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WO1998041536A9 - Glycoproteines enveloppes de vih et vis a glycosylation deficiente - Google Patents

Glycoproteines enveloppes de vih et vis a glycosylation deficiente

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Publication number
WO1998041536A9
WO1998041536A9 PCT/US1998/003374 US9803374W WO9841536A9 WO 1998041536 A9 WO1998041536 A9 WO 1998041536A9 US 9803374 W US9803374 W US 9803374W WO 9841536 A9 WO9841536 A9 WO 9841536A9
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amino acid
gpl20
hiv
cells
virus
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PCT/US1998/003374
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English (en)
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WO1998041536A1 (fr
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Priority to AU65358/98A priority Critical patent/AU6535898A/en
Publication of WO1998041536A1 publication Critical patent/WO1998041536A1/fr
Publication of WO1998041536A9 publication Critical patent/WO1998041536A9/fr

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  • Embodiments of the present invention relate to the human immunodeficiency virus and vaccines therefor. More particularly, embodiments of the present invention relate to selectively underglycosylated envelope glycoproteins useful as HtV-1 vaccines.
  • HIV Human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • the envelope (env) gene of HIV encodes a 160 kilodalton glycoprotein which is cleaved into an extracellular protein known as gpl20 and a transmembrane protein known as gp41.
  • the envelope glycoproteins contain conserved cysteine residues and N-linked carbohydrate sites.
  • the gpl20 molecule contains 5 variable regions referred to as VI - V5. These variable regions are designated as such because they exhibit amino acid sequence variability across HTV-1 isolates.
  • Gpl20 also contains constant regions, i.e., regions of relatively conserved amino acid sequence across HTV-1 isolates.
  • the HIV envelope protein gpl20 is heavily glycosylated, having about 55% of its molecular mass contributed by N-linked carbohydrates.
  • HTV-1 molecular clones contain an average of 23-24 potential N-linked glycosylation sites on gpl20.
  • Carbohydrate side chains of envelope glycoproteins of HTV-1 and other viruses have been postulated to interfere with binding of neutralizing antibodies To date, however, it has not been demonstrated that the absence of glycosylation sites enhances the antibody response to gpl20
  • HTV-1 gpl20 which did not appear to affect infectivity of a virus containing the gpl20 mutant in cell culture but which appeared to render the virus more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4
  • N-linked glycans within the amino-terminal portion of a recombinant gpl20 glycoprotein of immunodeficiency viruses such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) produces a selectively underglycosylated envelope glycoprotein capable of enhanced antibody responses useful as an HIV-1 vaccine
  • a compound which includes a recombinant human immunodeficiency virus type 1 envelope glycoprotein having an amino acid sequence which is altered with respect to a wild type HIV-1 envelope glycoprotein
  • the altered amino acid sequence includes a mutated consensus amino acid recognition sequence for N-linked carbohydrate attachment which, as a result of the mutated consensus amino acid recognition sequence, is not glycosylated in a mammalian host cell.
  • the resulting amino acid is referred to herein as being underglycosylated.
  • the mutated consensus amino acid recognition sequence is positioned between the N-terminus of gpl20 and the Cysteine at the N-terminal side of the gpl20 cysteine V3 loop.
  • the Cysteine is approximately at amino acid position 296.
  • the recombinant envelope glycoprotein has a mutated or otherwise altered consensus amino acid recognition sequence for N-linked carbohydrate attachment and is infective, i.e., when present as a component of a complete HIV virion, it supports viral infectivity.
  • Additional embodiments of the present invention are directed to pharmaceutical compositions and/or vaccines (both for protecting uninfected individuals or for treating infected individuals) that comprise such HIV-1 recombinant envelope proteins having altered sequences as described herein in pharmaceutically acceptable carriers or excipients. Methods including administering such pharmaceutical compositions or vaccines to humans to stimulate the production of antibodies against HIV are also contemplated. Still other embodiments of the present invention include DNA encoding the HIV-1 recombinant envelope proteins having altered sequences as described herein (particularly in an expression vector), recombinant cells comprising such DNA, and methods of making the recombinant mutant envelope glycoproteins by expressing such DNA.
  • Methods according to the present invention include delivering such DNA to cells to produce a translation polypeptide immunizing product capable of delivering an immune response.
  • the methods of the invention may be applied by direct injection of the DNA into cells of an animal, including a human, in vivo, or by in vitro transfection of some of the animal cells which are then reintroduced into the animal body.
  • the DNA may be delivered to various cells of the animal body, including muscle, skin, brain, lung, liver, spleen, or to cells of the blood. Delivery of the DNA directly in vivo is preferable to the cells of muscle or skin.
  • the DNA may be injected into muscle or skin using an injection syringe.
  • the DNA may also be delivered into muscle or skin using a vaccine gun.
  • Still further embodiments of the present invention include antibodies raised against, or preferentially binding to, the mutant envelope glycoprotein.
  • Fig. 1 is a schematic illustration of the HIV-1 envelope glycoprotein gpl20, with the hypervariable regions of the molecule indicated by darkened lines, designated VI -V5, wherein cysteine-cysteine disulfide bonds are represented by solid lines connecting each end of a loop. Numbers represent the first amino acid in each of the 24 potential N-linked glycosylation sites in the molecule.
  • Fig. 2 is a schematic illustration of gpl20 from HIV-1, showing the distribution and amount of conservation of N-linked glycosylation sites. Amino acids are numbered from the N- terminus of the molecule to the C-terminus. The numbers beneath the diagram denote the position of the first amino acid in the consensus sequence of an N-linked glycosylation site. Sites which are >90% conserved among HTV-1, HTV-2 and STV isolates are indicated by an arrow with an open head and are numbered sequentially with the prefix 'b'. Other sites which are conserved at a level of less than 50% are indicated by an arrow having a wavy tail. Fig.
  • FIG. 3 is a schematic representation of the location of the glycosylation sites in SIVmac239 (identified by the tree symbol at the top of the figure) and particularly, the 4th, 5th, and 6th glycosylation sites containing the consensus sequence Asn X Ser/Thr in the highly variable region 1 (VI) of the gpl20 sequence of SIVmac239 that were selected for mutagenesis All seven possible mutant forms of these sites were created and are referred to as g4, g5, g6, g45, g46, g56, and g456
  • Fig 4 is a graph showing the rate of virus production of CEMxl74 cells by SIV239mac mutant viruses with single glycosylation substitutions in gpl20
  • Fig 5 is a graph showing the rate of virus production of CEMxl74 cells by SIV239mac mutant viruses with multiple glycosylation substitutions in gpl20
  • Fig 6 is a graph showing rate of viral replication in Rhesus periferal blood mononuclear cells (PBMC's)
  • Fig 7 is a graph showing rate of replication of SIV glycosylation mutant g456 following transfection of CEMxl74 cells
  • Fig 8 is a graph showing rate of viral replication following infection of CEMxl74 cells with uncloned virus stock from g456 transfection
  • Fig 9 is a schematic showing the sequence of g456 revertant clones
  • Fig 10 is a graph showing rate of viral replication following infection of CEMxl74 cells
  • Fig 11 is a graph showing rate of viral replication following infection of Rhesus monkey cell line 221
  • Fig 12 is a sample of a gel electrophoresis showing migration of gpl ⁇ O precursor and gpl20 external surface subunit from wild type and g456 mutant viruses
  • Fig 13 is a graph showing rate of virus production of CEMxl74 cells by SIV239mac mutant viruses with five glycosylation substitutions in gp 120
  • Fig. 14 is a graph showing the results of an ELISA assay in which serum from monkeys immunized with a replication competant SIV containing a recombinant gpl20 protein having an altered amino acid sequence according to the invention or with a wild type SIV virion by was tested for the presence of antibodies able to bind to a peptide having the amino acid sequence NH2- Cys Asn Lys Ser Gle Thr Asp Arg T ⁇ Gly Leu -COOH (Peptide A) containing the altered
  • Fig 15 is a schematic representation of the amino acid sequence of SIVmac239 (residues).
  • Figs. 16-21 are graphs showing the immune response following infection for 16 weeks for each peptide for the indicated virus.
  • Fig. 22 is a graph showing animal sera antibody responses to peptide 14 following 24 weeks infection with mutant and wild-type SIVmac239 viruses.
  • Carbohydrates comprise about 50% of the mass of gpl20, the external envelope glycoprotein of the simian and human immunodeficiency viruses (SIV and HIV).
  • the envelope precursor of gpl20 When the envelope precursor of gpl20 is produced in mammalian cells in the presence of glycosylation inhibitors, the protein generally is not properly processed. Deficits imparted by lack of glycosylation include lack of proper folding, retention in the golgi, lack of proteolytic processing, and inability to bind to CD4. When fully glycosylated gpl20 is deglycosylated enzymatically in the absence of detergents, the deglycosylated gpl20 apparently retains its native structure and can bind CD4.
  • carbohydrates appear to be required to generate a properly folded, properly processed protein, but once formed the carbohydrates do not appear to be required to maintain native structure.
  • individual N-linked sites can be eliminated without impairing native structure or the ability of virus to replicate.
  • other N-linked sites are essential for the virus to replicate.
  • “Viral infectivity”, as used herein, refers to the ability of an infective virus containing an envelope gene of HIV, or an infectious DNA clone, that is engineered to encode the mutated consensus amino acid recognition sequence for N-linked carbohydrate attachment, to replicate in culture or in vivo.
  • Wild-type or native HIV-1 envelope glycoprotein refers to the envelope glycoprotein encoded by a naturally occurring HIV-1 isolate.
  • amino acid positions of the envelope glycoprotein such as the Cys at the N-terminal side of the cysteine loop containing V3 (approximately amino acid position 296) or the Cys at the C-terminal side of the cysteine loop containing VI and V2
  • cysteine cross-links form loops which contain hypervariable regions in gpl20 having widely accepted designations.
  • Recombinant glycoprotein refers to a glycoprotein produced by expression of a DNA sequence that does not occur in nature and which results from human manipulations of DNA bases.
  • the term recombinant envelope glycoprotein means gpl60, gpl20, or other env-encoded peptides containing at least the above-described N-terminal portion of gpl20 and containing at least one and if desired multiple mutated N-linked carbohydrate attachment sites as described herein
  • a recombinant protein or epitope of a protein is "immunogenic” or “antigenic” when it elicits an antibody response or is recognized by immunocompetent cells (i.e , cells of the immune system).
  • An antibody response is indicated by the formation in a mammal of antibodies to the protein and can be detected by conventional antibody detection assays on serum from the mammal; e g., an ELISA.
  • a protein or epitope is "non- immunogenic" (non-antigenic) when it is not able to elicit an antibody response or does not trigger the activation of immunocompetent cells, as explained above
  • a recombinant protein of the invention may be determined to be infective and therefore useful in further testing of infectivity in animals if it promotes syncytium formation as described herein.
  • Recombinant envelope glycoproteins according to the invention are recombinant human immunodeficiency virus envelope glycoproteins which are mutated with respect to a wild type (native) human immunodeficiency virus glycoprotein in the primary amino acid sequence to effect partial underglycosylation of the molecule.
  • envelope glycoproteins include the full length proteins or fragments thereof retaining the activity of the full length envelope glycoprotein. It is to be understood, however, that the term “underglycosylation” also refers to nonrecombinant HIV envelope glycoproteins which may undergo removal of glycans through standard known techniques to produce underglycosylated HIV envelope glycoproteins, rather than through recombinant techniques.
  • Proteins according to the invention will contain an amino acid sequence alteration which is introduced to positions in the N-terminal portion of gpl20 or useful fragments thereof (between the N-terminus of gpl20 and a specific cysteine at the N-terminal side of V3 which forms the loop containing V3).
  • an amino acid sequence alteration which is introduced to positions in the N-terminal portion of gpl20 or useful fragments thereof (between the N-terminus of gpl20 and a specific cysteine at the N-terminal side of V3 which forms the loop containing V3).
  • the virion is infective.
  • potential N-linked glycosylation sites in gpl20 itself or as a component of gpl60, gpl40 or other useful fragments thereof can be systematically mutated, either singly or in combination by site directed mutagenesis such that the consensus glycosylation sequence is disrupted.
  • Recombinant viruses are generated containing gpl20 genes that have such mutations.
  • the infectivity of each mutant virus is measured. Processing of gpl ⁇ O to gpl20 and gp41 may also be assessed as a rough measure of retention of conformation and infectivity.
  • HIV-1 strains or isolates in the practice of the present invention, e.g., MN, HXB2, LAI, NL43, MFA, BRVA, SC, JH3, ALAI, BALI, JRCSF, OYI,
  • the preferred mutation at the consensus N-linked glycosylation sequence is substitution of Asn, Ser, or Thr with a different amino acid defined as any amino acid other than the one occupying the position in the wild type.
  • sites of N-linked carbohydrate attachment located in the C-terminus of the gpl20 molecule are not mutated as described herein; that is, the majority of such sites in the C-terminal half of the molecule retain their function with respect to carbohydrate attachment, leaving the molecule substantially glycosylated in its carboxy terminal half.
  • HIV-1 envelope glycoprotein which contains a mutated N-linked carbohydrate attachment site in the N-terminal half of the molecule and which also contains one or several mutated N-linked carbohydrate attachment sites in the C-terminal half of the molecule.
  • a recombinant HIV-1 envelope glycoprotein according to the invention may contain a mutated N-linked carbohydrate attachment site within the N-terminal half of the molecule in combination with a mutated N-linked carbohydrate attachment site at one or more of the positions located in the C-terminal half of the envelope glycoprotein; such as one or more of sites 386, 392, 397, 406 or 463, and also optionally including mutated consensus sequences at approximately position 448 and/or position 392.
  • sites 386, 392, 397, 406 or 463 such as one or more of sites 386, 392, 397, 406 or 463, and also optionally including mutated consensus sequences at approximately position 448 and/or position 392.
  • the numbers given above for gpl20 refer to amino acid residues of the HXB2 envelope protein.
  • positions 386, 392, 397, 406 and 463 can be understood as a reference to theN-linked glycosylation sites positioned between the C-terminus of gpl20 and the Cys on the N-terminal side of the cysteine loop containing hypervariable region 4 (V4).
  • the reference to positions 289 and 356 can be applied to other strains with reference to Fig. 1 and Fig. 2.
  • the invention also provides mutated sites of N-linked carbohydrate attachment in an flV- 1 envelope glycoprotein such as gpl60, truncated forms of gp 160 such as gp 140, or gpl20, or fragments thereof which altered glycoproteins are effective HIV-1 vaccines.
  • flV- 1 envelope glycoprotein such as gpl60
  • truncated forms of gp 160 such as gp 140, or gpl20
  • fragments thereof which altered glycoproteins are effective HIV-1 vaccines.
  • N-linked glycosylation sites can be identified by locating the amino acid consensus sequence Asn-X-Ser/Thr in the glycoprotein.
  • the corresponding nucleotide sequence is located in the DNA sequence encoding the glycoprotein.
  • the corresponding nucleotide sequence to the amino acid consensus sequence is then mutated such that the codon specifying any one or more of the amino acids of the consensus sequence is altered so as to specify an amino acid other than the consensus amino acid.
  • the altered DNA sequence can then be used to produce an altered envelope glycoprotein or can be assembled into the DNA of the HIV-1 virion, along with the altered envelope protein, or into a vaccinia virus as known in the art and described herein.
  • Recombinant virions containing the altered glycoprotein and altered nucleotide sequence, wherein the mutations have substantially no effect on infectivity can then be identified according to methods and procedures well known in the art.
  • the molecular clone HXB2 which contains 24 N-linked glycosylation sites is used as the template DNA for site-directed mutagenesis as follows. Oligonucleotide- directed mutagenesis is performed on a selected fragment of HXB2 (Cohen et al., 1990 J. AIDS 13:11), which covers all 24 N-linked glycosylation sites of gpl20, using the method of Kunkel (Cohen et al., 1988, Nature 334:532). The oligonucleotide primers used for mutagenesis are synthesized using standard cyanoethyl phosphoamadite chemistry and are listed in Table I below.
  • Mutants are identified by the Sanger chain-termination method (Cullen, 1986, Cell 46:973). The fragment containing the desired mutation is excised from the replicative form of each mutant and used to replace the same fragment of HXB2. All HXB2-derived N-linked glycosylation site mutants containing the designated changes are further verified by DNA sequencing (Cullen, 1986, Cell 46:973).
  • HIV-1 envelope glycoprotein molecules which are candidate vaccine molecules will possess the following properties. 1) they will be altered in their primary amino acid sequence at one or more selected sites in the N-terminal portion of the molecule such that the site is no longer recognized in a mammalian, and preferably a human cell, as a site of carbohydrate attachment, 2) the sequence alterations to the protein will alter the protein to an extent which permits immune recognition of the protein, and 3) a sufficient amount of the wild type conformation of the molecule should be retained such that the mutant virus substantially retains infectivity It is believed that a recombinant gpl20 molecule which satisfies these criteria will be more likely to elicit a protective immune response against wild-type HIV-1 strains and thus to reduce infectivity of the natural virus
  • gpl20 molecules derived from any strain of HIV-1 which satisfy the criteria listed above can be generated using the methods described above
  • one of skill in the art needs to know the sequence of the gpl20/gpl60 gene in the particular strain of HIV- 1 of interest
  • the sequences of gp 120/gp 160 of many strains of HTV-1 are known, where new strains are discovered, the gpl 20/gp 160 sequence may be determined by a skilled artisan using ordinary cloning and sequencing technology such as that described in the Molecular Cloning Manual (Sambrook et al , 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY)
  • Potential vaccine molecules can be obtained by the skilled artisan without undue experimentation because the techniques and tests to be used are common and familiar to those knowledgeable in the art and are described herein to the extent that they are needed to practice the invention
  • gpl 20 molecules containing an altered amino acid sequence according to the invention Described herein are materials and methods for generating gpl 20 molecules containing an altered amino acid sequence according to the invention and then determining their ability to act as vaccines It is to be understood that altered gp 160 molecules or fragments thereof also are useful according to the invention as a vaccine candidate provided the N-terminal end of the gpl20 portion of the g ⁇ l60 molecule is underglycosylated Altered gpl60 molecules can be generated using the procedures described herein for gpl20
  • the invention contemplates alteration of the primary amino acid sequence of an HIV-1 envelope glycoprotein such that at least one site in the N-terminus of the envelope glycoprotein is no longer recognized as an N-linked carbohydrate addition site and therefore not glycosylated when the protein is synthesized in a mammalian, and preferably a human cell
  • the mobility of the recombinant protein on a gel is compared to the mobility of the wild type protein
  • the gel mobility of the recombinant protein differs from the wild type protein by a visible shift in band migration, it can be assumed that the recombinant protein is underglycosylated to an extent which is sufficient to test the recombinant molecule further for immunogenicity
  • chemical techniques for quantitating sugar content are well known See, e g , Chapin at al IRL Press (1986) pp 178-181 and Methods of Carbohydrate Chemistry Vol 7 (Whistler at
  • Recombinant gpl20 or gpl60 mutant glycoproteins can be obtained by expressing these proteins in any one of a number of expression systems These systems include but are not limited to the following
  • a baculovirus expression system can be used to obtain recombinant gpl 20 or gpl 60
  • a gene encoding the recombinant glycoprotein can be cloned into a commercially available baculovirus transfer plasmid
  • a recombinant baculovirus encoding such a protein can be generated as described by Summers and Smith (1988, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures: Texas Agricultural Experiment Station Bulletin No.
  • the virus can be used to infect cells, such as Sf9 cells, whereupon the recombinant glycoprotein will be expressed to high levels as the baculovirus replicates Protein is recovered from the culture using ordinary standard biochemical techniques jAlternatively, Chinese hamster ovary (CHO) cells can be transfected with a plasmid encoding a mutated gpl20 or gpl60 gene, using any number of transfection methods all of which are described in detail in Sambrook at al (supra) Recombinant proteins can be expressed in a constitutive manner under the control of its own promoter or under the control of another promoter such as another retrovirus LTR Alternatively, recombinant proteins can be expressed in an inducible manner, wherein expression is driven by a promoter that responds to the addition of an inducer molecule to the transfected cells Examples of such promoters can be found in
  • cell-free virions obtained from the culture supernatant of COS- 1 transfectants are collected at 48 hours post-transfection. Equal amounts of mutant and wild type viruses, as measured by RT activity, are used to infect CD4-positive SupTl cells. Virus-infected cultures are monitored for syncytium formation as determined by the presence of multinucleated cells as a measure of viral infectivity. As in the case of the wild type virus-infected SupTl cultures, syncytia and RT activity are expected to be detected in all the mutant virus-infected SupTl cultures.
  • the CD4 positive human T lymphoid cell line, SupTl is grown and maintained at 37° C in RPMI-1640 containing 10% heat-inactivated fetal bovine serum and 1% penicillin- streptomycin.
  • COS-1 cells are propagated in Dulbecco's minimal eagle medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin.
  • Cell-free supernatants are collected 48 hours after transfection.
  • Supernatants are filtered through 0.45 mm filters and assayed for virion-associated reverse transcriptase (RT) activity. Equal amounts of wild type and mutant virus, as measured by RT activity (100K cpm), is used to infect 1 x 10 6
  • SupTl cells One milliliter of the culture medium is collected every three or four days and assayed for RT. Cultures are monitored for 28 days to determine syncytium formation as a measure of viral infectivity. Reverse transcriptase assay to determine growth kinetics
  • One milliliter of culture medium is mixed with 0.5 ml 30% PEG and 0.4M NaCl on ice for 2 hours and spun at 2500 rpm at 4° C for 30 minutes.
  • the pellet is resuspended in 100 ml of RT buffer (0.5% Triton X-100, 15mM Tris pH 7.4, 3mM dithiothreitol, 500mM KCL, 30% glycerol).
  • HXB2-derived mutants each having one of the 24 N-linked glycosylation sites mutated by site-directed mutagenesis
  • the ability of HXB2-derived mutants to infect CD4-positive SupTl cells is compared with that of the wild type virus.
  • Most of the individual consensus N-linked glycosylation sites are dispensable for viral infectivity.
  • N-linked glycosylation sites that are likely to play important roles in HIV-1 infectivity are not randomly distributed in gpl 20; they are generally located in the N- terminal half of gpl 20.
  • a candidate vaccine for HTV-1 might be a partially glycosylated gpl 20 with most of the dispensable N-linked glycosylation sites removed, such that the conformation of the protein is largely unaltered and the CD4 binding site is retained.
  • Each of the 24 potential N-linked glycosylation sites in the gpl 20 coding region of the infectious molecular clone HXB2 is individually modified to generate 24 N-linked glycosylation site mutants (See Table 1). In these mutants, the Asn-X-Ser/Thr attachment sequence is replaced by either Gln-X-Ser/Thr or His-X-Ser/Thr.
  • each of mutant or wild type proviral DNA is transfected in 3-5 x 10 6 COS-1 cells using DEAE-dextran as described above. Cell lysates derived from COS-1 transfectants are then examined in standard western blots. It is expected from this example that no particular individual N-linked glycosylation site is indispensable for the expression of the envelope protein.
  • Recombinant envelope proteins can be used to generate antibodies using standard techniques, well known to those in the field. For example, the proteins are administered to challenge a mammal such as a monkey, goat, rabbit or mouse. The resulting antibodies can be collected as polyclonal sera, or antibody-producing cells from the challenged animal can be immortalized (e g by fusion with an immortalizing fusion partner) to produce monoclonal antibodies
  • the recombinant protein may be conjugated to a conventional carrier in order to increase its immunogenicity, and antisera to the peptide-carrier conjugate is raised
  • Coupling of a peptide to a carrier protein and immunizations may be performed as described in Dymecki, S M , et al , J Biol. Chem. 267 4815-4823, 1992
  • the serum is titered against protein antigen by ELISA or alternatively by dot or spot blotting (Boersma and Van Leeuwen, 1994, J. Neurosci. Methods
  • the antisera may be used in tissue sections
  • the sera is shown to react strongly with the appropriate peptides by ELISA following the procedures of Green et al , Cell, 28, 477-487 (1982) 2
  • Monoclonal antibodies Techniques for preparing monoclonal antibodies are well known, and monoclonal antibodies of this invention may be prepared using a recombinant envelope glycoprotein described herein or a synthetic peptide thereof containing the altered amino acid sequence, preferably bound to a carrier, as described by Arnheiter et al , Nature, 294, 278-280 (1981)
  • Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue was introduced Nevertheless, monoclonal antibodies may be described as being “raised to” or “induced by” the synthetic peptides or their conjugates
  • immunological tests rely on the use of either monoclonal or polyclonal antibodies and include enzyme linked immunoassays (ELISA), immunoblotting, immunoprecipitation and radioimmunoassays See Voller, A , Diagnostic Horizons 2 1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, MD, Voller, A et al., J. Clin. Pathol. 31 507-520 (1978), U S Reissue Pat No 31,006, UK Patent 2,019,408, Butler, J E , Meth.
  • ELISA enzyme linked immunoassays
  • mutant proteins can be detected using chromatographic methods such as SDS PAGE, isoelectric focusing, Western blotting, HPLC and capillary electrophoresis
  • Monoclonal antibody-producing hybridomas can be screened for antibody binding to the protein and to wild type envelope They can also be screened for the ability to neutralize infectivity of HTV-1 isolates, preferably multiple (e g , at least 3) isolates each having diverse sequences in the hypervariable VI or V2 regions
  • an antibody useful in the invention may comprise whole antibodies, antibody fragments, polyfunctional antibody aggregates, or in general any substance comprising one or more specific binding sites from an antibody
  • the antibody fragments may be fragments such as Fv, Fab and F(ab') 2 fragments or any derivatives thereof, such as a single chain Fv fragments
  • the antibodies or antibody fragments may be non-recombinant, recombinant or humanized
  • the antibody may be of any immunoglobulin isotype, e g , IgG, IgM, and so forth
  • aggregates, polymers, derivatives and conjugates e.g , aggregates, polymers, derivatives and conjugates
  • the wild-type envelope sequence was subcloned into the Xhol and BamHl sites of the expression vector pSVL (Pharmacia) following creation of a BamHl site 3' of the env coding sequence using the mutagenic primers #27 (9268-9302) 5'- GTATATGAAGGATCCATGGAGAAACCCAGCTGAAG-3' and #28 (9286-9253) 5'- CCATGGATCCTTCATATACTGTCCCTGATTGTAT-3'.
  • the mutant envelope sequences were subcloned into the resultant pSVLenv via the unique Xhol and Sacl sites.
  • STVmac239 were digested with Sphl and heated to 65 °C for 15 minutes. Each right-half clone was ligated together with the left- half clone p239SpSp5' using T4 DNA ligase. Three micrograms of the ligated DNA was used to transfect CEMxl74 cells treated with DEAE-dextran (Naidu, 1988).
  • the pSVL vector containing the wild-type or mutant envelope sequences were transfected into DEAE-dextran treated COS-1 cells, 1 microgram of DNA was used following the procedure of Levesque et al (Levesque, J -P , P Sanilvestri, A Hatzfeld, and J Hatzfeld (1991) DNA transfection in COS cells BioTechmques 11 313-318 )
  • Virus stocks and cell culture were prepared as follows Rhesus monkey peripheral blood mononuclear cells (PBMC's), CEMxl74, 221, and COS-1 cells were maintained as previously described
  • PBMC's peripheral blood mononuclear cells
  • CEMxl74 cells were transfected as described above The medium was changed every 2 days and the supernatants were harvested at or near the peak of virus production Cells and debris were removed by centrifugation and virus contained in the supernatant was aliquoted and stored at - 70° C
  • the concentration of p27 antigen was measured by antigen capture assay (Coulter Corporation, Hialeah, FL)
  • For virus infections five micrograms of p27 was used to infect 2 5 million pelletted cells
  • DNA sequencing and PCR amplification was performed as follows Cloned fragments containing mutated DNA were sequenced in their entirety manually or with an ABI377 automated DNA sequencer using dye-terminator cycle sequencing chemistry according to the instructions of the manufacturer (Perkin-Elmer Inc , Foster City, Calif) Total genomic DNA was isolated with the HRI AmpPrep kit (HRI Research, Inc , Concord, Calif) and used as a template for nested PCR amplification, using primers located outside of the viral env sequence Outer primers were #39 5'-GAGGGAGCAGGAGAACTCATTAGAATCCTCC-3' and #40 5'- GTTCTTAGGGGAACTTTTGGCCTCACTGATACC-3' The inner mutagenic primers created Xhol and BamHl sites used for cloning the PCR products into pSP72 and were #38 5'-CTCAGCTATACCTCCCTCGAGAAGCATGCTATAAC-3' and #32 5'-
  • reaction mix contained one microgram total DNA, 2mM Mg +2 , 200 ⁇ M each of the four deoxynucleoside triphosphates,
  • each primer 0 2 ⁇ M each primer, and 2 U of Vent polymerase (New England Biolabs, Beverly, Mass ) and were amplified for 30 cycles Each cycle consisted of denaturation at 93 °C for 1 min, annealing at 50 °C for 1 min, and elongation at 72 °C for 3 min 15 s ending with a 10 min final extension at 72 °C for the last cycle EXAMPLE V
  • Peptide 1 was purchased from Bio-Synthesis,Inc , (Lewisville, TX) and consisted of the amino acid sequence NH2- Cys Asn Lys Ser Gle Thr Asp Arg Trp Gly Leu -COOH
  • the 4th, 5th, and 6th glycosylation sites containing the consensus sequence Asn X Ser/Thr in the gpl 20 sequence of SIVmac239 were selected for mutagenesis These sites are located in the N-terminal half of the gpl 20 molecule and in the vicinity of the highly variable region 1 but nonetheless are strongly conserved among SIV sequences Therefore, the 4th, 5th and 6th sites are representative sites for mutation and testing of the resultant altered gpl 20 or gpl 60 molecule according to the invention
  • the Asn codon at all three sites of SIVmac239 is AAT
  • the AAT at sites 4 and 5 were changed to CAG (Gin) and at site 6 it was changed to CAA (Gin) Gin is structurally similar to Asn, differing only by a single CH 2 group Since only AAT and AAC can code for Asn, two nucleotides would be required in the codon to revert back to Asn All seven possible mutant forms of these sites were created. These will be
  • virus containing the M144V change in the g456 background replicated with only a slight delay when compared to STVmac239 upon both transfection and infection in both CEMxl74 and 221 cells.
  • the M144V mutant in the g456 background replicated with similar kinetics to the revertant recovered from the original transfection shown in Fig. 7.
  • the change of Met to Val at position 144 is able to compensate for the loss of the 4th 5th and 6th NXS/T sites.
  • Vectors for transient expression of STV 239 and g456 envelope proteins were constructed and transfected into COS-1 cells. Envelope protein was detected by Western blot. As shown in Fig. 12, the STV 239 clone yielded both the gpl 60 precursor and gpl 20 external surface subunit as expected during the 2-5 day time period that was examined. The g456 mutant yielded a precursor that migrated slightly faster than the gpl 60 precursor of the wild type. Such a faster mobility would be expected for a protein lacking some of its N- linked glycosylation. Little or no processed forms of env protein with a slightly faster mobility than gpl 20 were detected during the 2-5 days of measurement.
  • Fig. 14 This significant and unexpected result is shown in Fig. 14 and demonstrates that a virus with a gpl 20 containing an altered carbohydrate attachment site so as to prevent glycosylation at that site in the N-terminal half of the gpl 20 molecule is very immunogenic, whereas a wild type virion is much less immunogenic.
  • STV strain has been identified that is missing each of the 5th, 6th, 8th, 12th, and 13th sites of carbohydrate attachment.
  • This mutant virus is replication competent as show in Fig. 13. Studies have confirmed that mutants underglycosylated individually at the 4-13 glycosylation sites are replication competent.
  • SIV and HIV gpl 20 molecules have a high amino acid sequence similarity, with about 40% amino acid sequence identity. In addition, the molecules have the same organization of variable and constant regions. The glycosylation sites in HIV and SIV gpl 20 are located in similar positions along the length of the molecules. Therefore, results from the SIV experiments described herein are believed to be applicable to preparing candidate HIV-1 vaccines. In fact, infection of rhesus monkeys with SIV is generally believed to be a useful model for assessing novel vaccine strategies for AIDS. See Wyand et al. 1996, J. Virol. 70:3724-3733 hereby inco ⁇ orated by reference in its entirety.
  • Fig. 15 which shows the amino acid sequence of SIVmac239, residues 89-213
  • Twenty-three biotinylated peptides were purchased from Chiron Mimotypes (Victoria, Australia) and bound to strept-avidin 96-well plates (Boehringer Mannheim) over night at 4°C. Plates were washed 6 times in wash buffer (PBS and 0.1% Tween- 20) and animal sera was added at a 1 : 100 dilution for 90 minutes.
  • Figs. 16-21 show the reactivity of sera with each peptide.
  • Sera was from animals infected with the indicated viruses for 16 weeks. Sera from week 0 was used as a negative control.
  • the viruses lacking the 4th glycosylation site elicited an immune response against the corresponding peptide that spans the g4 site.
  • the wild-type virus was unable to elicit as strong an antibody response against this site.
  • a similar response was obtained with the antisera elicited by the viruses lacking the 5th or 6th glycosylation sites. Consequently, removal of carbohydrates from the SIV envelope protein allows exposure of previously unexposed antigenic sites.
  • Fig. 22 shows the reactivity of all sera to peptide 14 which contains an amino acid sequence which includes the 5th glycosylation site.
  • Vaccines comprising one or more HIV-1 gpl 20 molecules, as described herein, and variants thereof having antigenic properties, can be prepared by procedures well-known in the art. Procedures which are known for making wild-type envelope protein vaccines (e.g., such as those produced by Chiron or Genentech) can be used to make vaccines with a selectively underglycosylated envelope protein of the invention. Various modifications such as adjuvants and other viral or toxin components known for such vaccines or immunotherapeutics may be incorporated with the mutant molecule. For examples, such vaccines may be prepared as injectables, e.g., liquid solutions or suspensions. Solid forms for solution in or suspension in a liquid prior to injection also can be prepared.
  • injectables e.g., liquid solutions or suspensions. Solid forms for solution in or suspension in a liquid prior to injection also can be prepared.
  • the preparation also can be emulsified.
  • the active antigenic ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients are water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof.
  • the vaccine can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants, such as aluminum hydrazide or muramyl dipeptide, which enhance the effectiveness of the vaccine.
  • the vaccines are conventionally administered parenterally, by injection, for example either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and in some case oral formulations.
  • the peptides or proteins can be formulated into a vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium , or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • a method for immunizing an animal comprising the steps of obtaining a preparation comprising an expressible
  • DNA coding for recombinant immunogenic HIV-1 envelope mutant glycoprotein gpl 20 or gpl 60 molecule and introducing the preparation into an animal wherein the translation product of the DNA is formed by a cell of the animal, which elicits an immune response against the immunogen.
  • Further vaccines may be prepared using a live virus approach well known in the art.
  • the injectable preparation comprises a pharmaceutically acceptable carrier containing an expressible DNA coding the immunogenic HTV-l envelope mutant glycoprotein gpl20 or gpl 60 molecule or the live virus containing the DNA coding the immunogenic HIV-1 envelope mutant glycoprotein gpl20 or gpl 60 molecule, and on the introduction of the preparation into the animal, the polynucleotide or live virus is inco ⁇ orated into a cell of the animal wherein an immunogenic translation product of the DNA is formed, which elicits an immune response against the immunogen.
  • the preparation comprises one or more cells obtained from the animal and transfected in vitro with the DNA, whereby the DNA is inco ⁇ orated into the cells, where an immunogenic translation product of the DNA is formed, and whereby on the introduction of the preparation into the animal, an immune response against the immunogen is elicited.
  • the polynucleotide used for immunization may be an mRNA sequence, although a non- replicating DNA sequence may be used.
  • the DNA may be introduced into the tissues of the body using the injectable carrier alone; liposomal preparations are preferred for methods in which in vitro transfactions of cells obtained from the animal are carried out.
  • the carrier is preferably isotonic, hypotonic or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution.
  • the vaccines are administered in a manner compatible with dosage formulation an in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of about several hundred micrograms active ingredient per individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or two week intervals by a subsequent injection or other administration.
  • the efficacy of a vaccine according to the invention may be determined based on any clinical parameter which a medical doctor assesses for determining the onset and progress of HIV-1 infection or for determining whether an individual has AIDS.
  • Such parameters include, for example, measuring the level of T-cells in a patient.
  • Acceptable levels of T-cells in an uninfected patient are in the range of 1000-2000 T cells per mm 3 .
  • a composition comprising a recombinant human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein having an amino acid sequence which is altered with respect to a wild type HIV-1 envelope glycoprotein, said altered amino acid sequence including a mutated consensus amino acid recognition sequence for N-linked carbohydrate attachment which as a result of said mutated consensus amino acid recognition sequence is not glycosylated in a mammalian host cell, said mutated consensus amino acid recognition sequence being positioned between the N-terminus of gpl20 and the Cysteine at the N-terminal side of the gpl20 cysteine V3 loop, said Cysteine being approximately at amino acid position 296, said recombinant envelope glycoprotein having a mutated consensus amino acid recognition sequence for N-linked carbohydrate attachment being effective, when present as a component of a complete HIV virion, to support viral infectivity.
  • HIV-1 human immunodeficiency virus type 1
  • composition of claim 1, wherein said wild type HIV-1 envelope glycoprotein is gp 160 or a fragment thereof.
  • composition of claim 1 wherein said mutated consensus amino acid recognition sequence for N-linked carbohydrate attachment contains a substitution of Asn, Ser, or Thr as it occurs in the natural consensus sequence with a different amino acid.
  • composition of claim 1 wherein there is a mutated consensus amino acid sequence at an N-linked carbohydrate attachment site in the region between the N terminus of gpl20 and the Cys on the C-terminal side of the cysteine loop containing hypervariable region 2 (V2).
  • composition of claim 1 wherein there are multiple mutated consensus amino acid sequences at N-linked carbohydrate attachment sites in said region
  • a vaccine for use in protection of a human against infection with HIV-1 comprising the composition of claim 1
  • a vaccine for use in treatment of a human infected with HIV-1 comprising the composition of one of claim 1
  • the vaccine of claim 6 or 7, comprising an HIV-1 virion containing a gpl 20 molecule having an altered amino acid sequence comprising a mutated consensus amino acid recognition sequence for N-linked carbohydrate attachment which as a result of said mutated consensus amino acid recognition sequence is not glycosylated in a mammalian host cell, said mutated consensus amino acid recognition sequence being positioned between the N-terminus of g ⁇ l20 and the Cysteine at the N-terminal side of the gpl20 cysteine V3 loop, said Cysteine being approximately at amino acid position 296, said HIV-1 virion being infective
  • a method of inducing a protective immune response in a mammal comprising administering to the mammal an effective amount of the composition of claim 1 or the vaccine of any one of claims 6-8 sufficient to induce the protective immune response, prevent or delay infection by HIV-1.
  • a method of vaccinating a patient against HIV-1 comprising administering to the patient an effective amount of the composition of claim 1 or the vaccine of any one of claims 6-8 sufficient to prevent or delay infection by HIV-1.

Abstract

Selon la présente invention, en supprimant de manière sélective les glycanes liés à N à l'intérieur de la partie N-terminale d'une glycoprotéine recombinée gp120 d'un virus d'immunodéficience comme le virus d'immunodéficience humain de type I ou le virus d'immunodéficience simien, on produit une glycoprotéine enveloppe sélectivement sous-glycosylée capable de réactions immunitaires améliorées.
PCT/US1998/003374 1997-03-14 1998-03-13 Glycoproteines enveloppes de vih et vis a glycosylation deficiente WO1998041536A1 (fr)

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Cited By (2)

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US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins

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Publication number Priority date Publication date Assignee Title
WO1999024465A1 (fr) * 1997-11-10 1999-05-20 Dana-Farber Cancer Institute Glycoproteines d'enveloppe de lentivirus de primate stabilisees
EP1141314A2 (fr) 1998-12-31 2001-10-10 Chiron Corporation Polynucleotides codant pour des polypeptides antigeniques du type c du vih; polypeptides et leurs utilisations
JP2002533124A (ja) 1998-12-31 2002-10-08 カイロン コーポレイション Ｈｉｖポリペプチドの改善された発現およびウイルス様粒子の生成
EP1165798A2 (fr) 1999-03-29 2002-01-02 Statens Serum Institut Methode de production d'une construction de sequence de nucleotides a base de codons optimises, pour un vaccin genetique contre le vih, a partir d'un isolat primaire et precoce du vih, et constructions de l'enveloppe synthetique bx08
EP2065468A1 (fr) * 2000-08-14 2009-06-03 THE GOVERNMENT OF THE UNITED STATES OF AMERICA as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Modifications de la protéine du VIH augmentent l'immunogénicité dans le cadre d'une vaccination génétique
FR2829150B1 (fr) * 2001-09-06 2004-09-03 Bio Merieux Gene env mute codant pour une glypoproteine du vih-1 et applications
GB0225786D0 (en) * 2002-11-05 2002-12-11 Glaxo Group Ltd Vaccine
GB0225788D0 (en) 2002-11-05 2002-12-11 Glaxo Group Ltd Vaccine
AU2010229675B2 (en) 2009-03-27 2016-02-25 Academia Sinica Methods and compositions for immunization against virus
US20160122395A1 (en) * 2013-03-15 2016-05-05 The Macfarlane Burnet Institute For Medical Research And Public Health Ltd. Immunogenic compositions and a process for producing same

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof

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