Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/085—Staphylococcus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/002—Protozoa antigens
  • A61K39/005—Trypanosoma antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/002—Protozoa antigens
  • A61K39/008—Leishmania antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/002—Protozoa antigens
  • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6018—Lipids, e.g. in lipopeptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/6068—Other bacterial proteins, e.g. OMP
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• This invention relates to compositions and processes for inducing an immune response against a pathogenic organism such as a causative agent of a sexually-transmitted or mucosally-transmitted disease involving the nasal or respiratory administration of an antigen such as an envelope protein, e.g. an HIV oligomeric gpl 60, along with proteosomes and/or bioadhesive nanoemulsions.
• a pathogenic organism such as a causative agent of a sexually-transmitted or mucosally-transmitted disease involving the nasal or respiratory administration of an antigen such as an envelope protein, e.g. an HIV oligomeric gpl 60, along with proteosomes and/or bioadhesive nanoemulsions.
• peptide subunit or recombinant protein vaccines to protect against pathogenic microorganisms or malignancies has been impeded by lack of sufficient immunogenicity in the peptide and protein preparations. Often the undesired side effects resulting from exposure to an immunogen must be weighed against the adequacy of its immunogenic properties.
• the enhancement of immunogenicity of small peptides, protein fragments and polypeptide proteins without increasing undesired side effects from exposure to these agents is an important area of investigation. There exists a paucity of non-toxic and non- pyrogenic carriers and adjuvants for human use. Furthermore, carriers that are safe for human use f equently cannot be efficiently complexed to peptides to render them immunogenic without altering their antigenic structure.
• Hopp (Molec. Immunol, Vol 21, pp 13-16 (1984)) disclosed the addition of dipalmityl-lysine to a peptide to enhance its immunogenicity The immunopotentiation reported by Hopp was exceedingly short-lived and induced peak titers that were only system disclosed herein
• European patent publication EPA 1 1,748 published August 20, 1986 (inco ⁇ orated herein by reference) refers to an E. coh expression vector having a sequence coding for all or a portion of the repeat unit of the CS protein and discloses a process for purifying the immunogenic polypeptide from the E. coli culture.
• European patent publication EPA 192,626, published August 27, 1986 (inco ⁇ orated herein by reference) refers to an immunogenic polypeptide capable of conferring immunity to P. falciparum infection in mammals which comprises four or more tandem repeat units of the CS protein.
• the repeat unit is a tetrapeptide system having the sequence Asn Ala Asn Pro (as above).
• W087/06939 (November 19, 1987) teaches a process for isolating and purifying the CS protein expressed in recombinant E. coli.
• Symbol et al U.S. Patent 4,707,357, discloses an anti-malarial immunogenic stimulant comprising an immunogenic carrier and a peptide sequence of between 2 and 100 consecutive repeats of a sequence Asn X Y Pro, wherein X is Ala or Val and Y is Asn or Asp.
• the carriers include soluble molecules such as proteins and polysaccharides and particles such as liposomes and bacterial cells or membranes.
• the peptide is attached to the carrier by an amide bond formed between a carboxylate or amino groups of a carrier and, respectively, amino or carboxylate group of the peptide.
• the bonding may be through either an ether or ester linkage.
• Other disclosed carriers include terminal diamines with 1-10 methylene carbons joining the amines. Preferred carriers were said to be tetanus toxoid and amphiphilic proteins having a lipophilic portion and a hydrophilic portion.
• WO86/05790 discloses immunogenic antigen- carrier protein conjugates for use as vaccines against malaria.
• the conjugates contain the peptide H 2 N-(Asn Ala Asn Pro) 3 COOH, also designated (NANP) 3 .
• NANP amino acid-(Asn Ala Asn Pro) 3 COOH
• This document also describes a preferred carrier such as tetanus toxoid.
• Other carriers include diphtheria toxoid and synthetic peptides and polymers comprising lysine and arginine groups.
• the peptide is coupled to the carrier with the coupling reagent glutaraldehyde or by adding a cysteine residue to the N-terminus and using another conventional ester as a coupling reagent.
• WO86/00911 published February 13, 1986 refers to the use of a peptide having the amino acid sequence (Pro Asn Ala Asn) n (where n>23) adsorbed or coupled to a conventional vaccine carrier protein.
• Alum absorbed vaccines containing various forms of the CS epitope have not been sufficiently immunogenic for general human use.
• Many protein carriers and liposomes known in the art require lipid A or other adjuvants not acceptable for human use.
• HIV-1 Human Immunodeficiency Virus
• AIDS acquired immunodeficiency syndrome
• env HIV envelope
• gpl60 precursor molecule which is subsequently processed into the external envelope protein gpl20 and the transmembrane protein gp41.
• the precursor/product relationship between gp 160 and its products,gpl20 and gp41, as well as the amino acid sequences of all three, are well documented (Allan, et al, Science 225:1091-1094 (1985); Veronese et al. Science, 229: 1402-1405 (1985).
• HIV gpl20 and gp41 are the primary targets for immune recognition in HIV-infected subjects. These proteins have therefore received much attention in virus neutralization studies and vaccine development. Large segments of recombinantly expressed gpl20 (rgpl20), or native gpl20 purified from HIV-infected cells, elicit mostly type-specific neutralizing antibodies in animals.
• rgpl20 recombinantly expressed gpl20
• native gpl20 purified from HIV-infected cells, elicit mostly type-specific neutralizing antibodies in animals.
• Retrovir. 4: 319-329 (1988) describe the establishment of a process capable of producing the HIV glycoprotein gpl ⁇ O in its native form.
• native gpl60 breaks down into gpl20 and gp41. Consequently, the envelope protein obtained from cell culture media or from lysed virions is gp!20 and gp41.
• the glycoprotein gpl60 had only been produced through recombinant means.
• Recombinant gpl60 is different than the native gpl ⁇ O, particularly in regard to glycosylation, and these differences appeared critical in the search for an HIV vaccine, given that the HTV-1 envelope glycoproteins determine viral tropism and harbor epitopes which are essential for the development of neutralizing antibodies against the virus.
• US 5,116,740 disclosed a useful clone of HUT78 cells (termed 6D5451) which, when chronically infected with the HIV strain known as "HTLV-III 45 i,” released functionally intact viral glycoprotein gpl ⁇ O in its native form into the culture medium.
• the affinity of gpl ⁇ O was five times lower than that of gpl20 for binding CD4. Both gpl20 and gpl60 inhibited syncytia formation by HIV- 1 -infected cells when mixed with CD4+ cells. Both gpl20 and gpl ⁇ O had strong mitogenic effects on T cells from HIV- 1 -infected gibbons but not on cells from uninfected gibbons. US 5,116,740 (supra) and VanCott T.C et al, J Immunol Meth.
• the present inventors have discovered a novel vaccine composition which combines a protein or peptide antigen, optionally added hydrophobic material and an immunopotentiating membranous carrier which together preserve the antigenic integrity of the protein or peptide epitopes while at the same time enhancing their immunogenicity.
• Administration of this composition to a subject provokes a protective immune response comprising secretory neutralizing antibodies present in various mucosal sites in the body.
• This vaccine and the process for using it is preferably intended for use against pathogenic organisms, in particular those causing sexually transmitted diseases or mucosally transmitted diseases, including diseases spread by non-sexual spread through body fluids or mucosal secretions, including fecal spread.
• Such organisms include bacteria and enveloped viruses, particularly HI V-l.
• the present inventions provides a vaccine composition capable of eliciting neutralizing antibodies in a subject to a pathogenic organism which antibodies are present in vaginal secretions, intestinal secretions, lung secretions or feces, which composition comprises: (a) an antigen comprising a protein or peptide having
• an endogenous hydrophobic sequence of between about 3 and about 50 non-polar or uncharged amino acids (ii) added to the protein or peptide, an exogenous hydrophobic material comprising a sequence of between about 3 and about 50 non-polar or uncharged amino acids or a C8-C18 fatty acyl group; or
• the endogenous hydrophobic sequence or the exogenous hydrophobic sequence is an amino acid sequence is preferably between about 5 and about 29 residues.
• Preferred short exogenous hydrophobic sequences are Phe-Leu-Leu-Ala-Val or Val-Ala-Leu-Leu-Phe.
• the exogenous hydrophobic material may also be C8-C18 fatty acyl group, preferably lauroyl.
• a preferred protein is a viral envelope protein, such as oligomeric gpl ⁇ O from HIV- 1.
• the gp 160 has the sequence of residues 33-681 of SEQ ID NO: l .
• the antigen may be a peptide or a peptide oligomer.
• the protein or peptide naturally contains or has added to it at least one cysteine residue.
• the protein or peptide is chemically synthesized or recombinantly produced.
• the antigenic protein is complexed with proteosomes which are preferably hydrophobic, multimolecular membrane proteins.
• the vaccine composition is preferably formed by: (a) bonding the hydrophobic material to the protein or peptide to form a hydrophobic-hydrophilic compound; and (b) admixing the compound with the proteosomes, bioadhesive nanoemulsions, or both such that the antigen is complexed with the proteosomes or nanoemulsion.
• the admixing step is may be performed in the presence of a detergent, and is followed by the step of removing the detergent by dialysis. Alternatively the admixing step is performed lyophilization.
• the vaccine composition preferably is formulated for intranasal or respiratory administration, and preferably in a dosage form suitable for multiple inoculations. Also provided are processes for inducing a neutralizing antibody response in a subject against a pathogenic organism resulting in neutralizing antibodies in one or more of vaginal secretions, intestinal secretions, lung secretions and feces, which process comprises administering to the subject an effective amount of any of the vaccine compositions as described above.
• compositions and processes are particularly useful for inducing protective, neutralizing immunity to a pathogenic organism such as a causative agent of a mucosally transmitted or sexually transmitted disease.
• Administration of the vaccine may be prior to or after exposure (or both prior to and after exposure) to the pathogenic organism.
• the components of the vaccine composition can by complexed by any means known in the art. Any synthetic or cloned peptide which is to serve as the antigen can have a hydrophobic foot and/or a Cys added. Therefore any peptide can be made immunogenic by this approach. Hence this method differs from chemical covalent coupling of the prior art which depends on the correct chemistry to attach and orient a peptide epitope appropriately.
• the proteosomes used in the vaccine composition are preferably hydrophobic multimolecular membrane proteins. They may be obtained from any of a number of different organisms; in one embodiment, they are derived from meningococci. Complexing of the proteosomes with the antigenic component may be accomplished by any of a number of means, preferably by dialysis or lyophilization as noted above.
• Figure 1 is a schematic drawing illustrating a schedule of immunization and sample collection. The following abbreviations are used: SE - serum; VG - vaginal wash; FE - fecal pellets; LG - lung lavage; IN - intestinal lavage; SPL - spleen.
• Figures 2 A, 2B and 2C are a series of graphs showing the effects of intranasal immunization with oligo gp-160 formulated with proteosomes and/or bioadhesive nanoemulsions. Neutralizing antibodies were found in serum and vaginal and lung fluids and are shown in relative units. These antibodies recognize natively folded gp-120. Antibodies from serum (Fig. 2 A), vaginal wash (Fig.
• Fig. 2C lung wash
• SPR solid phase radioimmunoassay
• Ras3C is a lipid A containing adjuvant from RLBI; rgpl20MN (recombinant gpl20 from virus strain MN) and rcmgpl20MN (reduced, carboxymethylated rgpl20MN) were from Genentech. Recombinant gpl ⁇ O from MicroGenesys Mrgp-160:is not natively folded and therefore has much poorer expression of the native epitopes compared to oligo gpl ⁇ O, was another negative control.
• Figures 3 A and 3B are graphs showing the induction of neutralizing antibodies in vaginal wash (VG) (Fig. 3A) and in lung wash (LG) (Fig. 3B) with various of the antigenic preparations indicated.
• the symbols are as follows: — D — , — ⁇ — and — O — : saline controls; — • — o-gpl60/proteosomes;
• FIG. 4 A, 4B and 4C are graphs showing the induction of HTV- neutralizing antibodies in mice after intranasal immunization with o-gpl60.
• HTV- I MN neutralizing activity of sera Fig. 4A
• vaginal wash Fig. 4B
• lung wash Fig. 4C
• Fig. 4B and 4C respectively.
• the results are indicated as follows: Fig. 4A: — ⁇ — s.c.-Ras3C-pre; — D — s.c.-Ras3C-post (Ras3C is a commercial semisynthetic adjuvant); — • — in.-prot-prep;
• Fig. 4B — ⁇ — i n. -saline, — A — i n -saline, — • — i n. -saline;, — D — i.n.-prot;; — ⁇ — i.n.-prot/emul; — O — i.n.-prot/emul
• Fig. 4C — ⁇ — i.n. -saline; — • — i n. -saline; — A — i n -saline; — D — i.n.- prot,
• a suitable immunopotentiating system suitable for use with this invention renders peptides (including small peptides) immunogenic and enhances the immunostimulating properties of larger peptides, proteins, and protein fragments.
• immunostimulating is defined as the capacity to induce a T cell-mediated immune response, such as delayed hypersensitivity or cytotoxic T lymphocytes, and/or an antibody response.
• the desired amino acid sequences which comprise the epitope(s) to which immunity is desired may be made by chemical synthesis from amino acids and/or polymerization, by extraction from the pathogen, or by recombinant means.
• the antigenic peptides or proteins used in the vaccine may vary in sequence from the native antigenic or immunogenic sequences of the natural pathogens by addition, deletion, or insertion of other amino acids or by the attachment of additional sequences, preferably hydrophobic moieties.
• the antigenic peptide itself may be positively or negatively charged or neutral.
• the exogenous hydrophobic material also termed the "hydrophobic foot” or “hydrophobic anchor,” is optionally attached to the antigenic, immunostimulating sequence and may vary in structure.
• a preferred hydrophobic foot comprises a fatty acyl group containing from about 8 to 18 carbon atoms and bonded in an amide bond to the protein or peptide.
• the group is an alkanoyl group.
• a particularly preferred alkanoyl group is a lauroyl group. Hydrophobic groups of this type are easily added to the amino terminus of a synthetic peptide while the peptide is still on the resin used for synthesis.
• Peptides may be synthesized by the solid phase method (Merrifield, B., J. Amer. Chem. Soc. 55:2149-2154 (1963); Merrifield, B., Science 232:341-347 (1986); Wade, J.D. et al, Biopolymers 25:S21-S37 (1986); Fields, G.B., bit. J. Peptide Prot. Res. 35:161 (1990); MilliGen Report Nos. 2 and 2a, Millipore Co ⁇ oration, Bedford, MA, 1987).
• the fatty acyl group preferably as an alkanoyl chloride, can be reacted with the peptide on the resin.
• the fatty acyl group may also be added to the amino terminus by reaction of a fatty acid such as lauric acid. To avoid side reactions, free amino groups may be blocked to assure that the acyl group is attached to the end of the peptide. It is also possible to attach the acyl group on the carboxy terminus using Lys as the
• the exogenous hydrophobic anchor may also be a hydrophobic peptide of about 3 to 50 amino acids, preferably between about 5 and 24 amino acids in length.
• a hydrophobic peptide preferably consists of non-polar or neutral amino acids, although, the longer this peptide, the greater the tolerance for up to several charged amino acid residues.
• the amino acids that are particularly useful in providing hydrophobicity and are preferred for the hydrophobic peptide sequence of this invention are drawn from the following categories: 1. Small aliphatic, nonpolar residues: Ala, Thr or Pro
• Hydrophobic amino acids of longer chain length can also serve the function of the hydrophobic foot so long as the length of the hydrophobic foot does not exceed about 24-30 amino acids. It is important that the antigenic peptide bound to an exogenous hydrophobic foot not be rendered totally water insoluble in the presence of detergent.
• a specific example of endogenous hydrophobic regions associated with HIV gpl ⁇ O is the transmembrane domain of the gp41 portion of gpl ⁇ O. This region has a membrane spanning hydrophobic domain as well as other hydrophobic sequences, for example, amino acids 523-551 of the gpl ⁇ O sequence shown below (SEQ LD NO: 1). Such a sequence may be naturally rendered even more hydrophobic by palmitoylation (Yang et al, supra). Thus, for example in SEQ ID NO: 1, the Cys at position 776 would be palmitoylated (corresponding to position 764 in Yang et al).
• the hydrophobic peptide sequence may be added synthetically or recombinantly to either terminus of the antigenic peptide.
• a preferred hydrophobic peptide is a pentapeptide, most preferably Phe-Leu-Leu-Ala-Val or Val-Ala-Leu-Leu-Phe (SEQ ID NO: 2 and 3]
• Immunogenicity of longer peptides and proteins may also be potentiated by methods disclosed herein.
• Many extracted or cloned polypeptides (especially transmembrane polypeptides) have natural hydrophobic anchors which are frequently 15 to 30 amino acids long.
• the immunogenicity of such polypeptides may also be enhanced by extending a native hydrophobic anchor or by adding another hydrophobic anchor according the methods disclosed herein.
• a preferred decapeptide is Gly-Gly-Tyr-Cys-Phe- Val-Ala-Leu-Leu-Phe (SEQ ID NO:4) because it has appropriate size and composition to allow easy purification of a recombinant anchored protein.
• Native sequences may have length and composition which hinder extraction and purification.
• the hydrophobic anchor sequence is preferably added to the carboxy- terminus of the selected recombinant protein by genetic engineering methods.
• the polynucleotide that encodes the anchor can be added to the 3' end of the coding sequence for the desired recombinant protein.
• the polynucleotide that encodes the anchor may also be added at the 5' end of the selected coding sequence.
• the polynucleotide that encodes the anchor may be added to both the 5' and 3' termini of the sequence encoding the selected protein.
• the constructs can be complexed to the proteosomes by dialysis or lyophilization as described above in methods for preparation with peptides.
• the hydrophobic foot may be attached by the methods indicated for attachment to peptides as an alternative to production in a recombinant molecule as described above.
• Ratios of proteosomes to anchored recombinant protein may ranges between about 1 : 1 to about 1 :20. Preferred ratios are between about 1 : 1 and about 1 :3 for polypeptides or proteins.
• the complexing between the proteosomes or nanoemulsions and the antigenic protein or peptide is intended to be non-covalent. Although it is often referred to as hydrophobic, it includes ionic interactions and hydrogen bonding.
• hydrophobic complexing is more physical than chemical, and since hydrophilic protein epitopes remain conserved, exposed, and typically unaltered, antibodies generated against these epitopes will recognize the native protein or an epitope thereof and will therefore function in binding, attacking or removing the pathogen from which the protein or epitope is derived or with which it cross- reacts.
• Vaccine compositions may be introduced into a subject by conventional means, including parenteral routes (for example, subcutaneous, intradermal, intramuscular) and by direct application to mucous membranes. Lyophilized compositions may be "snorted" into the nasal cavity. Dosage will depend on the particular agent administered, and will be readily apparent to those skilled in the art.
• An oligomeric peptide may be synthesized as a repeating unit wherein the unit peptide sequence is repeated in a tandem array up to as many times as synthesis will allow. Tandem repeating units of 2-6 have been used with enhancing effects on the immune response. Epitope repetition enhances the immunogenicity of the peptide.
• Complexing the oligomeric peptide with an exogenous hydrophobic material prepared as the methodology described herein, can render a totally non-immunogenic peptide immunogenic without a need for added adjuvants, and in some case, even without a need for proteosomes as described herein. However, complexing the oligomeric peptides directly with proteosomes is also a preferred embodiment.
• a composition comprising an oligomeric peptide and at least one Cys residue which is complexed with proteosomes is a most preferred vaccine.
• each proteosome can be complexed with between about 6 and-30 protein or peptide molecules.
• Either dialysis or lyophilization may be used to prepare the vaccine as follows: 1. Dialysis Method a. Combination of Components in TEEN- 1 %
• proteosomes are stored in TEEN-1% buffer (0.05M Tris-HCl, 0.15M NaCl, 0.01M EDTA (ethylenediamine tetraacetate) and 1% Empigen-BB ® (Albricht and Wilson, Cumbria, England) at a concentration
• proteosome material mixed with the antigenic peptide which is also in a TEEN-1% solution.
• the peptide may have an endogenous or exogenous hydrophobic foot (with or without an added Cys residue and with or without tandemly repeating epitope, as desired) in a beaker or test tube.
• Ratios of proteosomal protein to antigenic peptide (weight: weight) that have been used have ranged from 1 : 1 to 1:40. The usual ratio is 1 : 1 although, depending on the circumstances, 1 :4 or higher may be preferable.
• the concentration of the peptide in solution prior to admixture with proteosomes must be high enough so that the concentration of both the peptide and the proteosomal protein in the combined mixture is >1 mg/ml when the components are at equal ratios.
• the concentration of the less concentrated component should be > .50 mg/ml and preferably, > .75 mg/ml.
• the proteosomes are at 1.1 mg protein/ml
• the peptide should be at 10 mg/ml prior to combining at a 1 : 1 ratio. While these minimal concentrations are not absolute, and although successful vaccines have been prepared using lower protein concentrations (when the peptide: protein ratio is significantly >1 : 1), the method described above is more consistently successful.
• the mixture is transferred to dialysis bags that, due to their low molecular weight cutoff, retain both the peptide and the protein while allowing the detergent (usually Empigen-BB) in the TEEN-1% to dialyze away.
• the detergent usually Empigen-BB
• Spectra-Por 6 dialysis tubing with a molecular weight cutoff of 1000 is routinely used to maximize the amount of peptide retained for complexing to the proteosomes.
• the dialysis tubing (closed using special Spectra-Por closures) is washed just prior to use with pyrogen-free distilled water and then phosphate buffered saline pH 8.5 (PBS-8.5) which consists of 0.025 M Na 2 HPO plus 0.15 M NaCl (normal saline).
• PBS-8.5 phosphate buffered saline pH 8.5
• the proteosome-peptide mixture is exhaustively dialyzed against this buffer, e.g. at a ratio of 200-250:1 for 10 days with daily changing the dialysis fluid).
• the buffer is changed to standard phosphate buffered saline, PBS (Na 2 HPO + NaH 2 PO 4 + NaCl at pH 7.4).
• the dialysis period may be shortened, for example to 5 days with 2 changes of fluid per day.
• Solution is collected from dialysis bag(s), and the bags are washed with 20% of their volume of PBS. The rinse is combined with vaccine.
• the vaccine is filtered through a 0.22 ⁇ m filter (the vaccine may need to be pre- filtered through a 0.8 or 0.45 ⁇ m filter) or through a 0.45 ⁇ m filter.
• the protein content is measured (e.g. by absorbance at 280 nm or by the Lowry assay), and samples taken for amino acid analysis, HPLC and other analyses, The samples are then diluted in PBS to a concentration of 0.4 mg protein per ml solution.
• the final vaccine is then diluted 1 : 1 with either normal saline (supplemented with 0.02% merthiolate as an antibacterial) to yield a 0.2 mg/ml solution.
• This solution may then be administered intramuscular (i.m.) at 0.5 ml per dose.
• the vaccine can be adsorbed to alum by diluting
• the peptide and proteosomes may be complexed and rendered immunogenic by simply lyophilizing these components together according to the following procedure.
• Proteosomes are removed from TEEN-1% by precipitation through the addition of three volumes of 100% ethanol to one volume of the proteosomes, allowing to stand at 4°C for one hour and then centrifuging them at 800-1000 g for 15 minutes; washing the proteosomes three times by adding the same amount of 100% ethanol as previously used and re-centrifuging as before, then resuspending the proteosomes in PBS to a concentration of 2 mg/ml
• the peptide (with its hydrophobic foot and, if desired, Cys and replicated epitopes) is then redissolved in PBS at 2 mg/ml (or another concentration as described above if a peptide protein ratio >1 1 is desired)
• the dissolved peptide is added to the proteosome suspension and mixed
• the mixture is lyophilized and, following lyophilization, resuspended to 1 mg protein/ml using distilled water
• the product is then filtered, analyzed, diluted and added to saline or alum as described above
• the antigenic peptides may be in the form of "peptide oligomers" which may be tandem or cross-linked oligomeric peptides wherein (1) the basic peptide unit is tandemly repeated a number of times, (2) the basic peptide unit contains at least one Cys residue (naturally or by addition), and two or more peptide units are cross-linked by disulfide linkages between Cys residues or (3) two or more basic peptide units are chemically cross-linked
• Epitopes may be repeated in tandem as many times as synthesis will allow Such structure enhances the immunogenicity of a peptide epitope
• a totally non-immunogenic peptide can be made immunogenic without added adjuvants and even without the proteosomes
• complexing the peptide oligomers with proteosomes is preferred
• Epitope repetition may be used in conjunction with the addition of Cys (see below) in the process in which an exogenous hydrophobic foot and this modified peptide is optionally complexed with proteosomes In either case, toxicity and side effects are minimal ADDITION OF CYSTEINE RESIDUES
• the peptides and proteins used in the present compositions and methods may natively contain Cys residues or have Cys residues added as described Thus, the native presence of Cys in the antigenic peptide or protein is not required
• Cys residues may be added during the synthesis Cys may also be added covalently to previously synthesized sequences by carbodiimide coupling
• the Cys is useful for effecting dimerization, oligomerization (if more than one Cys is present) or cyclization of the peptides Unless reducing agents are present, dimerization occurs spontaneously following deblocking and cleavage of the peptide when one Cys is present in the peptide In a preferred embodiment one Cys is located between the hydrophobic foot and the antigenic peptide epitope When the peptide contains two Cys residues, cyclization is accomplished spontaneously in dilute solution after de-blocking and cleavage of the peptide Ferricyanide oxidation of the peptide in the dilute solution causes formation
• a Cys residue can be added to provide for dimerization (or oligomerization) of either the hydrophilic antigenic epitope, the endogenous hydrophobic sequence or the exogenous hydrophobic group Dimerization appears to stabilize binding to the proteosome by providing two hydrophobic feet for the epitope
• the dimerized constructs also provide for more stable interaction with the antigen
• the Cys may be placed at either the C- or N- terminus of the antigenic peptide
• the nucleotide sequence encoding a hydrophobic foot peptide or any desired Cys residue may be attached in-frame to the nucleotide encoding the antigenic protein or peptide
• Cys When Cys is added, it is preferably done as part of the process of forming the vaccine compositions during the step of adding an exogenous hydrophobic material (such as during chemical synthesis or recombinant production) In another embodiment, the Cys is added to antigenic peptide that has its endogenous hydrophobic peptide. Two general approaches may be used. a. Dimerization
• Cys addition allows the enhancement of immunogenicity of an antigenic peptide when used in conjunction with (i) the hydrophobic foot plus proteosomes, (ii) tandemly repeated epitopes or (iii) both of the foregoing.
• dimerization provides two hydrophobic feet for the epitope, to provide more stable binding to the proteosomes or promote formation of auto- micelles. Furthermore, at least two epitopes created by the dimerization may improve conformation of the peptide epitope and allow a more stable interaction with antigen recognizing cells in the treated subject. b. Cyclization
• a preferred antigenic protein for use herein is an envelope protein of HTV- 1.
• the mature envelope proteins in virions and HIV-infected cells are gpl20 and gp41, which are derived from a single precursor, gp 160.
• the advantages of a gpl ⁇ O protein having antigenic epitopes in a native or undamaged form is important for a useful vaccine. This has been discussed above (See: U.S. Patent 5,116,740; VanCott T.C et al, J Immunol. Meth. 1995 , 753: 103-117).
• gpl ⁇ O is oligo-gpl ⁇ O (or o-gpl60), as disclosed in these references, because of its maintenance of antigenic epitopes, presumably in nativelike form, and due to the presence of gpl ⁇ O dimeric and tetrameric structures in this preparation.
• the o-gpl60 protein preparation can have a lower molecular weight.
• the gpl ⁇ O monomer appears to have a molecular mass of about 140 kDa due to a truncation which had not previously been recognized.
• Such a lower molecular mass truncation variant could be referred to as "gpl40" or "o-gpl40" due to its apparent molecular mass of about 140 kDa rather than 160 kDa.
• compositions of the present invention include variants gpl ⁇ O, whether they be amino acid substitution variants (either natural isolates or genetically engineered variants) as well as truncation variants which may have occurred inadvertently (as is believed to be the case for the 451 isolate) or have been deliberately prepared for any of a number of reasons, including improved secretion from cells.
• gpl ⁇ O is intended to encompass the disclosed truncation variant and other presently known or later discovered truncation variants and amino acid substitution variants of gpl ⁇ O.
• o-gpl60 was obtained from the HIN-1 isolate originally named HTLV-IIL451 This protein is listed on the SWISS-PROT database, (maintained by the National Center for Biotechnology
• Seq ID: 119434 was shown to have theamino acid sequence shown below (in single letter code).
• SEQ ID NO: 1 This sequence (SEQ ID NO: 1) is divided as follows: residues 1-32 are the signal peptide ending with the "/" mark. Residues 33-522 constitute gpl20, ending with the " ⁇ ” mark. Residues 523-868 constitute gp41. It was subsequently discovered that this clone was truncated, with the C-terminal 187 amino acids of gp41 missing. These are indicated by underscoring in the sequence below. Thus, the o-gpl60 protein as obtained from the cloned cell line described below has only 649 residues (from position 33 to 681 of SEQ LD NO: 1 ).
• HUT78 cells A single cell clone of HUT78 cells has been infected with human immunodeficiency virus type 1 (HIV-1), resulting in a cell line which continuously produces virus.
• HIV-1 human immunodeficiency virus type 1
• Clone 6D5 is susceptible to chronic infection with HIV- 1 , as described in Getchell, et al, J. Clin. Microhiol. 23:131-142 (1986).
• Clone 6D5 is infected with a specific strain of HIV- 1, HTLV-III 45 ⁇ , to produce the infected cell line 6D5451 (deposited with the American Type Culture Collection under the Budapest Treaty).
• the infected cell line is then grown in serum-free medium, by pelleting 6D5451 cells and resuspending them in serum-free medium (such as HB 101 or HB 104 medium, commercially available from Du Pont).
• serum-free medium such as HB 101 or HB 104 medium, commercially available from Du Pont.
• the medium also contains growth supplements such as transferrin, insulin, and bovine serum albumin.
• transferrin transferrin
• insulin insulin
• bovine serum albumin bovine serum albumin
• the amount of HIV proteins released into the media was nearly five-fold greater in serum-free medium than in serum-containing medium.
• the cell-free medium is used as the source of the glycoprotein.
• the medium is adjusted to 20 mM with sodium phosphate, pH 7 5, 0 5% Triton X-100, 0 1 mM phenylmethylsulfonyl fluoride, and 400 mM sodium chloride After incubation at room temperature for one hour, the medium was concentrated 30-fold with a Pellicon cassette system, commercially available from Millipore Extraneous proteins derived from the media supplement are removed from the concentrate by immunoaffinity absorption (overnight) with a Sepharose-bound goat antibody raised against the proteins in the growth supplement in the serum-free medium Proteins which bind to the goat antibody are removed, and the unbound material is passed through a lectin affinity column
• lectin-Sepharose® a lentil lectin column
• other lectins which will recognize mannose such as concanavalin-A
• PBS phosphate buffered saline
• the column is eluted with 400 mM ⁇ -methylmannoside to recover the viral glycoprotein (Any mannose, pyranoside, or saccharide which competes with the lectin in the affinity column may be used )
• Immunoblot analysis of the HIV glycoprotein is carried out by well known procedures, (e.g., Saragadharan, et al, Science 224 506-508 (1984) Essentially, the proteins are run on 7% SDS-polyacrylamide gels and transferred to commercially available nitrocellulose strips The strips are then treated with the appropriate antibodies, and the blots are developed with enzyme -conjugated secondary antibodies, the bands are visualized by reacting the strips with a chromogenic substrate for the enzyme gpl ⁇ O can be purified from the mixture of glycoproteins eluted from lentil-lectin Sepharose® column by immunoaffinity chromatography using a mAb to HIV-1 gp41 protein The mAb is produced and used employing procedures routine in the art The gpl ⁇ O elutes from the column in a nearly homogenous state as described in US 5, 116,740
• Glycoprotein gpl ⁇ O and its derivatives, prepared as described are used to prepare vaccine compositions in accordance with the methods disclosed herein
• PepG is an example of a cyclic peptide - it has two cysteines which have been joined in a disulfide bond to make a cyclic loop in the peptide
• PepMl is non-cyclic, is without an added Cys and contains the native epitope only once.
• PepCMl has an added Cys at the amino terminus as does pepCM3 and pepCM5
• PepCM3 has three replicates of the native M epitope and pepM5 and pepCM5 have five such replicates
• PepLl has an epitope of only seven amino acids as does its Cys-containing counterpart, CL1
• Proteosomes in their native hydrophobic state, have special lymphocyte activating properties which allow them to act as both a protein carrier and an adjuvant. Since they are not chemically modified, but retain their multimolecular hydrophobic and membranous structure in the present vaccine composition, their ability to potentiate the immunogenicity of the antigenic peptides to which they are complexed is due to these special properties which are retained by the methods set forth herein
• Proteosomes may be prepared from Group B type 2b meningococcal cells
• Proteosome preparation consists of two stages The first stage may be done by
• the direct cell extracts are typically obtained by extraction of packed bacterial cells for one hour at room temperature with 100 grams of cells per liter of a solution containing 0.1 M sodium acetate pH 5.0, 0 5 M CaCl 2 and 3% Empigen BB Ethanol is added to the mixture to a concentration of 20% v/v and the precipitate removed by centrifugation at 16,000 x g for 10 minutes Additional ethanol is added to the supernatant to a final concentration of 45% and the precipitate, constituting the direct cell extract, is collected by centrifugation
• the second stage of the proteosome preparation consists of separating the outer membrane proteins from other membrane components by dissolving either of the products from the first stage (the vesicles or the direct cell extract) at a concentration of approximately 2 mg protein/ml in a buffer (hereafter referred to as
• proteosomes prepared from bacteria other than those prepared from meningococci may also be prepared and used by the same methodology
• the hydrophobic foot attached through the Cys may be sufficient to provide needed antigenicity without use of proteosomes.
• C3H/HeJ mice are genetically non- responsive to lipopolysaccharide (LPS), an immunopotentiating substance and important component of many adjuvants Immunization with pepG alone, pepG in Freund's adjuvant, pepG with proteosomes (but without any hydrophobic foot), or either lauroyl-pepG or FLLAV-pepG without proteosomes indicated a lack of immunogenicity (group 1 , controls a-e) In marked contrast, pepG was made highly immunogenic by complexing with proteosomes via either a lauroyl hydrophobic foot (groups 2 and 4) or via the FLLAV pentapeptide hydrophobic foot (groups 3 and 5) This was demonstrated in both BALB/c mice (groups 2 and 3) and C3H/HeJ mice (groups 4 and
• pepCLl which is non-cyclic, was made immunogenic in both normal mice (group 15) and LPS non-responder mice (group 16) As expected, pepCLl controls were non-immunogenic (group 13, controls a-d)
• the Ml epitope was tested for immunogenicity in the system both with an added Cys (groups 9-12) and without the Cys (groups 6-8)
• the Cys was found to be exceedingly important High immunogenicity with either the standard 40 ⁇ g dose (group 1 1) or a sub-standard (8 ⁇ g) dose (group 12) of pepCMl complexed to proteosomes.
• This peptide, lauroyl-pepCMl was mildly immunogenic (after three immunizations) without proteosomes (group 10).
• pepMl lacking the Cys, exhibited only minimal immunogenicity even with proteosomes (groups 7 and 8).
• the Cys was considered to be important because its free sulfhydryl group causes dimerization of both the antigenic epitope and the hydrophobic foot. Dimerization of the epitope may allow better recognition by antigen presenting cells whereas dimerization of the hydrophobic foot promotes better complexing to proteosomes.
• R32RL recombinant protein
• R32Ft is immunogenic in vaccine testing when used alone and such immunogenicity is markedly enhanced when it is complexed to proteosomes via the added hydrophobic decapeptide anchor described above.
• the ligation mixture was transformed into E. coli strain MM294CI+ (SmithKline French]. AmpiciUin resistant colonies were obtained and screened for insertion of the Xho II gene fragment into pT17. A plasmid with the correct construction, pR16, was identified and transformed into E. coli strain MM294CI+ Expression vector pR16 was digested with restriction endonuclease BamHI as described above and a second Xho II CS protein gene fragment ligated into the vector. The ligation mixture was transformed into E.
• Expression vector pR32 (lO ⁇ g) was digested by restriction endonucleases Smal and Sail in 200 ⁇ l medium buffer (described above) for 1.5 hours at 37°C.
• the ligation mixture was transformed into E. coli strain MM294CI+. Ampicillin resistant colonies were obtained and screened for the insertion of the decapeptide into pR32. A plasmid with the correct construction, pR32Ft, was identified and transformed into E. coli strain AR58 (CI 857 ) and tested for expression of the gene product.
• the R32Ft peptide was purified from the above expression system as disclosed below All operations were performed on ice unless stated otherwise Three 20-g E coli frozen pellets (SmithKline Laboratories) were combined and thawed by suspending in 240 ml of 50 mM Tris , 2 mM EDTA, 5% glycerol [at pH
• the supernatant was heated in a boiling water bath for 5 minutes with stirring, cooled for one hour at ambient temperature, and then centrifuged at 12000 x g Crude antigen was precipitated in a 10% to 40% ammonium sulfate pellet
• the pellet was resuspended in 25 ml phosphate buffered saline (PBS) and dialyzed extensively against PBS (SpectroPor tubing, MW cut-off 3000)
• PBS phosphate buffered saline
• TFA trifluoroacetic acid
• the anchored recombinant proteins were complexed to the proteosomes via dialysis.
• Proteosomes in a concentration of 0.5-2.5 mg/ml were added to solution of the recombinant protein with the hydrophobic foot to provide ratios of proteosomes to anchored recombinant protein (w/w) range of 1 : 1 to 1 :20.
• the material was dialyzed in accord with the teachings above.
• mice were dosed with (a) 50 ⁇ g proteosomes with 50-100 ⁇ g R32Ft or (b) 50-100 g R32Ft without proteosomes. All injections were given using saline as the carrier. No additional adjuvants were used. Analysis of pooled sera from the groups of mice showed that, while the recombinant R32Ft alone was effective, the recombinant R32Ft complexed with the proteosome was at least 16 fold more effective as a vaccine. Both C57BL/6 strain and BALB/c mice responded to the vaccines. When the animals were given booster injections (up to two boosters) an improved immune response was seen in all instances.
• EXAMPLE VI Response to Leishmania Vaccine Mice immunized and then infected with Leishmania major in a murine model of cutaneous leishmaniasis having a lauryl or lauryl-Cys conjugated to the amino terminus was assessed for cell mediated immune response.
• Vaccines will consist of lauryl or lauryl-Cys conjugated to a selected synthetic gp63 peptide 467- 482 having the following sequence (SEQ ID NO:8): Gly-Asn- Val-Gln-Ala- Ala-Lys- Asp-Gly-Gly- Asn-Thr-Ala- Ala-Gly- Arg
• the peptide covalently conjugated to lauryl-Cys protected against severe Leishmania cutaneous lesions with an average of 81% reduction of lesions in 3 separate experiments. This occurred even when giving the lauryl-cysteinyl-peptide in saline without other adjuvants whereas the cysteinyl-peptide or the peptide without the added lauryl moiety was ineffective. Addition of proteosomes or other peptides did not further enhance protection. Studying of proliferation were negative. GeneBank analysis of this peptide revealed a striking homology with a human integrin molecule responsible for localization of cellular elements in the inflammatory process, indicating that the parasite may use immune mimicry to avoid host defenses. This peptide may therefore have wide application in ameliorating pathologic cellular immune responses caused by other forms of Leishmania or other parasites or bacteria such as mycobacteria where protective cell-medicated immunity is important.
• proteosomes confer intranasal immunogenicity on formalinized toxoid of Staphylococcal Enterotoxin B (SEB) when formulated with proteosomes.
• SEB Staphylococcal Enterotoxin B
• mice anti-SEB respiratory IgA and serum IgG were induced when the complexed compositions in saline were administered intranasally.
• the proteosome-toxoid vaccine also showed enhanced immunogenically when given parenterally.
• the proteosome-toxoid vaccine was made by the dialysis method as described The toxoid and proteosomes were mixed in the presence of 1% buffered detergent (Empigen) and dialyzed.
• Empigen buffered detergent
• mice immunized intranasally with proteosome-toxoid vaccines were significantly protected (p ⁇ 0.0117) against systemic challenge with >4 LDioo of
• hydrophobic foot As indicated, the methods disclosed herein are appropriate for use both with addition of the hydrophobic foot. However, when there is a hydrophobic moiety in or associated with the peptide, it is not necessary to synthetically add the hydrophobic foot
• proteosomes were constructed as indicated above and were stored at -70°C in small aliquots at concentration of >5 mg/ml (usually 6-7 mg/ml) in TEEN-0 1% buffer (or, on occasion, TEEN-1% having 1% Empigen BB) The proteosomes were defrosted immediately before use
• the antigenic material was prepared by one of the two following methods: (1) Dialysis
• the proteosomes were added to provide a 1 : 1 ratio (weight: weight) so that 10.7 mg of 6.7 mg/ml stock in 1.6 ml was added to produce a final concentration of 0.485 mg/ml of gpl ⁇ O and proteosomes.
• the resulting product was dialyzed across a 1000 Da cut-off SpectraPor 6 or 7 membrane for 10 days at 4°C against Tris buffered saline changing the buffer daily.
• Centriprep 30 tubes were used to simultaneously remove the TWEEN and concentrate the gpl ⁇ O stock from 0.7 mg/ml to >4 mg/ml by diluting 15 ml of the
• the resulting concentrate was rediluted with Tris buffered saline to 30 mis and recentrifuged as above to give a final volume of 3.2 mis with a gpl ⁇ O concentration of 4.25 mg/ml (analyzed spectrophotometrically at A 2 go) and with an estimated 99.999% TWEEN removal and 94% recovery of gpl ⁇ O.
• a gpl ⁇ O concentration 4.25 mg/ml (analyzed spectrophotometrically at A 2 go) and with an estimated 99.999% TWEEN removal and 94% recovery of gpl ⁇ O.
• 7.2 mg of gpl ⁇ O in 0.1 M Tris buffered saline was used of a stock of 4.2 mg/ml concentration in a volume of 1.7 mis.
• Empigen BB was added to give a final concentration of 1%.
• proteosomes were added to provide a l l ratio (weight: weight) so that 7.2 mg of 6.7 mg/ml stock in 1.1 ml was added to result in a final concentration of 2.5 mg/ml of gpl ⁇ O and proteosomes.
• the resulting product was dialyzed across a 1000 Da cut-off SpectraPor 6 or 7 membrane for 10 days at 4°C against Tris buffered saline changing the buffer daily.
• the gpl ⁇ O is a much larger than the R32ft discussed above.
• the gpl ⁇ O is also a transmembrane protein which naturally forms trimers that make its effective molecular weight even larger.
• the antigenic properties of compositions containing gpl ⁇ O complexed to proteosomes can be enhanced by addition of adjuvants such as alum. It has also been discovered that submicron emulsions enhance immunogenicity.
• TABLE 6 shows a comparison of ELISA analysis of sera from rabbits immunized 4 times i.m. with 85 ⁇ g of gpl ⁇ O formulated with alum, proteosomes plus alum, or proteosomes plus sub-micron emulsions.
• the results indicate that the proteosome complex formulation resulted in a much higher titer response to an important gpl20 epitope termed Alex 10.
• Induction of IgG and IgA antibodies in vaginal, intestinal, and lung lavage fluids, fecal extracts and sera occurs following intranasal immunization of mice with an oligomeric gpl ⁇ O vaccine.
• the vaccine can be delivered in saline, as a proteosome-oligo-gpl60 complex alone or in a solid fat nanoemulsion.
• Outer membrane protein proteosome preparations were purified as follows: Proteosomes were prepared from Group B type 2 Neisseria meningitides in two stages. The first stage was done by the collection of a bacterial cell extract precipitate. The direct cell extracts were obtained by extraction of packed bacterial cells by adding with one liter per 1000 grams of cell paste of a solution containing 1.0 M sodium acetate pH 5.0 mixing and then adding an equal volume of a solution of 1.0 M CaCl 2 with 6% Empigen BB. This suspension was then stirred for one hour at room temperature. Ethanol was added to the mixture to a concentration of 20%v/v, the precipitate removed by centrifugation at 10,800 x g for 15 minutes and the supernatant was filtered through cotton gauze. Ethanol was added to the filtrate to a final concentration of 45% and the precipitate, constituting the direct cell extract, was collected by centrifugation at 10,800 x g for 15 minutes.
• the second stage of the proteosome preparation consisted of isolating the outer membrane proteins from the other membrane components by dissolving the product from the first stage (using a Teflon paste homogenizer followed by stirring and sonication) in a buffer (TEEN-1%) containing 0.05 molar Tris-HCl (hydroxyacetyl amino methane), 0.15 M NaCl, 0.01 M EDTA (ethylene diamine tetra-acetate) and 1% Empigen BB (Albricht and Wilson, Cubria, England) brought to pH 8.0 using 1.5 ml TEEN-1% per gram of initial paste. The suspension was then centrifuged at 13,800 x g for 25 minutes at 4°C.
• the supernatant was saved and the dissolving process was repeated as above on the resultant pellet and subsequent pellets 2-4 times as needed, using 15-50% less volume as needed and saving and pooling all the supernatants which were then stored at 4°C.
• the proteins were then precipitated by the addition of solid ammonium sulfate at 500 g/1 of protein solution.
• the pellet and solid precipitates were collected after centrifugation at 20-30,000 x g for 20 minutes and redissolved in TEEN-1% using about 2 mg protein/ml or 1/4-1/5 the volume of the first Teen- 1% extraction This procedure was repeated on the pellets twice more using 600 g/1 of ammonium sulfate.
• the pellet from the last ammonium sulfate was dissolved using TEEN-1% at 1/4 the volume of the first Teen-1% extraction or 2 mg/ml with stirring and sonication. After centrifuging this solution at 28,400 x g for 25 minutes, the supernatant was saved at 4°C and the pellets were re-dissolved using less volume consonant with the size of the pellet. This process was repeated as needed until the pellet was negligible.
• the pooled dissolved pellets were dialyzed against TEEN-1% using an AG hollow fiber cartridge system with a 10,000 kDa cutoff A/G membrane using successive concentration and dilution 3-5 times.
• the final concentration of Empigen BB can be 0.1% to 1.0%. Products are stored at -20° or -70°C (or, for shorter periods at 4°C). The final product can be filtered through a 0.22 micron membrane and the concentration of the resultant proteosome preparations can be from 1-10 mg/ml (4-7 mg/ml is preferred).
• HIV (strain 451) Oligo-gpl60 as previously described in US patent 5,116,740.
• Mucoadhesive emulsion particles were prepared as previously described (see PCT/US/ 05589), 1 : 1 (w/w) fat/phospholipid mixture was dissolved in chloroform. The organic solvent was evaporated to complete dryness under reduced pressure using a rotary evaporator (Heidolph, Germany). To the dry lipid film, an aqueous solution containing 0.1% EDTA was added and the mixture was then hydrated by shaking for 30 min. Using a multiwrist shaker (Labline, USA) until all the lipids were homogeneously dispersed in the aqueous phase. The dispersion was homogenized using a Microlab 70 Gaulin Homogenizer (5 cycles at 800 bar).
• the resultant emulsome particles were determined to have a mean diameter of 140 +/- 50 nm.
• Carbocol 934 (BF Goodrich, Atlanta, Georgia) was added (0.1% final concentration) to confer mucoadhesive properties to the emulsion preparation.
• Pmax were vigorously mixed with equal parts of rgpl ⁇ O or proteosome-rgpl ⁇ O preparations to result in a 2.5% lipid concentration in the vaccines containing the emulsion.
• Proteosome-oligo gpl ⁇ O vaccine Preparation of Proteosome-oligo gpl ⁇ O vaccine.
• a portion of the stock oligo-gpl60 was concentrated using an Amicon filtration unit with a 30,000 MWCO filter as needed and then complexed to and formulated with proteosomes using dialysis.
• the oligo-gpl60 dissolved in saline buffered solution such as Dulbecco's PBS pH 7.4 containing Empigen BB (1%) and was then mixed with proteosomes at a 1 : 1 (wt/wt) ratio in the saline buffered 1% Empigen solution.
• the mixture was exhaustively dialyzed across a dialysis membrane with a 10,0000 Molecular Weight cutoff (SpectraPor 7; Spectrum Medical Industries, Los Angeles, California) against buffered saline for 16-21 days at 4°C exchanging at least 500 parts buffer each day and the vaccine was stored at 4°C prior to and during the immunizations.
• a 10,0000 Molecular Weight cutoff Spectrum Medical Industries, Los Angeles, California
• oligo-gpl ⁇ O or Proteosome-oligo-gpl60 Vaccines with bioadhesive nanoemulsion The oligo-gpl ⁇ O or the proteosome-oligo-gpl ⁇ O vaccine was vigorously mixed with equal volumes of the bioadhesive solid fat nanoemulsion prior to immunization. Intranasal Immunizations
• mice Female Balb/c mice were used throughout the experiment. Mice (five per group per experiment) were immunized intranasally and samples were collected according to the schedule shown in Figure 1. Intranasal immunization was in volumes of 60 ⁇ l (divided into 30 ⁇ l applications spaced 2-4 hours apart) for each of the three immunizations (at 3 week intervals) with preparations containing 10 or 50 ⁇ g or oligo-gpl ⁇ O formulated with either (1) saline, (2) bioadhesive nanoemulsion, (3) proteosomes in buffered saline, (4) proteosomes plus bioadhesive nanoemulsion.
• mice Prior to immunization, mice were mildly anaesthetized as previously described (Lowell et al. (1996) Infection and Immunity 64: 1706- 1713) or with a mixture of zylazine and ketamine or with methoxyflurane and then allowed to inhale 25-35 ⁇ l of the vaccine or saline (for non-immunized control animals) that was slowly instilled by micropipette into both nares.
• Vaginal secretions Samples were collected as previously described in detail using wicks (Polyfiltronic Group Inc., Rockland, Massachusetts) inserted prior to sacrifice, 10-14 days after the third immunization. The day of wick insertion was timed to coincide with the estimated time of ovulation which had been previously synchronized by placing a male mouse in a nearby cage on the appropriate day. briefly, the wicks were inserted after instillation of 25 ⁇ L PBS intravaginally and allowed to absorb secretions for 30-60 seconds after which the wick was removed, an additional 25 ⁇ L PBS was instilled and the opposite end of the wick was inserted into the vagina for another 30-60 seconds.
• wicks Polyfiltronic Group Inc., Rockland, Massachusetts
• the wick was transferred to a microfuge tube, immediately frozen with dry ice and stored individually at 70°C.
• Secretions from each mouse were individually eluted from the wicks by adding 0.8 mL of a solution of 0.5% each of BSA and casein plus protease inhibitors to the tube with the wick.
• the tube was then centrifuged at 10,000 rpm for 15 minutes prior to sampling for the ELISA. Intestinal and lung lavage fluids. Secretions were collected at sacrifice as previously described (Lowell et al. (1996) Infection and Immunity 64:1706-1713), 14 days after the third immunization.
• bronchial lavage samples immediately after sacrifice by CO 2 suffocation, the lungs were surgically exposed, a cannula was inserted in the trachea and, using a three-way stopcock, two lung lavage samples (1 mL each) using PBS containing 0.1% BSA were collected and combined. Intestinal lavage samples were then collected as described (Lowell et al. (1996) Infection and Immunity 64: 1706-1713) by passing 2 mL of PBS containing 0.1% BSA, 50 mM EDTA and 1 mg/mL of soybean trypsin inhibitor through a 20-25 cm section of small intestine. Lavage fluids from each mouse were vortexed and centrifuged to remove cell debris and then individually stored at
• Fecal pellets (25-30) were collected and pooled from groups of five mice one week after the last immumzation. Each collected pool of pellets was weighed and PBS containing 0.1% Sodium Azide was added to the pellets at a ratio of 1 mL per 0.1 g fecal pellets. The samples were vortexed vigorously for 15 minutes and then centrifuged in a microfuge at 14,000 RPM for 15 minutes to remove debris prior to storage of the supernatant at -20°C. Antibody Detection. ELISA
• HRP horseradish peroxidase
• Anti-oligo-gpl60 IgG and IgA antibodies in each of the fluids, sera and extracts collected from mice immunized with oligo-gpl ⁇ O delivered with proteosomes and/or nanoemulsions were analyzed by ELISA and compared to the collections of samples from saline immunized animals and from animals immunized with oligo-gpl ⁇ O delivered alone without proteosomes or the nanoemulsion. Results are shown in TABLE 6 A and 6B.
• proteosome formulation was preferred and the combination of proteosomes with the bioadhesive nanoemulsion was most preferred (Tables 1 and
• bioadhesive nanoemulsion emul or pmax
• proteosomes plus bioadhesive nanoemulsion induce antibodies in serum, vaginal wash and lung wash that preferentially reacted with the natively folded HIV gpl20 compared to their recognition of reduced and carboxymethylated gpl20 (rcmgpl20) ( Figures 2A-2C).
• Vaginal (VG) or lung (LG) fluids from saline controls or pre-immunization sera were unable to elicit antibodies that neutralized the virus as shown by the lack of reduction in viral titers (in the p24 assay) using these control samples.
• sequences within parentheses are identical to the sequences of the peptides in the native organism.
• mice were immunized ip on weeks 0, 3 and 7 with vaccines containing 40 ⁇ g of peptide; sera, obtained 2-3 weeks after each immunization, were tested in an ELISA for IgG antibodies against the homologous peptide (either pepG, pepMl or pepLl). Titers are the highest serum dilutions which had ELISA values that were a) more than 0.1 OD units and b) twice the value of pre- vaccination sera diluted 1:50.
• mice were immunized ip with vaccines containing 40 ⁇ g of peptide on weeks 0, 3 & 7; sera, obtained 2-3 weeks after each immunization, were tested in an ELISA for anti-pepMl IgG. Titers shown are the highest serum dilutions with ELISA values that were both a) >0.1 O D. units and b) twice the value of pre-vaccination sera diluted 1 :50.
• Control groups consisted of 5 mice immunized with either (a) peptide alone, (b) peptide in Freund's adjuvant, (c) peptide and proteosomes without hydrophobic feet, (d) lauroyl peptide without proteosomes, and (e) FLLAV-peptide without proteosomes.
• the detergent (Empigen) was removed from the proteosomes by ethanol precipitation and the proteosomes were washed and resuspended in saline prior to mixing (group 34) or lyophilization (group 35) with a saline solution of pepCMl .
• mice were immunized ip on weeks 0, 3 and 7 with 40 ⁇ g of peptide and the corresponding amount of proteosomes obtained 2-3 weeks after each immunization, were tested in an ELISA for IgG antibodies directed against the homologous peptide, pepMl; titers shown are the highest serum dilutions that had ELISA values that were both a) greater than 0.1 O.D. units and b) twice the value of pre-vaccination sera diluted 1:50.
• mice were immunized ip on weeks 0, 3 and 7 with vaccines containing 40 ⁇ g of peptide; sera, obtained 2-3 weeks after each immunization, were tested in an ELISA for IgG antibodies against meningococcal outer membrane proteins. Titers shown are the highest serum dilutions obtained after two or three immunizations which had ELISA values that were (a) more than 0.1 OD. units and (b) twice the value of pre-vaccination sera diluted 1 :50. TABLE 5
• Proteosome-Lauroyl-pepG 102,400 4 Proteosome-FLLAV-pepG 409,600
• mice were immunized ip on weeks 0, 3 and 7 with vaccines containing 40 ⁇ g of peptide; sera, obtained 2-3 weeks after each immunization, were tested in an ELISA for IgG antibodies against meningococcal outer membrane proteins. Titers shown are the highest serum dilutions obtained after two or three immunizations which had ELISA values that were (a) more than 0.1 OD unit and (b) twice the value of pre-vaccination sera diluted 1 :50.
• Alex 10 is a significant epitope of gpl20.

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• 1997
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  • 1997-07-10 WO PCT/US1997/012253 patent/WO1998001558A2/en active Application Filing
  • 1997-07-10 CA CA002259961A patent/CA2259961A1/en not_active Abandoned
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