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WO2004047861A1 - Vaccins contre des virus a substances cationiques utilisees en tant qu'additifs - Google Patents

Vaccins contre des virus a substances cationiques utilisees en tant qu'additifs Download PDF

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Publication number
WO2004047861A1
WO2004047861A1 PCT/EP2003/013349 EP0313349W WO2004047861A1 WO 2004047861 A1 WO2004047861 A1 WO 2004047861A1 EP 0313349 W EP0313349 W EP 0313349W WO 2004047861 A1 WO2004047861 A1 WO 2004047861A1
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Prior art keywords
hiv
vaccine according
expression vectors
dna
nucleotide sequence
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PCT/EP2003/013349
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English (en)
Inventor
Ulf SCHRÖDER
Jorma Hinkula
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Eurocine Ab
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Priority to AU2003292136A priority Critical patent/AU2003292136A1/en
Publication of WO2004047861A1 publication Critical patent/WO2004047861A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention provides a vaccine against a virus, such as, e.g. retro virus, the vaccine comprising at least two separate components, whereof at least one is a polynucleotide. Furthermore, at least one of the components comprises a novel adjuvant.
  • HIV-1 human immunodeficiency virus type 1
  • HIV-vaccines have been based on live attenuated or mutated non-pathogenic lentiviruses such as Simian/Human Immunodeficiency Virus (SHIV), Feline Immunodeficiency Virus ( FIV) and Simian Immunodeficiency Virus
  • SHIV Simian/Human Immunodeficiency Virus
  • FIV Feline Immunodeficiency Virus
  • Simian Immunodeficiency Virus Simian/Human Immunodeficiency Virus
  • FIV Feline Immunodeficiency Virus
  • Simian Immunodeficiency Virus Simian/Human Immunodeficiency Virus
  • the attraction in the DNA vaccine approach lies in its simplicity, its stability, the ease with which it can be modified and its expected low cost for large scale production.
  • the similarity with the attenuated, live vaccines is that the person vaccinated with a DNA- vaccine becomes his/her own endogenous vaccine antigen producer, thus inducing both a cell-mediated and a humoral immune response. This has been shown in small animal models, such as in mice, rats and chickens, with very good results, but so far less efficacy has been shown in larger animals and in man.
  • the DNA vaccine prime have so far shown to stimulate immunity towards correctly folded and presented antigens, in contrast to many classical protein/adjuvant vaccines.
  • the problem has been the lack of an effective adjuvant to combine with the DNA.
  • a good solution to the problem was seen when a DNA vaccinated individual obtained a booster immunization with a recombinant microorganism (Modified Vaccinia Virus Ankara (MVA), Semliki Forrest Virus (SFV), Salmonella) or a protein, such as a recombinant subunit protein or a peptide. Under these circumstances the immune response was greatly enhanced.
  • a recombinant microorganism Modified Vaccinia Virus Ankara (MVA), Semliki Forrest Virus (SFV), Salmonella
  • MVA Modified Vaccinia Virus Ankara
  • SFV Semliki Forrest Virus
  • Salmonella Salmonella
  • a protein such as a recombinant subunit protein or a peptide.
  • proteins may be the immune responses towards the carrier or the vector proteins, which may disturb the immunity against the vaccine antigen.
  • protein antigens are often temperature- sensitive and may have limited shelf or storage lifetime.
  • the present invention provides a vaccine against virus, wherein the antigens may be peptides and/or nucleotides. More specifically, the present invention relates to a vaccine against virus such as, e.g., retro virus including lenti virus, such as, e.g. HIV or SIV, the vaccine comprising at least two separate components, a first immunizing component comprising
  • DNA expression vectors comprising a nucleotide sequence encoding a viral protein or fragments thereof and/or one or more polypeptides encoded by at least part of the nucleotide
  • a second component comprising b) one or more DNA expression vectors comprising a nucleotide sequence encoding a viral protein or fragments thereof and/or one or more polypeptides encoded by at least part of the nucleotide
  • first and the second component are dispersed in a first and second medium, respectively, and the first and second medium comprise an adjuvant, same or different, at least one of the adjuvants being selected from cationic substances such as, e.g., acyl amines comprising from 4 to 30 carbon atoms, quaternary ammonium compounds derived from acyl amines, cationic acyl amides, amino acids conjugated to an acyl group, etc., and mixtures thereof.
  • cationic substances such as, e.g., acyl amines comprising from 4 to 30 carbon atoms, quaternary ammonium compounds derived from acyl amines, cationic acyl amides, amino acids conjugated to an acyl group, etc., and mixtures thereof.
  • One embodiment of the invention relates to a vaccine, wherein the first immunizing component comprises
  • DNA expression vectors comprising a nucleotide sequence encoding a viral protein or fragments thereof
  • the second component comprises b) one or more polypeptides encoded by at least part of the nucleotide.
  • first and the second components are dispersed in a first and second medium as described above.
  • acyl or “acyl group” encompasses natural or synthetic, branched or unbranched, cyclic or acyclic, substituted or unsubstituted acyl, alkyl, alkenyl and alkynyl chains of from 4 to 30 carbon atoms, such as, e.g., from 6 to 24 carbon atoms, from 8 to 20 carbon atoms or from 12 to 20 carbon atoms.
  • acyl amine encompasses acyl, alkyl, alkenyl or alkynyl chains onto which an amine group has been attached.
  • the term "antigen" is defined as anything that can serve as a target for an immune response.
  • the immune response can be cellular or humoral and be detected in systemic and/or mucosal compartments.
  • adjuvant is a substance that stimulates and prolongs the immune response when injected together with an antigen.
  • retrovirus covers any retrovirus including HIV-1 , HIV-2, SIV and Human T- cell Lymphotrophic virus (HTLV).
  • HIV means the human immunodeficiency virus. The term is intended to cover all clades and isolates of HIV-1 and HIV-2.
  • gp41 means the gp41 transmembrane HIV envelope glycoprotein and any fragments or variants, alleles, analogs and derivatives thereof.
  • gp41 nucleotide is intended to mean a nucleotide encoding the gp41 protein or fragments thereof.
  • glycoprotein 120 is intended to mean glycoprotein 120, a protein that protrudes from the surface of HIV and binds to CD4+ T cells.
  • glycoprotein 160 is intended to mean glycoprotein 160, a precursor of HIV envelope proteins gp41 and gp120.
  • L3 as used herein relates to an adjuvant comprising a monoglyceride and a fatty acid as described above and in PCT/SE97/01003.
  • N3 as used herein relates to an adjuvant according to the present invention comprising a cationic substance and, optionally a monoglyceride and/or a fatty acid.
  • the cationic substances in the adjuvant described above may comprise at least one group comprising a nitrogen atom, such as a primary, secondary or tertiary amine, a quaternary ammonium compound, a primary, secondary or tertiary amide or an amino acid.
  • the cationic substance may further comprise at least one acyl group, wherein the acyl group may be a natural or synthetic, branched or unbranched, cyclic or acyclic, substituted or unsubstituted acyl, alkyl, alkenyl and alkynyl chains of from 4 to 30 carbon atoms, such as, e.g., from 6 to 24 carbon atoms, from 8 to 20 carbon atoms or from 12 to 20 carbon atoms.
  • the cationic substance may be an acyl amine, such as, e.g. a lauryl amine (C12), palmityl amine (C16), palmitoleyl amine (C16:1 ), oleyl amine (C18:1) and linoleyl amine (C18:2).
  • acyl amine such as, e.g. a lauryl amine (C12), palmityl amine (C16), palmitoleyl amine (C16:1 ), oleyl amine (C18:1) and linoleyl amine (C18:2).
  • the cationic substance may be a mixture of oleyl amine and lauryl amine.
  • the w/w ratio of oleyl amine to lauryl amine may be from about 0.1 (i.e. 1 :10) to about 10 (i.e. 10:1 ), such as, e.g., from about 0.25 to about 9, from about 0.5 to about 8, from about 0.75 to about 7, from about 1 to about 6, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, from about 1 to about 2.
  • the adjuvant according to the invention which comprises a cationic substance, may further comprise a monoglyceride having the formula
  • R wherein R is selected from H and an acyl group containing from 6 to 30 carbon atoms with the proviso that two of the R groups are H.
  • acyl chains are normally placed on carbon atom 1 or 3 of the glycerol backbone, but there will often be a acyl migration between the carbon atoms 1 and 3, and the center carbon atom 2, resulting in that approximately 90% of the acyl chains will be positioned on the carbon atom 1 or 3, and about 10% will be positioned on the center carbon atom.
  • distilled 1 -monoglyceride from Danisco Ingredients (Denmark) with a purity of at least 80% w/w, such as, e.g., at least 90% w/w or at least 95% w/w.
  • the content of diglyceride in the 1 -monoglyceride is at the most 3% and the content of triglycerides and fatty acids are less than 1%.
  • the adjuvant may be a mixture of a monoglycerides and an acyl amine, such as, e.g., mono-olein and oleyl amine.
  • the w/w ratio of mono-olein to oleyl amine may be from about 0.1 to about 10, such as, e.g., from about 0.25 to about 9, from about 0.5 to about 8, from about 0.5 to about 7, from about 0.5 to about 6, from about 1 to about 5, from about 1 to about 4, from about 1 to about 3, from about 1 to about 2 and from about 1 to about 1.
  • the w/w ratio of mono-olein to oleyl amine may be about 0.45.
  • the adjuvant for use in a vaccine according to the present invention may also be a composition comprising
  • R is selected from H and an acyl group containing from 6 to 24 carbon atoms with the proviso that two of the R groups are H, and
  • One embodiment of the invention relates to a vaccine, wherein the first immunizing component dispersed in the first medium may comprise an adjuvant comprising one or more cationic acyl amines. This especially applies where the first immunizing component comprises one or more nucleic acid sequences.
  • the second component is dispersed in a medium, which may comprise an adjuvant comprising a monoglyceride and a fatty acid as describe above. This especially applies where the second component comprises one or more peptides.
  • the first and/or second component may further comprise an immune stimulating agent such as, e.g., a CpG motif or an immune stimulating small nucleotide sequence.
  • an immune stimulating agent such as, e.g., a CpG motif or an immune stimulating small nucleotide sequence.
  • the first and/or the second medium of the vaccine may be in the form of an aqueous suspension.
  • the first and/or second medium may comprise an adjuvant, which may comprise one or more cationic substances alone or together with monoglycerides and/or fatty acids.
  • the total concentration of cationic substances, either alone or if relevant together with monoglycerides and/or fatty acids, in the first or second medium is at the most 25% w/v, such as, e.g., at the most 20% w/v, at the most 15% w/v, at the most 10% w/v, at the most 5% w/v, at the most 4% w/v, at the most 3% w/v, at the most 2% w/v or at the most 1 % w/v.
  • a specific embodiment of the invention relates to a first and/or second medium comprising an adjuvant comprising one or more cationic substances and no monoglycerides, and wherein the total amount of cationic substances in the first or second medium is from about 0.1 % w/v to about 15% w/v, such as, e.g., from about 0.25% w/v to about 12.5% w/v, from about 0.5% w/v to about 10% w/v, from about 1% w/v to about 7.5% w/v, from about 1 % w/v to about 5% w/v, from about 1 % w/v to about 4% w/v, from about 1 % w/v to about 3% w/v, from about 1 % w/v to about 2% w/v or from about 0.5% w/v to about 4% w/v.
  • the invention also relates to a first and/or second medium comprising an adjuvant comprising one or more monoglycerides together with one or more cationic substances, wherein the total amount of cationic substances in the first or second medium is from bout 0.1 % w/v to about 10% w/v, such as, e.g., from about from about 0.25% w/v to about 9% w/v, from about 0.5% w/v to about 8% w/v, from about 1% w/v to about 7% w/v, from about 1 % w/v to about 6% w/v, from about 1 % w/v to about 5% w/v, from about 1 % w/v to about 4% w/v, from about 1 % w/v to about 3% w/v, from about 1 % w/v to about 2% w/v or from about 0.5% w/v to about 4% w/v.
  • the first and/or second medium may also comprise an adjuvant, which comprises monoglycerides and fatty acids.
  • the total concentration of monoglyceride and fatty acid is at the most 25% w/v, such as, e.g., at the most 20% w/v, at the most 15% w/v, at the most 10% w/v, at the most 5% w/v, at the most 4% w/v, at the most 3% w/v, at the most 2% w/v or at the most 1 % w/v.
  • first and/or the second medium may further comprise a surface-active agent, which may be hydrophilic and inert and biocompatible, such as, e.g., Pluronic F68.
  • a surface-active agent which may be hydrophilic and inert and biocompatible, such as, e.g., Pluronic F68.
  • the first and/or second medium may further comprise one or more physiologically acceptable additives, such as, e.g., buffering agents, stabilizing agents, osmotically active agents, preservatives and pH adjusting agents.
  • physiologically acceptable additives such as, e.g., buffering agents, stabilizing agents, osmotically active agents, preservatives and pH adjusting agents.
  • the pH of the medium should be within the physiologically acceptable range, such as from about pH 6 to pH 8. The exact value will be determined based on the pK a value of the cationic substances.
  • the human immunodeficiency virus is today one of the major human pathogens, and an efficient vaccine is highly needed.
  • the vaccine is against the HIV or SIV virus
  • the first component of the vaccine may comprise one or more DNA expression vectors comprising one or more gp41 nucleotide sequences or fragments thereof.
  • the first component may further comprise one or more DNA expression vectors comprising a nucleotide sequence encoding rev and/or other regulatory or accessory proteins, such as, e.g. tat or nef.
  • the DNA expression vectors may further comprise a promoter that enables expression of the nucleotides in eucaryotic cells, such as, e.g., a CMV IE promoter, a pRSV promoter or a pBRV promoter or the like.
  • the second component may comprise one or more gp41 peptides as defined above.
  • glycoprotein 120 mediates binding and entry steps in HIV-1 infection and that the gp120-CD4 co-receptor binding induces conformational changes that activate the gp41 transmembrane regions of the envelope. It is believed that the fusion structure of the envelope protein form after CD4 and co-receptor binding break down into two coils within the gp41 monomer, and that synthetic peptides against either gp41 helical coils are able to inhibit viral infectivity.
  • the gp41 coiled coil peptide may be used in order to obtain an antibody response that might inhibit fusion and are capable of recognizing several HIV-1 subtypes.
  • sequences suitable for use in a vaccine composition according to the invention are given specific examples of sequences suitable for use in a vaccine composition according to the invention, without limiting the invention hereto.
  • the sequences are selected from the HIV-1 database in Los Alamos. The selected sequences have been shown to be recognized by HIV neutralizing monoclonal antibodies and polyclonal antibodies from HIV-1 infected individuals. These epitopes belong to the repertoire of broadly HIV cross clade neutralizing epitopes, such as, e.g. the ELDKWAS amino acid sequence located on the gp41.
  • These sequences are combinations of 4-5 HIV-1 subtypes representing HIV variants which circulate in Europe, USA, Latin America but also and mostly in sub- Saharan Africa. This is the case especially for gp41 peptides representing HIV-1 clades/subtypes other than subtype B.
  • the vaccines may be administered in any convenient manner such as by mucosal administration, such as, e.g. nasal, oral, rectal, vaginal, lung, aural, or skin administration, or by intramuscular, subcutaneous, intradermal or topical routes, and combinations thereof.
  • mucosal administration such as, e.g. nasal, oral, rectal, vaginal, lung, aural, or skin administration, or by intramuscular, subcutaneous, intradermal or topical routes, and combinations thereof.
  • the nose is a very attractive route for immunization due to the fact that it is easily accessible, highly vascularized and contains a large absorptions surface. Also, as shown in the Experimental part, both mucosal and systemic immune responses can be induced and immune response can be induced at distant mucosal sites, such as the vagina and rectum. Furthermore large populations can easily be immunized, with less risk of infection.
  • the invention also provides HIV or SIV vaccines in the form of kits comprising at least two components.
  • the content of the two components can be combined in several ways a described below in order to achieve the best suitable immune response.
  • Both the first and the second component may further comprise one or more of the adjuvants described above.
  • One embodiment of the invention relates to a HIV or SIV vaccine in the form of a kit comprising at least two components, wherein each component comprises a) one or more DNA expression vectors comprising a gp41 nucleotide sequence or fragments thereof b) optionally, one or more DNA expression vectors comprising a nucleotide sequence comprising rev DNA and/or other regulatory or accessory proteins, such as, e.g. tat or nef, c) one or more gp41 peptides.
  • a HIV or SIV vaccine in the form of a kit comprising at least two separate components, the first immunizing component comprising a) one or more DNA expression vectors comprising a gp41 nucleotide sequence or fragments thereof, b) optionally, one or more DNA expression vectors comprising a nucleotide sequence comprising rev DNA and/or other regulatory or accessory proteins, such as, e.g. tat or nef,
  • the second component comprising c) one or more gp41 peptides.
  • Yet another embodiment of the invention relates to a HIV or SIV vaccine in the form of a kit comprising at least two separate components, the first immunizing component comprising a) one or more DNA expression vectors comprising a gp41 nucleotide sequence or fragments thereof, b) optionally, one or more DNA expression vectors comprising a nucleotide sequence comprising rev DNA and/or other regulatory or accessory proteins, such as, e.g. tat or nef, c) one or more gp41 peptides,
  • the second component comprising d) one or more gp41 peptides.
  • the invention relates to a HIV or SIV vaccine in the form of a kit comprising at least two separate components, the first immunizing component comprising a) one or more DNA expression vectors comprising a gp41 nucleotide sequence or fragments thereof, b) optionally, one or more DNA expression vectors comprising a nucleotide sequence comprising rev DNA and/or other regulatory or accessory proteins, such as, e.g. tat or nef,
  • the second component comprising c) one or more DNA expression vectors comprising a gp41 nucleotide sequence or fragments thereof, d) optionally, one or more DNA expression vectors comprising a nucleotide sequence comprising rev DNA and/or other regulatory or accessory proteins, such as, e.g. tat or nef.
  • the invention relates to a HIV or SIV vaccine in the form of a kit comprising at least two separate components, the first immunizing component comprising a) one or more gp41 peptides,
  • the second booster component comprising b) one or more DNA expression vectors comprising a gp41 nucleotide sequence or fragments thereof, c) optionally, one or more DNA expression vectors comprising a nucleotide sequence comprising rev DNA and/or other regulatory or accessory proteins, such as, e.g. tat or nef.
  • the invention also relates to a HIV or SIV vaccine in the form of a kit comprising at least two separate components, the first immunizing component comprising a) one or more gp41 peptides,
  • the second booster component comprising b) one or more DNA expression vectors comprising a gp41 nucleotide sequence or fragments thereof, c) optionally, one or more DNA expression vectors comprising a nucleotide sequence comprising rev DNA and/or other regulatory or accessory proteins, such as, e.g. tat or nef, d) one or more gp41 peptides.
  • the HIV or SIV vaccine according to the invention may further comprise a third or more booster components comprising a) one or more DNA expression vectors comprising a gp41 nucleotide sequence or fragments thereof and/or b ) optionally, one or more DNA expression vector comprising a nucleotide sequence comprising rev DNA and/or other regulatory or accessory proteins, such as, e.g. tat or nef and/or d) one or more gp41 peptides and any combinations thereof.
  • a third or more booster components comprising a) one or more DNA expression vectors comprising a gp41 nucleotide sequence or fragments thereof and/or b ) optionally, one or more DNA expression vector comprising a nucleotide sequence comprising rev DNA and/or other regulatory or accessory proteins, such as, e.g. tat or nef and/or d) one or more gp41 peptides and any combinations thereof.
  • a vaccine according to the invention may also be used for therapeutically purposes and in such cases it may be suitable to add an effective amount of either nucleotides encoding a CCR5 protein or fragments thereof or a CCR5 protein or fragments thereof, such as, e.g. the second external loop of the CCR5 chemokine receptor, which also serves as a co-receptor for HIV-1.
  • the CCR5 chemokine receptor have 5 external loop regions, but antibodies to the other loop regions do not seem to have much effect of the attachment to HIV-envelope.
  • the CCR5 component may be added to the first and/or the second component in a vaccine. However, for vaccine purposes the use of CCR5 may be undesired due to the risk of inducing inappropriate immune responses towards the body's CD4 cells.
  • the invention relates to a method for eliciting an immune response in an animal, the method comprising administration to the animal of a vaccine according to the invention, wherein the administration of the first immunizing component in an amount sufficient to induce an immune response in the animal is followed by at least one administration to the animal of the second component.
  • Figure 1 shows fecal IgA ELISA reactivity in four individual mice from group 927 at different time-points: pre-immunization (dotted bars), three weeks after primary (black bars) and 4 weeks booster immunization (striped bars).
  • Fecal IgA reactivity at dilution 1/8 is shown against the gp41 ELDKWAS-peptides representing clades A, B, D, E and the CCR5 2 nd loop peptide and the negative rev control peptide.
  • Figure 2 shows fecal IgA ELISA reactivity in four individual mice from group 928 at different time-points: pre-immunization (dotted bars), three weeks after primary immunization (black bars) and 4 weeks after booster immunization (striped bars).
  • Fecal IgA reactivity at dilution 1/8 is shown against the gp41 ELDKWAS-peptide representing clades A, B, D, E and the CCR5 2 nd loop peptide and the negative rev control
  • Figure 3 shows fecal IgA ELISA reactivity in three individual mice from group 929 at different time-points: pre-immunization (dotted bars), three weeks after primary immunization (black bars) and 4 weeks after booster immunization (striped bars).
  • Fecal IgA reactivity at dilution 1/8 is shown against the gp41 ELDKWAS-peptide representing clades A, B, D, E and the CCR5 2 nd loop peptide and the negative rev control peptide.
  • Figure 4 shows fecal IgA ELISA reactivity in six individual mice from group 930 at different time-points: pre-immunization (dotted bars), three weeks after being given saline (black bars) and 4 weeks a second saline dose (striped bars).
  • Fecal IgA reactivity at dilution 1/8 is shown against the gp41 ELDKWAS-peptide representing clades A, B, D, E and the CCR5 2 nd loop peptide and the negative rev control peptide.
  • Figure 5A-C shows the kinetics of vaginal wash IgA ELISA reactivity against gp41 ELDKWAS peptides representing HIV-1 clades A-C.
  • the median IgA reaction absorbance values are shown at vaginal wash dilution 1/2 at pre-, 3 months - 1 year after the booster immunization in groups 927-931.
  • the respective clade gp41 peptide amino acid sequences are shown on top of each graph.
  • Figure 5D shows the kinetics of vaginal wash IgA ELISA reactivity against the gp41 coiled coil peptide (LQAR) peptide representing HIV-1 clade B.
  • the median IgA reaction absorbance values are shown at vaginal wash dilution 1/2 at pre-, 3 months - 1 year after the booster immunization in mouse groups 927-931.
  • the gp41 coil-peptide amino acid sequence is shown on top of the graph.
  • Figure 5E shows the kinetics of vaginal wash IgA ELISA reactivity against the CCR5 2 nd loop peptide representing the human CCR5 HIV-1 co-receptor.
  • the median IgA reaction absorbance values are shown at vaginal wash dilution 1/2 at pre-, 3 months - 1 year after the booster immunization in mouse groups 927-931.
  • the CCR5-loop peptide amino acid sequence is shown on top of the graph.
  • Figure 6 illustrates HIV-1 SF2 SI (syncytia inducing HIV-1 phenotype) phenotype neutralization activity of two representative mouse sera from groups 927-931 collected 9 months after the booster immunization.
  • Mouse group numbers of individual mice (as presented in Table 1 ). Percent neutralization >50% was considered positive.
  • Figure 7 illustrates HIV-1 6920 NSI phenotype primary isolate neutralization activity of two representative mouse sera from groups 927-931 collected 9 months after the booster immunization.
  • Mouse group numbers of individual mice (as presented in Table 1 ). Percent neutralization >50% was considered positive
  • Figure 8 shows HIV-1 6920 NSI primary isolate neutralizing activity with pooled mouse 927 serum (A) and mouse 931 mouse serum (B) before and after absorption with uninfected and HIV-1 6920 infected U937 cells.
  • Figure 9 shows responders of IFN gamma secreting cells after intranasal HIV-1 gp160/rev DNA-N3 adjuvant (0-200 ⁇ g) immunization pre- and post HIV-1 gp41 peptide booster. Supernatant of rgp160 antigens stimulated cells in vitro, IFN gamma.
  • N3 adjuvant is an adjuvant according to the invention containing oleylamine
  • Figure 10 shows fecal IgA responses against HIV-1 gp160 envelope antigen pre- and post HIV-1 gp41 peptide booster immunization in HIV-1 gp160/rev DNA-N3 adjuvant (0-200 ⁇ g) immunized mice.
  • Figure 11 shows serum IgG response against HIV-1 gp160 envelope antigen after intranasal HIV-1 gp160/rev DNA-N3 adjuvant (0-200 ⁇ g) immunization pre and post HIV-1 gp41 peptide booster intranasally.
  • Figure 12 shows release of IL-4 in mice intranasally HIV-1 gp160/rev DNA-N3 adjuvant (0-200 ⁇ g) immunized pre and post HIV-1 gp41 peptide booster. Gp160 stimulated cells.
  • Figure 13 shows release of IL-2 in mice intranasally HIV-1 gp160/rev DNA-N3 adjuvant (0-200 ⁇ g) immunized pre and post HIV-1 gp41 peptide booster. Gp160 stimulated cells.
  • the following examples are intended to illustrate the invention without limiting it in any way.
  • N3 lipid emulsion was prepared by adding to a beaker 0.4 g of the mixture of mono-olein and oleylamine, 9.6 ml 0.1 M Tris buffer, pH 8.0 and 195 ⁇ l 5M HCI. The N3 emulsion was formed by sonication for 2 minutes, whereafter the pH was adjusted to 8.0.
  • the final vaccine formulation was a 1 :1 mixture of the obtained N3 emulsion and a DNA solution comprising naked DNA in a concentration suitable to obtain the amounts mentioned in Table 1.
  • the different doses of the N3 adjuvant used in the Examples were obtained by mixing different dilutions of the 4% N3 lipid emulsion described above.
  • mice of the H-2b haplotype Female 10-12 weeks old C57BI/6 mice of the H-2b haplotype (MTC, Karolinska Institute animal facility, Sweden) were immunized intranasally with combinations of HIV-1 rgp160BaL DNA, HIV-1 Rev/Lai DNA, human CCR5 DNA, gp41/MN coiled coil (aa 578-591 , GIKQLQARV- LAVERY) and gp41 ELDKWAS subtype A-D peptides (aa 661-675/MN, NEQLLELDKWASLWN, A/92UG31 aa 652- 665, EKDLLALDKWANLWN, C/92BR025 aa 651-665, NEQDLL-ALDSWNLWN, D/92UG021 aa 643-657, EQELLKLDQWASLWN) and peptides representing the human 2nd external loop of the human chemokine receptor CCR5 which also serves as a coreceptor for HIV
  • All DNA plasmids contain the CMV IE promoter for gene expression and the kanamycin gene.
  • mice As shown in Table 1 a group of 8 control mice were included. The other groups contained 6 mice.
  • the intranasal second booster immunization was performed with peptides (0,25 ⁇ g/ ⁇ l) admixed in 4% L3 adjuvant (monoolein/oleic acid at a molar ratio) prepared as previously described in PCT/SE97/01003 [24].
  • Synthetic peptides corresponding to the gp41 neutralizing epitope (aa 661- 675/ELDKWAS) (Hybaid T-peptides, Ulm, Germany) representing clade A/(92UG31), B/(MN), C/(92BR25), D/(92UG21); a gp41 clade B/(MN) peptide (Hybaid T-peptides, Ulm, Germany) located between aa 578-595 peptides representing the human and the simian CCR5 N-terminal region and 2nd loop aa 168-182, and aa 178-192 (Hybaid T- peptides, Ulm, Germany, 20, 21), a gp120 V3 loop region clade B aa 302-318 and as negative control a HIV-1 Lai rev-representing peptide aa 65-84 were synthesized by using solid phase F-moc chemistry.
  • the immunoreactions in the immunized mice were evaluated by the following methods.
  • 96-well plates (NUNC-Maxisorp, Odense, Denmark) were coated with a recombinant gp160.
  • This protein was solubilized in 0.1 M NaCO 3 buffer (pH 9.5 to 9.6) at coating concentration of 1 ⁇ g/ml. Plates were stored over night at room temperature and for a minimum of 24 h at 4°C. Patient sera was diluted in ten fold dilutions starting with 1 :100 in PBS (pH 7.4) with 0.5% bovine serum albumine (BSA, Boehring Mannheim, Mannheim, Germany) and 0.05% Tween 20 (Sigma, Aldrich, Sweden, AB).
  • BSA bovine serum albumine
  • the positive control used was a human HIV-lgG pool collected from HIV-1 infected Kenyan patients, the Kabi 62 serum, monoclonal antibodies against the gp41 ELDKWAS epitope Mab 2F5 (donated by Dr.H. Katinger), monoclonal anti-gp120 V3 antibody F58/H3 [30] and monoclonal antibodies against the 2nd CCR5 external loop Mab 2D7 (Coulter Pharmaceuticals, Palo Alto, CA).
  • As negative controls pre- immunization serum, pre-immunization vaginal washings and fecal pellets were used.
  • IgA and IgG Quantification of IgA and IgG was performed by capture ELISA with commercially available IgA (1 mg/ml) and IgG (2 mg/ml) standards (Sigma). Values above the mean optical density of the negative control plus 2 SD were used as the cut off and values above were considered as positive.
  • IgA purification and quantitation The fluids collected from the intestinal/fecal and lung washings were used to isolate and analyse the IgA content.
  • the Kaptive IgA/lgE reagents Biotech IgG, Copenhagen, Denmark) were purchased and used as recommended by the manufacturer. Reagents for preparing an in-house murine IgA capture ELISA were purchased and a commercial murine IgA (1 mg/ml, Sigma-Aldrich) was used for preparing a standard curve.
  • the substrate reaction was terminated by adding 100 ⁇ l/well of 2.5M H 2 SO 4 and the absorbance was measured at OD490.
  • the amounts of IgA in the mouse- samples were determined by comparing the OD-values of the test samples with the IgA standard.
  • Serum neutralization was evaluated in the virus dilution with TCID 50 (50% Tissue Culture Infective Dose) values in the range of 50 to 125 for 1 h at 37°C in round- bottomed culture plates (NUNC, Roskilde, Denmark). Seventy-five ⁇ l with 1x10 6 /ml
  • PHA phytohemagglutinine stimulated blood mononuclear cells
  • PBMC blood mononuclear cells
  • RPMI 1640 medium Life Technologies, Scotland, UK
  • 200 ⁇ l of fresh RPMI 1640 medium was added to each well. Every three days, 100 ⁇ l of medium was changed. After 6-7 days, 100 ⁇ l supernatant from each well was collected and the virus production was analyzed in a p24 antigen capture ELISA as previously described [30].
  • a HIV-1 positive serum pool - HIVIG- and the human monoclonal antibody 2F5, specific for the gp41 ELDKWAS epitope were used as a positive control.
  • Neutralization was defined as the sample titer resulting in 50% and 90% reduction of p24 antigen in the supernatant as compared to p24 antigen content when the virus was incubated in the presence of HIV antibody negative sera. All assays were repeated at least twice.
  • the HIV-1 neutralization assay was performed as previously described [32]. In brief, purified fecal IgA from control or immunized mice were incubated for 1 h at 37°C with 30-50 TCID 50 cell-free virus HIV-1 SF2 (T-cell line adapted subtype B isolate) or with the primary patient subtype B isolate 6920 in 200 ⁇ l volume in duplicate 96-well cell culture plate wells (Nunc, Aarhus, Denmark). The medium used was RPMI 1640 supplemented with 10% inactivated fetal calf serum (Life Technologies, Scotland, UK), 10 lU/ml IL2 (Amersham, Buckinghamshire, UK) and 50 ⁇ g/ml streptomycin and 50 IU penicillin (Life Technologies).
  • a HIV-1 positive serum - Kabi 62- was used, as positive control. Neutralization was defined as >50% reduction of p24 antigen in the supernatant as compared to p24 antigen content when the virus was incubated in the presence of HIV antibody negative sera. All assays were repeated at least twice.
  • the specificity of the neutralizing serum was tested by absorbing the HIV-1 neutralizing serum with uninfected U937 cell lysate or with HIV-1 6920 infected U937 cells [16-18] and then tested for their remaining neutralizing activity as described above.
  • heat-inactivated mouse serum was used at 1 :5 dilution in 106 uninfected or infected U937 cells and incubated for 1 h at room temperature. The cells were then spun down (450 x g) for 10 min and the cell-free supernatant was collected and used in the neutralization assay.
  • IgG/lgA synthesis in vitro 1x10 5 spleen cells were cultured in 200 ⁇ l of RPMI 1640 supplemented with 5% inactivated fetal calf serum at 37°C in 96-flat bottomed cell culture plates (NUNC) with rgp160 (1 ⁇ g/ml) or with peptides (10 ⁇ g/ml) for 72 hrs. After washing, antigen-specific immunoglobulins were measured by ELISA as mentioned above. A positive reactivity was considered if the ELISA substrate absorbance was higher than the mean OD+2SD of the negative control mice.
  • Lymph nodes were collected from sacrificed mice. The lymph nodes cells were pooled for each group of mice due to low total numbers of available cells from each individual mouse. 1x10° lymph node cells were cultured in 200 ⁇ l of RPMI 1640 supplemented with 5% inactivated fetal calf serum at 37°C in 96-flat bottomed cell culture plates with rgpl 60 (1 ⁇ g/ml) or with peptides (10 ⁇ g/ml) for 72 hrs. After washing, antigen-specific immunoglobulins were measured by ELISA as mentioned above. A positive reactivity was considered if the ELISA substrate absorbance was higher than the mean OD+2SD of the negative control mice.
  • MHC class I proteins were performed by using the prediction of MHC class I candidate sequences HYPERLINK "http://www-bimas.dcrt" http://www-bimas.dcrt.nih.gov/molbio/ hla_bind/index.html.
  • mice 3 days prior to the start of the study: Pre-bleed of mice.
  • each group contains 7 mice and each was immunized intranasally with 10 ⁇ of the vaccine formulations. By using only 10 ⁇ l of the vaccine formulation it is ensured that the vaccine formulation is only applied to the nose of the mice, and not to the mouth and lungs. Immunization schedule
  • WEEK 4 Blood sampling from mice for immunoanalysiss.
  • WEEK 7 Booster-immunization with 10 ⁇ g gp41 peptide in 4% L3-adjuvant intranasally
  • mice (6 ⁇ l/nostril) to all mice in groups 1-6.
  • mice in group 7 received the gp41 peptides diluted in saline (10 g/mouse, 6 l/nostril).
  • WEEK 10 Blood sampling and immunoassays.
  • WEEK 12 Mice sacrificed. Blood and spleen cell analyses.
  • Serum IgG responses against HIV-1 gp41 , gp120, gp160 and CCR5 were measured up to one year post-boost immunization.
  • High antibody titers were seen against all gp41 peptides.
  • HlV-specific IgG persisted over 12 months. No significant differences were detected between the 927, 928 and 929 groups (p>0.05) at 6 or 12 months and no significant serum IgG titer changes were seen during the two time points measured.
  • Table 2A Serum IgG titers against HIV-1 gp41, gp120 and CCR5 representing antigens 12 months post-boost immunization.
  • IgA serum antibody titers were detectable as late as one year post-boost immunization (see Table 2B). No significant geometric differences mean IgA antibody titers (p>0.05) were detected between the groups 927, 928, 929 against the gp41 clade A, B and C peptides while the IgA titer against gp41 clade D was lower in group 929 (p ⁇ 0,03). Similarly, the group 929 lacked detectable IgA antibodies against the V3-loop region. The IgA titers were in general ten-fold lower than the IgG titers.
  • HIV-1 subtype 6 months 12 months 6 months 12 months 6 months 12 months 6 months 12 months 6 months 12 months 12 months Mab 2F5 930
  • gpl60Lai B 420+/-120 140+/-45 160+/-45 105+/-20 200+/-60 140+/-35 ⁇ 50 ⁇ 50 65000 ⁇ 50
  • FIG. 1 shows the IgA ELISA antibody reactivity at different time points before and after immunization in four out of six individual mice representing group 927 mice.
  • Six out of six animals developed IgA against clades A, B, D, E and CCR5 after the second booster (927:1 , 927:2, 927:5 and 927:6).
  • the highest reactivity was detected against the CCR5 peptide. None of them developed IgA against the control rev peptide.
  • mice At 12 months follow up (see Table 3) when the mice were sacrificed the small intestine mucosa of all mice in group 927 was shown to contain IgA reactive with HIV-1 gp41.
  • Anti-ELDKWAS representing peptide reactivity of subtypes A, B and C was detectable in mice against the gp41 coil peptide. Further, 5/6 mice had intestinal IgA reactive with gp160 and 4/6 with the CCR5 2nd loop peptide.
  • Sample IgA titer cut off was calculated from mean absorbance values of negative control mice plus two SD.
  • IgA specific antibodies in fecal pellets were also found in the group 928 mice ( Figure 2). After the booster immunization all mice had developed IgA antibodies against gp41 peptides representing clades A, B and D while in two out of four the highest reactivity was detected against gp41 ELDKWAS clade B and CCR5 peptide (928:2 and 928:4). At 12 months (Table 3) small intestinal IgA was shown to react with gp41 ELDKWAS peptides of clade A in 6/6 mice, against clade B in 5/6 mice, clade C in 4/6 mice and against the gp41 coil peptide in 1/6 mice and against gp160 in 5/6 mice. None of the group 928 mice had IgA reactive with the CCR5 or Rev-peptides.
  • mice immunized with CCR5 peptides fecal IgA reactivity with the CCR5 2nd loop peptide was seen in 6/6 animals. At 12 months follow up 6/6 mice had detectable small intestine IgA reactive with the CCR5 peptide without reacting with tested HIV-1 antigens.
  • Figure 5A-E shows the IgA antibody reactivity in vaginal washes 3-12 months post- booster of all groups of immunized mice. High reactivities in group 927 were detected against all antigens. When studying the kinetics of vaginal HIV IgA reactivity, long-term reactivity (9 months) was seen against the gp41 peptides. Still 12 months after booster high median IgA absorbance reactivity was seen in vaginal samples against the gp41 ELDKWAS and the gp41 coiled-coil LQAR peptides were seen. Mice in group 928 developed IgA antibodies against all antigens except the CCR5 peptide although the response decreased with time more rapidly than for group 927 ( Figure 5).
  • mice in group 929 developed vaginal IgA antibodies against clade C and CCR5 peptide already after the first immunization (929:1 ). However, the group median reactivity was low or undetectable against all antigens at most time points.
  • the amount of sample that can be collected from each individual mouse is small (100 ⁇ l/sample occasion) and for this reason the samples were pooled within each group. Since each group of mice was analyzed against their own pool of pre-immunization samples, the amounts of IgA at the pre-immunization pool was not significantly different from the IgA amounts detected in the post-immunization samples within each study group.
  • the pre-immunization IgA amounts ranged between 3.3-8.1 ⁇ g/ml and the post- immunization samples between 2.7-11.5 ⁇ g/ml.
  • mice were shown to have lung IgA against gp41 ELDKWAS peptides from clades B and C only.
  • mice were shown to have CCR5 peptide specific lung IgA only (Table 3). Lung IgA from the negative control group (930) was detectable against all HIV and CCR5 antigens tested.
  • Table 4A summarizes the spleen IgG/ IgA B-cell responses a year after the final booster in spleen and lymph node B-cells.
  • the highest frequencies of spleen IgG B-cell cell responders were detected in groups 927, 928 and 929 against rgp160, gp41 ELDKWAS peptides clade A, B, C and among groups 929, 928 and 931 against the CCR5-peptide.
  • mice Three groups of mice (927, 928 and 929) had similarly high frequencies of spleen IgA B-cell responders against HIV-1 antigens, and two groups 927 and 931 had high frequencies of IgA responders against the CCR5 loop peptide.
  • One of the control group animals 930 reacted with the CCR5 peptide while none with the HIV-1 antigens.
  • Table 4B summarizes the lymph node IgG and IgA B cell responses against HIV-1 and CCR5 antigens 12 months after booster immunization. Due to low numbers of cells available in lymph nodes, all collected cells from each group of mice were pooled and analyzed in triplicate wells against each antigen. Lymph node IgG B-cell responses were seen against all HIV-1 and CCR5 antigens in groups 927, 928, 929 and 931. Due to low numbers of cells in group 927 analysis of the CCR5 peptide reactivity could not be done. Only the control group 930 cells reacted negative.
  • Lymph node IgA B-cell reactivity towards HIV-1 antigens was detected in groups 927, 928, 929 and 931 , with the strongest responses in groups 927 and 928.
  • B-cell IgA against the CCR5 peptide was seen in groups 928, 929 and 931. Due to low numbers of cells in group 927 analysis of the CCR5 peptide reactivity could not be done. Only the control group 930 cells reacted negative.
  • Antigens gp41 ELDKWAS peptides representing claades A-C, recombinant gpl60 antigen and CCR5 loop peptides.
  • fecal IgA purified from 6/6 mice in group 927 were shown to neutralize HIV-1 SF2 by ⁇ 80 % at 6 months (6/6 mice) and after 9 months (not shown) after booster immunization.
  • group 928 the fecal IgA neutralization capacity reached ⁇ 50 % neutralization titer at 6 months (4/6 mice) and ⁇ 50 % at 9 months post booster immunization (not shown).
  • Fecal IgA from all other groups failed to neutralize the HIV-1 SF2.
  • Fecal IgA purified from 4/4 mice in group 927 were shown to neutralize the primary HIV-1 isolate 6920 at 6 and 9 months after booster immunization.
  • group 928 the fecal IgA neutralization capacity was detectable at 6 months in 2/4 mice and undetectable at 9 months post booster immunization (not shown).
  • HIV-1 SF2 neutralizing lung lavage IgA was detected in 2/6 mice in groups 927 and 928 mice at 12 months post booster immunization (not shown).
  • HIV-1 neutralizing IgA titers resulting in 50 or 80% reduced HIV-1 p24 antigen detection are shown with 2-3 pooled IgA fractions from immunized mouse groups,
  • HIV isolate HIV-1 SF2 HIV-1 6920
  • results are seen as bars with two sets of data for each group of mice.
  • the first (lower datagroup) shows the results obtained 4 weeks after the DNA administration and the second shows the immune response three weeks after the booster vaccination. All results indicate that unless the DNA is co-administered with the N3 adjuvant, no immune response above cut-off level can be detected in any of the groups when only 10 ⁇ l vaccine formulation is used for the nasal administration. Also shown is the dose- dependent response of the N3 adjuvant according to the present invention.
  • the fusion structure of the envelope protein form after CD4 and co-receptor binding break down into two coils within the gp41 monomer and that synthetic peptides against either gp41 helical coils are able to inhibit viral infectivity [38,39].
  • the gp41 coiled coil peptide was included in order to obtain an antibody response that might inhibit fusion and are capable of recognizing several HIV-1 subtypes.
  • the ELDKWAS peptides were also included since it was previously shown that mucosal IgA, but not IgG, from HIV seropositive individuals neutralized HIV by recognizing the ELDKWAS epitope located on the gp41.
  • mice primarily immunized with rgp160/rev and CCR5 DNA and boosted with gp41- ELDKWAS and CCR5 peptides in 1 :1 (mohmol) monooleate/laurate.
  • CCR5 for therapeutic applications.
  • vaccine purposes it is believed that the use of CCR5 is undesired due to the risk of inducing inappropriate immune responses towards the body's CD4 cells.
  • High and long-term serum IgG and IgA titers was obtained against the neutralizing gp41 ELDKWAS epitopes from HIV-1 clades A, B, C and D.
  • Lower titers of serum IgG reacted with the gp41 coiled-coil peptide, gp120 V3 clade B and gp160/Lai still one year post-booster immunization.
  • Mice immunized with HIV-1 peptides only developed high serum IgG titers against the gp41 peptides, as seen also in the DNA-primed animals.
  • serum IgA titers were equal to titers obtained in DNA-primed animals against the gp41 neutralizing ELDKWAS epitopes from clades A, B and C, but significantly lower or undetectable 1 year after booster immunization against the gp41 ELDKWAS epitopes from clade D, gp41 coil, and gp120 V3 peptides.
  • Serum IgG and IgA titers against HlV-antigens did not differ significantly between the three groups of mice receiving HIV-1 antigens with or without inclusion of the CCR5 coreceptor antigen.
  • Serum IgG and IgA responses against the CCR5 representing peptide was seen in the three mouse groups immunized with CCR5 antigen with a significantly higher serum IgG against CCR5 in the two mouse groups receiving CCR5 antigens (p ⁇ 0,01 , p ⁇ 0,02) at 6 months post-last immunization. This difference was lost at 12 months follow-up.
  • mice immunized with HIV-envelope and CCR5 DNA were able to develop significantly higher mucosal lung IgA titers to gp41 peptides representing HIV-1 clades A-C than the other groups of mice.
  • mice were able to develop significantly higher titers of intestinal IgA recognizing the gp41 A and C and the gp41 B coil-coiled peptide, while gp41 clade B gp41 neutralizing epitope recognizing intestinal IgA was non-significant between mice given HIV-1 DNA or gp41 peptides.
  • Mucosal vaginal washings IgA were also substantially stronger reactive during 6-12 months in the HIV-1 gp160/rev CCR5 DNA mouse group during follow up than in the other groups even though the numbers of responsers were not significantly different between the groups.
  • vaginal anti-gp41 peptide specific IgA was only detectable in the HIV- and CCR5 DNA group. There seems to be a trend towards an enhanced anti-HIV antigen specific mucosal humoral immunity in this same group of mice.
  • the group differs in its immunization schedule by the inclusion of the CCR5-DNA and CCR5-peptide pooled with HIV-1 DNA and gp41 peptides.
  • the use of DNA-plasmid may have resulted in a higher amount of activated lymphocytes resulting in a longer and better preserved immune memory as the long-term humoral mucosal immunity indicates.
  • HIV-1 neutralizing serum antibodies were highly efficient in neutralizing HIV- 1 SF2 and the primary HIV-1 isolate in both plasmid-DNA primed mouse groups. In both these groups the HIV-1 neutralizing capacity could be removed by adsorbing the serum with HIV-1 infected U937 cells but only partially when uninfected cells were used. These results may suggest that the main functional antibodies were directed against HIV-1 neutralizing epitopes. However, it is important to remember that the way the neutralization assay was designed was not optimal for studying the anti-CCR5 blocking capacity of the serum. In spite of this the group no. 931 mouse serum was able to reach a HIV-1 SF2 neutralizing activity of between 60-70% against the primary HIV-1 isolate.
  • CCR5 coreceptor may be present at the virion envelope as has been previously suggested or that anti-idiotypic antibodies for the CCR5 peptide may have induced V3-neutralizing antibodies.
  • HIV-1 neutralizing IgA purified from fecal samples and from the lung was detected only in mice intranasally immunized with HIV-DNA with and without CCR5-plasmid.
  • the laboratory mouse has an average life span of between 2-3 years, and accordingly, a systemic and mucosal immunity obtained with only two immunizations intranasally, and which is still detectable 1 year after a booster immunization, appears as being a relatively long-term immunity.
  • the immunization strategy of the present invention includes relatively conserved gp41 envelope components and one of the main HIV-1 co- receptors of which some epitopes probably are exposed as cryptic and highly conserved determinants [36].
  • the two chosen gp41 epitopes included in immunization strategy in the present invention thus show a high or relatively high degree of conservation, as shown in a study by Dong et al. 2001 (41). They have shown comparisons of 862 HIV-1 strains and 90-97% of the isolates were shown to contain the LQAR sequence in the coiled- coil region. While the LELDKWAS region show a more variable mix of 50-97% conserved amino acids (with amino acids L-LD-W being >97% conserved) among the 862 isolates.
  • This combination of epitopes included in a vaccine may provide the conserved epitopes that have been so difficult to find in other regions of the HIV-1 envelope even though highly conserved cross-neutralization antibody inducing epitopes have been described in the gp120 V3 region (aa GPGR (42, 43, 44) and within the CD4-binding region of gp120 (45). Further evidence suggesting the importance of inducing antibodies against these gp41 epitopes are the low frequency of antibody reactivities against these regions in HIV-infected individuals [46,47]. With the inclusion of the CCR5-DNA in vaccine formulation in the present invention this combination has every crucial constituent involved in the fusion HIV-1 process.
  • HIV-1 gp41 specific IgA could be seen in some fecal pellets, lung lavages and vaginal washings still 12 months after immunization.
  • HlV-specific IgG and IgA memory B cells were detectable in spleen and draining lymph nodes was observed against gp41/MN, gp41 clade A, B, C, rgp160 and CCR5 loop peptide. Because memory B-cell are accumulated with every immunization even when antibodies are not detectable in serum, IgG and IgA B-cell stimulation in vitro in spleen and lymph nodes could be measured, which reflect the presence of long-term immunity. Memory B-cells is an important parameter to consider in the development of a preventive HIV vaccine. References
  • mice with recombinant gp41 in a systemic prime/mucosal boost protocol induces HIV-1 -specific serum IgG and secretory IgA antibodies.
  • Trkola A. Pomales AB., Yuan H., Korber B., Maddon PJ., Allaway GP., Katinger H., Barbas C 3rd , Burton DR., Ho DD.

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Abstract

L'invention concerne un vaccin contre un virus, par exemple un rétro virus comprenant un lentivirus, tel qu'un virus d'immunodéficience humain ou simien, ledit vaccin comprenant au moins deux composants séparés, un premier composant d'immunisation comprenant : a) un ou plusieurs vecteurs d'expression d'ADN comprenant une séquence de nucléotide codant une protéine virale ou ses fragments et/ou un ou plusieurs polypeptides codés par au moins une partie du nucléotide, et un second composant comprenant : b) un ou plusieurs vecteurs d'expression d'ADN comprenant une séquence de nucléotide codant une protéine virale ou ses fragments et/ou un ou plusieurs polypeptides codés par au moins une partie du nucléotide. Le premier et le second composant sont dispersés dans un premier et un second milieu, respectivement, et ces derniers comprennent un additif, identique ou différent, au moins un des additifs étant sélectionné parmi des substances cationiques telles que des acylamines comprenant entre 4 et 30 atomes de carbone, des composés d'ammonium quaternaire dérivés d'acylamines, des acylamides cationiques, des aminoacides conjugués à un groupe acyle, etc. et leurs mélanges.
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