WO2015007337A1 - Procédé de vaccination contre le vih - Google Patents

Procédé de vaccination contre le vih Download PDF

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WO2015007337A1
WO2015007337A1 PCT/EP2013/065289 EP2013065289W WO2015007337A1 WO 2015007337 A1 WO2015007337 A1 WO 2015007337A1 EP 2013065289 W EP2013065289 W EP 2013065289W WO 2015007337 A1 WO2015007337 A1 WO 2015007337A1
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xaa
seq
amino acid
peptide
arg
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PCT/EP2013/065289
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Lars H. HØIE
Anker LUNDEMOSE
Mats ÖKVIST
Arnt Ove HOVDEN
Maja Sommerfelt GRØNVOLD
Vidar Wendel HANSEN
Birger SØRENSEN
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Bionor Immuno As
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Priority to PCT/EP2013/065289 priority Critical patent/WO2015007337A1/fr
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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
    • 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
    • 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
    • C07K14/08—RNA viruses
    • C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
    • C07K14/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • C07K14/16—HIV-1 ; HIV-2
    • C07K14/161—HIV-1 ; HIV-2 gag-pol, e.g. p55, p24/25, p17/18, p7, p6, p66/68, p51/52, p31/34, p32, p40
    • 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
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2740/16034—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
    • 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
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to methods for treating reducing and/or delaying pathological effects of human immunodeficiency virus I (HIV) in a human infected with HIV or for reducing the risk of developing acquired immunodeficiency syndrome (AIDS) in a human.
  • HIV human immunodeficiency virus I
  • AIDS acquired immunodeficiency syndrome
  • Vacc-4x is a peptide-based therapeutic vaccine for human immunodeficiency virus (HIV)- l infection that aims to improve and sustain immune responses to conserved domains on the HIV- 1 core protein p24 Gag .
  • HIV human immunodeficiency virus
  • This rationale is based on observations that cell-mediated immune responses to Gag are associated with virus control and delayed disease progression [1] .
  • the Vacc-4x peptides are modified by amino acid substitution to improve antigen processing and presentation on diverse human leukocyte antigens (HLA) .
  • HLA human leukocyte antigens
  • the domains of p24 Gag represented in Vacc-4x are largely synonymous with the conserved immunologically vulnerable 'Sector 3' region critical for virus assembly, and where immune escape comes at a fitness cost [2] .
  • Vacc-4x consists of four modified peptides administered intradermally (i .d) using recombinant human granulocyte-macrophage colony stimulating factor (rhu-GM-CSF) as a local adjuvant.
  • Rhu-GM-CSF granulocyte-macrophage colony stimulating factor
  • Rhu-GM-CSF is used because it is a growth and differentiation factor for epidermal Langerhans cells and has adjuvant properties for the induction of immune responses [3] .
  • Vacc-4x has been found to be safe and well tolerated in previous smaller phase I [4] and II [5] clinical trials in HIV-infected subjects in Norway. It was shown to be immunogenic, to sustain CD4 T-cell counts, induce durable memory [6] and a transient reduction in viral load (VL) compared to preART values on treatment interruption [7] . However, these previous studies lacked placebo controls. Originally, this study was intended to be a pivotal placebo-controlled phase IIB Test of Concept trial enrolling 345 subjects. However, the inclusion criteria with respect to the pre- study CD4 count and CD4 nadir were raised in consideration of the SMART study [8] . Consequently, results from this study and the earlier phase I and II trials cannot be directly compared . Due to slow recruitment, this study was amended and reduced in size. It was thereafter redefined by the FDA as an exploratory phase II study for hypothesis-generating purposes only.
  • This study nevertheless represents one of the largest exploratory, prospective, randomized, double blind placebo-controlled phase II clinical trials for a therapeutic HIV vaccine recruiting 135 subjects (ITT-intention to treat) from 18 clinical trial sites in 4 European countries (Germany, UK, Spain and Italy) and the United States.
  • the primary objectives were to evaluate the effect of Vacc-4x immunizations versus placebo on CD4 counts, T-cell function and the response to interruption of ART (change in CD4 counts, rate of CD4 decline, VL, and the proportion of subjects restarting ART) .
  • the criteria for restarting ART were based on the ACTG 5197 clinical study of the Merck Adenovirus vectored preventative vaccine candidate Ad 5 [9] .
  • the secondary objectives were to evaluate the safety and tolerability of Vacc-4x as well as immunogenicity.
  • HLA human immunodeficiency virus I
  • AIDS acquired immunodeficiency syndrome
  • human subjects carrying the HLA B35 allele, associated with disease progression are surprisingly subject and responsive to a treatment with a composition that elicit a cell-mediated immune response in a subject, such as a composition comprising one or more peptides, such as HIV-specific peptides.
  • the present invention relates to a method for reducing and/or delaying pathological effects of human immunodeficiency virus I (HIV) in a human infected with HIV, or for reducing the risk of developing acquired immunodeficiency syndrome (AIDS) in a human, the method comprising the steps of: a) Determining the HLA genotype of said human and selecting said human carrying a HLA B35 epitope; b) administering to said human selected under step a) a composition comprising at least one peptide, such as a HIV-specific peptide, that elicit a cell-mediated immune response in a subject.
  • HIV human immunodeficiency virus I
  • AIDS acquired immunodeficiency syndrome
  • Figure 4 ELISPOT positive and negative responders to p24Gag peptide pool in relation to viral load for the Off-ART-Completer subgroup. P values were determined by Mann- Whitney U-Test. ⁇ denotes statistical significance, ns denotes not statistically significant.
  • FIG. 5 Viral load over time in Vacc-4x HLA B-35 positive and Vacc-4x non-HLA B-35 per protocol patients including long term follow-up (LTFU). Median values are shown with interquartile range. For the analysis, all viral load values after ART resumption were excluded. * No statistically significant difference in VL between groups (Mann- Whitney U-test).
  • VL Upon cessation of ART, VL normally peaks before host immune control establishes a steady state where a set point can be determined. In this study, VL peaked at approximately week 35-37, where the peak VL for the Vacc-4x group was lower than in the placebo group, but not significantly so. A lower peak VL could improve safety by reducing exposure to transient high level VL.
  • Peptide modification aims to increase HLA breadth and to improve uptake of peptides for presentation to T-cells.
  • a higher proportion of Vacc-4x subjects carried HLA B35, an allele associated with disease progression, which may in part explain the higher preART viral loads in this group [19] . These subjects nevertheless showed a reduction in VL from their preART set point compared to placebo (Table 4), ( Figure 5).
  • T-cell responses were measured ex vivo using IFN- ⁇ ELISPOT, proliferation and ICS. Since Vacc-4x aims to improve immune responses to immunologically vulnerable conserved domains on HIV-1 p24 Gag , responses to these conserved regions represented the major focus. T-cell responses were found to increase at weeks 44 and 52 (off ART) as a result of viral rebound. However the quality of immune responses was different in that ELISPOT responders in the Vacc-4x group had a lower viral load than ELISPOT responders in the placebo group. Earlier studies have emphasized quality versus quantity regarding effective immune responses to HIV [21] . Previous observations from the previous phase II study showed very low immune escape in these conserved regions in subjects that had received Vacc-4x [22] .
  • Vacc-4x was found to be immunogenic by inducing proliferative responses in both CD4 and CD8 T-cell populations (Table 5) even though the definition of positive was more stringent than in other studies [23] .
  • ICS showed greater cytokine expression in CD8+ T-cells compared to CD4+ T-cells and a tendency to higher polyfunctional T-cells amongst the Vacc-4x subjects where the main cytokines released were TNF-a and IFN- ⁇ .
  • Vacc-4x was found to be generally safe and well tolerated. Since the placebo was water alone, adverse events were assessed for Vacc-4x/GM-CSF.
  • HLA subtypes B27 associated with virus control
  • HLA B35 associated with disease progression
  • HLA-B57 associated with virus control
  • HLA-A2 intermediate control
  • Table 6 Peptide modification to extend the HLA repertoire. Modification adds HLA B35, B8, Al l epitopes not found in the natural sequence. Data from functional peptide/MHC binding assays carried out using the Prolmmune Ltd . (Oxford, UK), REVEALTM MHC-peptide Binding Assay.
  • the HLA class I tested comprised the most common Class I alleles; A01 : 01, A02 : 01, A03:01, All:01, A24: 02, A29: 02, B07 : 02, B08:01, B14: 02, B15 : 01, B27: 05, B35 : 01, B40: 01.
  • Human retroviruses that may be treated in accordance with the invention includes:
  • HTLV-1 originally called human T-cell leukaemia virus - now called human T-lymphotropic virus
  • HTLV-II human T-cell leukaemia virus also now called human T-lymphotropic virus
  • HIV-1 (earlier also called LAV - lymphadenopathy virus, also called Human T-lymphotropic virus III i.e. HTLV-III); and
  • HIV- 2 has also been called HTLV-IV; as well as any subtype of any of the above viruses.
  • HIV generally denotes human immunodeficiency virus 1.
  • HIV disease is composed of several stages including the acute HIV infection which often manifests itself as a flu-like symptoms and the early and medium stage symptomatic disease, which has several non-characteristic symptoms such as skin rashes, fatigue, night sweats, slight weight loss, mouth ulcers, and fungal skin and nail infections. Most HIV infected will experience mild symptoms such as these before developing more serious illnesses. It is generally believed that it takes five to seven years for the first mild symptoms to appear. As HIV disease progresses, some individuals may become quite ill even if they have not yet been diagnosed with AIDS (see below), the late stage of HIV disease.
  • AIDS is the late stage HIV disease and is a condition which progressively reduces the effectiveness of the immune system and leaves individuals susceptible to opportunistic infections and tumors.
  • gpl20 When using the term “gpl20” herein is meant the « 120 kDa N-terminal glycoprotein enzymatic cleavage product of gpl60, which in turn is the sole expression product of the HIV env gene. gpl20 forms the "spikes" on infective HIV virions and is non-covalently bound to gp41.
  • gp41 denotes the «41 kDa glycoprotein C-terminal enzymatic cleavage product of gpl60. gp41 is located in the membrane of HIV infected cells or virions. gp41 has an N-terminal transmembrane domain which binds non-covalently to gpl20. This transmembrane domain is termed “the transmembrane domain of gp41” or "tm-gp41” herein. The term includes within its scope naturally occurring mutated versions of the sequence as e.g . those set forth in Formula III. "C5" or the "C5 domain” denotes the 13 C-terminal amino acid residues of gpl20.
  • C2 or the “C2 domain” denotes a conserved region in gpl20. Regions in C2 form an antiparallel ⁇ -sheet with C5 in the inner proximal domain of gpl20.
  • Reducing and/or delaying pathological effect of HIV is in the present context meant to denote that use of the methods of the invention provides for a statistically significant reduction and/or delay in morbidity seen in individual infected with HIV which are treated according to the present invention. That is, the time of onset of manifest disease symptoms characterizing AIDS is later compared to non-treated controls and/or the number of pathological manifestations is reduced compared to controls not receiving the treatment of the present invention.
  • the expression "association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20” means that C5 can interact non- covalently with both or one of the tm-g41 and C2. The interaction with tm-gp41 is intermolecular, whereas the interacation with C2 is intramolecular.
  • An "agent capable of stabilising" association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 is a composition of matter which prevents or statistically reduces release of C5 from its intermolecular binding to gp41 and/or from its intramolecular binding to C2.
  • an agent is any substance of matter capable of exerting this effect, but important examples are antibodies, antibody fragments, and antibody analogues.
  • an agent according the present invention is an agent according the present invention - the precise molecular fomat is less important than the binding characteristics, and it is according to the invention also possible that such an agent may be a receptor or a receptor analogue, but also small molecule stabilisers are capable of functioning as an agen of the present invention.
  • the term "antibody” herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g ., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity, i .e. to function as an agent described above.
  • Various techniques relevant to the production of antibodies are provided in, e.g. , Harlow, et al ., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .Y., ( 1988) .) .
  • antibody fragment or antibody analogue comprises a portion of a full-length antibody, preferably antigen-binding or variable regions thereof. Examples of antibody
  • fragments/analogues include Fab, Fab', F(ab) 2 , F(ab') 2 , F(ab) 3 , Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CHI domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V- NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g ., Ill et al., Protein Eng 1997; 10: 949-57) ; camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional
  • antibody derivative comprises a full-length antibody or a fragment of an antibody, preferably comprising at least antigen-binding or variable regions thereof, wherein one or more of the amino acids are chemically modified, e.g. , by alkylation, PEGylation, acylation, ester formation or amide formation or the like, e.g ., for linking the antibody to a second molecule.
  • a “conjugate” as used herein comprises an agent to be used according to the invention such as an antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, etc.
  • a conjugate may be constituted of covalently linked peptides (an example of a conjugate is a fusion peptide comprising two peptides linked via peptide bonds so that the conjugate in that case may be an expression product from a nucleic acid fragment), but a conjugate can also be a combination of peptides covalent linked via chemical conjugation (a traditional example is conjugation using glutaraldehyde) .
  • Another example of a more complex conjugation is the example where an agent or peptide combination or other chemical substance of the present invention is linked to a carrier molecule, which in turn i coupled to other agents, peptide combinations or other chemical substances of the present invention (e.g . when such chemical substances are bound to a poly-lysine carrier (a lysine "tree")) .
  • a carrier molecule which in turn i coupled to other agents, peptide combinations or other chemical substances of the present invention (e.g . when such chemical substances are bound to a poly-lysine carrier (a lysine "tree")) .
  • humanized antibody is a human/non-human chimeric antibody that contains a minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity.
  • donor antibody such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin sequence.
  • the humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • An antibody having a "biological characteristic" of a reference antibody is one that possesses one or more of the biological characteristics of that antibody that distinguish it from other antibodies that bind to the same antigen.
  • peptide is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypep- tides of more than 100 amino acid residues.
  • amino acids in peptides it is intended that the amino acids are L-amino acids, unless other information is provided. Amino acids are referred to by their standard three letter or one letter designations unless otherwise stated .
  • Some unusual amino acids referred to herein includes homoarginine usually abbreviated by Har, norleucine usually abbreviated as Nle, Nl, or Nleu, ⁇ - ⁇ -methylated Lys usually abbreviated Lys(Me), Citrulline usually abbreviated Cit or with the single letter "B”, diaminopropionic acid usually abbreviated with Dpr and serinyl diaminopropionic acid usually abbreviated Dpr(Ser).
  • Har norleucine usually abbreviated as Nle, Nl, or Nleu
  • ⁇ - ⁇ -methylated Lys usually abbreviated Lys(Me)
  • Citrulline usually abbreviated Cit or with the single letter "B”
  • diaminopropionic acid usually abbreviated with Dpr and serinyl diaminopropionic acid usually abbreviated Dpr(Ser).
  • a “protein” is intended to denote a functional biomolecule comprising at least one peptide; when comprising at least two peptides, these may form complexes, be covalently linked, or may be non-covalently linked.
  • the polypeptide(s) in a protein can be glycosylated and/or lipidated and/or comprise prosthetic groups.
  • a "peptide combination” denotes one single molecule (a peptide multimer) or a mixture of molecules, where the single molecule or mixture includes at least two distinct peptides in a non-natural configuration relative to each other; the peptides may be from the same or different proteins..
  • a “peptide multimer” denotes a molecule which is constituted by at least two peptides in a non-natural configuration relative to each other. Examples are peptides from the same or from different proteins which are covalently linked via the side chains of at least one of their amino acids, or which are linked via their termini (e.g . via peptide bonds) but in a
  • peptide peptide multimers are detailed below. It will be understood that all peptide combinations of the present invention that are constituted by one single molecule are peptide multimers.
  • a “variant” or “analogue” of a peptide refers to a peptide having an amino acid sequence that is substantially identical to a reference peptide, typically a native or “parent” polypeptide.
  • the peptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.
  • Constant amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties.
  • Families of amino acid residues having similar side chains are known in the art, and include amino acids with basic side chains (e.g ., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g ., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g ., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g ., tyrosine, phenylalanine, tryptophan, histidine) .
  • basic side chains e.g lysine, arginine, histidine
  • acidic side chains e.
  • the acidic amino acids aspartic acid and glutamic acid may be substituted with amino acid residues having the side chains -(CH 2 ) n COOH, where n> 2.
  • a "retro form" of a peptide is a form of a peptide where the order of the amino acids in N- to C-terminal direction has been inverted.
  • the retro form of ALDFR is the peptide RFDLA.
  • an "inverso" form is characterized by the fact that each amino acid in the inverso form is in the opposite stereochemical configurational compared to the corresponding amino acid in the peptide. So, if the peptide is composed of L-amino acids, the inverso form is composed of D- amino acids.
  • a "retro-inverso" form of a peptide is a form of a peptide which is both an inverso form and a retro form.
  • the retro-inverso form of L-ala - L-Arg - L-Lys is D-Lys - D-Arg - D-ala.
  • substantially identical in the context of two amino acid sequences means that the sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 95, at least about 98, or at least about 99 percent sequence identity. In one embodiment, residue positions that are not identical differ by conservative amino acid substitutions.
  • Sequence identity is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • the publicly available GCG software contains programs such as "Gap” and "BestFit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g. , GCG Version 6.1. Polypeptide sequences can also be compared using FA ST A or ClustalW, applying default or recommended parameters.
  • FASTA2 and FASTA3 provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol . 1990; 183 : 63-98; Pearson, Methods Mol . Biol.
  • subsequence in general means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally occurring amino acid sequence or nucleic acid sequence, respectively.
  • the subsequence may be as short as 1 or 2 amino acids. This is because the inventive peptide combinations include amino acids from different peptide domains, where the amino acids together at least form a conformational epitope for an antibody.
  • such a conformational epitope could be composed of 4 amino acids from C5, but only 1 or 2 from tm-gp41 - the imporant point is here that this combined epitope from 2 domains is capable of being stabilised, i .e. that antibody binding to the same epitope in vivo will stabilise the configuration between C5 and tm-gp41 and/or C2.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome-binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
  • an "isolated" molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs (i .e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g., peptide, in the composition) .
  • composition of an antibody molecule will exhibit 98% - 99% homogeneity for antibody molecules in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use.
  • treatment refers to preventing, alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context.
  • treatment of a patient in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy, whereas “treatment” of a patient in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or prophylactic therapy.
  • antigen denotes a substance of matter which is recognized by the immune system's specifically recognizing components (antibodies, T-cells) .
  • immunogen is in the present context intended to denote a substance of matter, which is capable of inducing an adaptive immune response in an individual, where said adaptive immune response targets the immunogen.
  • an immunogen will induce antibodies that react with the immunogen .
  • an immunogen is an antigen, which is capable of inducing immunity.
  • epitope the region in an antigen or immunogen which is recognized by antibodies (in the case of antibody binding epitopes, also known as "B-cell epitopes") or by T-cell receptors when the epitope is complexed to an MHC molecule (in the case of T-cell receptor binding epitopes, i .e. "T-cell epitopes”) .
  • immunogenically effective amount has its usual meaning in the art, i.e. an amount of an immunogen, which is capable of inducing an immune response, which significantly engages pathogenic agents, which share immunological features with the immunogen.
  • vaccine is used for a composition comprising an immunogen and which is capable of inducing an immune response which is either capable of reducing the risk of developing a pathological condition or capable of inducing a therapeutically effective immune response which may aid in the cure of (or at least alleviate the symptoms of) a pathological condition.
  • T helper lymphocyte epitope (a T H epitope) is peptide, which binds an MHC Class II molecule and can be presented on the surface of an antigen presenting cell (APC) bound to the MHC Class II molecule.
  • an “immunological carrier” is generally a substance of matter which includes one or many T H epitopes, and which increase the immune response against an antigen to which it is coupled by ensuring that T-helper lymphocytes are activated and proliferate.
  • immunological carriers are the tetanus and diphtheria toxoids and keyhole limpet hemocyanin (KLH) .
  • KLH keyhole limpet hemocyanin
  • adjuvant has its usual meaning in the art of vaccine technology, i.e. a substance or a composition of matter which is 1) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen.
  • HLA B35 (B35) allele refers to any B35 serotype of the human leukocyte antigen (HLA) system.
  • the serotype identifies the HLA-B35 gene products, including any known nucleotide variants and polypeptide isoforms. This includes but is not limited to the HLA B35 alleles 3501, 3502, 3503, 3504, 3505, 3506, 3508, 3509, 3510, 3511, 3512, 3514, and 3520.
  • C I cell-mediated immunity
  • Vaccination aims to stimulate the immune response to a specific pathogen in advance of infection. When an individual is exposed to that pathogen, a memory response is triggered which prevents the establishment of infection. Vaccines therefore stimulate the adaptive immune response which unlike innate immunity, is long lived and has memory. There are two major arms to the adaptive immune system .
  • Humoral immunity which involves the development of antibodies that can bind virus particles and certain antibodies that can neutralize infection.
  • Cell mediated immunity that leads to the development of cytotoxic T- cells that kill infected cells exposing viral epitopes in the context of human leukocyte antigen (HLA) class I, in this way eliminating infected cells.
  • a peptide that elicits a cell-mediated immune response refers to any peptide that elicits an activation of antigen-specific cytotoxic T-lymphocytes. These peptides elicit a Cytotoxic T-lymphocyte immune (CTL) response that leads to the development of cytotoxic T-cells that kill infected cells exposing viral epitopes in the context of human leukocyte antigen (HLA) class I, in this way eliminating infected cells.
  • CTL Cytotoxic T-lymphocyte immune
  • HLA human leukocyte antigen
  • CLI Cytotoxic T-lymphocyte immune
  • HTL inducing peptide may be a helper T lymphocyte (HTL) inducing peptide comprising HTL epitopes.
  • HTL inducing peptide is a HLA Class II binding peptide that is capable of inducing a HTL response.
  • the peptides may in other embodiments be CTL inducing peptides comprising CTL epitopes in addition to or as an alternative to being a HTL inducing peptide.
  • CTL inducing peptide is a HLA Class I binding peptide that is capable of inducing a CTL response.
  • a peptide that elicit a cell-mediated immune response as used according to the present invention includes but is not limited to any peptide described in any one of international patent applications WO0052040, WO 2012/092934 or WO 2012/072088, which patent applications are hereby incorporated by reference.
  • compositions used according to the present invention comprise one or more peptide that elicits a cell-mediated immune response.
  • this peptide is at least one HIV-specific peptide.
  • HIV-specific peptides based on conserved regions of HIV gag p24, antigens in free or carrier- bound form comprising at least one of the said peptides.
  • the HIV-specific peptides to be used according to the present invention may originate from conserved areas of the HIV-1 core protein p24, having the properties of maintaining the uniqueness (sensitivity and specificity) of the HIV-l-epitope. Further the new peptides to be used according to the invention possess no recognized cytotoxic T lymphocyte (CTL) antagonistic effect and shall have at least one potential CTL epitope.
  • CTL cytotoxic T lymphocyte
  • the HIV-specific peptides, to be used according to the present invention, and which have met the above criteria are selected from the following groups;
  • Xaa in position 1 of the peptide derivate is Lys or Arg
  • Xaa in position 2 is Ala, Gly, Ser or Arg,
  • Xaa in position 3 is Leu or Met
  • Xaa in position 4 is Gly or Arg
  • Xaa in position 5 is Pro, Thr, Val, Ser, Gin or Ala,
  • Xaa in position 6 is Gly, Ala, Lys, Arg, Gin or Glu,
  • Xaa in position 8 is Thr or Ser
  • Xaa in position 9 is Leu or He
  • Xaa in position 14 is Thr, Ser or Val
  • Xaa in position 15 is Ala or Ser
  • Xaa in position 16 is Cys or Ser
  • Xaa in position 17 is Gin or Leu
  • Xaa in position 18 is Gly, Glu or Arg, and
  • Xaa in position 20 is Gly or Arg ;
  • Xaa in position 2 is Trp, Gly, Lys or Arg,
  • Xaa in position 3 is He, Leu, Val or Met
  • Xaa in position 4 is He, Val or Leu
  • Xaa in position 12 is Arg, Lys
  • Xaa in postion 13 is Met or Leu
  • Xaa in position 15 is Ser, Cys or Gin
  • Xaa in position 17 is Thr, Val, He, Ser or Ala
  • Xaa in position 18 is Ser, Gly or Thr,
  • Xaa in position 21 is Asp, Glu, Cys or Gly,
  • Xaa in position 22 is Gly or none
  • n 0, 1,2 or 3;
  • Xaa in position 2 is Asn, Ala or Lys
  • Xaa in position 3 is Pro, Gin, Gly, He or Leu
  • Xaa in position 7 is Val or Ala
  • Xaa in position 8 is Gly or Lys
  • Xaa in position 9 is Glu, Asp, Lys, Phe or Thr
  • Xaa in position 10 is He, Met, Val or Leu
  • Xaa in position 11 is Tyr, Leu or none
  • Xaa in position 12 is Ser or none
  • Xaa in position 13 is Arg or none
  • Xaa in position 14 is Asp, Arg, Trp, Ala or none
  • Xaa in position 15 is He or none Xaa in position 16 is Tyr or none
  • Xaa in position 17 is Lys or Arg
  • Xaa in position 18 is Arg, Lys or Asp
  • Xaa in position 19 is Trp or Gly
  • Xaa in position 20 is lie, Met, Val, Gin or Ala
  • Xaa in position 21 is He, Val or Ala
  • Xaa in position 22 is Leu, Met or Val
  • Xaa in position 23 is Gly or Cys
  • Xaa in position 24 is Leu or none
  • n 1,2 or 3
  • Xaa in position 2 is Glu, Arg, Phe or Lys
  • Xaa in position 5 is Pro or Thr
  • Xaa in position 6 is Met, Thr or NIeu
  • Xaa in position 7 is Phe or Leu
  • Xaa in position 8 is Ser, Thr, Ala or Met
  • Xaa in position 9 is Ala, Glu or Leu
  • Xaa in position 11 is Ser or none
  • Xaa in position 12 is Ala, Arg or none
  • Xaa in position 13 is He, Leu or none
  • Xaa in position 14 is Ser, Ala, Leu or none
  • Xaa in position 15 is Tyr, Glu or Asp
  • Xaa in position 16 is Gly or Asp
  • Xaa in position 17 is Ala or Leu
  • Xaa in position 18 is Thr, He, Val, Leu or Asn,
  • Xaa in position 19 is Pro, Thr or Ser
  • Xaa in position 20 is Tyr, Phe, NIeu, His or Gin
  • Xaa in position 21 is Asp, Asn, Leu or Ala
  • Xaa in position 22 is Leu, He, Val or Asn
  • Xaa in position 23 is Asn, Tyr, Cys or Gly
  • Xaa in position 24 is Thr, Met, lie, Ala, Val or none
  • Xaa in postion 25 is Gly or none
  • each HIV specific peptide may be free carboxyl- or amino groups, amides, acyls, acetyls; or salts of any of the HIV specific peptides.
  • the HIV-specific peptide sequences have the potential to serve as a good antigen wherein the antigen comprises at least one peptide selected from the group of sequences of SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 55 or SEQ ID NO: 61.
  • the antigenicity may be adapted through adjusting the ratio or concentration of different peptides or size of the peptides by for instance dimerisation or polymerisation and/or immobilisation to a solid phase.
  • the antigen comprises two or more polypeptide sequences, to be used according to the invention, which are either linked by a bridge for instance a disulphide bridge between the Cys residues of the chains or bridges like Ci-C 8 alkylen possibly intervened by one or more heteroatoms like O, S, or N or preferably they are unlinked.
  • the chains may be immobilized to a solid phase in monomeric, dimeric or oligomeric forms. Further amino acids may be added to the ends in order to achieve an «arm» to facilitate immobilization. All amino acids in the HIV-specific peptides to be used according to the invention can be in both D- or L-form, although the naturally occurring L- form is preferred .
  • the C- and N-terminal ends of the HIV-specific peptide sequences could deviate from the natural sequences by modification of the terminal NH 2 -group and/or COOH-group, they may for instance be acylated, acetylated, amidated or modified to provide a binding site for a carrier or another molecule.
  • the HIV-specific peptides to be used according to the invention are consisting of 6 to 50 amino acids, preferably between 10 and 30 amino acids. They are covering all natural variation of amino acids in the identified positions.
  • the polypeptide antigen to be used according to the invention is either in a free or in a carrier-bound form.
  • the carrier or solid phase to which the peptide is optionally bound can be selected from a wide variety of known carriers. It should be selected with regard to the intended use of the immobilized polypeptide as a diagnostic antigen or as an immunizing component in a vaccine.
  • Examples of carriers that can be used for e.g. diagnostic purposes are magnetic beads or latex of co-polymers such as styrene-divinyl benzene, hydroxylated styrene-divinyl benzene, polystyrene, carboxylated polystyrene, beads of carbon black, non-activated or polystyrene or polyvinyl chloride activated glass, epoxy-activated porous magnetic glass , gelatine or polysaccharide particles or other protein particles, red blood cells, mono- or polyclonal antibodies or fab fragments of such antibodies.
  • co-polymers such as styrene-divinyl benzene, hydroxylated styrene-divinyl benzene, polystyrene, carboxylated polystyrene, beads of carbon black, non-activated or polystyrene or polyvinyl chloride activated glass, epoxy-activated porous magnetic glass
  • RFIIPNIFTALSGGRRALLYGATPYAIG (SEQ ID NO: 64) (Nl in position 6 is Norleucine), or salts thereof, particularly acetate salts.
  • RFIIPNIeFTALSGGRRALLYGATPYAIG-NH 2 (SEQ ID NO: 64) (Nle in position 6 is Norleucine) or are salts thereof, particularly acetate salts. (In this application also referred to in the examples as Vacc-4x) .
  • One of the sequences contains a B-cell epitope and will activate the humoral immune system, whereas the other sequences contribute with CTL-epitopes and the amino acid changes implemented within the frame of the CTL-epitope are designed to achieve enhanced binding .
  • Other amino acid changes have been conducted in order to facilitate the synthesis of the peptide and/or increase the solubility of the peptide.
  • Compound to stimulate the humoral immunity in a subject such as C5 related compounds:
  • the present invention also relates to the use of compounds that stimulate the humoral immunity in a subject.
  • Humoral immunity involves the development of antibodies that can bind virus particles and certain antibodies that can neutralize infection.
  • a peptide that stimulate the humoral immunity in a subject as used according to the present invention includes but is not limited to any peptide described in any one of international patent applications WO2011/000962, WO0052040, WO 2012/092934 or WO 2012/072088, which patent applications are hereby incorporated by reference.
  • these compounds are agents capable of stabilising the association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.
  • these compounds are immunogens, which induces antibodies that stabilise association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.
  • One aspect of the invention relates to methods for reducing and/or delaying pathological effects of human retrovirus infection, such as immunodeficiency virus I (HIV) in a human infected with such virus, such as HIV, the method including administering an effective amount of an agent capable stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.
  • human retrovirus infection such as immunodeficiency virus I (HIV)
  • HIV immunodeficiency virus I
  • Another aspect is much similar, but relates to methods of reducing the risk of developing acquired immunodeficiency syndrome (AIDS), the method including administering an effective amount of an agent capable of stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.
  • AIDS acquired immunodeficiency syndrome
  • retrovirus infections such as HIV infected individuals with agents, which can mimic the antibodies which according to the present invention are characteristic for HIV infected long-term non-progressors - this is the most straightforward therapeutic utilisation of the findings underlying the present invention.
  • the one aspect aims at reducing pathological effects of retrovirus infections, such as HIV or prolonging the time it takes to develop manifest AIDS
  • the other aspect aims at reducing the risk of developing AIDS altogether and may therefore be used in individuals which are currently treated prophylactically with antiretroviral therapy.
  • the agent in these first aspects of the invention is a molecule comprising at least one amino acid sequence selected independently from an amino acid sequence derived from the transmembrane domain of gp41 and an amino acid sequence derived from the C2 domain, wherein the at least one amino acid sequence binds the C5 domain and optionally comprises at least one D-amino acid; in certain embodiments all the amino acids in the amino acid sequence are D-amino acids.
  • the molecule is preferably a peptide, and in certain embodiments this peptide consists of the at least one amino acid sequence.
  • the amino acid sequences typically include at most 10 amino acid residues, such as at most 9, at most 8, at most 7, at most 6, and at most 5 amino acid residues.
  • Preferred molecules are therefore peptides having 4, 5, 6, 7, 8, 9, or 10 amino acid residues.
  • Specific embodiments of the at least one molecule are therefore the peptides having or comprising SEQ ID NO: 34, 35, 36, 37, 39, 40, 42, 43 and 45, which may all be composed partly or entirely of D-amino acids.
  • molecules comprising peptides having Formula III are interesting embodiments of the at least one molecule.
  • the agent in these first aspects of the invention is selected from an antibody, an antibody fragment or an antibody analogue.
  • the antibody may be a fully human antibody, a humanized antibody, or a chimeric antibody, or a derivative thereof.
  • the antibody is an IgA, an IgD, an IgG, an IgE or an IgM - the antibody may be both monoclonal and polyclonal.
  • the antibody fragment is typically selected from a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab)2 fragment, a F(ab')2 fragment, an Fv fragment, a Heavy chain Ig (a llama or camel Ig), a V HH fragment, a single domain FV, and a single-chain antibody fragment, and the antibody analogue is typically selected from a scFV, a dsFV, a minibody, a diabody, a triabody, a kappa body, an IgNAR, a tandAb, a BiTE, and a multispecific antibody.
  • the agent binds to and stabilises association between one or more amino acid residues in the amino acid stretch
  • TZ 1 AKRRVVZ 2 REKR where Z 1 is K, R or E and where Z 2 is Q or E, and one or more amino acid residues in an amino acid stretch in the transmembrane domain of gp41 and/or in the constant C2 domain of gpl20.
  • This amino acid stretch from C5 is highly conserved across the multiple HIV clades known and effective interaction with this stretch by the agent is therefore believed to be highly advantageous.
  • a further aspect of the invention relates to a method for reducing the risk of or reducing and/or delaying pathological effects of human immunodeficiency virus I (HIV) in a human infected with HIV, the method including administering an effective amount of an immunogen, which induces antibodies that stabilise association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20, whereas other aspects relates to a prophylactic method using the same means.
  • one aspect relates to therapeutic active immunotherapy
  • another aspect relates to prophylactic immunotherapy of HIV disease, including AIDS. This also entails prophylaxis of HIV infection.
  • the immunogen is selected from a peptide combination detailed below when discussing these aspects of the invention, a composition detailed below, a nucleic acid fragment discussed in relation to other aspects, a virus or plasmid vector compositions discussed elsewhere.
  • the targeted association between the C5 domain and C2 and/or the transmembrane domain of gp41 involves at least one amino acid in the sequence TZ'AKRRVVZ 2 REKR, where Z 1 is K, R or E and where Z 2 is Q or E and an amino acid and involves at least one amino acid in the transmembrane domain of gp41 or at least one amino acid in the constant C2 domain of gpl20.
  • this particular sequence is extremely well-conserved across known HIV clades, and therefore it is the interaction between this sequence and tm-gp41 or C2 it is most feasible to target.
  • compositions comprising (1) a peptide combination, said multimer comprising a first peptide comprising the amino acid sequence of the 13 amino acid residue amino acid sequence of the C5 domain of HIV gpl20 including between 0 and 4 amino acid substitutions, a subsequence thereof, or an amino acid sequence comprising the inverso-, retro- or retro- in verso form of said amino acid sequence or subsequence, and at least one second peptide comprising an amino acid stretch present in the transmembrane domain of gp41 or present in the constant C2 domain of gpl20 or comprising an amino acid stretch present in any one of SEQ ID NOs.
  • this aspect relates to peptide combinations which have a resemblance in 3 dimensions with the epitopes which characterise the interacting areas in C5 on the one hand and tm-gp41 and/or C2 on the other.
  • the peptide combinations to be used according to the invention are useful immunogens that can induce antibodies having the same characteristics as the antibodies found in HIV LTNP individuals, but the peptide combinations also are promissing as diagnostic/prognostic tools.
  • the inclusion of retro-, inverso-, and retro-inverso peptides i .a. enables production of proteolytically stable peptides as well as peptides are truly foreign compared to the HIV counterpart.
  • said first peptide comprises the amino acid sequence having formula I :
  • X 1 -X 2 -X 3 -X 4 -X 5 -X 5 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 (I) wherein X 1 is Thr, X 2 is selected from Lys, Arg, Har and Glu, X 3 is selected from Ala and Val, X 4 is selected from Arg, Har, Lys and Cit (citrulline), X s is selected from Arg, Har, Lys and Cit, X 6 is selected from Arg, Har, Lys and Cit, X 7 is selected from Val, Leu, He and NIe (norleucin), X s is selected from Val, Leu He and NIe, X 9 is selected from Gin, Glu, Asn and Asp, X 10 is selected from Arg, Har and Cit, X 11 is selected from Glu and Asp, X 12 is Lys, and X 13 is selected from Arg, Har and Cit,
  • the first peptide may further comprise the dipeptide Ala-Pro linked to the N-terminus of the amino acid sequence having formula I and/or the first peptide may further comprise the dipeptide X 14 -X 15 linked to the C-terminus of the amino acid sequence having formula I, wherein X 14 is selected from Ala and Val, and wherein X 15 is selected from Val, Leu and NIe.
  • Examples and constitute embodiments of a first peptide of the peptide combinations to be used according to the invention are provided.
  • a number of naturally occurring mutants of gp41 and gpl20 has been observed, so when stating that the second peptide comprises an amino acid stretch present in the
  • the at least second peptide when derived from gp41, is in certain embodiments one which includes the amino acid sequence having the formula:
  • Z 1 is Asp
  • Z 2 is Arg
  • Z 3 is Pro
  • Z 4 is Glu or Gly
  • Z 5 is Gly or Arg
  • Z 6 is He
  • Z 7 is Glu
  • Z 8 is Glu
  • Z 9 is Glu
  • Z 10 is Gly
  • Z 11 is Gly
  • Z 12 is Glu or is absent
  • Z 13 is Arg or Gin
  • Z 14 is Asp or Gly
  • Z 15 is Arg or Lys
  • Z 16 is Asp or Gly and Z 17 is Arg
  • this embodiment of the second peptide may contain amino acid substitutions which result in a sequence identity of at least 80% with a corresponding amino acid sequence found in gp41.
  • Particularly interesting peptides derived from C2 and gp41, and gpl 20 are set forth in the preamble to the Examples and constitute embodiments of a second peptide of the peptide combinations to be used according to the invention.
  • the first peptide and the at least one second peptide are associated via a linker;
  • the linker can be any peptide linker, such as a glycine, a lysine or an arginine linker, a polyhistidinyi tag, Protein G, and Protein A but it is also possible to use a bis-maleimide linker, a disulfide linker, or a polyethylene glycol (PEG) linker.
  • PEG polyethylene glycol
  • the invention contemplates the use of "simple" linear peptides which are conjugated or fused to each other, but also peptide combinations where the individual peptides derived from C5 and other regions of gpl20 or gp41 are linked via non-peptide linkers e.g . complementary nucleic acids, nucleic acid derivatives or analogues e.g . PNA, LNA.
  • linker types are also within the scope of the present invention, and it is e.g. also a part to be used according to the invention to utilise linear peptides which include intrachain disulphide linkers.
  • At least one of the first and at least one second peptides in the peptide combination comprises an N- or C-terminal modification, such as an amidation, acylation, or acetylation.
  • suitable amides included those having the formula -C(0)-NR x R y , wherein R x and R y are independently selected from hydrogen and Ci- 6 alkyl, which alkyl group may be substituted with one of more fluoro atoms, for example -CH 3 , -CH 2 CH 3 and -CF 3 , a particular amide group which may be mentioned is -C(0)NH 2 .
  • suitable acetylated N-terminal ends include those of formula -NH-C(0) R z , wherein R z is hydrogen, Ci- 6 alkyl, which alkyl group may be substituted with one of more fluoro atoms, for example -CH 3 , -CH2CH3 and -CF 3 , or phenyl .
  • the peptide combinations are contemplated as vaccine agents or diagnostic agents, they are in certain embodiments coupled to a carrier molecule, such as an immunogenic carrier.
  • a carrier molecule such as an immunogenic carrier.
  • the peptides of the peptide combinations may thus be linked to other molecules either as recombinant fusions (e.g. via CLIP technology) or through chemical linkages in an oriented (e.g. using heterobifunctional cross-linkers) or nonoriented fashion.
  • Linking to carrier molecules such as for example diphtheria toxin, latex beads (convenient in diagnostic and prognostic embodiments), and magnetic beads (also convenient in diagnostic and prognostic embodiments), polylysine constructs etc, are all possible carrier molecules to be used according to the invention.
  • the immunogenic carrier is conveniently selected from carrier proteins such as those conventionally used in the art (e.g . diphtheria or tetanus toxoid, KLH etc.), but it is also possible to use shorter peptides (T-helper epitopes) which can induce T-cell immunity in larger proportions of a population. Details about such T-helper epitopes can e.g. be found in WO 00/20027, which is hereby incorporated by reference herein - all immunolgic carriers and "promiscuous" (i .e. universal) T-helper epitopes discussed therein are useful as immunogenic carriers in the present invention.
  • carrier proteins such as those conventionally used in the art (e.g . diphtheria or tetanus toxoid, KLH etc.)
  • T-helper epitopes shorter peptides
  • Details about such T-helper epitopes can e.g. be found in WO 00/
  • the carrier is a virus like particle, i .e. a particle sharing properties with virions without being infectious.
  • virus-like particles may be provided chemically (e.g. Jennings and Bachmann Ann Rev Pharmacol . Toxicol . 2009. 49: 303-26 Immunodrugs: Therapeutic VLP-based vaccines for chronic diseases) or using cloning techniques to generate fusion proteins (e.g . Peabody et al. J . Mol . Biol . 2008; 380: 252-63. Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2) .
  • Remune an HIV vaccine originally made by Immune Response Corporation, which consists of formalin inactivated HIV that has been irradiated to destroy the viral genome.
  • the company was started by Jonas Salk who used the same technique to generate the killed polio vaccine in widespread use today.
  • gpl20 fell off leaving only gp41 on the virion surface. This opens for the possibility of directly admixing C5-derived peptides disclosed herein with emune particles, because it should still be possible to obtain the binding between C5 and gp41 on a Remune particle.
  • Embodiments of the aspect related to peptide combinations also include those wherein the first peptide is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, and 5 or a fragment thereof, or the inverso-, retro- or retro-inverso form of a peptides selected from SEQ ID NO: 1, 2, 3, 4, and 5 or a fragment thereof, and wherein the second peptide is selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 46 or a fragment thereof or the inverso-, retro- or retro-inverso form of a peptides selected from SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 46 or a fragment thereof.
  • the fragment may be very short, as long as the peptide combination provides for the ability to induce antibodies which will stabilise association between C5 and gp41 and/or C2.
  • a number of interesting peptide combinations of the present invention are listed in the Preamble to the Examples.
  • the peptide combination to be used according to the invention comprises at most 70 amino acids, such as the most 69, at most 68, at most 67, at most 66, at most 65, at most 64, at most 63, at most 62, at most 61, at most 60, at most 59, at most 58, at most 57, at most 56, at most 55, at most 54, at most 53, at most 52, at most 51, at most 50, at most 49, at most 48, at most 47, at most 46, at most 45, at most 44, at most 43, at most 42, at most 41 , at most 40, at most 39, at most 38, at most 37, at most 36, at most 35, at most 34, at most 33, at most 32, at most 31, at most 30, at most 29, at most 28, at most 27, at most 26, at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 70 amino
  • the peptide combination to be used according to the invention comprises at least 6 amino acid residues, such as at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at
  • the peptide combination to be used according to the invention consists of 6 amino acid residues or 7 amino acid residues or 8 amino acid residues or 9 amino acid residues or 10 amino acid residues or 11 amino acid residues or 12 amino acid residues or 13 amino acid residues or 14 amino acid residues or 15 amino acid residues or 16 amino acid residues or 17 amino acid residues or 18 amino acid residues or 19 amino acid residues or 20 amino acid residues or 21 amino acid residues or 22 amino acid residues or 23 amino acid residues or 24 amino acid residues or 25 amino acid residues or 26 amino acid residues or 27 amino acid residues or 28 amino acid residues or 29 amino acid residues or 30 amino acid residues or 31 amino acid residues or 32 amino acid residues or 33 amino acid residues or 34 amino acid residues or 35 amino acid residues or 36 amino acid residues or 37 amino acid residues or 38 amino acid residues or 39 amino acid residues or 40 amino acid residues or 41 amino acid residues or 42 amino acid residues or 43 amino acid residues or 44 amino acid residues or 45 amino acid residues
  • an immunogenic composition (such as a vaccine composition) comprising a composition described herein in combination with a pharmaceutically acceptable diluent or vehicle and optionally one or more immunological adjuvant.
  • the present invention relates to the use of one or more peptide that elicit a cell-mediated immune response, which is at least one HIV-specific peptide selected from the group of amino acid sequences:
  • Xaa in position 1 of the peptide derivate is Lys or Arg
  • Xaa in position 2 is Ala, Gly, Ser or Arg,
  • Xaa in position 3 is Leu or Met
  • Xaa in position 4 is Gly or Arg
  • Xaa in position 5 is Pro, Thr, Val, Ser, Gin or Ala,
  • Xaa in position 6 is Gly, Ala, Lys, Arg, Gin or Glu,
  • Xaa in position 8 is Thr or Ser
  • Xaa in position 9 is Leu or He
  • Xaa in position 14 is Thr, Ser or Val
  • Xaa in position 15 is Ala or Ser
  • Xaa in position 16 is Cys or Ser
  • Xaa in position 17 is Gin or Leu
  • Xaa in position 18 is Gly, Glu or Arg, and
  • Xaa in position 20 is Gly or Arg ;
  • Xaa in position 2 is Trp, Gly, Lys or Arg,
  • Xaa in position 3 is He, Leu, Val or Met
  • Xaa in position 4 is He, Val or Leu
  • Xaa in position 12 is Arg, Lys
  • Xaa in postion 13 is Met or Leu
  • Xaa in position 15 is Ser, Cys or Gin
  • Xaa in position 17 is Thr, Val, He, Ser or Ala
  • Xaa in position 18 is Ser, Gly or Thr,
  • Xaa in position 21 is Asp, Glu, Cys or Gly,
  • Xaa in position 22 is Gly or none
  • n 0, 1,2 or 3; Xaa; Xaa 2 Xaa 3 Pro He Pro Xaa - Xaa 8 Xaa g Xaa i0 Xaa u Xaa i2 [Gly],, Xaa 13 Xaa i4 Xaa i 5 Xaa i6 Xaai 7 Xaa i8 Xaa i 9 Xaa 20 Xaa 2: Xaa 22 Xaa 23 Xaa 2 (SEQ ID NO: 55) wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or none
  • Xaa in position 2 is Asn, Ala or Lys
  • Xaa in position 3 is Pro, Gin, Gly, He or Leu
  • Xaa in position 7 is Val or Ala
  • Xaa in position 8 is Gly or Lys
  • Xaa in position 9 is Glu, Asp, Lys, Phe or Thr
  • Xaa in position 10 is He, Met, Val or Leu
  • Xaa in position 11 is Tyr, Leu or none
  • Xaa in position 12 is Ser or none
  • Xaa in position 13 is Arg or none
  • Xaa in position 14 is Asp, Arg, Trp, Ala or none Xaa in position 15 is He or none
  • Xaa in position 16 is Tyr or none
  • Xaa in position 17 is Lys or Arg
  • Xaa in position 18 is Arg, Lys or Asp
  • Xaa in position 19 is Trp or Gly
  • Xaa in position 20 is He, Met, Val, Gin or Ala
  • Xaa in position 21 is He, Val or Ala
  • Xaa in position 22 is Leu, Met or Val
  • Xaa in position 23 is Gly or Cys
  • Xaa in position 24 is Leu or none
  • n 1,2 or 3
  • Xaa in position 2 is Glu, Arg, Phe or Lys
  • Xaa in position 5 is Pro or Thr
  • Xaa in position 6 is Met, Thr or NIeu
  • Xaa in position 7 is Phe or Leu
  • Xaa in position 8 is Ser, Thr, Ala or Met
  • Xaa in position 9 is Ala, Glu or Leu
  • Xaa in position 11 is Ser or none
  • Xaa in position 12 is Ala, Arg or none
  • Xaa in position 13 is He, Leu or none
  • Xaa in position 14 is Ser, Ala, Leu or none
  • Xaa in position 15 is Tyr, Glu or Asp
  • Xaa in position 16 is Gly or Asp
  • Xaa in position 17 is Ala or Leu
  • Xaa in position 18 is Thr, He, Val, Leu or Asn,
  • Xaa in position 19 is Pro, Thr or Ser
  • Xaa in position 20 is Tyr, Phe, NIeu, His or Gin
  • Xaa in position 21 is Asp, Asn, Leu or Ala
  • Xaa in position 22 is Leu, He, Val or Asn
  • Xaa in position 23 is Asn, Tyr, Cys or Gly
  • Xaa in position 24 is Thr, Met, He, Ala, Val or none
  • Xaa in postion 25 is Gly or none
  • amino acid sequence of SEQ ID NO :47 is selected from the groups of SEQ ID NO:48 and SEQ ID NO:49.
  • amino acid sequence of SEQ ID NO : 50 is selected from the groups of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54.
  • amino acid sequence of SEQ ID NO : 55 is selected from the groups of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60.
  • amino acid sequence of SEQ ID NO : 61 is selected from the groups of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.
  • the at least one HIV-specific peptide comprises at least , two, three, or four peptides selected from each of the groups of SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO: 55 and SEQ ID NO: 61.
  • the at least one HIV-specific peptide consist of or comprises the peptides of the SEQ ID NO:49, SEQ ID NO: 52, SEQ ID NO: 57 and SEQ ID NO: 64.
  • immunogenic compositions include the use of state-of-the-art constituents such as immunological adjuvants. Apart from these adjuvants, which are detailed below, immunogenic compositions are prepared as generally taught in the art:
  • vaccines which contain peptide sequences as active ingredients are generally well understood in the art, as exemplified by U.S. Patents 4,608,251 ; 4,601,903; 4,599,231 ; 4,599,230; 4,596,792; and 4, 578,770, all incorporated herein by reference.
  • such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared .
  • the preparation may also be emulsified .
  • the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines; cf. the detailed discussion of adjuvants below.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously, intracutaneously, intradermal ⁇ , subdermally or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral, buccal, sublinqual, intraperitoneal, intravaginal, anal, epidural, spinal, and intracranial formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
  • the peptides and peptide combinations may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of immunity desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 pg to 2,000 pg (even though higher amounts in the 1-10 mg range are contemplated), such as in the range from about 0.5 pg to 1,000 pg, preferably in the range from 1 pg to 500 pg and especially in the range from about 10 pg to 100 pg.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • immunogenic molecules described herein can be therefore be formulated with adjuvants:
  • the adjuvants - to be combined are known to induce humoral responses and include: i) Salt suspensions (e.g. varieties of salts containing aluminum ions or calcium ions), ii) Oil-in-water emulsions (e.g.. varieties of squalane-based or squalene-based emulsions), iii) Water-in-oil emulsions (e.g.. Montanide ISA51 or ISA720), iv) Neutral liposomes, v) Cationic liposomes, vi) Microspheres, vii) Immunostimulating complexes (e.g...).
  • Salt suspensions e.g. varieties of salts containing aluminum ions or calcium ions
  • Oil-in-water emulsions e.g.. varieties of squalane-based or squalene-based emulsions
  • Water-in-oil emulsions e.g.. Montanide ISA51
  • Pattern-recognition receptor agonists e.g.. agonists for C-type lectin receptors (CLRs), NOD- like receptors (NLRs), RIG-like helicases (RLHs), Triggering receptor expressed on myeloid cells (TREMs) and Toll-like receptors (TLRs)
  • Saponins i.e. Any saponin derived from Quillaja saponaria or Platycodon grandiflorum
  • Virosomes/Virus-like particles e.g..
  • Enterotoxins i.e. Cholera toxin, CTA1-DD or Esherichia coli heat-labile enterotoxin
  • Suitable adjuvants include response-selective C5a agonists, such as EP54 and EP67 described in Hung CY et al.
  • An agonist of human complement fragment C5a enhances vaccine immunity against Coccidioides infection.
  • Vaccine (2012) and Kollessery G et al. Tumor- specific peptide based vaccines containing the conformationally biased, response-selective C5a agonists EP54 and EP67 protect against aggressive large B cell lymphoma in a syngeneic murine model.
  • Vaccine (2011) 29: 5904-10 are examples of response-selective C5a agonists, such as EP54 and EP67 described in Hung CY et al.
  • An agonist of human complement fragment C5a enhances vaccine immunity against Coccidioides infection.
  • Vaccine (2012) and Kollessery G et al. Tumor- specific peptide based vaccines containing the conformationally biased, response-selective C5a agonists EP54 and EP
  • this stable cell line which carries the vector to be used according to the invention and which expresses the nucleic acid fragment to be used according to the invention.
  • this stable cell line secretes or carries the peptide expression product, thereby facilitating purification thereof.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with the hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g ., Bolivar et al. , 1977).
  • the pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the prokaryotic microorganism for expression.
  • promoters most commonly used in recombinant DNA construction include the ⁇ - lactamase (penicillinase) and lactose promoter systems (Chang et al. , 1978; Itakura et al. , 1977; Goeddel et al. , 1979) and a tryptophan (trp) promoter system (Goeddei et al. , 1979; EP-A-0 036 776) . While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published .
  • eukaryotic microbes such as yeast cultures may also be used, and also here the promoter should be capable of driving expression.
  • Saccharomyces cerevisiase, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
  • the plasmid YRp7 for example, is commonly used (Stinchcomb et al , 1979; Kingsman et al. , 1979; Tschemper et al. , 1980) .
  • Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzman et al. , 1980) or other glycolytic enzymes (Hess et al. , 1968; Holland et al. , 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • 3-phosphoglycerate kinase Hitzman et al. , 1980
  • other glycolytic enzymes Hess et al. , 1968; Holland et al. , 1978
  • enolase glyceraldehyde-3
  • the termination sequences associated with these genes are also incorporated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • Other promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization .
  • Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.
  • cultures of cells derived from multicellular organisms may also be used as hosts.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293, Spodoptera frugiperda (SF) cells, Drosophila melanogaster cell lines (such as Schneider 2 (S 2 )), and MDCK cell lines.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
  • the control functions on the expression vectors are often provided by viral material.
  • commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40) .
  • SV40 Simian Virus 40
  • the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al. , 1978).
  • Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hindlll site toward the Bgll site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
  • An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., other Polyoma viruses, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • an exogenous origin such as may be derived from SV40 or other viral (e.g., other Polyoma viruses, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • nucleic acid vaccines can also be administered intraveneously and intraarterially.
  • nucleic acid vaccines can be administered by use of a so-called gene gun and/or by use of electroporation, and hence also these and equivalent modes of administration are regarded as part of the present invention.
  • nucleic acid fragment is introduced in the form of a vector wherein expression is under control of a viral promoter.
  • vectors to be used according to the invention cf. the discussion above.
  • detailed disclosures relating to the formulation and use of nucleic acid vaccines are available, cf. Donnelly JJ et al, 1997, Annu. Rev. Immunol. 15: 617-648 and Donnelly JJ et a/., 1997, Life Sciences 60: 163-172. Both of these references are incorporated by reference herein.
  • An alternative of using peptide immunogens or nucleic acid immunogens is the use of live immunogen technology.
  • the non-pathogenic microorganism can be any suitable attenuated bacterial strain (attenuated by means of passaging or by means of removal of pathogenic expression products by recom- binant DNA technology), e.g. Mycobacterium bovis BCG., non-pathogenic Streptococcus spp., E. coli, Salmonella spp., Vibrio cholerae, Shigella, etc. Reviews dealing with preparation of state-of-the-art live vaccines can e.g. be found in Saliou P, 1995, Rev. Prat.
  • nucleic acid fragment to be used according to the invention can be incorporated in a non-virulent viral vaccine vector such as a vaccinia strain or any other suitable poxvirus.
  • the non-pathogenic microorganism or virus is administered only once to a subject, but in certain cases it may be necessary to administer the microorganism/virus more than once in a lifetime in order to maintain protective immunity. It is even contemplated that immunization schemes as those detailed above for polypeptide vaccination will be useful when using live or virus vaccines.
  • live or virus immunization is combined with previous or subsequent polypeptide and/or nucleic acid immunization.
  • APTKAKRRVVEREKRAV SEQ ID NO : 2
  • DRPEGIEEEGGERDR (where amino acid 4 can be G and/or where amino acid 5 can be R and/or where amino acid 13 can be Q and/or where amino acid 14 can be G and/or where amino acid 15 can be K; SEQ ID NO: 6) ; DRPEGIENNGGERDR (SEQ ID NO: 7 where amino acid 4 can be G and/or where amino acid 5 can be R and/or where amino acid 13 can be Q and/or where amino acid 14 can be G and/or where amino acid 15 can be K);
  • DRPEGIENNGGERDRDR (where amino acid 4 can be G and/or where amino acid 5 can be R and/or where amino acid 13 can be Q and/or where amino acid 14 can be G and/or where amino acid 15 can be K and/or where amino acid 16 can be G); SEQ ID NO:46).
  • GGAIVNGSLADDDIVI (SEQ ID NO: 37, also termed 204d herein)
  • WWGCIEEEGCGGIEEEGGERDR SEQ ID NO:45: underlined amino acid residues are linked via a disulphide linker; the N-terminal W is preferably a D-amino acid and the C-terminal R may be amidated; the peptide is termed BI450-AdjBT 2, when having these two modifications).
  • n l, 2,3,4 Polypeptides II:
  • n l, 2,3,4
  • n l, 2,3,4 (Z-SEQ c5 -Z-SEQ c5 ) n
  • polypeptides I can be, but are not restricted to, the following sequences:
  • polypeptides II can be, but are not restricted to, the following sequences:
  • disulfide linked constructs can be, but are not restricted to, the following disulfide-bridge linked peptide sequences:
  • the above disulfide linked constructs may e.g. be synthesised by titration of 2- pyridinesulfenyl (SPyr)-protected cysteine-containing peptides with thiol-unprotected peptides.
  • SPyr 2- pyridinesulfenyl
  • This has proven to be a superior procedure to selectively generate disulfide-linked peptide heterodimers preventing the formation of homodimers (Schutz A ef a/., Tetrahedron, Volume 56, Issue 24, 9 June 2000, Pages 3889-3891).
  • linked constructs can be, but are not restricted to, the following linked peptide sequences, which have all been obtained from Bachem (UK) Ltd : AKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)
  • the Cys-Lys linker is typically established in the form of an amide bond between (2-oxo- ethyl) derivatized cysteine in one peptide and lysine in the other peptide.
  • SEQ ID NO: 38 is Cys-Lys linked to SEQ ID NOs 42, 43, 68, or where SEQ ID NO:41 is Cys-Lys linked to SEQ ID NOs: 39 or 40 are also within the scope of the present invention.
  • AKRRVV (SEQ ID NO: 35)
  • AEEEVV (SEQ ID NO: 36)
  • SEQ ID NOs 34-36 are preferably composed partly or completely of D-amino acids.
  • One preferred immunogen which induces antibodies that stabilise association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 is a compound of the following structure:
  • This compound may also be referred to as:
  • This preferred C5 compound consists of two linear peptide amide chains with 31 amino acids (A-chain) and 22 amino acids (B-chain) . Each chain has a free amino group at the
  • N-terminus and an amide group at the C-terminus are covalently linked via an amide bond between Cys(2-oxo-ethyl) i0 of the A-chain and Lys 2 of the B-chain. All amino acid residues except the achiral Gly are in the L-configuration.
  • the preferred C5 compound (also referred to in the examples as Vacc-C5) may be provided as an acetate salt.
  • the counter ion acetate is bound in ionic form to basic groups of the peptide molecule.
  • Immunomodulatory compounds encompasses certain small organic molecules that inhibit LPS induced monocyte TNF-a, IL- 1B, IL- 12, IL-6, MIP-la, MCP- 1, GM-CSF, G-CSF, and/or COX-2 production, COX-2 inhibitors, and Etoricoxib.
  • Other immunomodulatory compounds includes Viriostatics VS411 (didanosine + hydroxyurea). Specific immunomodulatory compounds are discussed herein elsewhere.
  • the immunomodulatory compounds used according to the present invention are co-stimulators of T cells and increase cell proliferation dramatically in a dose dependent manner.
  • immunomodulatory compounds provided herein may also have a greater co-stimulatory effect on the CD8+ T cell subset than on the CD4+ T cell subset.
  • the compounds have anti-inflammatory properties against myeloid cell responses, yet efficiently co-stimulate T cells to produce greater amounts of IL- 2, IFN- ⁇ , and to enhance T cell proliferation and CD8+ T cell cytotoxic activity.
  • immunomodulatory compounds provided herein may be capable of acting both indirectly through cytokine activation and directly on Natural Killer (“NK”) cells and Natural Killer T (“NKT”) cells, and increase the NK cells' ability to produce beneficial cytokines such as, but not limited to, IFN- ⁇ , and to enhance NK and NKT cell cytotoxic activity.
  • NK Natural Killer
  • NKT Natural Killer T
  • immunomodulatory compounds include, but are not limited to, cyano and carboxy derivatives of substituted styrenes such as those disclosed in US 5929117; l-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and I ,3-dioxo-2-(2,6- dioxo-3-fluoropiperidine-3-yl) isoindolines such as those described in US 5874448 and US 5955476; the tetra substituted 2-(2,6-dioxopiperdin-3-yl)-l-oxoisoindolines described in US 5798368; 1-oxo and I ,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines (e.g ., 4-methyl derivatives of thalidomide), substituted 2-(2,6-dioxopiperidin-3-y
  • immunomodulatory compounds do not include thalidomide.
  • the immunomodulatory compounds provided herein include, but are not limited to, 1-oxo-and 1,3 dioxo-2-(2,6-dioxopiperidin-3-yl) isoindo lines substituted with amino in the benzo ring as described in US 5635517 which is incorporated herein by reference.
  • other specific immunomodulatory compounds provided herein belong to a class of substituted 2-(2,6-dioxopiperidin-3-yl) phthalimides and substituted 2-(2,6- dioxopiperidin-3-yl)-l-oxoisoindoles, such as those described in US 6281230; US 6316471 ; US 6335349; US 6476052, and WO 98/03502), each of which is incorporated herein by reference.
  • other specific immunomodulatory compounds provided herein include, but are not limited to, l-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl) isoindolines and l,3-dioxo-2- (2,6-dioxo-3-fluoropiperidine-3-yl) isoindolines such as those described in US 5874448 and US 5955476, each of which is incorporated herein by reference.
  • other specific immunomodulatory compounds provided herein include, but are not limited to, the tetra substituted 2-(2,6-dioxopiperdin-3-yl)-l-oxoisoindolines described in US 5798368, which is incorporated herein by reference.
  • other specific immunomodulatory compounds provided herein include, but are not limited to, 1-oxo and l,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines disclosed in US 6403613, which is incorporated herein by reference.
  • other specific immunomodulatory compounds provided herein include, but are not limited to, 1-oxo and 1,3-dioxoisoindolines substituted in the 4- or 5-position of the indoline ring described in US 6380239 and U.S. application no. 10/900,270, filed July 28, 2004, which are incorporated herein by reference.
  • other specific immunomodulatory compounds provided herein include, but are not limited to, isoindoline-l-one and isoindoline-l,3-dione substituted in the 2-position with 2,6-dioxo-3-hydroxypiperidin-5-yl described in US 6458810, which is incorporated herein by reference.
  • Histone deacetylase inhibitors and other reservoir purging agents are Histone deacetylase inhibitors and other reservoir purging agents.
  • HDACi histone deacetylase inhibitor
  • These compounds are generally active (with depending on the compound, more or less specificity for a given HDAC) on histone deacetylases of Class I, II, and IV.
  • HDACi histone deacetylase
  • This chromatin remodeling results in modulation of gene expression, which can inhibit proliferation of cancer cells through biological processes such as cell cycle arrest, differentiation and/or apoptosis (for review see for instance Marks & Xu, J. Cell. Biochem., 107, 600-608, 2009; Federico & Bagella, J. Biomed. Biotechnol,
  • Reservoir purging agents such as histone deacetylase inhibitors, include but is not limited to e.g., Toll-like receptor-9 (TLR9) agonists, quercetin, trichostatin A, lipoic acid, an hydroxamic acid, valproic acid, sodium butyrate, vorinostat, romidepsin, TNF-a, PHA, Tat, interferon gamma (IFN- ⁇ ), CD154, IL-lbeta, IL-2, IL-6, IL-7 or IL-7R agonist, IL-9 or IL-9R agonists, 5- hydroxynaphtalene-l,4-dione (5HN), siRNA or antagomirs (anti-miRNA antisense) raised against miRNA controlling the expression of latent HIV, short hairpin RNAs (shRNAs) targeting transcription factor YY1 and/or cMyc, histone methyl transferase inhibitors as TLR9 (TLR9)
  • HDACi histone deacetylase inhibitors
  • M344 (4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide), chidamide (CS055/HBI-800), 4SC-202, (4SC), Resminostat (4SC), hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824, trichostatin A and panobinostat (LBH589); benzamides such as entinostat ( S-275), CI994, and mocetinostat (MGCD0103), cyclic tetrapeptides (such as trapoxin, such as trapoxin B), and the depsipeptides, such as romidepsin (ISTODAX), electrophilic ketones, and the aliphatic acid compounds such as phenylbutyrate, valproic acid, Oxam
  • a therapeutic amount of HDACi is administered to a mammal in the present method, e.g. an amount sufficient to enhance the immunological effect of the viral vaccine.
  • the HDACi may be administered using any suitable administrable form, including for example, oral, subcutaneous, intravenous, intraperitoneal, intranasal, enteral, topical, sublingual, intramuscular, intra-arterial, intramedullary, intrathecal, inhalation, ocular, transdermal, vaginal or rectal means.
  • Suitable reservoir purging agents includes, but are not limited to compounds that activate transcription factors including NF-KappaB, Prostratin, auranofin, bryostatin, a nontumorigenic phorbol ester, DPP (12-deoxyphorbol-13-phenylacetate), PMA, and Phorbol 12-myri state 13-acetate (PMA); Compounds that activate HIV mRNA elongation including P- TEF-b kinase and hexamethylbisacetamide (HMBA); T-cell stimulating factors including anti- CD3/CD28 - T-cell stimulating Ab's; Kinase inhibitors including Tyrphostin A, Tyrphostin B, and Tyrphostin C; TLR-7 agonists; PTEN (phosphatase and tensin homologue) gene inhibitors including SF1670 (Echelon Bioscience), Disulfiram (DSF), an inhibitor of acetaldehyde dehydrogenase, Protein Tyrosine Phosphatase Inhibitors
  • HDAC inhibitor SAHA HDAC inhibitor
  • Peripheral blood for the isolation of primary CD4+ and CD8+ T-cells was obtained from HIV-l-infected donors and healthy adult volunteers and used for Bcl-2 transduction and then HIV-1 reporter virus (GFP expressing) infection (Yang et al ., 2009, J Clin Invest., 119 ( l l) : 3473-3486) .
  • CD8+ T-cells were isolated, optionally stimulated with HIV-1 specific peptides, and cocultured with in vitro infected autologous CD4+ T-cells. The fraction of residual GFP+ CD4+ T-cells was measured by FACS. This procedure demonstrated CD8+ CTL mediated killing of the activated CD4+ T-cells.
  • An aspect of the present invention relates to a method for reducing and/or delaying pathological effects of human immunodeficiency virus I (HIV) in a human infected with HIV or for reducing the risk of developing acquired immunodeficiency syndrome (AIDS) in a human, the method comprising the steps of: a) Determining the HLA genotype of said human and selecting said human carrying a HLA B35 epitope; b) administering to said human selected under step a) a composition comprising one or more peptide, such as a HIV-specific peptide, that elicit a cell-mediated immune response in a subject
  • Xaa in position 2 is Ala, Gly, Ser or Arg,
  • Xaa in position 3 is Leu or Met
  • Xaa in position 4 is Gly or Arg
  • Xaa in position 5 is Pro, Thr, Val, Ser, Gin or Ala,
  • Xaa in position 6 is Gly, Ala, Lys, Arg, Gin or Glu,
  • Xaa in position 8 is Thr or Ser
  • Xaa in position 9 is Leu or He ,
  • Xaa in position 14 is Thr, Ser or Val
  • Xaa in position 15 is Ala or Ser
  • Xaa in position 16 is Cys or Ser
  • Xaa in position 17 is Gin or Leu
  • Xaa in position 18 is Gly, Glu or Arg, and
  • Xaa in position 20 is Gly or Arg ;
  • Xaa in position 2 is Trp, Gly, Lys or Arg,
  • Xaa in position 3 is He, Leu, Val or Met
  • Xaa in position 4 is He, Val or Leu
  • Xaa in position 12 is Arg, Lys
  • Xaa in postion 13 is Met or Leu
  • Xaa in position 15 is Ser, Cys or Gin
  • Xaa in position 17 is Thr, Val, He, Ser or Ala
  • Xaa in position 18 is Ser, Gly or Thr,
  • Xaa in position 21 is Asp, Glu, Cys or Gly,
  • Xaa in position 22 is Gly or none
  • n 0, 1,2 or 3;
  • Xaa in position 2 is Asn, Ala or Lys
  • Xaa in position 3 is Pro, Gin, Gly, He or Leu
  • Xaa in position 7 is Val or Ala
  • Xaa in position 8 is Gly or Lys
  • Xaa in position 9 is Glu, Asp, Lys, Phe or Thr
  • Xaa in position 10 is He, Met, Val or Leu Xaa in position 11 is Tyr, Leu or none
  • Xaa in position 12 is Ser or none
  • Xaa in position 13 is Arg or none
  • Xaa in position 14 is Asp, Arg, Trp, Ala or none
  • Xaa in position 15 is He or none
  • Xaa in position 16 is Tyr or none
  • Xaa in position 17 is Lys or Arg
  • Xaa in position 18 is Arg, Lys or Asp
  • Xaa in position 19 is Trp or Gly
  • Xaa in position 20 is He, Met, Val, Gin or Ala
  • Xaa in position 21 is He, Val or Ala
  • Xaa in position 22 is Leu, Met or Val
  • Xaa in position 23 is Gly or Cys
  • Xaa in position 24 is Leu or none
  • n 1,2 or 3
  • Xaa in position 2 is Glu, Arg, Phe or Lys
  • Xaa in position 5 is Pro or Thr
  • Xaa in position 6 is Met, Thr or NIeu
  • Xaa in position 7 is Phe or Leu
  • Xaa in position 9 is Ala, Glu or Leu
  • Xaa in position 11 is Ser or none
  • Xaa in position 12 is Ala, Arg or none
  • Xaa in position 13 is He, Leu or none
  • Xaa in position 14 is Ser, Ala, Leu or none
  • Xaa in position 15 is Tyr, Glu or Asp
  • Xaa in position 16 is Gly or Asp
  • Xaa in position 17 is Ala or Leu
  • Xaa in position 18 is Thr, He, Val, Leu or Asn,
  • Xaa in position 19 is Pro, Thr or Ser
  • Xaa in position 20 is Tyr, Phe, NIeu, His or Gin
  • Xaa in position 21 is Asp, Asn, Leu or Ala
  • Xaa in position 22 is Leu, He, Val or Asn
  • Xaa in position 23 is Asn, Tyr, Cys or Gly Xaa in position 24 is Thr, Met, He, Ala, Val or none
  • Xaa in postion 25 is Gly or none
  • each HIV specific peptide may be free carboxyl- or amino groups, amides, acyls, acetyls; or salts of any of the HIV specific peptides.
  • the amino acid sequence of SEQ ID NO : 47 is selected from the groups of SEQ ID NO: 48 and SEQ ID NO: 49.
  • amino acid sequence of SEQ ID NO : 50 is selected from the groups of SEQ ID NO: 51, SEQ ID NO : 52, SEQ ID NO: 53 and SEQ ID NO: 54.
  • amino acid sequence of SEQ ID NO : 55 is selected from the groups of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60
  • the amino acid sequence of SEQ ID NO : 61 is selected from the groups of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.
  • the at least one HIV-specific peptide comprises at least , two, three, or four peptides selected from each of the groups of SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO : 55 and SEQ ID NO: 61.
  • the at least one HIV-specific peptide consist of or comprises the peptides of the SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 57 and SEQ ID NO: 64.
  • the composition is an immunogenic composition in combination with a pharmaceutically acceptable diluent or vehicle and optionally one or more immunological adjuvant.
  • composition is in the form of a vaccine composition. In some embodiments the composition is in the form of a vaccine composition where the one or more adjuvant are provided either separately or in combination with the composition.
  • the method further comprises a preceding, simultaneous or subsequent step of administering an effective amount of at least one agent capable of stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.
  • the at least one agent capable of stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 is a molecule comprising at least one amino acid sequence selected independently from an amino acid sequence derived from the transmembrane domain of gp41 and an amino acid sequence derived from the C2 domain, wherein the at least one amino acid sequence binds the C5 domain and comprises at least one D-amino acid.
  • the molecule is a peptide.
  • the peptide consists of at least one amino acid sequence.
  • the amino acid sequence derived from the transmembrane domain of gp41 has an amino acid sequence of at most 10 amino acid residues.
  • the at least one agent capable of stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 is selected from an antibody, an antibody fragment or an antibody analogue.
  • the antibody which is a fully human antibody, a humanized antibody, or a chimeric antibody, or a derivative thereof.
  • the antibody is IgA, an IgD, an IgG, an IgE or an IgM.
  • the antibody fragment is selected from a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab)2 fragment, a F(ab')2 fragment, an Fv fragment, a Heavy chain Ig (a llama or camel Ig), a V HH fragment, a single domain FV, and a single-chain antibody fragment.
  • the antibody analogue is selected from a scFV, a dsFV, a minibody, a diabody, a triabody, a kappa body, an IgNAR, a tandAb, a BiTE, and a multispecific antibody.
  • the at least one agent capable of stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 binds to and stabilises association between one or more amino acid residues in the amino acid stretch TZ 1 AKRRVVZ 2 REKR, where Z 1 is K, R or E and where Z 2 is Q or E, and one or more amino acid residues in an amino acid stretch in the transmembrane domain of gp41 and/or in the constant C2 domain of gpl20.
  • the method further comprises a preceding, simultaneous or subsequent step of administering an effective amount of at least one immunogen, which induces antibodies that stabilise association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.
  • the immunogen is a peptide combination comprising a first peptide comprising the amino acid sequence of the 13 amino acid residue amino acid sequence of the C5 domain of HIV gpl20 including between 0 and 4 amino acid substitutions, a subsequence thereof, or an amino acid sequence comprising the inverso-, retro- or retro-inverso form of said amino acid sequence or subsequence, and - at least one second peptide comprising an amino acid stretch present in the transmembrane domain of gp41 or present in the constant C2 domain of gpl20 or comprising an amino acid stretch present in any one of SEQ ID NOs.
  • the first peptide comprises the amino acid sequence having formula I : x 1 -x 2 -x 3 -x 4 -x 5 -x 6 -x 7 -x 8 -x 9 -x 10 -x 11 -x 12 -x : (I) wherein X 1 is Thr, X 2 is selected from Lys, Arg, Har and Glu, X 3 is selected from Ala and Val, X 4 is selected from Arg, Har, Lys and Cit (citrulline), X 5 is selected from Arg, Har, Lys and Cit, X 6 is selected from Arg, Har, Lys and Cit, X 7 is selected from Val, Leu, He and Nle (norleucin), X 8 is selected from Val, Leu, He and Nle, X 9 is selected from Gin, Glu, Asn and Asp, X 10 is selected from Arg, Har and Cit, X 11 is selected from Glu and Asp, X 12 is Lys, and X 13
  • the first peptide further comprises the dipeptide Ala-Pro linked to the N-terminus of the amino acid sequence having formula I.
  • the first peptide further comprises the dipeptide X 14 -X 15 linked to the C-terminus of the amino acid sequence having formula I, wherein X 14 is selected from Ala and Val, and wherein X 15 is selected from Val, Leu and Nle.
  • the at least second peptide includes an amino acid sequence having the formula: wherein Z 1 is Asp, Z 2 is Arg, Z 3 is Pro, Z 4 is Glu or Gly, Z 5 is Gly or Arg, Z 6 is He, Z 7 is Glu, Z 8 is Glu, Z 9 is Glu, Z 10 is Gly, Z 11 is Gly, Z 12 is Glu or is absent, Z 13 is Arg or Gin, Z 14 is Asp or Gly, Z 15 is Arg or Lys, Z 16 is Asp or Gly and Z 17 is Arg,
  • the second peptide includes at least 5 consecutive amino acid residues from formula III.
  • first peptide and the at least one second peptide are associated via a linker.
  • the linker is selected from the group consisting of a bis-maleimide linker, a disulfide linker, a polyethylene glycol (PEG) linker, a glycine linker, a lysine linker, and an arginine linker.
  • the at least one of the first and at least one second peptides comprises an N- or C-terminal modification, such as an amidation, acylation, or acetylation.
  • the peptide combination is coupled to a carrier molecule, such as an immunogenic carrier.
  • a carrier molecule such as an immunogenic carrier.
  • the carrier is a virus like particle.
  • the first peptide is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 38, 41 and 44 or a fragment thereof, or the inverso-, retro- or retro-inverso form of a peptides selected from SEQ ID NO: 1, 2, 3, 4, 5, 38, 41 and 44 or a fragment thereof
  • the second peptide is selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 37, 39, 40, 42, 43, 45, 46 or a fragment thereof, or the inverso-, retro- or retro-inverso form of a peptides selected from SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 37, 39, 40, 42, 43, 45, 46 or a fragment thereof, and/or wherein the peptide combination is selected from the peptides having SEQ ID NOs: 1-46.
  • the peptide combination comprises at most 70 amino acids. In some embodiments the peptide combination comprises at least 6 amino acid residues.
  • the peptide combination consist of a number of amino acid residues selected from the group consisting of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 amino acid residues.
  • the peptide combination is selected from the group consisting of disulphide linked peptides between SEQ ID NO: 28 and any one of SEQ ID NOs: 29, 31, and 33, between SEQ ID NO: 30, and any one of SEQ ID NO: 29, 31, and 33, or between SEQ ID NO : 32 and any one of SEQ ID NO: 29, 31, and 33;
  • cysteine-lysine linked peptides between SEQ ID NO: 38 and any one of SEQ ID NO: 39, SEQ ID NO: 40; SEQ ID NO: 42, and SEQ ID NO: 43, or between SEQ ID NO: 41 and any one of SEQ ID NO: 39, SEQ ID NO: 40; SEQ ID NO: 42, and SEQ ID NO: 43.
  • the peptide combination is selected from the group consisting of: CGGAKRRVVGGAKRRVVGQREKRAV (SEQ ID NO: 28)
  • GKGGIEEEGGQDRDRGGRDRDR (SEQ ID NO: 42), GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO: 41 )
  • GKGGIEEEGGRDRDRGGQDRDR (SEQ ID NO: 73) .
  • the peptide combination is selected (H-Gly-Ala-Lys-Arg-Arg-Val-Val- Gly-Gly-Cys(2-oxo-ethyl)-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu- Arg-Glu-Lys-Arg-Ala-NH 2 ) (H-Gly-Lys-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg- Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH 2 ), acetate salt (amide bond between Cys(2-oxo-ethyl) 10 (A- chain) and Lys 2 (B-chain)) .
  • the method further comprises a preceding, simultaneous or subsequent step of administering an effective amount of at least one immunomodulatory compound and/or a reservoir purging agent, such as a histone deacetylase (HDAC) inhibitor.
  • a histone deacetylase (HDAC) inhibitor such as a histone deacetylase (HDAC) inhibitor.
  • the immunomodulatory compounds is selected from anti-PDl antibodies, such as MDX- 1106 (Merck), THALOMID® (thalidomide), anti-PDl antibodies, cyclophosphamide, Levamisole, lenalidomide, CC-4047 (pomalidomide), CC-11006 (Celgene), and CC-10015 (Celgene), and immunomodulatory compound described in any one of WO2007028047, WO2002059106, and WO2002094180.
  • anti-PDl antibodies such as MDX- 1106 (Merck), THALOMID® (thalidomide), anti-PDl antibodies, cyclophosphamide, Levamisole, lenalidomide, CC-4047 (pomalidomide), CC-11006 (Celgene), and CC-10015 (Celgene)
  • immunomodulatory compound described in any one of WO2007028047, WO2002059106, and WO2002094180 immunomodulatory
  • the immunomodulatory compound is selected from a 4-(amino)-2- (2,6-dioxo(3-piperidyl))-isoindoline-l ,3-dione and a 3-(4-amino-l-oxo-l,3-dihydro-isoindol-2- yl)-piperidine-2,6-dione.
  • the immunomodulatory compound is enantiomerically pure.
  • the reservoir purging agent such as a Histone deacetylase (HDAC) inhibitor is selected from M344 (4-(dimethylamino)-N-[7-(hydroxyamino)-7- oxoheptyljbenzamide), chidamide (CS055/HBI-800), 4SC-202, (4SC), Resminostat (4SC), hydroxamic acids such as vorinostat (SAHA), belinostat (PXD 101), LAQ824, trichostatin A and panobinostat (LBH589) ; benzamides such as entinostat (MS-275), CI994, and
  • HDAC Histone deacetylase
  • MCD0103 mocetinostat
  • cyclic tetrapeptides such as trapoxin, such as trapoxin B
  • the depsipeptides such as romidepsin (ISTODAX)
  • electrophilic ketones and the aliphatic acid compounds such as phenylbutyrate, valproic acid, Oxamflatin, ITF2357 (generic givinostat), Apicidin, MC1293, CG05, and CG06
  • compounds that activate transcription factors including NF-KappaB, Prostratin, auranofin, bryostatin, a nontumorigenic phorbol ester, DPP ( 12-deoxyphorbol-13-phenylacetate), PMA, and Phorbol 12-myristate 13-acetate (PMA)
  • Compounds that activate HIV mRNA elongation including P-TEF-b kinase and hexamethylbisacetamide (HMBA) ; IL-7; T-cell
  • CMI Cell-mediated immunity
  • peptides to be used according to the present invention that elicit Cell-mediated immunity (CMI) or peptides that stimulate the humoral immunity in a subject were synthesized using conventional techniques for linear sequences as described in international patent applications WO0052040, WO 2012/092934 or WO 2012/072088.
  • Immunomodulatory compound and a reservoir purging agent such as a histone deacetylase (HDAC) inhibitor used according to the present invention are well known to the person skilled in the art, and are commercially available.
  • HDAC histone deacetylase
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of F-moc synthesis (Atherton et al . 1978 J . Chem. Soc. Chem Commun 539), which is below termed "the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.
  • the peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis. Purity (HPLC) : more than 90 %.
  • the SPPS synthesis was started with 15 mmoles (A-chain) and 30 mmoles (B-chain) of the resin. After purification of a part of the crude C5-Peptide, 16.6 g of final product were obtained.
  • amino acid sequences of the A-chain H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys-Gly- Gly-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH 2
  • B-chain H-Gly-Lys-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-GIn- Asp-Arg-Asp-Arg-NH 2
  • SPPS solid phase peptide synthesis
  • the solvents and/or reagents are added, and the reaction mixture is stirred and then filtered to remove solvents and/or reagents from the resin.
  • Single steps of the SPPS cycle may be repeated in case the reaction is incomplete.
  • the SPPS cycle is repeated until the solid phase carries the complete amino acid sequence of the A-chain or the B-chain.
  • the B-chain is further modified with a bromoacetyl linker at Lys 2 .
  • This procedure consists of selectively cleaving the side-chain protecting group of Lys 2 and coupling bromoacetic acid to Lys 2 in the presence of a suitable activating agent. If the coupling reaction is incomplete, recoupling procedures can be performed. The SPPS is then completed by drying the peptide resin under reduced pressure.
  • Stage 2 Cleavage from the resin including cleavage of the acid labile protecting groups
  • Cleavage of the peptides from the resin and concomitant cleavage of the acid labile protecting groups is accomplished by treatment with TFA in the presence of water.
  • Scavengers are added as needed to trap reactive cations and to avoid alkylation of side-chain functions. After filtering off and washing the resin with TFA, the products are precipitated in IPE. They are filtered off, washed with IPE, and dried under reduced pressure.
  • the coupling of the two peptide chains is performed by the addition of a solution of the B- chain ( 1 equivalent) in aqueous TFA to a solution of the A-chain (1 equivalent) in TRIS buffer (adjusted to pH 8.5 by the addition of hydrochloric acid) . Additional TRIS buffer is added to maintain a pH > 8 in the reaction mixture. The reaction mixture is then stirred and the reaction progress is monitored by analytical HPLC. Upon completion, the pH of the reaction mixture is lowered to approx. pH 3 by the addition of TFA.
  • Portions of the C5-peptide are directly loaded onto the column. Subsequently, the ACN gradient of the TFA system is started . The collected fractions are checked by analytical HPLC and pooled accordingly.
  • the last stage of the manufacture of C5-Peptide is the ion exchange from the TFA salt, obtained in the previous stage, into the acetate salt.
  • the lyophilized material from the TFA purification is dissolved in 5% acetic acid and the solution loaded onto the ion exchange resin (acetate form) .
  • the elution is performed with 5% acetic acid and checked by TLC.
  • the product solution is filtered through a 0.2 pm membrane filter and lyophilized to yield the final product as a white to off-white material .
  • the peptides to be used according to the present invention can be produced by any known method of producing a linear amino acid sequence, such as recombinant DNA techniques.
  • a nucleic acid sequence which encodes a peptide to be used according to the invention or a multimer of the said peptides is introduced into an expression vector.
  • Suitable expression vectors are for instance plasmids, cosmids, viruses and YAC (yeast artifical chromosome) which comprise necessary control regions for replication and expression.
  • the expression vector may be stimulated to expression in a host cell .
  • Suitable host cells are for example bacteria, yeast cells and mammal cells.
  • the amino acid sequence is subjected to a chemical oxidation step in order to cyclize or link the two cysteine residues within one or between two peptide sequences, when the appropriate linear amino acid sequences are synthesized, see Akaji et al ., Tetrahedron Letter, 33, 8, p.1073-1076, 1992.
  • the product was then purified by reversed phase liquid chromatography on a column filled with Kromasil® 100 - 5 C8 (EKA Nobel, Surte, Sweden) in a suitable system containing acetonitrile in 0,1 % trifluoroacetic acid water solution.
  • the samples collected from the column were analyzed by analytical high performance liquid chromatography (HPLC) (Beckman System Gold, USA) equipped with a Kromasil® 100 - 5 C8 Column (EKA Nobel, Surte, Sweden). Fractions containing pure substance were pooled, the solvent was evaporated and the product freeze-dried from acetic acid.
  • the final HPLC analysis was performed on final product, and the structure of the peptide was confirmed by amino acid analysis and mass spectrometry (LDI-MS).
  • the amino acid derivatives was supplied by Bachem AG, Switzerland.
  • the peptide was synthesized in amide form, from corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).
  • the peptide was synthesized in amide form, from corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).
  • the peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).
  • the peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS). Purity (HPLC) : 85 %
  • the peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. Nl in the sequence is Norleucine. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).
  • the peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. Nl in the sequence is Norleucine. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).
  • the peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. Nl in the sequence is Norleucine. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).
  • the purity of the peptide was determined by HPLC analysis and the peptide structure was confirmed by amino acid analysis.
  • the peptide content (aminoacid free base) was 80%
  • a vaccine comprising the peptides of the SEQ ID NO: 49, 52, 57 and 64 (Also referred to herein as Vacc-4x) was prepared.
  • the freeze-dried peptides are dissolved in sterile water at a final concentration of 4 mg/ml .
  • the final salt concentration was 0,9 %.
  • a preparation of a granulocyte-macrophage-colony stimulating factor (GM-CSF) was also prepared, according to the manufacturers directions for use, to a final concentration of 0.3 mg/ml .
  • the two solutions are administered intracutaneously.
  • a typical injection dose is 100 ⁇ .
  • polypeptides to be used according to the invention includes at least one peptide specifically disclosed in any of international patent applications WO2011/000962,
  • WO0052040, WO 2012/092934 or WO 2012/072088 selected, including but not limited to any of SEQ ID NO:47, SEQ ID NO: 50, SEQ ID NO: 55 and SEQ ID NO: 61 to as well as peptide combinations that are disulphide linked peptides between SEQ ID NO: 28 and any one of SEQ ID NOs: 29, 31, and 33, between SEQ ID NO: 30, and any one of SEQ ID NO: 29, 31, and 33, or between SEQ ID NO: 32 and any one of SEQ ID NO: 29, 31, and 33; or cysteine-lysine linked peptides between SEQ ID NO: 38 and any one of SEQ ID NO: 39, SEQ ID NO:40; SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO: 68, or between SEQ ID NO:41 and any one of SEQ ID NO: 39, SEQ ID NO:40; SEQ ID NO:42, and SEQ ID NO:43, which form antigens and the active principle of
  • the vaccine may include compounds having beneficial effects in protecting or stimulating the host ' s immune system (human being or vertebrate animal) for instance interleukins, interferons, granulocyte macrophage growth factors, haematopoietic growth factors or similar.
  • the vaccine composition further contain an adjuvant or vehicle, more preferably the adjuvant or vehicle is Monophosphoryl Lipid A (MPL ®) possibly with alum, Freund ' s adjuvant (complete or incomplete) or aluminum hydroxyd.
  • MPL ® Monophosphoryl Lipid A
  • the optimal amount of adjuvant/vehicle will depend on the type(s) which is chosen.
  • the peptide or vaccine formulation can be freeze-dried prior to storage.
  • the vaccine may be stored preferably at low temperature, in ampoules containing one or more dosage units, ready for use.
  • a typical dosage unit of the peptide to be used according to the invention is within the concentration range : 1 pg-lmg per kg bodyweight, preferably within 2 ⁇ g-0.15 mg per kg body weight.
  • a suitable dose will depend on the body weight of the patient, the type of disease, severity of condition, administration route and several other factors.
  • the vaccine might be administered up to twelve times and through injection, typically it will be administered about three times. In preparation of an injection solution the peptides are dissolved in sterile sodium chloride solution at a final concentration of 1 mg/ml per peptide and 0,9% sodium chloride.
  • an injection volume is 100 ⁇ to 200 ⁇ (2 x 100 ⁇ ).
  • the peptide is preferably coadministered with a suitable adjuvant and/or a granulocyte-macrophage growth factor for instance Leucomax® (Shering Plough).
  • Suitable administration may be intracutane, subcutane, intravenous, peroral, intramuscular, intranasal, mucosal or any other suitable route.
  • Booster administrations may be required in order to maintain protection.
  • the vaccine compositions according to the invention are useful not only in prevention of infection, but also in treatment of infection.
  • Subjects were randomized into Vacc-4x and placebo groups in a 2: 1 ratio. Subjects were given a total of six immunizations with Vacc-4x (or placebo) as outlined in Figure 1. Following immunization, subjects remained on ART for 10 weeks to allow time to reduce immune activation associated with immunization. ART was stopped in eligible subjects if the CD4 count was >350 (determined at week 24) such that all subjects were ART -free at week 28. ART was resumed if CD4 counts fell below 350xl0 6 /L_ or decreased by >50% compared to week 28 or if viral load (VL) increased above 300,000 copies/mL The study formally ended at week 52 with a long-term follow-up (LTFU) until week 104.
  • LTFU long-term follow-up
  • ART resumption during LTFU was according to local guidelines and/or agreement between the subject and the investigator.
  • Study Participants Inclusion criteria selected subjects aged between 18 and 55 years, HIV- positive for at least one year, well controlled on ART (VL ⁇ 50 copies/m L for the last six months) , pre-study CD4 cell count ⁇ 400xl0 6 /L, nadir (lowest ever) CD4 cell count ⁇ 200xl 0 6 /L PreART VL values were requested where available. Since not all subjects had available pre ART VL values, these were not included in the randomization strategy.
  • Randomization and masking The randomization scheme was prepared by an independent statistician . Randomization was stratified by site, block size was 3 with a treatment ratio of 2 : 1 (Vacc4-x : Placebo) . This study was double- blind with respect to treatment assignment. Only the pharmacist was un-blinded to treatment.
  • Vacc-4x was manufactured by Bachem AG ( Bubendorf, Switzerland ), and distributed in vials by Penn Pharmaceutical Services Ltd ; (Gwent, United Kingdom ) .
  • Vacc-4x (or placebo) was reconstituted in water on site by a pharmacist and administered i .d . at a dose of O. l m L of a 12mg/mL solution approximately 10 minutes after administration of adjuvant.
  • the co-primary efficacy endpoints were a) to determine the proportion of subjects who met the criteria for resumption of ART between the interruption of ART at Week 28 and the end of the study at Week 52 and b) to determine the per cent change in CD4 counts between Week 28 (interruption of ART) and the last CD4 count assessment prior to resumption of ART.
  • Secondary efficacy endpoints were to compare time to restart of ART for Vacc-4x treated subjects versus placebo.
  • changes in CD4 and CD8 counts and H IV RNA levels were compared .
  • Immunogenicity was determined by T-cell responses to Vacc-4x and p24 Gag regions corresponding to Vacc-4x in peripheral blood mononuclear cells (PBMC) ex vivo using IFN- ⁇ ELI SPOT, T-cell proliferation and intracellular cytokine staining (ICS) .
  • Safety parameters included determining the proportion of subjects who regained virus suppression on ART resumption . Adverse events were recorded continuously throughout the study. Vital signs, physical examination and clinical laboratory evaluations were carried out at baseline and at selected visits throughout the study. The endpoint for subject reported outcomes (quality of life) was determined through the standard validated questionnaire, the SF-36 Health Survey at screening, week 32 and week 52.
  • IL-6 measurements (eBioscience) and detection of antibodies to rhu-G -CSF (USCN Life) were carried out by Bionor Laboratories (Skien, Norway).
  • the proportion of subjects who retrospectively demonstrated a four-fold increase in anti-rhu-GM-CSF antibody titer between baseline and week 6 was determined for the first 20 subjects.
  • T-cell responses were evaluated at central laboratories from PBMCs prepared and frozen at each participating site. All sites preparing PBMCs were accredited prior to the study start to ensure PBMCs with recovery > 70% and viability >80% on thawing. Overlapping 15-mer peptides (offset by 2 amino acids) for ELISPOT, proliferation and ICS were synthesized at Schafer-N, (Copenhagen, Denmark). Recombinant p24 core protein, HTLV IIIB was obtained from Bioprocess Pty Ltd. UK.
  • IFN- ⁇ ELISPOT analyses were carried at the University of Lausanne for all European and at UC Davis for all US samples as previously described [12] at weeks 1 (baseline), 6, 28, 44 and 52.
  • PBMCs were stimulated with antigens ⁇ g/well (15 mer peptide pools of Vacc peptides, 15 mer pool for p24 Gag corresponding to the same regions as Vacc-4x, as well as full length Vacc-4x peptides).
  • Staphylococcal enterotoxin B (SEB) and media constituted positive and negative controls respectively. Assays were considered valid if the negative control had ⁇ L50 spot forming units (sfu)/10 6 cells and the positive control had > 500 sfu/10 6 cells.
  • An assay was considered positive if there were > 55 sfu/10 6 cells and that this value was 4 fold above the negative control .
  • a subject responder was defined as a positive ELISPOT at any time point when the baseline ELISPOT was negative.
  • a responder was defined by a >2 fold increase in sfu/10 6 cells from baseline at any post immunization time point. All sfu were derived as the mean of triplicate wells.
  • an IFN- ⁇ ELISPOT assay using overlapping 15-mer p24 Gag peptides separately was carried out to identify new responses not present at baseline.
  • T-cell proliferation was carried out at the University of Lausanne for all samples and measured using carboxy-fluorescein diacetate succinimidyl ester (CFSE) labeling of both CD4+ and CD8+ T-cell subsets as well as total T-cells (CD3+) as previously described
  • CFSE carboxy-fluorescein diacetate succinimidyl ester
  • a positive proliferation assay was defined as the percentage of low dividing cells j>3 times the negative (medium only) control .
  • a subject was defined as a responder if, for a particular antigen, the subject had a negative assay response at baseline but a positive assay response at a later time point, or if the subject had a positive assay at baseline, that the assay response at a later time point was j>2 that of the baseline value.
  • ICS was used to identify polyfunctional T-cells [13] expressing IFN- ⁇ , IL-2, and/or TNF-a.
  • PBMCs were stimulated with Vacc-4x 15-mers, full length Vacc-4x peptides, native p24 15- mers and recp24.
  • a polyfunctional response was defined as cells expressing j> 2 cytokines.
  • HLA genotyping (Locus A and B) were performed to 2-digit specificity by PCR-SSO/Luminex
  • Co-primary endpoints The proportion of subjects who met the criteria for ART resumption was analysed using a logistic regression, adjusting for the country where subjects were enrolled. Percentage change in CD4 count was analysed using an analysis of variance
  • VL set point and Peak VL endpoints were analysed using the Wilcoxon rank sum test.
  • the VL set point was compared to the pre -ART VL set point, separately for each treatment group using the Wilcoxon matched pairs signed rank sum test.
  • the proportion of IFN- ⁇ ELI SPOT responders at any time point to p24Gag was compared using Mann Whitney U-test.
  • VL at Week 52 was compared for the subset of subjects defined to be ELI SPOT responders (and a separate analysis for ELI SPOT non-responders) using the Wilcoxon rank sum test. Data related to ICS was analysed using the Wilcoxon rank sum test.
  • VL set point in subjects in the Off-ART-Completer subgroup having HLA B35, B27, B57 and A02 alleles was compared with their preART VL set point (Table 4) .
  • n the number of subjects carrying HLA B57
  • VL was reduced from preART levels.
  • 7 subjects in the Vacc-4x carried HLA-B35 compared to none in the placebo group suggesting that Vacc- 4x provided benefit to subjects carrying HLA-B35.
  • the CD4+ T cell cytokine responses were low at all time points.
  • the response mainly consisted of IFN- ⁇ (median 1.4% at week 44/52), but also TNF-a (median 0.5% at week 44/52) .
  • TNF-a median 0.5% at week 44/52 .
  • There was a trend towards higher numbers of polyfunctional T cells in Vacc-4x subjects compared to placebo Week 44/52 (p 0.188, Mann-Whitney) .
  • Vacc-4x was found to be safe and well tolerated (ITT population). There were no statistically significant differences between the Vacc-4x and placebo groups as determined by the quality of life health survey (SF-36) .
  • Table 1A Subject demography and baseline characteristics for the ITT population
  • Viral load set point in the Off-ART-Completer subgroup is the mean of the two last VL measurements before ART resumption, p values Wilcoxon Rank Sum Test.
  • Table 5 Proliferative assay responses in CD4, CD8 and CD3 T-cell populations to Vacc-4x ( 15-mers and full length peptide) and p24 antigens (15-mers and recombinant p24) for the Off-ART-Completer subgroup that remained off ART until at least week 52.
  • HIV proteins have discordant associations with viral load. Nature Med. 2007 13:46-53 2. Dahirel V, Shekhar K, Pereyra F. et al . Coordinate linkage of HIV evolution reveals regions of immunological vulnerability. Proc. Natl Acad Sci USA 2011. 108: 11530-5
  • DTH delayed-type hypersensitivity
  • response diversity during and after immunization with a peptide-based HIV-1 immunotherapy candidate Vacc-4x.

Abstract

La présente invention concerne des procédés pour traiter, réduire et/ou retarder les effets pathologiques du virus de l'immunodéficience humaine I (VIH) chez un humain infecté par le VIH ou pour réduire le risque de développer le syndrome de l'immunodéficience acquise (SIDA) chez un humain.
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