshift modified gag/pol fusion coding sequence of HIV vector pNL4-3 as described in SEQ ID NO: 16,
  • the lentiviral vectors contemplated for use in the methods of the present invention may be pseudotyped.
  • "Pseudotyping" a virion is accomplished by co- transfecting a packaging cell with both the lentiviral vector of interest and a helper vector encoding at least one envelope protein of another virus or a cell surface molecule (see, for example, U.S. Patent Number 5,512,421, the entire text of which is herein incorporated by reference in its entirety).
  • One viral envelope protein commonly used to pseudotype lentiviral vectors is the vesicular stomatitis virus-glycoprotein G (VSV-G), which is derived from a rhabdovirus.
  • VSV-G vesicular stomatitis virus-glycoprotein G
  • viral envelopes proteins that may be used include, for example, rabies virus-glycoprotein G and baculovirus gp-64.
  • pseudotyping broadens the host cell range of the lentiviral vector particle by including elements of the viral entry mechanism of the heterologous virus used.
  • Pseudotyping of lentiviral vectors with, for example, VSV-G for use in the present invention results in lentiviral particles containing the lentiviral vector nucleic acid encapsulated in a nucleocapsid which is surrounded by a membrane containing the VSV-G envelope protein.
  • the nucleocapsid preferably contains proteins normally associated with the lentiviral vector.
  • the surrounding VSV-G protein containing membrane forms part of the viral particle upon its egress from the producer cell used to package the lentiviral vector.
  • the lentiviral particle is derived from HIV and pseudotyped with the VSV-G protein. Pseudotyped lentiviral particles containing the VSV-G protein can infect a diverse array of cell types with higher efficiency than amphotropic viral vectors.
  • the range of host cells includes both mammalian and non- mammalian species, such as humans, rodents, fish, amphibians and insects.
  • VSV-G pseudotyping has been described as being the most efficient for cutaneous transduction, a great advantage of using LV is that it is possible to target the vector to specific tissues or cells by replacing and/or modifying the virion envelope.
  • LVs are remarkably compatible with a broad range of viral envelope glycoproteins providing them with added flexibility; Rabies, Mokola, LCMV, Ross River, Ebola, MuLV, Baculovirus GP64, HCV, Sindai virus F protein, Feline Endogenous Retrovirus RD114 modified, Human Endogenous Retroviruses, Seneca virus, GALV modified and HA influenza glycoproteins or a combination thereof, to name a few of those viral envelope glycoproteins explored.
  • flexibility of LV platform for targeting different cell types is achieved by refining the surface of LV particles via the display of cell-specific ligands.
  • VSV-G as a
  • VSV-G could eventually be replaced by other envelopes if needed, for example in the case of multiple vector administration, although anti- VSV-G immunity does not seem to prevent repeated vector administrations.
  • the HIV inhibitor-based monotherapy comprises administration of a small molecule inhibitor, a protease inhibitor, an HIV CCR5 antagonist, a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor or a combination thereof.
  • the protease inhibitor contemplated for use in the methods of the present invention comprise indinavir, saquinavir, nelfinavir, darunavir, amprenavir, lopinavir, tripanivir, fosamprenavir or atazanavir, or any combination thereof.
  • the booster agent contemplated for use in the methods of the present invention comprise ritonovir or cobicistat, grapefruit juice extract, or a combination thereof.
  • nucleoside/nucleotide reverse transcriptase inhibitors comprise nucleoside/nucleotide analogs including zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), emtricitabine, stavudine (d4T), lamivudine (3TC), abacavir (ABC), tenofovir, apricitabine, stampidine, elvucitabine, and racivir, or any combination thereof.
  • nucleoside/nucleotide reverse transcriptase inhibitors comprise nucleoside/nucleotide analogs including zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), emtricitabine, stavudine (d4T), lamivudine (3TC), abacavir (ABC), tenofovir, apricitabine, stampidine, elvucitabine
  • the non-nucleoside reverse transcriptase inhibitors contemplated for use in the methods of the present invention comprise avirenz, nevirapine, etravirine, rilpivirine, loviride, delavirdine, efavirenz, tenofovir DF (TDF), or any combination thereof.
  • the excluded non- nucleoside reverse transcriptase inhibitors comprises avirenz, nevirapine, etravirine, rilpivirine, loviride, delavirdine, efavirenz, tenofovir DF (TDF), or any combination thereof.
  • the CCR5 antagonist comprises INCB9471, maraviroc, SCH532702, TBR-652, or any combination thereof.
  • the viral integrase inhibitor comprises elvitegravir, raltegravir, the second generation integrase inhibitor, MK- 2048, or any combination thereof.
  • the viral fusion inhibitor comprises enfuvirtide, maraviroc, PRO140, T-20, T-1249, vicriviroc, or any combination thereof.
  • the maturase inhibitor comprises bevirimat, becon, or any combination thereof.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in an amounts sufficient to produce and maintain virologic suppressed status.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV antiretroviral inhibitor monotherapy in an amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in an amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non- nucleoside reverse transcriptase inhibitors.
  • the HIV monotherapy comprises a small molecule inhibitor, a protease inhibitor, an HIV CCR5 antagonist, a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor or a combination thereof.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV virologically suppressed host in need thereof a therapeutically effective amount of an HIV vaccine and one or more HIV protease
  • the treatment regimen comprises a therapeutic HIV vaccine and an HIV protease inhibitor(s) regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non- nucleoside reverse transcriptase inhibitors.
  • the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a
  • an HIV vaccine and an HIV CCR5 antagonist a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor, or any combination thereof, in amounts sufficient to maintain virologic suppressed status
  • the treatment regimen comprises a therapeutic vaccine and a CCR5 antagonist, viral integrase inhibitor, maturation inhibitor, or viral fusion inhibitor regimen that specifically excludes
  • nucleoside/nucleotide reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors
  • a pharmaceutical composition comprising the aforementioned lentiviral vector.
  • a method for inducing an immune response in a subject comprising administering a pharmaceutical composition and a pharmaceutically acceptable carrier and/or a genetic adjuvant as are known in the art.
  • the lentiviral vector expresses one or more genes of interest to potentiate immunity, and wherein said immune response comprises a humoral immune response, a cell mediated immune response or a combination thereof.
  • the humoral immune response, cell-mediated immune response or a combination thereof is specific to a disease or condition of interest comprising cancer, Alzheimer's disease, autoimmune diseases, cardiovascular diseases, neurological diseases, fibrotic diseases, lipid metabolism diseases, extra-cellular matrix-related diseases, and chronic joint degenerative diseases, or any combination thereof.
  • HIV human immunodeficiency virus
  • the invention provides for a method for inducing an immune response in a subject, said method comprising administering a pharmaceutical composition comprising one or more of the lentiviral vector HIV vaccines disclosed herein, by various administration routes and protocols, e.g.
  • DNA prime/lentiviral vector boost DNA prime/two homologous lentiviral vector boosts, DNA prime/heterologous vector boost, lentiviral prime/lentiviral vector boost, lentiviral vector or DNA prime/lentiviral vector boost/adenoviral vector boost and vice versa, where the adenoviral vector is administered as the first boost and the lentiviral vector is administered as the second boost, each of the protocols being administered in combination with the aforementioned HIV inhibitor monotherapy regimen.
  • a first vector e.g., a nucleic acid plasmid construct, referred to as a DNA prime plasmid or using lentiviral vectors of the present invention, adenoviral-based vectors, pox-based vectors, including MVA and canary pox, VSV-based vectors, alphavirus-based vectors (e.g., VEE, Semliki Forest Virus), herpes virus-based vectors, among others known in the art) and subsequently administering a lentiviral vector of the present invention as a boost comprising the same payload, wherein one or both first and second vectors are the lentiviral vector of the present invention as a boost comprising the same payload as the DNA prime plasmid construct, or in some cases, a lentiviral vector of
  • a first vector e.g., a nucleic acid plasmid construct, referred to as a DNA prime plasmid or using lentiviral vectors of the present invention, adenovi
  • the HIV vaccine may be targeted to a specific cell, for example, by means of one or more tissue specific promoters as described supra.
  • a cell can be present as a single entity, or can be part of a larger collection of cells.
  • Such a "larger collection of cells” can comprise, for instance, a cell culture (either mixed or pure), a tissue (e.g. , endothelial, epithelial, mucosa or other tissue), an organ (e.g. , lung, liver, muscle and other organs), an organ system
  • the organs/tissues/cells being targeted are of the circulatory system (e.g. , including, but not limited to blood, including white blood cells), the mucosal system of the nose, trachea, bronchi, bronchioles, lungs, and the like), gastrointestinal system (e.g.
  • the cells being targeted are selected from the group consisting of antigen presenting cells.
  • Such an antigen presenting cell includes, but is not limited to, a skin fibroblast, a bowel epithelial cell, an endothelial cell, an epithelial cell, a dendritic cell, a plasmacytoid dendritic cell, Langerhan's cells, a monocyte, a mucosal cell and the like.
  • the target cells need not be normal cells and can be diseased cells.
  • diseased cells can be, but are not limited to, tumor cells, infected cells, genetically abnormal cells, or cells in proximity or contact to abnormal tissue such as tumor vascular endothelial cells.
  • the vector used in the methods of the present invention can be modified to include antigens from a variety of viruses and/or or bacteria in the payload to therapeutically treat said disease or as prophylaxis to said disease.
  • the vectors used in the methods of the present invention can be used to treat, in addition to HIV, a wide array of viral infections caused by various viruses including, but not limited to, viruses from the groups including dsDNA viruses (e.g. adenoviruses, herpesviruses, etc.); ssDNA viruses (e.g.
  • parvoviruses parvoviruses
  • dsRNA viruses e.g. reoviruses, rotavirus
  • (+)ssRNA viruses e.g.
  • the vectors used in the methods of the present invention can be used to treat, in addition to HIV, disease caused by viruses from a number of viral families including, but not limited to, Herpes Simplex Virus (HSV) - 1 (oral herpes simplex), HSV-2 (genital herpes simplex), Varicella Zoster virus (VZV) (chickenpox), Epstein-Barr virus
  • HSV Herpes Simplex Virus
  • HSV-2 human herpes simplex
  • VZV Varicella Zoster virus
  • EBV infectious mononucleosis
  • CMV Cytomegalovirus
  • Pox virus smallpox
  • other viruses as are known in the art.
  • the vectors used in the methods of the present invention can be used, in addition to treatment and management of HIV, to prophylatically or therapeutically treat various diseases including parasitic and microorganism ailments including, but not limited to, the disease malaria caused by mosquito transmission of the malarial parasite Plasmodium to humans.
  • the disease is caused by protozoan parasites of the genus Plasmodium. Only four types of the Plasmodium parasite can infect humans. The most serious forms of the disease are caused by Plasmodium falciparum and Plasmodium vivax, but other related species (Plasmodium ovale, Plasmodium malariae) can also affect humans.
  • Other diseases that can be treated by the method of the present invention are malaria, St. Louis encephalitis, dengue fever, yellow fever, West Nile virus; bubonic plague; Lyme disease, rocky mountain spotted fever, encephalitis; hantavirus; Chagas Disease, by way of example, not limitation.
  • the vectors used in the methods of the present invention can be used to treat, in addition to HIV, bacterial infections including, but not limited to, the following: human disease causing bacteria of the order Spirochaetales including T. pallidum (Syphillis), S. pilosicoli (diarrhea in humans especially in developing countries and in HIV patients), B. burgdorferi: (Lyme disease and related disorders), bacteria of the family
  • human disease causing bacteria of the order Spirochaetales including T. pallidum (Syphillis), S. pilosicoli (diarrhea in humans especially in developing countries and in HIV patients), B. burgdorferi: (Lyme disease and related disorders), bacteria of the family
  • Spirillaceae including C. jenuni (colitis, diarrhea, enteritis, enterocolitis, gastroenteritis, etc.), H. pylori (gastritis, duodenal and peptic ulcers, Taynaud's phenomenon, etc.); bacteria of the Family Spirosomaceae including Pseudomonas (cellulitis, cerebrospinal fluid shunt infections, acute cystitis, endocarditis, chronic eye infections, neonatal meningitis, peritonitis, pneumonia), of the family Legionellaceae including Legionella (pneumonia in humans (legionellosis, legionnaire's disease, Pittsburgh pneumonia, nonpneumonic Pontiac fever, rhabdomyolysis, bacteraemia and septicemia, endocarditis, systemic infections in cell- mediated immunity disorders); bacteria Rhizobiacea including Agrobacterium (peritonitis in continuous ambulatory peritoneal dialysis); Achromobacter (postoperative
  • compositions of the present invention contain a pharmaceutically and/or therapeutically effective amount of at least one nucleic acid construct, lentiviral vector, lentiviral vector system, viral particle/virus stock, or host cell (i.e., agents) of the invention.
  • the effective amount of an nucleic acid construct e.g., a nucleic acid construct, lentiviral vector, lentiviral vector system, viral particle/virus stock, or host cell (i.e., agents) of the invention.
  • the effective amount of an agent i.e., lentiviral vector system, viral particle/virus stock, or host cell (i.e., agents) of the invention.
  • the effective amount of an agent i.e., lentiviral vector system, viral particle/virus stock, or host cell (i.e., agents) of the invention.
  • the effective amount of an agent e.g., a nucleic acid construct, lentiviral vector, lentiviral vector system, viral particle/virus stock, or host cell (i.e
  • agent of the invention per unit dose is an amount sufficient to cause the detectable expression of the gene of interest.
  • effective amount of agent per unit dose is an amount sufficient to prevent, treat or protect against deleterious effects (including severity, duration, or extent of symptoms) of the disease or condition being treated.
  • the administration of the pharmaceutical compositions of the invention may be for either "prophylactic” or "therapeutic” purpose.
  • the compositions are provided in advance of any symptom, in pre-exposure (PrEP) or post-exposure prophylaxis (PEP) settings.
  • PrEP pre-exposure
  • PEP post-exposure prophylaxis
  • the prophylactic administration of the composition serves to prevent or ameliorate any subsequent deleterious effects (including severity, duration, or extent of symptoms) of the disease or condition being treated.
  • the specific use of the invention is in the settings of PrEP or PEP, at which time a subject would seek administration of the composition shortly (within few days of exposure) before or after a potential exposure to HIV, through any possible route.
  • compositions in such PrEP or PEP settings would in these cases be performed before the onset of any symptoms characteristic of the primary infection, before HIV becomes detectable by serologic assays, and in some cases even before the contamination event itself.
  • the composition is provided at (or shortly after) the onset of a symptom of the condition being treated.
  • kits for all therapeutic, prophylactic and diagnostic uses, one or more of the aforementioned HIV- vaccines, lentiviral vectors, lentiviral vector system, viral particle/virus stock, or host cell (i.e., agents) of the present invention, as well as other necessary reagents and appropriate devices and accessories, may be provided in kit form so as to be readily available and easily used.
  • a kit would comprise a pharmaceutical composition for in vivo administration comprising an HIV vaccine of the present invention, and a pharmaceutically acceptable carrier and/or a genetic adjuvant; and instructions for use of the kit.
  • HIV vaccine formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which
  • aqueous and non-aqueous sterile suspensions render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials. Extemporaneous injection solutions and suspensions may be prepared from purified nucleic acid preparations for the DNA plasmid priming compounds and/or purified viral vector compounds commonly used by one of ordinary skill in the art.
  • Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations may also include other agents commonly used by one of ordinary skill in the art.
  • the HIV vaccine formulation may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, intranasal, intramuscular, subcutaneous, intravenous, intraperitoneal, intraocular, intracranial, intradermal, transdermal (skin patches), topical, or direct injection into a joint or other area of the subject's body.
  • the HIV vaccine may likewise be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes. It is expected that from about one to about five dosages (e.g.
  • the initial prime and boost administrations may contain a quantity of antigen sufficient to induce a satisfactory immune response.
  • An appropriate quantity of prime and boost antigen(s) to be administered is determined for any of the prime/boost protocols disclosed herein by one skilled in the art based on a variety of physical characteristics of the subject or patient, including, for example, the patient's age, body mass index (weight), gender, health, immunocompetence, and the like.
  • the volume of administration will vary depending on the route of administration.
  • intramuscular injections may range from about 0.1 ml to 1.0 ml.
  • a patient has a normal immune system and is not infected with human immunodeficiency virus, although the vaccine may also be administered after initial HIV infection to ameliorate disease progression, or after initial infection to treat AIDS.
  • the HIV vaccine may be stored at temperatures of from about -80°C to about 37°C or less, depending on how the vaccine is formulated.
  • a variety of adjuvants known to one of ordinary skill in the art may be administered in conjunction with the viral vector in the HIV vaccine composition.
  • Non-limiting examples of "Genetic adjuvants” can be used with the HIV vaccine vector of the present invention to increase or enhance the immune response elicited by expression of the antigenic sequence of interest of the present invention.
  • These genetic adjuvants can be on different constructs but co-expressed or on the same vector construct yet contained in different expression cassettes.
  • Such desirable genetic adjuvants may include, without limitation, toll like receptors, CpG, cytokines (e.g., the interleukins (ILs) IL-l.beta., 11-2, 11-10, 11-12, 11-15, CTA1-DD, fas antigen, flagellin, etc.), or combinations thereof.
  • genetic adjuvants include, without limitation, the DNA sequences encoding GM-CSF, the interferons (IFNs) (for example, IFN-. alpha., IFN-.beta., and IFN-. gamma.), TNF-. alpha., and combinations thereof.
  • the genetic adjuvants may also be a immunostimulatory polypeptide from Parapox virus, such as a polypeptide of Parapox virus strain D1701 or NZ2 or Parapox immunostimulatory polypeptides B2WL or PP30 (see, e.g., U.S. Pat. No. 6,752,995). Still other such biologically active factors and
  • immunomodulators or immunostimulants that are known in the art that enhance the antigen- specific immune response may be readily selected by one of skill in the art, and a suitable plasmid vector containing the same factors constructed by known techniques.
  • One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired "effective level" in the individual patient.
  • One skilled in the art also can readily determine and use an appropriate indicator of the "effective level" of the compounds of the present invention by a direct (e.g. , analytical chemical analysis) or indirect (e.g. , with surrogate indicators of viral infection, such as p24 or reverse transcriptase in the case of HIV infection) analysis of appropriate patient samples (e.g. , blood and/or tissues).
  • mice are available and have been widely implemented for evaluating the in vivo efficacy against HIV of various gene therapy protocols (Sarver et al. (1993) AIDS Research and Human Retroviruses, 9(5), 483-487; Sarver et al. (1993) Antisense Research and Development, 3(1), 87-94.).
  • These models include mice, dogs, guinea pigs, monkeys, cats and rabbits. Even though these animals are not naturally susceptible to HIV disease, chimeric mice models (e.g.
  • SCID bg/nu/xid
  • NOD/SCID SCID-hu
  • immunocompetent SCID-hu bone marrow- ablated BALB/c
  • PBMCs peripheral blood mononuclear cells
  • lymph nodes fetal liver/thymus or other tissues
  • lentiviral vector or HIV employed as
  • the pharmaceutical composition can contain other pharmaceuticals, in conjunction with a vector according to the invention, when used to therapeutically treat AIDS. These other pharmaceuticals can be used in their traditional fashion (i.e., as agents to treat HIV infection).
  • the VRX1273 clinical indication is for patients who have a clinical need to switch to an NRTI-sparing regimen due to adverse effects, intolerance or compliance and are either currently virologically suppressed or not virologically suppressed.
  • VRX1273 may be co-administered with a boosted protease inhibitor (for example, boosted with ritonavir, cobicistat).
  • the initial therapeutic vaccine development strategy focuses on the vaccine as an NRTI-sparing agent. This clinical study strategy also incorporates the possibility of withdrawing or not withdrawing the boosted protease inhibitor monotherapy.
  • This Phase I study enrolls HIV-infected subjects with virological suppression for at least 6 months on a boosted protease inhibitor (LPV/r, ATV/r, or DRV/r)- based regimen containing 2 NRTIs. After screening tests are complete and all
  • PI boosted protease inhibitor
  • viral load, CD4+ counts and immunogenicity are assessed periodically.
  • contingencies are provided in the study to either remove the current boosted protease inhibitor monotherapy that the subject is on, or remove the monotherapy altogether at a time to be defined (treatment interruption).
  • treatment interruption similarly, viral load and CD4+ counts are further assessed on a periodic basis thereafter.
  • Immunization with VRX1273 vaccine in combination with boosted protease inhibitor (ritonavir-boosted PI (ATV, LPV or DRV)) in an NRTI-sparing regiment result in effective treatment and management of HIV-1 as revealed by reduced viral load less than 200 copies per cell in treated patients and an CD4 + T cell count of greater than 500 cells/ml in patients receiving such treatment.
  • the ultimate goal is to have a vaccine treatment regimen that is effective yet reduces drug toxicity exposure by sparing antiretroviral drugs in the HIV-1 treatment regimen.
  • MRKAd5 adenovirus-type 5 (Ad5) intramuscular vaccine expressing gag, pol, and nef gene
  • HIV vaccine in combination with boosted protease inhibitor (for example, and not by way of limitation, ritonavir-boosted PI (ATV, LPV or DRV)) in an NRTI sparing regiment resulted in effective treatment of HIV as revealed by reduced viral load less than 200 copies per cell and an elevated CD4 + count of greater than 500/ml.
  • boosted protease inhibitor for example, and not by way of limitation, ritonavir-boosted PI (ATV, LPV or DRV)

Abstract

This invention describes an improved therapeutic option for treatment and management of human immunodeficiency virus (HIV) infection. This invention more particularly relates to an NRTI-sparing regimen comprising administration of a therapeutic vaccine plus a monotherapy with an HIV antiretroviral in combinations that reduce drug- induced toxicities.

Description

HIV VACCINE THERAPY WITH CONCOMITANT ANTIVIRAL MONOTHERAPY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. provisional application Serial
No. 61/370,033, filed 2 August 2010, which is incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0002] The entire content of the following electronic submission of the sequence listing via the USPTO EFS-WEB server, as authorized and set forth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference in its entirety for all purposes. The sequence listing is identified on the electronically filed text file as follows:
Figure imgf000003_0001
FIELD OF THE INVENTION
[0003] This invention describes an improved therapeutic option for treatment and management of human immunodeficiency virus (HIV) infection. This invention more particularly relates to an NRTI-sparing regimen consisting of a therapeutic vaccine plus an HIV replication inhibitor-based monotherapy.
BACKGROUND OF THE INVENTION
[0004] More than 25 million people have died of AIDS worldwide and an estimated 33 million people worldwide are currently living with HIV/ AIDS [1]. In the United States, more people than ever are living with HIV, with current estimates at 1.1 million in 2009 [2]. Changes in incidence along with rising AIDS mortality have caused global HIV prevalence to level off. However, the number of people living with HIV has continued to rise due to population growth and, more recently, due to the life-prolonging effects of antiretroviral therapy (ART) [3].
[0005] The current standard of treatment for HIV/ AIDS is highly active antiretroviral therapy (HAART), consisting of a combination of several antiretroviral drugs [4]. Although this combination has been successful in reducing viral load (VL) in plasma and restoring some immune function, concerns regarding adverse effects associated with long-
- 1 - sd-564347 term usage of these antiretroviral drugs are growing. Specifically, a variety of metabolic disorders including HIV- associated lipodystrophy, central adiposity, dyslipidaemia, hyperlipidaemia, hyperglycemia and insulin resistance have been reported as resulting from combination therapies [3, 5-7]. These reactions, combined with complex and cumbersome dosing regimen, can have an adverse impact on subject adherence to therapy [8, 9]. As a result, poor adherence has led to an increased rate of HIV resistance, resulting in viral strains that have reduced sensitivity to the drugs [9, 10] and an increasing health care burden related to the treatment costs of these metabolic disorders.
[0006] Current HIV treatment guidelines recommend starting antiretrovirals
(ARV) with a combination of drugs including two nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) with either a protease inhibitor (PI) or a non-nucleoside reverse transcriptase inhibitor (NNRTI) [11]. Although these combinations are very effective in controlling HIV replication, resistance to NRTI can build up over time. Many NRTI and NNRTI resistance mutations confer cross-resistance to other drugs in that same class, thus limiting future viable antiretroviral options [12, 13]. Furthermore, NRTIs have poor tolerability and significant toxicities which can compromise compliance and promote drug resistance [14-16]. In addition, mitochondrial dysfunction induced by NRTIs produces a spectrum of illnesses including peripheral neuropathy, myopathies, steatohepatitis, pancreatitis, lipoatrophy, tubular acidosis and lactacidosis [17-20].
[0007] Because of issues relating to HAART, new chemotherapeutic approaches are needed. One possible approach is to elicit cytotoxic T lymphocyte response through vaccines.
[0008] Potent CTL activity has been shown to control viral replication during the acute (initial) phase of HIV infection [21]. However, the magnitude of responses gradually diminishes with disease progression and HAART alone cannot restore potent CTL activity [22]. In fact, prolonged use of HAART is associated with a decrease in CTL response [23]. Other evidence indicates that cytotoxic CD8 T-cell responses play a critical role in the control of viral replication and progression of disease in "long-term non-progressors" and "high-risk" sero-negative individuals who can maintain strong HIV-specific T-cell mediated immune responses. These T-cell responses delay disease progression over the long-term in the former group and prevents acquisition in the latter group [24-26].
[0009] In general, regimen simplification is a strategy used to decrease toxicities, and avoid drug interactions or improve antiretroviral compliance and convenience. Although regimen simplification using boosted protease inhibitor monotherapy (ΡΙ/r) is not
- 2 - sd-564347 the standard of care, a number of small studies have shown that this treatment regimen is adequate in maintaining virological suppression. However, large controlled trials are needed to confirm the safety and utility of this treatment as a recommended clinical strategy. The current Guidelines for Antiretro viral Treatment of Adult HIV Infections: 2010
Recommendations of the International AIDS Society USA Panel (JAMA, 304:321-333, 2010) does not recommend boosted protease monotherapy as part of the standard of care. At this time it is still considered experimental and may only be conducted under clinical trial conditions (DHHSS, Guidelines, 2010).
[0010] First generation HIV vaccines currently in development may offer some form of protection, but they are not entirely protective. Prophylaxis to disease is unlikely to occur with first- generation prototypes of AIDS vaccines. However, even a partially effective vaccine could be very promising in protecting some individuals against infection. In addition to reducing the rate of transmission of HIV, lowering viral load in infected individuals, would slow progression to AIDS and prolong life expectancy. More likely, these vaccines will affect the clinical course of the disease, not prevent infection, and will reduce the viral load and prolong symptom alleviation or symptom- free survival by slowing progression to AIDS.
[0011] A vaccine may affect person to person transmission, since high viral loads have been strongly correlated to increased rates of HIV transmission. Accordingly, a vaccine may reduce viral load to a level that results in decreased transmission. Regardless of the actual benefits to individual patients, then, use of a vaccine on an individual level may be extremely beneficial on an epidemiological level, at the scale of a whole population.
[0012] Because of the current shortcomings of antiretroviral therapy, a continuing and unmet medical need exists for new, improved, and alternative therapeutic treatment modalities for HIV infection. Because of the negative aspects of NRTIs, a NRTI- sparing regimen that is able to control viral replication would be a very useful addition to the HIV treatment armamentarium. Such additions could include treatment vaccines that are designed to induce potent cytotoxic T-cell responses. The present invention addresses these and other unmet needs by providing a novel HIV therapeutic vaccine co-administered with an antiretroviral backbone monotherapy regimen for treatment of HIV infection.
[0013] Citation of the above documents or any references cited herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information
- 3 - sd-564347 available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
SUMMARY OF THE INVENTION
[0014] The present invention provides improved methods for therapeutic treatment of human immunodeficiency virus (HIV) infection. This invention more particularly relates to methods for treatment of HIV-infected host in need thereof comprising administration of an HIV vaccine in combination with an HIV inhibitor-based monotherapy.
[0015] In one aspect, the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in amounts sufficient to produce and maintain virologic suppressed status.
[0016] In another aspect, the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors.
[0017] In another aspect, the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non- nucleoside reverse transcriptase inhibitors.
[0018] In yet another aspect, the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV virologically suppressed host in need thereof a therapeutically effective amount of an HIV vaccine and one or more HIV protease inhibitors in amounts sufficient to maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic HIV vaccine and an HIV protease inhibitor(s) regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.
[0019] In another aspect, the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV virologically suppressed
- 4 - sd-564347 host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV CCR5 antagonist, a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor, or any combination thereof, in amounts sufficient to maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and a CCR5 antagonist, viral integrase inhibitor, maturation inhibitor, or viral fusion inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non-nucleoside reverse
transcriptase inhibitors.
[0020] In one embodiment of the method of the present invention, the HIV vaccine comprises a viral vector-based HIV vaccine, an HIV protein-based HIV vaccine, a plasmid DNA-based HIV vaccine, an anti-HIV antibody-based vaccine, or any combination thereof.
[0021] In one embodiment of the method of the present invention, the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines, or a combination thereof.
[0022] In certain embodiments of the aforementioned methods, the HIV monotherapy comprises a small molecule inhibitor, a protease inhibitor, an HIV CCR5 antagonist, a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor or a combination thereof.
[0023] In yet another embodiment of the method of the present invention, the protease inhibitor further comprises a booster agent.
[0024] In one embodiment of the methods of the present invention, the administration of the NRTI-sparing regimen comprising administration of an HIV vaccine plus an HIV replication inhibitor-based monotherapy may be for either "prophylactic" or "therapeutic" purpose, or for both purposes.
[0025] Thus, in one embodiment of the methods of the present invention, the administration of the NRTI-sparing regimen comprising administration of an HIV vaccine plus an HIV replication inhibitor-based monotherapy is used in a therapeutic context.
[0026] In another embodiment of the methods of the present invention, the administration of the NRTI-sparing regimen comprising administration of an HIV vaccine plus an HIV replication inhibitor-based monotherapy is used in a prophylactic context in either in pre-exposure (PrEP) or post-exposure prophylaxis (PEP) settings.
[0027] In one embodiment of the methods of the present invention, the NRTI- sparing regimen comprising administration of an HIV vaccine plus an HIV replication inhibitor-based monotherapy in such PrEP or PEP settings would be performed, for example,
- 5 - sd-564347 prior to the onset of any symptoms characteristic of the primary infection, before HIV becomes detectable by serologic assays, and in some cases even before the HIV
contamination event itself.
[0028] In yet another embodiment of the present invention, the HIV vaccine further comprises an adjuvant or immune modulator, or a combination thereof.
[0029] In one embodiment of the present invention, the pre-vaccination virological status of the host is achieved by administration of a combination of antiretrovirals (ARV) comprising a combination of drugs including two nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) with either a protease inhibitor (PI), or boosted protease inhibitor, or a non- nucleoside reverse transcriptase inhibitor (NNRTI).
[0030] In one embodiment of the present invention, the host is an HIV first line treatment patient, an HIV second line treatment patient, an HIV third line treatment patient, or those HIV patients that harbor multi-drug resistant viruses (HIV salvage patients), or those patients that have less than two viable treatment options left, or any combination thereof.
[0031] In one embodiment of the methods of the present invention, the plasma viral load in the patient so treated is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 copies per milliliter.
[0032] In yet another embodiment of the aforementioned methods of the present invention, the CD4+ T lymphocyte count in the patient so treated is greater than about 500, 600, 700, 800, 900, 1000 cells per milliliter.
[0033] These and other aspects of some exemplary embodiments will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments without departing from the spirit thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 depicts an illustrative HIV vaccine candidate and various analogs thereof (SEQ ID NOs:l-2). This candidate is an HIV based lentiviral vector (LV) using the native HIV LTR as promoter. It expresses gag, pol and rev genes as immunogenic payloads. An important feature of this LV resides in the engineering or placement of the RRE-containing sequence upstream of the full length Rev coding sequence. Another
- 6 - sd-564347 important feature of this LV is the presence of a splice acceptor (SA) site located between the RRE-containing sequence and the Rev coding sequence. Another important feature of this LV is the immediate proximity of the immunogenic payload(s) to the RRE-containing sequence. This LV optionally contains a non-protein coding genetic sequence (Gtag) to allow its identification and discrimination from wt-HIV in an HIV therapeutic vaccine setting. This sequence can be placed either upstream of downstream of the SA site. The five other schematic representations depicted in Figure 1 are non-limiting representative examples of possible LV vaccine vector analogs. The second construct shows that the RRE element can be placed upstream or downstream of the immunogenic payload(s). The third construct shows that the Gtag sequence can be disposable. The fourth construct shows that downstream of the sequence encoding for the Rev protein another genetic payload(s) can be placed, payloads able to encode for genetic adjuvant, other immunogens, RNA antisense, ribozymes, etc. The fifth construct is a combination of the schematic vector construct representations described supra. The final and sixth construct contains an additional element which can be introduced for an additional regulation of transgene expression where SD/SA sites will be optional. An internal promoter/Rev combination could be used in all above listed constructs.
[0035] FIG. 2 depicts a generic vaccine HIV-based lentiviral vector construct and analogs thereof (SEQ ID NOs:3-4). The generic vaccine vector is an HIV based lentiviral vector (LV) using the native HIV LTR as promoter. It contains the minimal elements for production, encapsidation and integration in transduced host cells (psi, cPPT/cts and ppt elements). As for the HIV vaccine vector, the important feature of this LV resides in the engineering or placement of the RRE-containing sequence upstream of the full length Rev coding sequence, the placement of the splice acceptor (SA) site between the RRE-containing sequence and the Rev coding sequence, and the immediate proximity of the immunogenic payload(s) to the RRE sequence. This LV contains a non-protein coding genetic sequence (Gtag) to allow its identification and discrimination from wt-HIV in an HIV therapeutic vaccine setting. This sequence can be placed either upstream of downstream of the SA site. The three other schematic representations are several non- limiting examples of possible LV generic vector analogs. The second construct shows that the RRE element can be placed upstream or downstream of the genetic payload(s). The third construct shows that the Gtag sequence can be disposable. The fourth construct shows that downstream of the sequence encoding for the Rev protein another genetic payload(s) can be placed, payloads able to encode for genetic adjuvant, other immunogens, RNA antisense, ribozymes, etc. The final and fifth construct shows a configuration with an internal promoter regulated Rev and
- 7 - sd-564347 adjoined gene expression. As with the last example in Figure 1, the presence of SD/SA sites could be optional.
[0036] FIG. 3 depicts HIV vaccine candidate and analogs using a SIN configuration (SEQ ID NOs:5-6). This figure present schematic representation of the HIV vaccine vector candidate (first from top) described in the application. This candidate is an HIV based lentiviral vector (LV) with the native HIV LTR promoter activity disrupted either by deletion, mutation or insertion of elements such as Insulators. This SIN based HIV vaccine LV expresses gag, pol and rev genes as immunogenic payloads. As for the HIV vaccine vector, the important feature of this LV resides in the engineering or placement of the RRE- containing sequence upstream of the full length Rev coding sequence, the placement of the splice acceptor (SA) site between the RRE-containing sequence and the Rev coding sequence, and the immediate proximity of the immunogenic payload(s) to the RRE sequence. This LV contains a non-protein coding genetic sequence (Gtag) to allow its identification and discrimination from wt-HIV in HIV therapeutic vaccine setting. This sequence can be placed either upstream of downstream of the SA site. The four other schematic representations are non- limiting examples of possible SIN-based LV vaccine vector analogs. The second construct shows that the RRE element can be placed upstream or downstream of the immunogenic payload(s). The third construct shows that the Gtag sequence can be disposable. The fourth construct from the top shows that downstream of the sequence encoding for the Rev protein another genetic payload(s) can be placed, payloads able to encode for genetic adjuvant, other immunogens, RNA antisense, ribozymes, etc. The final and fifth construct is a combination of the schematic vector construct representations described supra.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0037] SEQ ID NO: 1 is an example of an HIV vaccine vector of FIG. 1.
[0038] SEQ ID NO: 2 is an example of a vaccine vector of FIG. 1.
[0039] SEQ ID NO: 3 is an example of a DNA prime plasmid construct of
FIG. 2.
[0040] SEQ ID NO: 4 is an example of FIG. 2.
[0041] SEQ ID NO: 5 is an example of FIG. 3.
[0042] SEQ ID NO: 6 is an example of FIG. 3.
[0043] SEQ ID NO: 7 is an exemplary packaging signal sequence.
[0044] SEQ ID NO: 8 is an example of 5' LTR of HIV of vector pNL4-3.
[0045] SEQ ID NO: 9 is an example of 3' LTR of HIV of vector pNL4-3. sd-564347 [0046] SEQ ID NO: 10 is an example of
[0047] SEQ ID NO: 11 is an example of
[0048] SEQ ID NO: 12 is an example of
HIV vector pNL4-3.
[0049] SEQ ID NO: 13 is an example of
[0050] SEQ ID NO: 14 is an example of
[0051] SEQ ID NO: 15 is an example of
vector pNL4-3.
[0052] SEQ ID NO: 16 is an example of
sequence having a frameshift mutation.
[0053] SEQ ID NO: 17 is an example of
receptor glycine residue of Gag of HIV vector pNL4-3.
DETAILED DESCRIPTION OF THE INVENTION
[0054] DEFINITIONS
[0055] As used herein, "HIV vaccine" includes both HIV-l-based vaccines and HIV-2-based vaccines.
[0056] As used herein, "HIV infection" refers to indications of the presence of the HIV virus in an individual including asymptomatic seropositivity, aids-related complex (arc), and acquired immunodeficiency syndrome (AIDS).
[0057] As used herein, "HIV viral load" refers to the number of viral particles in a sample of blood plasma HIV viral load is increasingly employed as a surrogate marker for disease progression. It is measured by PCR and cDNA tests and is expressed in number of HIV copies or equivalents per millilitre.
[0058] As used herein, "in amounts sufficient to maintain virologic suppressed status" refers to the amount of HIV vaccine and/or HIV inhibitor administered either solely or in combination to achieve a suppressed viral load of less than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 copies per milliliter and a CD4+ T lymphocyte count of greater than about 500, 600, 700, 800, 900, 1000 cells per milliliter.
[0059] As used herein, the term "reverse transcriptase" or "RNA-dependent
DNA polymerase" refers to a DNA polymerase enzyme that transcribes single- stranded RNA into single-stranded DNA. Normal transcription involves the synthesis of RNA from DNA; hence, reverse transcription is the reverse of normal transcription. Reverse transcriptases are ubiquitous to retroviruses. Common examples include HIV reverse transcriptase, M-MLV
- 9 - sd-564347 reverse transcriptase from the Moloney murine leukemia virus, AMV reverse transcriptase from the avian myeloblastosis virus, among others.
[0060] As used herein, the term "nucleoside and nucleotide reverse transcriptase inhibitors" or "NRTI" refers to nucleosides, nucleotides, and analogues thereof which mimic natural nucleoside and nucleotide bases. When a retrovirus, such as HIV type 1, replicates its viral RNA using reverse transcriptase, these mimics compete with natural nucleoside and nucleotide base pairs for DNA elongation. Incorporation of one of these mimics results in DNA chain termination.
[0061] As used herein, the term "non-nucleoside reverse transcriptase inhibitors" or "NNRTI" refers to compounds which bind to a retrovirus reverse transcriptase, such as HIV type l's reverse transcriptase, and inhibits its enzymatic activity. The binding by the "NNRTI" causes a conformational shift in the reverse transcriptase which prevents the enzyme from binding nucleoside and nucleotide bases, resulting in DNA chain termination.
[0062] As used herein, the term "protease inhibitor" or "PI" refers to compounds which bind to active site of a retroviral protease enzyme, such as HIV's protease enzyme. The binding by the "PI" causes a conformational shift in the retroviral protease enzyme, making it no longer able to cleave large viral precursor proteins into smaller functional proteins. Viruses that are produced are defective and unable to infect other cells.
[0063] As used herein, the term "entry/fusion inhibitor" or "entry or fusion inhibitor" refers to compounds which interfere with the binding, fusion and entry of an HIV virion to a human cell. By blocking this step in HIV's replication cycle, such agents slow the progression from HIV infection to AIDS.
[0064] As used herein, the term "integrase inhibitor" refers to compounds which interfere with the action of integrase, an enzyme that integrates genetic material from the virus into the host's DNA. Integrase inhibitors are also called strand transfer inhibitors. Strand transfer refers to the process by which the viral DNA strands are transferred from the viral genome to the host genome. Integrase inhibitors may be taken in combination with other types of retroviral drugs to minimize adaptation by the virus.
[0065] As used herein, the term "maturation inhibitor" refers to compounds which interfere with the assembly and budding of virion particles, by binding to the viral gag polyprotein. The bound gag polyprotein can no longer be processed to functional subunits by viral protease enzymes. The resulting virus particles are structurally defective and are incapable of spreading infection.
- 10 - sd-564347 [0066] As used herein, the terms "inhibitor of the cytochromes P450" or
"inhibitor of CYP3A4" or "CYP 450 inhibitor" refer to any member of the class of pharmaceuticals and/or natural products which inhibit at least the CYP3A4 isoform of the cytochromes P450. The class includes, but is not limited to, amprenavir, atazanavir, cobicistat, clarithromycin, cyclosporin, diltiazem, erythromycin, itraconazole, indinavir, ketoconazole, mibefradil, nefazodone, nelfinavir, ritonavir, vitamin E, bergamottin, dihydroxybergamottin and grapefruit juice. See GK Dresser et al. Clin. Pharmacokinetics 2000 January; 38(1): 41-57 for a review of clinically-relevant CYP3A4 inhibitors. In the context of the present invention, the preferred inhibitor of CYP3A4 is ritonavir.
[0067] As used herein, the term "treatment" means the administration of the antivirally active compounds according to this invention in combination or alternation according to the present invention to alleviate or eliminate symptoms of the viral infection and/or to reduce viral load in a patient.
[0068] Generally, the invention relates to improved methods for therapeutic treatment of human immunodeficiency virus (HIV) infection, and more specifically to treatment of an HIV infection in a host comprising administration of an HIV vaccine in combination with an HIV inhibitor-based monotherapy.
[0069] A non-limiting description of HIV vaccines and HIV inhibitors that may be used in combination therapy to achieve and maintain an HIV virologic suppressed status.
HIV VACCINES
[0070] According to one aspect of the methods of the present invention, the treatment regimen comprises administration of an HIV vaccine wherein the HIV vaccine comprises an LV-based HIV vaccine, an MLV-based HIV vaccine, an adenovirus-based HIV vaccine, an AAV-based HIV vaccine, a vaccinia virus-based HIV vaccine, a HIV protein- based HIV vaccine, a plasmid DNA-based HIV vaccine, an anti-HIV antibody-based vaccine, or any combination thereof.
[0071] Thus, according to one embodiment of the methods of the present invention, the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines, or a combination thereof.
[0072] According to one embodiment of the methods of the present invention, the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines comprising Vacc-4x (BioNor Pharma peptide vaccine), AGS 004
- 11 - sd-564347 (Argos), FIT-06 (FIT Biotech), LC002 (Dermavir-DNA expressing all HIV proteins except integrase formulated to a mannosilated particle to target APC), MVA BN-nef (Bavarian Nordic, MVA vector encoding subtype B HIV-nef gene), MVA BN32 (Affitech A/S, MVA vector encoding multiple CTL epitopes), MVA mBN32 (Bavarian Nordic, MVA vector encoding multiple CTL epitopes), tat vaccine (Instituto Superiore di Sanita, recombinant protein), GTU-nef DNA (FIT Biotech, DNA encoding sub-type b nef gene), GX-12 (Pohang University of Science and Technology in South Korea), Genexine, Dong-A, DNA expressing pol gene and inactivates the gene responsible for repressing the immune system), or any combination thereof.
[0073] According to yet another embodiment of the methods of the present invention, the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines comprising HIV AX (Genecure, replication-defective lentiviral based vaccine), GeoVax MVA boost (GeoVax, DNA prime expressing gag, pol, env plus boost with recombinant MVA plus GCSF adjuvant), VIR201 (Virax, Pty, LTD., expresses highly conserved regions of HIV and interferon-gamma), Vacc-5q (Bionor Immuno, HIV/ AIDS therapeutic vaccine), DP6-001 (Cytrx, polyvalent DNA prime vaccine followed by protein boost), LC-002 (Derma Vir) (Research Institute for Genetic & Human Therapy (RIGHT), NIAID/ACTG, Genetic Immunity), MRKAd5 (Merck & Co., adenovirus- type 5 (Ad5) intramuscular HIV vaccine expressing gag, pol, and nef gene), Neovacs (interferon- alpha polyclonal antibody inducer for treatment of HIV/ AIDS), ALVAC
(vCP1452)(Sanofi-Aventis Pasteur, canarypox vector encoding env, gag, the protease- encoding portion of the pol gene and CTL epitopes from the nef and pol gene products), NYVAC-HIV (Sanofi-Aventis Pasteur, attenuated vaccinia vector expressing multiple HIV genes), Lipopeptides (Aventis Pasteur, peptides from Gag, Nef and Pol proteins), VRC- HIVDNA009-00-VP (VRC/NIAID, DNA vaccine encoding gag, pol, nef, and multigene (A, B, and C) env genes together with an adjuvant gene encoding IL-2 fusion protein),
Autologous dendritic cells pulsed with ALVAC (vCP1452)(ACTG, Aventis, canarypox vector encoding env, gag, the protease-encoding portion of the pol gene and CTL epitopes from the nef and pol gene products), HIV Autologous dendritic cell HIV vaccination with conserved HIV-derived peptides (University of Pittsburgh), or any combination thereof.
[0074] According to yet another embodiment of the methods of the present invention, the HIV vaccine comprises one or more commercially available or developmental stage HIV therapeutic vaccines comprising Multi-epitope DNA vaccine (Epimmune, 21 CTL epitopes and proprietary, non-HIV derived "universal" CD4 T cell epitpe), DNA/MVA
- 12 - sd-564347 vaccine (Cobra Pharmaceuticals, Impfstoffwerk Dessau- Tornau GmbH (DT), Oxford University/MRC, DNA vaccine and an MVA vector encoding gag and multiple CTL epitopes), Remune +/- AmpliVax (Immune Response Corporation, whole-killed subtype A/G recombinant HIV isolate depleted of gpl20), TBC-M358 (MVA)/TBC-M335 (MVA)/ TBC- F357 (FPV)/TBC-F349 (FPV)(NIAID/Therion, MVA and fowlpox vectors encoding env, gag, tat, rev, nef and reverse transcriptase genes from HIV subtype B), Alphavax injectable AIDS vaccine against HIV type C (Alphavax, alphavirus replicon vector vaccine), LFn-p24 (Avant Therapeutics), V-l Immunitor (Immunitor, oral vaccine comprising pooled HIV antigens for treatment and prophylaxis of HIV/ AIDS), Novartis second generation candidate vaccine (Novartis, Clade B gag DNA/PLG and env DNA/PLG microparticles to prime immune response, followed by booster of recombinant envelope glycoprotein gpl40 and MR59 adjuvant), GSK Protein HIV Vaccine (GlaxoSmithKline, recombinant Tat, Nef, and gpl20 proteins in AS02A adjuvant), VRC-HIVADV014-00-VP (VRC/NIAID, Adevnovirus serotype 5 vector containing gag, pol, and multigene (A, B, and C) env genes), DNA016- 00VP, VRC-HIVDNAO 16-00 VP (NIH Vaccine Research Center, six separate DNA plasmids containing gag, pol, and nef genes from HIV subtypes A, B, and C), HIV gag DNA Vaccine IL-15 DNA IL-12 DNA (Wyeth, DNA vaccines encoding HIV subtype B, IL-12 and IL-15 (all formulated with bupivacaine), HIV CTL MEP + RC529-SE and GM-CSF adjuvants (Wyeth, DNA vaccine containing CTL epitopes from env or gag), or GW825780
(GlaxoSmithKline, DNA vaccine encoding a fusion protein incorporating epitopes from RT, Gag, Nef (delivered coated onto gold particles via gene gun), or any combination thereof.
[0075] In another embodiment of the methods of the present invention, the vaccine based vectors of the present invention can comprise at least one, but can optionally comprise two or more nucleotide sequences of interest. In order for two or more nucleotide sequences of interest to be expressed, there may be two or more transcription units within the vector genome, one for each nucleotide sequences of interest. In those instances, it is preferable to use one or more internal ribosome entry sites (IRESs) or FMDV 2A-like sequences for translation of the second (and subsequent) coding sequence(s) in a poly- cistronic (or as used herein, "multicistronic") message (Adam et al 1991 J. Virol. 65, 4985, the entire contents of which are incorporated herein by reference). The IRES/2 A(s) may be of viral origin (such as EMCV IRES, PV IRES, or FMDV 2A-like sequences) or cellular origin (such as FGF2 IRES, NRF IRES, Notch 2 IRES or EIF4 IRES). Non-limiting examples of lentiviral vector constructs of the present invention that utilize an IRES sequence may be found in Figures 2 and 3 infra.
- 13 - sd-564347 [0076] Accordingly, in certain embodiments of the methods of the present invention, in addition to the HIV vaccine-based immunogenic or antigenic sequence, other nucleotide sequences of interest or "payload" sequences can also be included in the HIV vaccine and comprise such nucleotide sequences encoding enzymes, cytokines, chemokines, growth factors, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin- like molecules, a single chain antibody, fusion proteins, immune co-stimulatory molecules, immunomodulatory molecules, a transdominant negative mutant of a target protein, a toxin, a conditional toxin, an antigen, a tumor suppresser protein and growth factors, membrane proteins, pro- and anti- angiogenic proteins and peptides, vasoactive proteins and peptides, anti-viral proteins and derivatives thereof (such as with an associated reporter group). The nucleotide sequences of interest may also encode pro-drug activating enzymes. When used in a research context, the nucleotide sequences of interest may also encode reporter genes such as, but not limited to, green fluorescent protein (GFP), luciferase, beta-galactosidase, or resistance genes to antibiotics such as, for example, ampicillin, neomycin, bleomycin, zeocin, chloramphenicol, hygromycin, kanamycin, among others. The nucleotide sequences of interest may also include those which function as anti-sense RNA, small interfering RNA (siRNA), other non-coding RNAs or ribozymes, or any combination thereof.
[0077] In addition, the additional antigenic or immunogenic sequences contemplated for use in the HIV vaccines of the methods of the present invention may further comprise any tumor antigens as are known in the art. A tumor antigen is a protein or protein or peptide fragment thereof produced in tumor cells that triggers an immune response in the host. Tumor antigens are useful in identifying tumor cells and are potential candidates for use in cancer therapy. Tumor antigens may include alphafetoprotein, carcinoembryonic antigen, CA-125, epithelial tumor antigen, tyrosinase, melanoma associated antigen, by way of example. Also, another aspect of the method of the present invention is antigenic compounds, proteins and protein fragments associated with Alzheimer's disease.
[0078] In addition, the level of payload sequence or antigen expression can be increased by using codon-optimized genes as well. Synthetic genes, created with the codon bias found in highly expressed human genes, have been shown to be much more efficiently expressed in human cells and to elicit higher and more reproducible levels of immune responses than did native coding sequences. Another implication of over-expression is that the amount of a lentiviral vector or DNA prime that needs to be administered to elicit an immune response can be greatly reduced.
- 14 - sd-564347 [0079] In yet another embodiment of the present invention, the HIV vaccine further comprises an adjuvant or immune modulator comprising lipid-A portion of gram negative bacteria endotoxin, trehalose dimycolate of mycobacteria, the phospholipid lysolecithin, dimethyldictadecyl ammonium bromide (DDA), certain linear
polyoxypropylene-polyoxyethylene (POP-POE) block polymers, aluminum hydroxide, and liposomes, immunostimulants (e.g., various interleukins and cytokines that are known to enhance the immune response including GM-CSF, IL-2, IL-12, TNF and IFN.gamma.), toll like receptors, CpG, immunomodulators and/or antibiotics (e.g., antibacterial, antifingal, anti- pneumocysitis agents), or any combination thereof.
LENTIVIRAL VECTORS
[0080] According to one aspect of the invention, the lentiviral vectors contemplated for use as HIV vaccines in the methods of the present invention may include, without limitation, those lentiviruses that infect primates (HIV, HIV-2, simian
immunodeficiency virus (SIV)) and non-primates (feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), Bovine Immunodeficiency Virus (BIV), caprine arthritis encephalitis virus (CAEV), visna maedi virus (VV), Jembrana disease virus (JDV)).
[0081] In yet another embodiment, lentiviral vectors for use as vaccines in the methods of the present invention could be also modified by removing the transcriptional elements of HIV LTR; such as in a so-called self-inactivating (SIN) vector configuration. The modalities of reverse transcription, which generates both U3 regions of an integrated provirus from the 3 ' end of the viral genome, facilitate this task by allowing the creation of so-called self-inactivating (SIN) vectors. Self-inactivation relies on the introduction of a disruption (employing for example, deletion, mutation and element insertion) in the U3 region of the 3 ' long terminal repeat (LTR) of the DNA used to produce the vector RNA. During reverse transcription, this deletion is transferred to the 5' LTR of the pro viral DNA. If enough sequence is eliminated to abolish the transcriptional activity of the LTR, the production of full-length vector RNA in transduced cells is abolished. This minimizes the risk that replication competent lentiviruses (RCLs) will emerge. Furthermore, it reduces the likelihood that cellular coding sequences located adjacent to the vector integration site will be aberrantly expressed, either due to the promoter activity of the 3' LTR or through an enhancer effect. Finally, a potential transcriptional interference between the LTR and the internal promoter driving the transgene is prevented by the SIN design. One example of a SIN based lentiviral vector is described in United States Patent 6,924,144, the entire contents
- 15 - sd-564347 of which are incorporated herein by reference in its entirety. Non- limiting representative examples of SIN-based lentiviral vectors of the present invention may be generated from one or more of the constructs specifically shown in Figures 1, 2, and/or 3 described herein or any combination thereof.
[0082] In yet another embodiment of the present invention, the lentiviral vector vaccine constructs of the present invention further comprise those lentiviral vectors in which the lentiviral integrase function has been deleted and/or abrogated by site directed mutagenesis. Insertional mutagenesis has been observed in clinical trials with oncoretro viral vectors and this has prompted detailed study of genotoxicty of all integrating vectors. The most straightforward approach for several vaccine applications would be avoiding the possibility of integration. Non-integrating lentiviral vectors have been developed by mutating the integrase gene or by modifying the attachment sequences of the LTRs. In particular, among the mutations studied, the D64V substitution in the catalytic domain has been frequently used because it shows the strong inhibition of the integrase without affecting proviral DNA synthesis. It has been reported that the mutation allows a transduction efficiency only slightly lower than integrative vectors but a residual integration that is about 1000-fold lower than an integrative vector at low vector doses. Another mutation described, D116N, resulted in residual integration about 2000 times lower than control vectors. In a couple of instances it has been shown that a single administration of an integrase (IN)- defective SIN LV elicits a significant immune response in the absence of vector integration and may be a safe and useful strategy for vaccine development. Thus, specifically contemplated within the scope of this invention is the modification to render the lentiviral vectors able to exist in episomal form yet still being able to provide transgene expression. Non-limiting representative examples of Integrase-deficient SIN-based lentiviral vectors of the present invention may be generated from one or more of the constructs specifically shown in Figures 1, 2, and/or 3 described herein or any combination thereof.
[0083] In one embodiment, the HIV vaccine for use in the methods of the present invention comprises the lentiviral vector described in Assignee's co-pending PCT application (PCT Application No. PCT/US09/038535) which was published as PCT
Publication No. WO 2009/120947 on October 1, 2009, the contents of which are incorporated by reference herein in their entirety.
[0084] In one particular embodiment of the methods of the present invention, the HIV vaccine comprises a lentiviral vector comprising
a 5' long terminal repeat (LTR) and a 3' LTR;
- 16 - sd-564347 a first nucleic acid sequence operably linked to said 5' LTR;
a second nucleic acid sequence operably linked to said 5' LTR comprising a functional REV coding sequence and a rev response element (RRE)-containing sequence wherein the RRE- containing sequence is located upstream of the REV coding sequence; and wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR.
[0085] In yet another particular embodiment of the present invention, the lentiviral vector employed in the methods of the present invention comprises one or more of the lentiviral vector constructs depicted in Figure 1, Figure 2 or Figure 3, or any combination thereof.
[0086] In yet another embodiment, the lentiviral vector employed in the methods of the present invention comprises a lentiviral vector for vaccine delivery comprising a 5' long terminal repeat (LTR) (SEQ ID NO: 8) and a 3' LTR (SEQ ID NO: 9); a first nucleic acid sequence operably linked to said 5' LTR, also referred to herein as the "payload"; and a second nucleic acid sequence, that is operably linked to said 5' LTR comprising a functional REV coding sequence (SEQ ID NO: 10) and a rev response element (RRE)(SEQ ID NO: ll)-containing sequence wherein the RRE-containing sequence is located upstream of the REV coding sequence, and wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR. The present invention is characterized in that expression of the payload is driven by the 5 'LTR and also where expression of the payload depends on the activity of Rev and the RRE-containing sequence. In other words, the 5' LTR can be a powerful enough promoter to drive expression of the payload and REV.
[0087] In yet another embodiment, the lentiviral vectors employed in the methods of the present invention may comprise a 5' LTR from a lenti virus; a 3' LTR from a lentivirus; a first nucleic acid sequence, i.e., a payload; and a second nucleic acid sequence, that is operably linked to said 5' LTR an RRE-containing sequence and a REV coding sequence wherein the RRE-containing sequence is located upstream of the REV coding sequence, wherein the first nucleic acid sequence expresses the full length wild-type sequence of either one or both of Gag and Pol; and wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR.
[0088] In yet another embodiment, the lentiviral vectors employed in the methods of the present invention may comprise a 5' long terminal repeat (LTR) and a 3' LTR; a first nucleic acid sequence operably linked to said 5' LTR; and a second nucleic acid
- 17 - sd-564347 sequence that is operably linked to said 5 ' LTR comprising a functional REV coding sequence and a rev response element (RRE)-containing sequence, wherein the RRE- containing sequence is located upstream of the REV coding sequence, and wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR, and wherein the lentiviral vector construct may further comprise one or more of the known lentiviral regulatory elements such as Tat, Nef, Vif, Vpu, Vpr or a combination thereof.
[0089] In yet another embodiment, the lentiviral vectors employed in the methods of the present invention specifically exclude all known lentiviral regulatory elements such as Tat, Nef, Vif, Vpu, and Vpr. In such an embodiment, the nucleic acid sequence(s) encoding at least one of Vpu, Vpr, Vif, Tat, Nef, or analogous lentiviral proteins are disrupted such that the nucleic acid sequence(s) are incapable of encoding functional Vpu, Vpr, Vif, Tat, Nef, or the nucleic acid sequence(s) encoding Vpu, Vpr, Vif, Tat, Nef, or analogous auxiliary proteins are removed from the lentiviral vector system of the present invention.
[0090] In yet another embodiment of the methods of the present invention, the lentiviral vector further comprises a nucleic acid sequence encoding one or more functionally active lentiviral RNA packaging elements.
[0091] HIV and other lentiviruses, as are known in the art, have the unique property to replicate in non-dividing cells. This property relies on the use of a nuclear import pathway enabling the viral DNA to cross the nuclear membrane of the host cell. In HIV reverse transcription, a central strand displacement event consecutive to central initiation and termination of plus strand synthesis creates a plus strand overlap; the central DNA flap. This central DNA flap is a region of triple- stranded DNA created by two discrete half-genomic fragments with a central strand displacement event controlled in cis by a central polypurine tract (cPPT) and a central termination sequence (CTS) during HIV reverse transcription. A central copy of the polypurine tract ds-active sequence (cPPT), present in all lentiviral genomes, initiates synthesis of a downstream plus strand. The upstream plus strand segment initiated at the 3 ' PPT will, after a strand transfer, proceed until the center of the genome and terminate after a discrete strand displacement event. This last event of HIV reverse transcription is controlled by the central termination sequence (CTS).
[0092] Accordingly, in yet another embodiment of the methods of the present invention, the lentiviral vector further comprises a nucleic acid sequence encoding functional central polypurine tract (cPPT), and central termination sequence (cTS) (SEQ ID NO: 13), and 3' LTR proximal polypurine tract (PPT) (SEQ ID NO: 9) elements.
- 18 - sd-564347 [0093] The vector further comprises expression of said one or more sequences of interest which is dependent upon REV-RRE activity. It is a feature of the invention that Rev and RRE as expressed by the same 5' LTR and are required for the transport of the payload to the cytoplasm after translation in the cell nucleus. Rev is a small regulatory protein of HIV that is essential for virus replication. The biological role of Rev is to control the pattern of viral gene expression by promoting the transition from the early phase of infection, during which small regulatory proteins are expressed, to the late stage, when larger structural proteins are synthesized and assembled into viral particles. Rev binds to a highly structured RNA region, the Rev response element (RRE), where it forms an oligomeric ribonucleoprotein complex with about 8 to 10 Rev proteins binding to the RRE. The most important region of the RRE consists of five stem-loop domains radiating from a central junction, with the core element for Rev binding consisting of a stem loop- structure containing a purine rich bulge region (SEQ ID NO: 11). Thus, according to yet another embodiment of the methods of the present invention, expression of said one or more antigenic sequences of interest in the lentiviral vector depends on REV-RRE activity.
[0094] According to yet another embodiment of the method of the present invention, the first nucleic acid sequence comprises a Gag/Pol coding sequence or derivative thereof.
[0095] According to yet another embodiment of the methods of the present invention, the first nucleic acid sequence of the lentiviral vector encodes one or more antigenic sequences of interest. According to yet another embodiment of the methods of the present invention, the first nucleic sequence is an unmodified sequence.
[0096] According to yet another embodiment of the method of the present invention, the Gag/Pol coding sequence comprises a modified Gag/Pol coding sequence.
[0097] Thus, in one embodiment of the methods of the present invention, the lentiviral vector also supports a high level of expression of gag-pol fusion protein not mediated by translational frameshifting. Myristylation is a key component in the HIV particle assembly process. Gag p55 undergoes posttranslational myristylation at the N- terminus of matrix, which allows the Gag complex to attach to the cell membrane via the myristic acid moiety. Disruption of the myristylation site results in an accumulation of Gag proteins in the cytoplasm. The higher concentration of proteins within the cytoplasm most likely increases proteosomal degradation of these polypeptides into the endogenous antigen pathway, resulting in the loading of Gag peptides on MHC class I molecules leading to higher
- 19 - sd-564347 immune responses. Accordingly, the invention further comprises at least one nucleotide substitution which alters the myristylation receptor glycine residue of Gag (SEQ ID NO: 17).
[0098] According to yet another embodiment of the method of the present invention, the cPPT/cTS is a part of a Pol coding sequence of said Gag/Pol coding sequence.
[0099] According to yet another embodiment of the method of the present invention, the functionally active lentiviral RNA packaging elements comprise a Gag packaging sequence or derivative thereof.
[00100] In yet another embodiment of the method of the present invention, the lentiviral vector may further comprise a heterologous promoter located 3' of said RRE, wherein said heterologous promoter comprises a viral promoter, a human promoter, or a synthetic promoter, or a synthetic promoter, or a combination thereof.
[00101] The lentiviral vector for use in the methods of the present invention further comprises a nucleic acid sequence encoding functionally active lentiviral RNA packaging elements (SEQ ID NO: 12). The full-length lentiviral RNA is selectively incorporated into the viral particles as a non-covalent dimer. RNA packaging into virus particles is dependent upon specific interactions between RNA and the nucleocapsid protein (NC) domain of the Gag protein. In nature, incorporation of the HIV genomic RNA into the viral capsid (referred to as "encapsidation") involves the so-called Psi region located immediately upstream of the Gag start codon and folded into four stem- loop structures, is important for genome packaging; SL1 to SL4. In particular, SL1 contains the dimerization initiation site (DIS), a GC-rich loop that mediates in vitro RNA dimerization through kissing - complex formation, presumably a prerequisite for virion packaging of RNA. Additional cis- acting sequences have also been shown to contribute to RNA packaging. Some of these elements are located in the first 50 nucleotides (nt) of the Gag gene, including SL4, whereas others are located upstream of the splice-donor site (SD1), and are actually mapped to a larger region covering the first 350^400 nt of the genome, including about 240 nt upstream of SL1. The SL1-4 region is an example of a simple sequence essential for RNA packaging. Other such sequences are known by those of skill in the art.
[00102] It is an aspect of the present invention that the transcription of the payload as well as the Rev coding sequence and RRE-containing sequence is driven by the same promoter, i.e., the 5' LTR (SEQ ID NO: 8). The 5' LTR has sufficient basal activity to drive transcription of a payload comprising nucleic acids that encode full length antigenic sequences, as well as packaging sequences, and Rev coding sequence and RRE-containing sequence on the same transcription unit, with the proviso that the RRE-containing sequence is
- 20 - sd-564347 located upstream of the REV coding sequence. The 5' LTR can be derived from various strains and clades of HIV, as are known in the art, and optimized for stronger basal promoterlike function. In particular, the 5 ' LTR from HIV Clade E can exhibit strong basal promoter activity. Various strains and clades of HIV are known in the art and may be used to generate the lentiviral vaccine vectors of the present invention including for example, without limitation, HIV groups: M (for major)(A, B, C, D, E, F, G, H, I, and J), O (outlier or "outgroup"), which is a relatively rare group currently found in Cameroon, Gabon, and France, and a third group, designated N (new group), and any circulating recombinant forms thereof. The 5' LTR further drives expression of the payload and Rev proteins. The HIV Rev protein directs the export of unspliced or partially spliced viral transcripts from the nucleus to the cytoplasm in mammalian cells. Rev contains the RNA binding domain, which binds the RRE present on target transcripts. Export activity is mediated by a genetically defined effector domain, which has been identified as a nuclear export signal.
[00103] In yet another embodiment of the present invention, the heterologous promoter comprises viral, human, and/or synthetic promoters or a combination thereof. In one embodiment, heterologous viral promoters comprise Mouse Mammary Tumor Virus (MMTV) promoter, Moloney virus, avian leukosis virus (ALV), Cytomegalovirus (CMV) immediate early promoter/enhancer, Rous Sarcoma Virus (RSV), adeno-associated virus (AAV) promoters; adenoviral promoters, and Epstein Barr Virus (EBV) promoters, or any combination thereof. In one embodiment, heterologous human promoters comprise
Apolipoprotein E promoter, Albumin promoter, Human ubiquitin C promoter, human tissue specific promoters such as liver specific promoter, prostate specific antigen (psa) promoter, Human phosphoglycerate kinase (PGK) promoter, Elongation factor-1 alpha (EF-la) promoter, dectin-2 promoter, HLA-DR promoter, Human CD4 (hCD4) promoter, or any combination thereof. In yet another embodiment, the synthetic promoters comprise those promoters described in US Patent 6,072,050, the contents of which are incorporated by reference in its entirety.
[00104] It is yet another aspect of the methods of the present invention that various elements of the lentiviral vector construct may include functional equivalent derivatives, fragments or modifications thereof that have been engineered into the nucleotide coding sequences and/or amino acid sequences of the first nucleic acid sequence or the second nucleic acid sequence of the lentiviral vectors of the present invention. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural genotypic, allelic variation, or that have been artificially engineered, and that
- 21 - sd-564347 do not alter the functional activity are intended to be within the scope of the invention. Thus, functional equivalent derivatives, fragments or modifications thereof of the first nucleic acid sequence or the second nucleic acid sequence of the lentiviral vectors of the present invention can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of marker nucleic acids, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidinea), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
[00105] Thus, in one embodiment, the lentiviral vector for use in the methods of present invention comprises a packaging signal sequence as described in SEQ ID NO: 7, a 5' LTR of HIV vector pNL4-3 as described in SEQ ID NO: 8, a 3' LTR of HIV vector pNL4- 3 as described in SEQ ID NO: 9, a REV gene of HIV vector pNL4-3 as described in SEQ ID NO: 10, a sequence of functional RRE of HIV vector pNL4-3 as described in SEQ ID NO: 11, a minimum packaging sequence of HIV vector pNL4-3 as described in SEQ ID NO: 12, a cPPT/cTS of HIV vector pNL4-3 as described in SEQ ID NO: 13, PPT sequence of HIV vector pNL4-3 as described in SEQ ID NO: 14, a gag/pol coding sequence of HIV vector pNL4-3 as described in SEQ ID NO: 15 a frameshift modified gag/pol fusion coding sequence of HIV vector pNL4-3 as described in SEQ ID NO: 16, a myristylation receptor glycine residue altered Gag (SEQ ID NO: 17), and functionally active derivatives, fragments or mutations thereof wherein the derivatives, fragments or mutations can be from about 60%
- 22 - sd-564347 to about 100% (or any integer value thereof) homologous to SEQ ID NOS: 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or any combination thereof.
[00106] Finally, the lentiviral vectors contemplated for use in the methods of the present invention may be pseudotyped. "Pseudotyping" a virion is accomplished by co- transfecting a packaging cell with both the lentiviral vector of interest and a helper vector encoding at least one envelope protein of another virus or a cell surface molecule (see, for example, U.S. Patent Number 5,512,421, the entire text of which is herein incorporated by reference in its entirety). One viral envelope protein commonly used to pseudotype lentiviral vectors is the vesicular stomatitis virus-glycoprotein G (VSV-G), which is derived from a rhabdovirus. Other viral envelopes proteins that may be used include, for example, rabies virus-glycoprotein G and baculovirus gp-64. The use of pseudotyping broadens the host cell range of the lentiviral vector particle by including elements of the viral entry mechanism of the heterologous virus used. Pseudotyping of lentiviral vectors with, for example, VSV-G for use in the present invention results in lentiviral particles containing the lentiviral vector nucleic acid encapsulated in a nucleocapsid which is surrounded by a membrane containing the VSV-G envelope protein. The nucleocapsid preferably contains proteins normally associated with the lentiviral vector. The surrounding VSV-G protein containing membrane forms part of the viral particle upon its egress from the producer cell used to package the lentiviral vector. In an embodiment of the invention, the lentiviral particle is derived from HIV and pseudotyped with the VSV-G protein. Pseudotyped lentiviral particles containing the VSV-G protein can infect a diverse array of cell types with higher efficiency than amphotropic viral vectors. The range of host cells includes both mammalian and non- mammalian species, such as humans, rodents, fish, amphibians and insects.
[00107] Even though VSV-G pseudotyping has been described as being the most efficient for cutaneous transduction, a great advantage of using LV is that it is possible to target the vector to specific tissues or cells by replacing and/or modifying the virion envelope. LVs are remarkably compatible with a broad range of viral envelope glycoproteins providing them with added flexibility; Rabies, Mokola, LCMV, Ross River, Ebola, MuLV, Baculovirus GP64, HCV, Sindai virus F protein, Feline Endogenous Retrovirus RD114 modified, Human Endogenous Retroviruses, Seneca virus, GALV modified and HA influenza glycoproteins or a combination thereof, to name a few of those viral envelope glycoproteins explored. In addition to modification or replacement of the entire envelope, flexibility of LV platform for targeting different cell types is achieved by refining the surface of LV particles via the display of cell-specific ligands. For vaccine applications, VSV-G as a
- 23 - sd-564347 pseudotyping envelope confers some important advantages, such as a broad cellular tropism (including dendritic cells) and low preexisting immunity in the human population. VSV-G could eventually be replaced by other envelopes if needed, for example in the case of multiple vector administration, although anti- VSV-G immunity does not seem to prevent repeated vector administrations.
HIV INHIBITORS
[00108] In the methods of the present invention, the HIV inhibitor-based monotherapy comprises administration of a small molecule inhibitor, a protease inhibitor, an HIV CCR5 antagonist, a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor or a combination thereof.
[00109] In one embodiment of the present invention, the protease inhibitor contemplated for use in the methods of the present invention comprise indinavir, saquinavir, nelfinavir, darunavir, amprenavir, lopinavir, tripanivir, fosamprenavir or atazanavir, or any combination thereof.
[00110] In one embodiment of the present invention, the booster agent contemplated for use in the methods of the present invention comprise ritonovir or cobicistat, grapefruit juice extract, or a combination thereof.
[00111] In one embodiment of the present invention, the excluded
nucleoside/nucleotide reverse transcriptase inhibitors comprise nucleoside/nucleotide analogs including zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), emtricitabine, stavudine (d4T), lamivudine (3TC), abacavir (ABC), tenofovir, apricitabine, stampidine, elvucitabine, and racivir, or any combination thereof.
[00112] In yet another embodiment of the present invention, the non-nucleoside reverse transcriptase inhibitors contemplated for use in the methods of the present invention comprise avirenz, nevirapine, etravirine, rilpivirine, loviride, delavirdine, efavirenz, tenofovir DF (TDF), or any combination thereof.
[00113] In yet another embodiment of the present invention, the excluded non- nucleoside reverse transcriptase inhibitors comprises avirenz, nevirapine, etravirine, rilpivirine, loviride, delavirdine, efavirenz, tenofovir DF (TDF), or any combination thereof.
[00114] In one embodiment of the present invention, the CCR5 antagonist comprises INCB9471, maraviroc, SCH532702, TBR-652, or any combination thereof.
- 24 - sd-564347 [00115] In one embodiment of the present invention, the viral integrase inhibitor comprises elvitegravir, raltegravir, the second generation integrase inhibitor, MK- 2048, or any combination thereof.
[00116] In one embodiment of the present invention, the viral fusion inhibitor comprises enfuvirtide, maraviroc, PRO140, T-20, T-1249, vicriviroc, or any combination thereof.
[00117] In one embodiment of the present invention, the maturase inhibitor comprises bevirimat, vivecon, or any combination thereof.
METHODS
[00118] In its broadest aspect, the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in an amounts sufficient to produce and maintain virologic suppressed status.
[00119] In another aspect, the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV antiretroviral inhibitor monotherapy in an amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors.
[00120] In another aspect, the present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in an amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non- nucleoside reverse transcriptase inhibitors.
[00121] In certain embodiments of the aforementioned methods, the HIV monotherapy comprises a small molecule inhibitor, a protease inhibitor, an HIV CCR5 antagonist, a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor or a combination thereof.
[00122] The present invention provides for a method for treating an HIV infection comprising co-administering to an HIV virologically suppressed host in need thereof a therapeutically effective amount of an HIV vaccine and one or more HIV protease
- 25 - sd-564347 inhibitors in amounts sufficient to maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic HIV vaccine and an HIV protease inhibitor(s) regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non- nucleoside reverse transcriptase inhibitors.
[00123] The present invention provides for a method for treating an HIV infection comprising co-administering to an HIV-infected host in need thereof a
therapeutically effective amount of an HIV vaccine and an HIV CCR5 antagonist, a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor, or any combination thereof, in amounts sufficient to maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and a CCR5 antagonist, viral integrase inhibitor, maturation inhibitor, or viral fusion inhibitor regimen that specifically excludes
nucleoside/nucleotide reverse transcriptase inhibitors and non-nucleoside reverse
transcriptase inhibitors.
[00124] In yet another embodiment of the method of the present invention, a pharmaceutical composition is provided comprising the aforementioned lentiviral vector.
[00125] In yet another embodiment of the method of the present invention, a method for inducing an immune response in a subject comprising administering a pharmaceutical composition and a pharmaceutically acceptable carrier and/or a genetic adjuvant as are known in the art.
[00126] In yet another embodiment of the method of the present invention, the lentiviral vector expresses one or more genes of interest to potentiate immunity, and wherein said immune response comprises a humoral immune response, a cell mediated immune response or a combination thereof.
[00127] In yet another embodiment of the method of the present invention, the humoral immune response, cell-mediated immune response or a combination thereof is specific to a disease or condition of interest comprising cancer, Alzheimer's disease, autoimmune diseases, cardiovascular diseases, neurological diseases, fibrotic diseases, lipid metabolism diseases, extra-cellular matrix-related diseases, and chronic joint degenerative diseases, or any combination thereof.
[00128] In yet another embodiment of the method of the present invention, method of inhibiting or controlling the replication of an infective or replicative human immunodeficiency virus (HIV) in a mammal in need thereof comprising administering a pharmaceutical composition comprising one or more of the aforementioned vectors or
- 26 - sd-564347 lentiviral vectors, in combination with the aforementioned HIV inhibitor monotherapy regimen.
[00129] In yet another aspect of the method of the present invention, the invention provides for a method for inducing an immune response in a subject, said method comprising administering a pharmaceutical composition comprising one or more of the lentiviral vector HIV vaccines disclosed herein, by various administration routes and protocols, e.g. , DNA prime/lentiviral vector boost, DNA prime/two homologous lentiviral vector boosts, DNA prime/heterologous vector boost, lentiviral prime/lentiviral vector boost, lentiviral vector or DNA prime/lentiviral vector boost/adenoviral vector boost and vice versa, where the adenoviral vector is administered as the first boost and the lentiviral vector is administered as the second boost, each of the protocols being administered in combination with the aforementioned HIV inhibitor monotherapy regimen.
[00130] It is yet another embodiment of the method of the present invention to provide for a prime/boost or heterologous or homologous boost approach to increase the effectiveness and/or immunogenicity of a vector comprising administering a first vector (e.g., a nucleic acid plasmid construct, referred to as a DNA prime plasmid or using lentiviral vectors of the present invention, adenoviral-based vectors, pox-based vectors, including MVA and canary pox, VSV-based vectors, alphavirus-based vectors (e.g., VEE, Semliki Forest Virus), herpes virus-based vectors, among others known in the art) and subsequently administering a lentiviral vector of the present invention as a boost comprising the same payload, wherein one or both first and second vectors are the lentiviral vector of the present invention as a boost comprising the same payload as the DNA prime plasmid construct, or in some cases, a lentiviral vector of the present invention as the prime of a prime/boost protocol followed by a viral vector boost (for example, employing adenoviral-based vectors, pox- based vectors, including MVA and canary pox, CMV-based vectors, VSV-based vectors, alphavirus-based vectors (e.g., VEE, Semliki Forest Virus), herpes virus-based vectors), or vice versa, or any combinations thereof, each of the protocols being administered in combination with the aforementioned HIV inhibitor monotherapy regimen.
[00131] In certain embodiments of the methods of the present invention, the HIV vaccine may be targeted to a specific cell, for example, by means of one or more tissue specific promoters as described supra. As used herein, a cell can be present as a single entity, or can be part of a larger collection of cells. Such a "larger collection of cells" can comprise, for instance, a cell culture (either mixed or pure), a tissue (e.g. , endothelial, epithelial, mucosa or other tissue), an organ (e.g. , lung, liver, muscle and other organs), an organ system
- 27 - sd-564347 (e.g. , circulatory system, respiratory system, gastrointestinal system, urinary system, nervous system, integumentary system or other organ system), or an organism (e.g. , a bird, mammal, or the like). Preferably, the organs/tissues/cells being targeted are of the circulatory system (e.g. , including, but not limited to blood, including white blood cells), the mucosal system of the nose, trachea, bronchi, bronchioles, lungs, and the like), gastrointestinal system (e.g. , including mouth, pharynx, esophagus, stomach, intestines, salivary glands, pancreas, liver, gallbladder, and others), urinary system (e.g. , such as kidneys, ureters, urinary bladder, urethra, and the like), nervous system (e.g. , including, but not limited to, brain and spinal cord, and special sense organs, such as the eye) and integumentary system (e.g. , skin, epidermis, and cells of subcutaneous or dermal tissue). Even more preferably, the cells being targeted are selected from the group consisting of antigen presenting cells. Such an antigen presenting cell includes, but is not limited to, a skin fibroblast, a bowel epithelial cell, an endothelial cell, an epithelial cell, a dendritic cell, a plasmacytoid dendritic cell, Langerhan's cells, a monocyte, a mucosal cell and the like. The target cells need not be normal cells and can be diseased cells. Such diseased cells can be, but are not limited to, tumor cells, infected cells, genetically abnormal cells, or cells in proximity or contact to abnormal tissue such as tumor vascular endothelial cells.
[00132] In certain embodiments, the vector used in the methods of the present invention can be modified to include antigens from a variety of viruses and/or or bacteria in the payload to therapeutically treat said disease or as prophylaxis to said disease.
[00133] For example, and not by way of limitation, the vectors used in the methods of the present invention can be used to treat, in addition to HIV, a wide array of viral infections caused by various viruses including, but not limited to, viruses from the groups including dsDNA viruses (e.g. adenoviruses, herpesviruses, etc.); ssDNA viruses (e.g.
parvoviruses); dsRNA viruses (e.g. reoviruses, rotavirus); (+)ssRNA viruses (e.g.
picornavirus, togavirus etc); (-)ssRNA viruses (e.g. Orthomyxoviruses, Rhabdoviruses, etc.); ssRNA-RT viruses (e.g. Retroviruses, HIV, SIV, etc.); and dsDNA-RT viruses (e.g.
Hepadnaviruses).
[00134] The vectors used in the methods of the present invention can be used to treat, in addition to HIV, disease caused by viruses from a number of viral families including, but not limited to, Herpes Simplex Virus (HSV) - 1 (oral herpes simplex), HSV-2 (genital herpes simplex), Varicella Zoster virus (VZV) (chickenpox), Epstein-Barr virus
(EBV)(infectious mononucleosis), Cytomegalovirus (CMV) (Toxoplasmosis, Rubella, Herpes simplex), Pox virus (smallpox), and other viruses as are known in the art.
- 28 - sd-564347 [00135] The vectors used in the methods of the present invention can be used, in addition to treatment and management of HIV, to prophylatically or therapeutically treat various diseases including parasitic and microorganism ailments including, but not limited to, the disease malaria caused by mosquito transmission of the malarial parasite Plasmodium to humans. The disease is caused by protozoan parasites of the genus Plasmodium. Only four types of the Plasmodium parasite can infect humans. The most serious forms of the disease are caused by Plasmodium falciparum and Plasmodium vivax, but other related species (Plasmodium ovale, Plasmodium malariae) can also affect humans. Other diseases that can be treated by the method of the present invention are malaria, St. Louis encephalitis, dengue fever, yellow fever, West Nile virus; bubonic plague; Lyme disease, rocky mountain spotted fever, encephalitis; hantavirus; Chagas Disease, by way of example, not limitation.
[00136] Furthermore, the vectors used in the methods of the present invention can be used to treat, in addition to HIV, bacterial infections including, but not limited to, the following: human disease causing bacteria of the order Spirochaetales including T. pallidum (Syphillis), S. pilosicoli (diarrhea in humans especially in developing countries and in HIV patients), B. burgdorferi: (Lyme disease and related disorders), bacteria of the family
Spirillaceae including C. jenuni (colitis, diarrhea, enteritis, enterocolitis, gastroenteritis, etc.), H. pylori (gastritis, duodenal and peptic ulcers, Taynaud's phenomenon, etc.); bacteria of the Family Spirosomaceae including Pseudomonas (cellulitis, cerebrospinal fluid shunt infections, acute cystitis, endocarditis, chronic eye infections, neonatal meningitis, peritonitis, pneumonia), of the family Legionellaceae including Legionella (pneumonia in humans (legionellosis, legionnaire's disease, Pittsburgh pneumonia, nonpneumonic Pontiac fever, rhabdomyolysis, bacteraemia and septicemia, endocarditis, systemic infections in cell- mediated immunity disorders); bacteria Rhizobiacea including Agrobacterium (peritonitis in continuous ambulatory peritoneal dialysis); Achromobacter (postoperative and posttraumatic wound infectious complications); bacteria Shigella (watery diarrhea, dysentery); bacteria Salmonella (enteric fever, typhoid, paratyphoid, gastroenteritis, food poisoning, diarrhea and/or vomiting) and other disease causing bacteria as are known to those of skill in the art.
PHARMACEUTICAL COMPOSITIONS
[00137] The pharmaceutical compositions of the present invention contain a pharmaceutically and/or therapeutically effective amount of at least one nucleic acid construct, lentiviral vector, lentiviral vector system, viral particle/virus stock, or host cell (i.e., agents) of the invention. In one embodiment of the invention, the effective amount of an
- 29 - sd-564347 agent of the invention per unit dose is an amount sufficient to cause the detectable expression of the gene of interest. In another embodiment of the invention, the effective amount of agent per unit dose is an amount sufficient to prevent, treat or protect against deleterious effects (including severity, duration, or extent of symptoms) of the disease or condition being treated.
[00138] The administration of the pharmaceutical compositions of the invention may be for either "prophylactic" or "therapeutic" purpose. When provided prophylactically, the compositions are provided in advance of any symptom, in pre-exposure (PrEP) or post-exposure prophylaxis (PEP) settings. The prophylactic administration of the composition serves to prevent or ameliorate any subsequent deleterious effects (including severity, duration, or extent of symptoms) of the disease or condition being treated. The specific use of the invention is in the settings of PrEP or PEP, at which time a subject would seek administration of the composition shortly (within few days of exposure) before or after a potential exposure to HIV, through any possible route. The administration of the composition in such PrEP or PEP settings would in these cases be performed before the onset of any symptoms characteristic of the primary infection, before HIV becomes detectable by serologic assays, and in some cases even before the contamination event itself. When provided therapeutically, the composition is provided at (or shortly after) the onset of a symptom of the condition being treated.
[00139] In yet another embodiment of the present invention, for all therapeutic, prophylactic and diagnostic uses, one or more of the aforementioned HIV- vaccines, lentiviral vectors, lentiviral vector system, viral particle/virus stock, or host cell (i.e., agents) of the present invention, as well as other necessary reagents and appropriate devices and accessories, may be provided in kit form so as to be readily available and easily used. Such a kit would comprise a pharmaceutical composition for in vivo administration comprising an HIV vaccine of the present invention, and a pharmaceutically acceptable carrier and/or a genetic adjuvant; and instructions for use of the kit.
[00140] HIV vaccine formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which
- 30 - sd-564347 render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials. Extemporaneous injection solutions and suspensions may be prepared from purified nucleic acid preparations for the DNA plasmid priming compounds and/or purified viral vector compounds commonly used by one of ordinary skill in the art. Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations may also include other agents commonly used by one of ordinary skill in the art.
[00141] The HIV vaccine formulation may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, intranasal, intramuscular, subcutaneous, intravenous, intraperitoneal, intraocular, intracranial, intradermal, transdermal (skin patches), topical, or direct injection into a joint or other area of the subject's body. The HIV vaccine may likewise be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes. It is expected that from about one to about five dosages (e.g. , two dosages - an initial inoculation, the prime of prime/boost, and a booster) may be required per immunization protocol. The initial prime and boost administrations may contain a quantity of antigen sufficient to induce a satisfactory immune response. An appropriate quantity of prime and boost antigen(s) to be administered is determined for any of the prime/boost protocols disclosed herein by one skilled in the art based on a variety of physical characteristics of the subject or patient, including, for example, the patient's age, body mass index (weight), gender, health, immunocompetence, and the like. Similarly, the volume of administration will vary depending on the route of administration. By way of example, intramuscular injections may range from about 0.1 ml to 1.0 ml. Preferably a patient has a normal immune system and is not infected with human immunodeficiency virus, although the vaccine may also be administered after initial HIV infection to ameliorate disease progression, or after initial infection to treat AIDS.
[00142] The HIV vaccine may be stored at temperatures of from about -80°C to about 37°C or less, depending on how the vaccine is formulated. A variety of adjuvants known to one of ordinary skill in the art may be administered in conjunction with the viral vector in the HIV vaccine composition.
- 31 - sd-564347 [00143] Non-limiting examples of "Genetic adjuvants" can be used with the HIV vaccine vector of the present invention to increase or enhance the immune response elicited by expression of the antigenic sequence of interest of the present invention. These genetic adjuvants can be on different constructs but co-expressed or on the same vector construct yet contained in different expression cassettes. Such desirable genetic adjuvants may include, without limitation, toll like receptors, CpG, cytokines (e.g., the interleukins (ILs) IL-l.beta., 11-2, 11-10, 11-12, 11-15, CTA1-DD, fas antigen, flagellin, etc.), or combinations thereof. Other desirable genetic adjuvants include, without limitation, the DNA sequences encoding GM-CSF, the interferons (IFNs) (for example, IFN-. alpha., IFN-.beta., and IFN-. gamma.), TNF-. alpha., and combinations thereof. The genetic adjuvants may also be a immunostimulatory polypeptide from Parapox virus, such as a polypeptide of Parapox virus strain D1701 or NZ2 or Parapox immunostimulatory polypeptides B2WL or PP30 (see, e.g., U.S. Pat. No. 6,752,995). Still other such biologically active factors and
immunomodulators or immunostimulants that are known in the art that enhance the antigen- specific immune response may be readily selected by one of skill in the art, and a suitable plasmid vector containing the same factors constructed by known techniques.
[00144] One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired "effective level" in the individual patient. One skilled in the art also can readily determine and use an appropriate indicator of the "effective level" of the compounds of the present invention by a direct (e.g. , analytical chemical analysis) or indirect (e.g. , with surrogate indicators of viral infection, such as p24 or reverse transcriptase in the case of HIV infection) analysis of appropriate patient samples (e.g. , blood and/or tissues).
[00145] Further, with respect to determining the effective level in a patient for treatment of HIV, in particular, suitable animal models are available and have been widely implemented for evaluating the in vivo efficacy against HIV of various gene therapy protocols (Sarver et al. (1993) AIDS Research and Human Retroviruses, 9(5), 483-487; Sarver et al. (1993) Antisense Research and Development, 3(1), 87-94.). These models include mice, dogs, guinea pigs, monkeys, cats and rabbits. Even though these animals are not naturally susceptible to HIV disease, chimeric mice models (e.g. , SCID, bg/nu/xid, NOD/SCID, SCID-hu, immunocompetent SCID-hu, bone marrow- ablated BALB/c) reconstituted with human peripheral blood mononuclear cells (PBMCs), lymph nodes, fetal liver/thymus or other tissues can be infected with lentiviral vector or HIV, and employed as
- 32 - sd-564347 models for HIV pathogenesis and gene therapy. Similarly, the simian immune deficiency virus (SIV)/monkey model can be employed, as can the feline immune deficiency virus (FIV)/cat model. The pharmaceutical composition can contain other pharmaceuticals, in conjunction with a vector according to the invention, when used to therapeutically treat AIDS. These other pharmaceuticals can be used in their traditional fashion (i.e., as agents to treat HIV infection).
[00146] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. The contents of any patents, patent applications, patent publications, or scientific articles referenced anywhere in this application are herein incorporated in their entirety
EXAMPLES EXAMPLE 1
CLINICAL RATIONALE AND INDICATION FOR HIV THERAPEUTIC VACCINE IN
AN NRTI-SPARING REGIMEN
[00147] This example demonstrates that highly immunogenic lenti viral vectors used according to the methods of the present invention elicit long-term anti-HIV immunity in humans with control of viremia and maintenance of CD4+ T cell counts.
CLINICAL INDICATION FOR VRX1273
[00148] The VRX1273 clinical indication is for patients who have a clinical need to switch to an NRTI-sparing regimen due to adverse effects, intolerance or compliance and are either currently virologically suppressed or not virologically suppressed. VRX1273 may be co-administered with a boosted protease inhibitor (for example, boosted with ritonavir, cobicistat).
[00149] The initial therapeutic vaccine development strategy focuses on the vaccine as an NRTI-sparing agent. This clinical study strategy also incorporates the possibility of withdrawing or not withdrawing the boosted protease inhibitor monotherapy.
- 33 - sd-564347 PHASE I PROTOCOL SYNOPSIS
STUDY
[00150] A Phase I Open-label Study to Evaluate the Safety, Tolerability, and Immunogenicity of a Lentiviral Vector-based Vaccine, VRX1273, in Virologically
Suppressed HIV-infected Subjects on Boosted Protease Inhibitor Maintenance Monotherapy.
DESIGN
[00151] This Phase I study enrolls HIV-infected subjects with virological suppression for at least 6 months on a boosted protease inhibitor (LPV/r, ATV/r, or DRV/r)- based regimen containing 2 NRTIs. After screening tests are complete and all
inclusion/exclusion criteria are met, subjects enter STEP 1 at week 2 where they discontinue their NRTIs but remain on a maintenance regimen of the original boosted protease inhibitor (PI) that they were on for the duration of the vaccination. During the post-vaccination period, viral load, CD4+ counts and immunogenicity are assessed periodically. Furthermore, as part of the clinical trial protocol, contingencies are provided in the study to either remove the current boosted protease inhibitor monotherapy that the subject is on, or remove the monotherapy altogether at a time to be defined (treatment interruption). In this treatment interruption period, similarly, viral load and CD4+ counts are further assessed on a periodic basis thereafter.
CONCLUSION
[00152] Immunization with VRX1273 vaccine in combination with boosted protease inhibitor (ritonavir-boosted PI (ATV, LPV or DRV)) in an NRTI-sparing regiment result in effective treatment and management of HIV-1 as revealed by reduced viral load less than 200 copies per cell in treated patients and an CD4+ T cell count of greater than 500 cells/ml in patients receiving such treatment. The ultimate goal is to have a vaccine treatment regimen that is effective yet reduces drug toxicity exposure by sparing antiretroviral drugs in the HIV-1 treatment regimen.
EXAMPLE 2
[00153] Immunization with Bionor Immuno's Vacc-4x HIV peptide-based vaccine in combination with boosted protease inhibitor (for example, and not by way of limitation, ritonavir-boosted PI (ATV, LPV or DRV)) in an NRTI sparing regiment resulted
- 34 - sd-564347 in effective treatment of HIV as revealed by reduced viral load less than 200 copies per cell and an elevated CD4+ count of greater than 500/ml.
EXAMPLE 3
[00154] Immunization with FIT Biotech's DNA vaccine consisting of the clade B HIV nef gene using GTU nuclear anchoring technology for the treatment of HIV in combination with boosted protease inhibitor (for example, and not by way of limitation, ritonavir-boosted PI (ATV, LPV or DRV)) in an NRTI sparing regiment resulted in effective treatment of HIV as revealed by reduced viral load less than 200 copies per cell and an elevated CD4+ count of greater than 500/ml.
EXAMPLE 4
[00155] Immunization with Merck & Co.'s MRKAd5 (adenovirus-type 5 (Ad5) intramuscular vaccine expressing gag, pol, and nef gene) HIV vaccine in combination with boosted protease inhibitor (for example, and not by way of limitation, ritonavir-boosted PI (ATV, LPV or DRV)) in an NRTI sparing regiment resulted in effective treatment of HIV as revealed by reduced viral load less than 200 copies per cell and an elevated CD4+ count of greater than 500/ml.
[00156] One skilled in the art will appreciate that numerous equivalents of the foregoing materials and equipment are readily available and that these Examples may be modified in accordance with the principles hereof using no more than routine
experimentation. All references cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entireties, whether previously specifically incorporated or not.
[00157] The foregoing description of some specific embodiments provides sufficient information that others can, by applying current knowledge, readily modify or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. In the drawings and the description, there have been disclosed exemplary embodiments and, although specific terms may have been employed, they are unless otherwise stated used in a generic and descriptive sense only and not for purposes of limitation, the scope of the claims therefore not being so limited. Moreover, one
- 35 - sd-564347 skilled in the art will appreciate that certain steps of the methods discussed herein may be sequenced in alternative order or steps may be combined. Therefore, it is intended that the appended claims not be limited to the particular embodiment disclosed herein.
- 36 - sd-564347 REFERENCES
1. UNAIDS/WHO, AIDS epidemic update. 2009.
2. CDC, HIV/AIDS surveillance report. 2007.
3. Vigouroux, C, et al., Adverse metabolic disorders during highly active antiretroviral treatments (HAART) of HIV disease. Diabetes Metab, 1999. 25(5): p. 383-92.
4. Guidelines for the use of antiretroviral agents in HIV- 1 infected adults and adolescents, HHS, Editor. 2009.
5. Behrens, G.M., et al., Clinical impact of HIV-related lipodystrophy and metabolic abnormalities on cardiovascular disease. Aids, 2003. 17 Suppl 1: p. S149-54.
6. Behrens, G.M., M. Stoll, and R.E. Schmidt, Lipodystrophy syndrome in HIV infection: what is it, what causes it and how can it be managed? Drug Saf, 2000. 23(1): p. 57- 76.
7. Powderly, W.G., Long-term exposure to lifelong therapies. J Acquir Immune Defic Syndr, 2002. 29 Suppl 1: p. S28-40.
8. Lucas, G.M., R.E. Chaisson, and R.D. Moore, Highly active antiretroviral therapy in a large urban clinic: risk factors for virologic failure and adverse drug reactions. Ann Intern Med, 1999. 131(2): p. 81-7.
9. Max, B. and R. Sherer, Management of the adverse effects of antiretroviral therapy and medication adherence. Clin Infect Dis, 2000. 30 Suppl 2: p. S96-116.
10. Nijhuis, M., S. Deeks, and C. Boucher, Implications of antiretroviral resistance on viral fitness. Curr Opin Infect Dis, 2001. 14(1): p. 23-8.
11. Services, U.D.o.H.a.H., Guidelines for the use of antiretroviral agents in HIV-1 infected adults and adolescents. 2009.
12. de Mendoza, C, O. Gallego, and V. Soriano, Mechanisms of resistance to antiretroviral drugs-clinical implications. AIDS Rev, 2002. 4(2): p. 64-82.
13. Whitcomb, J.M., et al., Broad nucleoside reverse-transcriptase inhibitor cross- resistance in human immunodeficiency virus type 1 clinical isolates. J Infect Dis, 2003. 188(7): p. 992-1000.
14. Lewis, W. and M.C. Dalakas, Mitochondrial toxicity of antiviral drugs. Nat Med, 1995. 1(5): p. 417-22.
- 37 - sd-564347 15. Shirasaka, T., et al., Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides. Proc Natl Acad Sci U S A, 1995. 92(6): p. 2398-402.
16. Simon, D.K. and D.R. Johns, Mitochondrial disorders: clinical and genetic features. Annu Rev Med, 1999. 50: p. 111-27.
17. Anderson, P.L., T.N. Kakuda, and K.A. Lichtenstein, The cellular pharmacology of nucleoside- and nucleotide- analogue reverse-transcriptase inhibitors and its relationship to clinical toxicities. Clin Infect Dis, 2004. 38(5): p. 743-53.
18. Chen, C.H., M. Vazquez-Padua, and Y.C. Cheng, Effect of anti-human
immunodeficiency virus nucleoside analogs on mitochondrial DNA and its implication for delayed toxicity. Mol Pharmacol, 1991. 39(5): p. 625-8.
19. Kakuda, T.N., Pharmacology of nucleoside and nucleotide reverse transcriptase inhibitor-induced mitochondrial toxicity. Clin Ther, 2000. 22(6): p. 685-708.
20. Pan-Zhou, X.R., et al., Differential effects of antiretroviral nucleoside analogs on mitochondrial function in HepG2 cells. Antimicrob Agents Chemother, 2000. 44(3): p. 496- 503.
21. Borrow, P., et al., Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol, 1994. 68(9): p. 6103-10.
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- 38 - sd-564347

Claims

CLAIMS What is claimed is:
1. A method for treating an HIV infection comprising co-administering to an HIV- infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in amounts sufficient to produce and maintain virologic suppressed status.
2. A method for treating an HIV infection comprising co-administering to an HIV- infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors.
3. A method for treating an HIV infection comprising co-administering to an HIV- infected host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV inhibitor monotherapy in amounts sufficient to produce and maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and an HIV inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.
4. A method for treating an HIV infection comprising co-administering to an HIV virologically suppressed host in need thereof a therapeutically effective amount of an HIV vaccine and one or more HIV protease inhibitors in amounts sufficient to maintain virologic suppressed status, wherein the treatment regimen comprises a therapeutic HIV vaccine and an HIV protease inhibitor(s) regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.
5. A method for treating an HIV infection comprising co-administering to an HIV virologically suppressed host in need thereof a therapeutically effective amount of an HIV vaccine and an HIV CCR5 antagonist, a viral integrase inhibitor, a maturation inhibitor, or a viral fusion inhibitor, or any combination thereof, in amounts sufficient to maintain virologic
- 39 - sd-564347 suppressed status, wherein the treatment regimen comprises a therapeutic vaccine and a CCR5 antagonist, viral integrase inhibitor, maturation inhibitor, or viral fusion inhibitor regimen that specifically excludes nucleoside/nucleotide reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.
6. The method according to Claim 1, 2, 3, 4, or 5, wherein the HIV vaccine comprises a n LV -based HIV vaccine, an MLV-based HIV vaccine, an adenovirus-based HIV vaccine, an AAV-based HIV vaccine, a CMV-based HIV vaccine, a vaccinia virus-based HIV vaccine, a HIV protein-based HIV vaccine, a plasmid DNA-based HIV vaccine, or any combination thereof.
7. The method according to Claim 1, 2, 3, 4, or 5, wherein the protease inhibitor comprises indinavir, saquinavir, nelfinavir, darunavir, amprenavir, lopinavir, tripanivir, fosamprenavir or atazanavir, or any combination thereof.
8. The method according to Claim 7, wherein the protease inhibitor further comprises a booster agent.
9. The method according to Claim 8, wherein the booster agent comprises ritonovir or cobicistat, grapefruit juice extract, or a combination thereof.
10. The method according to Claim 1, 2, 3, 4, or 5, wherein the excluded
nucleoside/nucleotide reverse transcriptase inhibitors comprise nucleoside/nucleotide analogs including zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), emtricitabine, stavudine (d4T), lamivudine (3TC), abacavir (ABC), tenofovir, apricitabine, stampidine, elvucitabine, and racivir, or any combination thereof.
11. The method according to Claim 4 or 5, wherein the excluded non-nucleoside reverse transcriptase inhibitors comprises avirenz, nevirapine, etravirine, rilpivirine, loviride, delavirdine; efavirenz, tenofovir DF (TDF), or any combination thereof.
12. The method according to Claim 5, wherein the CCR5 antagonist comprises
INCB9471, maraviroc, SCH532702, TBR-652, or any combination thereof.
- 40 - sd-564347
13. The method according to Claim 5, wherein the viral integrase inhibitor comprises elvitegravir, raltegravir, the second generation integrase inhibitor, MK-2048, or any combination thereof.
14. The method according to Claim 5, wherein the viral fusion inhibitor comprises enfuvirtide, maraviroc, PRO140, T-20, T-1249, vicriviroc, or any combination thereof.
15. The method according to Claim 5, wherein the maturase inhibitor comprises bevirimat, vivecon, or any combination thereof.
16. The method according to Claim 4 or 5, wherein the HIV vaccine further comprises an adjuvant, immunostimulants (e.g., various interleukins and cytokines), immunomodulators and antibiotics (e.g., antibacterial, antifingal, anti-pneumocysitis agents).
17. The method according to Claim 4 or 5, wherein the pre-vaccination virological status of the host is achieved by administration of a combination of antiretrovirals (ARV) comprising a combination of drugs including two nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) with either a protease inhibitor (PI), or boosted protease inhibitor, or a non- nucleoside reverse transcriptase inhibitor (NNRTI).
18. The method according to Claim 1, 2, 3, 4 or 5, wherein the host is an HIV first line treatment patient, an HIV second line treatment patient, an HIV third line treatment patient, or those HIV patients that harbor multi-drug resistant viruses (HIV salvage patients), or those patients that have less than two viable treatment options left, or any combination thereof.
19. The method according to Claim 1, 2, 3, 4 or 5, wherein the HIV vaccine comprises commercially available or developmental stage HIV therapeutic vaccines comprising Vacc- 4x (BioNor peptide vaccine), AGS 004 (Argos), FIT-06 (FIT Biotech), LC002 (Dermavir- DNA expressing all HIV proteins except integrase formulated to a mannosilated particle to target APC), MVA BN-nef (Bavarian Nordic, MVA vector encoding subtype B HIV-nef gene), MVA BN32 (Affitech A/S, MVA vector encoding multiple CTL epitopes), MVA mBN32 (Bavarian Nordic, MVA vector encoding multiple CTL epitopes), tat vaccine (Istituto Superiore di Sanita, recombinant protein), GTU-nef DNA (FIT Biotech, DNA encoding sub-type b nef gene), GX-12 (Pohang University of Science and Technology in
- 41 - sd-564347 South Korea, Genexine, Dong-A, DNA expressing pol gene and inactivates the gene responsible for repressing the immune system), HIV AX (Genecure, replication-defective lentiviral based vaccine), GeoVax MVA boost (GeoVax, DNA prime expressing gag, pol, env plus boost with recombinant MVA plus GCSF adjuvant), VIR201 (Virax, Pty, LTD., expresses highly conserved regions of HIV and interferon-gamma), Vacc-5q (Bionor Immuno, HIV/ AIDS therapeutic vaccine), DP6-001 (Cytrx, polyvalent DNA prime vaccine followed by protein boost), LC-002 (Derma Vir) (Research Institute for Genetic & Human Therapy (RIGHT), NIAID/ACTG, Genetic Immunity), MRKAd5 (Merck & Co., adenovirus- type 5 (Ad5) intramuscular vaccine expressing gag, pol, and nef gene), Neovacs (interferon- alpha polyclonal antibody inducer for treatment of HIV/AIDS), ALVAC (vCP1452)(Sanofi- Aventis Pasteur, canarypox vector encoding env, gag, the protease-encoding portion of the pol gene and CTL epitopes from the nef and pol gene products), NYVAC-HIV (Sanofi- Aventis Pasteur, attenuated vaccinia vector expressing multiple HIV genes), VILipopeptides (Aventis Pasteur, peptides from Gag, Nef and Pol proteins), VRC-HIVDNA009-00-VP (VRC/NIAID, DNA vaccine encoding gag, pol, nef, and multigene (A, B, and C) env genes together with an adjuvant gene encoding IL-2 fusion protein), Autologous dendritic cells pulsed with ALVAC (vCP1452)(ACTG, Aventis, canarypox vector encoding env, gag, the protease-encoding portion of the pol gene and CTL epitopes from the nef and pol gene products), HIV Autologous dendritic cell HIV vaccination with conserved HIV-derived peptides (University of Pittsburgh), Multi-epitope DNA.(Epimmune, 21 CTL epitopes and proprietary, non-HIV derived "universal" CD4 T cell epitpe), DNA/MVA (Cobra
Pharmaceuticals, Impfstoffwerk Dessau-Tornau GmbH (DT), Oxford University/MRC, DNA vaccine and an MVA vector encoding gag and multiple CTL epitopes), Remune +/- AmpliVax (Immune Response Corporation, whole -killed subtype A/G recombinant HIV isolate depleted of gpl20), TBC-M358 (MVA)/TBC-M335 (MVA)/ TBC-F357 (FPV)/TBC- F349 (FPV)(NIAID/Therion, MVA and fowlpox vectors encoding env, gag, tat, rev, nef and reverse transcriptase genes from HIV subtype B), Alphavax injectable AIDS vaccine against HIV type C (Alphavax, alphavirus replicon vector vaccine), LFn-p24 (Avant Therapeutics), V-l Immunitor (Immunitor, oral vaccine comprising pooled HIV antigens for treatment and prophylaxis of HIV/ AIDS), Novartis second generation candidate vaccine (Novartis, Clade B gag DNA/PLG and env DNA/PLG microparticles to prime immune response, followed by booster of recombinant envelope glycoprotein gpl40 and MR59 adjuvant), GSK Protein HIV Vaccine (GlaxoSmithKline, Marcus Atfield, M.D., Ph.D., recombinant Tat, Nef, and gpl20 proteins in AS02A adjuvant), VRC-HIVADV014-00-VP (VRC/NIAID, Adevnovirus
- 42 - sd-564347 serotype 5 vector containing gag, pol, and multigene (A, B, and C) env genes), DNA016- 00 VP, VRC-HIVDNAO 16-00 VP (NIH Vaccine Research Center, six separate DNA plasmids containing gag, pol, and nef genes from HIV subtypes A, B, and C), HIV gag DNA Vaccine IL-15 DNA IL-12 DNA (Wyeth, DNA vaccines encoding HIV subtype B, IL-12 and IL-15 (all formulated with bupivacaine), HIV CTL MEP + RC529-SE and GM-CSF adjuvants (Wyeth, DNA vaccine containing CTL epitopes from env or gag), or GW825780
(GlaxoSmithKline, DNA vaccine encoding a fusion protein incorporating epitopes from RT, Gag, Nef (delivered coated onto gold particles via gene gun), or any combination thereof.
20. The method according to Claim 6, wherein the HIV vaccine comprises a lentiviral vector comprising
a 5' long terminal repeat (LTR) and a 3' LTR;
a first nucleic acid sequence operably linked to said 5 ' LTR;
a second nucleic acid sequence operably linked to said 5' LTR comprising a functional REV coding sequence and a rev response element (RRE)-containing sequence wherein the RRE-containing sequence is located upstream of the REV coding sequence; and wherein transcription of said first nucleic acid sequence and said second nucleic acid sequence is driven by said 5' LTR.
21. The vector of claim 20, further comprising a nucleic acid sequence encoding one or more functionally active lentiviral RNA packaging elements.
22. The vector of claim 20, further comprising a nucleic acid sequence encoding functional central polypurine tract (cPPT), and central termination sequence (cTS), and 3' LTR proximal polypurine tract (PPT) elements.
23. The vector of claim 20, wherein said first nucleic acid sequence encodes one or more antigenic sequences of interest.
24. The vector of claim 23, wherein expression of said one or more antigenic sequences of interest depends on REV-RRE activity.
25. The vector of claim 20, wherein said first nucleic acid sequence comprises a Gag/Pol coding sequence or derivative thereof.
- 43 - sd-564347
26. The vector of claim 25, wherein said first nucleic sequence is an unmodified sequence.
27. The vector of claim 25, wherein said Gag/Pol coding sequence comprises a modified Gag/Pol coding sequence.
28. The vector of claim 25, wherein said cPPT/cTS is a part of a Pol coding sequence of said Gag/Pol coding sequence.
29. The vector of claim 21, wherein said functionally active lenti viral RNA packaging elements comprise a Gag packaging sequence or derivative thereof.
30. The vector of claim 23, further comprising a heterologous promoter located 3' of said RRE, wherein said heterologous promoter comprises a viral promoter, a human promoter, or a synthetic promoter, a tissue-specific promoter, or a combination thereof.
31. A pharmaceutical composition comprising a lentiviral vector according to claim 20.
32. A method for inducing an immune response in a subject comprising administering a pharmaceutical composition according to claim 31.
33. The method according to claim 32, wherein said lentiviral vector expresses one or more genes of interest to potentiate immunity, and wherein said immune response comprises a humoral immune response, a cell mediated immune response or a combination thereof.
34. The method according to claim 33 wherein said humoral immune response, cell mediated immune response or a combination thereof is specific to a disease or condition of interest comprising cancer, Alzheimer's disease, autoimmune diseases, cardiovascular diseases, neurological diseases, fibrotic diseases, lipid metabolism diseases, extra-cellular matrix-related diseases, and chronic joint degenerative diseases, or any combination thereof.
- 44 - sd-564347
35. A method of increasing the immunogenicity of a vector in a host cell comprising administering a first vector, followed by one or more sequential administrations of a lentiviral vector according to claim 20.
36. A method of inhibiting or controlling the replication of an infective, or replicative human immunodeficiency virus (HIV) in a mammal in need thereof comprising
administering a pharmaceutical composition comprising a lentiviral vector according to claim 20.
37. A method of increasing immunogenicity of a vector in a subject in need thereof comprising
a) administering a prime; and
b) sequentially administering a boost,
wherein at least one of said prime or said boost comprises a lentiviral vector according to claim 20.
38. The method wherein the plasma viral load in the patient so treated is less than 200 copies per milliliter and CD4 counts are greater than 500 cells per mm3.
- 45 - sd-564347
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