U.S. patent application number 15/787021 was filed with the patent office on 2018-04-26 for vaccine adjuvant.
The applicant listed for this patent is Wisconsin Alumni Research Foundation. Invention is credited to Tristan Theodore Brandhorst, Bruce Steven Klein, Huafeng Wang, Marcel Wuethrich.
Application Number | 20180110853 15/787021 |
Document ID | / |
Family ID | 61971618 |
Filed Date | 2018-04-26 |
United States Patent
Application |
20180110853 |
Kind Code |
A1 |
Klein; Bruce Steven ; et
al. |
April 26, 2018 |
Vaccine Adjuvant
Abstract
A Dectin-2 ligand vaccine adjuvant and a method of making and
using the Dectin-2 ligand vaccine adjuvant in a vaccine to immunize
a patient are disclosed. Also discloses is a vaccine composition
comprising a Bl-Eng2 antigen and methods of using the vaccine
composition to immunize a subject against a fungal infection.
Inventors: |
Klein; Bruce Steven;
(Madison, WI) ; Wang; Huafeng; (Madison, WI)
; Wuethrich; Marcel; (Madison, WI) ; Brandhorst;
Tristan Theodore; (Madison, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wisconsin Alumni Research Foundation |
Madison |
WI |
US |
|
|
Family ID: |
61971618 |
Appl. No.: |
15/787021 |
Filed: |
October 18, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62411281 |
Oct 21, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/57 20130101;
A61K 2039/55566 20130101; A61K 39/0002 20130101; A61K 2039/55516
20130101; A61K 39/39 20130101 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 39/00 20060101 A61K039/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under
AI093553 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A vaccine suitable to immunize a patient comprising an adjuvant,
wherein the adjuvant is a Dectin-2 ligand.
2. The vaccine of claim 1, wherein the Dectin-2 ligand is a
glycoprotein.
3. The vaccine of claim 1, wherein the Dectin-2 ligand is
Bl-Eng2.
4. The vaccine of claim 3, wherein Bl-Eng2 comprises SEQ ID
NO:1.
5. The vaccine of claim 3, wherein Bl-Eng2 comprises O-linked
glycosylations.
6. The vaccine of claim 1, wherein the vaccine immunizes a patient
against a fungal infection.
7. The vaccine of claim 1, wherein the vaccine comprises glucan
particles.
8. The vaccine of claim 1, wherein the vaccine immunizes a patient
against a bacterial infection.
9. The vaccine of claim 1, wherein the vaccine immunizes a patient
against a viral infection.
10. A method of preparing a vaccine comprising the steps of, (a)
preparing a pharmaceutically acceptable vaccine stabilizer; and (b)
introducing to the vaccine stabilizer a suitable antigen and an
adjuvant, wherein the adjuvant is a Dectin-2 ligand.
11. A method of protecting a patient from an infection comprising
the steps of: (a) obtaining the vaccine of claim 1, wherein the
vaccine comprises an adjuvant and a suitable antigen, wherein the
adjuvant is a Dectin-2 ligand; and (b) providing a therapeutically
effective amount of the vaccine to a subject, wherein the subject
is protected from the infection.
12. The method of claim 11 wherein the infection is a fungal
infection and the patient is protected from a fungi infection.
13. The method of claim 12, wherein the antigen is a fragment of
calnexin and the fungi is selected form the group consisting of
Histoplasma, Coccidiodes, Paracoccidioides, Penicillium,
Blastomyces, Sporothrix, Aspergillus, Pneumocystis, Magnaportha,
Exophiala, Neuroaspora, Cryptococcus, Schizophyllum, and
Candida.
14. A vaccine composition comprising Bl-Eng2 and a pharmaceutically
acceptable carrier.
15. The vaccine of claim 14, wherein Bl-Eng2 comprises SEQ ID
NO:1.
16. The vaccine of claim 14, wherein Bl-Eng2 comprises O-linked
glycosylations.
17. The vaccine of claim 14, wherein the vaccine is suitable to
immunize a subject against a fungal infection.
18. The vaccine of claim 14, wherein the vaccine additionally
comprises an adjuvant.
19. The vaccine of claim 18, wherein the vaccine comprises
incomplete Freunds adjuvant.
20. The vaccine of claim 14, wherein the vaccine comprises a
fragment of Bl-Eng2.
21. A method of protecting a patient from an infection comprising
the steps of: (a) obtaining the vaccine of claim 14, wherein the
vaccine comprises Bl-Eng2; and (b) providing a therapeutically
effective amount of the vaccine to a subject, wherein the subject
is protected from the infection.
22. The method of claim 21, wherein the infection is a fungal
infection.
23. The method of claim 21, wherein Bl-Eng2 comprises SEQ ID
NO:1.
24. The vaccine of claim 21, wherein Bl-Eng2 comprises O-linked
glycosylations.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application 62/411,281, filed Oct. 21, 2016, which is incorporated
by reference herein in its entirety.
BACKGROUND
[0003] Vaccines have been hailed as one of the greatest
achievements in public health during the past century. The global
eradication of Smallpox virus in humans and Rinderpest virus in
animals, and the near eradication or successful prevention of other
viral or bacterial infections, for example meningitis in children
due to Hemophilus influenze Type B, offer compelling examples. Yet,
the development of safe and efficacious vaccines against fungi has
been a major hurdle. This difficulty stems from the relative
genetic complexity and intractability of fungi in the laboratory,
limited knowledge of the mechanisms that underpin anti-fungal
protective immunity, and a lack of defined antigen (Ag) candidates
for vaccine protection against fungal pathogens.
[0004] To date, only two vaccines against fungi have moved into
clinical trials. An investigational candidate vaccine containing
rAls3p-N(NDV-3), directed against Candida (and also S. aureus), has
been tested for safety and immunogenicity in volunteers in a Phase
I trial. Another candidate vaccine containing rSap2p was found to
be tolerated and effective in inducing specific antibodies and B
cell memory in women with recurrent vulvovaginitis in a European
clinical trial. Highly conserved Ags that are shared across fungal
pathogens in a family or taxon would be preferable, but the only
such component that has shown promise is .beta.-glucan. Cassone et.
al. reported that this shared cell wall component served as the
basis for a glyco-conjugate vaccine against Candida and
Aspergillus. This preparation has not yet moved into clinical
trials, but .beta.-glucan particles (GPs) could serve as an
experimental platform for the delivery of candidate vaccines
against fungi.
[0005] The incidence of fungal infections and mycoses has increased
significantly in the past two decades, mainly due to the growing
number of individuals who have reduced immunological function
(immuno-compromised patients), such as cancer patients, patients
who have undergone organ transplantation, patients with AIDS,
patients undergoing hemodialysis, critically ill patients, patients
after major surgery, patients with catheters, patients suffering
from severe trauma or burns, patients having debilitative metabolic
illnesses such as diabetes mellitus, persons whose blood is exposed
to environmental microbes such as individuals having indwelling
intravenous tubes, and even in some elderly individuals. Fungal
infections are often also attributed to the frequent use of
cytotoxic and/or antibacterial drugs, which alter the normal
bacterial flora. Fungi include molds, yeasts and higher fungi. All
fungi are eukaryotic and have sterols but not peptidoglycan in
their cell membrane. They are chemoheterotrophs (requiring organic
nutrition) and most are aerobic. Many fungi are also saprophytes
(living off dead organic matter) in soil and water and acquire
their food by absorption. Characteristically fungi also produce
sexual and asexual spores. There are over 100,000 species
recognized, with 100 infectious members for humans.
[0006] Human fungal infections are uncommon in generally healthy
persons, being confined to conditions such as Candidiasis (thrush)
and dermatophyte skin infections such as athlete's foot.
Nevertheless, yeast and other fungi infections are one of the human
ailments which still present a formidable challenge to modern
medicine. In an immuno-compromised host, a variety of normally mild
or nonpathogenic fungi can cause potentially fatal infections.
Furthermore, the relative ease with which human can now travel
around the world provides the means for unusual fungal infections
to be imported from place to place. Therefore, wild and resistant
strains of fungi are considered to be one of the most threatening
and frequent causes of death mainly in hospitalized persons and
immuno-compromised patients.
[0007] The identity of conserved antigens among pathogenic fungi is
poorly understood.
[0008] This is especially true for immunologically significant
antigens that may serve as immunogens to vaccinate against
infection. There are currently no commercial vaccines against fungi
despite the growing problem of fungal infections. A vaccine against
pathogenic fungi, especially one that protects against multiple
fungal pathogens, would be of enormous clinical benefit, and of
commercial interest. Improved vaccines and methods of vaccination
against fungi are needed in the art.
[0009] Needed in the art is an improved adjuvant for a fungal,
bacterial and viral vaccines as well as novel vaccine antigens.
SUMMARY OF THE INVENTION
[0010] The present invention relates to a vaccine composition
comprising a Dectin-2 ligand.
[0011] In a first aspect, described herein is a vaccine suitable to
immunize a patient comprising anadjuvant, wherein the adjuvant is a
Dectin-2 ligand. In some embodiments, the Dectin-2 ligand is a
glycoprotein. In some embodiments, the Dectin-2 ligand is Bl-Eng2.
In one embodiment, Bl-Eng2 comprises SEQ ID NO:1. In some
embodiments, Bl-Eng2 comprises 0-linked glycosylations.
[0012] In some embodiments, the vaccine immunizes a patient against
a fungal infection. In some embodiments, the vaccine comprises
glucan particles. In some embodiments, the vaccine immunizes a
patient against a bacterial infection. In some embodiments, the
vaccine immunizes a patient against a viral infection.
[0013] In a second aspect, described herein is a method of
preparing a vaccine comprising the steps of, (a) preparing a
pharmaceutically acceptable vaccine stabilizer; and (b) introducing
to the vaccine stabilizer a suitable antigen and an adjuvant,
wherein the adjuvant is a Dectin-2 ligand.
[0014] In a third aspect, described herein is a method of
protecting a patient from an infection comprising the steps of: (a)
obtaining a vaccine suitable to immunize a patient, wherein the
vaccine comprises an adjuvant and a suitable antigen, wherein the
adjuvant is a Dectin-2 ligand; and (b) providing a therapeutically
effective amount of the vaccine to a subject, wherein the subject
is protected from the infection. In some embodiments, the infection
is a fungal infection and the patient is protected from a fungi
infection. In some embodiments, the antigen is a fragment of
calnexin and the fungi is selected form the group consisting of
Histoplasma, Coccidiodes, Paracoccidioides, Penicillium,
Blastomyces, Sporothrix, Aspergillus, Pneumocystis, Magnaportha,
Exophiala, Neuroaspora, Cryptococcus, Schizophyllum, and
Candida.
[0015] In a forth aspect, described herein is a vaccine composition
comprising Bl-Eng2 and a pharmaceutically acceptable carrier. In
some embodiment, Bl-Eng2 comprises SEQ ID NO:1. In some
embodiments, Bl-Eng2 comprises O-linked glycosylations. In some
embodiments, the vaccine is suitable to immunize a subject against
a fungal infection. In some embodiments, the vaccine additionally
comprises an adjuvant. In one embodiment, the vaccine comprises
incomplete Freunds adjuvant. In some embodiment, the vaccine
comprises a fragment of Bl-Eng2.
[0016] In a fifth aspect, described herein is a method of
protecting a patient from an infection comprising the steps of: (a)
obtaining a vaccine composition comprising Bl-Eng2 and a
pharmaceutically acceptable carrier; and (b) providing a
therapeutically effective amount of the vaccine to a subject,
wherein the subject is protected from the infection. In some
embodiments, the infection is a fungal infection. In some
embodiments, Bl-Eng2 comprises SEQ ID NO:1. In some embodiments,
Bl-Eng2 comprises O-linked glycosylations.
BRIEF DESCRIPTION OF DRAWINGS
[0017] The patent or patent application file contains at least one
drawing in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0018] FIGS. 1A-1F demonstrate identification of ligand activity
and enrichment by ConA. (A) Silver-stained SDS-PAGE gel of CWE
after water wash and sonication. (B) Dectin-2 reporter cells were
stimulated with plate-coated CWE treated with or without proteinase
K (pro-K), .alpha.-Mannosidase (.alpha.-M), or .beta.-Mannosidase
(.beta.-M). After 18 h, lacZ activity was measured. Data are the
mean.+-.SD of duplicate wells. (C) Flow chart of ligand enrichment
and purification. (D) CWE was incubated with ConA resin.
Flow-through (FL) and eluate (E) were run on SDS-PAGE gel, silver
stained and analyzed for ligand activity. (F) ConA eluate was
further separated by size exclusion using a BioLogic LP system
(Biorad) and Ultro Gel ACA44 resin (Pall Corporation) at a flow
rate of 1 ml/min (blue line represents the trace line of Amo
absorption). Fractions were tested by Dectin-2 reporter cells for
ligand activity. Fractions 4-6 contained most of the ligand
activity and were separated by a second run over the size exclusion
column (see FIG. 6C).
[0019] FIGS. 2A-2D show mass spec analysis identified Bl-Eng2 as a
Dectin-2 ligand candidate: (A) The ligand-negative and -positive
fractions (#9-13 and #1-7 from FIG. 6C, respectively) from the
second gel filtration were analyzed by Mass spectrometry. Numbers
on the right represent number of peptide specific fragments
detected. (B) Domains of native B. dermatitidis Eng2 (Bl-Eng2) and
recombinant Bl-Eng2 expressed in Pichia pastoris: SP denotes Signal
peptide; GH16 denotes glycosyl hydrolase catalytic domain;
Ser/Thr-rich domain harbors 68 potential O-linked glycosylatioin
sites; and Myc and His tags are placed at the C terminus for
purification. (C) 0.6 .mu.g Bl-Eng2 and 0.3 .mu.g PDIA1 were run on
SDS-PAGE gel under reducing conditions and stained for protein
(left) or carbohydrate (right). (D) Monosaccharide composition of
Bl-Eng2 measured by gas chromatography (GC). GC chromatogram of the
alditol acetate-derivatized monosugars of hydrolyzed Bl-Eng2 (top).
Monosaccharides are labeled as follows: Rha--rhamnose, Rib--ribose,
Xyl--xylose, Man--mannose, and Glu--glucose. Unlabeled peak at
5.953 min resulted from component degradation during alditol
acetate derivatization. Pie diagram shows the relative contribution
of monosaccharides (bottom).
[0020] FIGS. 3A-3D demonstrate that Bl-Eng2 is a bona-fide,
superior Dectin-2 ligand. (A) Pichia-expressed proteins were
plate-bound and tested for ligand activity using CLR expressing B3Z
reporter cells expressing FcR.gamma. chain, Dectin-2+FcR.gamma.,
MCL+FcR.gamma., and Mincle+FcR.gamma., and BWZ cells and a subline
expressing Dectin-1-CD3.zeta. (Dectin-1). (B) Supernatants from
murine BMDCs (2.times.10.sup.5 per well) co-cultured with
plate-bound Bl-Eng2 or PDIA1 were analyzed for IL-6 by ELISA.
Blastomyces vaccine yeast (4.times.10.sup.5 per well) was used as a
positive control. (C) Supernatants from BMDCs (10.sup.5 per well)
co-cultured with 1, 10, or 100 ng and 0.01, 0.1 or 1 pmol
plate-bound Bl-Eng2, Man-LAM, Furfurman or MP98 were analyzed for
IL-6 by ELISA. Blastomyces vaccine yeast (10.sup.4, 10.sup.5 or
10.sup.6 per well) was used as positive control. Data in A-C
represent the mean.+-.SEM of one representative experiment of 3
independent experiments. (D) Bl-Eng2 induces IL-6 and IL-1.beta. by
human PBMCs. Human PBMCs were stimulated with plate-bound Bl-Eng2
for 24 h and cytokines in cell culture supernatants were measured
by ELISA. Data represent the mean.+-.SEM of 5 healthy individuals.
*, p<0.05 vs. no Bl-Eng2.
[0021] FIGS. 4A-4F show Bl-Eng2 augments CD4.sup.+ T cell
development in vivo. Mice received 10.sup.6 adoptively transferred
naive 1807 T cells prior to vaccination (A-D) or no transfer (E+F).
Mice were subcutaneously vaccinated with 5 .mu.g calnexin and 10
.mu.g Bl-Eng2 or alum twice, two weeks apart, and then challenged
intratracheally with B. dermatitidis 26199 yeast two weeks
post-vaccination. At day 4 post-infection, the frequencies of IL-17
and IFN-.gamma. producing 1807 T cells (A) and the numbers of
activated (CD44.sup.+) and cytokine-producing 1807 cells in the
lung were enumerated by FACS (B). Almost all of the 1807 T cells
recruited to the lung were CD44.sup.+. Data represent the
average.+-.SEM of two independent experiments with 8-10 mice/group.
*, p<0.05 vs. control mice vaccinated with calnexin and IFA
alone and **, p<0.05 vs. control mice vaccinated with soluble
calnexin alone. Cytokines from lymph node cells stimulated ex vivo
with calnexin were measured by ELISA (D). The number indicates the
n-fold change of mice vaccinated with calnexin+Bl-Eng2 vs. mice
vaccinated with calnexin alone. *, p <vs. all other groups. Lung
CFU were counted at day 18 post-infection when naive mice were
moribund, (C+E). *, p<0.05 vs. all other groups. Numbers reflect
the n-fold change in lung CFU of mice vaccinated with calnexin and
Bl-Eng2 vs. control mice vaccinated with calnexin or IFA alone. The
survival of vaccinated mice was recorded for 30 days post-infection
(FIG. 4E). *, p<0.05 vs. all other groups. At day 4
post-infection, the number of calnexin-specific CD4.sup.+ T cells
was enumerated by tetramer staining (FIG. 4F). Data represent the
average.+-.SEM of tetramer positive cells from one of two
independent experiments with 4-5 mice/group. *, p<0.05 vs. all
other groups. Cnx denotes calnexin.
[0022] FIGS. 5A-5E show myeloid effector mechanisms by Bl-Eng-2.
Mice received 1807 cells prior to vaccination and were vaccinated
and boosted with indicated adjuvants and formulated calnexin. Two
weeks after the boost, mice were challenged i.t. with 10.sup.5
DsRed yeast and lungs were harvested 3 days later. The percentage
of dead (DsRed.sup.-Uvitex.sup.+)(blue) yeast among total
neutrophil-associated yeast (all Uvitex.sup.+ events)(blue and red
together) (see gating strategy in FIG. 11A) were analyzed and
calculated (dot plots are concatenates from 5 mice/group) to depict
the amount of killing by neutrophils (A+B). The percentage of
killing by alveolar macrophages is shown in (C). The number of live
yeast was depicted by showing the total number of DsRed.sup.+
events (D) or plating lung CFU (E). The numbers indicate the n-fold
reduction in live yeast (DsRed.sup.+ or CFU) vs. the calnexin
control groups. *p<0.05 control groups without Bl-Eng-2. Cnx
denotes calnexin.
[0023] FIGS. 6A-6C show separation, characterization, and
enrichment of Dectin-2 ligand activity. (A) 100 .mu.g CWE was
fractionated by a GELFREE (GF) 8100 system. The fractions were
separated by SDS-PAGE and silver stained. (B) Acetone-precipitated
fractions were assayed for ligand activity. (C) Fractions 4-6 from
the 1.sup.st gel filtration contained most of the ligand activity
(see FIG. 1F); they were separated by a second run over the size
exclusion column (blue line represents the trace line of Amo
absorption). Fractions were tested by Dectin-2 reporter cells for
ligand activity. Fractions 9-13 contained most of the ligand
activity and were determined the positive pool; fractions 1-7 were
the negative pool for the subsequent mass spec analysis.
[0024] FIGS. 7A-7B show mass spec analysis identifies Bl-Eng2 as a
candidate ligand for Dectin-2. (A) Complete list of Mass spec
candidates for Dectin-2 ligands. (B) Amino acid sequence of
recombinant Bl-Eng2 contains 637 amino acids (SEQ ID NO:2). Colored
aa match the protein domains illustrated in FIG. 2B.
[0025] FIGS. 8A-8D demonstrate that Aspergillus Eng2 is a Dectin-2
ligand. (A) 0.6 ug Pichia-expressed Aspergillus Eng-2 was
plate-coated and tested for ligand activity using CLR expressing
B3Z and BWZ reporter cells. (B) 30 ng plate-coated Pichia-expressed
Blastomyces Eng2 and Aspergillus Eng2 was tested for ligand
activity with Dectin-2 expressing B3Z reporter cells. (C) 30 ng
plate-coated Pichia-expressed Cryptococcus Eng2 was tested for
ligand activity with Dectin-2 expressing B3Z reporter cells. (D)
Supernatants from BMDCs (2.times.10.sup.5 per well) co-cultured
with plate-coated MP98 were analyzed for IL-6 by ELISA.
[0026] FIGS. 9A-9E demonstrate that Bl-Eng2 induces the development
of Th17 and Th1 cells in a Dectin-2 dependent manner and reduces
lung CFU concentration dependently. (A+C) Mice were subcutaneously
vaccinated twice with calnexin and Bl-Eng2, two weeks apart and
challenged intratracheally with B. dermatitidis 26199 yeast two
weeks post-vaccination. At day 4 post-infection, the numbers of
activated (CD44.sup.+) and cytokine producing 1807 T cells in wild
type (A) and Dectin-2.sup.-/- mice (C) were enumerated by FACS.
Data represent the average.+-.SEM of 5 mice/group. *, p<0.05 vs
calnexin-vaccinated control mice. Lymph node (LN) cells from the
draining brachial LN were stimulated ex vivo with calnexin and
cytokines in the cell culture supernatants were measured by ELISA
(D). (B+E) At day 4 post-infection, lung CFU of (B) wild type mice
and (E) Dectin-2.sup.-/- mice were determined by plating lung
homogenates. *, p<0.05 vs calnexin-vaccinated control mice.
(A-E) Numbers reflect the n-fold change of mice vaccinated with
calnexin and Bl-Eng2 vs. control mice vaccinated with calnexin. NS;
not statistically significant.
[0027] FIGS. 10A-10C show that Bl-Eng2 augments adjuvancy of Alum.
(A-C) Mice were subcutaneously vaccinated with 5 .mu.g calnexin and
10 .mu.g Bl-Eng2 or/and alum twice, two weeks apart, and then
challenged intratracheally with B. dermatitidis 26199 yeast two
weeks post-vaccination. At day 4 post-infection, the numbers of
activated (CD44.sup.+) and cytokine-producing 1807 cells in the
lung were enumerated by FACS (A+B). Data represent the
average.+-.SEM of 5 mice/group. *, p<0.05 vs. control mice
vaccinated with calnexin and Alum. The numbers indicate the n-fold
change of mice vaccinated with Alum+calnexin+Bl-Eng2 vs. mice
vaccinated with Alum+calnexin. *, p<vs. all other groups. Lung
CFU were counted at day 4 post-infection (C). The numbers indicate
the n-fold change in lung CFU of mice vaccinated with
Alum+calnexin+Bl-Eng2 vs. mice vaccinated with Alum+calnexin. *,
p<0.05 vs. all other groups. Cnx denotes calnexin.
[0028] FIGS. 11A-11D demonstrate gating strategy for tracking
neutrophil- and alveolar macrophage-associated with yeast,
activation of PMN and myeloid effector killing in the absence of
1807 T cell transfer. Viable cells (negative for fixable live/dead
dye) that were Siglec F.sup.-, CD11b.sup.+, Ly6G.sup.+ and
Ly6C.sup.int gated as neutrophils (PMNs) and SiglecF.sup.+,
CD11c.sup.+ gated as alveolar macrophages (A). Blastomyces yeast
have higher side scatter than most leukocytes, so Uvitex.sup.+,
SSC.sup.hi neutrophils are associated with yeast. Phagocytes in the
lungs that have phagocytosed inhaled chitin (from bedding/food)
stain with Uvitex when cells are permeabilized. The cells that have
phagocytosed chitin/cellulose have decreased Uvitex fluorescence
but tend to be autofluorescent in many channels including DsRed; an
additional gate was placed on Uvitex.sup.+ events to remove any
false positives in the neutrophil gate. Activated (CD11b.sup.hi)
neutrophils from the neutrophil gate were calculated and shown in
panel (B). Myeloid effector killing in the absence of 1807 T cells
(C+D). Mice did not receive adoptive transfer of 1807 cells prior
to vaccination and were vaccinated twice with calnexin+/-Bl-Eng-2
emulsified in IFA. Two weeks after the boost, mice were challenged
i.t. with 10.sup.5 DsRed yeast and lungs were harvested 3 days
later. The percentage of dead (DsRed.sup.-Uvitex.sup.+)(blue) among
total neutrophil- or macrophage-associated yeast (all Uvitex.sup.+
events)(blue and red together) (see gating strategy in FIG. 11A)
were analyzed and calculated (dot plots are concatenates from 5
mice/group) to depict the amount of killing by PMN and macrophages
(C). The number of live yeast was depicted by showing the total
number of DsRed.sup.+ events or plating lung CFU (D). The number
indicates the n-fold reduction in lung CFU vs. the calnexin control
group. *p<0.05 control groups without Bl-Eng-2.
[0029] FIG. 12 demonstrates a TB vaccine model. Mice were
subcutaneously vaccinated with 5 .mu.g Ag85B in IFA in the presence
or absence of 10 .mu.g Bl-Eng-2 twice, two weeks apart. Two weeks
after the boost, the mice were challenged with 150 CFU of M.
tuberculosis and three weeks later the lungs were harvested and
analyzed for T cell immune responses. The mean.+-.SEM number of
activated (CD44.sup.+) and cytokine producing CD4.sup.+ T cells
were enumerated by FACS. *, p value <0.05 vs. all other
groups.
[0030] FIG. 13 demonstrates an influenza vaccine model. Mice were
intranasally vaccinated with 5 .mu.g NP and 10 .mu.g Bl-Eng-2 or
not. 8 days after the boost, lung T cells were stimulated with NP
peptide and analyzed for the mean.+-.SEM number of tetramer
(NP396.sup.+) and cytokine producing CD8.sup.+ T cells by FACS. *,
p value <0.05 vs. all other groups.
[0031] FIG. 14 demonstrates that vaccination with Bl-Eng2 antigen
protects mice against fungal infection. Mice were vaccinated
subcutaneously with 5 .mu.g Bl-Eng2 protein formulated with
incomplete Freunds adjuvant (IFA, which consists of mineral oil)
twice two weeks apart. Two weeks after the boost, mice were
challenged with 2.times.10E4 wild type (ATCC 26199) B. dermatitidis
yeast. At day 4 and 11 post-infection animals were sacrificed their
lungs plated for colony forming units (CFU). Data represent an
average of 5-10 mice per group. Numbers indicate the n-fold change
vs. IFA control vaccinated mice.
[0032] FIG. 15 demonstrates T cell epitope identification of the
Bl-Eng2 protein. We synthesized 5 software predicted T cell
epitopes (peptides) and stimulated splenocytes from vaccinated mice
(FIG. 14) ex vivo. Peptide #1 (SEQ ID NO:4) stimulated splenocytes
from Bl-Eng2 vaccinated mice to produce IFN-.gamma. comparable to
full length Bl-Eng2 protein, whereas the other peptides and
Calnexin protein did not. Thus, peptide #1 (SEQ ID NO:4) is likely
harboring the protective T cell epitope.
[0033] FIG. 16 shows a sequence alignment of Eng2 (SEQ ID NO:3) and
Eng 3 (SEQ ID NO:12) from Aspergillus fumigatus, Eng2 (SEQ ID
NO:13) from Pseudogymnoascus destructans, Eng2 (SEQ ID NO:14) from
Coccidioides immitis, Eng2 (SEQ ID NO:15) from Coccidioides
posadasii, Eng2 (SEQ ID NO:1) from Blastomyces dermatitidis, and
Eng2 (SEQ ID NO:16) and Histoplasma capsulatum. The sequence of
conserved peptide #1 is shaded.
DETAILED DESCRIPTION OF THE INVENTION
In General
[0034] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, and patent
application was specifically and individually indicated to be
incorporated by reference.
[0035] In one embodiment, the present disclosure describes an
adjuvant for use in a vaccine. The adjuvant is a Dectin-2 ligand,
which stimulates an immune response when administered in a vaccine
composition. In another embodiment, the present disclosure
describes an antigen for use in a vaccine. The antigen is Bl-Eng2
or a variant thereof, which stimulates an immune response when
administered in a vaccine.
[0036] Due to changes in naming conventions and related homologs,
Bl-Eng2 of the current invention was named "Bl-Eng3" in
corresponding U.S. Provisional Application No. 62/411,281, which is
incorporated herein by reference. The polypeptide sequence of the
novel adjuvant and antigen has not changed.
[0037] In one embodiment, the Dectin-2 ligand is glycosylated. In
one embodiment, the Dectin-2 ligand comprises at least one N-linked
glycan, O-linked glycan or combinations thereof. In one embodiment,
the Dectin-2 ligand comprises at least one 0-ling glycan. In one
embodiment, the Dectin-2 ligand is Bl-Eng2, MP98, Furfurman, or
Man-LAM.
[0038] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive. It is specifically
contemplated that any listing of items using the term "or" means
that any of those listed items may also be specifically excluded
from the related embodiment.
[0039] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0040] As used herein the specification, "a" or "an" may mean one
or more, unless clearly indicated otherwise. As used herein in the
claims, when used in conjunction with the word "comprising," the
words "a" or "an" may mean one or more than one.
[0041] The terms "comprise," "have," and "include" are open-ended
linking verbs. Any forms or tenses of one or more of these verbs,
such as "comprises," "comprising," "has," "having," "includes," and
"including," are also open-ended. For example, any method that
"comprises," "has" or "includes" one or more steps is not limited
to possessing only those one or more steps and also covers other
unlisted steps.
[0042] The terms "polypeptide," "peptide," and "protein," as used
herein, refer to a polymer comprising amino acid residues
predominantly bound together by covalent amide bonds. By the term
"protein," we mean to encompass all the above definitions. The
terms apply to amino acid polymers in which one or more amino acid
residue may be an artificial chemical mimetic of a naturally
occurring amino acid, as well as to naturally occurring amino acid
polymers and non-naturally occurring amino acid polymers. As used
herein, the terms may encompass amino acid chains of any length,
including full length proteins, wherein the amino acids are linked
by covalent peptide bonds. The protein or peptide may be isolated
from a native organism, produced by recombinant techniques, or
produced by synthetic production techniques known to one skilled in
the art.
[0043] The term "post-translational modification," or "PMT" as used
herein, refers to the covalent and generally enzymatic modification
of proteins during or following protein biosynthesis.
Post-translational modifications may occur at the C- or N-termini
of the protein or on the amino acid side chains of the protein.
PTMs may include, but are not limited to, the addition of
phosphates, carbohydrates, acetates, amide groups, methyl groups,
lipid molecules, and combinations thereof. The addition of PTM to a
protein may be, but are not limited to, enzymatic phosphorylation,
glycosylation, acylation, alkylation, methylation and combinations
thereof. In one embodiment of the invention, PTMs include N- and
O-linked glycosylations.
[0044] The term "glycoprotein," as used herein, refers to a protein
in which a carbohydrate, monosaccharide or glycan is attached to a
hydroxyl or other functional group of the protein. The glycoprotein
is the result of a post-translational modification wherein a
carbohydrate has been covalently linked to the protein. The
glycosylation may be, but is not limited to, the covalent addition
of any glucan known in the art and may be one or more of a
monosaccharide, a carbohydrate, a glucose, a mannose, an
N-acetylglucosamine, and combinations thereof. In one embodiment of
the invention, the glycol protein comprises at least one O-linked
glycan.
[0045] The term "therapeutically effective amount," as used herein,
refers to an amount of an antigen or vaccine that would induce an
immune response in a subject receiving the antigen or vaccine which
is adequate to prevent signs or symptoms of disease, including
adverse health effects or complications thereof, caused by
infection with a pathogen, such as a virus or a bacterium. Humoral
immunity or cell mediated immunity or both humoral and cell
mediated immunity may be induced. The immunogenic response of an
animal to a vaccine may be evaluated, e.g., indirectly through
measurement of antibody titers, lymphocyte proliferation assays, or
directly through monitoring signs and symptoms after challenge with
wild-type strain. The protective immunity conferred by a vaccine
may be evaluated by measuring, e.g., reduction in clinical signs
such as mortality, morbidity, temperature number, overall physical
condition, and overall health and performance of the subject. The
amount of a vaccine that is therapeutically effective may vary
depending on the particular virus used, or the condition of the
subject, and may be determined by a physician.
[0046] The term "protected," as used herein, refers to immunization
of a patient against a disease. The immunization may be caused by
administering a vaccine comprising an antigen. Specifically, in the
present invention, the immunized patient is protected from a
fungal, bacterial, or viral infection.
[0047] The term "vaccine," as used herein, refers to a composition
that includes an antigen. Vaccine may also include a biological
preparation that improves immunity to a particular disease. A
vaccine may typically contain an agent, referred to as an antigen,
that resembles a disease-causing microorganism, and the agent may
often be made from weakened or killed forms of the microbe, its
toxins or one of its surface proteins. The antigen may stimulate
the body's immune system to recognize the agent as foreign, destroy
it, and "remember" it, so that the immune system can more easily
recognize and destroy any of these microorganisms that it later
encounters.
[0048] Vaccines may be prophylactic, e.g., to prevent or ameliorate
the effects of a future infection by any natural or "wild"
pathogen, or therapeutic, e.g., to treat the disease.
Administration of the vaccine to a subject results in an immune
response, generally against one or more specific diseases. The
amount of a vaccine that is therapeutically effective may vary
depending on the particular virus used, or the condition of the
patient, and may be determined by a physician. The vaccine may be
introduced directly into the subject by the subcutaneous, oral,
oronasal, or intranasal routes of administration.
[0049] A vaccine of the present invention will include a suitable
antigen to stimulate an immune response in a subject or patient. It
is envisioned that vaccines of the present invention are not
limited to a specific antigen or disease target, except where
specifically specified. In some embodiments, the vaccine of the
present invention provides immunity against a fungus, a parasite, a
bacteria, a microbe, or a virus. In one embodiment, the antigen is
Bl-Eng2 or a peptide fragment thereof and the vaccine composition
provides immunity against a fungus.
[0050] In some embodiments, the vaccine of the present invention
provides immunity against fungi. In one embodiment of the
invention, the vaccine comprises an antigen for the family of
ascomycetes in which the pan-fungal antigen Calnexin is highly
conserved, and has been shown to confer protection against
infection in experimental animal models. A non-limiting example of
an antigen of the present invention is the calnexin fragment
described in U.S. patent application Ser. No. 14/203,898 ("Method
of Treating Fungal Infection") and U.S. patent application Ser. No.
14/643,693 ("Peptide MHCII Tetramers to Detect Endogenous Calnexin
Specific CD4 T Cells"), both of which are incorporated herein in
their entirety.
[0051] In some embodiments, the vaccine of the present invention
provides immunity against a Blastomyces dermatitidis infection. In
one embodiment, the vaccine comprises Bl-Eng2 as an antigen to
confer protection against a fungal infection. In some embodiments,
the fungal infection is selected from the group consisting of
Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides
posadasii, Coccidioides immitis, Aspergillus fumigatus and
Pseudogymnoascus destructans. In some embodiments, the Bl-Eng2 is a
fragment of Bl-Eng2 comprising SEQ ID NO:4. Without wishing to be
bound by any particular theory, SEQ ID NO:4 is conserved among
Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides
posadasii, Coccidioides immitis, Aspergillus fumigatus and
Pseudogymnoascus destructans, and this conserved sequence may be
responsible for Bl-Eng2 mediated protection against fungal
infection.
Suitable Targets of the Present Invention
[0052] The term "fungi" or "funguses", as used herein, refers to a
member of a large group of eukaryotic organisms that may include
microorganisms, e.g., yeasts and molds. These organisms may be
classified as a kingdom of fungi, which is separate from plants,
animals, and bacteria. One major difference between fungi and the
others is that fungal cells have cell walls that contain chitin,
unlike the cell walls of plants, which contain cellulose.
[0053] These and other differences show that the fungi form a
single group of related organisms, named the Eumycota (true fungi
or Eumycetes), that share a common ancestor (a monophyletic group).
This fungal group may be distinct from the structurally similar
myxomycetes (slime molds) and oomycetes (water molds). Genetic
studies have shown that fungi are more closely related to animals
than to plants. In the present invention, the terms "fungi",
"funguses", or "fungal" may refer to fungi which may cause
infection in humans and animals.
[0054] In one preferred embodiment of the present invention, fungi
may include Candida albicans (using Candida Adh1 or Als3 protein as
an antigen), Aspergillus fumigatus, endemic systemic dimorphic
fungi including Coccidioides immitis and C. posadasii, Histoplasma
capsulatum, Blastomyces dermatitidis, Paracoccidioides
brasiliensis, Sporothrix schenkii and Penicillium marneffii) and
other ascomycetes using the shared and conserved antigenic domain
of Calnexin.
[0055] Aside from fungi, the present invention may be used as an
adjuvant for vaccination against any infectious disease that
requires the development of cellular immunity, in particular T
helper 1 and T helper 17 CD4+ cells and T cytotoxic 1 and T
cytotoxic 17 CD8+ T cells. This group of microorganisms may include
parasites, bacteria, and viruses.
[0056] In some embodiments, the present invention may be used as an
adjuvant for vaccination against a bacterial infection. The
bacteria may include, but is not limited to, Mycobacterium
tuberculosis, and other intracellular bacteria that require T cell
immunity for host protection. Any suitable antigen known in the art
to protect against the target bacterial infection can be used in a
vaccine composition with the adjuvant of the present invention. In
one embodiment, the vaccine composition comprises Bl-Eng2 and Ag85B
and protects against a Mycobacterium tuberculosis infection.
[0057] In some embodiments, the present invention may be used as an
adjuvant for vaccination against a viral infection. The virus may
include, but is not limited to, influenza A, and other viral
infections that require cell mediated immunity for host protection.
Any suitable antigen known in the art to protect against the target
viral infection can be used in a vaccine composition with the
adjuvant of the present invention. In one embodiment, the vaccine
composition comprises Bl-Eng2 and nucleoprotein (NP) and protects
against an influenza A infection.
Vaccine Administration
[0058] The term "administration," as used herein, refers to the
introduction of a substance, such as a vaccine, into a subject's
body through or by way of a route that does not include the
digestive tract. The administration, e.g., parenteral
administration, may include subcutaneous administration,
intramuscular administration, transcutaneous administration,
intradermal administration, intraperitoneal administration,
intraocular administration, intranasal administration and
intravenous administration.
[0059] The vaccine or the composition according to the invention
may be administered to an individual according to methods known in
the art. Such methods comprise application e.g. parenterally, such
as through all routes of injection into or through the skin: e.g.
intramuscular, intravenous, intraperitoneal, intradermal, mucosal,
submucosal, or subcutaneous. Also, the vaccine may be applied by
topical application as a drop, spray, gel or ointment to the
mucosal epithelium of the eye, nose, mouth, anus, or vagina, or
onto the epidermis of the outer skin at any part of the body.
[0060] Other possible routes of application are by spray, aerosol,
or powder application through inhalation via the respiratory tract.
In this last case the particle size that is used will determine how
deep the particles will penetrate into the respiratory tract.
[0061] Alternatively, application may be via the alimentary route,
by combining with the food, feed or drinking water e.g. as a
powder, a liquid, or tablet, or by administration directly into the
mouth as a: liquid, a gel, a tablet, or a capsule, or to the anus
as a suppository. The term "animal-based protein", as used herein,
refers to proteins that are sourced from ruminant milk, and other
sources, for example the muscle meat, of an animal, particularly a
mammal. Suitable animal-based proteins may include, but are not
limited to, digested protein extracts such as N-Z-Amine.RTM.,
N-Z-Amine AS.RTM. and N-Z-Amine YT.RTM. (Sheffield Products Co.,
Norwich, N.Y.), which are casein enzymatic hydrolysates of bovine
milk.
[0062] The term "vegetable-based protein," as used herein, refers
to proteins from vegetables. A vegetable-based protein may include,
without limitation, soy protein, wheat protein, corn gluten, rice
protein and hemp protein, among others. Preferred vegetable based
proteins in the present invention are soy proteins and corn gluten.
Corn gluten is a mixture of various corn-derived proteins. The soy
proteins can include 100% soy protein (available as VegeFuel.RTM.
by Twinlab), textured soy protein, and soybean enzymatic digest.
Textured soy protein is a soy protein that is made from defatted
soy flour that is compressed and processed into granules or chunks.
Soybean enzymatic digest describes soybean peptones that result
from the partial hydrolysis of soybean proteins.
Antibodies of the Present Invention
[0063] The term "antibody," as used herein, refers to a class of
proteins that are generally known as immunoglobulins. The term
"antibody" herein is used in the broadest sense and specifically
includes full-length monoclonal antibodies, polyclonal antibodies,
multi specific antibodies (e.g., bispecific antibodies), and
antibody fragments, so long as they exhibit the desired biological
activity. Various techniques relevant to the production of
antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A
LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1988).
[0064] The term "fusion protein," as used herein, refers to a
hybrid polypeptide which comprises protein domains from at least
two different proteins. Fusion proteins or chimeric proteins
(literally, made of parts from different sources) are proteins
created through the joining of two or more genes that originally
coded for separate proteins. Translation of this fusion gene
results in a single or multiple polypeptides with functional
properties derived from each of the original proteins. Recombinant
fusion proteins are created artificially by recombinant DNA
technology for use in biological research or therapeutics. Chimeric
or chimera usually designate hybrid proteins made of polypeptides
having different functions or physico-chemical patterns. Chimeric
mutant proteins occur naturally when a complex mutation, such as a
chromosomal translocation, tandem duplication, or
retrotransposition creates a novel coding sequence containing parts
of the coding sequences from two different genes. Naturally
occurring fusion proteins are commonly found in cancer cells, where
they may function as oncoproteins. In one embodiment of the present
invention, fusion proteins comprise at least one engineered
intein.
[0065] The term "immune status" or "immunocompetence," as used
herein, refers to the ability of the body to produce a normal
immune response following exposure to an antigen. Immunocompetence
is the opposite of immunodeficiency or immuno-incompetent or
immuno-compromised.
[0066] The present invention is generally applied to humans. In
certain embodiments, non-human mammals, such as mice and rats, may
also be used for the purpose of demonstration. One may use the
present invention for veterinary purpose. For example, one may wish
to treat commercially important farm animals, such as cows, horses,
pigs, rabbits, goats, and sheep. One may also wish to treat
companion animals, such as cats and dogs.
Adjuvants of the Present Invention
[0067] As used herein, "Th17 cells" refers to a population of CD4+
T cells which produce Il-17. As used herein, "Th1 cells" refers to
a population of CD4+ T cells which produce INF-.gamma.. As used
herein, "Tc1 cells" refers to a population of cytotoxic CD8+ T
cells that produce INF-.gamma.. Vaccine induced CD4+ T cells that
produce IL-17 (Th17 cells) and INF-.gamma. (Th1 cells) and CD8+Tc1
cells that produce INF-.gamma. are active in resistance against
fungal, bacterial and viral infections. In one embodiment of the
invention, the vaccine requires the activity of the Dectin-2
receptor on phagocytes that will trigger the development of
antigen-specific Th17 and Th1 cells to mediate resistance.
[0068] As used herein, the term "Dectin-2" or "Dec-2" refers to a
type II transmembrane C-type lectin receptor involved in the innate
immune response. Note to be bound by any particular theory,
Applicants working theory indicates that fungal vaccine recognition
by the Dectin-2/FcR.gamma./Syk/Card9 signaling axis is required for
the differentiation of Th17 and Th1 cells and the induction of
vaccine-induced resistance to fungal infection (Wang et. al 2014 J
Immunol).
[0069] As used herein, the term "Dectin-2 ligand" refers to a
molecule capable of binding to or activating the
Dectin-2/FcR.gamma./Syk/Card9 signaling axis to promote the
differentiation of Th17 and Th1 cells. The molecule may be a
protein, a lipid, a glycoprotein, a glycolipid or any glycan
capable of binding Dectin-2. A suitable Dectin-2 ligand of the
present invention is characterized by the ability to induce
Dectin-2 signaling using Dectin-2 expressing B3Z T cell reporter
cells (FIG. 2C) or to produce cytokines by bone marrow derived
dendritic cells (BMDC) in a Dectin-2 dependent manner (for example,
when comparing cytokine production by wild type vs.
Dectin-2-deficient BMDCs) (FIG. 2D+E) and increasing the activation
and differentiation of antigen-specific T cells in vivo (FIG. 3).
In one embodiment the Dectin-2 ligand of the present invention is
selected from the group consisting of Bl-Eng2, MP98, Furfurman from
Malassezia sp., and Man-LAM (Ishikawa et al. 2003, Yonekawa et al.
2014). In another embodiment of the invention, the Dectin-2 ligand
is a glycoprotein selected from the group consisting of MP98 and
Bl-Eng2.
[0070] As used herein, the term "Bl-Eng2" refers to the fungal
glycoprotein .beta.-1,3-endoglucansase from Blastomyces
dermatitidis. Bl-Eng2 has homology to Aspergillus fumigates
endoglucanase 2 (Eng2) at the C-terminal glycosylation site, and
endoglucanase 3 (Eng3) at the active site.
[0071] The predicted molecular weight of Bl-Eng2, based on amino
acid sequence alone, is 57 kDa. However, Bl-Eng2 may appear as a
115-130 kDa band on an SDS-PAGE gel based on post-translation
glycosylation. Bl-Eng2 comprises an 18 amino acid signal peptide,
an N-terminal GH16 glycosyl hydrolase catalytic domain, and a
C-terminal S/T-rich domain. Bl-Eng2 undergoes post-translational
modification and has a number of O-linked glycosylation sites,
which may be in the S/T-rich C-terminal domain. It is understood
that mannose is the major monosaccharide present in the PTM
glycosylation of Bl-Eng2 when it is expressed in Pichia pastoris.
In one embodiment B1-Eng2 comprises the sequence of SEQ ID NO: 1.
In one embodiment, the Bl-Eng2 is a fragment, single domain, or
short glycan fragment of the full-length Bl-Eng2.
[0072] As used herein, the term "MP98" refers to the chitin
deacetylase-like protein from Cryptococcus neoformans (Levitz et
al., 2001). MP98 comprises an N-terminal cleavable signal sequence,
a polysaccharide deacetylase domain found in fungal chitin
deacetylases, and a serine/threonine-rich C-terminal region. The
C-terminal region comprises N-liked glycosylation sites comprises
covalently linked mannose.
[0073] A Dectin-2 ligand suitable for use as a vaccine adjuvant in
the present invention may be in any form as discussed above. In one
embodiment, the Dectin-2 ligand may be expressed in commercially
available sources, e.g., Pichia pastoris. The Dectin-2 ligand maybe
expressed in any commercially available sources that is capable of
post-translational protein modifications. The Dectin-2 ligand
vaccine adjuvant may be then isolated and purified from these
sources. The protein expression, isolation, and purifications are
well known to a person having ordinary skill in the art. The
Examples demonstrate methods of expression, isolation, and
purifications of Bl-Eng2 according to one embodiment of the present
invention.
[0074] A vaccine comprising a Dectin-2 ligand adjuvant may also
comprise other suitable ingredients. In one embodiment, a vaccine
may also comprise a carrier molecule as a stabilizer component. As
the types of vaccines enclosed in the present invention may be
rapidly degraded once injected into the body, the vaccine may be
bound to a carrier molecule for stabilizing the vaccine during
delivery and administration. A suitable carrier or stabilizer may
comprise fusion proteins, polymers, liposomes, micro- or
nanoparticles, or any other pharmaceutically acceptable carriers. A
suitable carrier or stabilizer molecule may comprise a tertiary
amine N-oxide, e.g., trimethylamine-N-oxide, a sugar, e.g.,
trehalose, a poly(ethylene glycol) (PEG), an animal-based protein,
e.g., digested protein extracts such as N-Z-Amine.RTM., N-Z-Amine
AS.RTM. and N-Z-Amine YT.RTM. (Sheffield Products Co., Norwich,
N.Y.), a vegetable-based protein, e.g., soy protein, wheat protein,
corn gluten, rice protein and hemp protein, and any other suitable
carrier molecules.
[0075] As used herein "glucan particle" refers to a formulation of
the vaccine comprising .beta..sub.1,3 glucan particles as a solid
support. Glucan particles target Dectin-1, a key pattern
recognition receptor for anti-fungal immunity. Glucan particles
also serve as structural vessels or a type or structural scaffold
to deliver antigen as well as adjuvants in the vaccine formulation.
In one embodiment, .beta..sub.1,3 glucan particles (GPs) are used
as a solid support for Bl-Eng2. In one embodiment, glucan particles
in used in a vaccine formulation with calnexin fragments and a
Dectin-2 ligand adjuvant against pathogenic fungi.
[0076] In another aspect, the Dectin-2 ligand adjuvant of the
present invention may be administered in a formulation with any
known commercially available adjuvant in the art. Adjuvants to be
administrated may include, but are not limited to, aluminum
hydroxide (Alum), glucan particles engaging Dectin-1, Adjuplex, and
combinations thereof. It is also envisioned that when Bl-Eng3 is
administered in a vaccine composition as an antigen, it may be
administrated with a suitable adjuvant. Suitable adjuvants are any
commercially available adjuvant known in the art or an adjuvant
described herein.
Suitable Carrier or Vehicle
[0077] Suitable agents may include a suitable carrier or vehicle
for delivery. As used herein, the term "carrier" refers to a
pharmaceutically acceptable solid or liquid filler, diluent or
encapsulating material. A water-containing liquid carrier can
contain pharmaceutically acceptable additives such as acidifying
agents, alkalizing agents, antimicrobial preservatives,
antioxidants, buffering agents, chelating agents, complexing
agents, solubilizing agents, humectants, solvents, suspending
and/or viscosity-increasing agents, tonicity agents, wetting agents
or other biocompatible materials. A tabulation of ingredients
listed by the above categories, may be found in the U.S.
Pharmacopeia National Formulary, 1857-1859, (1990).
[0078] Some examples of the materials which can serve as
pharmaceutically acceptable carriers are sugars, such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
glycols, such as propylene glycol; polyols such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters such as ethyl
oleate and ethyl laurate; agar; buffering agents such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen free water;
isotonic saline; Ringer's solution, ethyl alcohol and phosphate
buffer solutions, as well as other nontoxic compatible substances
used in pharmaceutical formulations. Wetting agents, emulsifiers
and lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, release agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the compositions, according
to the desires of the formulator.
[0079] Examples of pharmaceutically acceptable antioxidants include
water soluble antioxidants such as ascorbic acid, cysteine
hydrochloride, sodium bisulfite, sodium metabisulfite, sodium
sulfite and the like; oil-soluble antioxidants such as ascorbyl
palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and
metal-chelating agents such as citric acid, ethylenediamine
tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid
and the like.
Stabilization Agent
[0080] In another configuration, the present formulation may also
comprise other suitable agents that stabilize the formulations. For
example, an approach for stabilizing solid protein formulations of
the invention is to increase the physical stability of purified,
e.g., lyophilized, protein. This will inhibit aggregation via
hydrophobic interactions as well as via covalent pathways that may
increase as proteins unfold. Stabilizing formulations in this
context may often include polymer-based formulations, for example a
biodegradable hydrogel formulation/delivery system. The critical
role of water in protein structure, function, and stability is well
known. Typically, proteins are relatively stable in the solid state
with bulk water removed. However, solid therapeutic protein
formulations may become hydrated upon storage at elevated
humidities or during delivery from a sustained release composition
or device. The stability of proteins generally drops with
increasing hydration. Water may also play a significant role in
solid protein aggregation, for example, by increasing protein
flexibility resulting in enhanced accessibility of reactive groups,
by providing a mobile phase for reactants, and by serving as a
reactant in several deleterious processes such as beta-elimination
and hydrolysis.
[0081] An effective method for stabilizing peptides and proteins
against solid-state aggregation for delivery may be to control the
water content in a solid formulation and maintain the water
activity in the formulation at optimal levels. This level depends
on the nature of the protein, but in general, proteins maintained
below their "monolayer" water coverage will exhibit superior
solid-state stability.
[0082] A variety of additives, diluents, bases and delivery
vehicles may be provided within the invention that effectively
control water content to enhance protein stability. These reagents
and carrier materials effective as anti-aggregation agents in this
sense may include, for example, polymers of various
functionalities, such as polyethylene glycol, dextran,
diethylaminoethyl dextran, and carboxymethyl cellulose, which
significantly increase the stability and reduce the solid-phase
aggregation of peptides and proteins admixed therewith or linked
thereto. In some instances, the activity or physical stability of
proteins may also be enhanced by various additives to aqueous
solutions of the peptide or protein drugs. For example, additives,
such as polyols (including sugars), amino acids, proteins such as
collagen and gelatin, and various salts may be used.
[0083] Certain additives, in particular sugars and other polyols,
may also impart significant physical stability to dry, e.g.,
lyophilized proteins. These additives may also be used within the
invention to protect the proteins against aggregation not only
during lyophilization but also during storage in the dry state. For
example sucrose and Ficoll 70 (a polymer with sucrose units)
exhibit significant protection against peptide or protein
aggregation during solid-phase incubation under various conditions.
These additives may also enhance the stability of solid proteins
embedded within polymer matrices.
[0084] Yet additional additives, for example sucrose, stabilize
proteins against solid-state aggregation in humid atmospheres at
elevated temperatures, as may occur in certain sustained-release
formulations of the invention. Proteins such as gelatin and
collagen also serve as stabilizing or bulking agents to reduce
denaturation and aggregation of unstable proteins in this context.
These additives can be incorporated into polymeric melt processes
and compositions within the invention. For example, polypeptide
microparticles can be prepared by simply lyophilizing or spray
drying a solution containing various stabilizing additives
described above. Sustained release of unaggregated peptides and
proteins can thereby be obtained over an extended period of
time.
[0085] Various additional preparative components and methods, as
well as specific formulation additives, are provided herein which
yield formulations for mucosal delivery of aggregation-prone
peptides and proteins, wherein the peptide or protein is stabilized
in a substantially pure, unaggregated form using a solubilization
agent. A range of components and additives are contemplated for use
within these methods and formulations. Exemplary of these
solubilization agents are cyclodextrins (CDs), which selectively
bind hydrophobic side chains of polypeptides. These CDs have been
found to bind to hydrophobic patches of proteins in a manner that
significantly inhibits aggregation. This inhibition is selective
with respect to both the CD and the protein involved. Such
selective inhibition of protein aggregation may provide additional
advantages within the intranasal delivery methods and compositions
of the invention.
[0086] Additional agents for use in this context include CD dimers,
trimers and tetramers with varying geometries controlled by the
linkers that specifically block aggregation of peptides and
protein. Yet solubilization agents and methods for incorporation
within the invention involve the use of peptides and peptide
mimetics to selectively block protein-protein interactions. In one
aspect, the specific binding of hydrophobic side chains reported
for CD multimers may be extended to proteins via the use of
peptides and peptide mimetics that similarly block protein
aggregation. A wide range of suitable methods and anti-aggregation
agents may be available for incorporation within the compositions
and procedures of the invention.
Stabilizing Delivery Vehicle, Carrier, Support or Complex-Forming
Species
[0087] In another embodiment, the present formulation may also
comprise other suitable agents such as a stabilizing delivery
vehicle, carrier, support or complex-forming species. The
coordinate administration methods and combinatorial formulations of
the instant invention may optionally incorporate effective lipid or
fatty acid based carriers, processing agents, or delivery vehicles,
to provide improved formulations for delivery of Dectin-2 ligand or
functionally equivalent fragment proteins, analogs and mimetics,
and other biologically active agents and antigens of the
composition. For example, a variety of formulations and methods are
provided for delivery which comprise one or more active agents,
including the Dectin-2 ligand adjuvant, such as a peptide or
protein, admixed or encapsulated by, or coordinately administered
with, a liposome, mixed micellar carrier, or emulsion, to enhance
chemical and physical stability and increase the half-life of the
biologically active agents (e.g., by reducing susceptibility to
proteolysis, chemical modification and/or denaturation) upon
mucosal delivery.
[0088] Within certain aspects of the invention, specialized
delivery systems for biologically active agents may comprise small
lipid vesicles known as liposomes or micelles. These are typically
made from natural, biodegradable, non-toxic, and non-immunogenic
lipid molecules, and can efficiently entrap or bind drug molecules,
including peptides and proteins, into, or onto, their membranes.
The attractiveness of liposomes as a peptide and protein delivery
system within the invention is increased by the fact that the
encapsulated proteins can remain in their preferred aqueous
environment within the vesicles, while the liposomal membrane
protects them against proteolysis and other destabilizing factors.
Even though not all liposome preparation methods known are feasible
in the encapsulation of peptides and proteins due to their unique
physical and chemical properties, several methods allow the
encapsulation of these macromolecules without substantial
deactivation.
[0089] Additional delivery vehicles carrier, support or
complex-forming species for use within the invention may include
long and medium chain fatty acids, as well as surfactant mixed
micelles with fatty acids. Most naturally occurring lipids in the
form of esters have important implications with regard to their own
transport across mucosal surfaces. Free fatty acids and their
monoglycerides which have polar groups attached have been
demonstrated in the form of mixed micelles to act on the intestinal
barrier as penetration enhancers. This discovery of barrier
modifying function of free fatty acids (carboxylic acids with a
chain length varying from 12 to 20 carbon atoms) and their polar
derivatives has stimulated extensive research on the application of
these agents as mucosal absorption enhancers.
[0090] For use within the methods of the invention, long chain
fatty acids, especially fusogenic lipids (unsaturated fatty acids
and monoglycerides such as oleic acid, linoleic acid, linoleic
acid, monoolein, etc.) provide useful carriers to enhance delivery
of Calnexin or a functionally equivalent fragment, and other
biologically active agents disclosed herein. Medium chain fatty
acids (C6 to C12) and monoglycerides have also been shown to have
enhancing activity in intestinal drug absorption and can be adapted
for use within the mucosal delivery formulations and methods of the
invention. In addition, sodium salts of medium and long chain fatty
acids are effective delivery vehicles and absorption-enhancing
agents for mucosal delivery of biologically active agents within
the invention. Thus, fatty acids can be employed in soluble forms
of sodium salts or by the addition of non-toxic surfactants, e.g.,
polyoxyethylated hydrogenated castor oil, sodium taurocholate, etc.
Other fatty acid and mixed micellar preparations that are useful
within the invention include, but are not limited to, Na caprylate
(C8), Na caprate (C10), Na laurate (C12) or Na oleate (C18),
optionally combined with bile salts, such as glycocholate and
taurocholate.
[0091] The vaccine formulation may additionally include a
biologically acceptable buffer to maintain a pH close to neutral
(7.0-7.3). Such buffers preferably used are typically phosphates,
carboxylates, and bicarbonates. More preferred buffering agents are
sodium phosphate, potassium phosphate, sodium citrate, calcium
lactate, sodium succinate, sodium glutamate, sodium bicarbonate,
and potassium bicarbonate. The buffer may comprise about 0.0001-5%
(w/v) of the vaccine formulation, more preferably about 0.001-1%
(w/v). The buffer(s) may be added as part of the stabilizer
component during the preparation thereof, if desired. Other
excipients, if desired, may be included as part of the final
vaccine formulation.
[0092] The remainder of the vaccine formulation may be an
acceptable diluent, to 100%, including water. The vaccine
formulation may also be formulated as part of a water-in-oil, or
oil-in-water emulsion.
[0093] Also provided as part of the invention is a method of
preparation of the vaccine formulation described herein.
Preparation of the vaccine formulation preferably takes place in
two phases. The first phase may typically involve the preparation
of the stabilizer component. The stabilizer component may comprise
any suitable components as discussed above. For example, a
vegetable-based protein stock solution may be prepared by
dissolving the vegetable-based protein in a diluent. The preferred
diluent may be water, preferably distilled and/or purified so as to
remove trace impurities (such as that sold as purified Super
Q.RTM.). In a separate vessel an animal-based protein may be
dissolved in a diluent, additionally with the sugar component and
buffer additives. Preferably, an equal volume of the
vegetable-based protein stock solution is added to the animal-based
protein solution. It is desirable that after HCl/KOH adjustment to
achieve a pH of approximately 7.2.+-.0.1, the stabilizer component
may be sterilized via autoclave. The stabilizer solution may be
refrigerated for an extended period prior to introduction of the
Dectin-2 ligand adjuvant and a suitable antigen.
[0094] The second phase of preparation of the vaccine formulation
may include introduction of the Dectin-2 ligand adjuvant and a
suitable antigen with the stabilizer component, thereby yielding
the vaccine formulation. Preferably, the Dectin-2 ligand adjuvant
may be diluted with a buffer solution prior to its introduction to
the stabilizer component.
[0095] Once this vaccine formulation solution has been achieved,
the formulation may be separated into vials or other suitable
containers. The vaccine formulation herein described may then be
packaged in individual or multi-dose ampoules, or be subsequently
lyophilized (freeze-dried) before packaging in individual or
multi-dose ampoules. The vaccine formulation herein contemplated
also includes the lyophilized version. The lyophilized vaccine
formulation may be stored for extended periods of time without loss
of viability at ambient temperatures. The lyophilized vaccine may
be reconstituted by the end user, and administered to a
patient.
[0096] The term "lyophilization" or "lyophilized," as used herein,
refers to freezing of a material at low temperature followed by
dehydration by sublimation, usually under a high vacuum.
Lyophilization is also known as freeze drying. Many techniques of
freezing are known in the art of lyophilization such as
tray-freezing, shelf-freezing, spray-freezing, shell-freezing and
liquid nitrogen immersion. Each technique will result in a
different rate of freezing. Shell-freezing may be automated or
manual. For example, flasks can be automatically rotated by motor
driven rollers in a refrigerated bath containing alcohol, acetone,
liquid nitrogen, or any other appropriate fluid. A thin coating of
product is evenly frozen around the inside "shell" of a flask,
permitting a greater volume of material to be safely processed
during each freeze drying run. Tray-freezing may be performed by,
for example, placing the samples in lyophilizer, equilibrating 1 hr
at a shelf temperature of 0.degree. C., then cooling the shelves at
0.5.degree. C./min to -40.degree. C. Spray-freezing, for example,
may be performed by spray-freezing into liquid, dropping by
.about.20 .mu.l droplets into liquid N.sub.2, spray-freezing into
vapor over liquid, or by other techniques known in the art.
[0097] The vaccine of the present invention may be either in a
solid form or in a liquid form. Preferably, the vaccine of the
present invention may be in a liquid form. The liquid form of the
vaccine may have a concentration of 50-4,000 nanomolar (nM),
preferably between 50-150 nM. In some embodiments, the
concentration will be between 1-50,000 nM.
[0098] To vaccinate a patient, a therapeutically effective amount
of vaccine comprising a suitable antigen and a Dectin-2 ligand
adjuvant may be administered to a patient. The therapeutically
effective amount of vaccine may typically be one or more doses,
preferably in the range of about 0.01-10 mL, most preferably 0.1-1
mL, containing 1-200 micrograms, most preferably 1-100 micrograms
of vaccine formulation/dose. The therapeutically effective amount
may also depend on the vaccination species. For example, for
smaller animals such as mice, a preferred dosage may be about
0.01-1 mL of a 1-50 microgram solution of antigen. For a human
patient, a preferred dosage may be about 0.1-1 mL of a 1-50
microgram solution of antigen. The therapeutically effective amount
may also depend on other conditions including characteristics of
the patient (age, body weight, gender, health condition, etc.), the
species of fungi, and others. In one embodiment the vaccine
formulation of the present invention comprises 1-100 micrograms of
Dectin-2 ligand adjuvant and 5-20 micrograms of Calnexin fragment
in either soluble or glucan particle formulation.
[0099] In another aspect, to vaccinate a patient against a fungal
infection, a therapeutically effective amount of a vaccine
comprising Bl-Eng2 as an antigen may be administered to a patient.
The therapeutically effective amount of vaccine my typically be one
or more doses, preferably in the range of about 0.01-10 mL, most
preferably 0.1-1 mL, containing 1-200 micrograms, most preferably
1-100 micrograms of vaccine formulation/dose. The vaccine my
comprise 1-100 micrograms of Bl-Eng2 as an antigen.
[0100] A vaccine of the present invention may be administered by
using any suitable means as disclosed above. Preferably, a vaccine
of the present invention may be administered by intranasal
delivery, transmucosal administration, subcutaneous or
intramuscular administration, e.g., needle injection. In some
embodiments, vaccine compositions for protection against a viral
infection are formulated for transmucosal delivery. In some
embodiments, vaccine compositions for protection against a
bacterial infection are formulated for subcutaneous
administration.
[0101] After vaccination using a vaccine of the present invention
comprising the Dectin-2 ligand adjuvant, a patient may be immunized
against at least one type of fungi, bacteria, or virus. In one
specific embodiment, a patient after vaccination may be immunized
against at least one species of dimorphic fungi. In one preferred
embodiment, a patient after vaccination may be immunized from
multiple dimorphic fungi including Histoplasma, Coccidiodes,
Paracoccidioides, Penicillium, Blastomyces, Sporothrix, and
Aspergillus fumigatus
[0102] The instant invention may also include kits, packages and
multicontainer units containing the above described pharmaceutical
compositions, active ingredients, and/or means for administering
the same for use in the prevention and treatment of diseases and
other conditions in mammalian subjects. Briefly, these kits include
a container or formulation that contains the Bl-Eng2 adjuvant or a
functionally equivalent fragment, and/or other biologically active
agents in combination with mucosal or subcutaneous delivery
enhancing agents disclosed herein formulated in a pharmaceutical
preparation for delivery.
[0103] As used herein, the term "pharmaceutically acceptable
carrier" refers to any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. Preferably, the carrier is suitable for intravenous,
intramuscular, subcutaneous, parenteral, spinal or epidermal
administration (e.g., by injection or infusion). Depending on the
route of administration, the antigentic peptide, i.e., the calnexin
protein may be coated in a material to protect the peptide from the
action of acids and other natural conditions that may inactivate
the peptide.
[0104] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see e.g.,
Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous and the like, as well as from
nontoxic organic acids such as aliphatic mono- and dicarboxylic
acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,
aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like,
as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0105] The composition can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable to high drug concentration.
[0106] In one embodiment, the composition may also comprise a
carrier molecule as a stabilizer component. As the types of
proteins or peptides enclosed in the present invention may be
rapidly degraded once injected into the body, the proteins or
peptides may be bound to a carrier molecule for stabilizing the
proteins or peptides during delivery and administration. A suitable
carrier or stabilizer may comprise fusion proteins, polymers,
liposome, micro or nanoparticles, or any other pharmaceutically
acceptable carriers. A suitable carrier or stabilizer molecule may
comprise a tertiary amine N-oxide, e.g., trimethylamine-N-oxide, a
sugar, e.g., trehalose, a poly(ethylene glycol) (PEG), an
animal-based protein, e.g., digested protein extracts such as
N-Z-Amine.RTM., N-Z-Amine AS.RTM. and N-Z-Amine YT.RTM. (Sheffield
Products Co., Norwich, N.Y.), a vegetable-based protein, e.g., soy
protein, wheat protein, corn gluten, rice protein and hemp protein,
and any other suitable carrier molecules. The composition may also
comprise any suitable carrier or vehicle, such as those as
discussed above. The composition may also comprise other
stabilization agents, such as those as discussed above.
[0107] In one embodiment, the composition may also comprise
suitable stabilizing delivery vehicle, carrier, support or
complex-forming species, such as those as discussed above. For
example, the composition may additionally comprise at least one of
a stabilizer, a buffer, or an adjuvant.
[0108] The present invention has been described in terms of one or
more preferred embodiments, and it should be appreciated that many
equivalents, alternatives, variations, and modifications, aside
from those expressly stated, are possible and within the scope of
the invention.
Example 1
[0109] Adaptive immunity is critical for the prevention and
resolution of fungal infections. The contribution of antibodies to
host defense is debated. In contrast, Ag-specific CD4.sup.+ T cells
play the major role in fungal resistance, as evidenced by the high
incidence of life-threatening fungal infections in patients with
impaired CD4.sup.+ T cells. CD4.sup.+ T cells confer resistance by
secretion of T-helper 1 (Th1) and Th17 cytokines such as
IFN-.gamma., TNF-.alpha., GM-CSF, and IL-17A, which activate
neutrophils, monocytes, macrophages and DCs for fungal clearance.
Since CD4.sup.+ T cells are germane to host defense against fungi,
the challenge is how best to elicit these T cells.
[0110] The transition from innate to adaptive immunity is fostered
by dendritic cells (DCs). These cells process and present Ag to
naive CD4.sup.+ T cells in the context of co-stimulatory factors
(e.g. cell surface ligands and cytokines) that provide the
combination of signals necessary to induce naive T cell activation
and proliferation. During their interactions with DCs, naive T
cells also become functionally specialized. Helper T cell
polarization occurs as a result of the cytokines produced by DCs:
Th1 polarization is associated with DC production of high levels of
IL-12p70, and Th17 polarization is associated with DC production of
IL-1.beta. and IL-6. While vaccine Ags typically have little impact
on the nature of the cytokines produced by DCs, the adjuvant can
have a dramatic effect. Alum (aluminum hydroxide), which is the
most commonly used adjuvant in current vaccine formulations,
suppresses DC production of pro-inflammatory cytokines such as
IL-12p70, creating an environment that polarizes T cells towards a
Th2 phenotype. Thus, a major weakness and central challenge in the
field of vaccinology is the lack of adjuvants that drive Th1 and/or
Th17 polarization and stimulate DCs to produce the appropriate
cytokines. Pathways that can differentially activate DC cytokine
profiles include toll-like receptors (TLRs), C-type lectin
receptors (CLRs), co-stimulatory ligands such as CD40, and cytokine
receptors.
[0111] C-type lectins are important in fungal recognition by DCs
and in inducing anti-fungal Th1 and Th17 responses. Dectin-1 and
Dectin-2 induce Th1/Th17 cells in response to Candida albicans and
Aspergillus fumigatus infection. While Dectin-1 is dispensable,
Dectin-2 is requisite for the development of protective Th1 and
Th17 cells and vaccine resistance against dimorphic fungi. Crude
fractions of mannoproteins isolated from Malassezia pachydermatis
as well as a lipoglycan (Man-LAM) of Mycobacterium tuberculosis
have been shown to trigger Dectin-2 signaling, however they have
not been evaluated as vaccine adjuvants, and glycans and lipids may
be difficult to express and scale.
[0112] The embodiment described here demonstrates a novel fungal
Dectin-2 ligand from an attenuated vaccine strain of Blastomyces
dermatitidis, Bl-Eng2. We tested whether the ligation of Dectin-2
effectively vaccinates mice against fungi. Our vaccination strategy
was to ligate Dectin-2 with Bl-Eng2 and assess the adjuvant
activity by combining it with the recently reported pan-fungal
vaccine calnexin. Fungal recombinant Bl-Eng2 was expressed and
scaled efficiently, it stimulated IL-6 and IL-1.beta. production in
vitro and Th1 and Th17 cells in vivo and, when used as an adjuvant
in combination with calnexin, it protected mice against pneumonia
in a model of lethal pulmonary fungal infection.
[0113] Results
[0114] B. dermatitidis vaccine yeast are bound by soluble Dectin-2
fusion protein and trigger NFAT signaling of Dectin-2 reporter
cells. Dectin-2.sup.-/- mice fail to develop Ag-specific Th1 and
Th17 cells or acquire vaccine resistance. We therefore sought to
identify the fungal pathogen-associated molecular pattern (PAMP)
that is recognized by Dectin-2. We used the NFAT-LacZ reporter
cells to enrich Dectin-2 ligand activity from the vaccine yeast
cell wall. We sonicated vaccine yeast, collected the water-soluble,
cell-wall extract (CWE) and analyzed it by SDS-PAGE. CWE displayed
a broad range of protein bands (FIG. 1A) and harbored Dectin-2
ligand activity (FIG. 1B). Digestion of CWE with proteinase K or
endo-mannosidases reduced this activity (FIG. 1B), suggesting that
both protein and glycan moieties may contribute to ligand activity.
To define the Mr of candidate proteins, we separated the CWE using
a GELFREE 8100 system (FIG. 6A). Fractions #5-6 ranging between 75
to 150 kDa in size contained ligand activity (FIGS. 6A-6B).
[0115] To enrich and identify glycoprotein with ligand activity, we
employed the lectin Concanavalin A (ConA), which binds
.alpha.-D-mannose and .alpha.-D-glucose moieties, gel filtration
and Mass spectrometry analysis (FIG. 1C). The majority of the
ligand activity was removed from CWE by a ConA resin, and the
eluate was highly enriched for ligand activity (FIGS. 1D-1E). The
enrichment by ConA suggested that the Dectin-2 ligand(s) in CWE are
mannoproteins. To further enrich ligand activity, the ConA eluate
was separated by size exclusion chromatography twice, sequentially.
Fractions F4-F6 after the first run contained ligand activity as
determined by the Dectin-2 reporter assay (FIG. 1F); F4-6 were
pooled and subjected to a second separation by gel filtration. The
positive fractions (F9-F13) and negative ones (F1-F7) after the
second gel filtration (FIG. 6C) were analyzed by mass spectrometry
(FIGS. 2A and 7A). Proteins that were more abundant in the positive
vs. negative fraction were considered candidates. Among the
candidates, an uncharacterized member (BDFG 08749) of the fungal
endo-1,3(4)-.beta.-D-glucanase family stood out in positive
fractions (FIG. 7A). The native 526-aa protein contains an 18-aa
signal peptide, an N-terminal GH16 glycosyl hydrolase (GH)
catalytic domain, and a C-terminal S/T-rich domain (FIGS. 2B and
7B) that could be responsible for the strong glycosylation (FIGS.
2C-2D). The GH16 catalytic domain of the endo glucanase has 60.1%
similarity (identical aa and conservative substitution) (45.8%
identity) and the entire glycoprotein has 45.2% similarity (28.8%
identity) to the GPI-anchored endo .beta.-1,3-glucanase Eng2 of A.
fumigatus. Thus, we named the protein ligand Blastomyces-Eng2
(B1-Eng2). PDIA1 is a protein that was more abundant in the
negative gel filtration fraction (e.g. a negative control) (FIG.
2A) and showed no reporter activity and little glycosylation (FIG.
2C).
[0116] Bl-Eng2 Protein is a Bona-Fide Ligand for Dectin-2--
[0117] To evaluate whether Bl-Eng2 is recognized by Dectin-2, we
cloned and expressed the recombinant protein in Pichia pastoris.
This eukaryotic expression system modifies recombinant proteins
with both O- and N-linked glycosylation. Full-length Bl-Eng2 was
fused to a N-terminal .alpha.-factor secretion signal and a
C-terminal Myc-6.times.His tag (FIG. 2B). Ni-NTA purified Bl-Eng2
showed a band of .about.120 kDa on SDS-PAGE gel (FIG. 2C), which
falls within the size range determined in FIG. 6A+B. Periodic
acid-Schiff (PAS) based glyco-stain of Bl-Eng2 showed strong
glycosylation (FIG. 2C), which likely accounts for the discrepancy
between predicted Mr of 57 kDa and apparent Mr of .about.120 kDa.
Gas chromatography (GC) analysis indicated that mannose is the
major monosaccharide, and constitutes 82.8% in glycan mass of
Pichia-expressed Bl-Eng2 (FIG. 2D).
[0118] To verify Bl-Eng2 ligand activity, B3Z reporter cells
expressing Dectin-2 or other distinct CLRs were incubated with
recombinant Bl-Eng2. Bl-Eng2 elicited strong NFAT-lacZ signalling
from Dectin-2 reporter cells, but not from the other CLR-expressing
cells (FIG. 3A), indicating a specific interaction between Dectin-2
and Bl-Eng2. Since Aspergillus Eng2 (Asp-Eng2) exhibits a high
degree of similarity to Bl-Eng2 and contains a Ser/Thr-rich C
terminus, we also tested whether Asp-Eng2 is recognized by
Dectin-2. Asp-Eng2 and Bl-Eng2 were similarly recognized by
Dectin-2 expressing reporter cells (FIGS. 8A-8B), hence Eng2 from
both fungal species are Dectin-2 ligands.
[0119] Dectin-2 is required for Bl-Eng2 ligand activity in primary
cells--To investigate whether Bl-Eng2 stimulates primary cells, we
examined pro-inflammatory cytokine production from bone
marrow-derived dendritic cells (BMDCs). BMDCs from wild type mice,
but not Dectin-2.sup.-/- or Card9.sup.-/- mice, produced a strong
IL-6 response when stimulated with recombinant Bl-Eng2, but not
PDIA1 (FIG. 3B), indicating ligand specificity for Dectin-2. Lack
of stimulation of BMDCs from knockout mice also excludes the
possibility of endotoxin contamination as the stimulus of IL-6 in
wild type cells. Thus, Pichia-expressed Bl-Eng2 triggers a cytokine
response in vitro that requires Dectin-2 and downstream Card9.
These results together indicate that Bl-Eng2 appears to be a
selective Dectin-2 ligand.
[0120] Bl-Eng2 is a Dectin-2 Ligand with Superior Capacity to
Elicit Cytokine Responses--
[0121] Dectin-2 recognizes several fungi including C. albicans, A.
fumigatus and Malassezia, which possess N- and O-linked mannan on
their surface. Thus, not surprisingly, there are two other Dectin-2
ligands described in the literature. They are Furfurman from
Malassezia spp. and Man-LAM from M. tuberculosis. In addition to
these ligands, by using B3Z reporter cells in the work here, we
observed that MP98 from Cryptococcus neoformans is also recognized
by Dectin-2 (FIG. 8C). MP98 also triggers IL-6 by BMDC in a
Dectin-2- and concentration-dependent manner (FIG. 8D). MP98 is a
mannoprotein of Mr of 98 kDa with 103 Ser/Thr residues at the
C-terminus that serve as potential O-linked glycosylation sites,
and 12 putative N-linked glycosylation sites.
[0122] To begin to evaluate the relative potency of Dectin-2
ligands, we compared the ability of Bl-Eng2 and the other three
Dectin-2 ligands to induce cytokine production by BMDCs. Bl-Eng2
induced the strongest IL-6 production by BMDCs when compared at
equal molar and mass ratios to the other ligands (FIG. 3C). These
results suggest that Bl-Eng2 is relatively potent for triggering
IL-6 and might be used as an adjuvant for vaccination to boost the
development of Ag-specific T cell responses.
[0123] Bl-Eng2 Induces the Production of IL-6 and IL-1,8 by Human
PBMCs--
[0124] A suitable adjuvant for vaccine formulation should ideally
stimulate human accessory cells. To test this capacity, we assessed
the effect of Bl-Eng2 on the function of human PBMCs. After
stimulation with plate-coated Bl-Eng2, human PBMCs from five
healthy subjects produced up to 17 ng/ml IL-6 and 9 ng/ml
IL-1.beta. as measured in the cell culture supernatants by ELISA
(FIG. 3D). These data suggest that recombinant Bl-Eng2 has the
capacity to induce the production of Th17 cell priming cytokines by
human antigen-presenting cells (APC) in vitro.
[0125] Bl-Eng2 Promotes T Cell Development In Vivo and Imparts
Vaccine Efficacy--
[0126] To investigate whether Bl-Eng2 could be harnessed as a
vaccine adjuvant, we performed preclinical studies in mice. We
first tested whether Bl-Eng2 augments the development of vaccine
Ag-specific T cells. To assess these T cell responses in vivo, we
vaccinated mice with the pan-fungal Ag calnexin and enumerated
CD4.sup.+ T cell responses by TCR Tg 1807 cells, which are specific
for calnexin. Calnexin was suspended with incomplete freund's
adjuvant (mineral oil) and injected subcutaneously. The addition of
Bl-Eng2 into the formulation sharply increased the frequency of
IL-17 producing 1807 T cells (FIG. 4A) and the number of activated
(CD44.sup.+) and IL-17 and IFN-.gamma. producing 1807 T cells, as
measured by ex vivo stimulation with anti-CD3 and anti-CD28 mAb
(FIGS. 4B and 9A). Ex vivo stimulation with the vaccine Ag calnexin
also yielded sharp increases in the amount of IL-17 produced by T
cells from the draining lymph nodes (FIG. 4D). Thus, Bl-Eng2
promoted the development of Th17 cells more so than Th1 cells.
[0127] Addition of Bl-Eng2 to the vaccine also reduced lung CFU as
early as four days after mice received a lethal experimental
challenge, and did so in a concentration-dependent manner (FIG.
9B). In a parallel group, at the time unvaccinated control mice
were moribund (day 18 post-infection), the addition of Bl-Eng2 to
the vaccine reduced lung CFU by more than two logs (FIG. 4C).
Combining the vaccine with commercial alum as an adjuvant did not
increase the frequency and numbers of cytokine producing T cells or
reduce the fungal burden (FIGS. 4A-4D). However, combining Bl-Eng-2
together with Alum increased the adjuvancy of Alum as measured by
the number of activated (CD44.sup.+), IL-17 and IFN-.gamma.
producing 1807 T cells and the reduction in lung CFU (FIG. 10).
These results suggest that Bl-Eng-2 can work in concert with other
(commercially available and FDA approved) adjuvants and augment
vaccine efficacy.
[0128] Bl-Eng2 failed to increase the development of Th17 and Th1
cells, the production ex vivo of IL-17 and IFN-.gamma. or reduce
lung CFU in Dectin-2.sup.-/- mice, verifying that the adjuvant
effect is Dectin-2-dependent in vivo (FIGS. 9C-9E). Thus, Bl-Eng2
exhibits adjuvant-like properties by increasing the development of
Ag-specific (1807) Th17 and Th1 cells and protecting mice from
lethal pulmonary infection with B. dermatitidis.
[0129] The studies above exploited TCR Tg T cells to sensitively
report the ability of Bl-Eng2 to enhance development of calnexin
Ag-specific Th17 and Th1 cells upon vaccination. However, adoptive
transfer of these cells into mice artificially enhances the number
of CD4.sup.+ T cell precursors in the animal. To investigate
whether Bl-Eng2 also has the capacity to induce the development of
endogenous calnexin-Ag specific CD4.sup.+ T cells and similarly
protect animals, we vaccinated wild type mice in the absence of
adoptive transfer. The formulation of Bl-Eng2 with the calnexin
subunit vaccine again reduced lung CFU by over two logs vs. control
mice vaccinated with calnexin in mineral oil alone (IFA), and by
over 3 logs vs. mice that got IFA alone (FIG. 4E). The addition of
Bl-Eng2 to the calnexin vaccine formulation also increased survival
significantly vs. control mice vaccinated with calnexin alone (FIG.
4E). This is remarkable since the number of Ag-specific T cell
precursors before vaccination was far lower in the absence than in
the presence of transferred of naive 1807 cells, indicating that
Bl-Eng2 is a powerful adjuvant that drives the development of
protective endogenous calnexin-specific CD4.sup.+ T cells (FIG.
4F).
[0130] Bl-Eng-2 Augments In Vivo Killing of Fungi by Neutrophils
(PMN) and Alveolar Macrophages--
[0131] To investigate the downstream myeloid effector mechanisms of
Bl-Eng-2 adjuvancy we used red fluorescent B. dermatitidis yeast to
report phagocytic uptake and fungal viability during cellular
interactions with the murine leukocytes. The concept of using
fluorescence to monitor microbial fate and investigate functional
outcomes of individual microbial cell-host cell encounters has been
introduced recently and provides a powerful strategy to measure
effector mechanisms in vivo. At day 4 post-infection, mice
vaccinated with calnexin+Bl-Eng-2 and calnexin+Alum+Bl-Eng-2 showed
increased activation and killing by neutrophils and alveolar
macrophages vs. calnexin and calnexin+Alum controls (FIGS. 5A-5C
and FIG. 11). The increase in in vivo fungal killing by neutrophils
and macrophages correlated with reduced numbers of DsRed.sup.+
yeast in the lung (FIGS. 5D and 11D) and CFU by plating (FIGS. 5E
and 11C). Bl-Eng-2 mediated effects were observed in the presence
of adoptively transferred 1807 T cells (FIG. 5) and by endogenous
CD4.sup.+ T cells without adoptive transfer (FIGS. 11C-11D). Thus,
the addition of Bl-Eng-2 augments the activation and killing by
myeloid effector cells such as the neutrophils and alveolar
macrophages in the lung.
[0132] Discussion
[0133] We describe a novel ligand for Dectin-2: Bl-Eng2. Discovery
of a potent CLR ligand may address a limitation of current
vaccines: the lack of adjuvants that elicit protective
cell-mediated immunity. The approach we took to identify Bl-Eng2
was based on prior work from our group and other laboratories.
Dectin-2 recognizes and mediates host defense against several fungi
including C. albicans, C. glabrata, A. fumigatus, Malassezia spp.,
Coccidiodes posadasii, Histoplasma capsulatum and B. dermatitidis.
Additionally, Dectin-2.sup.-/- mice vaccinated with attenuated B.
dermatitidis yeast fail to prime Ag-specific Th1 and Th17 cells or
acquire vaccine resistance to pulmonary infection. Thus, Dectin-2
regulates innate recognition of the fungal vaccine, and the
development of a protective cellular immune response. Hence, we
sought to identify the Dectin-2 ligand from the vaccine strain. We
hypothesized that the ligand would prime APC to produce cytokines
(e.g. IL-6) that are known to foster the development of Th17 cells
that protect against lethal fungal challenge.
[0134] By using Dectin-2 reporter cells as a probe, we enriched and
identified Bl-Eng2 by ConA binding, gel filtration and Mass
spectrometry. The identification of Bl-Eng2 also led us to unveil
the unappreciated role of Asp-Eng2 in binding Dectin-2. Both Eng2
proteins are bona fide Dectin-2 ligands since they trigger NFAT
signaling in Dectin-2 reporter cells. Bl-Eng2 features a 45.2%
overall and 60.1% GH16 domain sequence similarity to Eng2 from A.
fumigatus (Asp-Eng2) and contains a Ser/Thr-rich C-terminus that
both proteins have in common. Bl-Eng2 and Asp-Eng2 respectively
harbor 68 and 74 potential O-linked glycosylation sites within
their respective 134-aa and 234-aa long Ser/Thr-rich C-terminus,
but display no consensus sites for N-linked glycosylation
(Asn-X-Ser/Thr). In addition to the Eng2 glycoproteins, we now also
establish here that MP98 from C. neoformans serves as a ligand for
Dectin-2.
[0135] Dectin-2 has been reported to recognize high mannose
structures of fungi, such as .alpha.-1,2-mannan from C. albicans
and furfurman, which is a mannoprotein from Malassezia spp. Man-Lam
from M. tuberculosis consists of four components: a
mannosyl-phophatidyl-myo-inositol (MPI) anchor, a mannose backbone,
an arabinan domain, and a .alpha.1,2-mannose cap. MP98 from C.
neoformans is a mannoprotein with a Mr of 98 kDa; it contains 12
possible N-linked glycosylation sites, and 103 Ser/Thr residues at
the C-terminus that serve as potential 0-linked glycosylation
sites. The minimal unit of Bl-Eng2 that confers ligand activity is
uncertain. Since both mannosidase and proteinase K digestion of CWE
starting material reduced Dectin-2 ligand activity, both the
protein and glycan moieties of Bl-Eng2 may contribute to its
action, perhaps explaining its superior stimulation of cytokine
responses compared to the other ligands.
[0136] We found that recombinant Bl-Eng2 elicits potent downstream
functions. It induces the production of IL-6 by BMDC in a Dectin-2-
and Card9-dependent manner. In addition, Bl-Eng2 induces the
production of IL-6 and IL-1.gamma. by human PBMC, which may have
strong implications for the translational aspect of our discovery.
In comparison to previously described Dectin-2 ligands, Bl-Eng2
triggers superior cytokine production by murine BMDC. Ligand
induced IL-6 production was >100 fold higher for Bl-Eng2 than
the other Dectin-2 ligands: Furfurman from Malassezia spp. and
Mannose-capped lipoarabinomannan (Man-Lam) from M. tuberculosis and
MP98 from C. neoformans.
[0137] Bl-Eng2 induction of T cell priming cytokines by APCs
efficiently promoted the development of calnexin Ag-specific Th17
cells (more so than Th1 cells), and recall of these cells to the
lung upon fungal challenge of vaccinated mice. The large numbers of
pro-inflammatory T cells sharply reduced lung CFU and increased
survival after infection of Bl-Eng2 vaccinated vs. control mice. In
comparison, combining commercial Alum with the calnexin subunit
vaccine did not show an adjuvant effect. However, Bl-Eng-2 combined
with Alum augmented its adjuvancy indicating that Bl-Eng-2 has the
potential to improve T cell priming by the commercially available
and FDA approved Alum. Thus, in our subunit vaccine model,
Bl-Eng2-induced Dectin-2 signaling was associated with cellular
immune responses that protected mice against lethal pulmonary
fungal infection. Although not experimentally addressed in this
manuscript, it is conceivable that Bl-Eng-2 can also augment the
induction of CD4.sup.+ T cell-dependent antibody responses that
promote host protection against fungi, especially when combined
with Alum since the latter is known to stimulate both T and B cell
immune responses. It remains to be investigated whether antibody
will be protective in our vaccine setting.
[0138] We previously reported that mice vaccinated with calnexin
and other adjuvants (glucan particles engaging Dectin-1, Adjuplex,
or the combination) were optimally protected when we adoptive
transferred naive 1807 cells to increase the pool of Ag-experienced
CD4.sup.+ T cells. Here, the addition of Bl-Eng2 to the same
calnexin vaccine reduced lung CFU by more than two to three logs
vs. control mice even without adoptive transfer of large numbers of
naive 1807 T cell precursors. These results imply that engagement
of Dectin-2 by Bl-Eng2 may be better than engagement of Dectin-1 by
glucan particles and other previously used adjuvants at expanding
the pool of endogenous calnexin-specific CD4.sup.+ T cell
precursors or that Bl-Eng2 induced individual Ag-experienced cells
to produce larger amounts of effector cytokines. Thus, Bl-Eng2 may
be a powerful vaccine adjuvant in situations where T cell
precursors are low in number and adoptive transfer of naive T cell
precursors is either not feasible or too costly.
[0139] In contrast to the protective effects of Bl-Eng2
vaccination, Man-Lam induced Dectin-2 responses that caused Th17
cell-mediated autoimmune disease pathology and EAE. Man-Lam
stimulation of Dectin-2 lead to the development of MOG.sub.35-55
peptide-specific T cells that produced IL-17, IFN-.gamma. and
GM-CSF upon ex vivo stimulation. This could simply relate to model
selection rather than adjuvant efficiency. Thus, it is unclear
whether Man-Lam is capable of inducing protective T cell immunity
in an infectious disease setting. Although C. neoformans MP98 and
its glycan modifications also promoted T cell activation, the
T-helper phenotype and functional role in resistance by primed T
cells were not investigated.
[0140] In conclusion, among the few Dectin-2 ligands reported to
date, or newly discovered here, Bl-Eng2 is the most potent at
stimulating murine and human cells to produce cytokines known to
foster the development of protective Th17 and Th1 cells e.g. IL-6
and IL-1.beta.. The production of IL-17 and IFN-.gamma. by Th17 and
Th1 cells then promotes the activation and killing of fungi by
myeloid effector cells such as neutrophils and alveolar
macrophages. Since Bl-Eng2 also greatly augments protective
immunity mediated by a subunit vaccine, Bl-Eng2 could potentially
be harnessed as an adjuvant for vaccination against infectious
disease that requires cellular immunity for host defense. The
structural basis underpinning Bl-Eng2 potency as an adjuvant will
be important to investigate and understand so that those features
can be harnessed for vaccine development in the fight against
infectious disease due to intracellular pathogens.
[0141] Material and Methods
[0142] Fungi--
[0143] Strains used were wild-type, virulent B. dermatitidis ATCC
strain 26199, DsRed26199 and strain #55, the isogenic, attenuated
mutant lacking BAD1. B. dermatitidis was grown as yeast on
Middlebrook 7H10 agar with oleic acid-albumin complex (Sigma) at
39.degree. C.
[0144] Mouse Strains--
[0145] Inbred wild type C57BL/6 and congenic B6. PL-Thy1.sup.a/Cy
(stock #00406) mice carrying the Thy 1.1 allele were obtained from
Jackson Laboratories, Bar Harbor, Me. Blastomyces-specific TCR Tg
1807 mice were generated in our lab and were backcrossed to
congenic Thy1.1.sup.+ mice as described elsewhere. Dectin-2.sup.-/-
mice were bred at our facility. Mice were 7-8 weeks old at the time
of these experiments. Mice were housed and cared for as per
guidelines of the University of Wisconsin Animal Care Committee who
approved all aspects of this work.
[0146] Preparation of CWE--
[0147] Blastomyces dermatitidis yeast were harvested from 7H10
agar, washed with H.sub.2O, and sonicated for 3 min on ice. After
centrifuging, the soluble extract was collected, passed through a
0.45-.mu.m pore-size filter and used as CWE. The protein level was
measured with the Pierce BCA assay (Thermo Fisher Scientific).
[0148] Enrichment of Mannosylated Proteins and Mass Spectrometry
Analysis--
[0149] To enrich the mannosylated proteins, CWE was incubated with
Concanavalin A (ConA) Sepharose resin (Sigma-Aldrich), and the
bound fraction was eluted with methyl-.alpha.-D-mannopyranoside as
described previously. The ConA-enriched proteins were then applied
to a size exclusion column of Ultragel AcA 44 resin (Pall) and
eluted with PBS. The ConA enrichment and size exclusion fractions
were assessed using SDS-PAGE and silver staining. Size exclusion
fractions that contained Dectin-2 ligand activity were analyzed by
mass spectrometry as previously described at the Mass Spectrometry
Facility, University of Wisconsin-Madison. Briefly, peptides were
analyzed by nano-LC-MS/MS using the Agilent 1100 nanoflow system
(Agilent Technologies) connected to a hybrid linear ion
trap-orbitrap mass spectrometer (LTQ-Orbitrap XL, Thermo Fisher
Scientific) equipped with a nanoelectrospray ion source.
[0150] Generation and Purification of r-Bl-Eng2--
[0151] Bl-Eng2 was cloned and expressed in P. pastoris using
standard recombinant techniques. Total RNA was extracted from B.
dermatitidis yeast and transcribed to cDNA as previously described.
Using the cDNA as a template, the Bl-ENG2 coding sequence was
amplified using KOD Hot Start DNA Polymerase (Toyobo) with primers
5'-GGCTCGAGAAAAGAGAGGCTGAAGCTAGGGCTACCAAGCTCGCGTT (SEQ ID NO:9) and
5'-GTTTCTAGACCGTACTTGTCATTTGTGGGGTATCCCG (SEQ IN NO:10), and
inserted in-frame into the XhoI/XbaI sites of the pPICZ.alpha.A
vector (Invitrogen). The resulting expression vector was then
linearized with PmeI and transformed into Pichia pastoris strain
X-33 (Invitrogen) by electroporation. Yeast colonies were screened
for Bl-Eng2 protein expression by Western blot analysis using an
anti-His antibody (Cell Signaling Technology). Bl-Eng2 protein
secreted from methanol-induced yeast cells was purified using
Ni-NTA agarose (Qiagen) according to the manufacturer's protocol,
and dialyzed against PBS. Purity of recombinant Bl-Eng2 was
assessed by SDS-PAGE and silver staining. Without being bound to
any particular theory, it is believed that the alpha-factor signal
peptide is excised from the recombinant Bl-Eng2 upon secretion of
the protein from the yeast. The predicted sequence of the Bl-Eng2
recombinant protein after excision of the alpha-factor signal
peptide is included below.
[0152] Predicted recombinant Bl-Eng2 protein: alpha-factor signal
peptide excised during secretion (SEQ ID NO:11).
TABLE-US-00001 RATKLALLAALAKLSTGAYVLQDDYQPSNFFDDFAFFDGPDPSNAYVTYV
DKSKALRDGLASNNNDFVYLGVDHQNVARGRGRESVRLETKKSYKHGLIV
ADISHMPGNICGTWPAFWATGATWPDDGEFDIIEGVNKQNKNVVALHTTA
GCKVEDNNKYSGILVTKDCDVYSPNQPSNQGCLFRAPSATSYGTAFNSIG
GGVYATEWTSDSISVWFFPRYQIPSNINDENPDPSTWPRPIAHFTGCEFD
KFFQEQRIIFNTAFCGDWAKATWNENGCAAGGRTCEDYVKNNPWAFSEAF
WSINYMKVFQNKQGDTSTSTTTSSTSSTSSSSTEAPTTTMTTSSTYEPSV
SSSTAPEPSQSASTPSEYPQPSTAEPTASSSSYPKSSFASTDSPVPTDYP
VPSSDEPTVPSATYSESSPVPTDYPVPSSDEPTVPSATYSESLPSASAPS
EYPTGTASVDPTDVSSCTPPPTQSCITYTTKTTIAIVVTAPESYKEAIQT
ESAEDETEPAAYPTEPAGYPTNDKYGLEQKLISEEDLNSAVDHHHHHH
[0153] Carbohydrate Analysis--
[0154] Bl-Eng2 protein glycosylation was assessed using the Pierce
Glycoprotein Staining Kit (Thermo Fisher Scientific).
Monosaccharide composition was determined by gas chromatography as
described elsewhere.
[0155] CLR Reporter Assay--
[0156] B3Z/BWZ reporter cells expressing Dectin-2, Mincle, MCL and
Dectin-1 have been described previously. For B3Z/BWZ cell
stimulation, 10.sup.5 B3Z/BWZ cells per well in a 96-well plate
were incubated for 18 h with heat-killed fungal cells or
plate-coated ligands. .beta.-galactosidase (lacZ) activity was
measured in total cell lysates using CPRG (Roche) as a substrate.
OD 560 was measured using OD 620 as a reference.
[0157] Stimulation of Mouse BMDCs or Human PBMCs and Cytokine
Detection--Generation of bone marrow-derived dendritic cells
(BMDCs) has been described previously. Peripheral blood mononuclear
cells (PBMCs) were isolated from heparinized whole blood collected
over Ficoll-Paque Plus (GE). 1-2.times.10.sup.5 BMDCs or
5.times.10.sup.5 PBMCs per well in a 96-well plate were incubated
with plate-bound Bl-Eng2. After 24 h, supernatants were collected
and cytokine levels were measured by ELISA (R&D Systems or
Biolegend) according to the manufacturer's specifications.
[0158] Vaccination with Calnexin and Bl-Eng2 and Enumeration of
Rare Epitope-Specific T Cells--
[0159] Prior to vaccination, mice received adoptively transferred
naive 1807 T cells or not. Mice were vaccinated twice
subcutaneously with 10 .mu.g recombinant calnexin and 10 .mu.g
Bl-Eng2 formulated in incomplete Freund's adjuvant (IFA), two weeks
apart. Two weeks after the boost, mice were challenged with
2.times.10.sup.4 26199 yeast and analyzed for lung T cell responses
(at day 4 post-infection) and lung CFU (at day 4 or two weeks
post-infection). 1807 T cell responses were detected with the
congenic Thy1.1 marker and endogenous, calnexin-specific T cells by
tetramer. T cells were detected using the following antibodies:
tetramer-PE, CD4-BUV395, CD8-PeCy7, CD3-BV421, CD90.2-BV785,
CD44-BV650, Live-dead Near IR, IFN-.gamma.-A488 and IL-17-A647.
[0160] Intracellular Cytokine Stain--
[0161] Lung cells were harvested at day 4 post-infection. Cells
(0.5.times.10.sup.6 cells/ml) were stimulated for 5 hours with
anti-CD3 (clone 145-2C11; 0.1 .mu.g/ml) and anti-CD28 (clone 37.51;
1 .mu.g/ml) in the presence of Golgi-Stop (BD Biosciences).
Stimulation with fungal ligands yielded comparable cytokine
production by transgenic T-cells compared to CD3/CD28 stimulation.
After cells were washed and stained for surface CD4 and CD8 using
anti-CD4 BV395, anti-CD8 PeCy7, and anti-CD44-FITC mAbs
(Pharmingen), they were fixed and permeabilized in Cytofix/Cytoperm
at 4.degree. C. overnight. Permeabilized cells were stained with
anti-IL-17A PE and anti-IFN-.gamma. Alexa 700 (clone XMG1.2)
conjugated mAbs (Pharmingen) in FACS buffer for 30 min at 4.degree.
C., washed, and analyzed by FACS. Cells were gated on CD4 and
cytokine expression in each gate analyzed. The number of cytokine
positive CD4.sup.+ T cells per lung was calculated by multiplying
the percent of cytokine-producing cells by the number of CD4.sup.+
T cells in the lung.
[0162] The Generation of Bone Marrow Dendritic Cells--
[0163] Bone marrow-derived dendritic cells (BMDCs) were obtained
from the femurs and tibias of individual mice. Each bone was
flushed with 10 ml of 1% FBS in RPMI through a 22G needle. Red
blood cells were lysed followed by wash and re-suspension of cells
in 10% FBS in RPMI medium. In a petri dish, 2.times.10.sup.6 bone
marrow cells were plated in 10 ml of RPMI containing 10% FBS plus
penicillin-streptomycin (P/S) (HyClone), 2-mercaptoethanol and 20
ng/ml of rGM-CSF. The culture media were refreshed every three days
and BMDCs were harvested after 10 days for in vitro co-culture
assays.
[0164] Ex vivo stimulation of primed T cells for cytokine protein
measurement--Ex vivo cell culture supernatants were generated using
the brachial and inguinal draining lymph nodes harvested from mice
28 days post-vaccination and at day 4 post-infection, washed and
resuspended in complete RPMI containing 10 .mu.g/ml recombinant
calnexin, and plated in 96-well plates at a concentration of
5.times.10.sup.5 cells/well. Supernatants were collected from ex
vivo co-cultures after three days of incubation at 37.degree. C.
and 5% CO.sub.2. IFN-.gamma. and IL-17 (R&D System) were
measured by ELISA according to manufacturer specifications
(detection limits, 0.05 ng/ml and 0.02 ng/ml, respectively).
[0165] Tracking Association of Yeast with Neutrophils and Alveolar
Macrophages In Vivo--
[0166] Mice were euthanized three days after challenge i.t. with
10.sup.5 DsRed yeast and hearts were perfused with PBS to remove
blood from the lungs to improve staining. Lungs were dissociated,
digested and stained as described previously. In summary, lungs
were dissociated and digested in buffer containing collagenase D
and DNase I. After erythrocyte lysis, cells were stained for
myeloid cell markers and then fixed in Cytofix/Cytoperm (BD
Biosciences, San Jose, Calif.). Cells were stained for 30 minutes
at room temperature with 1 .mu.g/ml Uvitex-2B (Polysciences,
Warrington, Pa.) diluted in BD perm/wash buffer and then
subsequently washed with BD perm/wash buffer and fixed with 2%
paraformaldehyde.
[0167] Statistics--
[0168] Differences in the number of cells and lung CFU were
analyzed using Wilcoxon rank and Mann Whitney test for
non-parametric data or a T-test if data were normally distributed.
A Bonferroni adjustment was used to correct for multiple tests. A
value of P<0.05 is considered significant.
Example 2
[0169] The embodiment described herein demonstrates the use of
Bl-Eng2 as a vaccine adjuvant in bacterial and viral vaccines. As
demonstrated in FIG. 12 and FIG. 13, Bl-Eng2 functions as an
adjuvant in bacterial and viral vaccine formulations and increased
the number of activated (CD44+) CD4+ and CD8+ producing T cells.
Mice were vaccinated with Ag85B from Mycobacterium tuberculosis and
Nucleoprotein (NP) from Influenza A in the presence and absence of
Bl-Eng-2 and compared the number of corresponding cytokine
producing CD4.sup.+ and CD8.sup.+ T cells, respectively. In the TB
vaccine model, Ag-specific IL-17 and IFN-.gamma. producing
CD4.sup.+ T cells and in the Influenza model, IFN-.gamma. producing
cytotoxic CD8.sup.+ T cells (CTL) are thought to be most protective
against bacterial and viral infection, respectively. The addition
of Bl-Eng-2 to Ags 85B or NP augmented the number of cytokine
producing Ag-specific CD4.sup.+ and CD8.sup.+ T cells in both
models. Thus, Bl-Eng-2 augments immunity also in response to
non-fungal (bacterial and viral) Ags and does not only increase
CD4.sup.+ but also CD8.sup.+ T cell immune responses.
Example 3
[0170] The embodiment described herein demonstrates the use of
Bl-Eng2 as a novel antigen for use in vaccine compositions. As
demonstrated in FIGS. 14A-14B and FIGS. 15A-15B, Bl-Eng2 functions
as an antigen when used in vaccine compositions to raise antifungal
T cells in the subject mice. Mice subcutaneously vaccinated with
Bl-Eng2 formulated in IFA had significantly reduced lung CFU at 4
(15-fold) and 11 post-infection (>5,000 fold) compared to
control mice (FIG. 14). Splenocytes from Bl-Eng2 vaccinated mice
produced >10 ng/ml INF-.gamma. when stimulated in vitro with
full length Bl-Eng2 protein or peptide 1 which is comprised of the
following amino acid sequence: AFFDGPDPSNAYV (SEQ ID NO:4).
Therefore vaccination with this peptide alone will engender a
similar level of protection as full length Bl-Eng2 protein.
[0171] This peptide could also be used to expand autologous,
endogenous Bl-Eng2-specific T cells of patients that will undergo
transplantation, chemotherapy or other immunocompromising
treatments to boost their immunity against fungal infection (e.g.
cellular immunotherapy following stem cell transplantation). The
lethality of invasive pulmonary infection with Aspergillus
fumigatus is 50-90% in that patient population.
REFERENCES
[0172] 1. Brown G D, Denning D W, Gow N A, Levitz S M, Netea M G,
et al. (2012) Hidden killers: human fungal infections. Sci Transl
Med 4: 165rv113. [0173] 2. Brown G D, Denning D W, Levitz S M
(2012) Tackling human fungal infections. Science 336: 647. [0174]
3. Romani L (2011) Immunity to fungal infections. Nat Rev Immunol
11: 275-288. [0175] 4. Wuthrich M, Deepe G S, Jr., Klein B (2012)
Adaptive immunity to fungi. Annu Rev Immunol 30: 115-148. [0176] 5.
Leibundgut-Landmann S, Wuthrich M, Hohl T M (2012) Immunity to
fungi. Curr Opin Immunol 24: 449-458. [0177] 6. Wuthrich M, Gem B,
Hung C Y, Ersland K, Rocco N, et al. (2011) Vaccine-induced
protection against 3 systemic mycoses endemic to North America
requires Th17 cells in mice. J Clin Invest 121: 554-568. [0178] 7.
Zelante T, De Luca A, Bonifazi P, Montagnoli C, Bozza S, et al.
(2007) IL-23 and the Th17 pathway promote inflammation and impair
antifungal immune resistance. Eur J Immunol 37: 2695-2706. [0179]
8. Mori A, Oleszycka E, Sharp F A, Coleman M, Ozasa Y, et al.
(2012) The vaccine adjuvant alum inhibits IL-12 by promoting PI3
kinase signaling while chitosan does not inhibit IL-12 and enhances
Th1 and Th17 responses. Eur J Immunol 42: 2709-2719. [0180] 9.
Gringhuis S I, Wevers B A, Kaptein T M, van Capel T M, Theelen B,
et al. (2011) Selective C-Rel activation via Malt1 controls
anti-fungal T(H)-17 immunity by dectin-1 and dectin-2. PLoS Pathog
7: e1001259. [0181] 10. Gringhuis S I, den Dunnen J, Litjens M, van
der Vlist M, Wevers B, et al. (2009) Dectin-1 directs T helper cell
differentiation by controlling noncanonical NF-kappaB activation
through Raf-1 and Syk. Nat Immunol 10: 203-213. [0182] 11.
Geijtenbeek T B, Gringhuis S I (2009) Signalling through C-type
lectin receptors: shaping immune responses. Nat Rev Immunol 9:
465-479. [0183] 12. LeibundGut-Landmann S, Gross O, Robinson M J,
Osorio F, Slack E C, et al. (2007) Syk- and CARDS-dependent
coupling of innate immunity to the induction of T helper cells that
produce interleukin 17. Nat Immunol 8: 630-638. [0184] 13. Robinson
M J, Osorio F, Rosas M, Freitas R P, Schweighoffer E, et al. (2009)
Dectin-2 is a Syk-coupled pattern recognition receptor crucial for
Th17 responses to fungal infection. J Exp Med 206: 2037-2051.
[0185] 14. Saijo S, Ikeda S, Yamabe K, Kakuta S, Ishigame H, et al.
(2010) Dectin-2 recognition of alpha-mannans and induction of Th17
cell differentiation is essential for host defense against Candida
albicans. Immunity 32: 681-691. [0186] 15. Zhu L L, Zhao X Q, Jiang
C, You Y, Chen X P, et al. (2013) C-type lectin receptors Dectin-3
and Dectin-2 form a heterodimeric pattern-recognition receptor for
host defense against fungal infection. Immunity 39: 324-334. [0187]
16. Rivera A, Hohl T M, Collins N, Leiner I, Gallegos A, et al.
(2011) Dectin-1 diversifies Aspergillus fumigatus-specific T cell
responses by inhibiting T helper type 1 CD4 T cell differentiation.
J Exp Med 208: 369-381. [0188] 17. Loures F V, Rohm M, Lee C K,
Santos E, Wang J P, et al. (2015) Recognition of Aspergillus
fumigatus hyphae by human plasmacytoid dendritic cells is mediated
by dectin-2 and results in formation of extracellular traps. PLoS
Pathog 11: e1004643. [0189] 18. Carrion Sde J, Leal S M, Jr.,
Ghannoum M A, Aimanianda V, Latge J P, et al. (2013) The RodA
hydrophobin on Aspergillus fumigatus spores masks dectin-1- and
dectin-2-dependent responses and enhances fungal survival in vivo.
J Immunol 191: 2581-2588. [0190] 19. Wang H, Lebert V, Hung C Y,
Galles K, Saijo S, et al. (2014) C-type lectin receptors
differentially induce th17 cells and vaccine immunity to the
endemic mycosis of north America. J Immunol 192: 1107-1119. [0191]
20. Yonekawa A, Saijo S, Hoshino Y, Miyake Y, Ishikawa E, et al.
(2014) Dectin-2 is a direct receptor for mannose-capped
lipoarabinomannan of mycobacteria. Immunity 41: 402-413. [0192] 21.
Wuthrich M, Brandhorst T T, Sullivan T D, Filutowicz H, Sterkel A,
et al. (2015) Calnexin induces expansion of antigen-specific CD4(+)
T cells that confer immunity to fungal ascomycetes via conserved
epitopes. Cell Host Microbe 17: 452-465. [0193] 22. Hartl L,
Gastebois A, Aimanianda V, Latge J P (2011) Characterization of the
GPI-anchored endo beta-1,3-glucanase Eng2 of Aspergillus fumigatus.
Fungal Genet Biol 48: 185-191. [0194] 23. McGreal E P, Rosas M,
Brown G D, Zamze S, Wong S Y, et al. (2006) The
carbohydrate-recognition domain of Dectin-2 is a C-type lectin with
specificity for high mannose. Glycobiology 16: 422-430. [0195] 24.
Sato K, Yang X L, Yudate T, Chung J S, Wu J, et al. (2006) Dectin-2
is a pattern recognition receptor for fungi that couples with the
Fc receptor gamma chain to induce innate immune responses. J Biol
Chem 281: 38854-38866. [0196] 25. Ishikawa T, Itoh F, Yoshida S,
Saijo S, Matsuzawa T, et al. (2013) Identification of Distinct
Ligands for the C-type Lectin Receptors Mincle and Dectin-2 in the
Pathogenic Fungus Malassezia. Cell Host Microbe 13: 477-488. [0197]
26. Taylor P R, Roy S, Leal S M, Jr., Sun Y, Howell S J, et al.
(2014) Activation of neutrophils by autocrine IL-17A-IL-17RC
interactions during fungal infection is regulated by IL-6, IL-23,
RORgammat and dectin-2. Nat Immunol 15: 143-151. [0198] 27. Levitz
S M, Nong S, Mansour M K, Huang C, Specht C A (2001) Molecular
characterization of a mannoprotein with homology to chitin
deacetylases that stimulates T cell responses to Cryptococcus
neoformans. Proc Natl Acad Sci USA 98: 10422-10427. [0199] 28.
Wuthrich M, Ersland K, Sullivan T, Galles K, Klein B S (2012) Fungi
subvert vaccine T cell priming at the respiratory mucosa by
preventing chemokine-induced influx of inflammatory monocytes.
Immunity 36: 680-692. [0200] 29. Jhingran A, Mar K B, Kumasaka D K,
Knoblaugh S E, Ngo L Y, et al. (2012) Tracing conidial fate and
measuring host cell antifungal activity using a reporter of
microbial viability in the lung. Cell Rep 2: 1762-1773. [0201] 30.
Ifrim D C, Bain J M, Reid D M, Oosting M, Verschueren I, et al.
(2014) Role of Dectin-2 for host defense against systemic infection
with Candida glabrata. Infect Immun 82: 1064-1073. [0202] 31. Lin
L, Ibrahim A S, Xu X, Farber J M, Avanesian V, et al. (2009)
Th1-Th17 cells mediate protective adaptive immunity against
Staphylococcus aureus and Candida albicans infection in mice. PLoS
Pathog 5: e1000703. [0203] 32. Spellberg B, Ibrahim A S, Lin L,
Avanesian V, Fu Y, et al. (2008) Antibody titer threshold predicts
anti-candidal vaccine efficacy even though the mechanism of
protection is induction of cell-mediated immunity. J Infect Dis
197: 967-971. [0204] 33. Lam J S, Mansour M K, Specht C A, Levitz S
M (2005) A model vaccine exploiting fungal mannosylation to
increase antigen immunogenicity. J Immunol 175: 7496-7503. [0205]
34. Specht C A, Nong S, Dan J M, Lee C K, Levitz S M (2007)
Contribution of glycosylation to T cell responses stimulated by
recombinant Cryptococcus neoformans mannoprotein. J Infect Dis 196:
796-800. [0206] 35. Sterkel A K, Lorenzini J L, Fites J S,
Subramanian Vignesh K, Sullivan T D, et al. (2016) Fungal Mimicry
of a Mammalian Aminopeptidase Disables Innate Immunity and Promotes
Pathogenicity. Cell Host Microbe 19: 361-374. [0207] 36. Brandhorst
T T, Wuthrich M, Warner T, Klein B (1999) Targeted gene disruption
reveals an adhesin indispensable for pathogenicity of Blastomyces
dermatitidis. J Exp Med 189: 1207-1216. [0208] 37. Marty A J,
Wuthrich M, Carmen J C, Sullivan T D, Klein B S, et al. (2013)
Isolation of Blastomyces dermatitidis yeast from lung tissue during
murine infection for in vivo transcriptional profiling. Fungal
Genet Biol 56: 1-8. [0209] 38. Zarnowski R, Westler W M, Lacmbouh G
A, Marita J M, Bothe J R, et al. (2014) Novel entries in a fungal
biofilm matrix encyclopedia. MBio 5: e01333-01314. [0210] 39.
Wuthrich M, Wang H, Li M, Lerksuthirat T, Hardison S E, et al.
(2015) Fonsecaea pedrosoi-induced Th17-cell differentiation in mice
is fostered by Dectin-2 and suppressed by Mincle recognition. Eur J
Immunol 45: 2542-2552. [0211] 40. Wuthrich M, Filutowicz H I, Klein
B S (2000) Mutation of the WI-1 gene yields an attenuated
Blastomyces dermatitidis strain that induces host resistance. J
Clin Invest 106: 1381-1389.
Sequence CWU 1
1
161526PRTBlastomyces dermatitidis 1Met Arg Ala Thr Lys Leu Ala Leu
Leu Ala Ala Leu Ala Lys Leu Ser 1 5 10 15 Thr Gly Ala Tyr Val Leu
Gln Asp Asp Tyr Gln Pro Ser Asn Phe Phe 20 25 30 Asp Asp Phe Ala
Phe Phe Asp Gly Pro Asp Pro Ser Asn Ala Tyr Val 35 40 45 Thr Tyr
Val Asp Lys Ser Lys Ala Leu Arg Asp Gly Leu Ala Ser Asn 50 55 60
Asn Asn Asp Phe Val Tyr Leu Gly Val Asp His Gln Asn Val Ala Arg 65
70 75 80 Gly Arg Gly Arg Glu Ser Val Arg Leu Glu Thr Lys Lys Ser
Tyr Lys 85 90 95 His Gly Leu Ile Val Ala Asp Ile Ser His Met Pro
Gly Asn Ile Cys 100 105 110 Gly Thr Trp Pro Ala Phe Trp Ala Thr Gly
Ala Thr Trp Pro Asp Asp 115 120 125 Gly Glu Phe Asp Ile Ile Glu Gly
Val Asn Lys Gln Asn Lys Asn Val 130 135 140 Val Ala Leu His Thr Thr
Ala Gly Cys Lys Val Glu Asp Asn Asn Lys 145 150 155 160 Tyr Ser Gly
Ile Leu Val Thr Lys Asp Cys Asp Val Tyr Ser Pro Asn 165 170 175 Gln
Pro Ser Asn Gln Gly Cys Leu Phe Arg Ala Pro Ser Ala Thr Ser 180 185
190 Tyr Gly Thr Ala Phe Asn Ser Ile Gly Gly Gly Val Tyr Ala Thr Glu
195 200 205 Trp Thr Ser Asp Ser Ile Ser Val Trp Phe Phe Pro Arg Tyr
Gln Ile 210 215 220 Pro Ser Asn Ile Asn Asp Glu Asn Pro Asp Pro Ser
Thr Trp Pro Arg 225 230 235 240 Pro Ile Ala His Phe Thr Gly Cys Glu
Phe Asp Lys Phe Phe Gln Glu 245 250 255 Gln Arg Ile Ile Phe Asn Thr
Ala Phe Cys Gly Asp Trp Ala Lys Ala 260 265 270 Thr Trp Asn Glu Asn
Gly Cys Ala Ala Gly Gly Arg Thr Cys Glu Asp 275 280 285 Tyr Val Lys
Asn Asn Pro Trp Ala Phe Ser Glu Ala Phe Trp Ser Ile 290 295 300 Asn
Tyr Met Lys Val Phe Gln Asn Lys Gln Gly Asp Thr Ser Thr Ser 305 310
315 320 Thr Thr Thr Ser Ser Thr Ser Ser Thr Ser Ser Ser Ser Thr Glu
Ala 325 330 335 Pro Thr Thr Thr Met Thr Thr Ser Ser Thr Tyr Glu Pro
Ser Val Ser 340 345 350 Ser Ser Thr Ala Pro Glu Pro Ser Gln Ser Ala
Ser Thr Pro Ser Glu 355 360 365 Tyr Pro Gln Pro Ser Thr Ala Glu Pro
Thr Ala Ser Ser Ser Ser Tyr 370 375 380 Pro Lys Ser Ser Phe Ala Ser
Thr Asp Ser Pro Val Pro Thr Asp Tyr 385 390 395 400 Pro Val Pro Ser
Ser Asp Glu Pro Thr Val Pro Ser Ala Thr Tyr Ser 405 410 415 Glu Ser
Ser Pro Val Pro Thr Asp Tyr Pro Val Pro Ser Ser Asp Glu 420 425 430
Pro Thr Val Pro Ser Ala Thr Tyr Ser Glu Ser Leu Pro Ser Ala Ser 435
440 445 Ala Pro Ser Glu Tyr Pro Thr Gly Thr Ala Ser Val Asp Pro Thr
Asp 450 455 460 Val Ser Ser Cys Thr Pro Pro Pro Thr Gln Ser Cys Ile
Thr Tyr Thr 465 470 475 480 Thr Lys Thr Thr Ile Ala Ile Val Val Thr
Ala Pro Glu Ser Tyr Lys 485 490 495 Glu Ala Ile Gln Thr Glu Ser Ala
Glu Asp Glu Thr Glu Pro Ala Ala 500 505 510 Tyr Pro Thr Glu Pro Ala
Gly Tyr Pro Thr Asn Asp Lys Tyr 515 520 525 2637PRTArtificial
Sequencesynthetic 2Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe
Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr
Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly
Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro
Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr
Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu
Glu Lys Arg Glu Ala Glu Ala Arg Ala Thr Lys Leu Ala Leu 85 90 95
Leu Ala Ala Leu Ala Lys Leu Ser Thr Gly Ala Tyr Val Leu Gln Asp 100
105 110 Asp Tyr Gln Pro Ser Asn Phe Phe Asp Asp Phe Ala Phe Phe Asp
Gly 115 120 125 Pro Asp Pro Ser Asn Ala Tyr Val Thr Tyr Val Asp Lys
Ser Lys Ala 130 135 140 Leu Arg Asp Gly Leu Ala Ser Asn Asn Asn Asp
Phe Val Tyr Leu Gly 145 150 155 160 Val Asp His Gln Asn Val Ala Arg
Gly Arg Gly Arg Glu Ser Val Arg 165 170 175 Leu Glu Thr Lys Lys Ser
Tyr Lys His Gly Leu Ile Val Ala Asp Ile 180 185 190 Ser His Met Pro
Gly Asn Ile Cys Gly Thr Trp Pro Ala Phe Trp Ala 195 200 205 Thr Gly
Ala Thr Trp Pro Asp Asp Gly Glu Phe Asp Ile Ile Glu Gly 210 215 220
Val Asn Lys Gln Asn Lys Asn Val Val Ala Leu His Thr Thr Ala Gly 225
230 235 240 Cys Lys Val Glu Asp Asn Asn Lys Tyr Ser Gly Ile Leu Val
Thr Lys 245 250 255 Asp Cys Asp Val Tyr Ser Pro Asn Gln Pro Ser Asn
Gln Gly Cys Leu 260 265 270 Phe Arg Ala Pro Ser Ala Thr Ser Tyr Gly
Thr Ala Phe Asn Ser Ile 275 280 285 Gly Gly Gly Val Tyr Ala Thr Glu
Trp Thr Ser Asp Ser Ile Ser Val 290 295 300 Trp Phe Phe Pro Arg Tyr
Gln Ile Pro Ser Asn Ile Asn Asp Glu Asn 305 310 315 320 Pro Asp Pro
Ser Thr Trp Pro Arg Pro Ile Ala His Phe Thr Gly Cys 325 330 335 Glu
Phe Asp Lys Phe Phe Gln Glu Gln Arg Ile Ile Phe Asn Thr Ala 340 345
350 Phe Cys Gly Asp Trp Ala Lys Ala Thr Trp Asn Glu Asn Gly Cys Ala
355 360 365 Ala Gly Gly Arg Thr Cys Glu Asp Tyr Val Lys Asn Asn Pro
Trp Ala 370 375 380 Phe Ser Glu Ala Phe Trp Ser Ile Asn Tyr Met Lys
Val Phe Gln Asn 385 390 395 400 Lys Gln Gly Asp Thr Ser Thr Ser Thr
Thr Thr Ser Ser Thr Ser Ser 405 410 415 Thr Ser Ser Ser Ser Thr Glu
Ala Pro Thr Thr Thr Met Thr Thr Ser 420 425 430 Ser Thr Tyr Glu Pro
Ser Val Ser Ser Ser Thr Ala Pro Glu Pro Ser 435 440 445 Gln Ser Ala
Ser Thr Pro Ser Glu Tyr Pro Gln Pro Ser Thr Ala Glu 450 455 460 Pro
Thr Ala Ser Ser Ser Ser Tyr Pro Lys Ser Ser Phe Ala Ser Thr 465 470
475 480 Asp Ser Pro Val Pro Thr Asp Tyr Pro Val Pro Ser Ser Asp Glu
Pro 485 490 495 Thr Val Pro Ser Ala Thr Tyr Ser Glu Ser Ser Pro Val
Pro Thr Asp 500 505 510 Tyr Pro Val Pro Ser Ser Asp Glu Pro Thr Val
Pro Ser Ala Thr Tyr 515 520 525 Ser Glu Ser Leu Pro Ser Ala Ser Ala
Pro Ser Glu Tyr Pro Thr Gly 530 535 540 Thr Ala Ser Val Asp Pro Thr
Asp Val Ser Ser Cys Thr Pro Pro Pro 545 550 555 560 Thr Gln Ser Cys
Ile Thr Tyr Thr Thr Lys Thr Thr Ile Ala Ile Val 565 570 575 Val Thr
Ala Pro Glu Ser Tyr Lys Glu Ala Ile Gln Thr Glu Ser Ala 580 585 590
Glu Asp Glu Thr Glu Pro Ala Ala Tyr Pro Thr Glu Pro Ala Gly Tyr 595
600 605 Pro Thr Asn Asp Lys Tyr Gly Leu Glu Gln Lys Leu Ile Ser Glu
Glu 610 615 620 Asp Leu Asn Ser Ala Val Asp His His His His His His
625 630 635 3652PRTAspergillus fumigatus 3Met Ala Pro Ser Ser Leu
Leu Leu Ser Val Gly Ser Leu Ile Thr Ser 1 5 10 15 Ser Leu Val Ser
Ala Thr Ala Leu Glu Ala Arg Gln Ser Gln Thr Tyr 20 25 30 Gln Leu
Ala Glu Ser Trp Gln Gly Glu Ser Phe Ile Asn Asp Trp Asn 35 40 45
Phe Phe Asp Gly Ala Asp Pro Thr Asn Gly Tyr Val Thr Tyr Val Asn 50
55 60 Gln Ser Phe Ala Lys Gln Ser Gly Leu Val Lys Val Thr Glu Ser
Gly 65 70 75 80 Ser Phe Tyr Met Gly Val Asp Tyr Glu Ser Thr Leu Asn
Pro Asn Gly 85 90 95 Ala Gly Arg Glu Ser Val Arg Ile Glu Ser Lys
Asn Tyr Tyr Thr Glu 100 105 110 Gly Leu Tyr Val Ile Asp Ile Glu His
Met Pro Gly Ser Ile Cys Gly 115 120 125 Thr Trp Pro Ala Phe Trp Ser
Val Gly Lys Asn Trp Pro Asn Asp Gly 130 135 140 Glu Ile Asp Ile Ile
Glu Gly Val Asn Leu Gln Lys Ala Asn Lys Ile 145 150 155 160 Val Leu
His Thr Ser Gly Ser Cys Asp Val Ser Gly Ser Asn Asp Met 165 170 175
Thr Gly Thr Leu Ser Ser Ser Glu Cys Gly Glu Ala Ser Gly Thr Val 180
185 190 Gly Cys Val Val Lys Gly Thr Asn Gly Ser Ser Gly Asp Pro Phe
Asn 195 200 205 Glu Ser Gly Gly Gly Val Tyr Ala Met Glu Trp Thr Asp
Thr Phe Ile 210 215 220 Lys Ile Trp Phe Phe Pro Arg Ser Gln Ile Pro
Ala Ser Leu Ala Ser 225 230 235 240 Gly Asn Pro Asp Thr Ser Ser Phe
Gly Thr Pro Met Ala His Leu Gln 245 250 255 Gly Ser Cys Asp Phe Ala
Glu Arg Phe Lys Ala Gln Lys Leu Ile Ile 260 265 270 Asp Thr Thr Phe
Cys Gly Asp Trp Ala Gly Asn Val Phe Ala Glu Ser 275 280 285 Thr Cys
Pro Met Ser Asp Pro Ser Ser Pro Met Gln Ser Cys Val Asn 290 295 300
Tyr Val Ala Gln Asn Pro Ala Ala Phe Lys Glu Ala Tyr Trp Glu Ile 305
310 315 320 Asn Ser Ile Lys Ile Tyr Gln Tyr Gly Val Ser Ala Ala Ser
Ser Ala 325 330 335 Ala Val Ser Gln Ala Thr Ala Ser Lys Val Glu Gly
Thr Arg Val Ser 340 345 350 Ala Gln Ala Ala Asn Thr Ala Thr Pro Thr
Val Pro Ala Pro Val Glu 355 360 365 Thr Thr Thr Val Pro Gln Pro Ala
Gln Thr Asn Thr Val Ala Thr Ser 370 375 380 Ala Ala Asp His Ala Thr
Pro Ser Ser Ala Glu Thr Thr Thr Val Pro 385 390 395 400 Ala Ala Thr
Gly Ala Pro Ser Val Ser Ala Thr Glu Gly Gly Asp Ser 405 410 415 Glu
Leu Glu Ser Thr Ser Thr Val Tyr Val Thr Ser Thr Thr Thr Ile 420 425
430 Cys Pro Val Ala Glu Ser Ser Ser Ala Ala Ala Ala Gly Gly Lys Glu
435 440 445 Asp Ala Pro Ser Asn Gly Thr Ser Gly Ala Glu Val Ala Ala
Thr Ser 450 455 460 Val Ala Ala Ala Ala Pro Ala Ala Ala Thr Ser Gly
His Pro Gly Ala 465 470 475 480 Asp Ala Ile Ala Asn Ser Ala Ala Ala
Thr Ser Thr Asp Ala Gln Ser 485 490 495 Glu Ser Ala Thr Ser Arg Leu
Thr Ala Gly Ala Leu Ser Glu Ile Pro 500 505 510 Thr Ala Pro Pro Glu
Pro Val Ser Gln Ala Val Ser Thr Gly Ser Phe 515 520 525 Asp Asp Ser
Asp Thr Ala Gln Gly Asp Ser Glu Glu Gln Gly Ser Ile 530 535 540 Ala
Ser Ala Ser Val Ala Pro Ser Thr Ile Pro Val Pro Ala Ser Ser 545 550
555 560 Ser Ala Ala Ala Leu Gly Gly Ser Ser Ile Ala Ser Ser Phe Ala
Ser 565 570 575 Ser Arg Leu Ile Pro Arg Pro Thr Gly Ser Ser Thr Ala
Ala Ser Ala 580 585 590 Thr Ala Ile Ala Thr Trp Ser Pro Thr Ala Gly
Glu Ser Ala Ser Gly 595 600 605 Thr Ala Lys Glu Ser Ala Thr Leu Thr
Thr Pro Ser Glu Val Phe Phe 610 615 620 Thr Pro Gly Leu Ser Asn Gly
Ala Asn Arg Met Ser Val Gly Leu Ser 625 630 635 640 Gly Leu Ile Gly
Val Met Phe Ile Ala Ala Leu Ala 645 650 413PRTBlastomyces
dermatitidis 4Ala Phe Phe Asp Gly Pro Asp Pro Ser Asn Ala Tyr Val 1
5 10 513PRTBlastomyces dermatitidis 5Val Leu Phe Ala Ala Ser Ser
Ala Leu Ala Ala Pro Val 1 5 10 613PRTBlastomyces dermatitidis 6Ala
Thr Tyr Ser Glu Ser Leu Pro Ser Ala Ser Ala Pro 1 5 10
713PRTBlastomyces dermatitidis 7Ser Glu Tyr Pro Thr Gly Thr Ala Ser
Val Asp Pro Thr 1 5 10 813PRTBlastomyces dermatitidis 8Ser Glu Tyr
Pro Gln Pro Ser Thr Ala Glu Pro Thr Ala 1 5 10 946DNAArtificial
Sequencesynthetic 9ggctcgagaa aagagaggct gaagctaggg ctaccaagct
cgcgtt 461037DNAArtificial Sequencesynthetic 10gtttctagac
cgtacttgtc atttgtgggg tatcccg 3711548PRTArtificial
Sequencesynthetic 11Arg Ala Thr Lys Leu Ala Leu Leu Ala Ala Leu Ala
Lys Leu Ser Thr 1 5 10 15 Gly Ala Tyr Val Leu Gln Asp Asp Tyr Gln
Pro Ser Asn Phe Phe Asp 20 25 30 Asp Phe Ala Phe Phe Asp Gly Pro
Asp Pro Ser Asn Ala Tyr Val Thr 35 40 45 Tyr Val Asp Lys Ser Lys
Ala Leu Arg Asp Gly Leu Ala Ser Asn Asn 50 55 60 Asn Asp Phe Val
Tyr Leu Gly Val Asp His Gln Asn Val Ala Arg Gly 65 70 75 80 Arg Gly
Arg Glu Ser Val Arg Leu Glu Thr Lys Lys Ser Tyr Lys His 85 90 95
Gly Leu Ile Val Ala Asp Ile Ser His Met Pro Gly Asn Ile Cys Gly 100
105 110 Thr Trp Pro Ala Phe Trp Ala Thr Gly Ala Thr Trp Pro Asp Asp
Gly 115 120 125 Glu Phe Asp Ile Ile Glu Gly Val Asn Lys Gln Asn Lys
Asn Val Val 130 135 140 Ala Leu His Thr Thr Ala Gly Cys Lys Val Glu
Asp Asn Asn Lys Tyr 145 150 155 160 Ser Gly Ile Leu Val Thr Lys Asp
Cys Asp Val Tyr Ser Pro Asn Gln 165 170 175 Pro Ser Asn Gln Gly Cys
Leu Phe Arg Ala Pro Ser Ala Thr Ser Tyr 180 185 190 Gly Thr Ala Phe
Asn Ser Ile Gly Gly Gly Val Tyr Ala Thr Glu Trp 195 200 205 Thr Ser
Asp Ser Ile Ser Val Trp Phe Phe Pro Arg Tyr Gln Ile Pro 210 215 220
Ser Asn Ile Asn Asp Glu Asn Pro Asp Pro Ser Thr Trp Pro Arg Pro 225
230 235 240 Ile Ala His Phe Thr Gly Cys Glu Phe Asp Lys Phe Phe Gln
Glu Gln 245 250 255 Arg Ile Ile Phe Asn Thr Ala Phe Cys Gly Asp Trp
Ala Lys Ala Thr 260 265 270 Trp Asn Glu Asn Gly Cys Ala Ala Gly Gly
Arg Thr Cys Glu Asp Tyr 275 280 285 Val Lys Asn Asn Pro Trp Ala Phe
Ser Glu Ala Phe Trp Ser Ile Asn 290 295 300 Tyr Met Lys Val Phe Gln
Asn Lys Gln Gly Asp Thr Ser Thr Ser Thr 305 310 315 320 Thr Thr Ser
Ser Thr Ser Ser Thr Ser Ser Ser Ser Thr Glu Ala Pro 325 330 335 Thr
Thr Thr Met Thr Thr Ser Ser Thr Tyr Glu Pro Ser Val Ser Ser 340 345
350
Ser Thr Ala Pro Glu Pro Ser Gln Ser Ala Ser Thr Pro Ser Glu Tyr 355
360 365 Pro Gln Pro Ser Thr Ala Glu Pro Thr Ala Ser Ser Ser Ser Tyr
Pro 370 375 380 Lys Ser Ser Phe Ala Ser Thr Asp Ser Pro Val Pro Thr
Asp Tyr Pro 385 390 395 400 Val Pro Ser Ser Asp Glu Pro Thr Val Pro
Ser Ala Thr Tyr Ser Glu 405 410 415 Ser Ser Pro Val Pro Thr Asp Tyr
Pro Val Pro Ser Ser Asp Glu Pro 420 425 430 Thr Val Pro Ser Ala Thr
Tyr Ser Glu Ser Leu Pro Ser Ala Ser Ala 435 440 445 Pro Ser Glu Tyr
Pro Thr Gly Thr Ala Ser Val Asp Pro Thr Asp Val 450 455 460 Ser Ser
Cys Thr Pro Pro Pro Thr Gln Ser Cys Ile Thr Tyr Thr Thr 465 470 475
480 Lys Thr Thr Ile Ala Ile Val Val Thr Ala Pro Glu Ser Tyr Lys Glu
485 490 495 Ala Ile Gln Thr Glu Ser Ala Glu Asp Glu Thr Glu Pro Ala
Ala Tyr 500 505 510 Pro Thr Glu Pro Ala Gly Tyr Pro Thr Asn Asp Lys
Tyr Gly Leu Glu 515 520 525 Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn
Ser Ala Val Asp His His 530 535 540 His His His His 545
12356PRTAspergillus fumigatus 12Met Tyr Ile Arg Ser Thr Leu Pro Ile
Leu Gly Phe Ser Ala Thr Gly 1 5 10 15 Met Ala Ala Tyr Val Leu Glu
Asp Asp Tyr Gly Thr Ser Thr Ser Phe 20 25 30 Phe Asp Lys Phe Ser
Phe Phe Thr Asp Pro Asp Pro Thr Gly Gly Phe 35 40 45 Val Ser Tyr
Val Asp Arg Asn Thr Ala Gln Asp Thr Gly Leu Ile Phe 50 55 60 Ala
Asn Gly Ala Val Tyr Met Gly Val Asp His Thr Asn Val Ala Gly 65 70
75 80 Ser Ser Gly Arg Gln Ser Val Arg Leu Thr Ser Thr Lys Ser Tyr
Thr 85 90 95 His Gly Leu Ile Ile Leu Asp Leu Glu His Met Pro Gly
Gly Ile Cys 100 105 110 Gly Thr Trp Pro Ala Phe Trp Met Leu Gly Pro
Asp Trp Pro Ser His 115 120 125 Gly Glu Ile Asp Ile Ile Glu Gly Val
Asn Thr Gln Pro Val Asn Gln 130 135 140 Met Thr Leu His Ser Thr Asp
Gly Cys Ser Ile Ala Asn Gly Gly Phe 145 150 155 160 Thr Gly Thr Pro
Thr Asp Ile Arg Ala Gly Thr Pro Asn Pro Thr Asn 165 170 175 Trp Gly
Pro Pro Leu Ala Lys Phe Ala Pro Gly Ser Cys Ser Phe Asp 180 185 190
Ala His Phe Ser Glu Met Gln Ile Val Phe Asp Thr Thr Phe Cys Gly 195
200 205 Gly Trp Ala Gly Ser Val Trp Gly Ser Gly Ser Cys Ala Ser Leu
Leu 210 215 220 Thr Ser Asn Cys Tyr Asp Tyr Ala Pro Ser Gln Asp Thr
Asn Ala Gly 225 230 235 240 Cys Gly Ile Ala Ala Thr Ser Ser Arg Thr
Tyr Gly Thr Gly Phe Asn 245 250 255 Asn Ala Gly Gly Gly Ile Tyr Ala
Thr Glu Trp Thr Ser Ala Gly Ile 260 265 270 Ser Ile Trp Phe Phe Pro
Arg Gly Ser Thr Val Ala Pro Ser Cys Gln 275 280 285 Asp Phe Val Ala
Asn Asn Pro Ser Ala Phe Arg Glu Ala Tyr Trp Leu 290 295 300 Ile Glu
Ser Leu Lys Val Tyr Gln Asp Ala Pro Gly Glu Ser Asn Asn 305 310 315
320 Met Arg Met Asn Ala Thr Ser His Leu Asn Val Gln Leu Pro Arg Lys
325 330 335 Gly Gly Arg Arg Ser Pro Gly Leu His Gly Arg Gly Phe Leu
Glu Gly 340 345 350 Thr Gly Lys Trp 355 13311PRTPseudogymnoascus
destructans 13Met Pro Ser Leu Gln Thr Leu Ile Pro Ala Ala Ala Ile
Ala Trp Leu 1 5 10 15 Val Gly Thr Ala Ser Ala Ala Tyr Thr Leu Gln
Asp Val Tyr Asp Ser 20 25 30 Thr Asn Phe Phe Asp Gly Phe Asn Phe
His Asp Gly Pro Asp Pro Thr 35 40 45 Asn Gly Phe Val Asp Tyr Ala
Asn Ala Glu Thr Ala Asn Asn Ala Gly 50 55 60 Leu Ala Gly Leu Ser
Gln Asp Gly Val Tyr Met Gly Val Asp His Thr 65 70 75 80 Thr Met Ser
Pro Pro Asn Gly Arg Ala Ser Val Arg Val Glu Ser Gln 85 90 95 Lys
Gln Tyr Thr Leu Gly Leu Phe Ile Ala Asp Ile Lys His Met Pro 100 105
110 Gly Ala Glu Cys Gly Ser Trp Pro Ala Phe Trp Thr Tyr Gly Pro Asp
115 120 125 Trp Pro Asn Ala Gly Glu Ile Asp Ile Met Glu Gly Val Asn
Thr Gln 130 135 140 Leu Thr Asn Asp Val Thr Leu His Thr Ser Gly Ser
Cys Ser Met Asn 145 150 155 160 Asn Pro Asn Ser Gln Leu Gly Ser Val
Leu Ser Asn Ala Asp Cys Ser 165 170 175 Gly Thr Arg Gly Cys Gly Gln
Ala Thr Ile Asp Pro Ser Asn Tyr Gly 180 185 190 Thr Gly Phe Asn Thr
Ile Gly Gly Gly Val Tyr Ala Met Glu Trp Thr 195 200 205 Asn Glu Val
Ile Ala Val Tyr Phe Phe Pro Arg Tyr Ala Ile Pro Asp 210 215 220 Asp
Ile Asn Ser Gly Asn Pro Asp Pro Ser Thr Trp Gly Thr Pro Leu 225 230
235 240 Thr Asn Phe Val Gly Asp Ser Cys Asn Ile Gly Ser His Phe Lys
Asn 245 250 255 His Tyr Ile Val Phe Asp Thr Thr Phe Cys Gly Asp Trp
Ala Gly Gly 260 265 270 Val Trp Gly Asp Gln Cys Gly Ala Arg Ala Ala
Thr Cys Glu Asp Phe 275 280 285 Val Ser Gln Asn Pro Ala Ala Tyr Glu
Glu Ser Tyr Trp Leu Val Asn 290 295 300 Ser Val Lys Val Tyr Thr Asn
305 310 14495PRTCoccidioides immitis 14Met Arg Ala Ala Lys Val Thr
Leu Leu Ala Ala Leu Ala Gln Leu Ala 1 5 10 15 Ala Ala Ser Tyr Glu
Leu Met Asp Asp Tyr Asn Pro Ser Asn Phe Phe 20 25 30 Asp Lys Phe
Glu Phe Phe Ser Gly Arg Asp Pro Ser Asn Gly Tyr Val 35 40 45 Ala
Tyr Gln Gly Lys Glu Ala Ala Leu Ser Ser Asn Leu Ala Gln Lys 50 55
60 Leu Glu Asn Ser Ile Arg Ile Gly Ser Asp Ser Thr Asp Ile Ala Thr
65 70 75 80 Gly Pro Gly Arg Arg Ser Val Arg Leu Glu Thr Lys Ala Arg
Tyr Lys 85 90 95 His Gly Leu Ile Val Ala Asp Ile Lys His Met Pro
Gly Ser Ile Cys 100 105 110 Gly Ile Trp Pro Ala Phe Trp Thr Val Gly
Ser Arg Trp Pro Glu His 115 120 125 Gly Glu Met Asp Ile Ile Glu Gly
Val Asn Arg Gln Ser Ile Asn Lys 130 135 140 Met Ala Leu His Thr Thr
Ala Gly Cys Lys Ile Asn Ser Asn Gly Asp 145 150 155 160 Phe Thr Gly
Val Val Glu Thr Pro Asp Cys Asp Val Asn Ser Pro Asn 165 170 175 Gln
Ala Pro Asn Gln Gly Cys Leu Phe Thr Ser Ser Gln Gly Asn Ser 180 185
190 Tyr Gly Thr Asn Phe Asn Asn Arg Asn Gly Gly Val Tyr Ala Met Glu
195 200 205 Trp Thr Ser Asp Glu Ile Thr Val Trp Phe Phe Pro Arg Gly
Asn Ile 210 215 220 Pro Asp Asp Val Asn Ser Gln Asn Pro Asp Pro Ser
Lys Trp Gly Lys 225 230 235 240 Pro Ser Ala Arg Phe Ser Gly Asp Cys
Asp Leu Asp Arg Phe Val Gln 245 250 255 Asp Gln Arg Ile Ile Phe Asn
Thr Ala Phe Cys Gly Asp Trp Ala Lys 260 265 270 Gly Leu Trp Asn Ser
Asp Ser Val Cys Arg Ala Lys Gly Pro Ser Cys 275 280 285 Glu Asp Tyr
Val Lys Asn Asn Pro Lys Asp Phe Ala Glu Ala Tyr Trp 290 295 300 Glu
Ile Tyr Gly Met Lys Val Tyr Ser Lys Gly Gln Gly Gln Lys Ile 305 310
315 320 Ser Ser Ala Ala Thr Ser Pro Thr Gln Ala Ser Thr Thr Gln Val
Ser 325 330 335 Thr Thr Gln Ile Ser Ser Ala Gln Ser Ala Ser Ala Ser
Ala Ser Val 340 345 350 Ser Asp Gly Pro Asp Thr Ser Ser Asn Thr Pro
Pro Ser Ala Thr Glu 355 360 365 Ser Gly Asn Ala Ser Ser Ile Glu Ser
Arg Ser Thr Asp Val Glu Pro 370 375 380 Thr Lys Thr Pro Thr Gly Thr
Asp Gly Gly Ala Ser Leu Thr Asn Ala 385 390 395 400 Pro Cys Asn Gly
Pro Asn Cys Pro Ser Gln Ser Pro Thr Thr Ser Gly 405 410 415 Gly Val
Ser Pro Thr Asp Lys Ser Glu Tyr Pro Ala Asn Pro Gly Thr 420 425 430
Thr Gly Gly Ser Pro Leu Pro Thr Asn Lys Pro Glu Ile Pro Ser Ser 435
440 445 Cys Thr Pro Arg Thr Thr Cys Val Thr Tyr Thr Arg Ile Glu Thr
Val 450 455 460 Thr Tyr Ile Asn Lys Asn Pro Ala Pro Phe Gln Thr Gly
Val Gln Ser 465 470 475 480 Pro Thr Lys Ala Ser Gly Asp Asp Glu Ser
Ile Ile Pro Ile Arg 485 490 495 15495PRTCoccidioides posadasii
15Met Arg Ala Ala Lys Val Thr Leu Leu Ala Ala Leu Ala Gln Leu Ala 1
5 10 15 Ala Ala Ser Tyr Glu Leu Met Asp Asp Tyr Asn Pro Ser Asn Phe
Phe 20 25 30 Asp Lys Phe Glu Phe Phe Ser Gly Arg Asp Pro Ser Asn
Gly Tyr Val 35 40 45 Ala Tyr Gln Gly Lys Glu Ala Ala Leu Ser Ser
Asn Leu Ala Gln Lys 50 55 60 Leu Glu Asn Ser Ile Arg Ile Gly Ser
Asp Ser Thr Asp Ile Ala Thr 65 70 75 80 Gly Ser Gly Arg Arg Ser Val
Arg Leu Glu Thr Lys Ala Arg Tyr Lys 85 90 95 His Gly Leu Ile Val
Ala Asp Ile Lys His Met Pro Gly Ser Ile Cys 100 105 110 Gly Val Trp
Pro Ala Phe Trp Thr Val Gly Ser Arg Trp Pro Glu His 115 120 125 Gly
Glu Met Asp Ile Ile Glu Gly Val Asn Arg Gln Ser Ile Asn Lys 130 135
140 Met Ala Leu His Thr Thr Ala Gly Cys Lys Ile Asn Ser Asn Gly Asp
145 150 155 160 Phe Thr Gly Val Val Glu Thr Pro Asp Cys Asp Val Asn
Ser Pro Asn 165 170 175 Gln Ala Pro Asn Gln Gly Cys Leu Phe Thr Ser
Ser Gln Gly Asn Ser 180 185 190 Tyr Gly Thr Asn Phe Asn Asn Arg Asn
Gly Gly Val Tyr Ala Met Glu 195 200 205 Trp Thr Ser Asp Glu Ile Thr
Val Trp Phe Phe Pro Arg Gly Asn Ile 210 215 220 Pro Asp Asp Val Asn
Ser Gln Asn Pro Asp Pro Ser Lys Trp Gly Lys 225 230 235 240 Pro Ser
Ala Arg Phe Ser Gly Asp Cys Asp Leu Asp Arg Phe Val Gln 245 250 255
Asp Gln Arg Ile Ile Phe Asn Thr Ala Phe Cys Gly Asp Trp Ala Lys 260
265 270 Gly Leu Trp Asn Ser Asp Ser Val Cys Arg Ala Lys Gly Pro Ser
Cys 275 280 285 Glu Asp Tyr Val Lys Asn Asn Pro Lys Asp Phe Ala Glu
Ala Tyr Trp 290 295 300 Glu Ile Tyr Gly Met Lys Val Tyr Ser Lys Gly
Gln Gly Gln Lys Ile 305 310 315 320 Ser Ser Ala Ala Thr Ser Pro Thr
Gln Ala Ser Thr Thr Gln Val Ser 325 330 335 Thr Thr Gln Ile Ser Ser
Ala Gln Ser Ala Ser Ala Ser Ala Ser Val 340 345 350 Ser Asp Gly Pro
Asp Thr Ser Ser Asn Thr Pro Pro Ser Ala Thr Gly 355 360 365 Ser Gly
Asn Ala Ser Ser Ile Glu Ser Arg Ser Thr Asp Ala Glu Pro 370 375 380
Thr Lys Ala Pro Thr Gly Thr Asp Gly Gly Ala Ser Pro Thr Asn Ala 385
390 395 400 Pro Cys Asn Gly Pro Asn Cys Pro Ser Gln Ser Pro Thr Thr
Ser Gly 405 410 415 Gly Val Ser Pro Thr Asp Lys Ser Glu Tyr Pro Ala
Asn Pro Gly Thr 420 425 430 Thr Asp Gly Ser Pro Leu Pro Thr Asn Lys
Pro Glu Ile Pro Thr Ser 435 440 445 Cys Thr Pro Arg Thr Thr Cys Val
Thr Tyr Thr Arg Ile Glu Thr Val 450 455 460 Thr Tyr Ile Asn Lys Asn
Pro Ala Pro Phe Gln Thr Gly Val Gln Ser 465 470 475 480 Pro Thr Lys
Ala Ser Gly Asp Asp Glu Ser Ile Ile Pro Ile Arg 485 490 495
16490PRTHistoplasma capsulatum 16Met Arg Thr Thr Lys Leu Thr Leu
Leu Ala Thr Leu Ala Lys Leu Ser 1 5 10 15 Ala Gly Thr Tyr Val Leu
Lys Asp Asp Tyr Gln Pro Ser Asn Phe Phe 20 25 30 Asp Asn Phe Asn
Phe Phe Asn Gly Pro Asp Pro Ser Asn Gly Tyr Val 35 40 45 Thr Tyr
Leu Asp Lys Ser Asn Ala Val Asn Asn Gly Leu Ala Ser Asn 50 55 60
Glu Asn Asp Phe Val Tyr Leu Gly Val Asp Ser Lys Asn Val Ala Lys 65
70 75 80 Gly Leu Gly Arg Glu Ser Val Arg Leu Glu Thr Lys Lys Thr
Tyr Lys 85 90 95 His Gly Leu Ile Val Val Asp Ile Ser His Met Pro
Gly Gly Ile Cys 100 105 110 Gly Thr Trp Pro Ala Leu Trp Ser Thr Gly
Ala Thr Trp Pro Glu Asp 115 120 125 Gly Glu Leu Asp Ile Ile Glu Gly
Val Asn Ser Gln Thr Lys Asn Val 130 135 140 Val Ala Leu His Thr Thr
Ala Gly Cys Lys Val Glu Asp Asn Ser Asn 145 150 155 160 Tyr Ser Gly
Glu Leu Val Thr Lys Asp Cys Asp Ile Asn Ser Pro Thr 165 170 175 Gln
Pro Gly Asn Gln Gly Cys Leu Phe Arg Ala Pro Ser Ser Met Ser 180 185
190 Tyr Gly Asn Ser Phe Asn Ser Ile Gly Gly Gly Ile Tyr Ala Ala Glu
195 200 205 Trp Thr Thr Asp Ser Ile Ser Val Trp Phe Phe Pro Arg Tyr
Arg Ile 210 215 220 Pro Ser Asp Ile Asn Ser Glu His Pro Asp Pro Ser
Ser Trp Ala Arg 225 230 235 240 Pro Ile Ala His Phe Thr Gly Cys Glu
Phe Asp Lys Phe Phe Gln Glu 245 250 255 Gln Arg Ile Ile Ile Asn Thr
Ala Phe Cys Gly Asp Trp Ala Lys Asn 260 265 270 Thr Trp Ser Gln Asp
Ala Glu Cys Ala Ala Lys Ala Asp Ser Cys Glu 275 280 285 Ala Tyr Val
Gln Asn Asn Pro Ser Ala Phe Ser Glu Ala Tyr Trp Ser 290 295 300 Ile
Asn Tyr Met Lys Val Phe Gln Asp Glu Val Val Asp Tyr Pro Gly 305 310
315 320 Asp Ser Thr Thr Thr Thr Thr Ser Thr Thr Ala Ser Gln Thr Asp
Ser 325 330 335 Thr Glu Pro Thr Thr Thr Thr Thr Thr Thr Ser Thr Thr
Thr Thr Thr 340 345 350 Ser Thr Gln Pro Ala Asp Thr Gly Ala Thr Asn
Thr Asp Ser Ser Pro 355 360 365 Ser Ala Ser Ala Thr Asn Glu Tyr Pro
Thr Gly Ser Ala Ser Val Glu 370 375 380 Pro Thr Asp Ile Ala Ser Cys
Thr Pro Pro Pro Thr Glu Ser Cys Ile 385 390 395 400 Thr Tyr Thr Thr
Lys Thr Thr Ile Ala Val Val Val Thr Pro Thr Gly 405 410 415 Tyr Asn
Glu Ala Ile Ile Pro Ile Pro Thr Glu Ser Ala Glu Tyr Glu
420 425 430 Thr Glu Pro Thr Glu Asn Pro Ile Glu Pro Ser Glu Tyr Pro
Thr Ala 435 440 445 Pro Val Gly Tyr Pro Thr Glu Pro Ile Gly Tyr Pro
Thr Glu Pro Ile 450 455 460 Gly Tyr Pro Thr Asn Asp Gln Asp Val Pro
Leu Lys Arg Arg Gln His 465 470 475 480 Ile Lys Lys His Ile Ala Gly
Thr His His 485 490
* * * * *