EP0436634A1 - Proteines et peptides du vih utiles pour le diagnostic, la prophylaxie ou la therapie du sida - Google Patents

Proteines et peptides du vih utiles pour le diagnostic, la prophylaxie ou la therapie du sida

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Publication number
EP0436634A1
EP0436634A1 EP89911309A EP89911309A EP0436634A1 EP 0436634 A1 EP0436634 A1 EP 0436634A1 EP 89911309 A EP89911309 A EP 89911309A EP 89911309 A EP89911309 A EP 89911309A EP 0436634 A1 EP0436634 A1 EP 0436634A1
Authority
EP
European Patent Office
Prior art keywords
hiv
compound
peptide
neutralizing
present
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP89911309A
Other languages
German (de)
English (en)
Inventor
James R. Rusche
Scott D. Putney
Kashayar Javaherian
John Farley
Raymond Grimaila
Debra Lynn
J. Inst.Pasteur Petro-Breyer
Thomas O'keeffe
Gregory Larosa
Albert T. Profy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Repligen Corp
Original Assignee
Repligen Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Repligen Corp filed Critical Repligen Corp
Publication of EP0436634A1 publication Critical patent/EP0436634A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • HIV Human immunodeficiency virus
  • HTLV-III human T-cell lymphotropic virus HI
  • LAV lymphadenopathy-associated virus
  • ARV AIDS-associated retrovirus
  • the virus displays tropism for the OKT4 + lymphocyte subset (Klatzmann, D., F. Barre-Sinoussi, M.T. Nugeyre, C Dauget, E. Vilmer, C. Griscelli, F. Brun-Vezinet, C Rouzioux, J.D. Gluckman, J.C. Chermann, and L. Montagnier [1984] Science 225:59-63).
  • Antibodies against HIV proteins in the sera of most AIDS and AIDS related complex (ARC) patients, and in asymptomatic people infected with the virus (Sarngadharan, M.G., M. Popovic,
  • HIV envelope protein refers to chemical compounds having more than one amino acid.
  • compound refers to chemical compounds in general.
  • compound includes proteins, peptides, and polypeptides.
  • fusion compounds where polypeptides are combined with non-polypeptide moieties.
  • naturally occurring HIV envelope protein refers to the proteins gpl60, gpl20, and gp41 only.
  • HIV refers to any HIV virus, including HIV-1 and HIV-2.
  • HIV is known to undergo amino acid sequence variation, particularly in the envelope gene
  • the principal neutralizing domain is located between the cysteine residues at amino acids 296 and 331 of the HIV envelope.
  • the numbering of amino acids follows the published sequence of HIV-III B (Ratner, L. et al. [1985] Nature 313:277- 284). This domain is known to be hypervariable but retains the type-specific antigenic and immunogenic properties related to virus neutralization.
  • a further aspect of the subject invention is the discovery of highly conserved amino acids within the principal neutralizing domain. Although certain sequences from this region have been published (see, for example, Southwest Foundation for Biological Research, published PCT application, Publication No. WO 87/02775; Genetic Systems Corporation, Published United Kingdom Application
  • Diagnostic kits or therapeutic agents using viral proteins isolated from virus infected cells or recombinant proteins would contain epitopes specific to the viral variant from which they were isolated. Reagents containing proteins from multiple variants would have the utility of being more broadly reactive due to containing a greater diversity of epitopes. This would be advantageous in the screening of serum from patients or therapeutic treatment of patients. Synthetic peptides can be advantageous as the active ingredient in a vaccine, therapeutic agent or diagnostic reagent due to the ease of manufacture and ability to modify their structure and mode of presentation.
  • Synthetic peptides have been used successfully in vaccination against foot and mouth disease virus (Bittle et al. [1982] Nature 29830-33); poliovirus (Emini et al. [1983] Nature 305:699); hepatitis B (Gerin et al. [1983] Proc. Natl. Acad. Sci. 80:2365-2369); and influenza (Shapira et al. [1984] Proc. Natl. Acad. Sci. 81:2461-2465).
  • Such a vaccine advantageously, would be effective to immunize a host against the variant AIDS viruses.
  • the subject invention defines the location of the HIV principal neutralizing domain and discloses methods to utilize this segment of the HIV envelope protein for developing diagnostic, therapeutic, and prophylactic reagents. More specifically, the HIV principal neutralizing domain is located between cysteine residues 296 and 331 of the HIV envelope protein. The location of this domain is shown in Table 1. Although the specific amino acid sequence of the principal neutralizing domain is known to be highly variable between variants, we have found that peptides from this domain are invariably capable of raising, and/or binding with, neutralizing antibodies. This unexpected discovery provides a basis for designing compositions and strategies for the prevention, diagnosis, and treatment of AIDS.
  • the discovery of the principal neutralizing domain resulted from extensive research involving a multitude of HIV envelope proteins and peptides from many HIV variants. Proteins and peptides capable of raising, and/or binding with, neutralizing antibodies are disclosed here. These novel HIV proteins and peptides, or their equivalents, can be used in the diagnosis, prophylaxis, and/or therapy of AIDS. Further, the peptides can be used as immunogens or screening reagents to generate or identify polyclonal and monoclonal antibodies that would be useful in prophylaxis, diagnosis and therapy of HIV infection.
  • a further aspect of the invention is the discovery of highly conserved regions within the principal neutralizing domain. This discovery was quite unexpected because of the known variability of the amino acids within this segment of the HIV envelope protein.
  • proteins and peptides of the invention are identified herein by both their amino acid sequences and the DNA encoding them. Thus, they can be prepared by known chemical synthetic procedures, or by recombinant DNA means.
  • peptides or peptides having the antigenic or immunogenic properties of these peptides, can be used, advantageously, in a vaccine, e.g., a cocktail of peptides, to elicit broad neutralizing antibodies in the host. Further, these peptides can be used sequentially, e.g., immunizing initially with a peptide equivalent to the principal neutralizing domain of an HIV variety followed by immunization with one or more of the above peptides. Polyclonal or monoclonal antibodies that bind to these peptides would be advantageous in prophylaxis or therapy against HIV, the causative agent of AIDS.
  • Figure 1 shows commonly occurring sequences of the principal neutralizing domain.
  • Figure 2 is a schematic for multi-epitope gene construction.
  • Figure 3 depicts the steps in the construction of a specific multi-epitope gene.
  • Figure 4 shows the sequences of four synthesized single-stranded oligomers for construction of a multi-epitope gene.
  • Described here is a segment of the HIV envelope protein which raises, and/or binds with, neutralizing antibodies. This unique and highly unexpected property has been observed in each HIV variant that has been examined.
  • the segment of interest has been named the "principal neutralizing domain.”
  • the principal neutralizing domain is bounded by cysteine residues which occur at positions
  • the principal neutralizing domain is also referred to as the "loop.”
  • the segment of the protein envelope identified here as the principal neutralizing domain is known to be highly variable between HIV variants. Thus it is surprising that, for each variant, this segment is capable of eliciting, and/or binding with, neutralizing antibodies.
  • the principal neutralizing domain identified here is a small segment of the HIV envelope protein. This small segment may be combined with additional amino acids, if desired, for a specific purpose. All such proteins are claimed here except where such proteins constitute a naturally occurring HIV envelope protein.
  • naturally occurring envelope protein refers only to gp160, gp120, and gp41.
  • Table 1 Listed in Table 1 are sequences of the principal neutralizing domain for some of the variants tested. Table 9 contains a complete list of the principal neutralizing domains.
  • Amino acids may be referred to using either a three-letter or one-letter abbreviation system.
  • HIV 10 Kd fusion protein denoted Sub 1.
  • the amino acid sequence of the HIV portion of Sub 1 is shown in Table 2 and the DNA sequence of the HIV portion of Sub 1 in Table 2A.
  • the amino acid sequence of Sub 1 is shown in Table 2B and the DNA sequence in Table 2C.
  • HIV 18 Kd fusion protein denoted Sub 2.
  • the amino acid sequence of the HIV portion of Sub 2 is shown in Table 3 and the DNA sequence of the HIV portion of
  • HTV 27 Kd fusion protein denoted PB1 RF .
  • the amino acid sequence of the HIV portion of PBIRF is listed in Table 4 and the DNA sequence of the HIV portion of PB1 RF is listed in Table 4A.
  • the entire amino acid sequence and DNA sequence of PB1 RF are in Tables 4B and 4C, respectively.
  • HIV 28 Kd fusion protein denoted PB1 MN .
  • the amino acid sequence of the HIV portion of PBIMN is shown in Table 5 and the DNA sequence of the HIV portion of PB1 MN is shown in Table 5A.
  • the entire amino acid sequence and DNA sequence of PBIMN are shown in Tables 5B and 5C, respectively.
  • HIV 26 Kd fusion protein denoted PB1 SC .
  • the amino acid sequence of the HIV portion of PB1 SC is listed in Table 6 and the DNA sequence of the HIV portion of PBlsc is shown in Table 6A.
  • the entire amino acid sequence and DNA sequence of PB1 SC are shown in Tables 6B and 6C, respectively.
  • HIV 26 Kd fusion protein denoted PB1 WMJ2 .
  • the amino acid sequence of the HIV portion of PB1 WMJ2 is listed in Table 7 and the DNA sequence of the HIV portion of PB1- WMJ2 is shown in Table 7A.
  • the entire amino acid sequence and DNA sequence of PB1- WMJ2 are shown in Tables 7B and 7C, respectively.
  • the amino acid cysteine in parentheses is added for the purpose of crosslinking to carrier proteins. Also, where the peptides have cysteines at or near both ends, these cysteines can form a disulfide bond, thus giving the peptides a loop-like configuration. For any of these peptides which do not have cysteines at or near both ends, cysteines may be added if a loop-like configuration is desired. The loop configuration can be utilized to enhance the immunogenic properties of the peptides. Other amino acids in parentheses are immunologically silent spacers.
  • Pentide 135 (from isolate HIV-III B ):
  • Peptide 139 (from isolate HIV-RF):
  • Peptide 142 (from isolate HIV-MN):
  • Peptide 143 (from isolate HIV-SC):
  • Peptide 131 (from isolate HIV-IIIB):
  • Peptide 132 (from isolate HIV-IIIB):
  • Peptide 134 (from isolate HIV-IIIB):
  • RP74 from isolates HIV-IIIB, HIV-RF, HIV-MN, HIV-SC:
  • RP80 from isolates HIV-IIIB, HIV-RF:
  • RP81 (from isolates HIV-IIIB, HIV-RF, HIV-WMJ1, HIV-MN):
  • RP88 from isolates HIV-MN, HIV-SC:
  • RP140 from isolates HIV-IIIB, HIV-RF:
  • Peptide 64 (from isolates HIV-IIIB, HIV-RF, HIV-MN, HIV-SC):
  • Peptide 338 (from isolates HIV-IIIB, HIV-RF):
  • proteins and peptides exemplifying the subject invention can be made by well-known synthesis procedures. Alternatively, these entities can be made by use of recombinant DNA procedures. Such recombinant DNA procedures are disclosed herein since they were, in fact, the procedures initially utilized to obtain the novel proteins and peptides of the invention. However, once these entities were prepared and their molecules sequenced, it is apparent to a person skilled in the art that the preferred method for making them would now be by chemical synthesis means. For example, there are available automated machines which can readily make proteins and peptides of the molecular sizes disclosed herein.
  • an expression vector plasmid denoted pREV2.2 was used. This plasmid was initially constructed from a plasmid denoted pBG1.
  • Plasmid pBGl is deposited in the E. coli host MS371 with the Northern Regional Research Laboratory (NRRL, U.S. Department of Agriculture, Peoria, Illinois, USA). It is in the permanent collection of this repository. E. coli MS371(pBG1), NRR1 B-15904, was deposited on November 1, 1984. E. coli MS371, NRRL B-15129 is now available to the public.
  • Plasmid pREV2.2 was deposited in the E. coli JM103 host with NRRL on July 30, 1986.
  • E. coli JM103(pREV2.2) received the accession number NRRL B-18091.
  • NRRL B-15904 and NRRL B-18091 will be available, without restrictions, to the public upon the grant of a patent which discloses them.
  • E. coli strains disclosed herein, were deposited as follows:
  • This latter deposit can be subjected to standard techniques to separate the plasmid from the host cell, and, thus, use the host E. coli CAG629 as disclosed herein.
  • the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures.
  • the depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
  • novel HIV proteins and peptides of the subject invention can be expressed in Saccharomvces cerevisiae using plasmids containing the inducible galactose promoter from this organism (Broach, J.R., Y. Li, L.C. Wu, and M. Jayaram [1983] in Experimental Manipulation of Gene Expression, p. 83, ed. M. Inouye, Academic Press). These plasmids are called YEp51 and YEp52 (Broach, J.R. et al [1983]) and contain the E.
  • Yeast promoters such as galactose and alcohol dehydrogenase (Bennetzen, J.L and B.D. Hall [1982] J. Biol. Chem. 257:3018; Ammerer, G. [1983] in Methods in Enzymology Vol. 101, p. 192), phosphoglycerate kinase (Derynck, R., R.A. Hitzeman, P.W. Gray, D.V. Goeddel [1983] in
  • triose phosphate isomerase (Alber, T. and G. Kawasaki [1982] J. Molec and Applied Genet. 1:419), or enolase (Innes, M.A. et al. [1985] Science 226:21) can be used.
  • genes disclosed herein can be expressed in simian cells. When the genes encoding these proteins are cloned into one of the plasmids as described in Okayama and Berg (Okayama, H. and
  • mammalian cell gene expression/protein production systems can be used. Examples of other such systems are the vaccinia virus expression system (Moss, B. [1985] Immunology Today 6:243; Chakrabarti, S., K. BrechUng, B. Moss [1985] Molec. and Cell. Biol. 5:3403) and the vectors derived from murine retroviruses (Mulligan, R.C [1983] in Experimental Manipulation of Gene Expression, p. 155, ed. M. Inouye, Academic Press).
  • HIV proteins and peptides of the subject invention can be chemically synthesized by solid phase peptide synthetic techniques such as BOC and FMOC (Merrifield, R.B. [1963] J. Amer. Chem. Soc 85:2149; Chang, C. and J. Meienhofer [1978] Int. J. Peptide Protein Res. 11:246).
  • solid phase peptide synthetic techniques such as BOC and FMOC (Merrifield, R.B. [1963] J. Amer. Chem. Soc 85:2149; Chang, C. and J. Meienhofer [1978] Int. J. Peptide Protein Res. 11:246).
  • the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA. Because of the redundancy of the genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid. Thus, the genetic code can be depicted as follows: Phenylalanine (Phe) TTK Histidine (His) CAK
  • Threonine (Thr) ACL Tryptophan (Trp) TGG
  • Each 3-letter deoxynucleotide triplet corresponds to a trinucleotide of mRNA, having a 5'-end on the left and a 3'-end on the right. All DNA sequences given herein are those of the strand whose sequence corresponds to the mRNA sequence, with thymine substituted for uracil. The letters stand for the purine or pyrimidine bases forming the deoxynucleotide sequence.
  • QR TC if S is A, G, C or T; alternatively
  • novel amino acid sequences of the HIV proteins and peptides of the subject invention can be' prepared by nucleotide sequences other than those disclosed herein.
  • Functionally equivalent nucleotide sequences encoding the novel amino acid sequences of these HIV proteins and peptides, or fragments thereof having HIV antigenic or immunogenic or therapeutic activity can be prepared by known synthetic procedures. Accordingly, the subject invention includes such functionally equivalent nucleotide sequences.
  • scope of the subject invention includes not only the specific nucleotide sequences depicted herein, but also all equivalent nucleotide sequences coding for molecules with substantially the same HIV antigenic or immunogenic or therapeutic activity.
  • scope of the subject invention is intended to cover not only the specific amino acid sequences disclosed, but also similar sequences coding for proteins or protein fragments having comparable ability to induce the formation of and/or bind to specific HIV antibodies possessing the properties of virus neutra ⁇ zation.
  • nucleotide sequences encoding HIV antigenic or immunogenic or therapeutic activity of the subject invention to produce HIV proteins via microbial processes. Fusing the sequences into an expression vector and transforming or transfecting into hosts, either eukaryotic (yeast or mammalian cells) or prokaryotic (bacterial cells), are standard procedures used in producing other well-known proteins, e.g., insulin, interferons, human growth hormone, IL-1, IL-2, and the like. Similar procedures, or obvious modifications thereof, can be employed to prepare HIV proteins or peptides by microbial means or tissue-culture technology in accord with the subject invention.
  • eukaryotic eukaryotic
  • prokaryotic bacterial cells
  • nucleotide sequences disclosed herein can be prepared by a "gene machine” by procedures well known in the art. This is possible because of the disclosure of the nucleotide sequence.
  • restriction enzymes disclosed can be purchased from Bethesda Research Laboratories, Gaithersburg, MD, or New England Biolabs, Beverly, MA The enzymes are used according to the instructions provided by the supplier.
  • Immunochemical assays employing the HIV proteins or peptides of the invention can take a variety of forms.
  • One preferred type is a liquid phase assay wherein the HIV antigen and the sample to be tested are mixed and allowed to form immune complexes in solution which are then detected by a variety of methods.
  • Another preferred type is a solid phase immunometric assay.
  • solid phase assays an HIV protein or peptide is immobilized on a solid phase to form an antigenimmunoadsorbent. The immunoadsorbent is incubated with the sample to be tested. After an appropriate incubation period, the immunoadsorbent is separated from the sample, and labeled anti-
  • human IgG antibody is used to detect human anti-HIV antibody bound to the immunoadsorbent.
  • the amount of label associated with the immunoadsorbent can be compared to positive and negative controls to assess the presence or absence of anti-HIV antibody.
  • the immunoadsorbent can be prepared by adsorbing or coupling a purified HIV protein or peptide to a solid phase.
  • Various solid phases can be used, such as beads formed of glass, polystyrene, polypropylene, dextran or other material.
  • Other suitable solid phases include tubes or plates formed from or coated with these materials.
  • the HIV proteins or peptides can be either covalently or non-covalently bound to the solid phase by techniques such as covalent bonding via an amide or ester linkage or adsorption. Afrer the HIV protein or peptide is affixed to the solid phase, the solid phase can be post-coated with an animal protein, e.g., 3% fish gelatin. This provides a blocking protein which reduces nonspecific adsorption of protein to the immunoadsorbent surface.
  • the immunoadsorbent is then incubated with the sample to be tested for anti-HIV antibody.
  • blood plasma or serum is used.
  • the plasma or serum is diluted with normal animal plasma or serum.
  • the diluent plasma or serum is derived from the same animal species that is the source of the anti-(human IgG) antibody.
  • the preferred anti-(human IgG) antibody is goat anti-(human IgG) antibody.
  • the diluent would be goat serum or plasma.
  • incubation e.g., pH and temperature
  • duration of incubation are not crucial. These parameters can be optimized by routine experimentation. Generally, the incubation will be run for 1-2 hr at about 45°C in a buffer of pH 7-8.
  • the immunoadsorbent and the sample are separated. Separation can be accomplished by any conventional separation technique such as sedimentation or centrifugation.
  • the immunoadsorbent then may be washed free of sample to eliminate any interfering substance.
  • the immunoadsorbent is incubated with the labeled anti-(human IgG) antibody (tracer) to detect human antibody bound thereto.
  • the immunoadsorbent is incubated with a solution of the labeled anti-(human IgG) antibody which contains a small amount (about 1%) of the serum or plasma of the animal species which serves as the source of the anti-(human IgG) antibody.
  • Anti- (human IgG) antibody can be obtained from any animal source. However, goat anti-(human IgG) antibody is preferred.
  • the anti-(human IgG) antibody can be an antibody against the Fc fragment of human IgG, for example, goat anti-(human IgG) Fc antibody.
  • the anti-(human IgG) antibody or anti-(human IgG) Fc can be labeled with a radioactive material such as 125 I; labeled with an optical label, such as a fluorescent material; or labeled with an enzyme such as horseradish peroxidase.
  • the anti-human antibody can also be biotinylated and labeled avidin used to detect its binding to the immunoadsorbent.
  • the immunoadsorbent After incubation with the labeled antibody, the immunoadsorbent is separated from the solution and the label associated with the immunoadsorbent is evaluated. Depending upon the choice of label, the evaluation can be done in a variety of ways.
  • the label may be detected by a gamma counter if the label is a radioactive gamma emitter, or by a fluorimeter, if the label is a fluorescent material. In the case of an enzyme, label detection may be done colorimetrically employing a substrate for the enzyme.
  • the amount of label associated with the immunoadsorbent is compared with positive and negative controls in order to determine the presence of anti-HIV antibody.
  • the controls are generally run concomitantly with the sample to be tested.
  • a positive control is a serum containing antibody against HIV;
  • a negative control is a serum from healthy individuals which does not contain antibody against HIV.
  • kits for screening blood can include:
  • an immunoadsorbent e.g., a polystyrene bead coated with an HIV protein or peptide
  • a diluent for the serum or plasma sample e.g. normal goat serum or plasma
  • an anti-(human IgG) antibody e.g., goat anti-(human IgG) antibody in buffered, aqueous solution containing about 1% goat serum or plasma;
  • a positive control e.g., serum containing antibody against at least one of the novel HIV proteins or peptides
  • a negative control e.g., pooled sera from healthy individuals which does not contain antibody against at least one of the novel HIV proteins or peptides.
  • an additional element of the kit can be the substrate for the enzyme.
  • Another type of assay for anti-HIV antibody is an antigen sandwich assay.
  • a labeled HIV protein or peptide is used in place of anti-(human IgG) antibody to detect anti-HIV antibody bound to the immunoadsorbent.
  • the assay is based in principle on the bivalency of antibody molecules. One binding site of the antibody binds the antigen affixed to the solid phase; the second is available for binding the labeled antigen.
  • the assay procedure is essentially the same as described for the immunometric assay except that after incubation with the sample, the immunoadsorbent is incubated with a solution of labeled HIV protein or peptide. HIV proteins or peptides can be labeled with radioisotope, an enzyme, etc. for this type of assay.
  • the bacterial protein, protein A which binds the Fc segment of an IgG molecule without interfering with the antigen-antibody interaction can be used as the labeled tracer to detect anti-HIV antibody adsorbed to the immunoadsorbent.
  • Protein A can be readily labeled with a radioisotope, enzyme, or other detectable species.
  • Immunochemical assays employing an HIV protein or peptide have several advantages over those employing a whole (or disrupted) virus. Assays based upon an HIV protein or peptide will alleviate the concern over growing large quantities of infectious virus and the inherent variability associated with cell culturing and virus production. Further, the assay will help mitigate the real or perceived fear of contracting AIDS by technicians in hospitals, clinics and blood banks who perform the test.
  • Immunochemical assays employing recombinant envelope proteins from multiple viral variants have additional advantages over proteins from a single HIV variant. Assays incorporating protein sequences from multiple variants are more likely to accurately survey antibodies in the human population infected with diverse HIV variants. Also, solid phase enzyme-Hnked immunosorbent assay (ELISA) utilizing different HIV variant proteins would allow determination of prevalent serotypes in different geographic locations. This determination has not been possible until now as no available antibody detection kit utilizes more than one HIV variant
  • variant-specific antisera Another use of recombinant proteins from HIV variants is to elicit variant-specific antisera in test animals. This antiserum would provide a reagent to identify which viral variant infected an individual.
  • "virus typing” can only be done by viral gene cloning and sequencing. Binding of variant-specific serum to a patient viral isolate would provide a means of rapid detection not currently available. For example, sera raised to the proteins denoted PB1 llIB , PB1 RF , PB1 MN , PB1 SC , and PB1 WMJ2 can be used to screen viral isolates from patients to determine which HIV variant the clinical isolate most closely resembles. This "screening" can be done by a variety of known antibody- antigen binding techniques.
  • Vaccines comprising one or more of the HIV proteins or peptides, disclosed herein, and variants thereof having antigenic properties, can be prepared by procedures well known in the art.
  • such vaccines can 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.
  • the preparation also can be emulsified.
  • the active antigenic ingredient or ingredients can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants such as aluminum hydroxide or muramyl dipeptide or variations thereof. In the case of peptides, coupling to larger molecules such as KLH sometimes enhances iramunogenicity.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants such as aluminum hydroxide or muramyl dipeptide or variations thereof.
  • peptides In the case of peptides, coupling to larger molecules such as KLH sometimes enhances iramunogenicity.
  • 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 cases, oral formulations.
  • traditional binders and carriers include, for example, polyalkalene glycols or triglycerides.
  • Suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1 to about 2%.
  • Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain from about 10% to about 95% of active ingredient, preferably from about 25% to about 70%.
  • the compounds can be formulated into the 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 carboxy groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the Hke.
  • a vaccine composition may include peptides containing T helper cell epitopes in combination with protein fragments containing the principal neutralizin domain.
  • T helper cell epitopes Several of these epitopes have been mapped within the HIV envelope, and these regions have been shown to stimulate proUferation and lymphokine release from lymphocytes. Providing both of these epitopes in a vaccine may result in the stimulation of both the humoral and the cellular immune responses.
  • a vaccine composition may include a compound which functions to increase the general immune response.
  • a compound which functions to increase the general immune response is interleukin-2 (IL-2) which has been reported to enhance immunogenicity by general immune stimulation (Nunberg et al. [1988] In New Chemical and
  • IL- 2 may be coupled with an HIV peptide or protein comprising the PND to enhance the efficacy of vaccination.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, 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 foUowed in one or two week intervals by a subsequent injection or other administration.
  • HIV is known to undergo amino acid sequence variation, particularly in the envelope gene (Starcich, B.R. [1986] Cell 45:637-648; Hahn, B.H. et al. [1986] Science 232:1548-1553). Over 100 variants have been analyzed by molecular cloning and restriction enzyme recognition analysis, and several of these have been analyzed by nucleotide sequencing. Some of these are the HIV isolates known as RF (Popovic, M. et al. [1984] Science 224:497-500), WMJ-1 (Hahn, B.H. et al. [1986] Science 232:1548-1553), LAV (Wain-Hobson, S. et al.
  • HIV peptides from different viral isolates can be used in vaccine preparations to protect against infections by different HIV isolates.
  • a vaccine preparation can be made using more than one envelope protein fragment corresponding to the principal neutralizing domain of more than one HIV isolate to provide immunity and thus give better protection against AIDS.
  • the vaccine preparation can be made using a single protein fragment that is comprised of a tandem arrangement of principal neutralizing epitopes from more than one HIV isolate. By identifying the principal neutralizing domain of HIV, this polypeptide region can be applied to formulate valuable vaccine, diagnostic, and therapeutic reagents.
  • Antibodies to recombinant peptides disclosed herein are useful as therapeutic and prophylactic reagents.
  • the generation of polyclonal or monoclonal antibodies capable of neutralizing a variety of HIV variants could be used to reduce the incidence of accidental infection and treat HIV infected people that are immuno-compromised. Additionally, immunization regimens may elicit polyclonal sera capable of broadly neutralizing several variants of HIV.
  • the ability to neutralize multiple HIV variants is termed broadly neutralizing antibody. Broadly neutralizing antibody may neutralize two or more HIV variants or all HIV variants. Therefore, a mixture of broadly neutralizing antibodies that neutralize different groups of HIV variants would be useful for diagnosis, prophylaxis, and therapy of AIDS.
  • Example 22 shows that immunization with five peptides elicits broadly neutralizing sera. Broadly neutralizing sera may also be generated if several sequences from the hypervariable region of diverse HIV variants are presented as a single synthetic peptide. Additionally, one may elicit this broadly neutralizing sera by reimmunization of animals primed with RP136 or equivalent proteins with peptides containing only the conserved amino acids within this hypervariable region. These immunization regimens would be useful for vaccination and for deriving antibodies useful as therapeutic agents.
  • Polyvalent immune globulin for use in passive immunization can be prepared by immunization of horses or by poofing immune human sera and fractionation of the IgG component from plasma or sera.
  • Human or mouse monoclonal antibody producing cell lines may be prepared by standard transformation and hybridoma technology (Methods in Enzvmoloev, Vol. 121, Sections I and II [1986] eds. J.J. Langone and H.V. Vunakis, Academic Press).
  • HIV monoclonal antibody can be prepared in accord with the procedures disclosed by Matsushita et al. (Matsushita et al. [1988] Journal of Virology
  • antibody is meant to encompass monoclonal or polyclonal antibodies, whole, intact antibodies or antibody fragments having the immunological activity of the whole antibody. Also encompassed within the term “antibody” are chimeric antibodies having the variable and constant regions from different host species, or those wherein only the CDRs are replaced.
  • compositions comprising antibodies may be administered to an individual or animal in need of treatment.
  • the HIV antigens described here may be administered in order to stimulate the recipient's own immune response.
  • a single antigen may be administered or, preferably, a broadly neutralizing anligen or mixture of antigens may be administered.
  • peptides made by organic synthesis can be advantageous for diagnostic, therapeutic, and prophylactic use by improving efficiency of immobilization, increasing protein stability, increasing immunogenicity, altering immunogenicity, reducing toxicity, or allowing multiple variations simultaneously.
  • peptides can be modified to increase or decrease net charge by modification of amino or carboxyl groups (carbamylation, trifluoroacetylation or succinylation of amino groups; acetylation of carboxyl groups).
  • Peptides can be made more stable by, for example, inclusion of D-amino acids or circularization of the peptide. Reductive state of peptides can be altered by, for example, sulfonation of cystinyl groups.
  • Peptides can also be modified covalently or non-covalently with non-proteinaceous materials such as lipids or carbohydrates to enhance immunogenicity or solubility.
  • Polyethylene glycol can be used to enhance solubility.
  • the subject invention includes all such chemical modifications of the proteins and peptides disclosed herein so long as the modified protein and/or peptide retains substantially all the antigenic/immunogenic properties of the parent compound.
  • Peptides can also be modified to contain antigenic properties of more than one viral variant. This has been done, for example, with Foot and Mouth Disease virus.
  • Foot and Mouth Disease virus is similar to HIV in that multiple variants exist and immunization with one variant does not lead to protection against other variants.
  • the real utility of peptides as immunogens is demonstrated by eliciting immunity to more than one variant by modification of the peptide to possess properties of both natural variants. When such a modified variant was used to immunize test animals, they were protected against both Foot and Mouth virus strains A10 and A12 (Brown, F. in Virus Vaccines, ed. G. Dreesman, J. Branson, R. Kennedy, pp.49- 54 [1985]).
  • HIV peptides or proteins containing a PND epitope can also be coupled with or incorporated into an unrelated virus particle, a replicating virus, or other microorganism in order to enhance immunogenicity.
  • the HIV epitope may be genetically or chemically attached to the viral particle or microorganism or an immunogenic portion or component thereof.
  • Antigenic epitopes have been attached to viral proteins or particles to enhance the immune response.
  • the VP6 capsid protein of rotavirus has been used as an immunologic carrier protein for an epitope of interest either in the monomeric form or as oligomers of VP6 in the form of particles (EP Publication No. 0 259 149).
  • Example 18 shows that a peptide containing amino acid sequences from two HIV variants can block virus neutralization activity of two virus specific neutralizing antisera. This suggests that a peptide or protein containing sequences of two or more HIV variants can elicit an immune response effective against two or more HIV variants.
  • Example 19 shows that co-immunization with envelope proteins from two HIV isolates elicits an immune response capable of neutralizing two HIV isolates. This suggests that co-immunization with proteins from two or more HIV variants can eHcit an immune response effective against two or more HIV variants.
  • the pREV2.2 plasmid expression vector was constructed from plasmid pBG1.
  • Plasmid pBG1 can be isolated from its E. coli host by weH known procedures, e.g., using cleared lysate-isopycnic density gradient procedures, and the Hke.
  • pREV2.2 expresses inserted genes behind the E. coli promoter. The differences between pBGl and pREV2.2 are the following:
  • pREV2.2 lacks a functional replication of plasmid (rop) protein.
  • pREV2.2 has the trpA transcription terminator inserted into the Aatll site. This sequence insures transcription termination of over-expressed genes.
  • pREV2.2 has genes to provide resistance to ampicillin and chloramphenicol, whereas pBGl provides resistance only to ampicillin.
  • pREV2.2 contains a sequence encoding sites for several restriction endonucleases.
  • TAGGCCATGGGCCCTCGAGCTTAA 5' This fragment has Bell and EcoRI sticky ends and contains recognition sequences for several restriction endonucleases.
  • This fragment has Aatll sticky ends and contains the trp A transcription termination sequence.
  • 3a 5 ⁇ g of pREVITT, prepared as disclosed above (by standard methods) was cleaved with Ndel and Xmnl and the approximately 850 base pair fragment was isolated.
  • 3b 5 ⁇ g of plasmid pBR325 (BRL, Gaithersburg, MD), which contains the genes conferring resistance to chloramphenicol as well as to ampicillin and tetracycline, was cleaved with Bell and the ends blunted with Klenow polymerase and dexoynucleotides. After inactivating the enzyme, the mixture was treated with Ndel and the approximately 3185 base pair fragment was isolated. This fragment contains the genes for chloramphenicol and ampicillin resistance and the origin of replication.
  • This fragment contains recognition sequences for several restriction enzyme sites.
  • Plasmid was purified from several clones and screened by digestion with Mlul or Clal.
  • Recombinant clones with the new multiple cloning site will give one fragment when digested with either of these enzymes, because each cleaves the plasmid once.
  • This plasmid is called pREV2.2.
  • Plasmid pREV2.1 was constructed using plasmid pREV2.2 and a synthetic oligonucleotide. The resulting plasmid was used to construct pPB1-Sub 1 and pPB1-Sub 2.
  • Plasmid pREV2.2 is cleaved with restriction enzymes Nrul and BamHI and the 4 Kb fragment is isolated from an agarose gel.
  • the fragments from 1 and 2 are Ugated in 20 ⁇ l using T4 DNA ligase, transformed into competent R coli cells and chloramphenicol resistant colonies are isolated.
  • Plasmid clones are identified that contain the oligonucleotide from 2. spanning the region from the Nrul site to the BamHI site and recreating these two restriction sites.
  • This plasmid is termed pREV2.1.
  • Plasmid pPB1-Sub 1 which contains approximately 165 base pairs (bp) of DNA encoding essentially the HIV env gene from the PvuII site to the Dral site, and from which is synthesized an approximately 12 Kd fusion protein containing this portion of the gpl20 envelope protein can be constructed as follows:
  • CeUs were grown in a 10-liter volume in a Chemap (Chemapec, Woodbuiy, NY) fermentor in 2% medium (2% yeast extract, bacto-tryptone, casamino acids pifco, Detroit, MI], 0.2% potassium monobasic, 0.2% potassium dibasic, and 0.2% sodium dibasic). Fermentation temperature was 30°C, the pH was 6.8, and air was provided at 1 wm. Plasmid selection was provided 20 ⁇ g/ml chloramphenicol.
  • Typical cell yield (wet weight) was 30 g/l.
  • the column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCl.
  • the HIV protein (12 Kd) eluted at approximately 0.2 M NaCl as assayed by SDS-polyacrylamide gel electrophoresis.
  • Plasmid pPB1-Sub 2 which contains approximately 320 bp of DNA encoding essentially the HIV env gene from the PvuII site to the Seal site, and from which is synthesized an approximately 18 Kd fusion protein containing this portion of the gpl20 envelope protein, can be constructed as follows:
  • This material was lysed using a BEAD-BEATERTM containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 6 M guanidine-hydrochloride, 20 mM Tris-Cl pH 8.0, 5 mM DTT, 15 mM ⁇ -mercaptoethanol, 5 mM PMSF, and 1 mM KEDTA The pellet was solubiUzed using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.
  • the supernate (90 ml) was dialyzed against 4 liters of 8 M urea, 20 mM sodium formate, pH 4.0, 1 mM EDTA and 15 mM /3-mercaptoethanol. Dialysis was done each time for 8 hr or longer with three changes of buffer. Spectraphor dialysis tubing (S/P, McGraw Park, IL) with a 3.5 Kd MW cut-off was used.
  • CM Chromatography The dialysate was loaded onto a 100 ml column (2.5 cm x 20 cm) packed with CM FAST FLOW SEPHAROSETM (Pharmacia) equilibrated in 8 M urea, 20 mM sodium formate pH 4.0, 15 mM /3-mercaptoethanol, and 1 mM Na
  • the column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCl.
  • the HIV protein (18 Kd) eluted at approximately 0.2 M NaCl as assayed by SDS- polyacrylamide gel electrophoresis.
  • Synthesis of peptides can be done by a variety of estabUshed procedures, for example, automated peptide synthesis.
  • Peptides were assembled by soUd-phase synthesis on cross-linked polystyrene beads starting from the carboxyl terminus and adding amino acids in a step-wise fashion (Merrifield, R.B. [1963] S. Am. Chem. Soc. 85:2149). Each synthesis was performed on an automated peptide synthesizer (Applied Biosystems 430-A) using standard t-Boc chemistry. Amino acids were coupled as highly reactive symmetric anhydrides formed immediately prior to use. To minimize coupling difficulties, dimethylformamide was used as the coupling buffer.
  • the crude peptides were purified by reverse-phase chromatography on a 1.0 cm x 25 cm Vidac semi-preparative C 18 column.
  • the buffers employed were: (A) 0.1% trifluoroacetic acid in H 2 O, and (B) 0.1% trifluoroacetic acid in 80% acetonitrile/20% H 2 O. Gradient elution was utilized to elute the bound peptide and collected fractions were further analyzed to identify pure product. Peptide identity was confirmed by amino acid analysis following 6 N HC1 hydrolysis. The synthesis included the addition of terminal amino acids not homologous to HIV for purposes of labeling, cross-linking, or structure of the peptide. These non-HIV amino acids are indicated in parenthesis.
  • the product of synthesis can be further purified by a number of established separatory techniques, for example, ion exchange chromatography.
  • Plasmid pPB1Rp which contains approximately 565 bp of DNA encoding essentially the HIV RF env gene from the PvuII site to th Beg1ll site, and from which is synthesized an approximately 27 Kd fusion protein containing this portion of the gpl20 envelope protein can be constructed as follows:
  • Cell lysis 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-Cl pH 8.0, 5 mM KEDTA, 5 mM DDT, 15 mM /3-mercaptoethanol, 0.5% TRITONTMX- 100, and 5 mM PMSF. 300 mg lysozyme was added and the suspension incubated for 30 min at room temperature,
  • This material was lysed using a BEAD-BEATERTM containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 8 M urea, 20 mM Tris-Cl pH 8.0, 5 mM DTT, 15 mM ⁇ - mercaptoethanol, 5 mM PMSF, and 1 mM KEDTA The pellet was solubUized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.
  • the supernate (90 ml) was dialysed against 4 liters of 8 M urea, 20 mM HEPES, pH 6.5, 1 mM EDTA and 15 mM ⁇ -mercaptoethanol. Dialysis was done each time for 8 hr or longer with three changes of buffer. Spectrophor dialysis tubing with a 3.5 Kd MW cut-off was used.
  • CM Chromatography The dialysate was loaded onto a 100 ml column (2.5 cm x 20 cm) packed with CM FAST FLOW SEPHAROSETM equilibrated in 8 M urea, 10 mM HEPES pH 6.5, 15 mM ⁇ -mercaptoethanol, and 1 mM Na EDTA at room temperature. The column was washed with 200 ml equiUbrium buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCl. The HIV protein (26 Kd) eluted at approximately 0.2 M NaCl as assayed by SDS-polyacrylamide gel electrophoresis.
  • Plasmid pPB1 MN which contains approximately 600 bp of DNA encoding essentially the HIV MN env gene from the Bg1ll site to the Bg1ll slitle, and from which is synthesized an approximately 28 Kd fusion protein containing this portion of the gpl20 envelope protein, can be constructed as follows:
  • Cell lysis 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-Cl pH 8.0, 5 mM KEDTA, 5 mM DTT, 15 mM ⁇ -mercaptoethanol, 0.5% TRITONTMX- 100, and 5 mM PMSF. 300 mg lysozyme was added and the suspension incubated for 30 min at room temperature.
  • This material was lysed using a BEAD-BEATERTM containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 8 M urea, 20 mM Tris-Cl pH 8.0, 5 mM DTT, 15 mM ⁇ - mercaptoethanol, 5 mM PMSF, and 1 mM KEDTA The peUet was solubilized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.
  • the supernate (90 ml) was dialysed against 4 liters of 8 M urea, 20 mM
  • HEPES pH 6.5, 1 mM EDTA, and 15 mM /3-mercaptoethanol. Dialysis was done each time for 8 hr or longer with three changes of buffer. Spectraphor dialysis tubing with a 3.5 Kd MW cut-off was used.
  • CM Chromatography The dialysate was loaded onto a 100 ml column (2.5 cm x 20 cm) packed with CM FAST FLOW SEPHAROSETM equilibrated in 8 M urea, 10 mM HEPES pH 6.5, 15 mM ⁇ -mercaptoethanol, and 1 mM KEDTA at room temperature. The column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 Uter linear gradient from 0-0.4 M NaCl. The HIV protein (28 Kd) eluted at approximately 0.2 M NaCl as assayed by SDS-polyacrylamide gel electrophoresis.
  • Plasmid pPB1 SC which contains approximately 570 bp of DNA encoding essentially the HIV SC env gene from the PvuII site to the Bg1ll site, and from which is synthesized an approximately 26 Kd fusion protein containing this portion of the gp120 envelope protein, can be constructed as follows:
  • Cell lysis 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-Cl pH 8.0, 5 mM KEDTA 5 mM DTT, 15 mM ⁇ -mercaptoethanol, 0.5% TRITONTMX- 100, and 5 mM PMSF. 300 mg lysozyme was added and the suspension incubated for
  • This material was lysed using a BEAD-BEATERTM containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 8 M urea, 20 mM Tris-Cl pH 8.0, 5 mM DTT, 15 mM ⁇ - mercaptoethanol, 5 mM PMSF, and 1 mM KEDTA The pellet was solubilized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.
  • the supernate (90 ml) was dialysed against 4 liters of 8 M urea, 20 mM HEPES, pH 6.5, 1 mM EDTA and 15 mM /3-mercaptoethanol and 1 mM KEDTA at room temperature.
  • the dialysate was loaded onto a 100 ml column (2.5 cm x20 cm) packed with CM FAST FLOW SEPHAROSETM equilibrated in 8 M urea, 10 mM HEPES pH 6.5, 15 mM ⁇ -mercaptoethanol, and 1 mM KEDTA at room temperature.
  • the column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCl.
  • Plasmid pPB1 WMJ 2 which contains approximately 560 bp of DNA encoding essentially the HIV WMJ2 env gene and from which is synthesized an approximately 26 Kd fusion protein containing this portion of the gpl20 envelope protein, can be constructed as follows: 1. Synthesizing DNA fragment in Table 7A
  • Cell lysis 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-Cl pH 8.0, 5 mM KEDTA 5 mM DTT, 15 mM ⁇ -mercaptoethanol, 0.5% TRITONTMX- 100, and 5 mM PMSF. 300 mg lysozyme was added and the suspension incubated for 30 min at room temperature.
  • This material was lysed using a BEAD-BEATERTM containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 8 M urea, 20 mM Tris-Cl pH 8.0, 5 mM DTT, 15 mM ⁇ - mercaptoethanol, 5 mM PMSF, and 1 mM KEDTA The pellet was solubilized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.
  • the supernate (90 ml) was dialysed against 4 Uters of 8 M urea, 20 mM HEPES, pH 6.5, 1 mM EDTA and 15 mM /3-mercaptoethanol. Dialysis was done each time for 8 hr or longer with three changes of buffer. Spectraphor dialysis tubing with a 3.5 Kd MW cut-off was used.
  • CM Chromatography The dialysate was loaded onto a 100 ml column (2.5 cm x 20 cm) packed with CM FAST FLOW SEPHAROSETM equilibrated in 8 M urea, 10 mM HEPES pH 6.5, 15 mM ⁇ -mercaptoethanol, and 1 mM Na EDTA at room temperature. The column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCl. The HIV protein (26 Kd) eluted at approximately 0.2 M NaCl as assayed by SDS-polyacrylamide gel electrophoresis.
  • proteins or peptides contain the epitope recognized by antibodies that are responsible for cell fusion inhibition. For example, fusion inhibition of HIV lllB infected cells by antiserum to the PB1-III B protein of the parent application is abated by addition of PB1-III B protein to 5 ⁇ gfml. Using antiserum to PB1-III B and adding any one of the proteins or peptides, for example, Sub 2, Sub 1, CNBrl, peptide 135 or peptide
  • peptide 139 A peptide containing only the central portion of the peptide 139, e.g., peptide 339, also can block the fusion inhibition activity of antiserum to PB1-RF. This, for the first time, localizes the critical amino acids necessary to elicit neutralization or block fusion inhibiting antibody to a ten amino acid sequence (e.g., peptide 339).
  • Antisera from an animal immunized with two PB1 proteins from HIV IIIB and HIV RF isolates were capable of blocking cell fusion of both HIV lllB - and HIV RF -infected cells. This demonstrates that co-immunization with separate proteins containing envelope sequences of two HIV isolates elicits an immune response capable of neutralizing both isolates. This novel property of small proteins or peptides blocking immune serum has not been described before.
  • proteins and peptides of the subject invention contain the entire epitope for raising humoral immune responses that neutralize HIV infection and block HIV infected cell fusion. This is shown by these proteins and peptides competing these activities out of serum from animals immunized with the entire HIV envelope. More specifically, proteins and peptides that can compete the activities from anti-gp160 or anti-PB1 sera are Sub 2, Sub 1, CNBrl, peptide 135, and peptide 136.
  • proteins and peptides of the invention also can be used to stimulate a lymphocyte proliferative response in HIV infected humans. This then would stimulate the immune system to respond to HIV in such individuals.
  • Plasmid pPB1 which contains approximately 540 bp of DNA encoding essentially the HIV env gene from the PvuII site to the Bg1ll site, and from which is synthesized an approximately 26 Kd fusion protein containing this portion of the gpl20 envelope protein, can be constructed as follows:
  • This DNA fragment can be synthesized by standard methods and encodes a portion of gpl20. It has a blunt end on the 5' end and an end which will ligate with a BamHI overhang on the 3' end.
  • This material was lysed using a BEAD-BEATERTM (Biospec. Products, Bartlesville, OK) containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 6 M guanidine-hydrochloride, 20 mM Tris- Cl pH 8.0, 5 mM DTT, 15 mM ⁇ -mercaptoethanol, 5 mM PMSF, and 1 mM KEDTA. The pellet was solubilized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.
  • the supernate (90 ml) was dialysed against 4 liters of 8 M urea, 20 mM Tris- Cl, pH 8.0, 1 mM EDTA and 15 mM ⁇ -mercaptoethanol. Dialysis was done each time for 8 hr or longer with three changes of buffer. Spectraphor dialysis tubing (S/P, McGraw Park, IL) with a 3.5 Kd MW cut-off was used.
  • CM Chromatography The dialysate was loaded onto a 550 ml column (5 cm x 28 cm) packed with CM FAST FLOW SEPHAROSETM equilibrated in 8 M urea, 10 mM potassium phosphate pH 7.0, 15 mM /3-mercaptoethanol, and 1 mM KEDTA at room temperature. The column was washed with 2 liters equilibration buffer, and the protein eluted with a 5.0 liter linear gradient from 0-0.4 M NaCl. The HIV protein (26 Kd) eluted at approximately 0.2 M NaCl as assayed by SDS-polyacrylamide gel electrophoresis.
  • Example 21 Immunization with two or more peptides to obtain broadly neutralizing antisera
  • Five peptides i.e., peptide 135, peptide 139, peptide 141, peptide 142 and peptide 143, were cross-linked individually to carrier proteins and used to immunize goats. Each peptide is capable of eliciting type specific neutralization when used individually as an immunogen.
  • Synthetic peptides were cross-Unked through a sulfhydiyl bond to keyhole limpet hemocyanin (KLH) by using Succinimidyl- 4-(n-Maleimidomethyl)Cyclohexane 1-Carboxylate (Pierce).
  • the ratio of peptide to KLH was 1:2 by weight. 200 ⁇ g of each cross-linked peptide was used in the immunization cocktail (a total of 1 mg of 5 peptides, 2 mg of KLH). This method of crosslinking or immunization regimen is but an example and not meant to be limiting. After four immunizations, immune sera was tested for neutralization of these five HIV isolates as well as distinctly different isolates. The immune serum could block fusion of cells infected with any of five isolates from which the peptide sequences were derived. In addition, the serum neutralized other variants not used in the immunization.
  • Equivalent broad neutralizing sera may also be obtained by variations of this immunogen. For example, using more than five peptides having the amino acid sequence derived from the principal neutralizing domain from more than five variants. Alternatively, a single peptide (e.g., peptide 64 or peptide 74) containing segments homologous to diverse HIV variants may also be used to elicit broad neutralizing antibody.
  • a single peptide e.g., peptide 64 or peptide 74
  • segments homologous to diverse HIV variants may also be used to elicit broad neutralizing antibody.
  • An immunization protocol capable of eliciting broad neutralizing antibodies may take the form of initial immunization with a peptide or protein antigenically equivalent to the principal neutralizing domain, or segments thereof.
  • the initial immunization is followed with a second immunization.
  • the initial immunization could be done with, for example, peptide 135, peptide 139, peptide 141, peptide 142, or peptide 143, with subsequent immunization with, for example, one or more of the following peptides:
  • the method is to immunize with a protein or peptide and then boost the immune response to a defined subset of the original immunogen.
  • This immunization method may be useful in vaccine methodology and also to generate broad neutralizing polyclonal or monoclonal antibodies for therapeutic applications.
  • Certain segments of the principal neutralizing domain have been found to be capable of eliciting the antigenic and immunogenic responses which are associated with the principal neutralizing domain as a whole.
  • a region of the principal neutralizing domain known as the "tip of the loop” has been shown to be capable of raising, and/or binding with, neutralizing antibodies. This capabUity is observed for the "tip of the loop" of a variety of HIV variants.
  • the tip of the loop comprises a three amino acid segment which is highly conserved between HIV variants, together with various amino acids which occur on either side of the three conserved amino acids.
  • the three conserved amino acids which are Gly Pro Gly, usually occur at, or about, positions 311, 312, and 313 of the HIV envelope protein.
  • the "tip of the loop” comprises the Gly Pro Gly segment together with the 2 to 8 amino acids which flank either or both sides of this segment in any given HIV variant.
  • the amino acids which flank the conserved segment may be any of the 20 natural amino acids, in any sequential order.
  • PNDs principal neutralizing domains
  • the present research shows that HIV viruses such as IIIB and LAV-BRU having the Gln-Arg (Q-R) dipeptide to the left of the Gly-Pro-Gly sequence are relatively uncommon.
  • the MN- like sequence in this region (...I H I G P G9) is the most common.
  • the present research shows that prindpal neutralizing domains of other commonly studied variants comprise relatively uncommon sequences.
  • compositions which can predictably elicit and/or bind with neutralizing antibodies to a broad range of HIV variants.
  • the generalized formula for such a composition can be as follows:
  • x is 0 to 13 amino acids in length
  • y is 0 to 17 amino acids in length
  • z is P, A S, Q, or L
  • either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV prindpal neutralizing domain, a peptide capable of stimulating T-cells, or a general immune stimulant
  • Table 12 is a compilation of the common sequence patterns that occur in the region at the tip of the loop. For example, approximately 60% of the HIV isolates contain the core sequence I a I G P G R (a represents several different residues), approximately 50% contain the sequence I G P G R A and approximately 40% contain G P G R A F. When a His residue is present at the a position in I a I G P G R, this sequence occurs in approximately 30% of the HIV isolates.
  • a vaccine composition comprising a mixture of peptides having the sequence I a I G P G R where all of the possible replacements for the a are present, is capable of eliciting antibodies which neutralizes a majority of HIV variants.
  • the peptides are linked to carrier proteins or adjuvants as described in Example 21.
  • a potential cocktail might contain peptides from each of the eight groups represented.
  • the peptide sequences may be presented as a hybrid polypeptide containing the principal neutralizing domain from two or more of these groups.
  • such a cocktail will contain peptides which will be capable of raising antibodies which neutralize at least 70% and most preferably at least 90% of HIV variants.
  • the antigens of the subject invention can be identified by their ability to raise antibodies which bind to certain amino acid sequences.
  • particularly advantageous antigenic compounds would raise antibodies which bind to common amino add sequences such as G-P-G-R- A-F, I-G-P-G-R-A-F, I-G-P-G-R-A I-a-I-G-P-G-R, I-a-I-G-P-G-R-A and I-a-I-G-P-G-R-A-F, where a is any of the 20 amino adds.
  • a polypeptide representing the occurrence of amino acids in all of the variants can be represented as follows: x 13 x 12 x 11 x 10 x 9 x 8 x 7 x 6 x 5 x 4 x 3 x 2 x 1 G z G y 2 y 3 y 4 y 5 y 6 y 7 y 8 y 9 y 10 y 11 y 12 y 13 y 14 y 15 y 16 y 17 , wherein x 1 is I, R, M, IQR, V, L, K, F, S, G, Y, SRG, or YQR;
  • x 2 is H, R, Y, T, S, P, F, N, A K, G, or V;
  • x 3 is I, L, M, T, V, E, G, F, or Y;
  • x 4 is R, S, G, H, A, K, or not present;
  • X 5 is K, R, I, N, Q, A TR, RQ, or not present;
  • x 6 is R, K, S, I, P, Q, E, G, or T;
  • x 7 is T, K, V, I, A R, P, or E;
  • x 8 is N, NV, Y, KI, I, T, DK, H, or K;
  • x 9 is N, S, K, E, Y, D, I, or Q;
  • x 10 is N, Y, S, D, G, or H;
  • x 12 is R, I, or K
  • x 13 is T, I, M or A;
  • z is P, A Q, S, or L;
  • y 1 is R, K, Q, G, S, or T;
  • y 2 is A V, N, R, K, T, S, F, P, or W;
  • y 3 is F, I, V, L, W, Y, G, S, or T;
  • y 4 is Y, V, H, L, F, S, I, T, M, R, VH, or FT;
  • y 5 is T, A V, Q, H, I, S, Y, or not present;
  • y 6 is T, R, I, Q, A M, or not present
  • y 7 is G, E, K, R, T, D, Q, A, H, N, P, or not present;
  • y 8 is R, Q, E, K, D, N, A G, S, I, or not present;
  • y 9 is I, V, R, N, G, or not present
  • y 10 is I, T, V, K, M, R, L, S, E, Q, A, or not present;
  • y 1 1 is G, R, E, K, H, or not present;
  • y 12 is D, N, I, R, T, S, or not present
  • y 13 is I, M, ME, L, or not present
  • y 14 is R, G, K, S, E, or not present
  • y 15 is Q, K, or R
  • y 17 is H, Y, R, or Q.
  • Monoclonal and/or polyclonal antibodies with broad neutralizing activity can be generated using the commonly occurring peptide sequences for use in prophylactic or therapeutic compositions.
  • the commonly occurring sequences described here can be used in much the same way as the other peptides described in this application.
  • these peptides can be modified in order to provide T-lymphocyte stimulation, general immune stimulation, to enhance immunogenicity or solubility, or to reduce toxidty.
  • the peptides may also be modified by addition of terminal cysteine residue(s) or by conjugation to a carrier protein, adjuvant, spacer, and/or Unker.
  • the peptides may be fused with other HIV epitopes to produce a multiepitope polypeptide which could be useful with an even greater number of HIV variants.
  • the peptides can be circularized by bonding between cysteine residues.
  • the cysteine residues used to make such drcularized peptides could be the naturally occurring cysteine residues at the ends of the prindpal neutralizing domain, or cysteine residues may be added to the terminal ends of the peptides.
  • vaccine compositions may include peptides containing T helper cell epitopes in combination with protein fragments containing the prindpal neutralizing domain.
  • T helper cell epitopes Several of these epitopes have been mapped within the HIV envelope, and these regions have been shown to stimulate proliferation and lymphokine release from lymphocytes. Providing both of these epitopes in a vaccine composition may result in the stimulation of both humoral and cellular immune responses.
  • Synthetic genes can be constructed which encode proteins comprised of the neutralizing epitopes from more than one HIV isolate.
  • the synthetic gene exemplified here comprises a tandem arrangement of DNA sequences encoding neutralizing epitopes from HIV isolates IIIB, RF, SC, MN, and WMJ1.
  • Each epitope-encoding domain within the gene was designed to encode the 11 amino acids centered at the common Gly-Pro-Gly sequence at the tip-of-the-loop for each of the isolates.
  • the multi-epitope gene contained 5 different coding regions, each of which encoded a neutralizing epitope from a different isolate.
  • the epitope which was chosen for each of the 5 isolates consisted of the Gly-Pro-Gly sequence along with the 4 amino acids on either side of the Gly-Pro-Gly sequence from each of the 5 isolates. Domains coding for othe neutralizing epitopes from these isolates could have been incorporated into the multi-epitope gene. Also, genes coding for neutralizing epitopes from other isolates can be used.
  • the genes were constructed such that the domains were linked by DNA sequences encoding four glycine residues.
  • the composition or length of the linking sequence can be varied but preferably it is a sequence that is non-immunogenic itself.
  • the DNA sequence of the synthetic gene described here was designed such that restriction sites were encoded at either end of the fragment to facilitate cloning into the vector or, alternatively, to permit the construction of longer multi-epitope genes by attachment of 2 or more shorter genes ( Figure 2).
  • a methionine residue was encoded at the 5' end of the gene to fadUtate cleavage when produced as part of a fusion protein.
  • Figure 3 depicts the steps in the construction of the multi-epitope gene described here.
  • the amino add sequence encoded by this gene is shown in Table 13.
  • the portions of this amino acid sequence which correspond to each of the 5 isolates are identified in Table 13.
  • Double-stranded subfragments of the full-length gene were first constructed starting with single-stranded synthetic oUgomers designed to encode tandem neutralizing epitopes and Unking amino add sequences. Any number of subfragments can be used. In this experiment the gene was divided into two portions, but three, four, or more portions can be used.
  • Four single-stranded oligomers of between 67 and 78 nucleotides in length were synthesized (HEO-1, HEO-2, HEO-3, and HEO-4) ( Figure 3).
  • the oligomers were designed in pairs (HEO-1 and 2; HEO-3 and 4) as opposite and adjacent strands of the double-stranded subfragments having 10 (HEO-1 + 2) or 11 (HEO-3 +4) bases of complementary overlap.
  • the oUgomers of each pair were mixed and heated 65°C for 5 minutes, then incubated at 37°C for 1 hour to anneal.
  • HEO- 1+2 comprised the coding sequences for 3 epitopes plus adjacent linker amino acids; HEO-3+4 extended from the fourth epitope to the end of the gene.
  • the samples were extracted with phenol/chloroform and precipitated with ethanol by standard procedures.
  • the resulting double-stranded DNAs were digested with Hindlll (HEO-1 +2) or Sacl (HEO-3+4) and purified on a 3% NuSieve agarose gel.
  • the purified fragments were Ugated with Hindlll + Sacl digested pUC19 (New England Biolabs) in a 3- component ligation and transformed into E. coli JM105 cells.
  • the resulting plasmid was designated pUC/MEP-1.
  • the MEP-1 insert was removed from pUC/MEP-1 and recloned into Hindlll + Sacl digested pRev2.1 for high-level expression of a fusion protein comprised of a leader portion from the E. coli BG gene fused to the multi-epitope protein.
  • the resulting plasmid designated pMEP-1-8342, was transformed into E. coli strain SG20251 and the 12.9 Kd multi-epitope fusion protein was identified by coomassie blue staining or Western blot analysis using a probe selected from antisera to the looptip peptides from each of the 5 HIV isolates.
  • the fusion protein can be used intact or, alternatively, the leader portion can be cleaved off by cyanogen bromide which cleaves on the carboxy-terminal side of methionine residues.
  • the amino add sequence of the fusion protein is shown in Table 13A
  • the multiepitope peptide can be purified from recombinant cells by methods described above.
  • Other synthetic genes can be constructed which encode tandem neutralizing epitopes from any number of different HIV isolates using the procedure described above.
  • variations on the above procedure can be made which are meant to be included in the present invention.
  • the lengths of the neutralizing epitopes encoded by a gene can vary, and there can be variation in the length of the individual epitopes within a single gene.
  • the number of neutralizing epitopes within a multi-epitope gene can vary, and the composition or the length of the amino add sequences of the epitopes or the linking sequences can be varied from the example that is described herein.

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Abstract

L'invention concerne l'identification d'une partie de la protéine d'enveloppe du VIH appelée domaine de neutralisation principal. Des polypeptides comprenant ce domaine ont la capacité de développer des anticorps neutralisants et/ou de se lier à ceux-ci. L'invention concerne également de nouveaux polypeptides du VIH utilisables dans le diagnostic, la prophylaxie ou la thérapie du SIDA. On peut préparer ces polypeptides selon des techniques synthétiques chimiques connues, ou au moyen d'ADN de recombinaison. Ces polypeptides appartiennent à la sous-unité gp 120, amino acides 298-320, comprenant la séquence ......gly-pro-gly...... et des variantes de celle-ci. L'invention concerne en outre des polypeptides multiépitopes comprenant des analogues de cet épitope peptidique provenant de différentes variantes du VIH.
EP89911309A 1988-10-03 1989-09-29 Proteines et peptides du vih utiles pour le diagnostic, la prophylaxie ou la therapie du sida Withdrawn EP0436634A1 (fr)

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