Note: Descriptions are shown in the official language in which they were submitted.
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PEPTIDES WHICH MIMIC CANDIDA CARBOHYDRATE EPITOPES
AND THEIR USE IN A VACCINE
This application contains subject matter related to
Serial Nos. and 08/247,972, filed May 23, 1994,
08/483,558, filed June 7, 1995, and claims priority of
Serial No. 08/756, 014, filed November 25, 1996 and Serial
No. 60/045,030, filed April 28, 1997, and incorporated
herein by reference in their entireties.
Technical Field
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.
Backaround of the Invention
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 1):43-49; Matsumoto, M.S.,
et al. 1991. Effect of combination therapy with
recombinant human granulocyte colony-stimulating factor
(rG-CSF) and antibiotics in neutropen.ic mice unresponsive
to antibiotics alone. J. Antimicrob. Chemother. 28:447-
453; Meunier, F. 1987. Prevention of mycoses in
immunocompromised patients. Rev. Infect. Dis. 9:408-416;
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Meunier, F., et al. 1992. Candidemia in immunocom-
promised patients. Clin. Infect. Dis. 14 (Suppl 1) :S120-
5125; and Van't Wout, J.W. et al. 1989. Comparison of
the efficacies of amphotericin B, Fluconazole, and
Itraconazole against a systemic Candida albicans
infection in normal and neutropenic mice. Antimicrob.
Agents Chemother. 33: 147-151).
Several attempts have been made in the prior art to
achieve immunostimulating compounds for the treatment of
candidiasis as evidenced below.
U.S. Patent No. 5,288,639 to Bernie et al. discloses
the use of antibodies specific for stress proteins of C.
al.bicans 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 33o 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. The 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 burette et al.
discloses immunologically active dipeptidyl 5-0,6-O-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 burette provide non-
specific host protection against infectious organisms
such as C. albicans.
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U.S. Patent No. 4,315,913 to burette discloses
immunologically active dipeptidyl 2-amino-1,2-dideoxy-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 immunologically active dipeptidyl 4-0-6-O-acyl-2-
amino-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 inhibitive to infections caused
by fungi such as C. albicans.
U.S. Patent No. 5,032,404 to Lopez-Berestein et al.
disclose a liposomal agent for treating disseminated
fungal infection in an animal. Because of the nature of
polysaccharide fungal cell walls, it is expected that all
medically important fungi activate complement. The
patent indicated that there is a positive correlation
between animals deficient in late-acting complement
components and increased susceptibility to fungi such as
C. albicans. 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
r 30 phosphatidyl choline. Liposomes incorporate an effective
amount of a polyene macrolide anti-fungal compound such
a as hamycins or lucensomycin, filipin) lagosin and
natamycin.
U.S. Patent No. 4,678,748 to Sutka et al. discloses
a process for the production of the immunobiological
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preparations applicable in the diagnosis, prevention and
treatment of Candida guilliermondii infections. Strains
of C. guilliermondii 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 the 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 (Soll, D.R. 1992. High-frequency
switching in C. albicans. Clin. Microbiol. Rev. 5:183-
203). Perhaps related to the genetic instability are
findings that 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.
1990. Wall mannoproteins in cells from colonial
phenotypic variants of C. albicans. J. Gen. Microbiol.
136:2421-2432). Some of these antigens include putative
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virulence factors such as adhesins and enzymes (Cutler,
J.E. 1991. Putative virulence factors of C. albicans.
Ann. Rev. Microbiol. 45:187-218).
Studies on adherence properties of C. albicans are
5 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. 61:3244-3249;
and Kanbe, T. , et al. 1992. Evidence that C. albicans
binds via a unique adhesion system on phagocytic cells in
the marginal zone of the mouse spleen. Infect. Immun.
60:1972-1978), and extracellular matrix proteins (ECM)
and endothelial cells (Filler, S.G., et al. 1991. C.
albicans stimulates endothelial cell eicosanoid
production. J. Infect. Dis. 164:928-035; Klotz, S.A.
1992. Fungal adherence to the vascular compartment: A
critical step in the pathogenesis of disseminated
candidiasis. Clin. Infect. Dis. 14:340-347; Mayer,
C.L., et al. 1992. Technical report: C. albicans
adherence to endothelial cells. Microvascular Res.
43:218-226; Rotrosen, D. et al. 1985. Adherence of
Candida to cultured vascular endothelial cells:
mechanisms of attachment and endothelial cell
penetration. J. Infect. Dis. 153:1264-1274).
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
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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 mannoproteins (8, 11).
Both mannan and protein moieties may function as
adhesins.
Some adhesins have integrin-like activity in that
they act as receptors for mammalian proteins such as
iC3b, fibronectin, laminin and fibrinogen; one adhesin
has lectin-like activity; and a C3d receptor has been
described (Bendel, C.M., et al. 1993. Distinct
mechanisms of epithelial adhesion for C. albicans and
Candida tropicalis. 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 in C. albicans.
Microbiol Rev. 55:1-20; Cutler, J.E. 1991. Putative
virulence factors of C. albicans. Ann. Rev. Microbiol.
45:187-218; Gilmore, B.J., et al. 1988 An iC3b receptor
on C. albicans: structure, function, and correlates for
pathogenicity. J. Infect. Dis. 157:38-46; Klotz, S.A.,
et al. 1993. Adherence of Candida to immobilized
extracellular matrix proteins is mediated by C. albicans
calcium-dependent surface glycoproteins. Microbiol.
14:133-147). The surface of hydrophilic yeast cells of
C. aLbicans has a fibrillar appearance both in vitro and
in vivo (Hazen, K.C. et al. 1993. Surface hydrophobic
and hydrophilic protein alterations in C. albicans. FEMS
Microbiol. Lett. 107:83-88; Marrie, 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 in C.
albicans observed by rapid-freezing technique, J.
Electron Microsc. 35:237-246).
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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) .
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.
40:266-272). It is not clear, however, if these proteins
differ from the major PMP or are the same proteins, but
with a trunca~ed version of the glycan portion.
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.
Disclosure of the Invention
Accordingly, 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.
In a preferred embodiment 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.
In an alternative embodiment of the invention 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,
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(where S, serine), 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 ,Q-1,2-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 ccmprising a pharmaceutical effective amount
of ar~ 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 in the acid stable portion of the mannan
epitope and ~-1,2-linked 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 ,Q-1,2-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.
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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 ,Q 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
5 for the ,Q 1,2-trimannose or others epitopes in the acid
stable and acid labile regions of the mannan portion of
the phosphomannan complex of Candida alhicans 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 albicans peptides specific for phosphomannan
complex-neutralizing antibodies.
Finally 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. In a preferred embodiment the peptide has a
consensus sequence of amino acids selected from the group
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consisting of, ArXXAr(Z)ZZArAr; where: Ar, aromatic amino
30 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.
The above and other objects of the invention will
become readily apparent to those of skill in the relevant
art from the following detailed description, wherein only
the preferred embodiments of the invention are shown and
5 described, simply by way of illustration of the best mode
of carrying out the invention. As is readily recognized
the invention is capable of modifications within the
skip of the relevant art without departing from the
spirit and scope of the invention.
Brief Description of the Figures
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).
Figure 2 shows MAb specific for a phosphomannan
fraction that contains candidal adhesins protects mice
against disseminated candidiasis. BALB/cByJ mice were
giver. 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
5x10' viable yeast cells, and susceptibility to
disseminated candidiasis was assessed by determining
candidal CFU in the kidneys (A) or by survival curves
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(B). In both cases, significant differences (P<0.01)
between that received either polyclonal antiserum or MAb
B6.1 and DPBS controls were found.
Figure 3 shows MAb B6.1 protects SCID mice against
disseminated candidiasis. BALB/cByJSmn-scid/J male mice
were given MAb B6.1 intraperitoneally and challenged i.v.
with 5x10' C. albicans 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 5x10' yeast cells i.v.
One hour later they received MAb B6.1 or buffer (DPGS)
i.p. MST = mean survival time MST (days); DPGS 9.0~2.0
86.1 16.4+8.3.
Figure S 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 vulvovaginal 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, i.p. Four h after the i.p., animals received
5x10' C. albicians. Intravaginally, 20 h later they
received MAb 86.1 or buffer again i.p. for C. albicians
cfu.
Figure 7 shows the prophylactic effect of candidal
MAbs on mice to vulvovaginal candidiasis. All mice were
pretreated with estradiol before the mAb treatments.
Same design as B-3 , but one group of animals received MAb
B6.
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Figure 8 shows the effect of active immunization
witr. L-adhesion to mice against vulvovaginal candidiasis
L - liposome; L02ME - liposome-2ME vaccine prep.
Animals received 0.2 ml i.v. (178mg 2ME in 0.2m1) weekly
for 5 weeks. Estiadiol was given subcu, 72 h later C.
albicia (5x10') gives intravaginally, 48 h after
infection vaginal cfu determined.
Figure 9 shows the proposed structure of the
phosphomannan complex (PMC)-in this case, n-linkage to
cell wall protein is shown.
Figure 10 shows a P-2 size exclusives column.
Frac~ions 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
IOmM HCl, 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
Figure 13 shows 2-DNMR of B6.1 epitope
Figure 14 (4) 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.
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Statement of Deposit
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.
HB11925.
Description of the Invention
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 ~i
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. albicans yeast cells.
By use of Ab-affinity chromatography and a phage
display peptide library (PDPL) , made by James Burritt and
Clifford Bond, 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.
As shown below, PS76p induces an antibody response
in mice and the antibodies react with whole yeast cells
of C. albicans, and with a ~i-mercaptoethanol extract (2ME
. 30 extract) of the fungus. The 2-ME extract contains the
phosphomannoprotein complexes. By examination of several
_ peptides that are recognized by MAb B6.1, a generalized
formula for peptides is given that may serve in the
formulation of a protective vaccine against various forms
of candidiasis.
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Example 1
Selection of mimetopes. 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
l0 rosettes with B6.1-Sepharose, but not with S10-Sepharose.
The affinity matrices were pre-blocked in phosphate
buffered saline (PBS) plus to bovine serum albumin (BSA)
prior to incubation with the PDPL.
To remove phage that display nonapeptides reactive
with IgM epitopes outside of the antibody combining site,
the PDPL was reacted first with the S10 affinity matrix.
An aliquot of the nonapeptide PDPL (approx. 7.5 x 1011)
phage or 1500 redundants of each nonpeptide represented
in the library) was diluted in phage buffer (50 mM
Tris-HC1, pH 7 . 5, 150 mM NaCl, 0 . 5 o v/v Tween-20 and 0.1 0
BSA) and incubated with S10-Sepharose in a small
polystyrene tube (16 h, 4°C, with gentle rotation). 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 15.5% polyethylene glycol (PEG), 3.3 M
NaCl. The S10 matrix was regenerated and blocked in PBS
+ to BSA. S10 preadsorption of the library was repeated
three more times until the number of S10 adsorbent phage
decreased substantially (by a factor of 105).
To obtain the peptide ao, 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 101° pfu/ml) was incubated
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with MAb B6.1-Sepharose beads (16h, 4°C), transferred to
a column and non-adherent phage were removed by extensive
washing with phage buffer. Bound phage were eluted in 2
ml 0.1 M glycine buffer, pH 2.2 and the pH immediately
5 neutralized. A few microliters of the eluted phage were
removed for phage titering and the remaining phage were
mixed with "starved" E. coli K91 for amplification. The
infected cel is were incubated briefly in Luria broth (LB)
plus 0.75 ~g kanamycin (kan)/ml and spread onto LB agar
10 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
15 phage were diluted in 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.
Results show that 0 . 008 0 of the input phage from the
preadsorbed PDPL were selected in the first round of MAb
B6.1 selection. 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 H6.1-specific PDPL clones. The observed
increase in elution titer with each successive round of
selection indicated that the Ab selection and
amplification provides enrichment of MAb B6.1-reactive
clones with each round. DNA sequencing and western blot
analysis on the third selection pool of phage was done
for further analysis.
Example 2
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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 pIII protein of each phage. From the
randomly selected phage, 29 of the 60 phage clones
exhibited nonapeptide sequences that were unique, but had
areas of homology with each other. Importantly, these 29
phage clones reacted in dot blot analysis with MAb B6.1,
but not with the other control IgM MAbs B5 or 510. The
other 31 phage clones displayed nonapeptide sequences
with the common IgM binding motif that is not associated
with the paratope (antibody combining site) on MAb 86.1.
In order to identify additional clones reactive with
only MAb B6.1, duplicate plaque lifts were prepared from
plates containing well-separated phage from the third
selection pool. The NCM filters were incubated with
either MAbs S10 or B6.1, aligned and compared, and MAb
86.1 positive plaques excised from the plates and
prepared for DNA sequencing. The results as shown in
Table 1 indicate that the MAb B6.1 -specific clones (n =
54) were represented by five unique nonapeptide displays
in the PDPL. A type clone {PS2, PS76, PS31, PS28, and
PS55) is designated for each of the five displays.
Aromatic amino acids appear in bold text, and the P-P-G
carboxy-terminal region of the pIII protein in the
Ml3KBst construct has been included to show the
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orientation of the displayed nonapeptides from the clones
(Table 1) .
I. Table 1. Peptide Secruences from MAb B6 1 Reactive
PDPL Clones
Type No. Peptide
cloneout segue
of 54 rom
clones MAb
H6.1-reactive
PDPL
clones.
pS2 8 P P G L Y W S G P P V W
PS76 4 P p G W F G T V F Q R Y
PS31 38 P P G W Y G G Y T K Y H
pS28 2 P P G W F ~ G T T L ~ S
PS55 2 I I I I S W Y E G L R L I G P P
~ I I I I I
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Example 3
Evidence that the selected clones/peptides react
with the MAb B5.1 binding site. Three different
inhibition formats were used to test for phage/peptide
reactivity with the MAb B6.1 binding site.
One liter cultures of each selected phage clone and
the parent Ml3KBst phage (as a control) were harvested
and the phage titers determined. Phage-coated latex bead
samples for PS76, PS2, PS31, and Ml3KBst (control) which
agglutinated strongly with a rabbit anti-M13 phage
polyclonal antiserum (a gift from A. Jesaitis, MSU) were
prepared, but MAb B6.1 did not agglutinate any of the
phage-latex bead conjugates. Given the small copy number
(five) and the end orientation of pIII proteins by M13,
this result was not surprising.
Various concentrations of the harvested phage were
assayed for their ability to inhibit agglutination by
either MAb B6.1-coated latex beads and soluble 2-ME
extract, or 2-ME extract-coated latex beads and MAb B6.1
were tested. This approach was also unsuccessful. Since
the pIIi protei n is a minor surface molecule on the phage
particle, we calculate that an inhibition may require a
minimum of 1014 phage particles which makes this approach
untenable.
Two immunoblot-dot assays were examined. These
assays provide the necessary sensitivity to screen phage
clones by inhibition. Each ef the dot blot inhibition
assays provide different information about the candidate
peptides. Method one identifies which peptides compete
well with 2-ME extract for binding to MAb B6.1, and
method two confirms that lower affinity binding clones
actually interact with the antibody combining site.
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
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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
S anti-mouse u-chain specific Ab (Sigma A-9688). This
method allowed for detection of MAb B6.1 at 0.005 ug
MA.b/ml, which was chosen for the phage inhibition
studies. MAb B6.1 at S ng/ml was preincubated (1 h,
22-24 C, gentle agitation) with the various selected
plage clones or with the parent phage, Ml3KBst (3.5 x
10=~ pfu or 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/MgCl2 buffer (pH 9.5) and immersed
in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate. Using this approach we found that two of
the phage clones, PS76 and PS2 gave moderate to strong
inhibition of MA.b B6.1 binding to the blotted 2-ME
extract. Inhibition by the phage clones was dose
dependent, as similar assays performed with 2 x 1012
pfu/ml demonstrated only moderate inhibition by phage
clone PS76 and weak inhibition by PS2. Ml3KBst-
containing solutions did not inhibit antibody binding
indicating that parent viral proteins are not
responsible for the inhibition activity shown by PS76
and PS2. Lack of inhibition by clone PS31 may indicate
lower binding affinity to antibody than 2-ME extract.
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 Ml3KBst were prepared at various concentrations
and dot blotted onto nitrocellulose (pfu per dot ranged
from 2 x 101° up to 8 x 101°) to determine the
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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 1011
pfu were chosen. The clones were applied to
5 nitrocellulose and preblocked in phage buffer. In
separate tubes, MAb B6.1 was mixed with the 2-ME
extract at 5 or 50 ug carbohydrate/ml. For inhibition
studies done with soluble reactants, we used
acid-hydrolyzed 2-ME extract to free the B6.1 epitope
10 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.
15 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
20 lower concentration of extract (5 ~g/ml) did not
inhibit. No antibody bound to the Ml3KBst parent phage.
Phage clones PS55 and PS28 samples are currently being
tested in similar assays.
Results from dot blot studies with various phage
clones demonstrate that affinity selected phage will
inhibit the interaction of MAb B6.1 with PM.
Example 4
Synthetic peptide inhibits binding of MAb B6.1 to
its' carbohydrate epitope. On the basis of inhibition
studies with intact phage clones, the nonapeptide
displayed by phage clone PS76 was chosen for synthesis
and used in inhibition studies. We obtained a synthetic
13-mer peptide (Bio-Synthesis, Lewisville, TX),
YRQFVTGFWGPPC, which was designed to include the PS76
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nonapeptide sequence (designated as PS76p) plus the 3
amino acid pIII tether (GPP) and an added cysteine (C}
to facilitate peptide coupling to a carrier protein,
such as keyhole limpet hemocyanin (KLH). 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, 20o 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), 15o v/v dimethylformamide,
chloroform, or methanol.
Because pH below 5 or above 8.5 is required to
solubilize PS76p, the reactivity of MAb B6.1 at low and
high pH was investigated. The antibody (at 0.01 ~g/ml)
maintained its capacity to recognize dot blots of 2-ME
extract (0.5 ~.g/ml) between pH 4 and 5 and between pH 8
and 9. Although MAb B6.1 is functional at these pH
conditions, the PS76p may or may not retain proper
epitope conformation at this pH and may require
tethering to a carrier molecule to enable inhibition
studies to be done at pH 7. Initial inhibition assays
at pH 8.6 with up to 200 ~M PS76p were negative,
suggesting that confirmation could be a factor.
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-f-maleimidobutyrloxyl) succinimide ester (GMBS). The
procedure was carried out in degassed, nitrogen-sparged
0.05 M citrate-phosphate buffer pH 5 to maintain PS76p
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solubility. Briefly, 10 mg carrier protein in citrate-
phosphate buffer was stirred with 2 mg crosslinker (lh,
under nitrogen, room temperature) and then passed over
a small Sephadex G25 column to isolate the carrier
protein-linker product from unreacted crosslinker. The
KLH-MBS and OVA-GMBS fractions were placed into fresh
glass tubes and nitrogen sparged. Peptide PS76p (4 or
5 mg, dissolved in 50 ul dimethylformamide) was added
to each protein-linker solution and stirred at room
temperature under nitrogen atmosphere for 4 hours.
Samples were dialyzed at 4°C against two changes of
citrate-phosphate buffered pH 5 to remove free peptide,
and then dialyzed against four changes of phosphate
buffered saline pH 7.2.
Samples of the final conjugates, PS76p-MBS-KLH
(i.e. PS76-KLH) and PS76p-GMBS-OVA (i.e. PS76-OVA) were
tested by enzyme linked immunosorbent assay (ELISA) for
reactivity with MAb B6.1 at physiologic pH. Microtiter
plate wells were coated with the peptide conjugates or
the carrier proteins alone, washed (Tris buffered
saline with O.lo Tween-20), and blocked in 50
BLOTTO/phage buffer. Wells were washed and incubated
with N'~Ab B6.1, an irrelevant IgM (MAb S10) or block
only, washed, and a horseradish peroxidase-conjugated
goat anti-mouse Ig (G and M) was added. Wells were
washed and an o-phenylenediamine substrate solution was
added, incubated 10 min and color development stopped
with 10% sulfuric acid, and the plate read at OD =
490nm. Both 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 ~.1/ml
in block) was pre-incubated with or without either 50
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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
in a dose-dependent fashion.
Example 5
Antibody response of mice immunized against PS76p.
If PS76p truly mimics the B6.1 carbohydrate epitope
(i.e., ,Q-1,2 'rimannose), then anti-PS76p antibodies
would react with the surface of C. al,bicans and with
the 2-ME extract. Immunizations were done by four
methods described below.
Method 1. Administration of peptide intraperi-
toneally (i.p.) BALB/c mice (4 animals) were immunized
i.p. with PS76p (1 mg per dose) mixed into sterile PBS
(degassed and nitrogen sparged) with and without RS-700
MPL+TDM Ribi Adjuvant (Ribi Immunochem, Hamilton, MT).
The insolubility of PS76p in PBS, as noted above,
produced a particulate inoculum which was sonicated
briefly (10 sec) ice cold) to minimize particulate size
prior to injection. Animals were boosted with 1 mg
PS76p at day 21 and serum samples were obtained on day
28. Sera from all four mice agglutinated hydrophilic C.
albicans cells and agglutinated 2-ME extract-latex
beads. The anti-Candida response appeared somewhat
stronger in the animals receiving peptide with
adjuvant.
Reactivity of the anti-PS76p sera from the mice
was also tested in dot blot assays against 2-ME extract
(1 ~,g/dot). Immune sera were diluted 1:10 in 50
BLOTTO-phage buffer, incubated 12-16 h with preblocked
dot blots, washed and incubated with peroxidase-
conjugated goat anti-mouse Ig (G, M, and A). All four
immunized mice showed reactivity to the 2-ME extract
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whereas the normal mouse serum control (also 1:10 dil)
was negative. Additional dot blots were incubated in
primary antibodies as above and reacted with either 'y-
chain specific or ~-chain specific secondary
antibodies. These blots indicate that the response
appears to be primarily IgM.
Animals were administered booster immunizations of
FS76p every two weeks and serum samples obtained 1 week
after each. An ELISA assay was utilized to determine
any change in titer for the anti-PS76p antiserum
samples. Briefly, 2ME extract-coated wells or wells
coated with an irrelevant carbohydrate were blocked in
5o BLOTTO/phage buffer and incubated with dilutions of
either pre-immune sera or various anti-PS76p antiserum
samples. Subsequent steps with the secondary antibody
and substrate solution were as described above for
ELISA. All of the anti-PS76p serum samples reacted
with 2ME extract and recognized the peptide-carrier
protein conjugates, but not the irrelevant carbohydrate
or the carrier proteins alone. Titers against 2ME
extract did not increase much above 40 for the
intraperitoneal (i.p.) immunizations. Other BALB/C
mice (2 animals) were immunized with PS76p plus Ribi
adjuvant (1 mg/dose subcutaneous inoculation (s.c.),
and reached higher anti-PS76 antibody titers (e.g. 160
by ELISA) after the second boost. ELISA tests
performed with class-specific secondary antibody
reagents confirm that the response is primarily IgM.
Method 2
Administration of 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
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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-
5 specific secondary antibody reagents confirm that anti-
conjugate responses are primarily IgM.
Method 3
Administration of PS76p as a multiple antigen
10 peptide (MAP) construct. The nonapeptide PS76p
(YRQFVTGFW; where: Y, tyrosine; R. arginine; Q,
glutamine; F, phenylalanine; V, valine; T, threonine;
G, glycine; W, tryptophan) was synthesized on a
branched-lysine core to produce eight, radically
15 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.
20 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
25 anti-PS76-MAP response is primarily IgM.
Method 4
Administration of individual phage clones PS76,
PS2, PS31, PS28, and PS55. BALB/C mice (1 animal per
each phage clone) were immunized s.c. with 2 X 1011 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. Against 2ME
extract, the ELISA titers for the anti-phage antibody
samples were: 320 for anti-PS55; 640 for anti-PS76,
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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.
Examble 6
Peptide sequences with potential vaccine and
therapeutic applications.
From the above experiments and results the
following amino acid sequences have vaccine and
therapeutic potential.
Model sequence derived from PS76 clone (expressed
as N-terminal - C-terminal direction):
YRQFVTGFW; where: Y, tyrosine; R, arginine; Q,
glutamine; F, phenylalanine; V, valine; T, threonine;
G, glycine; W, tryptophan.
Consensus sequences of amino acids and amino acid
positions based upon several clones with reactivity to
MAb B6.1:
ArXXAr(Z)ZZArAr; where: Ar, aromatic amino acid
(F, w or Y); X, any amino acid; Z, equals S (where S,
serine), T or G; (Z), is S, T, or G which may or may
not be present. As is clear to those of skill in the
art, one can devise functional equivalents to any of
the above sequences and routinely test the amino acid
sequences to determine if they maintain their
functional integrity and properties. The functional
equivalents may be longer or shorter in length than the
disclosed nonapeptide. In one embodiment the sequence
has 4-12 amino acids. In an alternative embodiment the
sequence has 5-9 amino acids.
Example 7
Vaccine and therapeutic uses of above amino acids.
The amino acids may be coupled to carrier proteins,
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such as keyhole limpet hemocyanin (KLH), tetanus
toxoid, or a cell wall protein from C. albicans. 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. 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.
In addition, once a mimetic peptide of the
invention is identified and sequenced, it can be
reverse translated into the DNA coding sequence, which
itself can be used as a vaccine. Techniques for
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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 phosphomannoprotein 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 adhesin-
specific antibodies in host resistance to disseminated
candidiasis and define the effects of these antibodies
on fungal attachment phenomena as measured by several
in vitro adherence systems, and by in vivo analysis.
The invention focuses on the phosphomannoprotein
complex which the inventors have shown to contain
adhesin sites.
The adhesin(s) responsible for adherence of C.
albicans hydrophilic yeast cells to the splenic
marginal zone was isolated, and presentation of the
adhesin (as part of the phosphomannoprotein complex) to
mice resulted in induction of specific antibody
responses. Mice were induced to produce polyclonal
antisera specific for the phosphomannoprotein and a few
mAbs have been isolated. Mice who develop anti-
phosphomannoprotein responses show increased survival
against disseminated candidiasis. Sera from vaccinated
mice specifically react with phosphomannoprotein.
Immune serum has been shown to passively transfer
resistance to naive animals. The invention addresses
the role of antibodies in host defense against
disseminated candidiasis.
An understanding of mechanisms by which blood-
borne C. albicans yeast cells disseminate in the host
CA 02272632 1999-OS-20
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may be gained through knowledge of fungal adhesins and
host ligand molecules to which these adhesins bind.
The findings by Klotz and others that C. albicans
attaches to exposed basement membrane (ECM) and
platelet aggregates on the ECM, led to speculation that
damaged endothelial cells expose the ECM and allow
attachment of C. albicans from the circulatory system
(Klotz, S.A. 1992. Fungal adherence to the vascular
compartment: A critical step in the pathogenesis of
disseminated candidiasis. Clin. Infect. Dis. 14:340-
347). Perhaps relevant to these findings is that
indwelling venous catheters are responsible for
increased susceptibility to candidiasis and it is
believed that venous catheters damage endothelia.
Importantly, 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.
Edwards has demonstrated that C. albicans 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), and 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.
CA 02272632 1999-OS-20
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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
5 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
10 macrophages. Infect. Immun. 61:3244-3249; Hazen,
K.C., et al. 1991. Differential adherence between
hydrophobic and hydrophilic yeast cells of C. albicans.
Infect. Immun. 59:907-912; and Kanbe, T., et al. 1992.
Evidence that C. a.Ibicans binds via a unique adhesion
15 system on phagocytic cells in the marginal zone of the
mouse spleen. Infect. Immun. (60:1972-1977)).
The adhesins responsible for the yeast/macrophage
interaction have been isolated and characterized
(Kanbe, T., et al. 1994. Evidence for adhesin activity
20 in the acid-stable moiety of the phosphomannoprotein
cell wall complex of C. albicans. Infect. Immun.
62:1662-1668); and Kanbe, T., et al. 1993. Evidence
that rnannans of C. albicans are responsible for
adherence of yeast forms to spleen and lymph node
25 tissue. Infect. Immun. 61:2578-2584).
One of the adhesin sites has been identified to
structure (Li, R.K., et al. 1993. Chemical definition
of an epitope/adhesin molecule on C. albicans. J.
Biol. Chem. 268:18293-18299), and the nature of the
30 macrophage ligand is under investigation (Han, Y., et
al. 1994. Mouse sialoadhesin is not responsible for C.
albicans yeast cell binding to splenic marginal zone
macrophages. Infect. Immun. (62: 2115-2118).
The present inventors set out to determine whether
antibodies are protective against disseminated
CA 02272632 1999-OS-20
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31
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 in 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. albicans by neutrophils in the absence of
serum in vitro. J. Clin. Invest. 61:349-359), others
concluded that specific antibodies may block
phagocytosis of C. albicans (LaForce, F.M., et al.
1975. Inhibition of leukocyte candidacidal activity by
serum for patients with disseminated candidiasis. J.
Lab. Clin. Med. 86:657-666; and Walker, S.M. et al.
1980. A serum-dependent defect of neutrophil function
in chronic mucocutaneous candidiasis., J. Clin. Pathol.
33:370-372).
The suggestion by some that 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 Staphylococcus aureus and C.
albicans in 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. In one study on susceptibility of various
kinds of immunodeficient mice to hematogenous
disseminated candidiasis, the importance of candida-
CA 02272632 1999-OS-20
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32
specific antibodies was dismissed and, instead, T-cell-
mediated immunity was concluded as the important
acquired-specific host defense (Cantorna, M.T., et al.
1991. Acquired Immunity to Systemic Candidiasis in
Immunodeficient Mice. J. Infect. Dis. 164:936-943).
The conclusions were, however, contended by others
(Matthews, R. et al. 1992. Acquired immunity to
systemic candidiasis in immunodeficient mice: Role of
antibody to heat-shock protein 90. J. Infect. Dis.
166:1193-1194) because an alternative interpretation is
that specific antibodies were not induced in the
immunodeficient animals.
However, antibodies appear to assist the host in
resisting disseminated candidiasis. Mourad and
Friedman showed that mice with high antibody titers
against C. albicans 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).
These findings were corroborated by Pearsall who
reported that serum could transfer protection to naive
animals against a deep seated infection with C.
albicans (Pearsall, N.N., et al. 1978. Immunologic
responses to C. albicans. III Effects of passive
transfer of lymphoid cells or serum on murine
candidiasis. J. Immunol. 120:1176-1180). Sensitized
lymphoid cells transferred cutaneous delayed
hypersensitivity to naive mice, but did not protect
these animals against the deep seated disease.
In 1978, Domer's group determined that C. albicans
cutaneous infection provoked mice to produce antibodies
specific for the fungus, and such animals were less
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33
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. Nonspecific and Candida-
specific immune responses in mice suppressed by chronic
administration of anti-~. J. Leukocyte Biol. 44:422-
433). These experiments were confirmed by other
investigators (Maiti, P.K., et al. 1985. Role of
antibodies and effect of BCG vaccination in
experimental candidiasis in mice. Mycopathologia
91:79-85).
In unrelated observations, production of
antibodies against conserved epitopes of candida and
human heat-shock protein (hsp) 90 correlated with the
ability of experimental animals to resist disseminated
candidiasis. Patients who recovered from disseminated
disease produced this antibody (Matthews, R. et al.
1992. Acquired immunity to systemic candidiasis in
immunodeficient mice: Role of antibody to heat-shock
protein 90. J. Infect. Dis. 166:1193-1194) and anti-
hsp 90 from patient sera protected recipient mice
against disseminated candidiasis (Matthews R.C., et al.
1991. Autoantibody to heat-shock protein 90 can
mediate protection against systemic candidosis.
Immunol. 74:20-24). Although the authors claimed that
the patient's sera contained antibodies only against
hsp 90, the detection method used (i.e., PAGE and
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Western blotting) was unlikely to show antibodies
against the candida cell surface PMP.
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. albicans interactions, such as adherence
events. For example, a major antigen expressed on the
surface of serotype A strains is not an adhesin. Since
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. 29:23-34).
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.
Whereas the bulk of clinical studies indicate an
importance of T-cell dependent cell mediated immunity
(CMI) in host resistance to mucosal candidiasis,
neither clinical observations nor most animal
experimental studies show that CMI plays a major role
in resistance to disseminated candidiasis. (See
Brawner, D.L., et al. 1992. Oral candidiasis in HIV-
infected patients. AIDS Reader July/August:117-124;
Fidel, P.L., et al. 1993. Candida-specific cell-
mediated immunity is demonstrable in mice with
experimental vaginal candidiasis. Infect. Immun.
61:1990-199520; Odds, F. C. 1988. Candida and
candidiasis. Bailiere Tindall, London.)
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T-cell dependent cell mediated immune (CMI)
responses appear not to be involved in host resistance
to disseminated candidiasis. A possible explanation is
that CMI is overshadowed in importance by the action of
5 neutrophils, macrophages, specific antibodies and other
factors.
The inventors have studied disseminated
candidiasis, and immune responses to C. albicans, in
normal and immunocompromised mice for over twenty
10 years. Recently the variable nature of the cell
surface of ~. albicans 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.
15 Work progressed from characterizing the surface of C.
albicans tc an understanding of functions of cell
surface moieties as they relate to candida-host
interactions.
Events that occur within 30-45 min after yeast
20 cells of C. albicans gain access to the circulation of
the host and become attached to deep tissue sites where
the fungal cells may adhere either to a host phagocytic
cell or to a non-phagocytic cell site, such as an
endothelial cell were studied.
25 Clinical isolates of 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)
30 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
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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 50o glycerol
at -20°C, and as cell pellets in sterile water at -
20°C. Fresh new working cultures will be prepared form
the frozen stocks every week. For preparation of
hydrophilir_ cells, a loopful of the glycerol stock will
be used to inoculate 25 ml of GYEPB (2% glucose, 0.30
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 1000 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.
Alternatively, 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-
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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. albicans
populations. J. Microbiol. Methods. 6:289-299). Equal
volumes (100 ~.l) of yeast cells (2 x 106/ml) and
hydrophobic (i.e., low sulfate) blue polystyrene
microspheres (diameter, 0.801 ~,m; ca. 9 x 105
microspheres per ml (Serva Fine Biochemicals, Wesburg,
NY), each suspended in sodium phosphate buffer (0.05 M,
pH 7.2), will be placed into acid-washed glass tubes
(12 x 75 mm), equilibrated to 23°C for 2 min and
vigorously mixed for 30 sec. Yeast cells with three or
more attached microspheres are considered to by
hydrophobic.
The protocol for f~-mercaptoethanol extraction of
the adhesins as part of the cell wall phosphomanno-
protein complex (2ME extract) is the same as previously
defined in our laboratory and further detailed below
(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).
Example 9
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.
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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.
1991. Differential adherence between hydrophobic and
hydrophilic yeast cells of C. albicans. Infect. Immun.
59:907-91212).
It was found that 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.
1992. Evidence that C. albicans binds via a unique
adhesion system on phagocytic cells in the marginal
zone of the mouse spleen. Infect. Immun. 60:1972-
1978). An essentially identical binding pattern of
yeast cells to the mouse spleen occurs in vivo
following an intravenous (i.v.) presentation of fungal
cells (Tripe, D.L. et al. 1994. Evidence for
complement independent in vivo adherence of C.
albicans. Abstr. Annu. Meet. ASM.).
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). However, if yeast
cells are opsonized in fresh mouse serum without
detectable antibodies against C. albicans (Morrison,
R.P., et al. 1981. In vitro studies of the interaction
of murine phagocytic cells with C. albicans. J.
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39
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 vitro studies of the interaction of murine
phagocytic cells with C. albicans. J. Reticuloendo-
thel. Soc. 29:23-34). The opsonization is due to
activation cf the alternative complement cascade and is
required for optimal phagocytosis by mouse peritoneal
macrophages. When 8 x 108 yeast cells are complement
opsonized and given i.v. to mice, the number of yeast
cells that bind to the splenic marginal zone is
essentially the same as compared to binding of non-
opsonized yeast cells. Furthermore, 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. later the animals were
sacrificed and the number of fungal colony forming
units (cfu) in the spleen of C3 depleted mice was
similar to splenic cfu of normal controls. A very
interesting finding, however, was that C3 depleted mice
had higher counts in the lungs as compared to normal
controls, implying that complement may play a role in
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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 i3-mercaptoethanol (2ME
extract) (Kanbe, T., et al. 1993. Evidence that
10 mannans of C. albicans are responsible for adherence of
yeast forms tc spleen and lymph node tissue. Infect.
Immun. 61:2578-2584).
Example 10
15 Preparation of Antigen (2ME extract or
phosphomannoprotein, which contains the adhesins)
2-ME extract of C. albicans strain CA-1 was
isolated and used for immunization by inserting the 2ME
extract within liposomes.
20 1. Medium:GYEP broth
Glucose 20
Yeast extract 0.30
Peptone lo/per liter
25 2. C, albicans strain
Strain CA-1 culture by 4 to 6 times trar_-~Lerring
into a fresh medium (GYEP) was used as a starter
culture. 5 ml of the culture was inoculated into 1.2
liter GYEP broth medium, incubated at 37°C under
30 constant aeration by rotation of flasks at 180 rpm,
incubated 22-28 h.
3. Extract (how to prepare the 2-ME extract.)
2-ME Extraction of the surface of C albicans
35 Recommended tubes, rotors, etc. vary with batch size.
1. Count a 1:100 dilution of the GYEP yeast culture.
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Estimate the total number of cells and wet weight
in the culture 101 cells/g wet weight).
Alternatively weigh the centrifuge tube before and
after collecting the pellet to determine yeast wet
weight. [Grams wet weight is used in steps 8 and
9 below to determine the required volumes of .1M
EDTA pH 9.0 and mercaptoethanol.]
2. Pellet Candida for 10 min. by centrifugation at
2,500xg, 4-6C.
3. Wash the pelleted cells 2X with cold deionized
water (dH.,O) .
4. Suspend the washed cells in 250 ml of dHzO.
5. Pellet the cells by centrifugation at 5,000 xg for
10 min. and discard the supernatant liquid.
6. Suspend the cells in 250 ml of cold O.1M
ethylenediamine tetraacetic acid (EDTA), pH 7.5.
7. Pellet the cells at 5,000 xg for 5 min and discard
supernatant material.
8. Suspend to 2.0 ml/g. wet weight in O.1M EDTA pH
9.0, at room temperature.
9. In a fume hood, add 2-mercaptoethanol to 0.3M to
the cell suspensions, cap tightly and invert to
mix.
10. At room temperature, mix (by inverting the tube)
every 5 min. for 30 min.
11. Pellet the cells at 5,000 xg for 10 min.
12. Collect the supernatant material and centrifuge
the supernatant at 5,000 xg until the supernatant
material (2-ME extract) is clear.
13. Dialyze the 2-ME extract against cold dH20, change
the wash every 2-6 hours until the odor of 2-
mercaptoethanol is no longer apparent.
14. Concentrate by lyophilization. The dried product
is referred to as the 2-ME extract. The 2-ME
extract contains the phosphomannoprotein complex.
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Candida adhesins are contained within the mannan
portion of the complex.
At concentrations less than l~,g/ml, the 2ME
extract blocks binding of hydrophilic yeast cells to
the splenic marginal zone macrophages. In addition,
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 a-
mannosidase digestion.
These data strongly indicate that the adhesins are
glycans, probably mannan, and not proteins. In
addition, 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. &1:2578-2584).
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.
alhicans. J. Biol. Chem. 258:18293-18299).
By use of mAb lOG (available from the lab of Dr.
Cutler), a tetramannosyl chain was identified as the
epitope to which mAb lOG is specific. The
tetramannosyl is a f~-1,2-linked straight-chained
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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.
Further analysis of the acid-stable portion of the
PMP revealed that adhesin activity is also associated
with this part of the complex (Kanbe, T., et al. 1994.
evidence for adhesin activity in the acid-stable moiety
of the phosphomannoprotein cell wall complex of C.
albicans. Infect. Immun. 62:1662-1668).
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 1H and 13C 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.
Example 11
Preparation of multilamellar liposomes contained
2-ME extract (L-2ME) or PBS (L-PBS).
Materials:
1. Cell wall antigens (2-ME extract)
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2. L-a-phosphatidylcholine (L-a-lechithin): Type
XI-E, from frozen egg yolk, P-2772 (Lot#- 112H8362),
chloroform solution (100 mg/ml), Sigma, St. Louis, MO.
3. cholesterol: 99o grade, FW = 386.7, C-8567
(Lot # - IlOH8473), chloroform solution (100 mg/ml),
Sigma.
4. chloroform: 9180-O1, ,1.T. Baker, Phillipsburg,
NJ.
5. methanol: A452-4, Fisher Chem.
6. PBS: Dulbecco's phosphate buffered saline, pH
- 7.4, Sigma.
Procedures:
1. Put 200 ~.1 of phosphatidylcholine and 30 ~1 of
cholesterol in chloroform solutions into 10 ml
methanol/chloroform (1:1} contained in a 500 ml round
bottom flask.
2. Evaporate at 37°C (indicator = set at 2) at
low vacuum rotation until a thin layer film forms on
the interior of the flask.
3. Dissolve the dried lipid film in 10 ml
chloroform and remove the chloroform by low vacuum
rotary evaporation at 37°C.
4. Add 5 ml of PBS containing 10 mg of the
solubilized cell wall antigen (2-ME extract) to the
flask.
5. Disperse the lipid film layer into the 2-ME
extract solution by gentle rotation at room temperature
for 10 min. For empty control liposome (L-PBS),
disperse the thin film in 10 ml PBS only.
6. Hold the suspension at room temperature for 2
hrs. and then sonicate at 20°C in a water bath
sonicator (FS5, Fisher Scientific) for 3 min.
7. Maintain the suspension at room temperature
for another 2 hrs. to allow swelling of the liposomes.
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8. Centrifuge at 1,000 X g (3,000 rpm with SS34,
Sorvall RC-5B Refrigerated Superspeed Centrifuge,
DuPont) for 30 min at 16°C to remove non-liposome
associated antigen from liposome encapsulated 2ME-
5 extract.
9. Suspend the liposome in 10 ml PBS and
centrifuge again. Repeat these procedures two more
times.
10. The liposome-encapsulated 2-ME extract is
10 finally suspended in 4 ml PBS and stored at 4°C under
nitrogen.
11. Determine the amount of 2-ME extract
entrapped in liposomes. The liposome-2-ME extract
complex should show a yellowish color by the phenol-
15 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 ~.1 of the liposome-2ME extract
preparation into a well of a microtiter plate and mix
20 with 30 ~.1 of 5% phenol solution. Incubate the mixture
at 21-23°C for 2 min and add 120 ~,1 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 nm. By use of this
25 optical density (OD) was compared to the standard
dilutions of 2-ME-extract in PBS. The results were as
follows:
Amt. of 2-ME per 5m1 PBS O.D. at 490nm
30 1. 10 mg/5m1 0.318
2. 5 mg/5m1 0.159
3. 2.5 mg/5m1 0.078
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As determined by the Dubois (phenol-sulfuric acid)
test for carbohydrates the amount of 2-ME extract
entrapped in liposomes was 178 ~g per 0.2 ml of the
preparation. Varying amounts of adhesin fractions may
be added during formation of the liposomes to determine
the effect on final adhesin concentration that becomes
complexed.
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. Each week,
the animals 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
(agglutinin titers were consistently about 40). Mice
immunized against 2ME extract in PBS produced titers
less than 5, or none at all.
Thus 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.)
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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.
A very significant advance was made upon the
finding that liposome-encapsulated 2ME extract promotes
a strong antibody response in 100% of the immunized
mice. The mechanism by which liposomes cause a
heightened antibody response is unknown, but in work
unrelated to ours, others have also obtained excellent
results with this approach (Livingston, P.O., et al.
1993. GD3/proteosome vaccines induce consistent IgM
antibodies against the ganglioside GD3. Vaccine
II:1199-2004; Wetzler, L.M. et al. 1992. Gonococcal
sporin vaccine evaluation: comparison for proteosomes,
liposomes, and blobs isolated from rmp deletion
mutants., J. Infect. Dis. 166:551-555).
Example 13
Production of mAbs against cell surface antigens
of C. albicans. One of the mAbs (mAb lOG) 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.
CA 02272632 1999-OS-20
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48
43:966-972; Cutler, J.E., et al. 1994. Production of
monoclonal antibodies against mannan determinants of C.
albicans, B. Maresca and G. S. Kobayashi (ed.), In:
Molecular Biology of pathogenic fungi; A Laboratory
Manual. Telos Press, p.197-206; and Li, R.K., et al.
1991. A cell surface/plasma membrane antigen of C.
albicans. J. Gen. Microbiol. 137:455-464).
Cell-mediated immunity may not be important in
resistance to disseminated candidiasis. Some
investigators have reported that macrophages are
important, while others have found no evidence that
macrophages protect (Qian, Q. et al. 1994. Elimination
of mouse splenic macrophages correlates with increased
susceptibility to experimental disseminated
candidiasis. J. Immunol. 152:5000-5008). Perhaps the
biggest pitfall in many of these works is that the
approaches used to eliminate macrophages were non-
specific.
In the present studies (Qian, Q. et al. 1994.
Elimination of mouse splenic macrophages correlates
with increased susceptibility to experimental
disseminated candidiasis. J. Immunol. 152:5000-5008),
mouse splenic macrophages were eliminated by
intravenous (i.v.) delivery of liposome-entrapped
dichloromethylene diphosphonate (L-C12MDP). This
liposome conjugate becomes selectively taken up by
macrophages, which causes their elimination.
Splenic tissue sections immunoperoxidase stained
with mAbs against marginal zone macrophages (mAB MONTS-
4), red pulp macrophages (mAB SK39) and neutrophils
(mAB SK208) showed that 36 h after L-C12MDP treatment,
macrophages but not neutrophils were depleted, and
circulating neutrophils responded normally to an
irritated peritoneum and showed normal phagocytic
ability. That is, in response to thioglycollate in the
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49
peritoneum, neutrophils migrated in normal numbers to
the peritoneal cavity and expressed the normal
activation phenotype of high mac-1 (integrin) and low
Mel-14 (L-selectin) antigen levels. These neutrophils
also showed normal ability to ingest C. albicans yeast
cells in vitro and in vivo. However, the spleens from
L-CIzMDP-treated mice lost their ability to bind
yeasts, which agrees with our previous findings that
hydrophilic yeast cells bind specifically to marginal
zone macrophages.
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-
C1,MDP recovered most of their macrophage function by
56 days and became normal in their resistance to C.
albicans.
These results indicate that macrophages play an
important role in host resistance to disseminated
candidiasis. The similar results obtained with normal
mice and the congenitally thymic deficient (nude) mouse
indicate that the mechanism of protection by
macrophages does not involve activation of T-cell
functions. This result is important, because it is
consistent with earlier reports indicating that cell-
mediated immunity may not be critical in resistance of
mice to deep-seated or disseminated candidiasis
(Mourad, S., et al. 1968. Passive immunization of mice
against C. albicans. Sabouraudia 6:103-105 ; Pearsall,
N.N., et al. 1978. Immunologic responses to C.
albicans. III Effects of passive transfer of lymphoid
cells or serum on murine candidiasis. J. Immunol.
120:1176-1180). These results did not, however, negate
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the hypothesis that antibodies play a role in host
defense against disseminated candidiasis.
Example 14
5 2ME extract from a Cryptococcus neoformans
acapsular mutant does not have adhesin activity and
serves as a negative control. For negative control
purposes a fungal 2ME extract that does not contain
candida-like adhesin activity was obtained. Extensive
10 investigations were done on various strains of
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.
15 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. These experiments are
summarized by stating that S. cerevisiae produces some,
20 but not all, of the candida adhesins responsible for
yeast cell binding to the splenic marginal zone. To
obtain a fungal 2ME extract that did not have the
ability to block adherence of C. albicans to splenic
tissue, the acapsular mutant strain 602 of C.
25 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
30 . 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 i3-mercaptoethanol method gave a water soluble
35 cell wall material that did not affect binding of C.
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albicans 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
95a 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.
Example 15
To test whether C. albicans serotype differences
are important, the inventors prepared 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. Observation of two antigenic groups in C.
albicans. J. Bacteriol. 82:570-573; and Hasenclever,
H.F., et al. 1961. Antigenic studies of Candida II.
Antigenic relation of C. albicans group.A and group B
to Candida stellatoidea and Candida tropicalis. J.
Bacteriol. 82:574-577). Most cf the adhesin isolations
are from the serotype A strain.
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Example 16
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.
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-1(1CR)BR)F1, and
henceforth referred to as CD-1, 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.
Assessment of the adhesin-liposome preparations in
mice. The vaccine preparations are assessed by
determining their relative ability to induce antibody
responses in mice. In studies it was found that 0.1-
0.2m1 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. albicans in genetically distinct
mice. Infect. Immun. 38:1020) and females are somewhat
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more resistant (Ashman, R.B., et al. 1991. Murine
candidiasis; Sex differences in the severity of tissue
lesions are not associated with levels of serum C3 and
C5, Immunol. Cell Biol. 69:7-10; Dourer, J.E. 1988.
Intragastric colonization of infant mice with C.
albicans induces systemic immunity upon challenge as
adults. J. Infect. Dis. 157:950-958.).
Control groups: It was found that liposome-buffer
(PBS) preparations neither induce antibody responses
nor cause increased resistance in mice to disseminated
disease, thus in work with BALB/c mice, these controls
are omitted. As a control in all studies, mice are
immunized against the adhesin fractions prepared in
buffer (0.01 M PBS) alone. Doses of adhesin for
controls are determined by assessing the concentration
of adhesins in the final liposome preparation. The
results from these control animals, when compared with
liposome-adhesin test mice, provide a better indication
of the advantage offered by liposome encapsulation. A
reliable determination of 2ME extract adhesin content
can be made by the phenol-sulfuric acid method of
Dubois for carbohydrate. For adhesins with a high
protein content, such as the hydrophobins or adhesins
responsible for adherence to endothelial cells, protein
assays (such as the BCA, Pierce), are used.
Example 17
Immunization of 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
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produced an agglutinin antibody titer from 20-40 in
1000 of the mice as measured by agglutination of 2ME
extract-coated latex beads.
Mice immunized against the adhesin fraction showed
increased survival times, 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' yeast
cells (i.e., 0.2 ml i.v. of a concentration of yeast
cells of 12.5 x 105/ml PBS), slight prolongation of
survival was also noted in mice challenged with four
times more yeast cells. In a repeat experiment, an
additional group of mice was added that received 2ME
extract ;n 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.
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. Immunologically 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 in vivo intravital 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
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glycoproteins. The effect of immune sera and mAbs on
adherence characteristics of complement opsonized cells
and unopsonized cells is examined. These results lead
to preventative and therapeutic strategies for
5 disseminated candidiasis.
To determine effectiveness of the vaccine, mice
were immunized for the five week period to induce
antibody responses against the adhesin fraction. They
were then rendered immunocompromised by treatment with
10 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;
15 and Jensen, J.T., et al. 1993. Resistance of SCID
mice to C. albicans administered intravenously or
colonizing the gut rule of polymorphonuclear leukocytes
and macrophages. J. Infect. Dis. 167:912-919), or
cyclophosphamide given subcutaneously at 200 mg/kg
20 mouse (Steinshamn, S. et al. 1992. Tumor necrosis
factor and interleukin-6 in C. albicans infection in
normal and granulocytopenic mice. Infect. Immun.
60:4003-4008).
The neutrophil suppressive effects of both
25 treatments were confirmed by monitoring peripheral
blood neutrophil counts, thioglycollate elicited
peritoneal exudates, and assessing by FACScan analysis
integrins and L-selectins (these techniques are defined
in Qian, Q. et al. 1994. Elimination of mouse splenic
30 macrophages correlates with increased susceptibility to
experimental disseminated candidiasis. J. Immunol.
152:5000-5008).
At a low dose yeast challenge mice that were first
vaccinated, then treated with mAB RB6-8C5 to make them
35 neutropenic, and then challenged with C. albicans were
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still protected against disseminated candidiasis (as
compared to the control mice that received treatment of
mAB RB6-8C5 without prior vaccination.
Exams 1 a 19
Immune serum neutralizes adhesins.
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. When splenic sections are pretreated
with 0.1 ~g or more of the 2ME extract, 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).
However, 2ME extract will not inhibit yeast
adherence if the extract is treated with antiserum from
vaccinated animals. In this experiment, antiserum from
BALB/c mice vaccinated against the 2ME extract was heat
inactivated (56°C, 30min) and produced a specific
aggiuti.nin titer of 40 against the 2ME extract-coated
latex beads. In the test condition, 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. 100 ~l of each was overlaid
onto splenic cryosections for 15 min at 4°C, the
mixtures were decanted, 100 ~.l of a suspension of yeast
cells (1.5 x 10'/0.1 ml DMEM) was added to each tissue
section for 15 min at 4°C, and yeast cell binding was
quantified as previously described (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). Binding was compared with control sections
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pretreated with the 2ME extract concentrations but
without antiserum, and with sections pretreated with
normal mouse serum (NMS) (positive binding control).
On control sections pretreated with 2ME extract at all
three concentrations, binding of yeast cells to the
marginal zone areas was less than 30 of positive
binding control sections in which the pretreatment was
NMS alone. An additional control, in which sections
were pretreated with antiserum alone, showed that
binding was not affected.
Ir~ the neutralization test, adherence of yeast
cells to tissues pretreated with a combination of
either 1 ~g 2ME extract or 2 ~g 2ME extract + anti-2ME
extract antiserum was essentially the same as the
positive binding control, and adherence was slightly
reduced when tissues were pretreated with a combination
of 4 ~g 2ME extract + the antiserum.
When the mouse polyclonal anti-adhesin serum is
mixed with yeast cells during their addition to the
splenic tissues, yeast cell binding to the marginal
zone macrophages is reduced. Addition of 25 or 50 ~1
of the anti-adhesin per 100 ~,1 total of yeast cell
suspension reduced by over 80o yeast cell binding in
the ex vivo assay. Addition of 10 ~,1 reduced binding
by about 300. NMS controls had no effect on binding.
The data from the above experiments indicate that
the polyclonal antiserum produced in 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
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Evidence that immune serum transfers protection.
Ir~ an experiment, immune serum (i.e., anti-2ME extract)
was obtained from 20 vaccinated (the five week
protocol) BALB/c mice. NMS was collected from mice
that received an equal number of injections of PBS.
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 105 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. In this experiment,
however, the animals were sacrificed 48h after
challenge and the spleen, kidneys, liver and lungs were
removed, homogenized in sterile saline (hand-held glass
tissue homogenizes) and plated onto Mycosel agar for
cfu. The tissue homogenization does not cause
measurable death of fungal elements (Poor, A.H. et al.
1981. Analysis of an in vivo model to study the
interaction of host factors with C. albicans. Infect.
Immun. 31:1104-1109).
As can be seen in Table 2, cfu from organs of mice
that received immune serum were less in all organs with
the most striking differences noted in the kidneys.
These data suggest that immune serum contains factors
that may protect mice against hematogenous disseminated
candidiasis.
Table 2. Evidence that anti-adhesin serum transfers
protection against disseminated candidiasis to naive
mice.
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Organs Colony forming
units (cfu)
(SD of
coefficient)/g
tissue homogenate
Immune Serum Normal Serum No Serum
S leen 0.6x1040.5 1.5x1040.4 1.3x104+0.1
Kidneys 2.1x101.4 5.2x10''1.7 4.2x10'+0.3
Liver 1.5x1030.3 3.1x1031.8 2.5x103+0.6
Lungs 2.3x1020.4 2.9x1020.6 2.8x1020.4
Example 21
Measurement of antibody res~onses~ Mouse
polyclonal anti-2ME extract caused agglutination of
whole yeast cells. Latex beads coated with the 2ME
extract as previously reported, (Li, R.K., et al. 1993.
Chemical definition of an epitope/adhesin molecule on
C. albicans. J. Biol. Chem. 268:18293-18299),
agglutinate strongly in the presence of the polyclonal
antisera, whereas no agglutination occurs in the
presence of normal mouse serum (NMS), and agglutination
of the 2ME extract-latex is blocked by addition of
soluble 2ME extract. Latex agglutination titers of the
various sera are determined by adding 25 ~1 of the 2ME
latex conjugate, mixing by rotation for 2-5 min and
determining the agglutination end-point.
An anti-adhesin ELISA assay was also developed.
Because of its sensitivity and ability to
simultaneously test many different samples, the ELISA
will be especially useful in characterizing the
predominant class of immunoglobulins produced in
protective sera as indicated below. Coating microtiter
plates with 2ME extract or Fr.II readily occurs in the
presence of 0.06 M carbonate buffer (pH 9.6); 3o BSA
neutralizes non-specific binding. Confirmation of
adhesin binding to the plates is accomplished by
demonstrating specific reactivity with the adhesin-
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specific mAb lOG as detected by commercial secondary
anti-mouse Ig-enzyme and substrate; showing that mAb
lOG does not bind to the plates in the presence of
soluble lOG antigen or 2ME extract; and, binding of an
5 irrelevant mAb or NMS is low. It was found that 2ME
extract-coated plates may be stored indefinitely at
20°C.
Taii vein blood from vaccinated mice was evaluated
for antibody titers (anti-Ig) on a weekly basis during
10 the five weeks of vaccinations-boosters. After that
time titers will be determined every three weeks until
antibody levels decline near background. Various
classes/subclasses of antibodies in the antisera will
also be titered by use of the ELISA assay.
15 Commercially available enzyme-labeled antibodies
specific for the various mouse Ig heavy chains will be
used. This experiment will be of interest later if,
for example, IgM anti-adhesins are found in high titer
in mice that are protected, as opposed to IgG2b that
20 might predominate in mice poorly protected.
Example 22
Pools of mAbs specific for candida adhesins are
also used for passive transfer. Ascites fluid of each
25 mAb and their concentrated Ig fractions obtained by use
of an ABx HPLC preparative column are available. This
column works very well for isolation of IgM and IgG
classes of mAbs. Dr. Hazen provided the mAbs specific
for hydrophobic adhesins of C. albicans.
30 Mice (initially BALB/c females) are given various
doses of pools of mAbs against the various adhesins.
The protocols chosen are roughly deduced from results
obtained with polyclonal antiserum experiments. After
establishing antibody titers, the animals axe
35 challenged with appropriate doses of C. albicans and
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organ cfu determined at various times after challenge.
Control animals.receive mAbs known to react with the
cell surface of C. albicans, but have been shown not to
react with adhesin sites (e.g., mAb 2B3.1). Mab H9
(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; and Brawner, D.L., et al:
1985. Variability in expression of cell surface
antigens of C. albicans during morphogenesis. Infect.
Immun. ~~:337-343) reacts with a candida carbohydrate
surface epitope not involved in adhesion events (our
unpublished data).
Control mAbs (2B3.1 and H9) and our anti-adhesin
mAb (mAb lOG) 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.
Example 23
The effects of anti-adhesins on attachment
phenomena was investigated. Sera from vaccinated mice
inhibits the adhesins (2ME extract) from binding to
splenic marginal zone tissue and the antiserum also
prevents attachment of hydrophilic yeast cells to the
spleen.
As Ig fractions of antisera and mAbs become
available, approaches are used similar to those already
applied in the ex vivo assay to test the effect of
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antisera (anti-sMB extract) on yeast cell adherence to
the splenic marginal zone. The various polyclonal
antisera and mAbs are selected based upon preliminary
results. Their effects, either singly or in
combination, on tissue adherence of hydrophilic and
hydrophobic yeast cells, complement-coated yeast cells
and adhesin-producing recombinant C. albicans strains
are determined. The systems used for study and
comparison include the ex vivo assay, the endothelial
assay and the capillary shear-dependent adhesion assay.
Intravital microscopy is used to follow yeast cell
endothelial interactions within mice that have been
vaccinated and in non-sensitized mice given polyclonal
and mAbs. In all of these methods, careful
consideration is given to appropriate controls.
Depending on the experiment, 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
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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. The effects of polyclonal and mAbs on
adherence interactions with various tissues are
extensively evaluated by adherence assays.
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.OOa 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.
Example 24
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
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phosphate buffered saline (DPBS) (Sigma Chem. Co., St.
Louis, MO), and used to challenge mice.
The PMC (referred to as the adhesin extract) was
obtained in crude form, as before (19,20}, by a ~i-
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
3.5o protein. Following proteinase digestion, the
protein content dropped to 0.47%, yet all adhesin
activity was retained (20).
I.II.Liposome encapsulation of the adhesin extract.
The adhesin extract was encapsulated into
multilamellar liposomes as described previously (11).
Briefly, 200 ul of phosphatidylcholine (100 mg
phosphatidylcholine/ml chloroform) and 30 ~C1 of
cholesterol (100 mg cholesterol/ml chloroform) (molar
ratio of phosphatidylcholine/cholesterol at
approximately 3.2:1) were combined into 10 ml of
chloroform-methanol (1:1) in a 500 ml round bottom
flask. The solution was dried as a thin film by rotary
evaporation at 37 C under reduced pressure. 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. To separate non-
liposome associated antigen from liposome encapsulated
antigen, the preparation was sedimented by centrifu-
gation 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,
was finally suspended in 4 ml DPBS and stored at 4 C
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under nitrogen for up to 2 weeks. 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
5 buffer (DPBS) without adhesin extract was added during
the preparation. These control liposomes are referred
to as L-PBS.
Vaccination and challenge of mice.
10 In all experiments mice were used and housed in
accordance with institutional regulations in an AAALAC
certified animal facility. BALB/cByJ (Jackson Labs,
Bar Harbor, ME) female mice, 6-7 weeks old, received
the initial vaccine and weekly booster immunizations.
15 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
20 done as before (19,20,27). 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
25 challenged i.v. with viable yeast cells prepared to the
appropriate concentration in 0.2 ml DPBS.
Treatment of polyclonal antiserum.
To characterize the nature of the protective
factors) in antiserum, polyclonal antiserum was
30 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
35 volume DPBS-washed packed dead yeast cells.
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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. Briefly,
buffer A consisted of 25 mM MES (2-(N-
Morpholino]ethanesulfonic acid) (Sigma), pH 5.2-5.8 and
buffer B was 1M sodium acetate, pH7Ø One part of
polyclonai 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/min and each fraction
was 40 drops. At ten minutes, the percent of buffer B
was brought to 20, at 15 min buffer B was brought to
500, at 20 min ~,. was brought to 700, at 25 min it was
brought to 1000 and was retained at 1000 until 55 min
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
ultrafiltration (PM30 Diaflo Ultrafiltration 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.
Passive transfer experiments.
Normal mouse serum (control), polyclonal antisera,
antisera heated at 56 C, C. albicans-adsorbed antisera
and HPLC-fractionated polyclonal antisera were tested
for their ability to transfer resistance against
disseminated candidiasis to naive mice. For each
condition, 7-8 week old female or male BALB/cByJ mice
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(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 106 yeasts/ml
DPBS and 20 h later they were given i.p. another 0.2 ml
of test serum. Forty-eight hours after challenge,
candida cfu/g kidney were determined as described
below. In some experiments, 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 ) .
Isolation and characterization of monoclonal antibodies
( mAb s ) .
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
B6.1 as evidenced by Ouchterlony lines of non-identity
against candida cell wall extracts. Both of the mAbs
agglutinate C. albicans yeast cells and both are IgM as
indicated by reactions with commercial Ig-heavy chain
specific antibodies (Sigma).
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.
In these experiments, the 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. In one
experiment, mAb B6 was obtained from ascites fluid,
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adjusted to an agglutinin titer of 320 and compared to
the effect of mAb B6.1 at a titer of 20.
Assessment of resistance/susceptibility to disseminated
candidiasis.
To determine relative susceptibility or resistance
to disseminated candidiasis, we used survival curves
and/or colony forming units (cfu) per g kidney tissue
in mice challenged i.v. with yeast form cells of C.
albicans. For survival curves, groups of test and
control animals consisted of a minimum of five mice per
group. Survival differences between the groups were
calculated for statistical significance by the
Kolmogorov-Smirnov two sample test (9). The kidney is
a target organ in experimental disseminated
candidiasis, therefore, C. albicans cfu in 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 in 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) in 0.2 ml DPBS, but without liposomes. The mean
survival times of these animals did not differ from
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animals that received only DPBS (data not shown).
Serum from vaccinated mice transfers protection.
Pooled polyclonal antiserum from vaccinated mice
protected both naive normal BALB/cByJ and SCID mice
from disseminated disease. Whereas heat treatment (56
C, 30 min) had no effect on the protective ability of
the antiserum, adsorption with C. albicans yeast cells
removed the activity.
To determine if the vaccine induces protection
against both serotypes cf C. albicans and against other
Candida species, 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
(5x10' yeast cells) or a strain of C. tropicalis (1x106
yeast cells). Kidneys were removed 48 h later for efu
determinations. Antiserum-treated mice challenged
with tre serotype B strain had 11.3 (~2.7) x 103 cfu/g
kidney tissue, while normal mouse serum (NMS)- treated
mice (controls) had 41 .4 (~7.0) x 103 cfu/g (p<0.001)
(~ are standard error values). Likewise, antiserum-
treated mice challenged with C. tropicalis developed
145 (~16) x 10' cfu/g kidney as compared to 267 (~34) x
10' cfu/g for NMS-treated controls (p<0.001) (~ are
standard error values).
Two fractions from antiserum transfer protection.
Antiserum separated by use of an ABx HPLC column
yielded three major fractions, Fr. I, Fr. II, Fr. III.
All of the detectable agglutinin activity was
associated with Fr. III. Fr. III gave the strongest
evidence for ability to transfer protection, but
protective activity was also associated with Fr. II.
That is, mice given either normal mouse serum (negative
control;, unfractionated polyclonal antiserum (positive
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control), Fr.I, Fr.II or Fr.III and challenged with C.
albicans i.v. resulted in 109 (+33.3), 40.9 (~ 2.3),
93.2 (~9.5), 59.2 (~11.4) and 50.9 (~9.7) x 103 cfu/g
kidney, respectively (+ are standard error values).
5 The differences were significant to p<0.05 when
polyclonal antiserum, Fr. II or Fr. III were each
compared to cfu/g tissue for animals treated with
normal mouse serum. In these experiments, the total
amount of protein received by each mouse was 22.4 mg of
10 Fr.I, 3.8 mg of Fr.II and 22.6 mg of Fr.III. The
antibody activity (agglutination titer) of Fr. III (22.6
mg) was the same as the agglutinin activity of
unfractionated polyclonal antiserum.
15 MAb B6.1 transfers protection, but mAb B6 does not.
Although 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
20 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
25 B6.1 and administered to mice in the volumes and
schedules as indicated. Even though the agglutinin
titers at day 2 after administration were 10 for
animals that received mAb B6 and 2 for mice that
received mAb B6.1, no protection was observed due to
30 mAb B6 as compared to mice given DPBS prior to yeast
cell challenge (data not shown). In the survival
experiments, of ten BALB/cByJ mice treated with mAb
B6.1, six survived the entire 67 day observation
period, whereas all mAb B6 treated mice died by day 25
35 and all control (DPBS treated) mice died by day 19.
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Likewise, SCID mice treated with mAb B6.1 survived
significantly (p<0.01) longer than control mice. In
experiments on BALB/cByJ mice, the 67 day survivors
were sacrificed and their kidneys and spleens were
plated fcr candida cfu. No cfu in splenic tissues were
detected in any of the animals. However, the kidneys
from two of the mice showed cfu development (97.7 x
103/g and 207.7 x 103/g), whereas no cfu were detected
in undiluted homogenates of kidneys from four of the
mice.
This work provides strong evidence that antibodies
specific for certain cell surface determinants on C.
albicans aid the host in resistance against
disseminated candidiasis. First, mice with enhanced
resistance were those that were L-adhesin vaccinated
and had agglutinin titers of 40-80. Second, mice
vaccinated with only the adhesin extract developed low
anti-adhesin titers (less than 5) and showed no
enhanced resistance. Third) 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). Fourth, heat treatment (56 C, 30
min) had no effect on the protective ability of the
polyclonal antiserum, but adsorption with C. albicans
removed the activity. Fifth, fractionation of the
antiserum by an HPLC ABx column yielded a fraction
that contained all of the agglutinin activity and this
fraction transferred protection to naive animals.
Sixth, 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 SLID mice
survived significantly longer than control animals. In
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this experiment, six out of ten of the treated
BALB/cByJ animals survived the entire 67 day
observation period and four out of six of the survivors
appeared to be cured as evidenced by the lack of cfu
recoverable from their spleen and kidneys.
The results also show that an antibody with
specificity for a cell surface determinant of C.
albicans may not necessarily protect animals against
disseminated disease. These findings explain the
variable results earlier workers have obtained
regarding the role of antibodies in protection against
disseminated candidiasis. Animals that received the
agglutinating IgM mAb B6 were just as susceptible as
controls to disseminated candidiasis, even when mAb B6
was given at about 16 times the titer of mAb B6.1 with
resulting higher in vivo titers than in animals that
received mAb B6.1. The inventors have also found two
additional mAbs specific for surface determinants that
also do not protect (unpublished data). These results
support the hypothesis that antibodies of only certain
specificities against C. albicans are protective.
Strains of C. albicans are either serotype A or B
and both types can cause disseminated disease (41). In
addition, there is an increasing number of candidiasis
cases due to other candida species such as C.
tropicalis (26,32). 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. These data
suggest that antiserum from vaccinated mice contains
antibodies that are broadly protective.
The inventors have determined that 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
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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. In
fact, it causes larger agglutinates at a given titer
than mAb B6.1 ;unpublished observations). In animals
that received the mAbs) the agglutinin titers'in the
serum of mice that received mAbs B6 or B6.1 were
essentially the same. The lower cfu in mAb B6.1-
treated animals, but not mAb B6-treated mice, also
militates against the argument that cfu are
artificially reduced because of the presence of serum
agglutinins. In addition, animals that passively
received mAb B6.1 had enhanced survival as compared to
mice tha. received mAb B6.
Two other possibilities are that 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. However, mAb B6.1 may promote complement
opsonization more efficiently than the non-protective
IgM agglutinin, mAb B6.
Example 25
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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. When 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. The results in
Table 3 show that mice immunized with L-adhesion were
protected against the Candida challenge.
TABLE 3
Mice vaccinated against candida adhesin extract have
greater resistance to disseminated candidiasis than
control animalsa
Challenge Vaccine Mean (SE) survival
time
dose (CFU) preparation (days)b
Expt 1 Expt
2
1 x 10 DPBS 12.5(1.0) Not done
L-PBS 12.0(0.0) Not done
L-adh 20.0(2.2) Not done
P<0.05
5 x 105 DPBS 21.8 (4.2) 19.4 (7.9)
L-PBS 17.3 (4.1) 20.6 (9.8)
L-adh 31.8(4.3) 46.0(12.8)
P<0.05 P<0.05
2.5 x 105 DPBS 25.0(6.7) 38.2(21.6)
L-PBS 23.8(8.3) 46.2(25.5)
L-adh 32.5(3.8) 65.8(13.9)
P<0.1 P<0.05
aNormal 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
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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.
5 °SE, standard error; Expt l, four mice per group;
Expt 2, five mice per group.
Pooled polyclonal antiserum from vaccinated mice
protected both BALB/cByJ and SCID mice. The protective
ability was heat stable (56°C for 30 min.). Similar
10 experiments were performed to demonstrate that the
protective ability of the antibodies was not strain- or
species-specific. Antiserum from mice vaccinated with
C. albicans serotype A protected normal mice challenged
with serotype B of C. albicans (5 X 10' cells) or a
15 strain of C. tropicalis (106 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 103 and normal serum-treated mice
(41.4 +!- 7.0) x 103. Kidneys of mice challenged with
20 C. tropicalis contained the following CFU per gram:
antiserum-treated mice (145 +/- 16) x 103 and normal
serum-treated mice (267 +/- 34) x 103. Both of these
differences were statistically significant
(p < 0.001) .
Example 26
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,
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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.
albicans when used therapeutically.
Example 27
BALB/cByJ female mice, 7 weeks old, were given 5 x
10' 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.
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' 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.
The experiment above was repeated with a second
MAb. MAb B6 also protected against mucocutaneous
candidiasis.
Example 28
BALB/cByJ female mice received 5 weekly i.v.
injections of L-adhesion vaccine preparation (0.2 ml
containing 178 ug of L-adhesion). Estradiol was given
s.c. and 72 hours later 5 x 105 C. albicans were given
intravaginally. The vaginas were dissected 48 hours
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after infection and plated to determine C. albicans
CFU. The results demonstrate that protection was
achieved against mucocutaneous candidiasis by active
immunization. These results have been confirmed in
repeated experiments.
Example 29
Therapeutic immunization with C. albicans adhesins
protects against preestablished mucocutaneous C.
albicans infections.
BALB/cBy,: female mice were given estradiol s.c.
and 72 hours later 5 x 10' 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.
Example 30
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
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was concluded that fraction M3 in the acid labile
portior_ 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.
Examoie 31
Monoclonal antibodies (MAb 6.1) against C.
albicans adhesion molecules can protect against
mucocutaneous C. albicans infection when given
therapeutically.
BALB/cBYJ female mice were given estradiol s.c.
and 72 hours later 5 x 105 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. albicans cfu counts. The
results are shown in figures 15 and 16. MAb b6.1
protected in both cases. MAb 6 was without effect.
These experiments show that certain monoclonal
antibodies against C. albicans adhesions can provide
therapeutic protection against preestablished
mucocutaneous C. albicans infection.
Electrospray-mass spectrometry (MS) 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 86.1 with the
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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.
Regarding the sugar linkage of the MAb B6.1
epitope, signals at 4.937, 4.880, 4.845, and A.4.823
ppm indicate that 'H protons of the non-reducing
terminal mannose are of the ,Q-configuration. Signals
at 5.265 and 4.964 ppm indicate the a- and ~3-
configurations of the reducing terminal mannose (r),
respectively. These 1H n.m.r. spectra of the MAb B6.1
epitope almost exactly match the intensity resonances
of the phsophomannan complex reported by Kobayashi
(Arch. Bio. Bio. 278: 195-204 (1990)). The spectral
pattern shows that the epitope is (3-linked. Two
dimensional n.m.r. showed 13C chemical shifts of the MA
B6.1 epitope that also matched the downfield shifts of
fraction M3 from Kobayashi's data. These results
indicate that the MAb B6.1 epitope is a ~3-1,2 linked
trimannose. Data are presented showing that the MAb
B6 epitope is located in the acid stable part of the
adhesion complex (Tables 5 and 6). It is also shown by
dot-blct analysis that the acid stable portion of the
adhesion complex reacts with MAb B6 but not with MAb
B6.1.
Example 32
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 z20ug/ml. Fraction M3 (or
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MIII) 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
5 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
10 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 concs) with constant
15 amount of Ab-beads to determine agglutination of beads.
The acid-stable fractions react with MAb B.6.
Each fraction at indicated concentration was mixed
with MAb-B6.1-latex beads to which was added an amount
of PMC which is known to cause agglutination of the Ab-
20 coated beads ( 2~.g) .
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.
25 Fraction M3 (or MIII) 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.
30 Table 4 shows a determination of agglutinin
activity with MAb B6.1-beads by indirect measurement.
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TABLE 4
Sample # Concentration each Fraction
of
(microaram/ml )
2000 200 20 2
M7 (MVII) + + + +
M6 (MVI) + + + +
M5 (MV) +/- + + +
M4 (MIV) - - + +
M3 (MIII) - - _ +
M2 (MII) + + + +
M1 (MI) + + + +
M3&4 (MIII, IV) - - - +
all - - + +
TABLE 5
Sample Concentration of each fraction
(microgram/ml)
2000 200 20 2
A - - _ -
B - - _ _
C - - - _
D - - - _
None of the acid-stable fractions react with MAb-B6.1.
TABLE 6
Sample Concentration of each fraction
(microaram/ml )
2000 200 20 2
A + + + _
B + + +
C + + +
D + + +
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
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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. Typically, an effective
dosage of a compound according to the present invention
is about O.l~g to 500 mg when administered either
orally, subcutaneously or intramuscularly, as required
to confer immunity.
Example 33
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
~3-1,2-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.
On the basis of the fractionation profile of the
2ME extract-BSA conjugate sample eluted from the
Sephacryl-S-300 size-exclusion column, two pools of
fractions were collected. When the fraction profile
was compared to the eluting locations (fraction
numbers) of unconjugated 2-ME extract and unconjugated
BSA, the first pool, referred to as peak I, appeared to
represent the conjugate because this peak eluted much
earlier (i.e., it had a higher molecular weight) than
either of the unconjugated materials.
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I). Determination of concentrations of carbohydrate
and protein in peak I of the conjugate sample.
1. The conjugate sample was analyzed by SDS-PAGE
(7.50). 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).
2. The amount of protein in peak I, as determined by
Pierces BCA protein assay, was approximately 540.
II). Test to determine if peak I conjugate material
induces antibodies in test animals.
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 ~Cg 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.
Conjugation of 2-ME to bovine serum albumin (BSA)
(The following method is based on Schneerson, et al.,
work (1986, Infect. Immun. 52:19-528), and some parts
are *modified.)
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84
Materials:
1. 2-ME extract isolated from Candida albicans CA1
strain
2. cyanogen bromide (CNBr) (Sigma, C-6388, FW=15.9)
3. adipic acid dihydrazide (Sigma, A-0638, FW=174.2)
4. Sephacryl-S-300 (Sigma, Lot# 98F0424)
5. 1-ethyl-3 (3-dimethylaminoprophyl) carbodimide.HCl
(EDC)(Sigma, E-6383)
6. bovine serum albumin (BSA), (Sigma, A-8022,
Fraction
V)
7. dialysis tubing (MWCO=6-8,000, Spectrum Medical
Industries Co.)
Methods:
(I) Activation of 2-ME extract by cyanogen bromide
1. Activate 2-ME extract at pH 10.5 at 4C for 6 min
with 1.0 mg of CNBr per mg of 2-ME extract.
Monitor pH continuously; maintain at pH 10.5 by
dropwise addition of 0.1 M NaOH. (2-ME extract is
dissolved in (10) ml of pyrogen-free water.)
2. Add adipic acid to CNBr-activated 2-ME extract to
a final concentration of 0.3M. Adjust pH to 8.5
with 0.2 N HCl. The adipic acid is dissolved in
0.5 M NaHC03.
3. Allow the reaction mixture to tumble overnight at
4C.
4. Centrifuge the resultant solution at 16,000 x g at
4C for 1 hr.
5. Collect supernatant and dialyze it against
*deionized-water for 72 hr at 4C. (*Chromatography
is not used in our method, but is described in the
I&I paper.)
6. Lyophilize (free-dry) dialyzed supernatant
material. This material is denoted as the 2-ME
hydrazide compound.
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(II) Coupling oz 2-ME hydrazide to BSA
The BSA is covalently bound to the 2-ME hydrazide
derivative by carbodiimide-mediated condensation using
1-ethyl-3(3-dimezhylaminopropyl carbodiimide (EDC).
5 1. Mix 80 mg 2-ME hydrazide and 80 mg BSA in 2.5 ml
deion-water. (1:1 ratio of 2-ME hydrazide: BSA by
weight ) .
2. Keep the mixture on ice during the entire
procedures.
10 3. Stir the mixture continuously.
4. Add 4.9 mg of EDC to a final concentration of 0.1
M.
S. The reaction mixture is stirred for 3 hr at 4C, pH
S.0 and dialyzed against 0.2 M NaCl (pH 7.0) at 4C
15 overnight, (**When 0.1 M EDC is added, it is pH
5Ø )
6. Centrifuge the resultant mixture of 2-ME extract
conjugated with BSA at 10,000 x g at 4C for 1 hr.
20 7. Pass the supernatant through a 1.6 x 100 cm size-
exciusion column of Sephacryl-S-300 equilibrated
in 0.2 M NaCl.
8. Test each of eluted fractions by the Dubois'
carbohydrate assay and by protein assays and also
25 measure absorbance at 220 nm.
9. Fractions) containing the 2ME extract-BSA
conjugate is lyophilized.
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The purpose of the above description and examples
is to illustrate some embodiments of the present
invention without implying any limitation. It will be
apparent to those of skill in the art that various
modifications and variations may be made to the
composition and method of the present invention without
departing from the spirit or scope of the invention.
All publications and patents cited herein are hereby
incorporated by reference in their entireties.
CA 02272632 1999-OS-20
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Appucanrs or aeent's tile ; lntemauonat appncatto~
reference number 8-044CPCT
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Ruie l3bls)
A. l'~e indtcauons mane below relate to the microoreantsm reterrea to m the
desenpuon
on page 13 . line 2
B. IDENTIFICATION OF DEPOSIT Further depos«s are tdenulied on an additional
sheet Q
Name of deoos«arv tnsutuuon
American Type Culture Collection (ATCC)
Address of depos«ary tnst«utton mncruamq posrai cone and counrrm
12301 Parkla~n Drive
Rockville) Maryland
USA
Date of aepos« 07 June 1995 (07.06.95) ~ccesston W tuber X11925
C. ADDITIONAL INDICATIONS ~ieuae olonx y nor oopircabler This intotma«on is
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For recetvme Office use oniv For intemauonal Bureau use only
This sheet was received with the mtcrnanonal application Q This sheet was
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