WO2016065252A2 - Conception d'un immunogène env trimère natif - Google Patents

Conception d'un immunogène env trimère natif Download PDF

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WO2016065252A2
WO2016065252A2 PCT/US2015/057098 US2015057098W WO2016065252A2 WO 2016065252 A2 WO2016065252 A2 WO 2016065252A2 US 2015057098 W US2015057098 W US 2015057098W WO 2016065252 A2 WO2016065252 A2 WO 2016065252A2
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env
hiv
glycoprotein
clade
vector
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PCT/US2015/057098
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WO2016065252A3 (fr
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Bimal K. CHAKRABARTI
Saikat BOLIAR
Supratik DAS
Tripti SHRIVASTAVA
Charles Richter King
Jayanta Bhattacharya
Sweety SAMAL
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International Aids Vaccine Initiative
Translational Health Science & Technology Institute
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Publication of WO2016065252A2 publication Critical patent/WO2016065252A2/fr
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • This application relates to a novel HIV-1 envelope glycoprotein that is soluble, cleavable and maintains a native conformation which may be utilized as an HIV-1 vaccine immunogen, as a native Env trimer mimic, for identification of small molecules, for use as an immunogen that binds specific HIV-1 broad neutralizing antibodies, for identification of small molecules for use as anti-viral compound that binds specific HIV-1 envelope glycoprotein monomer and/or trimer, as antigens for crystallization and electron microscopy (EM) structural analysis and for the identification of broad neutralizing antibodies from HIV- 1 infected individuals or vaccinated subjects or antibody or ligand libraries.
  • HIV-1 vaccine immunogen as a native Env trimer mimic
  • an immunogen that binds specific HIV-1 broad neutralizing antibodies for identification of small molecules for use as anti-viral compound that binds specific HIV-1 envelope glycoprotein monomer and/or trimer, as antigens for crystallization and electron microscopy (EM) structural analysis and for the identification of broad neutralizing antibodies from HIV- 1 infected
  • HIV human immunodeficiency virus
  • SIV simian immunodeficiency viruses
  • An infectious HIV particle consists of two identical strands of RNA, each approximately 9.2 kb long, packaged within a core of viral proteins. This core structure is surrounded by a phospholipid bilayer envelope derived from the host cell membrane that also includes virally-encoded membrane proteins (Abbas et al, Cellular and Molecular Immunology, 4th edition, W.B. Saunders Company, 2000, p. 454).
  • the HIV genome has the characteristic 5'-LTR-Gag-Pol-Env-LTR-3 ' organization of the retrovirus family.
  • Long terminal repeats (LTRs) at each end of the viral genome serve as binding sites for transcriptional regulatory proteins from the host and regulate viral integration into the host genome, viral gene expression, and viral replication.
  • the HIV genome encodes several structural proteins.
  • the gag gene encodes structural proteins of the nucleocapsid core and matrix.
  • the pol gene encodes reverse transcriptase (RT), integrase (IN), and viral protease (PR) enzymes required for viral replication.
  • the tat gene encodes a protein that is required for elongation of viral transcripts.
  • the rev gene encodes a protein that promotes the nuclear export of incompletely spliced or unspliced viral RNAs.
  • the vz/gene product enhances the infectivity of viral particles.
  • the vpr gene product promotes the nuclear import of viral DNA and regulates G2 cell cycle arrest.
  • the vpu and nef genes encode proteins that down regulate host cell CD4 expression and enhance release of virus from infected cells.
  • the env gene encodes the viral envelope glycoprotein that is translated as a 160-kilodalton (kDa) precursor (gpl60) and cleaved by a cellular protease to yield the external 120-kDa envelope glycoprotein (gpl20) and the transmembrane 41-kDa envelope glycoprotein (gp41), which are required for the infection of cells (Abbas et al, Cellular and Molecular Immunology, 4th edition, W.B. Saunders Company, 2000, pp. 454-456).
  • kDa 160-kilodalton
  • gp41 transmembrane 41-kDa envelope glycoprotein
  • gpl40 is a modified form of the Env glycoprotein, which contains the external 120-kDa envelope glycoprotein portion and the extracellular part of the gp41 portion of Env and has characteristics of both gpl20 and gp41.
  • the nef gene is conserved among primate lentiviruses and is one of the first viral genes that is transcribed following infection. In vitro, several functions have been described, including down- regulation of CD4 and MHC class I surface expression, altered T-cell signaling and activation, and enhanced viral infectivity.
  • HIV infection initiates with gpl20 on the viral particle binding to the CD4 and chemokine receptor molecules (e.g., CXCR4, CCR5) on the cell membrane of target cells such as CD4 + T-cells, macrophages and dendritic cells.
  • CD4 and chemokine receptor molecules e.g., CXCR4, CCR5
  • the bound virus fuses with the target cell and reverse transcribes the RNA genome.
  • the resulting viral DNA integrates into the cellular genome, where it directs the production of new viral RNA, and thereby viral proteins and new virions. These virions bud from the infected cell membrane and establish productive infections in other cells. This process also kills the originally infected cell.
  • HIV can also kill cells indirectly because the CD4 receptor on uninfected T-cells has a strong affinity for gpl20 expressed on the surface of infected cells.
  • the uninfected cells bind, via the CD4 receptor-gpl20 interaction, to infected cells and fuse to form a syncytium, which cannot survive.
  • Destruction of CD4 + T-lymphocytes, which are critical to immune defense, is a major cause of the progressive immune dysfunction that is the hallmark of AIDS disease progression.
  • the loss of CD4 + T cells seriously impairs the body's ability to fight most invaders, but it has a particularly severe impact on the defenses against viruses, fungi, parasites and certain bacteria, including mycobacteria.
  • HIV-1 uses a trimeric Env complex containing gpl20 and gp41 subunits (Burton et al, Nat Immunol. 2004 Mar;5(3):233-6).
  • the fusion potential of the Env complex is triggered by engagement of the CD4 receptor and a coreceptor, usually CCR5 or CXCR4.
  • Neutralizing antibodies seem to work either by binding to the mature trimer on the virion surface and preventing initial receptor engagement events, or by binding after virion attachment and inhibiting the fusion process (Parren & Burton, Adv Immunol. 2001 ;77: 195-262). In the latter case, neutralizing antibodies may bind to epitopes whose exposure is enhanced or triggered by receptor binding. However, given the potential antiviral effects of neutralizing antibodies, it is not unexpected that HIV-1 has evolved multiple mechanisms to protect it from antibody binding (Johnson & Desrosiers, Annu Rev Med. 2002;53 :499-518).
  • HIV-1 envelope glycoprotein (Env) is the main viral protein involved in the entry of the virus and is also the primary target for neutralizing antibodies, but due to immune evasion strategies and extreme sequence variability of Envs, generation of bNabs has been daunting task (Phogat S, Wyatt R. Curr Pharm Des. 2007; 13 :213-27, Phogat S, et al. J Intern Med. 2007 262:26-43, Karlsson Hedestam GB, et al Nat Rev Microbiol. 2008 6: 143-55).
  • Env is the focus of global HIV-1 vaccine immunogen design efforts. In natural HIV-1 infections, approximately 10-20% of chronically infected individuals develop "neutralization breadth". Serum antibodies from these individuals can potently neutralize an array of HIV-1 isolates of different clades.
  • This broad neutralizing activity is mediated by monoclonal antibodies whose target epitopes have been mapped to various sub-regions of the Env such as the CD4 binding site, various glycans and the membrane proximal external region (MPER) (Li Y, et al, Nat Med 2007, 13 : 1032-1034; Li Y, et al, J Virol 2009, 83 : 1045-1059; Moore PL, et al., J Virol 201 1, 85:3128-3141; Sather DN, et al, Vaccine 2010, 28 Suppl 2:B8-12; Walker LM, et al, Science 2009, 326:285-289; Scheid JF, et al, Science 201 1, 333 : 1633-1637; Walker LM, et al, Nature
  • MPER membrane proximal external region
  • Neutralizing monoclonal antibodies isolated from clade B infected donors tend to display less breadth and potency against non- clade B viruses, and they recognize epitopes on the virus that so far have failed to elicit broad neutralizing responses when incorporated into a diverse range of immunogens.
  • Env spike is formed by cleavage of gpl60 glycoprotein by the furin protease into two subunits: the exterior gpl20 and the transmembrane gp41. The cleaved oligomeric subunits of the envelope glycoprotein remain non-covalently bound to form the mature, meta-stable Env structure.
  • Env Efficient cleavage of Env plays an important role in imparting its native trimeric conformation (Ringe RP, et al, Proc Natl Acad Sci U S A 2013, 110: 18256-18261). Many broadly neutralizing antibodies (bnAb) are dependent on Env conformation and one recently isolated bnAb, PGT151 is also cleavage specific (Falkowska E, et al, Immunity 2014, 40:657-668; and Blattner C, et al, Immunity 2014, 40:669-680).
  • the invention relates to an engineered or non-naturally occurring HIV-1 clade C envelope glycoprotein (env).
  • env HIV-1 clade C envelope glycoprotein
  • the glycoprotein binds broadly neutralizing antibodies isolated from HIV-1 infected individuals with high specificity in contrast to the binding of non-neutralizing antibodies.
  • the glycoprotein may be expressed on a cell surface or expressed in soluble form.
  • the present invention encompasses methods to express the glycoprotein in a soluble form and in a membrane bound form. The ability of the glycoprotein to bind with specificity to broadly neutralizing antibodies is dependent upon the protein maintaining its cleaved native trimeric state.
  • the glycoprotein comprises the amino acid sequence comprising SEQ ID NO: 2, 6, or 10.
  • the present invention includes an engineered or non-naturally occurring nucleic acid molecule encoding the HIV-1 clade C envelope glycoprotein.
  • the nucleic acid molecule may be SEQ ID NO: 1, 3, 7, or 1 1.
  • the engineered or non-naturally occurring nucleic acid molecule is codon optimized.
  • the engineered or non-naturally occurring Clade C Env glycoprotein is soluble and maintains a native gpl60 trimeric cleaved conformation when expressed.
  • the glycoprotein includes a deletion of the transmembrane region of g l60.
  • the glycoprotein includes a deletion of the C- terminus, wherein the Kennedy and MPER sequences are conserved.
  • an in frame linker connects the region upstream of the deletion of the transmembrane domain to the C-terminal region downstream of the deletion of the transmembrane domain.
  • the engineered or non-naturally occurring Clade C Env glycoprotein includes stabilization mutations, wherein the stabilization mutations result in cysteine residues on either side of the cleavage site of gpl60, and wherein upon cleavage a disulfide bond can be formed.
  • the stabilization mutations correspond to mutations of A501C and T605C of gpl60 with reference to the position numbering of SEQ ID NO: 2.
  • the engineered or non-naturally occurring Clade C Env glycoprotein includes a proline mutation, wherein the proline mutation is I559P of gpl60 with reference to the position numbering of SEQ ID NO: 2.
  • soluble, stabilized, proteolytically cleaved, trimeric gp41 proteins can be generated by engineering an intermolecular disulphide bond between gpl20 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (SOSIP).
  • the present invention relates to homologous glycoproteins that are modified, but share the characteristics of the glycoproteins of the present invention. Not being bound by a theory, small deviations from the specific sequences will result in a glycoprotein with the same characteristics.
  • the engineered or non- naturally occurring HIV-1 clade C envelope glycoprotein has at least 70%, 80%, 90%, preferably 95% identity to the protein of SEQ ID NO: 2, 6, or 10, wherein the native trimeric cleaved conformation of gpl60 is maintained when the glycoprotein is expressed, and wherein the glycoprotein has specificity to the binding of broadly-neutralizing antibodies.
  • the present invention relates to a vector comprising a nucleic acid encoding the glycoprotein.
  • the vector is configured to express the protein encoded by the nucleic acid in a mammal.
  • the vector is a plasmid.
  • the vector is a virus.
  • the virus is a lentivirus, adenovirus, adeno associated virus (AAV), or poxvirus.
  • the nucleic acid molecule is operably linked to one or more expression control elements.
  • an isolated host cell line maintains the vector.
  • the host cell line may be stably transformed with the vector.
  • the host cell line may be a prokaryotic host cell.
  • the present invention relates to a vaccine or immunogenic composition.
  • the vaccine or immunogenic composition preferably includes an excipient.
  • a genetic vaccine comprises a vector for expression of the envelope glycoprotein and a pharmaceutical excipient.
  • An "excipient” is a natural or synthetic substance formulated alongside the active ingredient of a medication, included for the purpose of bulking-up formulations that contain potent active ingredients (thus often referred to as “bulking agents,” “fillers,” or “diluents”), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility.
  • Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life.
  • the selection of appropriate excipients also depends upon the route of administration and the dosage form, as well as the active ingredient and other factors.
  • the vaccine or immunogenic composition may include a vector that is a virus.
  • the virus may be a retrovirus, adenovirus, AAV, and a poxvirus. Most preferably, MVA is used as the viral vector.
  • the vaccine or immunogenic composition may be a combination of the envelope glycoprotein of the present invention with another HIV antigen. This may include any number of HIV antigens encoded by the gag, pol, and env genes. Additionally, envelope glycoproteins from other strains of HIV can be used in the vaccine or immunogenic composition.
  • the vaccine may also include another vector that includes a nucleic acid encoding a clade B envelope glycoprotein.
  • the clade B envelope glycoprotein may be JRFL.
  • the present invention relates to a method of eliciting an immune response in a mammal comprising introducing the engineered or non-naturally occurring HIV-1 clade C envelope glycoprotein to a mammal.
  • the introducing may be by a viral vector.
  • the immune response can be elicited by use of a genetic vaccine.
  • An immune response may be elicited by introducing protein to the mammal.
  • the protein may be cleaved and solubilized before administration.
  • the protein may become cleaved after administration.
  • the protein may be introduced with an adjuvant.
  • the adjuvant may be a lecithin and may optionally be combined with an acrylic polymer, a lecithin coated oil droplet in an oil-in- water emulsion or a lecithin and an acrylic polymer in an oil-in-water emulsion.
  • the adjuvant may be ISCOMATRIX or Adjuplex.
  • the adjuvant may comprise alum.
  • the soluble envelope glycoproteins of the present invention have about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to any of the sequences described.
  • the envelope glycoproteins have the characteristic of being cleaved in vivo and having a native conformation.
  • the cleaved envelope glycoproteins are preferentially bound by broadly neutralizing antibodies.
  • the envelope glycoprotein of the present invention is used as a tool to determine other envelope glycoproteins that can bind to broadly neutralizing antibodies.
  • Crystal structures of antibodies bound to the envelope glycoprotein indicate epitopes important to bind broadly neutralizing antibodies.
  • structures of unknown envelope glycoproteins can be inferred based on the structure of the envelope glycoprotein of the present invention. In this way, envelope glycoproteins most likely to elicit broadly neutralized antibodies can be selected for further testing.
  • the envelope glycoprotein can be used to make therapeutic broadly neutralizing antibodies for treatment of patients in need thereof.
  • the envelope glycoproteins of the present invention are used to generate antibodies in an animal.
  • the animal expresses human light chain and heavy chain variable domain genes and is used to generate humanized antibodies.
  • the antibodies are administered to a patient in need thereof.
  • Figure 1 Illustrates screening of Indian clade C Envs for cleavage.
  • A Representative Western blot analysis of cleavage-competent full-length JRFL and 4-2. J41 Env from total cell lysate. The Env glycoproteins were probed with anti-HIV human polysera (HIVIG).
  • B Graphic presentation of FACS-based cell surface antibody binding assay.
  • C Representative FACS-based cell surface staining curves with Mean Fluorescent Intensity (MFI) values of cleavage-competent JRFL(+), cleavage-defective JRFL(-) and 4-2.
  • MFI Mean Fluorescent Intensity
  • the JRFL(+) and JRFL(-) Env clones are cytoplasmic -tail truncated at a.a. 709, unless mentioned otherwise.
  • Figure 2. Illustrates efficient cleavage of 4-2.J41 Env on the cell surface.
  • A FACS-based cell surface binding curves of wild type and cleavage-defective JRFL and 4- 2.J41 Env. The wild type and cleavage-defective Envs are designated by (+) and (-), respectively. Mean fluorescence intensities (MFI) of binding of cleavage-dependent antibody PGT151 are shown. The graphs shown here are derived from the same representative experiments. Bars at each antibody concentration indicate the SEM values for duplicate samples.
  • B Western blot analysis of Env glycoproteins from cell surface biotinylation. Cell surface expressed JRFL(+) and 4-2.
  • J41 Envs were biotinylated, lysed and immunoprecipitated with neutravidin-agarose before analyzed by Western blot and probed with anti-HIV human polysera (HIVIG).
  • C Western blot analysis for spontaneous shedding of gpl20. Cell-free supernatant of Env transfected 293T cells were precipitated with lectin- agarose and then analyzed by Western blot and probed with anti-HIV human polysera (HIVIG).
  • Figure 3 Illustrates antibody binding curves of cleavage-competent JRFL (+), cleavage-defective JRFL (-) and 4-2.J41 Env.
  • Mean fluorescence intensities (MFI) of FACS- based binding assay for neutralizing (VRCOl, 10E8, PG9, PG16, PGT121, PGT145) and non-neutralizing (b6, 17b, 39F, 22B) antibodies are shown in panel (A) and (B), respectively.
  • the graphs shown here are derived from the same representative experiments. Bars at each antibody concentration indicate the SEM values for duplicate samples.
  • Figure 4 Illustrates effects of cleavage-defective mutations in 4-2. J41 Env to antibody binding.
  • the wild type and cleavage-defective Envs are designated by (+) and (-), respectively.
  • Mean fluorescence intensities (MFI) of binding of neutralizing (VRCOl, PGT121, PG9, 4E10) and non-neutralizing antibodies (F105, 17b, b6, 5 and 7B2) are shown in panel (A) and (B), respectively.
  • the graphs shown here are derived from the same representative experiments. Bars at each antibody concentration indicate the SEM values for duplicate samples.
  • Figure 5 Illustrates CD4-induced shedding of gpl20 from 4-2.J41 Env spikes.
  • (B) 293T cells expressing 4-2.J41 were treated with 25g of sCD4 for 1 hour at room temperature and the cell supernatants were analyzed by ELISA for the presence of "shed" gpl20. The gpl20 present in cell supernatants were captured on the ELISA plates by pre- coating with lectin and then detected with anti-gpl20 clad C rabbit polyclonal antibody.
  • FIG. Illustrates effects of increased cell surface expression of 4-2.J41 Env to antibody binding.
  • A Binding curves of VRCOl, F105 and b6 antibodies to wild-type and cytoplasmic tail deleted 4-2.J41delCT Env.
  • B Binding curves of VRCOl, F 105 and b6 antibodies to wild-type and codon-optimized 4-2. J41 Env. The graphs shown here are derived from the same representative experiments. Bars at each antibody concentration indicate the SEM values for duplicate samples.
  • Figure 7 Illustrates the strategy for screening of cleaved native clade-C Env.
  • Figure 8. Illustrates the differential binding of neutralizing vs. non-neutralizing antibodies.
  • Figure 9. Illustrates that Clade C Env 4-2.J41 is efficiently cleaved similar to JRFL.
  • Figure 10 Illustrates that PGT151 selectively binds to cleaved Envs.
  • Figure 11 Illustrates that 4-2. J41 binds selectively to broadly neutralizing antibodies.
  • Figure 12 Illustrates that structural occlusion inhibits binding of non-neutralizing antibodies to 4-2. J41 env.
  • Figure 13 Illustrates that codon-optimized 4-2. J41 Env maintains its native conformation.
  • Figure 14 Illustrates western blot analysis of neuraminidase and endo H treated Env. Spontaneously shed 4-2. J41 and JRFL Env proteins in cell culture supernatant were deglycosylated with Neuraminidase and Endo H for analysis by Western blot with HIVIG antibodies as probe.
  • FIG. 15 Illustrates increased cell surface expression of tail-truncated and codon optimized 4-2.
  • J41 Env. (A-B) Binding curves of PGT121 antibody to wildtype and tail- truncated (delCT) or codon-optimized (CO) 4-2. J41 Env. The graphs shown here are derived from the same representative experiments. Bars at each antibody concentration indicate the SEM values for duplicate samples.
  • Figure 16 (A & B). Illustrates comparative binding ability of 4-2. J41 and 4- 2.J41delCT Envs to neutralizing and non-neutralizing antibodies. Deletion of the C terminal sequence of 4-2.J41 Env enhanced the binding with non-neutralizing antibodies.
  • Figure 17 Illustrates comparative binding ability of 4-2. J41 Env to neutralizing and non-neutralizing antibodies, (B) Illustrates that extension of C-terminal sequences beyond 745 restored the conformation similar to wild type 4-2. J41 without compromising the cleavage.
  • the invention provides a non-naturally occurring or engineered cleavage competent clade C HIV-1 Env, 4-2.J41, derived from the plasma of an HIV-1 infected individual in India.
  • This Env is stable and efficiently exposes epitopes for an array of known broadly neutralizing antibodies.
  • the Env can also be expressed in a soluble, natively cleaved form that maintains the native conformation of native Env.
  • Soluble, stabilized, proteolytically cleaved, trimeric proteins may be generated by engineering an intermolecular disulphide bond between gpl20 and gp41 (SOS), combined with a single residue change, I559P, within gp41 (SOSIP).
  • 7,939,083 are the determinants of this enhanced stability which are located in the N-terminal region of KNH1 1144 gp41 and that, when substituted into heterologous Env sequences (e.g., JR-FL and Ba-L) they have a similarly beneficial effect on trimer stability.
  • These stabilized trimers retain the epitopes for several neutralizing antibodies and related agents (CD4-IgG2, bl2, 2G12, 2F5 and 4E10) and the CD4-IgG2 molecule, so that the overall antigenic structure of the gpl40 protein has not been adversely impaired by the trimer-stabilizing substitutions.
  • the protein is optimized for expression in mammals.
  • Env protein for use as an antigen in eliciting and identifying broadly neutralizing antibodies against HIV-1.
  • the Env protein can be used in a vaccine or immunogenic composition.
  • a DNA or genetic vaccine is used.
  • the vaccine includes a virus.
  • isolated Env protein is used as an immunogen.
  • the protein can be used as an antigen to produce broadly neutralizing antibodies in an animal.
  • the animal expresses human heavy and light chain variable regions to produce fully humanized antibodies. Such antibodies may be used as a therapeutic for HIV infected patients in need thereof.
  • the Env protein also can be used in screening for broadly neutralizing antibodies. Crystal structures of the envelope glycoprotein bound by broadly neutralizing antibodies can be used to determine important epitopes for envelope glycoproteins from other strains of HIV.
  • the present invention refers to various domains and sites present in all HIV- 1 Env proteins. Such sequences and domains are well known in the art and can be found by accessing P04578-ENV_HV1H2 on the UniProt website
  • the Kennedy sequence refers to a hydrophilic sequence centered around position 740 of the gpl60 protein sequence and is C-terminal to the transmembrane or membrane spanning domain.
  • the transmembrane domain is generally located at position 685-705 in the gpl60 protein sequence.
  • the cleavage site is centered around position 512 of gpl60.
  • the MPER is located around position 662-683. Modification of any of these sites by mutation is understood to include modifications occurring to sites slightly deviating from these exact positions.
  • the terms, "Env protein”, Env", and "glycoprotein” all refer to the envelope glycoprotein of HIV.
  • protein protein
  • peptide polypeptide
  • amino acid sequence amino acid sequence
  • the terms are used interchangeably herein to refer to polymers of amino acid residues of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
  • the terms "antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject.
  • the term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular type directed against that protein.
  • antibody includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, Fv and scFv which are capable of binding the epitope determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and include, for example:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • F(ab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;
  • scFv including a genetically engineered fragment containing the variable region of a heavy and a light chain as a fused single chain molecule.
  • a "neutralizing antibody” may inhibit the entry of HIV-1 virus with a neutralization index >1.5 or >2.0. Broad and potent neutralizing antibodies may neutralize greater than about 50% of HIV-1 viruses (from diverse clades and different strains within a clade) in a neutralization assay. The inhibitory concentration of the monoclonal antibody may be less than about 25 mg/ml to neutralize about 50% of the input virus in the neutralization assay.
  • proteins including the antibodies and/or antigens of the invention may differ from the exact sequences illustrated and described herein.
  • the invention contemplates deletions, additions and substitutions to the sequences shown, so long as the sequences function in accordance with the methods of the invention.
  • particularly preferred substitutions are generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids.
  • amino acids are generally divided into four families: (1) acidic— aspartate and glutamate; (2) basic— lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • nucleotide sequences and “nucleic acid sequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA/RNA hybrids, or synthetic nucleic acids.
  • the nucleic acid can be single-stranded, or partially or completely double-stranded (duplex).
  • Duplex nucleic acids can be homoduplex or heteroduplex.
  • transgene may be used to refer to "recombinant" nucleotide sequences that may be derived from any of the nucleotide sequences encoding the proteins of the present invention.
  • the term “recombinant” means a nucleotide sequence that has been manipulated “by man” and which does not occur in nature, or is linked to another nucleotide sequence or found in a different arrangement in nature. It is understood that manipulated “by man” means manipulated by some artificial means, including by use of machines, codon optimization, restriction enzymes, etc.
  • the nucleotide sequences of the envelope glycoproteins may be codon optimized, for example the codons may be optimized for human use.
  • the nucleic acid molecules of the invention have a nucleotide sequence that encodes the antigens of the invention and can be designed to employ codons that are used in the genes of the subject in which the antigen is to be produced.
  • Many viruses, including HIV and other lentiviruses use a large number of rare codons and, by altering these codons to correspond to codons commonly used in the desired subject, enhanced expression of the antigens can be achieved.
  • the codons used are "humanized" codons, i.e., the codons are those that appear frequently in highly expressed human genes (Andre et al, J. Virol. 72: 1497-1503, 1998) instead of those codons that are frequently used by HIV.
  • Such codon usage provides for efficient expression of the transgenic HIV proteins in human cells. Any suitable method of codon optimization may be used. Such methods, and the selection of such methods, are well known to those of skill in the art.
  • there are several companies that will optimize codons of sequences such as Geneart (geneart.com).
  • Geneart geneart.com
  • the invention further encompasses nucleotide sequences encoding functionally and/or antigenically equivalent variants and derivatives of the envelope glycoproteins of the invention and functionally equivalent fragments thereof.
  • These functionally equivalent variants, derivatives, and fragments display the ability to retain antigenic activity. For instance, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide.
  • Conservative amino acid substitutions are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tyrosine/tryptophan.
  • the variants have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity to the antigen, epitope, immunogen, peptide or polypeptide of interest.
  • the soluble envelope glycoproteins of the present invention have about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence identity to any of the sequences depicted in the figures and/or specification.
  • sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • sequence identity may be determined using any of a number of mathematical algorithms.
  • a nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993;90: 5873-5877.
  • Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988;4: 1 1-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.
  • WU-BLAST Woodington University BLAST
  • WU-BLAST version 2.0 executable programs for several UNIX platforms can be downloaded from ftp://blast.wustl.edu/blast/executables.
  • the various recombinant nucleotide sequences and antibodies and/or antigens of the invention are made using standard recombinant DNA and cloning techniques. Such techniques are well known to those of skill in the art. See for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al. 1989).
  • the nucleotide sequences of the present invention may be inserted into “vectors.”
  • the term “vector” is widely used and understood by those of skill in the art, and as used herein the term “vector” is used consistent with its meaning to those of skill in the art.
  • the term “vector” is commonly used by those skilled in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule.
  • any vector that allows expression of the envelope glycoprotein of the present invention may be used in accordance with the present invention.
  • the envelope glycoprotein of the present invention may be used in vitro (such as using cell-free expression systems) and/or in cultured cells grown in vitro in order to produce the encoded HIV envelope glycoprotein which may then be used for various applications such as in the production of proteinaceous vaccines.
  • any vector that allows expression of the envelope glycoprotein in vitro and/or in cultured cells may be used.
  • any vector that allows for the expression of the envelope glycoprotein of the present invention and is safe for use in vivo may be used.
  • the vectors used are safe for use in humans, mammals and/or laboratory animals.
  • the protein coding sequence should be "operably linked" to regulatory or nucleic acid control sequences that direct transcription and translation of the protein.
  • a coding sequence and a nucleic acid control sequence or promoter are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the nucleic acid control sequence.
  • nucleic acid control sequence can be any nucleic acid element, such as, but not limited to promoters, enhancers, IRES, introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto.
  • promoter will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for R A polymerase II and that when operationally linked to the protein coding sequences of the invention lead to the expression of the encoded protein.
  • the expression of the transgenes of the present invention can be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals.
  • the promoter can also be specific to a particular cell-type, tissue or organ.
  • suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for expression of the transgenes of the invention.
  • suitable promoters and/or enhancers can be selected from the Eukaryotic Promoter Database (EPDB).
  • the present invention relates to a recombinant vector expressing an envelope glycoprotein.
  • the present invention may encompass additional HIV antigens, epitopes or immunogens.
  • the additional HIV epitope is an HIV antigen, HIV epitope or an HIV immunogen, such as, but not limited to, the HIV antigens, HIV epitopes or HIV immunogens of U.S. Patent Nos. 7,341,731;
  • HIV in another embodiment, HIV, or immunogenic fragments thereof, may be utilized as the HIV epitope.
  • any epitope recognized by an HIV antibody may be used in the present invention.
  • the anti-HIV antibodies of U.S. Patent Nos. 6,949,337, 6,900,010, 6,821,744, 6,768,004, 6,613,743, 6,534,312, 6,511,830, 6,489, 131, 6,242, 197, 6, 114,143, 6,074,646, 6,063,564, 6,060,254, 5,919,457, 5,916,806, 5,871,732, 5,824,304, 5,773,247, 5,736,320, 5,637,455, 5,587,285, 5,514,541, 5,317,009, 4,983,529, 4,886,742, 4,870,003 and 4,795,739 are useful for the present invention.
  • the vectors used in accordance with the present invention should typically be chosen such that they contain a suitable gene regulatory region, such as a promoter or enhancer, such that the envelope glycoprotein of the invention can be expressed.
  • any suitable vector can be used depending on the application.
  • plasmids, viral vectors, bacterial vectors, protozoal vectors, insect vectors, baculovirus expression vectors, yeast vectors, mammalian cell vectors, and the like can be used.
  • Suitable vectors can be selected by the skilled artisan taking into consideration the characteristics of the vector and the requirements for expressing the envelope glycoprotein under the identified circumstances.
  • the aim is to express the envelope glycoprotein of the invention in vivo in a subject, for example in order to generate an immune response against an HIV-1 antigen and/or protective immunity against HIV-1
  • expression vectors that are suitable for expression on that subject, and that are safe for use in vivo, should be chosen.
  • any vectors that are suitable for such uses can be employed, and it is well within the capabilities of the skilled artisan to select a suitable vector.
  • the vectors used for these in vivo applications are attenuated to prevent the vector from amplifying in the subject.
  • plasmid vectors preferably they will lack an origin of replication that functions in the subject so as to enhance safety for in vivo use in the subject.
  • viral vectors preferably they are attenuated or replication-defective in the subject, again, so as to enhance safety for in vivo use in the subject.
  • the present invention also encompassed the use of the soluble envelope glycoprotein described herein as an immunogen, advantageously as HIV-1 vaccine components.
  • a vaccine is used to produce broadly neutralizing antibodies in a subject.
  • An essential component of a HIV-1 vaccine design is to mimic the native Env spikes on the virion in the immunogen.
  • the Env, protein 4-2.J41 when expressed on the cell surface, likely assumes a native conformation. This Env is stable, selectively exposes epitopes for only neutralizing antibodies and presents native trimeric conformation in the membrane-anchored form.
  • a trimeric envelope glycoprotein that is fully cleaved and assumes a native conformation is the immunogen for eliciting neutralizing antibodies.
  • JRFL was the only naturally cleaved Env available on which such immunogens could be designed.
  • a vaccine or immunogenic composition includes both 4-2. J41 and JRFL.
  • An effective system for presenting HIV-1 Env as a membrane-anchored immunogen is through a genetic vaccine method such as a virus vector or plasmid DNA. Since it would be important for an HIV-1 Env vaccine candidate to bear structural resemblance and similar physio-chemical properties as the Env present on infectious viruses; expression of an efficiently cleaved Env in its native trimeric conformation on the cell surface through genetic vaccination, described herein, could prompt the immune system towards elicitation of potent neutralizing antibodies.
  • viral vectors are used.
  • Viral expression vectors are well known to those skilled in the art and include, for example, viruses such as adenoviruses, adeno-associated viruses (AAV), alphaviruses, herpesviruses, retroviruses and poxviruses, including avipox viruses, attenuated poxviruses, vaccinia viruses, and particularly, the modified vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566).
  • viruses when used as expression vectors are innately non-pathogenic in the selected subjects such as humans or have been modified to render them non-pathogenic in the selected subjects.
  • replication-defective adenoviruses and alphaviruses are well known and can be used as gene delivery vectors.
  • the nucleotide sequences and/or envelope glycoproteins of the invention are administered in vivo, for example where the aim is to produce an immunogenic response in a subject.
  • a "subject" in the context of the present invention may be any animal.
  • the subject is a human, for example a human that is infected with, or is at risk of infection with, HIV- 1.
  • the nucleotide sequences and/or envelope glycoproteins of the invention are preferably administered as a component of an immunogenic composition comprising the nucleotide sequences and/or envelope glycoproteins of the invention in admixture with a pharmaceutically acceptable carrier.
  • the immunogenic compositions of the invention are useful to stimulate an immune response against HIV-1 and may be used as one or more components of a prophylactic or therapeutic vaccine against HIV-1 for the prevention, amelioration or treatment of AIDS.
  • the nucleic acids and vectors of the invention are particularly useful for providing genetic vaccines, i.e. vaccines for delivering the nucleic acids encoding the envelope glycoproteins of the invention to a subject, such as a human, such that the envelope glycoproteins are then expressed in the subject to elicit an immune response.
  • compositions of the invention may be injectable suspensions, solutions, sprays, lyophilized powders, syrups, elixirs and the like. Any suitable form of composition may be used.
  • a nucleic acid or vector of the invention having the desired degree of purity, is mixed with one or more pharmaceutically acceptable carriers and/or excipients.
  • the carriers and excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobul
  • An immunogenic or immunological composition can also be formulated in the form of an oil-in-water emulsion.
  • the oil-in-water emulsion can be based, for example, on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane, squalene, EICOSANETM or tetratetracontane; oil resulting from the oligomerization of alkene(s), e.g., isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, such as plant oils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryl tri(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, e.g., isostearic acid esters.
  • the oil advantageously is used in combination with emulsifiers to form the emulsion.
  • the emulsifiers can be nonionic surfactants, such as esters of sorbitan, mannide (e.g., anhydromannitol oleate), glycerol, polyglycerol, propylene glycol, and oleic, isostearic, ricinoleic, or hydroxystearic acid, which are optionally ethoxylated, and poly oxypropylene-polyoxy ethylene copolymer blocks, such as the Pluronic® products, e.g., L121.
  • the adjuvant can be a mixture of emulsifier(s), micelle-forming agent, and oil such as that which is commercially available under the name Provax® (IDEC Pharmaceuticals, San Diego, CA).
  • the immunogenic compositions of the invention can contain additional substances, such as wetting or emulsifying agents, buffering agents, or adjuvants to enhance the effectiveness of the vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.) 1980).
  • Adjuvants may also be included.
  • Adjuvants include, but are not limited to, mineral salts (e.g., A1K(S04)2, AlNa(S04)2, A1NH(S04)2, silica, alum, Al(OH)3, Ca3(P04)2, kaolin, or carbon), polynucleotides with or without immune stimulating complexes (ISCOMs) (e.g., CpG oligonucleotides, such as those described in Chuang, T.H. et al, (2002) J. Leuk. Biol. 71(3): 538- 44; Ahmad-Nejad, P. et al (2002) Eur. J. Immunol.
  • mineral salts e.g., A1K(S04)2, AlNa(S04)2, A1NH(S04)2, silica, alum, Al(OH)3, Ca3(P04)2, kaolin, or carbon
  • ISCOMs immune stimulating complexes
  • saponins such as QS21, QS17, and QS7 (U.S. Patent Nos. 5,057,540; 5,650,398; 6,524,584; 6,645,495), monophosphoryl lipid A, in particular, 3-de-O- acylated monophosphoryl lipid A (3D-MPL), imiquimod (also known in the art as IQM and commercially available as Aldara®; U.S. Patent Nos. 4,689,338; 5,238,944; Zuber, A.K. et al (2004) 22(13-14): 1791-8), and the CCR5 inhibitor CMPD167 (see Veazey, R.S. et al (2003) J. Exp. Med. 198: 1551-1562).
  • Aluminum hydroxide or phosphate are commonly used at 0.05 to 0.1% solution in phosphate buffered saline.
  • cytokines such as, but not limited to, IL-2, IL-4, GM-CSF, IL-12, IL-15 IGF-1, IFN-a, IFN- ⁇ , and IFN- ⁇
  • immunoregulatory proteins such as CD40L (ADX40; see, for example, WO03/063899)
  • CD 1 a ligand of natural killer cells also known as CRONY or a-galactosyl ceramide; see Green, T.D. et al, (2003) J. Virol.
  • immunostimulatory fusion proteins such as IL-2 fused to the Fc fragment of immunoglobulins (Barouch et al, Science 290:486- 492, 2000) and co-stimulatory molecules B7.1 and B7.2 (Boyer), all of which can be administered either as proteins or in the form of DNA, on the same expression vectors as those encoding the antigens of the invention or on separate expression vectors.
  • the adjuvants may be lecithin combined with an acrylic polymer (Adjuplex-LAP), lecithin coated oil droplets in an oil-in-water emulsion (Adjuplex-LE) or lecithin and acrylic polymer in an oil-in-water emulsion (Adjuplex-LAO) (Advanced BioAdjuvants (ABA)).
  • Adjuplex-LAP acrylic polymer
  • Adjuplex-LE lecithin coated oil droplets in an oil-in-water emulsion
  • Adjuplex-LAO Advanced BioAdjuvants
  • the immunogenic compositions can be designed to introduce the nucleic acids or expression vectors to a desired site of action and release it at an appropriate and controllable rate.
  • Methods of preparing controlled-release formulations are known in the art.
  • controlled release preparations can be produced by the use of polymers to complex or absorb the immunogen and/or immunogenic composition.
  • a controlled-release formulation can be prepared using appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled release characteristics or release profile.
  • Another possible method to control the duration of action by a controlled-release preparation is to incorporate the active ingredients into particles of a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers.
  • a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Suitable dosages of the nucleic acids and expression vectors of the invention in the immunogenic composition of the invention can be readily determined by those of skill in the art.
  • the dosage of the immunogens can vary depending on the route of administration and the size of the subject.
  • Suitable doses can be determined by those of skill in the art, for example by measuring the immune response of a subject, such as a laboratory animal, using conventional immunological techniques, and adjusting the dosages as appropriate.
  • Such techniques for measuring the immune response of the subject include but are not limited to, chromium release assays, tetramer binding assays, IFN- ⁇ ELISPOT assays, IL-2 ELISPOT assays, intracellular cytokine assays, and other immunological detection assays, e.g., as detailed in the text "Antibodies: A Laboratory Manual” by Ed Harlow and David Lane.
  • the immunogenic compositions of the invention are ideally administered to a subject in advance of HIV infection, or evidence of HIV infection, or in advance of any symptom due to AIDS, especially in high-risk subjects.
  • the prophylactic administration of the immunogenic compositions can serve to provide protective immunity of a subject against HIV-1 infection or to prevent or attenuate the progression of AIDS in a subject already infected with HIV-1.
  • the immunogenic compositions can serve to ameliorate and treat AIDS symptoms and are advantageously used as soon after infection as possible, preferably before appearance of any symptoms of AIDS but may also be used at (or after) the onset of the disease symptoms.
  • the immunogenic compositions can be administered using any suitable delivery method including, but not limited to, intramuscular, intravenous, intradermal, mucosal, and topical delivery. Such techniques are well known to those of skill in the art. More specific examples of delivery methods are intramuscular injection, intradermal injection, and subcutaneous injection. However, delivery need not be limited to injection methods. Further, delivery of DNA to animal tissue has been achieved by cationic liposomes (Watanabe et al, (1994) Mol. Reprod. Dev.
  • delivery routes can be oral, intranasal or by any other suitable route. Delivery also be accomplished via a mucosal surface such as the anal, vaginal or oral mucosa.
  • Immunization schedules are well known for animals (including humans) and can be readily determined for the particular subject and immunogenic composition.
  • the immunogens can be administered one or more times to the subject.
  • there is a set time interval between separate administrations of the immunogenic composition typically it ranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks.
  • the interval is typically from 2 to 6 weeks.
  • the immunization regimes typically have from 1 to 6 administrations of the immunogenic composition, but may have as few as one or two or four.
  • the methods of inducing an immune response can also include administration of an adjuvant with the immunogens. In some instances, annual, biannual or other long interval (5-10 years) booster immunization can supplement the initial immunization protocol.
  • the present methods also include a variety of prime-boost regimens, for example DNA prime-Adenovirus boost regimens.
  • one or more priming immunizations are followed by one or more boosting immunizations.
  • the actual immunogenic composition can be the same or different for each immunization and the type of immunogenic composition (e.g., containing protein or expression vector), the route, and formulation of the immunogens can also be varied.
  • an expression vector is used for the priming and boosting steps, it can either be of the same or different type (e.g., DNA or bacterial or viral expression vector).
  • Prime-boost regimen provides for two priming immunizations, four weeks apart, followed by two boosting immunizations at 4 and 8 weeks after the last priming immunization. It should also be readily apparent to one of skill in the art that there are several permutations and combinations that are encompassed using the DNA, bacterial and viral expression vectors of the invention to provide priming and boosting regimens.
  • a specific embodiment of the invention provides methods of inducing an immune response against HIV in a subject by administering an immunogenic composition of the invention, preferably comprising an adenovirus vector containing DNA encoding one or more of the epitopes of the invention, one or more times to a subject wherein the epitopes are expressed at a level sufficient to induce a specific immune response in the subject.
  • an immunogenic composition of the invention preferably comprising an adenovirus vector containing DNA encoding one or more of the epitopes of the invention, one or more times to a subject wherein the epitopes are expressed at a level sufficient to induce a specific immune response in the subject.
  • Such immunizations can be repeated multiple times at time intervals of at least 2, 4 or 6 weeks (or more) in accordance with a desired immunization regime.
  • the immunogenic compositions of the invention can be administered alone, or can be co-administered, or sequentially administered, with other HIV immunogens and/or HIV immunogenic compositions, e.g., with "other" immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or "cocktail” or combination compositions of the invention and methods of employing them.
  • the ingredients and manner (sequential or co-administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration.
  • the other HIV immunogens can be administered at the same time or at different times as part of an overall immunization regime, e.g., as part of a prime-boost regimen or other immunization protocol.
  • HIV immunogens are known in the art, one such preferred immunogen is HTVA (described in WO 01/47955), which can be administered as a protein, on a plasmid (e.g., pTHr.HIVA) or in a viral vector (e.g., MVA.HIVA).
  • HTVA plasmid
  • RENTA viral vector
  • one method of inducing an immune response against HIV in a human subject comprises administering at least one priming dose of an HIV immunogen and at least one boosting dose of an HIV immunogen, wherein the immunogen in each dose can be the same or different, provided that at least one of the immunogens is an epitope of the present invention, a nucleic acid encoding an epitope of the invention or an expression vector, preferably a VSV vector, encoding an epitope of the invention, and wherein the immunogens are administered in an amount or expressed at a level sufficient to induce an HIV-specific immune response in the subject.
  • the HIV-specific immune response can include an HIV- specific T-cell immune response or an HIV-specific B-cell immune response.
  • Such immunizations can be done at intervals, preferably of at least 2-6 or more weeks.
  • Pseudotyped viruses may be generated by co-transfecting cells with at least two plasmids encoding the soluble Env cDNA of the present invention and the rest of the HIV genome separately.
  • the Env gene may be replaced by the firefly luciferase gene.
  • Transfectant supernatants containing pseudotyped virus may be co-incubated overnight with B cell supernatants derived from activation of an infected donor's primary peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • Cells stably transfected with and expressing CD4 plus the CCR5 and CXCR4 coreceptors may be added to the mixture and incubated for 3 days at 37° C. Infected cells may be quantified by luminometry.
  • the soluble envelope glycoproteins of the present invention may be crystallized in combination with PG9, VRCOl, PGT121, 10E8, PGT145 or PG16 or with any other neutralizing antibodies, including those described in the examples, to determine the exact molecular surface where the soluble envelope glycoprotein binds with the neutralizing antibody to design HIV-1 immunogens.
  • Crystals of the invention may be obtained by conventional means as are well- known in the art of protein crystallography, including batch, liquid bridge, dialysis, vapor diffusion and hanging drop methods (see, e.g., Johnson et al., Biochemistry. 1982 Sep 28;21(20):4839-43; Brayer & McPherson, J Biol Chem. 1982 Apr 10;257(7):3359-61 ; McPherson &Weickmann, J Biomol Struct Dyn. 1990 Apr;7(5): 1053-60; and Koszelak et al, J Mol Biol. 1989 Sep 20;209(2):323-5; Weber et al., Acta Crystallogr B. 1991 Feb 1;47 ( Pt l): 116-27 and Weber, Methods Enzymol. 1991;202:727-41).
  • the crystals of the invention are grown by dissolving a substantially pure neutralizing antibody, such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16, and soluble envelope glycoprotein in an aqueous buffer containing a precipitant at a concentration just below that necessary to precipitate the protein. Water is removed by controlled evaporation to produce precipitating conditions, which are maintained until crystal growth ceases.
  • a substantially pure neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16
  • the crystals of the invention and particularly the atomic structure co-ordinates obtained therefrom, have a wide variety of uses.
  • the crystals and structure co-ordinates are particularly useful for identifying protein domains that bind to a neutralizing antibody, such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16, and thus are useful to elicit anti-HIV antibodies.
  • a neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16
  • Such protein domains may be useful in eliciting clade C anti-HIV antibodies, however variants may be useful in eliciting clade A, B, D or E anti-HIV antibodies.
  • the structure co-ordinates may be used as phasing models in determining the crystal structures of a synthetic or mutated neutralizing antibody, such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16, domains, as well as the structures of co-crystals of such domains with ligands.
  • a synthetic or mutated neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16
  • the method may use the co-ordinates of atoms of interest of a neutralizing antibody which are in the vicinity of the binding region in order to model the pocket in which the ligand binds. These coordinates may be used to define a space which is then screened "in silico" against a candidate molecule.
  • the invention provides a computer-based method of rational drug or compound design or identification which comprises: providing the coordinates of at least selected co-ordinates; providing the structure of a candidate compound; and fitting the structure of the candidate to the selected co-ordinates.
  • a neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16, as defined by its co-ordinates which represent the active site or binding region.
  • a neutralizing antibody such as PG9, VRCOl, PGT121, 10E8, PGT145 or PG16
  • One advantage of the envelope glycoprotein of the invention is that the codon- optimized 4-2. J41 Env, unlike JRFL, displays similar properties as its non-codon optimized counterpart. These findings have important implications in HIV-1 vaccine research as 4- 2.J41 can provide an additional platform for Env based immunogen design.
  • Applicants have identified and optimally engineered a clade C primary isolate Env, 4-2. J41, which when expressed on the cell surface is efficiently cleaved and likely conforms to the native structure as evidenced from its binding properties to several neutralizing and non-neutralizing antibodies (see examples). Applicants have additionally, engineered the Env protein to retain the characteristics described herein in a soluble form.
  • 4-2.J41 Env provides a novel vaccine or immunogenic composition with multiclade cleaved Env immunogens.
  • Such multiclade Env vaccination strategy has been shown to increase neutralization breadth (Chakrabarti BK, et al, Vaccine 2005, 23 :3434-3445). Therefore, utilization of this cleavage-competent Env, 4-2.J41, from subtype C, will serve as an additional platform and broaden the approaches of designing potential immunogens for development of an effective HIV- 1 vaccine.
  • Applicants disclose the use of a cleavage competent clade C HIV-1 Env, 4-2. J41, from the plasma of an HIV infected individual in India. This Env is stable and efficiently exposes epitopes for an array of known broadly neutralizing antibodies. Results indicate that 4-2.J41, when expressed on the cell surface, likely assumes a native conformation. A codon- optimized 4-2. J41 Env, unlike JRFL, displays similar properties as its non-codon optimized counterpart. The soluble form also is stable and efficiently exposes epitopes for an array of known broadly neutralizing antibodies and likely assumes a native conformation. These findings have important implications in HIV-1 vaccines and can provide an additional platform for Env based immunogen design.
  • the selected 10 Env clones, which showed some degree of cleavage in Western blot were further investigated for their cleavage efficiency by another relatively more sensitive method, cell surface expression followed by FACS based antibody binding assay (FIG. IB).
  • the Envs tested were found to bind the neutralizing and non-neutralizing antibodies at varying concentrations.
  • MFI ratio of binding
  • CD4bs directed neutralizing vs. non- neutralizing antibody
  • VRCOl vs. F105 Supplementary Table 1
  • Table 1 Ratio of binding to neutralizing (VRCOl) versus non-neutralizing (F 105) antibodies by different Indian clade C Envs in cell-surface binding assay. The data is the average of three independent experiments.
  • Env 4-2.J41 is efficiently cleaved on the cell surface
  • 4-2.J41 Env Applicants used cell surface labeling with biotin that is impermeable to cells. The biotinylated Envelope glycoproteins in cell lysate were precipitated by neutravidin-agarose.
  • Applicants observed only gpl20 protein in the gel indicating that 4-2.J41 Env present on the cell surface was only in cleaved form (Fig 2B).
  • Applicants also tested Env transfected cell culture supernatants by Western blot for presence of gpl20 proteins.
  • the gpl20 subunit of Env is "spontaneously" shed from the membrane and can be detected in the supernatant.
  • gpl20 protein was also detected in the supernatant of 4-2.
  • J41 transfected cells providing evidence of cleavage and "spontaneous" shedding of cell surface expressed 4-2.
  • J41 Env (Fig 2C). In comparison to JRFL, a slightly higher migrating band was observed for 4-2. J41 gpl20.
  • J41 presents a cleaved native structure that can be recognized by PGT151 antibody.
  • biochemical analysis of the Env glycoprotein also demonstrated that 4-2. J41 Env on the cell surface is mostly cleaved as only a gpl20 protein species was detected in biotinylated samples.
  • Results of the neutralization assays as well as cell surface binding assays confirmed that 4-2. J41 Env glycoproteins were recognized more efficiently by neutralizing antibodies compared to non-neutralizing antibodies on virion as well as cell surface. This antigenic property of 4-2.J41 Env is suitable for immunogen design.
  • Env 4-2.J41 similar to JRFL, bound to CD4bs-directed neutralizing antibody VRCOl, but does not bind to non-neutralizing antibodies F105 and b6 (Fig 3).
  • Fig 3 A 4-2.J41 Env bound to trimer conformation-dependent antibodies PG9 and PG16 efficiently (Fig 3 A).
  • JRFL is known to lack the epitopes for these two antibodies and as expected did not bind PG9 and PG16 antibodies (Fig 3 A).
  • J41 as only epitopes for broadly neutralizing antibodies but not non-neutralizing antibodies are exposed. Binding of PG9, PG16 and PGT145 to this Env also suggests that 4-2. J41 Env likely assumes a native conformation when expressed on the cell surface. Table 2: Average neutralization IC5 0 values of antibodies for 4-2. J41 and 4-2.J41delCT Env- pseudotyped viruses.
  • Soluble CD4 induces shedding of 4-2.J41 gpl20 from cell surface [00128]
  • One of the functional characteristics of HIV- 1 envelope is that in response to CD4 binding, the gpl20 subunit is shed from the trimer as part of the virus entry process.
  • sCD4 soluble CD4
  • HIV-1 One of the immune evasion strategies of HIV-1 is to maintain less number of Env spikes on virion.
  • An essential component of HIV- 1 vaccine design is to mimic the native Env spikes on the virion in the immunogen with higher level of expression.
  • the cytoplasmic tail of gp-41 of Env carries trafficking signal that allows the internalization of Env from the plasma membrane followed by degradation or recycling, and thus a lesser number of spikes express on the surface of virion.
  • Env Indian clade C HIV-1 Env, cell lines and antibodies.
  • the full-length Env (g l60) clones were obtained from National AIDS Research Institute, Pune, India and have been described previously (Ringe R, et al, Retrovirology 2010, 7:76).
  • Tzm-bl (NIH AIDS Reagent Program) and 293T cells (ATCC) were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% heat inactivated fetal bovine serum (HIFBS), 20 mM L- glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin.
  • DMEM Dulbecco's modified Eagle medium
  • HIFBS heat inactivated fetal bovine serum
  • Anti-HIV-1 broadly neutralizing antibodies (VRCOl, PGT121, PGT145, PGT151, PG9, PG16, 4E10, 10E8), non-neutralizing antibodies (F105, b6, 22B, 7B2, 17B, 39F) and HIVIG antibodies were obtained from the IAVI Neutralizing Antibody Center (TSRI, La Jolla, California).
  • the anti-gpl20 (clade C) rabbit polyclonal antibody was purchased from Immunetech, USA.
  • the supernatant was collected and proteins in the supernatant were then precipitated on ice with 20% trichloroacetic acid for 30 minutes, followed by centrifugation at 13000 rpm and 4°C for 15 minutes, washing with acetone and air-drying.
  • the precipitate was finally resuspended in IX SDS-sample loading buffer by heating at 100 C.
  • the samples were then analyzed for protein expression and cleavage by Western blot analysis using HIVIG human antibodies as probes.
  • Biotinylation of cell surface expressed Env was carried out using Pierce Cell Surface Protein Isolation kit (Thermo Scientific). The cells were lysed and biotin-labeled proteins were pooled with NeutrAvidin agarose according to the manufacturer's instructions for further analysis by Western blot.
  • the washed agarose-beads were resuspended in IX SDS-sample loading buffer, heated at 100°C and analyzed by Western blot using HIVIG human antibodies as probes.
  • the precipitated and washed agarose-beads were resuspended in IX deglycosylation buffer (G5 reaction buffer, New England Biolabs), digested with 500U of Endo H and 50U of Neuraminidase (New England Biolabs) overnight at 37 °C and then analyzed by Western blot using HIVIG human antibodies as probes.
  • Soluble CD4 induced shedding of gpl20 was measured by FACS and ELISA.
  • Env transfected 293T cells were harvested 48 hours post-transfection, washed extensively with FACS buffer, resuspended in 1ml of FACS buffer (12-20 X 10 6 cells per reaction) and incubated in the absence or presence of sCD4 (50 g) for 1 hour at 4°C with intermittent mixing. The cells were centrifuged and the pellet was resuspended in FACS buffer and then added in a round-bottomed 96-well tissue culture plate (2.5X10 5 cells/well) for staining with the indicated antibodies (VRCOl, PGT121, b6, 7B2) for lhour. The cells were then washed
  • Viruses pseudotyped with 4- 2.J41 Env were produced by co-transfection of envelope expressing plasmid (pSVIII-env) with env-deleted HIV-1 backbone plasmid (pSG3AEnv) into 293T cells in 6-well tissue culture plates using FuGENE®6 Transfection Reagent (Promega Inc). Cell supernatants containing pseudotyped viruses were harvested 48 hours post-transfection and then stored at - 80 °C until further use.
  • the infectivity assays were done in Tzm-bl cells (1 X 10 5 cells/ml) containing DEAE-Dextran (25 ⁇ g/ml) in 96-well microtiter plates and infectivity titers were determined by measuring luciferase activity using Britelite luciferase substrate (Perkin Elmer) with a Victor X2 Luminometer (Perkin Elmer).
  • Viruses pseudotyped with 4-2. J41 Env were assessed for their neutralization sessitivity against neutralizing and non-neutralizing antibodies in a Tzm-bl neutralization assay. Briefly, pseudoviruses (1 X 10 5 RLU/well) were incubated with serial dilutions (2- fold) of monoclonal antibodies in duplicate wells of a 96-well flat-bottom culture plate. After 1 hour of incubation at 37°C, 1 X 10 5 TZM-bl cells containing 25g/ml DEAE-Dextran were added to each well. After 48 hours, luciferase activity was measured using the Britelite luciferase substrate (Perkin Elmer). The IC50 values were defined as antibody concentrations that caused a 50% reduction in RLU compared to the virus control.

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Abstract

La recherche en matière de vaccins contre le VIH-1 se concentre principalement sur la conception d'un immunogène pouvant susciter la production d'anticorps largement neutralisants efficaces contre les glycoprotéines d'enveloppe (Env). L'invention concerne une Env de clade C qui est stable et efficacement clivée de la surface cellulaire. Des formes solubles de cette Env sont également décrites. Cette Env met à nu des épitopes dirigés contre de nombreux anticorps largement neutralisant ciblant des zones distinctes de cette Env, tout en masquant ceux dirigés contre des anticorps non neutralisants. La liaison de plusieurs anticorps dépendant de la conformation suggère que cette Env adopte une conformation native à la surface des cellules et sous forme soluble. L'efficacité du clivage et des propriétés de liaison des anticorps de cette Env en font un immunogène approprié pour une utilisation dans un vaccin et pour servir de plate-forme efficace pour une conception rationnelle d'immunogènes pour le vaccin contre le VIH-1.
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