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WO2004046168A2 - Glycoproteines d'enveloppe recombinantes du vih-1 de sous-type d - Google Patents

Glycoproteines d'enveloppe recombinantes du vih-1 de sous-type d Download PDF

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Publication number
WO2004046168A2
WO2004046168A2 PCT/US2003/036653 US0336653W WO2004046168A2 WO 2004046168 A2 WO2004046168 A2 WO 2004046168A2 US 0336653 W US0336653 W US 0336653W WO 2004046168 A2 WO2004046168 A2 WO 2004046168A2
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Prior art keywords
htv
protein
seq
isolated
nucleic acid
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WO2004046168A3 (fr
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Thomas C. Vancott
Mathew E. Harris
Vaniambadi S. Kalyanaraman
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Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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Henry M Jackson Foundation for Advancedment of Military Medicine Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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

Definitions

  • the present invention relates to HTV-1 envelope proteins and peptides derived from the donor of the Neutralizing Reference Human Serum (2) which is noted for its capacity to neutralize primary HIV isolates of varied subtypes.
  • the present invention arose in part from research funded by the U.S. Military HIV Research Program Strategic Technical Objective.
  • HTV-1 envelope glycoprotein gpl ⁇ O is known to exist as a multimer (trimers or tetramers) on the surface of a virion (Earl et al. (1990) Proc. Natl. Acad. Sci. USA 87, 648-652; Pinter et al. (1989) J. Virol. 2674-2679; Schawaller et al. (1989) Virology 172, 367-369; Thomas et al. (1991) J.
  • gp41 derived from gpl ⁇ O expression in mammalian cells forms tetramers indicating the possibility that regions outside of the alpha helical gp41 sequences may impact on overall quaternary structure of gp41 (Mclnerney et al. (1998) J. Virol. 72, 1523-1533). It has been shown that immunization of mice with oligomeric gp 140 results in the induction of a number of mAbs with specificity to oligomeric-specific or sensitive epitopes within gp41 (Broder et al. (1994) Proc Natl Acad Sci USA 91, 11699-11703; Earl et al. (1994) J. Virol.
  • Vaccine-induced broadly neutralizing antibodies have been difficult to achieve. Recent encouraging developments have shown the ability of DNA and recombinant viral vaccination strategies to induce viral-specific CD8 T cell responses (Amara et al. (2001) Science 292, 69-74; Barouch et al. (2001) J. Virol. 75, 5151-5158; Barouch et al (2000) Science 290, 486-492). These responses, in the absence of measurable neutralizing antibody, have provided some level of protection (not sterilizing) from disease after pathogenic challenge. The current goal of inducing more potent neutralizing antibody and combining these with vaccination strategies inducing CDS T cell responses may provide increased levels of protection. The goal remains to continue research into novel subunit envelope vaccines towards the induction of neutralizing antibody.
  • the present invention is based on the identification of novel, isolated nucleic acid molecules which comprise a sequence selected from the group consisting of SEQ TD NO: 1, 3, 5 or 7 and which encode an HTV-1 subtype D envelope protein or a fragment thereof. Also encompassed in the present invention is the identification of a nucleic acid sequence encoding an isolated HTV-1 subtype D envelope protein selected from the group consisting of SEQ ID NO: 2, 4, 6 and 8. In a further embodiment of the invention, the nucleic acid molecule consists essentially of the nucleic acid sequence of SEQ ID NO: 1, 3, 5 or 7 or the complement thereof.
  • the isolated nucleic acid molecule comprises a sequence selected from the group consisting of nucleotides 190-2463 of SEQ ID NO: 1, nucleotides 190-2469 of SEQ ID NO: 3, nucleotides 157-2469 of SEQ ID NO: 5, and nucleotides 190-2427 of SEQ ID NO: 7.
  • the present invention is also based on the identification of an isolated protein or " fragment thereof capable of generating a broad HTV-1 neutralization following administration in a mammal selected from the group consisting of (a) an isolated protein comprising the amino - acid sequences depicted in SEQ TD NO: 2, 4, 6 and 8; (b) an isolated protem fragment comprising at least ten amino acids of any of the sequences depicted in SEQ ID NO: 2, 4, 6, and 8; and (c) an isolated protein comprising conservative amino acid substitutions of any of the sequences depicted in SEQ ID NO: 2, 4, 6 and 8.
  • the isolated protein capable of generating a broad HTV-1 neutralization following adrriinistration in a mammal has an amino acid sequence consisting essentially of SEQ D NO: 2, 4, 6 or 8.
  • the protein is glycosylated.
  • the protein or fragment thereof is fused to a second protein.
  • the present invention is also based on vaccine compositions comprising nucleic acid molecules encoding an HTV-1 subtype D envelope protein or a fragment thereof, wherein the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5 or 7.
  • the vaccine compositions comprise nucleic acid molecules that comprise a sequence which encodes an isolated HTV-l subtype envelope protein selected from the group consisting of SEQ ID NO: 2, 4, 6 or 8.
  • the vaccine composition may further comprise a pharmaceutically acceptable carrier and an isolated protein or fragment thereof capable of generating a broad HTV-1 neutralization following administration in a mammal selected from the group consisting of (a) an isolated protem comprising the amino acid sequences depicted in SEQ ID NO: 2, 4, 6 and 8; (b) an isolated protein fragment comprising at least ten amino acids of any of the sequences depicted in SEQ ID NQ: 2, A, 6, and 8; and (c) an isolated protein comprising conservative amino acid substitutions of any of the sequences depicted in SEQ ID NO: 2, A, 6 and 8.
  • the isolated protein or fragment thereof may further be fused to a second protein.
  • the isolated protein or fragment thereof may be linked to an adjuvant.
  • the present invention is also based on the identification of recombinant vectors comprising the nucleic acid molecules of the invention.
  • the recombinant vectors comprise nucleic acid molecules encoding HTV-1 subtype D envelope proteins or fragments thereof comprising nucleic acid sequences selected from the group consisting of SEQ ID NO: 1, 3, 5 or 7.
  • the recombinant vectors comprise nucleic acid molecules comprising sequences that encode isolated HTV-1 subtype D envelope proteins or fragments thereof selected from the group consisting of SEQ ID NO: 2, 4, 6 or 8.
  • the present invention is also based on isolated cells comprising any one of the recombinant vectors of the invention.
  • vaccine compositions comprise any one of the vectors of the invention.
  • the present invention is further based on the identification of isolated cells comprising the nucleic acid molecules of the invention.
  • the isolated cells comprise nucleic acid molecules encoding HTV-1 subtype D envelope proteins or fragments thereof comprising nucleic acid sequences selected from the group consisting of SEQ ID NO: 1, 3, 5 or 7.
  • the isolated cells comprise nucleic acid molecules comprising sequences that encode isolated HTV-1 subtype D envelope proteins or fragments thereof selected from the group consisting of SEQ ID NO: 2, 4, 6 or 8.
  • the isolated cells are eukaryotic cells.
  • the present invention is also based on the identification of methods of generating a broadly cross-reactive neutralizing immune.response against multiple strains of HTV-1 in a mammal infected with HTV-1.
  • the method comprises administering an effective amount of any one of the vaccine compositions of the invention to the mammal.
  • the vaccine is effective for the prevention and treatment of HIV-1 Mection and Acquired Immune Deficiency Syndrome (AIDS).
  • a goal of immunization against HTV is to induce neutralizing antibody (NA) responses broadly reactive against diverse strains of virus.
  • NA neutralizing antibody
  • the gpl40 sequences presented here provide a component useful in the manufacture of vaccine compositions for the prophylaxis, treatment, and/or attenuation of HTV-1 Mection in at-risk or affected subjects.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a polyadenylation signal and transcription termination sequence will . usually be located 3' to the coding sequence.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • naked DNA means nucleic acid molecules that are free from viral particles, particularly retroviral particles. This term also means nucleic acid molecules which are free from facilitator agents including but not limited to the group comprising: lipids, liposomes, extracellular matrix-active enzymes, saponins, lectins, estrogenic compounds and steroidal hormones, hydroxylated lower al yls, dimethyl sulfoxide (DMSO) and-urea.
  • DMSO dimethyl sulfoxide
  • nucleic acid molecule refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, and/or cytosine) or ribonucleotides (adenine, guanine, uracil, and/or cytosine) and may include in either its single stranded form, or in double-stranded helix as well as RNA. This term refers only to the primary and secondary- structure of the molecule and is not limited to any particular tertiary form.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 ' direction) coding sequence.
  • the promoter sequence is bounded (inclusively) at its 3 ' terminus by the transcription initiation site and extends upstream (5 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • a "signal sequence” can be included before the coding sequence or the native amino acid signal sequence from the envelope protein may be used. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media. This signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes. For instance, alpha-factor, a native yeast protem, is secreted from yeast, and- its signal sequence can be attached to heterologous proteins to be secreted into the media (see, for example, U.S. Patent 4,546,082).
  • alpha-factor and its analogs have been found to secrete heterologous proteins from a variety of yeast, such as Saccharomyces and Kluyveromyces, (see, for example, EP 88312306.9 and EP 0301669).
  • yeast such as Saccharomyces and Kluyveromyces
  • An example for use in mammalian cells is the tPA signal used for expressing Factor VHIc light chain.
  • DNA sequences are "substantially homologous" when at least about
  • -Sequences that are .substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art (see, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press).
  • a cell has been "transformed" by exogenous or heterologous DNA when such DNA as been introduced inside the cell.
  • the transforming DNA may or may not be mtegrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid or viral vector.
  • a stably transformed cell is one in which the tiansforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the tiansforming DNA.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "vector” is a replicon, such as plasmid, virus, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • the present invention provides nucleic acid molecules that encode recombinant HTV-1 subtype D gpl40 envelope glycoproteins.
  • said nucleic acid molecules are selected from the group consisting of SEQ ID NO: 1, 3, 5 and 7.
  • Such nucleic acid molecules can be in an isolated form, or can be operably linked to expression control elements or vector sequences.
  • the present invention further provides host cells that contain the vectors via transformation, transfection, electroporation or any other art recognized means of introducing a nucleic acid into a cell.
  • the present invention further provides proteins or peptides which are the products of the nucleic acid molecules of the invention.
  • said proteins are selected from the group consisting of SEQ ID NO: 2, A, 6 and 8.
  • Proteins and peptides of the invention may be prepared by any available means, including recombinant expression of the desired protein or peptide in eukaryotic or prokaryotic host cells (see, for example, U.S. Patent 5,696,238).
  • Methods for producing proteins or peptides of the invention for purification may employ, conventional molecular biology, .microbiology, andxecombinantDNA-techniques- within the ordinary skill level of the art. Such techniques are explained fully in the literature (see, for example, Sambrook et al.
  • Vectors are used to simplify manipulation of the DNA which encodes the HTV proteins or peptides, either for preparation of large quantities of DNA for further processing (cloning vectors) or for expression of the HTV proteins of peptides (expression vectors).
  • Vectors comprise plasmids, viruses (including phage), and integrated DNA fragments (i.e., fragments that are integrated into the host genome by recombination).
  • Cloning vectors need not contain expression control sequences.
  • control sequences in an expression vector include transcriptional and trans ⁇ ational control sequences such as a transcriptional promoter, a sequence encoding suitable ribosome binding sites, and sequences which control termination of transcription and translation.
  • the expression vector should preferably include a selection gene to facilitate the stable expression of HTV gene and/or to identify transformants.
  • the selection gene for maintaining expression can be supplied by a separate vector in cotransformation systems using eukaryotic host cells.
  • Suitable vectors generally will contain replicon (origins of replication, for use in non-integrative vectors) and control sequences which are derived from species compatible with the intended expression host.
  • replicable vector as used herein, it is intended to encompass vectors containing such replicons as well as vectors which are replicated by integration into the host genome.
  • Transformed host cells are cells which have been transformed or transfected with vectors containing HTV peptide or protein encoding DNA.
  • the expressed HTV proteins or peptides may be secreted into'the"cultur ⁇ supernatant " , " u ⁇ der the control of suitable processing signals in the expressed peptide, e.g. homologous or heterologous signal sequences.
  • Expression vectors for host cells ordinarily include an origin of replication, a promoter located upstream from the HTV protein or peptide coding sequence, together with a ribosome binding site, a polyadenylation site, and a transcriptional termination sequence. Those of ordinary skill will appreciate that certain of these sequences are not required for expression in certain hosts.
  • An expression vector for use with microbes need only contain an origin of replication recognized-by the-host, a promoter-which-will function in-the-host, and a selection- gene.
  • promoters are derived from polyoma, bovine papilloma virus, CMV (cytomegalovirus, either murine or human), Rouse sarcoma virus, adenovirus, and simian virus 40 (SV40).
  • Other control sequences e.g, terminator, polyA, enhancer, or amplification sequences can also be used.
  • An expression vector is constructed so that the HTV protein or peptide coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed and translated under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence).
  • the control sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site. If the selected host cell is a mammalian cell, the control sequences can be heterologous or homologous to the HTV coding sequence, and the coding sequence can either be genomic DNA containing introns or cDNA.
  • Higher eukaryotic cell cultures may be used to express the proteins of the present invention, whether from vertebrate or invertebrate cells, including insects, and the procedures of propagation thereof are known (see, for example, Boulton et al. (1992) Practical cell culture techniques, Humana Press).
  • Suitable host cells for expressing HTV proteins or peptides in higher eukaryotes include: monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL1651); baby hamster kidney cells (BHK, ATCC CRLl 0); Chinese hamster ovary (CHO) cells-DHFR (Urlaub & Chasin (1980) Proc. Natl. Acad. Sci. USA 77, 4216-4220); mouse Sertoli cells (Mather (1980) Biol. Reprod.
  • monkey kidney cells CVI, ATCC CCL70); African green monkey kidney cells (VER076, ATCC CRL1587); human cervical carcinoma cells (HeLa, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BR A7ATCC - CRL1442); human lung cells (W138, ATCC CCL75); human liver cells (HepG2, HB8065); mouse mammary tumor (MMT 060652, ATCC CCL51); rat hepatoma cells (Baumann et al. (1980) J. Cell. Biol. 85, 1-8) andTRI cells (Mather et al. (1982) Ann. NY Acad. Sci. 383, 44- 68).
  • HTV-1 gene products when expressed in mammalian tissue, the recombinant HTV-1 gene products may have higher molecular weights than expected due to glycosylation. It is therefore intended that partially or completely glycosylated forms of HTV-1 preproteins or peptides having-moleGular-weights-somewhat different from.160, 140, 120 or_4J,kDa are-within-- the scope of this invention.
  • eukaryotic expression system An exemplary eukaryotic expression system is that employing vaccinia virus, which is well-known in the art (see, for example, WO 86/07593).
  • Yeast expression vectors are known in the art (see, for example, U.S. Patents 4,446,235; 4,443,539; 4,430,428).
  • vector pHSI transforms Chinese hamster ovary cells
  • Mammalian tissue may be cotransformed with DNA encoding a selectable marker such as dihydrofolate reductase (DHFR) or thymidine kinase and DNA encoding the HTV protein or peptide.
  • DHFR dihydrofolate reductase
  • thymidine kinase DNA encoding the HTV protein or peptide.
  • wild type DHFR gene it is preferable to select a host cell which is deficient in DHFR, thus permitting the use of the DHFR coding sequence as marker for successful transfection in hgt medium, which lacks hypoxanthine, glycine, and ymidine.
  • An appropriate host cell in this case is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub & Chasin (1980) Proc. Natl. Acad. Sci. USA 77, 4216-4220.
  • CHO Chinese hamster ovary
  • HTV-1 proteins or peptides are produced by growing host cells transformed by an exogenous or heterologous DNA construct, such as an expression, vector described above under conditions whereby the HTV-1 protein is expressed.
  • the HTV-1 envelope glycoprotein or peptide is then isolated from the host cells and purified. If the expression system secretes the protein or peptide into the growth media, the protein can be purified directly from cell-free media. The selection of the appropriate growth conditions and initial crude recovery methods are within the skill of the art. ⁇
  • a coding sequence for an HTV-1 protein or peptide of the invention can be cloned into any suitable vector and thereby maintained in a composition of cells which is substantially free of cells that do not contain an HTV-1 • coding sequence.
  • Numerous cloning vectors are known to those of skill in the art. Examples of recombinant DNA vectors for cloning and host cells which they can transform include the various bacteriophage lambda vectors (E. coli), pBR322 (E. coli), pACYG177 (Ei coli), pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), pLAFRI (gram-negative bacteria), pME290 (non-E.
  • Fusion proteins of the present invention comprise, fused to at least one polypeptide partner sequence, a selected HTV-1 subtype D envelope glycoprotein encoded by SEQ ID NO: 1, 3, 5 or 7, or a fragment thereof.
  • the fusion protein comprises, fused to at least one polypeptide partner sequence, a selected HTV-1 subtype D envelope glycoprotein of SEQ ID NO: 2, A, 6 or 8, or a fragment thereof.
  • said at least one polypeptide partner sequence may be the same sequence as the selected HTV-1 subtype D envelope glycoprotein, or a fragment thereof.
  • said at least one polypeptide partner sequence is a different HTV-1 subtype D envelope glycoprotein of SEQ ID NO: 2, 4, 6 or 8 from the selected HTV-1 subtype D envelope glycoprotein; or a fragment thereof.
  • said at least one polypeptide partner sequence is a protein or peptide which is heterologous to the selected HIV-1 subtype D envelope glycoprotein.
  • said at least one polypeptide partner sequence comprises more than one partner sequence which are selected from various combinations of heterologous proteins or peptides, the same sequence as the selected HTV-1 subtype D envelope glycoprotein, and different HTV-1 subtype D envelope glycoproteins of SEQ ID NO: 2, 4, 6 or- 8 from the selected HTV-1 subtype D envelope glycoprotein, or fragments thereof.
  • Such cells act as hosts and may include, for the fusion proteins of the present invention, yeasts, fungi, insect cells, plants cells or animals cells.
  • Expression vectors for many of these host cells have been isolated and characterized, and are used as starting materials in the construction, through conventional recombinant DNA techniques, of vectors having a foreign DNA insert of interest. Any DNA is foreign if it does not naturally derive from the host cells used to express the DNA insert.
  • the foreign DNA insert may be expressed on extrachromosomal plasmids or after integration in whole or in part in the host cell chromosome(s), or may actually exist in the host cell as'a combination of more "than one molecular form.
  • the choice of host cell and expression vector for the expression of a desired foreign DNA largely depends on availability of the host cell and how fastidious it is, whether the host cell will support the replication of the expression vector, and other factors readily appreciated by those of ordinary skill in the art.
  • the foreign DNA insert of interest comprises any DNA sequence coding for fusion proteins including any synthetic sequence with this coding capacity or any such cloned sequence or combination thereof.
  • fusion proteins coded and expressed by an entirely recombinant DNA-sequence is encompassed -by this invention but-not to the. exclusion . of fusion proteins peptides obtained by other techniques.
  • Vectors useful for constructing eukaryotic expression systems for the production of fusion proteins comprise the fusion protein's DNA sequence, operatively linked thereto with appropriate transcriptional activation DNA sequences, such as a promoter and/or operator.
  • appropriate transcriptional activation DNA sequences such as a promoter and/or operator.
  • Other typical features may include appropriate ribosome binding sites, termination codons, enhancers, terminators, or replicon elements. These additional features can be inserted Mo the vector at the appropriate site or sites by conventional splicing techniques such as restriction endonuclease digestio ⁇ .and ligation.
  • Yeast expression systems which are the preferred variety of recombinant eukaryotic expression system, generally employ Saccharomyces cerevisiae as the species of choice for expressing recombinant proteins.
  • Saccharomyces cerevisiae Other species of the genus Saccharomyces are suitable for recombinant yeast expression system, and include but are not limited to carlsbergensis, uvarum, rouxii, montaniw, kluyveri, elongisporus, norbensis, oviformis, and diastaticus. Saccharomyces cerevisiae and similar yeasts possess well known promoters useful in the construction of expression systems active in yeast, including but not limited to GAP, GAL10, ADH2, PH05, and alpha mating factor.
  • Yeast vectors useful for constructing recombinant yeast expression systems for expressing fusion proteins include, but are not limited to, shuttle vectors, cosmid plasmids, chimeric plasmids, and those having sequences derived from two micron circle plasmids. Insertion of the appropriate DNA sequence coding for fusion proteins into these vectors will, in principle, result in a useful recombinant yeast expression system for fusion proteins where the modified vector is inserted into the appropriate host cell, by transformation or other means.
  • Recombinant mammalian expression system are another means of producing the fusion proteins for the vaccines/immunogens of this invention.
  • a host mammalian cell can be any cell that has been efficiently cloned in cell culture.
  • mammalian expression options can be extended to include organ culture and transgen ⁇ c animals
  • Host mammalian cells useful for the purpose of constructing a recombinant mammalian expression system include, but are not limited to, Vero cells, NIH3T3, GH3, COS, murine C127 or mouse L cells.
  • Mammalian expression vectors can be based on virus vectors, plasmid vectors which may have SV40, BPV or other viral replicons, or vectors without a replicon for animal cells. Detailed discussions on mammalian expression vectors can be found in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press.
  • Fusion proteins may possess additional and desirable structural modifications not shared with the same organically synthesized peptide,- such as adenylation,-carboxylation, glycosylation, hydroxylation, methylation, phosphorylation or myristylation. These added features may be chosen or preferred as the case may be, by the appropriate choice of recombinant expression system. On the other hand, fusion proteins may have its sequence extended by the principles and practice of organic synthesis.
  • a vaccine composition of the present invention induces at least one of a humoral and a cellular immune response in a mammal who has been administered the vaccine or is effective in enhancing at least one immune response to at least one strain of HTV, such that the vaccine administration is suitable for vaccination purposes.
  • a vaccine of the present invention delivers to a subject in need thereof a recombinant HTV-1 env protein or proteins encoded by at least one of SEQ ID NO: 1, 3, 5 and 7.
  • a vaccine of the present invention may farther comprise additional HTV-1 env protein or proteins which may correspond to HTV-1 env proteins different from SEQ ED NO: 2, 4, 6 and 8 may fiirther potentiate the immunization methods of the invention.
  • Viral vaccines Various genetically engineered virus hosts (“recombinant viruses”) can be used to prepare viral vaccines for administration of at least one HTV-1 env protem encoded by SEQ ID NO: 1, 3, 5 or 7.
  • recombinant viruses are particularly advantageous, in that the viral Mection component promotes a vigorous immune response that targets activation of B lymphocytes, helper T lymphocytes, and cytotoxic T lymphocytes.
  • Numerous virus species can be used as the recombinant virus hosts for the vaccines of the invention.
  • a preferred recombinant virus for a viral vaccine is vaccinia virus (see U.S. Patents 4,603,112 and 5,762,938; WO 87/06262; Cooney et al. (1993) Proc. Natl.
  • any recombinant virus can be used to express antigens for a vaccm6 " 6f the invention.
  • the use of multiple viral vaccines can obviate anti-viral immune responses that may render a booster with the viral vaccine less effective (due to possible potentiation of a vigorous anti-virus response).
  • Any available vaccine vector may be used, including live Venezuelan Equine Encephalitis virus (see, for example, U.S.
  • Patent 5,643,576) poliovirus (see, for example, U.S. Patent 5,639,649), pox virus (see, for example, U.S. Patent 5,770,211).
  • recombinant canarypox can be used (Pialoux et al. (1995) AIDS Res. Hum. Retroviruses 11, 373-81, erratum in Pialoux et al. (1995) ADDS Res. Hum. Retroviruses 11, 875; Andersson et al (1996) J. Meet. Dis. 174,'977-985;-Fries etal. (1-996) Vaccine 14, 428-434; Gonczol et al (1995) Vaccine-13, 1080-1885).
  • adenovirus or adenovirus Another alternative is defective adenovirus or adenovirus (Gilardi-Hebenstreit et al. (1990) J. Gen. Virol. 71, 2425-31; Prevec et al. (1990) J. Meet. Dis. 161, 27-30; Lubeck et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6763-6767; Xiang et al. (1996) Virology 219, 220- 227).
  • suitable viral vectors include retroviruses that are packaged in cells with amphotropic host range (see, for example, Miller (1990) Human Gene Ther. 1, 5-14; Ausubel et al.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • vaccine or immunogenic compositions may be prepared as vaccine vectors which express the HTV-l envelope glycoprotein or peptide of the invention in the host animal.
  • DNA vaccines An alternative to a traditional vaccine comprising an antigen and an adjuvant involves the direct in vivo introduction of DNA encoding at least one gene product of SEQ ID NO: 1, 3, 5 or 7 into tissues of a subject for expression of the antigen by the cells of the subject's tissue.
  • Such vaccines are termed herein "DNA vaccines” or “nucleic acid-based vaccines” throughout the specification.
  • DNA vaccines, including naked DNA are described, for example, in U.S. Patent 5,739,118 and WO 95/20660 and WO 93/19183, the disclosures of which are hereby incorporated by reference in their entirety.
  • DNA vaccine can be used as a boost, e.g., as described above with respect to the recombinant HTV-1 proteins encoded by at least one of SEQ ID NO: 1, 3, 5, and 7.
  • the DNA vaccine can be used to prime immunity, with the recombinant viral vaccine or vaccines used to boost the anti-HTV immune response.
  • the DNA vaccine may comprise one or more vectors for expression of one or more of the HTV-l-env f SEQ TD NO: 1, 3, 5, and 7.
  • the HTV-1 env genes may correspond to genes expressed by the recombinant virus vaccine, or they may be different.
  • vectors are prepared for expression in the recombinant virus vaccine and in transfected mammalian cells as part of a DNA vaccine.
  • the ability of directly injected DNA that encodes a viral protem to elicit a protective immune response has been demonstrated in numerous experimental systems (Conry et al (1994) Cancer Res. 54, 1164-1168; Cox et al (1993) Virol 67, 5664-5667; Davis et al (1993) Hum. Mol. Genet.2, 1847-1851; Sedegah et al (1994) Proc. Natl. Acad. Sci. 91, 9866-9870; Montgomery et al (1993) DNA Cell Biol.
  • Vaccination through directly introducing DNA that encodes at least one HTV-1 env protein encoded by SEQ ID NO: 1, 3, 5 or 7 to elicit a protective immune response produces both cell-mediated and humoral responses.
  • This is analogous to results obtained with live viruses (Raz et al (1994) Proc. Natl. Acad. Sci. 91, 9519-9523; Ulmer et al (1993) Science 259, 1745-1749; Wang et al (1993) Proc. Natl. Acad. Sci. 90, 4156-4160; Xiang et al. (1994) Virology 199, 132-140).
  • promoters differ in tissue specificity and efficiency in initiating mRNA synthesis (Xiang et al. (1994) Virology 209, 564-579; Chapman et al. (1991) Nuc. Acid. Res. 19, 3979- 3986).
  • CMV cytomegalovirus
  • Vectors containing the nucleic acid-based vaccine of the invention may also be introduced into the desired host by other methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), or a DNA vector transporter (see, for example, Wu et al. (1992) J. Biol. Chem. 267, 963-967; Wu et al. (1988) J. Biol. Chem. 263, 14621-14624).
  • a preferred aspect of the present- invention concerns engineering of bi-functional plasmids that can serve as a DNA vaccine and a recombinant virus vector.
  • Direct injection of the purified plasmid DNA i.e., as a DNA vaccine comprising at least one nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, 3, 5 and 7, would elicit a broadly reactive neutralizing immune response.
  • the plasmid would also be useful in live, recombinant viruses as immunization vehicles.
  • the bi-functional plasmid of the invention provides a heterologous gene, or an insertion site for a heterologous gene, under control of two different expression control sequences: an animal expression control sequence, and a viral expression control sequence.
  • the term "under control” is used in its ordinary sense, i.e., operably or operatively associated with, in the sense that the expression control sequence, such as a promoter, provides for expression for expression of a heterologous gene.
  • the animal expression control sequence is a mammalian promoter (avian promoters are also contemplated by the present invention); in a specific embodiment, the promoter is cytomegalovirus immediate early (CMV) promoter.
  • CMV cytomegalovirus immediate early
  • the virus promoter is a vaccinia virus early promoter, or a vaccinia virus late promoter, or preferably both.
  • Subjects could be vaccinated with a multi-tiered regimen, with the bi-functional plasmid administered as DNA and, at a different time, but in any order, as a recombinant virus vaccine.
  • the invention contemplates single or multiple a ⁇ lrninistrations of the bi-functional plasmid as a DNA vaccine or as a recombinant virus vaccine, or both.
  • This vaccination regimen may be complemented with administration of recombinant protein vaccines, or may be used with additional vaccine vehicles.
  • HTV-1 env protein or proteins can prime or boost a cellular or humoral immune response.
  • An effective amount of the HTV-1 env protem or proteins of SEQ ID NO: 2, A, 6, or 8, or encoded by SEQ ID NO: 1, 3, 5, or 7, or antigenic fragments thereof, can be prepared in an admixture with an adjuvant to prepare a vaccine.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antige and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al.
  • Adjuvants include, but are not hmited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useM human adjuvants such as BGG (bacille Calmette-Guerin) and Colynebacteriumparvum.-- Selection of an adjuvant depends on the subject to be vaccinated. Preferably, a pharmaceutically acceptable adjuvant is used.
  • a vaccine for a human should avoid oil or hydrocarbon emulsion adjuvants, including complete and incomplete Freund's adjuvant.
  • an adjuvant suitable for use with humans is alum (alumina gel).
  • infra, recombinant HTV-1 env protein is adrr ⁇ istered intramuscularly in alum.
  • the recombinant HTV-1 env protein vaccine can be administered subcutaneously, intradermally, intraperitoneally, or via other acceptable vaccine administration routes.
  • Recombinant proteins of the invention or peptide fragments thereof may also be circularized in order to mimic the geometry of those portions as they occur in the envelope protein.
  • Circularization may be facilitated by disulfide bridges between existing cysteine residues. Cysteine residues may also be included in positions on the peptide which flank the portions of the peptide which are derived from the envelope protein. Alternatively, cysteine residues within the portion of a peptide derived from the envelope protein may be deleted and/or conservatively substituted to eliminate the formation of disulfide bridges involving such residues. Other means of circularizing peptides are also well known. The peptides may be circularized by means of covalent bonds, such as amide bonds, between amino acid residues of the peptide such as those at or near the amino and carboxy termini (see, for example, U.S. Patent 4,683,136).
  • Vaccine administration can be accomplished with a recombinant viral vaccine, a DNA vaccine a recombinant protein vaccine, or any combination thereof.
  • recombinant HTV-1 env protein in alum is provided i.m. to boost the immune response.
  • Each dose of vaccine may contain or encode the same protein or proteins encoded by SEQ ID NO: 1, 3, 5 or 7, or different combinations thereof.
  • subsequent vaccines may express different HTV-1 env genes.
  • the subsequent vaccines may have or encode some HTV-1 env proteins in common, and others that are different, from the earlier vaccine.
  • the administration of a vaccine comprising or encodinglhe proteuror proteins encoded by SEQ ID NO: 1, 3, 5 or 7 can be for either a "prophylactic” or “therapeutic” purpose, and preferably for prophylactic purposes.
  • the vaccine composition is provided in advance of any detection or symptom of HTV Mection or AIDS.
  • the prophylactic administration of an effective amount of the compound(s) serves to prevent or attenuate any subsequent HTV Mection.
  • the vaccine is provided in an effective amount upon the detection of a symptom of actual Mection.
  • a composition is said to be "pharmacologically acceptable” if its admMstration can be tolerated by a-recipient patient.
  • Such an agent is said to-be administered in a "therapeutically or - prophylactically effective amount” if the amount administered is physiologically significant.
  • a vaccine or composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, preferably by enhancing a broadly reactive humoral or cellular immune response to HTV-1.
  • the "protection” provided need not be absolute (i. e. , the HTV Mection or AIDS need not be totally prevented or eradicated), provided that there is a statistically significant improvement relative to a control population. Protection can be limited to mitigating the severity or rapidity of onset of symptoms of the disease.
  • a vaccine of the present invention can confer resistance to one or more strains of HTV- 1.
  • the present invention thus concerns and provides a means for preventing or attenuating infection by at least one HTV-1 strain.
  • a vaccine is said to prevent or attenuate a disease if its administration to an individual results either in the total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or in the total, or partial immunity of the individual to the disease.
  • At least one vaccine of the present invention can be administered by any means that achieve the intended purpose, using a pharmaceutical composition as described herein.
  • administration of such a composition can be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, tiansdermal, or buccal routes.
  • Subcutaneous administration is preferred.
  • Parenteral adrninistration can be by bolus injection or by gradual perfusion over time.
  • a typical regimen for preventing, suppressing, or treating a disease or condition which can be alleviated by a cellular immune response by active specific cellular i munotherapy comprises administration of an effective amount of a vaccine composition as described above, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including one week to about twenty-four months.
  • an "effective amount" of a vaccine composition is “one whichis sufficient to achieve a desired biological effect, in this case at least one of cellular or humoral immune response to HTV-1. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the ranges of effective doses provided below are not intended to limit the invention and represent preferred dose ranges.
  • the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation (see, for example, Beers (1999) Merck Manual of Diagnosis and Therapy, Merck & Company Press; Hardman et -al. (2001), Goodman & Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill; Katzung (1988) Clinical Pharmacology, Appleton & Lange; which references and references . cited therein, are entirely incorporated herein by reference).
  • the dosage for a human adult will be from about 10 5 -10 9 plaque forming units (pfu)/kg or colony forming units (CFUykg per dose, with 10 6 to 10 8 preferred. Whatever dosage is used, it should be a safe and effective amount as dete ⁇ nined by known methods, as also described herein.
  • the vaccine dosages administered will typically be, with respect to an individual protein encoded by SEQ ID NO: 1 , 3; 5 or 7, a minimum of about 0.1 mg/dose, more typically a minimum of about 1 mg/dose, and often a minimum of about 10 mg/dose.
  • the maximum dosages are typically not as critical.
  • the dosage will be no more than 500 mg/dose, often no more than 250 mg/dose.
  • These dosages can be suspended in any appropriate pharmaceutical vehicle or carrier in sufficient volume to carry the dosage.
  • the final volume including carriers, adjuvants, and the like, typically will be at least 0.1 ml, more typically at least about 0.2 ml.
  • the upper limit is governed by the practicality of the amount to be administered, generally no more than about 0.5 ml to about 1.0 ml.
  • the recipients of the vaccines of the present invention can be any mammal which can acquire specific immunity via a cellular or humoral immune response to HTV-1 , where the cellular response is mediated by an MHC class I or class II protein.
  • the preferred recipients are mammals of the Orders Primata (including humans, chimpanzees, apes and monkeys).
  • the most preferred recipients are humans.
  • the subjects preferably are infected with HTV or provide a model of HTV-1 Mection (see, for example, Hu et al. (.1987) Nature 328, 721-723, which reference is entirely incorporated herein by reference).
  • the present invention identifies and provides immunogens capable of eliciting an antibody response with neutralizing-activity against primary HTV-1 isolates.
  • - Nucleic-acid molecules and the various constructs of the present invention are derived from a CCR5-using primary subtype D HTV-1 isolate from recently HTV-1 Mected individuals living in Kenya and supplied by the U.S. Military HTV Research Program.
  • HEK293 and CHO cell lines optimized for the expression of gpl40 are used for the production and purification of these proteins for use in pre-clinical studies.
  • CHO cells optimized for gpl40 expression. also provide GMP production of these proteins for phase I clinical vaccine safety and immunogenicity studies.
  • a two-step process facilitates the selection and testing of appropriate genes and gene products.
  • Wild-type gpl40 genes from different subtype D strains of HTV-1 are used to develop HEK293 cells optimized for protein expression. Based on the levels of expression of gpl40 by the HEK293 cells lines generated, cell lines expressing gpl40 are prepared for protein production and purification.
  • the selected gp 140 genes have their signal peptide deleted, incorporate an hel site at the beginning and EcoRl and BamHl sites at the end, and are codon-optimized in order to improve their expression 10-20 fold in CHO cells. These codon-optimized gp 140 genes, SEQ ED NO:, 1, 3, 5, and 7, are then used to develop and optimize CHO cell lines expressing gpl40.
  • Expression vectors are constructed with subtype D env gene clones of SEQ ED NO: 1, 3, 5, and 7. These constructs have gpl40 genes comprising mutated gpl20/gp41 cleavage sites.
  • Vector DNA is transfected into HEK293 cells and selected with G418. Cell colonies are screened with a g l20 capture ELISA and the highest secreting cell lines selected by cloning. Sequencing of the viral DNA in the vector and the viral DNA in selected clones of HEK293 cells to " verifies the insertion of the correct env genes. The cell lines are then be evaluated both' by RDPA and HTV-1 gpl20 antigen capture assays. The highest producing cell clones are then • adapted for growth in serum-free media.
  • CHO cell lines secreting HTV-1 subtype D g ⁇ l40 The gp 140 genes with the highest expression levels in HEK293 cells are selected for codon-optimization and cloning Mo dihydrofolate reductase (DHFR)-deficient CHO cells. Expression-vectors containing the DHFR gene-are constructed and the codon-optimized genes — are introduced Mo the DHFR-deficient CHO cells. Env-secreting CHO cells are selected and cloned in G418 with increasing concentration of Methotrexate. The cloned cells are then adapted for growth in serum-free medium. The cloned, selected env-secreting cell lines are characterized for env expressing by g l20 capture ELISA.
  • DHFR dihydrofolate reductase
  • Purification procedures acceptable for transition to GMP production are evaluated and optimized for gpl40. These include: GNA chromatography, size exclusion by gel filtration chromatography and affinity purification. Selected cell lines are scaled up for the production of quantities of supernatant fluids that yield approximately 5 mg of purified g l40.
  • Unfractionated and fractionated gpl40, gpl20 or oligomeric gp41 is loaded onto 5-20% sucrose gradients containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5% Triton x-100 and centrifuged in an SW40 rotor for twenty hours at 4°C at 40,000 rpm (Earl et al. (1994) J. Virol. 68, 3015-3026). Fractions are collected and half of each immunoprecipitated with Rl 60, a polyclonal rabbit antisera (Willey et al. (1991) Virology 184, 319-329).
  • Proteins are separated by SDS- polyacrylamide gel electrophoresis (10%) followed by Western blotting using a cocktail of mAbs (three anti-gpl20 specific and three anti-gp41 specific) and 125 I-labeled anti mouse IgG.
  • mAbs three anti-gpl20 specific and three anti-gp41 specific
  • 125 I-labeled anti mouse IgG 125 I-labeled anti mouse IgG.
  • -Peak fractions are pooled, concentrated approximately ten-fold with Centricon-30 microconcentrators (Amicon), cross-linked with ethylene glycol bis(succinimidylsuccinate) (EGS; Pierce) and analyzed by SDS-polya ' crylamide gel electrophoresis (4%) and Western blotting as above.
  • HTV-1 envelope glycoproteins binding to sCD4 and monoclonal antibodies
  • Binding of the various fractions to a panel of gpl20 and gp41 specific mAbs to include those specific for conformational, linear and oligomeric-specific epitopes as well as sCD4 are measured using surface plasmon resonance (BIAcore) (VanCott et al. (1995) J. Immunol. Meth. 183, 103-117). Unfractionated and fractionated gpl40 and gp41 are captured by mAbs specific for gp41 (1G2), gpl20 (2C6) or sCD4 immobilized within a biosensor -matrix as previously described (VanCott et al. (1995) J. Immunol. Meth.
  • the gp41 mAb binds to a linear epitope within gp41 and the gp!20 mAb binds to a conformational epitope within gpl20.
  • the various HTV-1 env preparations are injected across each of the mAb or sCD4 matrices and the amount captured is measured. Subsequently HTV-1 sera (pooled sera from thirty-five HTV-1 seropositive individuals), sCD4 or the panel of gp41, gpl20 and o-gpl40-specific mAbs are injected across the gpl40/ gpl20/gp41 captured matrix and binding ratios are measured.
  • These mAbs include those specific for V1/V2 or V3 regions of env, the CD4 binding site, the C5 region of env or gp41.
  • both the Ras3C and PCPP adjuvants formulations with ogpl40 elicit antibodies with preferential reactivity with conformationally-Mact gpl20 suggesting minimal disruption of overall tertiary env structure (VanCott et al (1997) J. Virol. 71, 4319-4330).
  • Ribi adjuvant system composed of a 2.0% v/y squalene oil-in-water emulsion containing 250 ⁇ g/ml of 4' monophosphoryl lipid A derived from lipopolysaccharide (LPS) of Salmonella Minnesota R595 (MPL ® ), 250 ⁇ g/ml cell wall skeleton (CWS) from Mycobacterium phlei and synthetic dicorynomycolate (S-TDCM) is used as the adjuvant (Szentivanyi et al. (1986) Immunobiology and immunopharmacology of bacterial endotoxins, Plenum Press, pages 407-420; Powell et al.
  • LPS lipopolysaccharide
  • MPL ® Salmonella Minnesota R595
  • CWS cell wall skeleton
  • S-TDCM synthetic dicorynomycolate
  • PCPP polyphosphazene
  • PCPP polyphosphazene
  • Payne et al. a synthetic microsphere hydrogel adjuvant with a molecular weight of 2000 KDar(Payne e ⁇ al " (1998) Adv. Drug Deliv. Rev. 31, 185-196; Payne et al. (1995) Pharm. Biotechnol. 6, 473-493; Lu et al. (1996) J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 12, 99-106; Payne et al. (1998) Vaccine 16, 92-98; McNeal et al.
  • o-gpl40-UGD is captured by antibodies to the C-terminus of gpl20 (D7324) previously adsorbed overnight to ELISA plates. Plates are washed twice with wash buffer (PBS with 0.1% Tween 20, pH 7.4) prior to the incubation with two-fold dilutions of serum diluted in serum diluent (wash buffer with 5% skim milk, pH 7.4) for one hour at 37°C. Plates are washed three times with wash buffer and incubated with HRP-conjugated goat-anti-rabbit IgG (diluted in serum diluent). After a one hour incubation at 37°C, plates are washed three times after which ABTS substrate is added.
  • wash buffer PBS with 0.1% Tween 20, pH 7.4
  • HRP-conjugated goat-anti-rabbit IgG diluted in serum diluent
  • the reaction is stopped with 0.5% SDS after thirty minutes at 37°C.
  • Data is expressed as a serum endpoint titer; the maximal serum dilution at which the specific binding (measured by optical density units) are greater then 2x mean plus 2x standard deviation of both the pre-immune and control sera.
  • T-cell-line-adapted and primary HTV-1 viral stocks from clades A, B, C, D, E and F (U.S. Military HIV Research Program) and T-cell laboratory- adapted (TCLA) HTV-1 isolates DIB, MN and RF (NTH ADDS Research and Reagent Repository) are used in HTV-1 neutralizing antibody assays. All primary isolates have been initially cultured on PHA-stimulated PBMC and are low passage. None have been passaged through neoplastic cell lines. TCLA virus isolates are obtained as previously described and the HTV-1 neutralizing antibody assays to be used are as described previously (Mascola et al. (1996) J. Meet. Dis.
  • H9 cells are used as target cells and virus- growth kinetics and median tissue culture Mective dose (TCTD50) is determined within the assay format. All sera and mucosal washes are heat-inactivated at 56°C for forty minutes prior to use. Culture media without antibody, pooled normal human serum (NHS) and pre-immune sera (macaque) or mucosal washes serve as controls for baseline virus growth, hihibition of H9 Mection is assessed by quantitative p24 measurement of cell supernatants during the early virus growth phase (day 4, 5 or 6).
  • the serum dilution causing 50% and 90% reduction in p24 antigen is calculated by linear regression analysis. Assessment of neutralizing antibody activity of serum is performed as previously described with minor modifications (Mascola-et al (1996) J. Meet. Dis. 173, 340-348; Mascola et al (1994) J. Meet. Dis. 169, 48-54; Mascola et al (1997) J. Virol. 71, 7198-7200).
  • PHA-stimulated PBMC are used as target cells for primary strains and virus growth kinetics and median tissue culture Mective dose (TCID50) is determined within the assay format. The same donor cryopreserved PBMC are used in both the titration and neutralization assays.

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Abstract

La présente invention concerne des molécules d'acides nucléiques recombinantes codant pour des glycoprotéines d'enveloppe gp140 recombinantes du VIH-1 de sous-type D, ainsi que les glycoprotéines codées par ces molécules. L'invention concerne également des vecteurs comprenant ces molécules d'acides nucléiques, ainsi que des compositions vaccinales comprenant lesdites molécules d'acides nucléiques ou lesdites glycoprotéines. L'invention concerne en outre un procédé permettant d'induire une réponse immunogène étendue vis-à-vis du VIH-1 pour neutraliser le virus VIH-1.
PCT/US2003/036653 2002-11-15 2003-11-17 Glycoproteines d'enveloppe recombinantes du vih-1 de sous-type d Ceased WO2004046168A2 (fr)

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US5696238A (en) * 1991-08-20 1997-12-09 Chiron Corporation Purified GP120 composition retaining natural conformation
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EP2527367A1 (fr) * 2007-08-20 2012-11-28 Glaxo Group Limited Procédé de production
JP2015027298A (ja) * 2007-08-20 2015-02-12 グラクソ グループ リミテッドＧｌａｘｏ Ｇｒｏｕｐ Ｌｉｍｉｔｅｄ 製造方法
US9163249B2 (en) 2007-08-20 2015-10-20 Glaxo Group Limited Production methods
EP3216802A1 (fr) * 2007-08-20 2017-09-13 Glaxo Group Limited Procédé de production
US10633673B2 (en) 2007-08-20 2020-04-28 Glaxo Group Limited Production methods

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