EP0964698A1 - Peptide, welche candida-kohlenhydratepitope nachahmen und ihre anwendungen in einem impfstoff - Google Patents

Peptide, welche candida-kohlenhydratepitope nachahmen und ihre anwendungen in einem impfstoff

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Publication number
EP0964698A1
EP0964698A1 EP97950697A EP97950697A EP0964698A1 EP 0964698 A1 EP0964698 A1 EP 0964698A1 EP 97950697 A EP97950697 A EP 97950697A EP 97950697 A EP97950697 A EP 97950697A EP 0964698 A1 EP0964698 A1 EP 0964698A1
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EP
European Patent Office
Prior art keywords
albicans
mice
mab
candidiasis
candida
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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.)
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EP97950697A
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English (en)
French (fr)
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EP0964698A4 (de
Inventor
Jim E. Montana State University Cutler
Yongmoon Montana State University Han
Pati Montana State University Glee
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Montana State University
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Research and Development Institute Inc of Montana State University
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Publication of EP0964698A1 publication Critical patent/EP0964698A1/de
Publication of EP0964698A4 publication Critical patent/EP0964698A4/de
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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • A61K39/0002—Fungal antigens, e.g. Trichophyton, Aspergillus, Candida
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

Definitions

  • the present invention relates to peptides which mimic carbohydrate epitopes (mimotopes) of Candida and to a vaccine comprising the peptides or antibodies to the peptides and a method for the treatment of disseminated candidiasis due to infection by Candida albicans .
  • Candida albicans is a fungus responsible for various forms of candidiasis, a condition which may be found in normal and immunocompromised patients, such as those with acquired immune deficiency syndrome. Humans and mice who are neutropenic are especially at risk of developing disseminated candidiasis (Denning, D.W., et al . 1992. Antifungal prophylaxis during neutropenia or allogeneic bone marrow transplantation: what is the state of the art? Chemotherapy 38(suppl l):43-49; Matsumoto, M.S., et al . 1991. Effect of combination therapy with recombinant human granulocyte colony-stimulating factor
  • U.S. Patent No. 5,288,639 to Bernie et al discloses the use of antibodies specific for stress proteins of C. albi cans for the treatment of systemic candidiasis.
  • Bernie et al isolated a 47 kilo-dalton immunodominant antigen from C. albicans and found that serum from patients with systemic candidiasis reacts with the antigen.
  • Monoclonal antibodies raised against the fungal stress proteins produced a 33% survival at 24 hours in animals challenged with a lethal dose of the C. albicans .
  • U.S. Patent No. 4,397,838 to d'Hinterland discloses preparations of purified proteoglycans extracted from bacterial membranes.
  • proteoglycans serve as immuno- adjuvants and have an immunostimulating activity without being immunogenic themselves. They are useful in serving as adjuvants with ribosomal vaccines such as a vaccine containing the ribosomes of C. albicans .
  • U.S. Patent No. 4,310,514 to Durette et al discloses immunologically active dipeptidyl 5-0, 6-0-acyl- 2-amino-2-deoxy-D-glucofuranose derivatives. The compounds are used to delay the release of an antigen and stimulate the immune response of the host in conjunction with a vaccine. Compounds of Durette provide nonspecific host protection against infectious organisms such as C. albicans .
  • U.S. Patent No. 4,315,913 to Durette discloses immunologicaily active dipeptidyl 2-ammo-l , 2-d ⁇ deoxy-D- glucose derivatives. These derivatives are also useful as immunological adjuvants and themselves provide non- specific host protection against C. albicans .
  • U.S. Patent No. 4,368,910 to Shen et al . is directed to immunologicaily active dipeptidyl 4-0-6-0-acyl-2- ammo-2-deoxy-D-glucose derivatives. These derivatives are indicated to be useful as immunogenic agents and vaccines and by themselves provide non-specific host protection against infectious organisms such as C. albicans .
  • U.S. Patent No. 4,323,560 to Baschang et al . is directed to phosphorylmuramyl peptides.
  • the peptides are used to stimulate immunity.
  • the compounds of Baschang et al . have been found to be mhibitive to infections caused by fungi such as C. albicans .
  • U.S. Patent No. 5,032,404 to Lopez-Berestem et al disclose a liposomal agent for treating disseminated fungal infection m an animal. Because of the nature of polysaccharide fungal cell walls, it is expected that all medically important fungi activate complement.
  • the patent indicates that disseminated fungal infection can be treated with liposomal agent comprised of lipids, a polyene macrolide anti-fungal compound and cholesterol .
  • Lipids can include phosphatidyl choline.
  • Liposomes incorporate an effective amount of a polyene macrolide anti-fungal compound such as hamycms or lucensomycm, filipm, lagosm and natamycm.
  • U.S. Patent No. 4,678,748 to Sutka et al discloses a process for the production of the immunobiological preparations applicable in the diagnosis, prevention and treatment of Candida guilliermondii infections. Strains of C. guilli ermondii are killed and used to formulate a vaccine . Early attempts at obtaining compounds which provide non-specific host protection against C. albicans are generally in uhe form of immuno adjuvants used in conjunction with vaccines.
  • More specific vaccine approaches include targeting aspects of C. albicans pathogenesis.
  • An important aspect of pathogenesis is adherence of C. albicans to host tissue. Discussion below provides an understanding of adherence as it relates to pathogenesis of disseminated candidiasis.
  • C. albicans is an organism that may show considerable variability of certain characteristics. Genetics studies show that the organism is diploid, but apparently without the ability to undergo meiosis, yet it has impressive genetic variability between and within strains (Scherer, S. et al . 1990. Genetics of C. albicans . Microbiol. Rev. 54:226-241). Chromosomal aberrations unpredictably occur (Rustchenko-Bulgac et al . 1990.
  • Chromosomal rearrangements associated with morphological mutants provide a means for genetic variation of C. albicans . J. Bacteriol, 172:1276-1283), and may be related to high frequency phenotypic (colony) changes in some strains (Soil, D.R. 1992. High-frequency switching in C. albicans . Clin. Microbiol. Rev. 5:183- 203) .
  • strains of C. albicans variably express cell surface antigens (Cutler, J.E., et al . 1994. Antigenic variability of C. albicans cell surface. Curr. Top. Med. Mycol. 5:27-47, and Martinez, J.P., et al .
  • C. albicans Studies on adherence properties of C. albicans are important in gaining an understanding of C. albicans interactions with its host. The ability to bind to mucus and epithelial surfaces likely plays a critical role in maintaining C. albicans at these locations.
  • the fungus also shows adherence specificities for selected populations of splenic and lymph node macrophages (Cutler, J.E., et al . 1990. Characteristics of C. albicans adherence to mouse tissue. Infect. Immun. 58:1902-1908; Han, Y., et al . 1993. Binding of C. albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages . Infect. Immun.
  • the fungal adhesins range in properties from hydrophilic to hydrophobic molecules (Hazen, K.C. 1990. Cell surface hydrophobicity of medically important fungi, especially Candida species, p. 249-295. In R.J. Doyle and M. Rosenberg (ed.), Microbial Cell Surface Hydrophobicity American Society of Microbiology, Washington, Kennedy, M.J. 1988. Adhesion and association mechanisms of C. albicans . Curr. Top. Med. Mycol 2.73-169) and all may be mannoprotems (8, 11) . Both mannan and protein moieties may function as adhesins
  • adhesins have mtegrin-like activity m that they act as receptors for mammalian proteins such as C3b, fibronectm, lammin and fib ⁇ nogen, one adhesm has lectm-like activity, and a C3d receptor has been described (Bendel, CM., et al 1993. Distinct mechanisms of epithelial adhesion for C. albicans and Candida tropi calis Identification of the participating ligands and development of inhibitory peptides. J. Clin. Invest 92.1840-18492, Calderone, R.A. , et al . 1991. Adherence and receptor relationships m C. albicans .
  • albicans has a fibrillar appearance both m vitro and m vivo (Hazen, K.C. et al . 1993. Surface hydrophobic and hydrophilic protein alterations in C. albicans . FEMS Microbiol. Lett. 107:83-88; Mame, T.J., et al . 1981. The ultrastructure of C. albicans infections. Can. J. Microbiol. 27:1156-1164; and Tokunaga, M. et al . 1986. Ultrastructure of outermost layer of cell wall m C. albicans observed by rapid-freezing technique, J. Electron Microsc. 35:237-246). A major component that makes up the fibrils on the cell surface of C.
  • albicans and extends deeper into the cell surface appears to be the phosphomannoprotein (PMP) .
  • PMP phosphomannoprotein
  • the cell surface is probably more complex than this, as additional proteins with relatively small amounts of carbohydrate may also be present (Hazen, K.C., et al . 1994. Hydrophobic cell wall protein glycosylation by the pathogenic fungus C. albicans . Can. J. Microbiol.
  • the present inventors have overcome the deficiencies and inability of the prior art to obtain a vaccine against disseminated candidiasis by directing their attention to a composition comprising C. albicans adhesins .
  • an object of the present invention is to provide a vaccine for treatment of candidiasis comprising a pharmaceutically effective amount of peptides specific to the mannan portion of the phosphomannan complex of Candida which elicits an immune response .
  • the peptide is a nonapeptide with the amino acid sequence YRQFVTGFW; where: Y, tyrosine; R, arginine ; Q, glutamine; F, phenylalanine; V, valine; T, threonine; G, glycine; W, tryptophan.
  • the peptide which has a consensus amino acid sequence for peptides with reactivity to MAb B6.1, selected from the group consisting of , ArXXAr (Z) ZZArAr; where: Ar, aromatic amino acid (F, W or Y) ; X, any amino acid; Z, equals S, (where S, se ⁇ ne) , T or G; (Z) , is S, T, or G which may or may not be present .
  • the invention also encompasses a vaccine wherein the mannan active portion comprises a composition structure selected from the group consisting of ⁇ - l , 2-linked straight chain t ⁇ , tetra- and penta-mannosyl residues m the acid labile part of the mannan portion of the pnosphomannan complex.
  • Still another ob ect of the invention provides a vaccine for treatment of disseminated and mucocutaneous Candidiasis comprising a pharmaceutical effective amount of an epitope of Candida Albicans comprising a beta 1,2- trimannose or acid stable epitopes that elicit an immune response .
  • the invention provides isolated protective antibodies for passive protection against hematogenous disseminated candidiasis and mucocutaneous candidiasis.
  • the antibodies may be monoclonal antibodies specific for mannan epitopes m the acid stable portion of the mannan epitope and ⁇ - l , 2-lmked tri, tetra- and penta-mannosyl residues in the acid labile part of the mannan portion of the phosphomannoprotein complex.
  • the invention also encompasses a vaccine wherein the mannan active portion comprises a composition structure selected from the group consisting of ⁇ - l , 2-linked straight chain tri, tetra- and penta-mannosyl residues in the acid labile part of the mannan portion of the phosphomannan complex.
  • Still another object of the invention provides a vaccine for treatment of disseminated and mucocutaneous Candidiasis comprising a pharmaceutical effective amount of an epitope of Candida Albicans comprising a beta 1,2- trimannose or acid stable epitopes that elicit an immune response .
  • the invention also encompasses a vaccine for treatment of disseminated candidiasis comprising a pharmaceutical effective amount of a peptide specific for Candida albicans epitopes, either ⁇ 1,2-linked oligomannose or acid stable epitopes in the phosphomannan complex, or a DNA vaccine comprising the sequences of said peptide, that elicit an immune response.
  • Still another embodiment provides a therapeutic composition for treatment of disseminated candidiasis comprising a pharmaceutical effective amount of passive humoral antibodies directed against a peptide specific for the ⁇ 1 , 2 -trimannose or others epitopes in the acid stable and acid labile regions of the mannan portion of the phosphomannan complex of Candida albicans that elicits an immune response. Also provided are isolated protective antibodies for passive protection against hematogenous disseminated candidiasis and mucocutaneous candidiasis .
  • the invention advantageously provides a method for the treatment of disseminated candidiasis and mucocutaneous candidiasis comprising administering an effective amount of the monoclonal antibodies of the invention to provide protection.
  • Still another embodiment provides a method for immunization against candidiasis comprising generating Candida albi cans peptides specific for phosphomannan complex-neutralizing antibodies.
  • the invention provides a peptide specific to the mannan portion of the phosphomannan complex of candidiasis wherein said peptide has the amino acid sequence YRQFVTGFW; where: Y, tyrosine; R, arginine; Q, glutamine; F, phenylalanine; V, valine; T, threonine ; G, glycine; W, tryptophan, or function equivalents of said peptide.
  • the peptide has a consensus sequence of amino acids selected from the group consisting of , ArXXAr (Z) ZZArAr; where: Ar, aromatic amino acid (F, W, or Y) ; X, any amino acid; Z, equal S (S, serine) , T or G; (Z) is S, T or G which may or may not be present .
  • Figure 1 shows polyclonal antiserum (Ab) protects normal and SCID mice against disseminated candidiasis.
  • Polyclonal antiserum from L-adhesin-vaccinated mice was administered to BALB/cByJ (A') and SCID (B) mice, the animals were challenged i.v. with C. albicans, and the resulting kidney candidal CFU per gram of tissue were determined. Bars, standard errors. Differences between the values obtained from mice that received polyclonal antiserum and control mice that received NMS were significant (P ⁇ 0.01).
  • FIG. 2 shows MAb specific for a phosphomannan fraction that contains candidal adhesins protects mice against disseminated candidiasis.
  • BALB/cByJ mice were given polyclonal antiserum (pAb) , MAbs specific for either the mannan adhesin fraction (Mab B6.1) or some other cell surface determinant (Mab B6) , or buffer (DPBS) as a control.
  • the animals were challenged i.v. with 5xl0 5 viable yeast cells, and susceptibility to disseminated candidiasis was assessed by determining candidal CFU in the kidneys (A) or by survival curves (B) . In both cases, significant differences (P ⁇ 0.01) between that received either polyclonal antiserum or MAb B6.1 and DPBS controls were found.
  • FIG. 3 shows MAb B6.1 protects SCID mice against disseminated candidiasis.
  • BALB/cByJSmn-sci /J " male mice were given MAb B6.1 mtraperitoneally and challenged i.v. w th 5xl0 5 C albi cans cells.
  • the resulting survival curves were plotted and found to significantly (P ⁇ 0.01) differ from those of mice given buffer (DPBS) instead of the MAb.
  • Figure 4 shows disseminated Candidias By Survival Time Measurements.
  • Therapeutic effect of MAb B6.1 on Candida infected mice one hour infection
  • BALB/cByJ female mice, N35da old were given 5xl0 5 yeast cells i.v.
  • Figure 5 shows the therapeutic effect of MAb B6.1 on candidal infected mice (one hour infection) . Same as Figure 4 design except that kidney cfu 48h after the i.v. infection was used as the indicator of disease severity.
  • Figure 6 shows the Prophylactic effect of MAb B6.1 on mice to vulvovagmal candidiasis.
  • DPBS Dulbecco phosphate buffered saline
  • E estradiol Mice (BALB/cByJ, penale N35-45da old) were given estradiol subcu, 72h later they received buffer (DPBS) or MAb B6.1, l.p.
  • MAb B6.1 l.p.
  • animals received 5xl0 5 C. albicians .
  • Intravagmally 20 h later they received MAb B6.1 or buffer again l.p. for C. albicians cfu.
  • Figure 7 shows the prophylactic effect of candidal MAbs on mice to vulvovagmal candidiasis. All mice were pretreated with estradiol before the mAb treatments. Same design as B-3, but one group of animals received MAb B6.
  • Figure 9 shows the proposed structure of the phosphomannan complex (PMC) -in this case, n- linkage to cell wall protein is shown.
  • PMC phosphomannan complex
  • Figure 10 shows a P-2 size exclusives column. Fractions A-D are the void volume and all react with MAb B.6, but not MAb B6.1.) The 2-M extract was treated with lOmM HC1, 100°C, 60 min. before placing outo column.
  • Figure 11 shows a mass spectra for the mannan portion of the vaccine.
  • Figure 12 shows one dimension H-nmv of B6.1 epitope
  • FIG. 13 shows 2-DNMR of B6.1 epitope
  • Figure 14 shows the protective or prophylactic effect of the liposome-2ME extract (L-2ME) as a vaccine against disseminated candidiasis.
  • Mice were vaccinated with the L-2ME, with liposomes alone (L-PBS) or buffer alone (PBS), then challenged i.v. with various doses of C. albicans .
  • Figure 15 shows the therapeutic effect of MAb B6.1 on mice against vulvovaginal candidiasis .
  • Figure 16 shows the therapeutic effect of MAb B6.1, MAb 6 and DPBS on mice against vulvovaginal candidiasis.
  • Figure 17 shows the fractionation profile of the 2ME extract-BSA conjugate sample eluted from the Sephacryl-S- 300 size-exclusion column, two peaks were formed.
  • Figure 18 show the eluting locations (fraction numbers) of unconjugated 2 -ME extract and unconjugated BSA.
  • Monoclonal Antibody B6.1 (930610) was deposited under the terms of the Budapest Treaty on June 7, 1995 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA. ATCC Accession No.
  • MAb B6 Monoclonal antibody, MAb B6. 1, enhances resistance of mice against hematogenous disseminated candidiasis (Han and Cutler. 1995. Infect. Immun. 63:2714-2719) and against Candida vaginitis.
  • MAb B6.1 is specific for a ⁇ - 1, 2-trimannose carbohydrate moiety that is phosphodiester linked to the other mannan complexes, all of which are part of the phosphomanno-protein complex expressed on or near the surface of C. albi cans yeast cells.
  • a family of peptides that are recognized by MAb B6.1 has been defined.
  • Each of these peptides is nine amino acids in length and are referred to as nonapeptides .
  • Each nonapeptide that appears to mimic a carbohydrate epitope, as evidenced by reactivity with MAb B6.1 is referred to as a mimotope .
  • a model example of one mimotope is PS76p and its amino acid sequence is given below.
  • PS76p induces an antibody response in mice and the antibodies react with whole yeast cells of C. albicans , and with a -mercaptoethanol extract (2ME extract) of the fungus.
  • the 2 -ME extract contains the phosphomannoprotein complexes.
  • Sepharose 4B (CL4B-200, Sigma) was activated with CNBr and coated with either the IgM antibody MAb B6.1 (3 mg per ml packed beads) or with an irrelevant IgM MAb control.
  • the irrelevant IgM (from S. Pincus, MSU) was designated S10 and is specific for a protein antigen of group B streptococcus.
  • Ab-coated beads were washed and tested for functional activity by demonstrating their ability to form Candida yeast cell rosettes with B6.1-Sepharose, but not with SlO-Sepharose .
  • the affinity matrices were pre-blocked in phosphate buffered saline (PBS) plus 1% bovine serum albumin (BSA) prior to incubation with the PDPL.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • the PDPL was reacted first with the S10 affinity matrix.
  • An aliquot of the nonapeptide PDPL (approx. 7.5 x 10 11 ) phage or 1500 redundants of each nonpeptide represented in the library) was diluted in phage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% v/v Tween-20 and 0.1% BSA) and incubated with SlO-Sepharose in a small polystyrene tube (16 h, 4°C, with gentle rotation) .
  • phage buffer 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% v/v Tween-20 and 0.1% BSA
  • the mixture was transferred to a small polystyrene column and the non-adsorbed phage were recovered in the void volume and from the 50 ml phage buffer wash by precipitation with 0.15 vol of 16.5% polyethylene glycol (PEG), 3.3 M NaCl .
  • PEG polyethylene glycol
  • the S10 matrix was regenerated and blocked in PBS + 1% BSA. S10 preadsorption of the library was repeated three more times until the number of S10 adsorbent phage decreased substantially (by a factor of 10 5 ) .
  • the remaining PDPL i.e, those phage that did not react with S10 were reacted with the MAb B6.1 affinity matrix and clones reactive with MAb B6.1 were obtained as follows.
  • the preadsorbed PDPL (about 2 ml at 4.62 X 10 10 pfu/ml) was incubated w th MAb B6.1-Sepharose beads (16h, 4°C), transferred to a column and non-adherent phage were removed by extensive washing with pnage buffer.
  • Bound phage were eluted m 2 ml 0 1 M glycine buffer, pH 2.2 and the pH immediately neutralized A few microliters of the eluted phage were removed for phage titenng and the remaining phage were mixed with "starved" E. coli K91 for amplification.
  • the infected cells were incubated briefly Luna broth (LB) plus 0 75 ⁇ g ⁇ anamyc ⁇ n (kan) /ml and spread onto LB agar containing 75 ⁇ g kan/ml (LBkan) for overnight growth.
  • Colonies were scraped from the LBkan surface into Tris- buffered saline (TBS) and centrifuged to obtain the phage-rich supernatant fluid. Phage were precipitated and recovered by centrifugation. Half of the amplified phage were diluted m phage buffer and incubated with a fresh aliquot of the MAb B6.1- Sepharose for a second round of affinity selection. The eluted phage were titered and amplified as above, and half were subjected to a third round of selection with fresh MAb B6.1-Sepharose
  • This number (20-fold less than if the PDPL was not preadsorbed on an S10 affinity matrix) indicates that our preadsorption removed nonparatope specific clones, which should enhance the chances of isolating MAb B6.1-spec ⁇ fIC PDPL clones.
  • DNA sequencing and western blot analysis on the third selection pool of phage was done for further analysis.
  • Example 2 Analysis of the MAb B6.1 selected phage clones.
  • the third selection pool of phage was analyzed initially by random sequencing of phage clones, and then by a plaque lift step before sequencing as follows. An appropriate dilution of phage pool was plated, and single plaques were randomly isolated and stored individually in phage buffer. Phage minipreps were prepared in LBkan broth and harvested to provide single-stranded template DNA for sequencing with Sequenase 2.0 (USB/Amersham) .
  • the phage templates were primed with a gene III specific primer which anneals approximately 50 nucleotides (nt) from the 27-mer insert that codes for the nonapeptide expressed on the end of the pill protein of each phage.
  • Method 1 Inhibition of MAb B6.1 binding to blotted 2 -ME extract by intact phage. To determine the sensitivity, dot blots of 2-ME extract (0.5 ⁇ g/dot on nitrocellulose) were blocked in phage buffer, and surveyed with different concentrations of MAb B6.1 (from 0.001-20 ⁇ g Ab/ml). The secondary Ab was a 1:1000 dilution of alkaline phosphatase conjugated goat anti-mouse ⁇ -chain specific Ab (Sigma A-9688) . This method allowed for detection of MAb B6.1 at 0.005 ⁇ g MAb/ml, which was chosen for the phage inhibition studies.
  • MAb B6.1 at 5 ng/ml was preincubated (1 h, 22-24 C, gentle agitation) with the various selected plate clones or with the parent phage, M13KBst (3.5 x 10" pfu of each clone/ml) .
  • the Ab/phage mixture was added to pre-blocked 2-ME extract dots in separate tubes and incubated overnight at 4°C. Blots were washed, incubated with secondary antibody for 4 h, and washed in Tris/NaCl/MgCl 2 buffer (pH 9.5) and immersed in nitroblue tetrazolium/5-bromo-4-chloro-3 -indolyl phosphate.
  • Method 2 Inhibition of MAb B6.1 binding to phage dot blots by soluble 2 -ME extract.
  • MAb B6.1-selected phage clones PS2 , PS76, PS31 and the parent control phage M13KBst were prepared at various concentrations and dot blotted onto nitrocellulose (pfu per dot ranged from 2 x 10 10 up to 8 x lO 10 ) to determine the sensitivity of immunoblot detection with MAb B6.1 (0.5 ⁇ g Ab/ml phage buffer) and with a 1:1000 dilution of secondary antibody as above. Phage dots with 4 x 10 11 pfu were chosen. The clones were applied to nitrocellulose and preblocked in phage buffer.
  • MAb B6.1 was mixed with the 2 -ME extract at 5 or 50 ⁇ g carbohydrate/ml.
  • 2 -ME extract For inhibition studies done with soluble reactants, we used acid-hydrolyzed 2 -ME extract to free the B6.1 epitope from the remainder of the PM molecule in order to reduce the possible stearic hindrance preventing an inhibition from taking place.
  • the pre-blocked phage dots were added to the various solutions of antibody with or without extract and incubated overnight, 4°C. The blots were washed, incubated in alkaline phosphatase-labeled secondary antibody and detection was done as described above.
  • the 2 -ME extract at 50 ⁇ g/ml inhibited binding of antibody to all the phage clones. This inhibition was dose dependent, as the lower concentration of extract (5 ⁇ g/ml) did not inhibit. No antibody bound to the M13KBst parent phage. Phage clones PS55 and PS28 samples are currently being tested in similar assay
  • Synthetic peptide inhibits binding of MAb B6.1 to its' carbohydrate epitope.
  • the nonapeptide displayed by phage clone PS76 was chosen for synthesis and used in inhibition studies.
  • a synthetic 13-mer peptide (Bio-Synthesis, Lewisville, TX) , YRQFVTGFWGPPC , which was designed to include the PS76 nonapeptide sequence (designated as PS76p) plus the 3 amino acid pill tether (GPP) and an added cysteine (C) to facilitate peptide coupling to a carrier protein, such as keyhole limpet hemocyanin (KLH) .
  • PS76p the PS76 nonapeptide sequence
  • GPP 3 amino acid pill tether
  • C cysteine
  • PS76p Due to the high number of hydrophobic amino acids in the synthesized PS76p, solubility tests were run to determine conditions for dot blot inhibition studies.
  • the PS76p was soluble in trifluoroacetic acid, dimethylsulfoxide, 20% v/v acetic acid, citrate and acetate buffers below pH 5.4, borate buffer above pH 8.5, but not soluble in deionized water, phosphate buffered saline (PBS) , 15% v/v dimethylformamide, chloroform, or methanol .
  • PBS phosphate buffered saline
  • Inhibition assays at physiologic pH were performed with peptide attached to carrier protein.
  • the PS76p with the added cysteine as described above, was conjugated to KLH and to ovalbumin (OVA) by use of two different heterobifunctional , N-hydroxysuccinimide- ester crosslinkers (Pierce Chemical Co.), specifically m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and N- ⁇ -maleimidobutyrloxyl) succinimide ester (GMBS) .
  • MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester
  • GMBS N- ⁇ -maleimidobutyrloxyl
  • PS76-KLH PS76-KLH
  • PS76p-GMBS-OVA PS76-OVA
  • ELISA enzyme linked immunosorbent assay
  • PS76-KLH and PS76-OVA conjugates reacted with MAb B6.1, but not with MAb S10.
  • MAb B6.1 did not react with the KLH or OVA alone.
  • the PS76-KLOH conjugate was utilized in an ELISA-based inhibition assay as follows. Microtiter plate wells were coated with PS76-KLH and blocked as above.
  • MAb B6.1 (20 ⁇ l/ml in block) was pre-incubated with or without either 50 or 500 ⁇ g of acid-hydrolyzed 2ME extract per ml of solution, and then added to the conjugate coated well.
  • the 2ME extract inhibited MAb B6.1 binding to PS76-KLH a dose -dependent fashion.
  • PS76-KHL and PS76-OVA conjugates BALB/C mice (4 animals) were immunized s.c. with either PS76-KLH or PS76-OVA (250 ⁇ m per dose) . Intervals for booster immunization and obtaining serum samples were as above.
  • ELISA titer values against 2ME extract for both the anti-PS76-KLH and anti-PS65-OVA antisera were 320.
  • the antiserum samples also recognized the opposite carrier-peptide conjugate but not the opposite carrier protein.
  • ELISA tests performed with class- specific secondary antibody reagents confirm that anti- conjugate responses are primarily IgM.
  • PS76p as a multiple antigen peptide (MAP) construct.
  • the nonapeptide PS76p (YRQFVTGFW; wnere : Y, tyrosine; R. arginine ; Q, glutamine; F, phenylalanine ; V, valine; T, threonine; G, glycine; W, tryptophan) was synthesized on a branched- lys e core to produce eight, radically displayed peptides (Bio-synthesis, Lewisville, TX) .
  • This MAP construct, PS76-MAP when mixed with PBS is slightly soluble compared to the PS76p alone.
  • PS76-MAP was administered to BALB/C mice (4 animals, 25 ⁇ g per dose) by s.c. immunizations with Ribi adjuvant. Intervals for booster immunization and obtaining serum samples were as above.
  • the ELISA titer for pooled anti-PS76-MAP antiserum after the first boost was 40 against 2ME extract. ELISA tests performed with class- specific secondary antibody reagents confirm that the anti-PS76-MAP response is primarily IgM.
  • mice (1 animal per each phage clone) were immunized s.c. with 2 X 10 11 pfu of PS76, PS2, PS31, PS28, or PS55 mixed with Ribi adjuvant. Intervals for the first booster immunization and obtaining serum samples were as above.
  • the ELISA titers for the anti-phage antibody samples were: 320 for anti-PS55; 640 for anti-PS76, anti-PS2 and anti-PS28; and 1280 for anti-PS31.
  • Responses, tested against 2ME extract were primarily IgM class antibodies except for anti-PS31 which demonstrated a predominant IgG response .
  • YRQFVTGFW YRQFVTGFW
  • Y tyrosine
  • R arginine
  • Q glutamine
  • F phenylalanine
  • V valine
  • T threonine
  • G glycine
  • W tryptophan.
  • the amino acids may be coupled to carrier proteins, such as keyhole limpet hemocyanin (KLH) , tetanus toxoid, or a cell wall protein from C. albicans .
  • KLH keyhole limpet hemocyanin
  • the conjugate administered in combination with an appropriate adjuvant, such as the Ribi MPOL, will induce a protective antibody response and/or cell mediated response against hematogenous disseminated candidiasis and against Candida vaginitis.
  • an appropriate adjuvant such as the Ribi MPOL
  • Such vaccine preparations can be administered to patients who will be at high risk of developing hematogenous disseminated candidiasis and to women who experience recurrent Candida vaginitis .
  • Antibodies specific for the peptides could be used prophylactically to prevent hematogenous disseminated candidiasis and Candida vaginitis, and protective antibodies could be used therapeutically against Candida vaginitis.
  • the invention also investigates a vaccine induced alteration of pathogenesis of candidiasis generally, particularly hematogenous disseminated candidiasis and mucocutaneous candidiasis.
  • the invention focuses on optimizing a vaccine against Candida adhesins and determining the effect of immune serum on its ability to protect mice against candidiasis.
  • the inventors show that 1) the Candida vaccine can be used to protect naive individuals against Candida infections before they are infected; 2) the Candida vaccine can be used to treat previously infected individuals; 3) the antibodies can be used to protect naive individuals before they are infected; and 4) the antibodies can be used to treat previously infected individuals .
  • a mimetic peptide of the invention can be reverse translated into the DNA coding sequence, which itself can be used as a vaccine.
  • Techniques for preparing specified DNA sequences are well established. Also, the use of DNA fragments as vaccines, especially those intended to induce cell mediated immunity have been described and tested successfully.
  • Data of the invention indicates that I) immune responses against Candida phosphomannoprotem moieties protect mice against disseminated and mucocutaneous candidiasis, (ii) sera from immune animals transfer protection to naive mice.
  • the underlying emphasis of studies leading to the present invention was to determine the role of adhesm- specific anti odies host resistance to disseminated candidiasis and define the effects of these antibodies on fungal attachment phenomena as measured by several m vitro adherence systems, and by m vivo analysis.
  • the invention focuses on the phosphomannoprotem complex which the inventors have shown to contain adhesm sites.
  • the adhesm (s) responsible for adherence of C. albicans hydrophilic yeast cells to the splenic marginal zone was isolated, and presentation of the adhesm (as part of the phosphomannoprotem complex) to mice resulted in induction of specific antibody responses
  • Mice who develop anti- phosphomannoprotem responses show increased survival against disseminated candidiasis Sera from vaccinated mice specifically react with phosphomannoprotem.
  • Immune serum has been shown to passively transfer resistance to naive animals.
  • the invention addresses the role of antibodies host defense against disseminated candidiasis.
  • indwelling venous catheters are responsible for increased susceptibility to candidiasis and it is believed that venous catheters damage endothelia.
  • the kidney is a target organ for systemic disease and this organ normally has an exposed basement membrane (ECM) as part of the glomerular apparatus.
  • ECM basement membrane
  • Edwards has demonstrated that C. albi cans binds directly to the endothelial cells (Filler, S.G., et al . 1987. An enzyme-linked immunosorbent assay for quantifying adherence of Candida to human vascular endothelium. J. Infect. Dis. 156:561-566; and Rotrosen, D. et al . 1985.
  • Adherence of Candida to cultured vascular endothelial cella mechanisms of attachment and endothelial cell penetration. J. Infect. Dis. 153:1264-1274
  • this event may well initiate host inflammatory changes (Filler, S.G., et al . 1994. Mechanisms by which C. albicans induces endothelial cell prostaglandin synthesis. Infect. Immun. 62:1064- 1069; and Filler, S.G., et al . 1991. C. albicans stimulates endothelial cell eicosanoid production. J. Infect. Dis. 164:928-035).
  • a shear dependent adherence assay has allowed observations that corroborate some of the endothelial binding interactions.
  • the adherence of C. albicans hydrophilic yeast cells to mouse splenic marginal zone macrophages and macrophages within the subcapsular and medullary sinuses of peripheral lymph nodes has been characterized by the present inventors (Cutler, J.E., et al . 1990. Characteristics of C. albicans adherence to mouse tissue. Infect. Immun. 58:1902-1908; Han, Y., et al . 1993. Binding of C. albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages. Infect. Immun.
  • the adhesins responsible for the yeast/macrophage interaction have been isolated and characterized (Kanbe, T., et al . 1994. Evidence for adhesin activity in the acid-stable moiety of the phosphomannoprotem cell wall complex of C. albicans . Infect. Immun. 62:1662-1668); and Kanbe, T., et al . 1993. Evidence that mannans of C. albicans are responsible for adherence of yeast forms to spleen and lymph node tissue. Infect. Immun. 61:2578-2584).
  • the present inventors set out to determine whether antibodies are protective against disseminated candidiasis Given the complexity of adhesins and variable character of the cell surface of C. albicans, the role of antibodies in host defense against disseminated candidiasis has remained a contentious issue. Evidence that argues against a protective role for antibodies is derived mostly from clinical observations showing that precipitin antibodies specific for Candida antigens can be detected m the sera of most patients with disseminated or deep-seated candidiasis Experimentally, while some investigators reported that human antibodies specific for C. albicans enhance phagocytic cell uptake of fungal elements (Diamond, R.D., et al . 1978. Damage to pseudohyphal forms of C.
  • IgE responses may inhibit phagocytosis by human neutrophils of C. albicans indicates the importance of investigating the protective nature of Ig subtypes (Berger, M., et al . 1980. IgE antibodies to Staphylo coccus aureus and C. albicans m patients with the syndrome of hyperimmuno- globulin E and recurrent infections. J. Immunol. 125:2437-2443). In addition, none of the early investigators addressed the issue of antibody specificity.
  • mice with high antibody titers against C. albi cans were relatively resistant against hematogenously disseminated disease, and immunity was transferrable to naive mice via the anti-serum (Mourad, S., et al . 1961. Active immunization of mice against C. albicans . Proc . Soc . Exp . Biol . Med. 106:570-572; and Mourad, S., et al . 1968. Passive immunization of mice against C. albicans . Sabouraudia 6:103-105).
  • Domer's group determined that C. albicans cutaneous infection provoked mice to produce antibodies specific for the fungus, and such animals were less susceptible to disseminated candidiasis than control (Giger, D.K., et al . 1978 Experimental murine candidiasis: pathological and immune responses to cutaneous inoculation with C. albicans . Infect. Immun. 19:499-509) . Further experiments supported a specific protective effect. If B-cells were depleted by anti- ⁇ therapy, the mice were unable to make antibody in response to the cutaneous infection, their T-cell activities appeared unaffected, but these animals were more susceptible to disseminated disease than controls (Kuruganti, U., et al . 1988.
  • C. albicans The surface of C. albicans is variable, and the inventors have obtained evidence that immunodominant antigens may not necessarily be involved in critical host-C. albi cans interactions, such as adherence events.
  • a major antigen expressed on the surface of serotype A strains is not an adhesin.
  • C. albicans readily activates the alternative complement cascade and C3 deposition on the Candida cell surface promotes ingestion by phagocytic cells, an opsonic role for specific antibodies may not be very important (Morrison, R.P., et al . 1981. In vitro studies of the interaction of murine phagocytic cells with C. albicans . J. Reticuloendothel . Soc .
  • the present inventors show that the vaccine protected mice by production of antibodies specific for Candida adhesins. Perhaps the ideal protective antibody response would prevent adherence of circulating yeast cells to endothelial and subendothelial surfaces, while enhancing or not affecting an interaction with phagocytic cells.
  • CMI T-cell dependent cell mediated immunity
  • CMI T-cell dependent cell mediated immune
  • the inventors have studied disseminated candidiasis, and immune responses to C. albicans, in normal and immunocompromised mice for over twenty years. Recently the variable nature of the cell surface of C . albi cans and antibody responses by mice to C. albicans cell wall antigens have been analyzed. The function of the moieties on the fungal cell surface and adherence properties was investigated. Work progressed from characterizing the surface of C. albicans to an understanding of functions of cell surface moieties as they relate to candida-host interactions .
  • C. albicans are either serotype A or B, but one or the other serotype may predominate in human subjects depending on the immunological status of the patient.
  • the prototype strains used are CA-1 (serotype A) and A-9 (serotype B) that have been extensively studied in the laboratory.
  • An important consideration in all work on C. albicans is the inherent variability potential of the species. Culture conditions and handling of the strains have been standardized to stabilize their characteristics and allow for long-term reproducible results .
  • Example 8 Culturing of C. albicans to maintain constant characteristics. Strains of C. albicans show genetic instabilities and antigenic variability. To maintain constancy in surface characteristics throughout the experiments, the strains will be stored in 50% glycerol at -20°C, and as cell pellets in sterile water at -
  • Fresh new working cultures will be prepared form the frozen stocks every week.
  • a loopful of the glycerol stock will be used to inoculate 25 ml of GYEPB (2% glucose, 0.3% yeast extract, 1% peptone broth) in a 50 ml Erlenmeyer flask, the culture will be incubated for 24 h at 37°C under aeration by rotation at 160-180 rmp, then serially transferred to fresh GYEPB (e.g., 3 drops of culture may be transferred to 25 ml GYEPB three to six times at 24 h intervals and incubated as above) .
  • This procedure produces almost 100% hydrophilic yeast forms in stationary phase of growth.
  • Yeasts are harvested by centrifugation, the pelleted cells are washed three times in ice-cold deionized water, held on ice as pelleted cells until use (up to 2h) , and suspended to the appropriate working concentration in the appropriate medium.
  • yeast cells may be grown to have a hydrophobic cell surface (Hazen, K.C., et al . 1991. Differential adherence between hydrophobic and hydrophilic yeast cells of C. albicans . Infect. Immun. 59:907-91212; Hazen, K.C., et al . 1986. Influence of growth conditions on cell surface hydrophobicity of C. albicans and Candida glabrata . Infect. Immun. 54:269- 271) . The cultures are grown exactly as above, except that incubations are at 24 °C.
  • a microsphere assay is used to monitor the percentage of cells that have a hydrophobic or hydrophilic cell surface (Hazen, K.C., et al . 1987. A polystyrene microsphere assay for detecting surface hydrophobicity variations within C. albi cans populations. J. Microbiol. Methods. 6:289-299). Equal volumes (100 ⁇ l) of yeast cells (2 x 10 6 /ml) and hydrophobic (i.e., low sulfate) blue polystyrene microspheres (diameter, 0.801 ⁇ m; ca .
  • Tissue adherence characteristics of C. albicans and adhesin isolation By use of an ex vivo adherence assay, the adherence characteristics of hydrophilic and hydrophobic yeast cells to mouse splenic and lymph node tissue was examined (Cutler, J.E., et al., 1990, Characteristics of Candida albicans adherence to mouse tissues. Infec . Immun. 58:1902-1908); Han, Y., et al . 1993. Binding of C. albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages. Infect. Immun. 61:3244-3249; and Hazen, K.C., et al .
  • C. albicans hydrophilic yeast cells specifically adhere to mouse splenic marginal zone macrophages (Cutler, J.E., et al . 1990. Characteristics of C. albicans adherence to mouse tissue. Infect. Immun. 58:1902-1908; Kanbe, T., et al .
  • Complement may play a role in organ distribution of C . albicans from the blood.
  • the pattern of yeast cell adherence to the spleen is not influenced by the presence of fetal bovine serum, or the absence of serum in the ex vivo assay (Cutler, J.E., et al . 1990. Characteristics of C. albicans adherence to mouse tissue. Infect. Immun. 58:1902-1908; and Riesselman, M.H. et al. 1991. Improvements and important considerations of an ex vivo assay to study interactions of C. albicans with splenic tissue., J. Immunol. Methods 1450:153-160).
  • yeast cells are opsonized in fresh mouse serum without detectable antibodies against C. albicans (Morrison, R.P., et al . 1981. In vi tro studies of the interaction of murine phagocytic cells with C. albicans . J. Reticuloendothel . Soc . 29:23-34) binding to the marginal zone is enhanced by 50-200% (Tripp, D.L. et al . 1994. Evidence for complement independent in vivo adherence of C. albicans . Abstr. Annu . Meet. ASM.) . In vivo binding of yeast cells to the splenic marginal zone appears unaffected by complement opsonization .
  • Yeast cells become opsonized by incubation for 30 min at 37°C in the presence of 2.5% (or more) fresh mouse serum (Morrison, R.P., et al . 1981. In vi tro studies of the interaction of murine phagocytic cells with C. albicans . J. Reticuloendothel. Soc. 29:23-34) .
  • the opsonization is due to activation of the alternative complement cascade and is required for optimal phagocytosis by mouse peritoneal macrophages.
  • 8 x 10 8 yeast cells are complement opsonized and given i.v.
  • mice made complement C3 deficient by treatment with cobra venom factor still show the same yeast cell adherence in vivo as in complement sufficient animals (Tripp, D.L. et al . 1994. Evidence for complement independent in vivo adherence of C. albicans . Abstr. Annu. Meet. ASM). These results have been confirmed by Kozel's group who used a different approach. Cobra venom depleted C3 mice and normal control animals were given viable yeast cells. Forty-five min.
  • C3 depleted mice had higher counts in the lungs as compared to normal controls, implying that complement may play a role in the organ distribution of C. albicans yeast cells from the circulation.
  • Adhesins responsible for attachment of hydrophilic yeasts to splenic marginal zone are glycans (mannans) and not protein.
  • the adhesins responsible for attachment of hydrophilic yeast cells to the marginal zone macrophages are solubilized from the fungal cell surface by extraction with ⁇ -mercaptoethanol (2ME extract) (Kanbe, T., et al . 1993. Evidence that mannans of C. albicans are responsible for adherence of yeast forms to spleen and lymph node tissue. Infect. Immun. 61:2578-2584) .
  • Pellet Candida for 10 min. by centrifugation at 2,500xg, 4-6°C. 3. Wash the pelleted cells 2X with cold deionized water (dH,0) .
  • the 2 -ME extract contains the phosphomannoprotein complex.
  • Candida adhesins are contained within the mannan portion of the complex.
  • the 2ME extract blocks binding of hydrophilic yeast cells to the splenic marginal zone macrophages.
  • latex beads coated with the 2ME extract bind to the splenic macrophages in a pattern identical to that of whole yeast cells.
  • the activity of the adhesins in the 2ME extract is not affected by boiling or proteolytic enzymes, but is destroyed by periodate oxidation and ⁇ - mannosidase digestion.
  • the adhesins are glycans, probably mannan, and not proteins.
  • the 2ME extract can be fractionated further by proteinase K digestion and con A-affinity chromatography to yield an adhesin fraction, termed Fr.II that is practically devoid of detectable protein, yet retains full adhesin activity (Kanbe, T., et al . 1993. Evidence that mannans of C. albicans are responsible for adherence of yeast forms to spleen and lymph node tissue. Infect. Immun. 61:2578-2584).
  • the mannan nature of the adhesins is further supported by subsequent purification work which showed the adhesin activities to be associated with the mannan portions of the phosphomannoprotein (PMP) .
  • the PMP was degraded by mild acid hydrolysis, and the released oligomannosyl side chains were size separated by P-2 column chromatography (Li, R.K., et al . 1993. Chemical definition of an epitope/adhesin molecule on C. albicans . J. Biol. Chem. 268:18293-18299).
  • mAb 10G available from the lab of Dr. Cutler
  • the tetramannosyl is a ⁇ -l,2-linked straight-chained tetramannose and is one of the adhesin sites in the PMP.
  • the purified tetramannose blocks binding of yeast cells to the splenic marginal zone, and latex beads coated with the epitope bind to the marginal zone in a pattern essentially identical to yeast cell binding. This work represents the first identification to structure of an adhesin on the surface of C. albicans .
  • the inventors induced in mice an antibody response against 2 -ME extract and have obtained nine mAbs specific for this fraction.
  • a simplified model of cell wall phosphomannoprotein (PMP) of C. albicans serotype B based on a structure by others is available (Kobayashi, H., et al . 1990. Structural study of cell wall phosphomannan of C. albicans NIH B-792 (serotype B) strain, with special reference to X H and 13 C NMR analyses of acid-labile oligomannosyl residues. Arch. Biochem. Biophys . 278:195-204) . The number of mannose units in each oligomannosyl side chain ranges from 1-7.
  • the 2ME extract was then formulated into liposomes to test its effectiveness as a vaccine. The method of preparing the liposomes is set forth below.
  • L- ⁇ -phosphatidylcholine (L- ⁇ -lechithin) Type XI-E, from frozen egg yolk, P-2772 (Lot#- 112H8362) , chloroform solution (100 mg/ml), Sigma, St. Louis, MO.
  • the liposome-encapsulated 2-ME extract is finally suspended in 4 ml PBS and stored at 4°C under nitrogen.
  • the liposome-2-ME extract complex should show a yellowish color by the phenol- sulfuric acid test for carbohydrates, thus indicating the presence of 2 -ME extract in the liposomes.
  • the phenol-sulfuric acid procedure (Dubois) is done as follows: place 60 ⁇ l of the liposome-2ME extract preparation into a well of a microtiter plate and mix with 30 ⁇ l of 5% phenol solution. Incubate the mixture at 21-23°C for 2 min and add 120 ⁇ l of concentrated sulfuric acid. Observe a color change from colorless to yellow for the positive reaction. Read the color change at an optical density of 490 ran. By use of this optical density (OD) was compared to the standard dilutions of 2-ME-extract in PBS. The results were as follows :
  • Example 12 Liposomes made of phosphatidycholine/cholesterol which contained 178 ⁇ g of 2ME extract per 0.2 ml preparation were used as the vaccine preparation. Mice were immunized by giving 5 weekly intravenous (i.v.) injections of varying doses (0.1-0.3 ml) of the liposome-2ME extract per animal. One group of mice received 0.2 ml of the preparation on days 1, 3, 5 and 10, and then weekly for two more weeks. Control mice received either liposomes prepared with the 2ME extract diluent (phosphate-buffered saline, PBS) , PBS alone, or an equivalent amount of 2ME extract in PBS.
  • PBS phosphate-buffered saline
  • mice were bled and tested for agglutinins by determining if the sera agglutinated whole yeast cells or latex beads coated with the 2ME extract. Mice immunized weekly for 5 weeks with 0.1 ml or 0.2 ml of the preparation gave the highest agglutinin titers
  • mice immunized against 2ME extract in PBS produced titers less than 5, or none at all.
  • the liposome-encapsulation method of antigen presentation induces in mice polyclonal antisera against antigens within the 2ME extract including Candida adhesins, and will allow for subsequent isolation of mAbs against these antigens. (The inventors have been able to perfect the vaccine such that a liposome is not required.)
  • Liposome-encapsulated 2ME extract promotes strong antibody responses, but the 2ME extract alone is not very immunogenic in mice.
  • Adjuvants such as those of Ribi (Ribi Adjuvant System) and Hunter (TiterMax) are not very effective in inducing mice to make antibody against the glycan moieties with the 2ME extract. Less than 50% of the mice sensitized against the 2ME extract-Ribi adjuvant combination produced a slight antibody response, and none of the animals responded when the Hunter adjuvant was used.
  • mAb 10G is specific for an adhesin site in the acid- labile portion of the PMP contained in the 2 -ME extract and the inventors have obtained nine new mAbs against the 2 -ME extract. Fusion, cloning and selection methods have been used extensively and described in detail (Brawner, D.L., et al . 1984. Variability in expression of a cell surface determinant on C. albicans as evidenced by an agglutinating monoclonal antibody. Infect. Immun. 43:966-972; Cutler, J.E., et al . 1994.
  • mouse splenic macrophages were eliminated by intravenous (i.v.) delivery of liposome-entrapped dichloromethylene diphosphonate (L-C1 2 MDP) . This liposome conjugate becomes selectively taken up by macrophages, which causes their elimination.
  • i.v. intravenous delivery of liposome-entrapped dichloromethylene diphosphonate
  • mice When macrophage depleted mice were systemically challenged with C. albicans , clearance of viable fungal elements from blood was slower, their kidneys had higher recoverable cfu, and neither BALB/c nor nu/nu mice survived as long as control mice. Mice given L- Cl 2 MDP recovered most of their macrophage function by 56 days and became normal in their resistance to C. albicans .
  • Example 14 2ME extract from a Cryptococcus neofor ans acapsular mutant does not have adhesin activity and serves as a negative control.
  • a fungal 2ME extract that does not contain candida-like adhesin activity was obtained.
  • Saccharomyces cerevisiae and on the Ballou mutant strains mnnl, mnn2 and mnn4 (Raschke, W.C. et al . 1973. Genetic control of yeast mannan structure, Isolation and characterization of yeast mannan mutants, J. Biol . Chem. 248:4660-4666).
  • the strains were grown at various temperatures and yeast from different phases of growth were analyzed for their binding characteristics to mouse splenic tissue.
  • S . cerevisiae produces some, but not all, of the Candida adhesins responsible for yeast cell binding to the splenic marginal zone.
  • the acapsular mutant strain 602 of C. neoformans was examined (Kozel, T.R., et al . 1971.
  • Nonencapsulated variant of Cryptococcus neoformans 1 Virulence studies and characterization of soluble polysaccharide . Infect. Immun. 3:287-294).
  • C. neoformans strain 602 log and stationary phase cells were removed form the various growth conditions and tested for adherence to splenic tissue in the ex vivo assay. None of these growth conditions yielded adherent yeast cells. Stationary phase cells extracted by the ⁇ -mercaptoethanol method gave a water soluble cell wall material that did not affect binding of C. albi cans yeast cells. That is, in the ex vivo assay, pretreatment of splenic sections with 10 ⁇ g, 25 ⁇ g and 100 ⁇ g of the cryptococcal 2ME extract had no detectable effect on binding of C. albicans hydrophilic yeast cells to the marginal zone, as compared to over 95% inhibition of binding due to pretreatment of splenic tissues with 1 ⁇ g of 2ME extract from C. albicans yeast cells.
  • the chemical nature of the cryptococcal 2ME extract is apparently mostly glucan (James, P.G., et al . 1990. Cell-wall glucans of Cryptococcus neoformans CAP 67. Carbohyd. Res. 198:23-38) which serves as a non-specific control material.
  • adhesin fractions from serotype A and B strains (CA-1 and A- 9, respectively) . Both adhesin fractions cause identical dose response inhibition of binding of either serotype A or B strain yeast cells. Data show animals vaccinated against serotype A 2ME extract became protected against disseminated candidiasis by the serotype B strain. Because serotype B strains apparently contain all antigens found on serotype A strains, but serotype A strains have one (or more) cell surface antigens not found on serotype B strains (Hasenclever, H.F., et al . 1961. Antigenic studies of Candida I.
  • mice represent the simplest and most accepted experimental mammalian model of human candidiasis. Work derived from the survival and cfu experiments is more directly applicable to human needs than other non- animal studies proposed.
  • mice Male and female BALB/c and BALB/c outbred crosses are used to test the ability of various non-toxic vaccine to induce antibody responses. These mouse strains and thymic deficient (nude) mice on a BALB/c background and SCID mice are used for testing the ability of antibodies to protect animals against disseminated candidiasis. In addition, colonies of BALB/c mice crossed with an outbred mouse to yield the vigorous strain (BALB/c ByJ x Cri : CD-I (1CR) BR) FI, and henceforth referred to as CD-I, are also available from Montana State University. Initially, groups of three animals are used to assess the efficacy of the immunizations in terms of antibody titers.
  • the number of animals used is based upon numbers required for statistical analysis.
  • the experiments are evaluated by either fungal colony forming units (cfu) in animal organs retrieved well before ill-effects of the disease are apparent, or by animal survival.
  • the vaccine preparations are assessed by determining their relative ability to induce antibody responses in mice. In studies it was found that 0.1- 0.2ml of the liposome-2ME extract complex is more immunogenic than other doses, and weekly boosters work best. Work was performed primarily on female BALB/c mice which have relatively high innate resistance to disseminated candidiasis (Hector, R.F., et al . 1982. Immune responses to C.
  • a reliable determination of 2ME extract adhesin content can be made by the phenol-sulfuric acid method of Dubois for carbohydrate.
  • protein assays such as the BCA, Pierce
  • mice against liposome-encapsulated 2-ME extract protects the animals against disseminated candidiasis.
  • BALB/c female mice were immunized against the 2ME extract containing the mannan adhesins by encasing the extract in liposomes as indicated above.
  • Each mouse from groups of 4 mice each were immunized against the liposome-2ME extract conjugate by giving 0.2 ml i.v. once each week for five weeks. All mice pro ⁇ uced an agglutinin antibody titer from 20-40 m 100% of the mice as measured by agglutination of 2ME extract-coated latex beads.
  • mice immunized against the adhesin fraction showed increased survival Limes, as compared to PBS controls, when challenged with a lethal dose of C. albicans yeast form cells Although increased survival was more apparent when mice were challenged with 2.5 x 10 5 yeast cells (i.e , 0 2 ml i.v. of a concentration of yeast cells of 12.5 x 10 5 /ml PBS), slight prolongation of survival was a.so noted mice challenged with four times more yeast cells. In a repeat experiment, an additional group of mice was added that received 2ME extract PBS (the same amount of 2ME extract as complexed within the 2ME extract -liposome vaccine) . These animals, which did not produce antibodies, did not show increased survival .
  • 2ME extract PBS the same amount of 2ME extract as complexed within the 2ME extract -liposome vaccine
  • Example 18 The inventors use passive transfer experiments to determine if antibodies are responsible for immunity. Immune sera from vaccinated animals, mAbs specific for the 2 -ME extract of C. albicans , and mAbs against hydrophobic proteins of C. albicans are tested for their ability to protect naive animals against disseminated candidiasis. Immunologicaily competent mice, T-cell deficient (nu/nu) , T- and B- cell deficient (SCID) , and mice with induced neutropenia (by use of the anti-neutrophil antibody, mAb RB6-8C5) are tested.
  • the ex vivo assay, the capillary tube shear- dependent adhesin assay, the endothelial adherence assay, and m vivo mtravital microscopic methods are used to determine the effect of immune sera and protective mAbs on adherence characteristics of C. albicans to various host cells, tissues and glycoproteins .
  • the effect of immune sera and mAbs on adherence characteristics of complement opsonized cells and unopsonized cells is examined.
  • mice were immunized for the five week period to induce antibody responses against the adhesin fraction. They were then rendered immunocompromised by treatment with either mAb RB6-8C5, at 100 ⁇ g antibody/mouse i.v., that severely depletes neutrophils in vivo (Czuprynski, C.J., et al. 1994. Administration of anti-granulocyte mAb RB6-8C5 impairs the resistance of mice to Listeria monocytogenes infection. J. Immunol. 152:1836-1846; and Jensen, J.T., et al . 1993. Resistance of SCID mice to C.
  • yeast challenge mice that were first vaccinated, then treated with mAB RB6-8C5 to make them neutropenic, and then challenged with C. albicans were still protected against disseminated candidiasis (as compared to the control mice that received treatment of mAB RB6-8C5 without prior vaccination.
  • Sera from immune animals neutralize adhesin activity and blocks yeast attachment. Sera from vaccinated mice react with the adhesin fraction as evidenced by specific agglutination of adhesin-latex bead conjugates.
  • Sera from vaccinated mice react with the adhesin fraction as evidenced by specific agglutination of adhesin-latex bead conjugates.
  • C. albicans yeast cells will not bind to the tissues (Kanbe, T., et al . 1993. Evidence that mannans of C. albicans are responsible for adherence of yeast forms to spleen and lymph node tissue. Infect. Immun. 61:2578-2584, and our unpublished data) .
  • 2ME extract will not inhibit yeast adherence if the extract is treated with antiserum from vaccinated animals.
  • antiserum from BALB/c mice vaccinated against the 2ME extract was heat inactivated (56°C, 30min) and produced a specific agglutinin titer of 40 against the 2ME extract-coated latex beads.
  • 1 ⁇ g, 2 ⁇ g and 4 ⁇ g of 2ME extract was each mixed for 1 h on ice with a 1:4 dilution of antiserum.
  • Binding was compared with control sections pretreated with the 2ME extract concentrations but without antiserum, and with sections pretreated with normal mouse serum (NMS) (positive binding control) .
  • NMS normal mouse serum
  • mice against the 2ME extract contains antibodies that neutralize Candida adhesins responsible for yeast cell binding to the marginal zone, the antibodies also block yeast cell attachment and the blocking ability of the antiserum appears to be dose dependent .
  • Example 10 Evidence that immune serum transfers protection.
  • immune serum i.e., anti-2ME extract
  • 20 vaccinated mice 20 vaccinated mice.
  • NMS was collected from mice that received an equal number of injections of PBS.
  • mice Three groups of normal naive BALB/c mice (three/group) were given the following: Group 1 received 0.5 ml of immune serum i.p. on Day 1; Group 2 mice received 0.5 ml NMS from PBS-treated animals; Group 3 mice did not receive serum. Four hours later, each mouse was challenged i.v. with 5 x 10 5 yeast cells. The following day, the appropriate mice received either 0.2 ml antiserum, NMS or PBS. At the yeast cell challenge dose, it was expected that normal mice would begin to die of disseminated candidiasis by day 9 or 10 and all mice should die by day 20.
  • Control mAbs (2B3.1 and H9) and our anti-adhesin mAb (mAb 10G) are of the IgM class.
  • Isotype switching work can be performed as known in the art (Schlageter, A.M. et al. 1990. Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide . Infect. Immun. 58:1914-1918) if required to provide specificity for monoclonal antibodies of the invention.
  • the number of different kinds of mAbs in the pooled mAb preparations are systematically dissected to determine the minimum number required for protection.
  • non-binding yeasts such as C. neoformans or S . cerevisiae transformed with plasmid only are used as negative binding controls.
  • NMS and isotype-matched irrelevant mAbs are used as negative controls for immune polyclonal antisera and mAbs, respectively.
  • the detailed use of these various adherence techniques and data acquisition/evaluation methods are given in the respective proposal from each investigator and/or their publications.
  • the pathogenesis of hematogenous disseminated candidiasis appears to involve adhesion events between yeast cells of C. albicans and specific host tissues. Host antibodies specific for Candida adhesins alter the pathogenesis and may aid host survival.
  • Candida adhesins have been isolated that cause specific yeast cell adherence to mouse splenic marginal zone macrophages. These adhesins are part of the phosphomannoprotein (PMP) complex on the Candida cell surface. Vaccines made of solubilized adhesins encapsulated in liposomes provoke antibody responses in mice against the adhesins. Vaccinated animals have increased resistance against disseminated candidiasis, their serum neutralizes adhesin activity, prevents yeast cell attachment to the spleen and appears to transfer protection. Monoclonal antibodies (mAbs) against the PMP-derived adhesins are available from Dr. Cutler.
  • mAbs Monoclonal antibodies
  • the vaccine may be formulated in liposome formulations as set forth above. Additional formulations may be prepared as with formulations and adjuvants as known in the art (see Remingtons Pharmaceutical Sciences, 18th ed., Mack Publishing Co., 1990) . Vaccines may include from 0.01 to 99.00% by weight adhesin composition.
  • the vaccine of the present invention may, in a preferred embodiment, be formulated in an effective amount of about 0.5g per human of 1501bs.
  • Organisms culture conditions and isolation of the adhesin fraction.
  • C. albicans serotypes A (strain CA-1) and B (strain A9) were used and previously characterized (8,20,21,49).
  • C. tropicalis strain CT-4 is from Montanta State University stock collection and species identification was confirmed by API 20C Yeast Identification Strips (Analytab Products, Plainview, NY) .
  • Stock cultures were stored and maintained as described (19,20) and grown to stationary phase in GYEP broth (19,20) at 37 C.
  • the yeast cells were washed three times in sterile deionized water, suspended to the appropriate concentration in sterile Dulbecco's phosphate buffered saline (DPBS) (Sigma Chem. Co., St. Louis, MO), and used to challenge mice.
  • DPBS Dulbecco's phosphate buffered saline
  • the PMC (referred to as the adhesin extract) was obtained in crude form, as before (19,20), by a ⁇ - mercaptoethanol extraction of the serotype A isolate of C. albicans . Less than 1 mg of this extract inhibited adherence of yeast cells to splenic and lymph node macrophages, hence, it contains the adhesins (17,20) . Chemically, the extract is primarily mannan with about 3.5% protein. Following proteinase digestion, the protein content dropped to 0.47%, yet all adhesin activity was retained (20) .
  • the film was dissolved in 10 ml of chloroform, evaporated again, dispersed at room temperature for 10 min in 5 ml DPBS containing 10 mg of the adhesin extract, allowed to stand for 2 h, sonicated for 3 min and held at room temperature for an additional 2 h.
  • the preparation was sedimented by centrifugation at 1,000 x g for 30 min. The pelleted liposomes were suspended in 5 ml DPBS, pelleted again and this process was repeated two more times.
  • the liposome- encapsulated adhesin extract referred to as L-adhesin
  • the amount of adhesin extract within the L-adhesin was 178 mg/ml as determined by the phenol-sulfuric acid reaction (12) .
  • Control liposomes were prepared exactly as above, but buffer (DPBS) without adhesin extract was added during the preparation. These control liposomes are referred to as L-PBS.
  • mice were used and housed in accordance with institutional regulations in an AAALAC certified animal facility.
  • BALB/cByJ Jackson Labs, Bar Harbor, ME
  • mice 6-7 weeks old, received the initial vaccine and weekly booster immunizations.
  • Each injection consisted of 0.2 ml of the liposome- adhesin complex (L-adhesin) administered intravenously (i.v.) .
  • Anti-adhesin titers in mouse sera were assessed by slide agglutination against latex beads coated with the adhesin extract. Adhesin coating was done as before (19,20,27).
  • mice When the agglutinin titers reached 40 or more (usually by the 4th booster) , the animals were challenged. Control mice received an equal volume and number of injections consisting of diluent (DPBS) only prior to challenge. The mice were challenged i.v. with viable yeast cells prepared to the appropriate concentration in 0.2 ml DPBS. Treatment of polyclonal antiserum.
  • DPBS diluent
  • polyclonal antiserum was obtained and pooled from vaccinated mice.
  • the serum fraction was either immediately stored at -20 C, heated at 56 C for 30 min prior to use, or adsorbed five times with formalin killed washed C. albicans strain 1 yeast cells at a ratio of ten volumes antiserum to one volume DPBS-washed packed dead yeast cells.
  • the antiserum was also fractionated by passage through an ABx HPLC column (J T. Baker, Phillipsburg, NJ) as described (40) to obtain pools of various separated serum components, including a fraction which contained all of the agglutinin activity.
  • buffer A consisted of 25 mM MES (2-[N-[N-
  • buffer B was 1M sodium acetate, pH7.0.
  • One part of polyclonal antiserum was mixed with two parts buffer A and the mixture was loaded onto the ABx column with buffer A at a flow rate of 1.5 ml/mm and each fraction was 40 drops At ten minutes, the percent of buffer B was brought to 20, at 15 mm buffer B was brought to 50%, at 20 mm it was brought to 70%, at 25 mm it was brought to 100% and was retained at 100% until 55 mm at which time the run was terminated.
  • each of the peaks detected by absorption at 280 nm was collected, dialyzed against at least 100 volumes of DPBS at 4 C with a minimum of four changes of DPBS over a 36 h period, and each pooled fraction was concentrated by ultraflltration ⁇ PM30 Diaflo Ultraflltration membrane, Amicon Division, Beverly, MA) Each concentrated fraction was brought to approximately one-half of the original starting volume of antiserum applied to the column Each was tested for the ability to agglutinate whole yeast cells and latex beads coated with the adhesin fraction.
  • mice Normal mouse serum (control) , polyclonal antisera, antisera heated at 56 C, C. albi cans-adsorbed antisera and HPLC-fractionated polyclonal antisera were tested for their ability to transfer resistance against disseminated candidiasis to naive mice.
  • test serum 7-8 week old female or male BALB/cByJ mice (Jackson Labs) were given 0.5 ml of the test serum intraperitoneally (i.p.), 4 h later they were given 0.2 ml i.v. of a suspension containing 2.5 x 10 6 yeasts/ml DPBS and 20 h later they were given i.p. another 0.2 ml of test serum.
  • Candida cfu/g kidney were determined as described below.
  • passive transfer of immune serum and challenge with live yeast cells were done in 18-20 weeks old male SCID mice (BALB/cByJSmn- scid/J, Jackson Labs) .
  • mAbs monoclonal antibodies
  • Mice were immunized with whole yeast cells (4) or the L-adhesin (11) and two mAbs specific for yeast surface epitopes were isolated as before (4,11) .
  • MAb B6 has the same specificity as mAb C6 (6) and mAb B6.1 is specific for an epitope in the PMC of C. albicans .
  • the epitope specificity of mAb B6 differs from mAb
  • the mAbs were produced in serum free medium, concentrated by ammonium sulfate precipitation, and suspended and diluted in DPBS to give identical agglutinin titers. The same strategy as described above for polyclonal antiserum was used to determine the ability of mAbs B6 and B6.1 to transfer protection.
  • agglutinin titers of each mAb was diluted to 20 (approximately 220 mg/ml for mAb B6.1 and 290 mg/ml for mAb B6) before administration to the BALB/cByJ mice.
  • mAb B6 was obtained from ascites fluid, adjusted to an agglutinin titer of 320 and compared to the effect of mAb B6.1 at a titer of 20.
  • albicans cfu m kidney tissue may be used as an indicator of disease severity (28,43,46)
  • the cfu determinations were done by homogenizing the kidneys with glass tissue homogenizers as described (43) except that the kidneys were homogenized 1 ml DPBS and plated onto Mycosel agar (BBL Microbiology Systems, Becton Dickinson and Co., Cockeysville, MD) Statistical significance of difference between test and control groups was determined by the Student t-test.
  • Vaccinated mice have increased survival rates . Vaccinated mice showed more resistance to disseminated candidiasis than did control mice as indicated by an increase in mean survival times following challenge (Table 3) To demonstrate a requirement for liposome delivery, some animals were given i.v. an equivalent amount of adhesin extract (178 mg) m 0.2 ml DPBS, but without liposomes. The mean survival times of these animals did not differ from animals that received only DPBS (data not shown) .
  • Serum from vaccinated mice transfers protection.
  • mice were passively given, as above, the anti -serotype A polyclonal antiserum and challenged i.v. with either a serotype B strain of C. albicans (5xl0 5 yeast cells) or a strain of C. tropicalis (lxlO 6 yeast cells) .
  • Kidneys were removed 48 h later for cfu determinations.
  • Antiserum-treated mice challenged with the serotype B strain had 11.3 (+ . 2.7) x 10 3 cfu/g kidney tissue, while normal mouse serum (NMS)- treated mice (controls) had 41.4 ( ⁇ 7.0) x 10 3 cfu/g (p ⁇ 0.001) ( ⁇ are standard error values) .
  • antiserum- treated mice challenged with C. tropicalis developed 145 ( +16) x 10 3 cfu/g kidney as compared to 267 ( ⁇ 34) x 10 3 cfu/g for NMS-treated controls (p ⁇ 0.001) ( ⁇ are standard error values) .
  • MAb B6.1 transfers protection, but mAb B6 does not.
  • both mAbs are strong agglutinins and are of the same class, only mAb B6.1 transferred protection against disseminated candidiasis to naive BALB/cByJ mice. This result was demonstrated by both cfu/g kidney counts and by survival curve analysis. In these experiments, both mAbs were standardized to have the same agglutinin titers as indicated in the Materials and Methods. In one experiment, the titer of mAb B6 was increased to approximately 16 times that of mAb B6.1 and administered to mice in the volumes and schedules as indicated.
  • mice with enhanced resistance were those that were L-adhesin vaccinated and had agglutinin titers of 40-80.
  • mice vaccinated with only the adhesin extract developed low anti-adhesin titers (less than 5) and showed no enhanced resistance.
  • polyclonal antiserum from vaccinated mice protected naive normal BALB/cByJ and SCID mice from disseminated disease. SCID mice, however, did not make antibodies or develop a protective response as a result of the vaccinations (data not shown) .
  • mice that received mAb B6.1 which is specific for the adhesin extract and is a strong agglutinin of whole yeast cells, developed fewer cfu in their kidneys following challenge and both normal and SCID mice survived significantly longer than control animals.
  • mAb B6.1 which is specific for the adhesin extract and is a strong agglutinin of whole yeast cells
  • C. albicans are either serotype A or B and both types can cause disseminated disease (41) .
  • the vaccine of the present invention induces in mice a response that also protects against disseminated disease due to a serotype B strain of C. albicans and against C. tropicalis .
  • mAb B6.1 also protects mice against serotype B and C. tropicalis strains.
  • the explanation for the broad protection of polyclonal antiserum appears to involve antibodies with varying specificities, antibodies with specificity for the B6.1 epitope. Since it has been found that mAb B6.1 also protects SCID mice, neither T nor B cells appear to be involved in the protection. Not being bound by any one theory, one possible mechanism is that antibodies in the mouse cause simple agglutination of the yeast cells which effectively reduces the number of independent infection units. This explanation does not seem likely because mAb B6 does not protect, but it is a strong agglutinin.
  • mAb B6.1 alters adherence of yeast cells in vivo, and/or enhances phagocytosis of yeast cells by neutrophils and macrophages.
  • the first possibility is under investigation. The mechanism would not involve Fc receptors on phagocytic cells because mAb B6.1 is an IgM.
  • mAb B6.1 may promote complement opsonization more efficiently than the non-protective IgM agglutinin, mAb B6.
  • Example 25 BALB/cByJ female mice 6 to 7 weeks old received an initial injection of 0.2 ml of liposome encapsulated Candida adhesion complex (L-adhesion) containing III.178 ug/0.2 ml of adhesion complex and subsequent weekly injections administered i.v.
  • adhesion agglutination titers reached 40 (usually by the fourth booster injection) the animals were challenged i.v. with viable yeast cells.
  • Control mice received the same volumes of buffer (DPBS) or liposome-PBS (L-PBS) in the same numbers of injections.
  • DPBS buffer
  • L-PBS liposome-PBS
  • mice were give buffer (DPBS) alone, liposome-buffer (L-PBS) , or the liposome-adhesin complex (L-adh) and challenged with various doses of C. albicans .
  • Mean survival times for two separate experiments (Expt 1 and Expt 2) were determined, and results from the DPBS and L-adh groups were compared for statistical significance by the Kolmogorov-Smironov one-sample test.
  • D SE standard error
  • Expt 1 four mice per group;
  • mice five mice per group.
  • mice vaccinated with C. albi cans serotype A protected normal mice challenged with serotype B of C. albicans (5 X 10 5 cells) or a strain of C. tropicalis (10 6 cells) .
  • Kidneys of mice challenged with serotype B contained the following colony forming units (CFU) per gram: antiserum-treated mice (11.3 +/- 2.7) x 10 3 and normal serum-treated mice (41.4 +/- 7.0) x 10 3 .
  • Kidneys of mice challenged with C. tropicalis contained the following CFU per gram: antiserum-treated mice (145 +/- 16) x 10 3 and normal serum-treated mice (267 +/- 34) x 10 3 . Both of these differences were statistically significant (p ⁇ 0.001) .
  • Monoclonal antibodies prepared against the phosphomannan complex of C. albicans were passively protective prophylactically .
  • Monoclonal antibodies were prepared by standard procedures from mice immunized with whole yeast cells or with L-adhesion (Brawner and Cutler, Infect. Immun. 51 : 337-343 (1986); Cutler, Han, and Li, In B. Maresca and G.S. Kobayashi (eds), Molecular Biology of Pathogenic Fungi: a laboratory manual, Telos Press, NY, 1994, pp. 197-206) .
  • Female or male BALB/cByJ mice, 7-8 weeks old, were given 0.5 ml of MAb B6.1 (220 ug/mouse) i.p. and 4 hours later were given 0.2 ml of a suspension containing 2.5 x 106 yeast cells i.v. MAb B6.1 protected the mice as demonstrated by CFU counts and survival times. A similar experiment showed that MAb B6.1 protected SCID mice.
  • Monoclonal antibodies also protected against C. albi cans when used therapeutically .
  • MAb B6.1 BALB/cByJ female mice, 7 weeks old, were given 5 x 10 5 yeast cells i.v. One hour later they received MAb B6.1 or buffer (DPBS) i.p. MAb B6.1 showed therapeutic protection by reduced kidney CFU and by increased survival time of treated mice over controls.
  • mice BALB/cByJ female mice, 7-9 weeks old were given estradiol s.c; 72 hours later they received control buffer (DPBS) or 0.5 ml MAb B6.1 i.p. Four hours later they received 5 x 10 5 C. albicans intravaginally and 20 hours later they were given a second injection of MAb or buffer. The vaginas were dissected 48 hours after infection, homogenized, and plated for C. albicans CFU. The result shows that MAb B6.1 protected mice against mucocutaneous candidiasis.
  • DPBS control buffer
  • MAb B6.1 i.p.
  • MAb B6 also protected against mucocutaneous candidiasis .
  • mice BALB/cByJ female mice were given estradiol s.c. and 72 hours later 5 x 10 5 C. albicans were given intravaginally. One hour later the mice were either vaccinated with the L-adhesion vaccine preparation or with liposome buffer (DPBS) (L-DPBS) as a control. After 7 days the animals were sacrificed and the vaginal tissue processed to determine the C. albicans cfu. The results demonstrate that vaccination after mucocutaneous infection has occurred and has therapeutic value.
  • DPBS liposome buffer
  • the C. albicans adhesion complex was treated with 10 mM HC1 at 100 C for 60 minutes. Then it was chromatographed on P-2 size exclusion columns. The complex was separated into two major parts; one was acid stable and the other was the acid labile region. Samples from each peak in the acid labile region were tested for ability to block agglutination of MAb B6.1- coated latex beads by the C. albicans adhesion complex. As shown on Table 4, Fractions M3 and M4 were active. Fraction M3 has the highest concentration of the MAb B6.1 epitope (or materials with the strongest affinity for the MAb) . M4 also reacted with MAb B6.1, but because fraction M4 also contains some fraction 3, it was concluded that fraction M3 in the acid labile portion of the adhesion complex contains the epitope for MAb B6.1
  • the acid stable portion of the adhesion complex contains larger antigenic fragments and can be tested by direct capacity to agglutinate MAb-coated latex beads.
  • Tables 5 and 6 show that MAb B6, but not MAb B6.1, is directed against the acid stable portion of the C. albicans adhesion complex.
  • Monoclonal antibodies (MAb 6.1) against C. albi cans adhesion molecules can protect against mucocutaneous C. albi cans infection when given therapeutically .
  • mice BALB/cBYJ female mice were given estradiol s.c. and 72 hours later 5 x 10 5 C. albicans were given intravaginally. One hour later or 4 hours later the mice were given monoclonal antibody or (DPBS) intravaginally. At 24 hours the MAbs or DPBS were given again. At 48 hours the vaginal tissue was processed to determine the C. albi cans cfu counts. The results are shown in figures 15 and 16. MAb b6.1 protected in both cases. MAb 6 was without effect.
  • MS Electrospray-mass spectrometry revealed that fractions M3 and M4 contained a trimannose and tetramannose plus trimannose. Reference sugars raffinose (trimer) and stachyose (tetramer) are exactly matched to the sizes of the test fractions. Fractions M3 and M4 reacted with MAb B6.1 as evidenced by their ability to block the interaction of MAb B6.1 with the adhesion complex. However, with equal amounts of each fraction, fraction M3 could block 10 -times more adhesion complex interaction with MAb B6.1 than fraction M IV (Table 4) . Since fraction M3 is essentially al trimannose, and fraction M4 contains mostly tetramannose and some trimannose, it was concluded that the MAb B6.1 epitope is a trimannose.
  • Table 4 shows MAb B6.1-beads by indirect measurement. Fraction M7, even at 2000 ⁇ g/ml, does not prevent agglutination of the Ab-coated beads. Fraction M3 inhibits agglutination of this fraction is present in the mixture at >20 ⁇ g/ml. Fraction M3 (or Mill) has the highest concentration of the MAb B6.1 specific epitope; or, M3 binds with strongest affinity. M4 also reacts with MAb B6.1. Because fraction M4 also contains fraction M3 , the inventors conclude that M3 is the epitope for MAb B6.1.
  • Table 5 shows a determination of agglutinin activity of the acid-stable part with MAb B6.1-beads. This table shows a direct measurement i.e., each fraction was mixed with Ab-beads to determine agglutination of beads.
  • Table 6 shows a determination of agglutinin activity of the acid-stable part with MAb B.6-beads. This table shows a direct measurement i.e., each fraction was mixed (at indicated cones) with constant amount of Ab-beads to determine agglutination of beads. The acid-stable fractions react with MAb B.6.
  • Fraction M7 even at 2000 ⁇ g/ml, does not prevent agglutination of the Ab-coated beads. Fraction M3 inhibits agglutination of this fraction is present in the mixture at > 20 ⁇ g/ml. Fraction M3 (or Mill) has the highest concentration of the MAb B6.1 specific epitope. Or, M3 binds with strongest affinity. M4 also reacts with MAb B6.1. Because fraction M4 also contains fraction M3 , it is concluded that M3 is the epitope for MAb B6.1. Table 4 shows a determination of agglutinin activity with MAb B6.1 -beads by indirect measurement. TABLE 4
  • the acid-stable fractions react with MAb B.6.
  • the mannan complex or its components may be conjugated to proteins (for example Bovine Serum Albumin), polysaccharides, a vector, including a phage vector or other know carrier molecule.
  • proteins for example Bovine Serum Albumin
  • polysaccharides for example polysaccharides
  • a vector including a phage vector or other know carrier molecule.
  • the mannan complex does not require liposome delivery for an active vaccine.
  • the effective dosage for mammals may vary due to such factors as age, weight activity level or condition of the subject being treated.
  • an effective dosage of a compound according to the present invention is about 0. l ⁇ g to 500 mg when administered either orally, subcutaneously or intramuscularly, as required to confer immunity.
  • Applicants have been able to omit the use of liposomes in the vaccine formulation by conjugating the 2 -ME extract to a carrier protein, thus, increasing the immunogenicity of the 2-ME extract.
  • the protein, BSA used in these preliminary experiments was chosen as a prototypic carrier molecule because BSA is readily available and inexpensive.
  • the goal of this work is to purify the 2-ME extract protective epitope (i.e., the ⁇ - l , 2 -trimannose) , and couple this epitope to an appropriate protein carrier molecule, such as tetanus toxoid or other protein carrier that is acceptable for human use .
  • an appropriate protein carrier molecule such as tetanus toxoid or other protein carrier that is acceptable for human use .
  • the conjugate sample was analyzed by SDS-PAGE
  • Peak I contained protein (as determined by silver staining) and carbohydrate (as determined by periodic acid staining) . Peak II also contained both carbohydrate (due mostly to 2- ME extract) and protein (due mostly to BSA) , but the electrophoretic position was similar to the position of unconjugated BSA (not shown) .
  • mice BALB/cBy female mice (7 week old) from NCI were vaccinated with the conjugate (peak I material) mixed in the Ribi Adjuvant System (R-700) by an i.p. injection. Three different doses of the conjugate were tested; 10, 50, and 250 ⁇ g per mouse. Control mice received the adjuvant only by the same route. Three weeks later, the animals were boosted with same formula of vaccine or control adjuvant by the same route. Five days after the booster, blood was drawn from a tail vein, and agglutinin activity in sera was determined against 2 -ME coated latex beads. Result: a positive agglutination reaction occurred. Agglutinin titers will be determined.
  • bovine serum albumin (BSA), (Sigma, A-8022, Fraction V)
  • inaicanons ma ⁇ e below relate to the microorganism reterre ⁇ to in tne ⁇ sc ⁇ mion on oaee 13 . line 2
  • ATCC American Type Culture Collection

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