U.S. patent application number 16/201585 was filed with the patent office on 2019-03-21 for polypeptides having immunoactivating activity and methods of producing the same.
The applicant listed for this patent is University of Louisville Research Foundation, Inc.. Invention is credited to Krystal Hamorsky, Nobuyuki Matoba.
Application Number | 20190085036 16/201585 |
Document ID | / |
Family ID | 46831316 |
Filed Date | 2019-03-21 |
United States Patent
Application |
20190085036 |
Kind Code |
A1 |
Matoba; Nobuyuki ; et
al. |
March 21, 2019 |
Polypeptides Having Immunoactivating Activity And Methods Of
Producing The Same
Abstract
Isolated polypeptides are provided that comprise a cholera toxin
B subunit variant having one or more modifications to increase the
expression of the polypeptide in a plant cell. Nucleic acids
sequences, vectors, and plant cells for expressing the cholera
toxin B subunit variant polypeptides are also provided. Further
provided are methods for producing the cholera toxin B subunit
variant polypeptides that include the steps of transforming a plant
cell with a nucleic acid encoding the cholera toxin B subunit
variant polypeptides; expressing the variant polypeptides; and
purifying the polypeptides. Still further provided are methods of
isolating the variant polypeptides that include the steps of
obtaining a plant cell expressing the cholera toxin B subunit
variant polypeptides; extracting the cholera toxin B subunit
variant polypeptides from the plant cell; and purifying the cholera
toxin B subunit variant polypeptides. Methods of eliciting an
immune response are also provided.
Inventors: |
Matoba; Nobuyuki;
(Louisville, KY) ; Hamorsky; Krystal; (Louisville,
KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Louisville Research Foundation, Inc. |
Louisville |
KY |
US |
|
|
Family ID: |
46831316 |
Appl. No.: |
16/201585 |
Filed: |
November 27, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14005388 |
Oct 9, 2013 |
10160789 |
|
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PCT/US2012/029072 |
Mar 14, 2012 |
|
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16201585 |
|
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61452308 |
Mar 14, 2011 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61K 2039/542 20130101; C07K 2319/04 20130101; C12N 15/8258
20130101; C07K 2319/02 20130101; A61K 39/107 20130101; A61P 37/06
20180101; C07K 14/28 20130101; A61K 38/00 20130101 |
International
Class: |
C07K 14/28 20060101
C07K014/28; C12N 15/82 20060101 C12N015/82; A61K 39/02 20060101
A61K039/02 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with government support under Grant
No. W81XWH-10-2-0082 awarded by the United States Department of
Defense. The government has certain rights in the invention.
Claims
1. An isolated nucleic acid molecule, comprising a sequence that
encodes a polypeptide comprising a cholera toxin B subunit variant
having one or more modifications to increase the expression of the
polypeptide in a plant cell.
2. An expression vector, comprising the nucleic acid molecule of
claim 1 operably linked to an expression cassette.
3. A plant cell transfected with the vector of claim 2, or a
progeny of the plant cell, wherein the cell or the progeny thereof
expresses the polypeptide.
4. A method of producing a cholera toxin B subunit variant
polypeptide, comprising: transforming a plant cell with a nucleic
acid encoding a cholera toxin B subunit variant polypeptide having
one or more modifications to increase the expression of the variant
polypeptide in the plant cell; expressing the cholera toxin B
subunit variant polypeptide in the plant cell; and purifying the
cholera toxin B subunit variant polypeptide.
5. The method of claim 4, wherein the plant cell comprises a
Nicotiana plant cell.
6. The method of claim 5, wherein the Nicotiana plant cell is a
Nicotiana benthamiana plant cell.
7. The method of claim 4, wherein the nucleic acid encoding the
cholera toxin B subunit variant comprises the sequence of SEQ ID
NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or
SEQ ID NO: 13.
8. The method of claim 4, wherein the cholera toxin B subunit
variant polypeptide comprises the amino acid sequence of SEQ ID NO:
4, SEQ ID NO: 6, or SEQ ID NO: 25.
9. The method of claim 4, wherein the one or more modifications
comprise a secretory signal peptide selected from the group
consisting of a rice alphaamylase secretory signal peptide, a
Nicotiana plumbagenifolia calreticulin secretory signal peptide, an
apple pectinase secretory signal peptide, and a barley
alpha-amylase secretory signal peptide.
10. The method of claim 4, wherein the one or more modifications
comprise a secretory signal peptide having an amino acid sequence
selected from the group consisting of SEQ ID NOS: 18, 20, 22, and
24.
11. The method of claim 9, wherein the secretory signal polypeptide
comprises the rice alpha-amylase secretory signal peptide.
12. The method of claim 4, wherein polypeptide comprises an amino
acid sequence selected from the group consisting of SEQ ID NOS:
26-29.
13. The method of claim 4, wherein the one or more modifications
comprise an endoplasmic reticulum retention signal having the amino
acid sequence of SEQ IDNO: 31.
14. The method of claim 4, wherein the cholera toxin B subunit
variant polypeptide includes two or more N-linked glycosylation
sequons.
15. The method of claim 4, wherein the cholera toxin B subunit
variant polypeptide comprises the amino acid sequence of SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
16. A method of isolating a cholera toxin B subunit variant
polypeptide from a plant tissue, comprising: obtaining a plant cell
expressing a cholera toxin B subunit variant polypeptide having one
or more modifications to increase the expression of the polypeptide
in a plant cell; extracting the cholera toxin B subunit variant
polypeptide from the plant cell; and purifying the cholera toxin B
subunit variant polypeptide from the plant cell.
17. The method of claim 16, wherein the step of extracting the
cholera toxin B subunit variant polypeptide from the plant cell
comprises homogenizing the plant tissue in an aqueous buffer having
an acidic pH.
18. The method of claim 17, wherein the pH of the aqueous buffer is
about 5.
19. The method of claim 16, wherein purifying the cholera toxin B
subunit variant polypeptide from the plant cell comprises purifying
the variant polypeptide using chromatography.
20. The method of claim 16, wherein the plant tissue comprises a
Nicotiana plant tissue.
21. The method of claim 20, wherein the plant tissue is a Nicotiana
benthamiana plant tissue.
22. The method of claim 16, wherein the cholera toxin B subunit
variant polypeptide comprises the amino acid sequence of SEQ ID NO:
4, SEQ ID NO: 6, or SEQ ID NO: 25.
23. The method of claim 16, wherein the one or more modifications
comprise a secretory signal peptide selected from the group
consisting of a rice alphaamylase secretory signal peptide, a
Nicotiana plumbaginifoha calreticulin secretory signal peptide, an
apple pectinase secretory signal peptide, and a barley
alpha-amylase secretory signal peptide.
24. The method of claim 16, wherein the one or more modifications
comprise a secretory signal peptide having an amino acid sequence
selected from the group consisting of SEQ ID NOS: 18, 20, 22, and
24.
25. The method of claim 23, wherein the secretory signal peptide is
the rice alpha-amylase secretory signal peptide.
26. The method of claim 16, wherein the cholera toxin B subunit
variant polypeptide comprises an amino acid sequence selected from
the group consisting of SEQ ID NOS: 26-29.
27. The method of claim 16, wherein the one or more modifications
comprise an endoplasmic reticulum retention signal having the amino
acid sequence of SEQ IDNO: 31.
28. The method of claim 16, wherein the cholera toxin B subunit
variant polypeptide includes two or more N-linked glycosylation
sequons.
29. The method of claim 16, wherein the cholera toxin B subunit
variant polypeptide comprises the amino acid sequence of SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
30. A method for eliciting an immune response in a subject,
comprising administering to a subject in need thereof an effective
amount of a cholera toxin B subunit variant polypeptide having one
or more modifications to increase the expression of the polypeptide
in a plant cell.
31. The method of claim 30, wherein administering an effective
amount of the cholera toxin B subunit variant polypeptide increases
an amount of IgG, IgA, IgM, effector T cells, regulatory T cells,
or combinations thereof in a subject.
32. The method of claim 30, wherein administering an effective
amount of the cholera toxin B subunit variant polypeptide comprises
orally administering the cholera toxin B subunit variant
polypeptide.
33. The method of claim 30, wherein the cholera toxin B subunit
variant polypeptide comprises the amino acid sequence of SEQ ID NO:
4, SEQ ID NO: 6, or SEQ ID NO: 25.
34. The method of claim 30, wherein the cholera toxin B subunit
variant polypeptide includes two or more N-linked glycosylation
sequons.
35. The method of claim 34, wherein the cholera toxin B subunit
variant polypeptide comprises the amino acid sequence of SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/005,388, filed Oct. 9, 2013, which is the U.S. National
Stage of International Application No. PCT/US2012/029072, filed on
Mar. 14, 2012, published in English, which claims the benefit of
U.S. Provisional Application No. 61/452,308, filed on Mar. 14,
2011. The entire teachings of the above applications are
incorporated herein by reference.
INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE
[0003] This application incorporates by reference the Sequence
Listing contained in the following ASCII text file being submitted
concurrently herewith: [0004] a) File name:
56001007001SEQUENCELISTING.txt; created Nov. 12, 2018, 23 KB in
size.
TECHNICAL FIELD
[0005] The presently-disclosed subject matter relates to
polypeptides having immunoactivating activity and methods of
producing the same. In particular, the presently-disclosed subject
matter relates to immunoactivating polypeptides comprising a
cholera toxin B subunit variant having one or more modifications to
increase the expression of the variant in the plant cell, as well
as methods of producing those polypeptides in a plant cell.
BACKGROUND
[0006] Cholera is a serious diarrheal disease caused by the
pathogenic strains of Vibrio cholerae, which leads to severe
dehydration and even death within 18 hours if left untreated.
Indeed, the World Health Organization (WHO) has estimated that 3 to
5 million cases of cholera occur each year with approximately
100,000 to 130,000 of those cases ending in death.
[0007] Despite the severity of cholera, cholera is generally no
longer a concern in developed countries. However, it is still a
major threat in many developing countries, where a safe water
supply and advanced sanitation systems are generally not available.
In fact, large outbreaks of cholera sporadically occur every year,
as recently seen in Papua New Guinea (2009-2010), Zimbabwe and
other African countries (2008-2010), as well as, most recently, in
Haiti (2010-present).
[0008] Due to recurring outbreaks, implementation of a mass
vaccination program for cholera has now been proposed as part of
global cholera prevention strategies. Dukoral (Crucell,
Netherlands) is an internationally licensed, World Health
Organization-prequalified oral cholera vaccine, which contains
killed Vibrio cholerae bacteria and a recombinant cholera toxin B
subunit polypeptide (rCTB; SEQ ID NO: 2) produced in
genetically-modified bacterium. This vaccine has been shown to
provide protection in greater than 80% of subjects to which it is
administered, which is higher than killed V. cholerae alone.
Nevertheless, production of sufficient doses of the vaccine for
mass vaccination campaigns has proven to be challenging,
particularly for the rCTB, whose production is significantly
hindered by the limited scalability of fermentation-based
production.
SUMMARY
[0009] The presently-disclosed subject matter meets some or all of
the above-identified needs, as will become evident to those of
ordinary skill in the art after a study of information provided in
this document.
[0010] This Summary describes several embodiments of the
presently-disclosed subject matter, and in many cases lists
variations and permutations of these embodiments. This Summary is
merely exemplary of the numerous and varied embodiments. Mention of
one or more representative features of a given embodiment is
likewise exemplary. Such an embodiment can typically exist with or
without the feature(s) mentioned; likewise, those features can be
applied to other embodiments of the presently-disclosed subject
matter, whether listed in this Summary or not. To avoid excessive
repetition, this Summary does not list or suggest all possible
combinations of such features.
[0011] The presently-disclosed subject matter relates to
polypeptides having broad immunoactivating activity and methods of
producing the same. In particular, the presently-disclosed subject
matter relates to polypeptides comprising a cholera toxin B subunit
variant having one or more modifications to increase the expression
of the polypeptide in the plant cell, as well as methods of
producing those polypeptides in a plant cell.
[0012] In some embodiments of the presently-disclosed subject
matter, an isolated polypeptide is provided that comprises a
cholera toxin B subunit variant having one or more modifications to
increase the expression of the polypeptide in a plant cell. In some
embodiments, the cholera toxin B subunit variant comprises the
sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 25.
[0013] In some embodiments of the presently-disclosed polypeptides,
the one or more modifications to the cholera toxin B subunit
variant polypeptide comprise the addition of a secretory signal
peptide selected from the group consisting of a rice alpha-amylase
secretory signal peptide, a Nicotiana plumbaginifolia calreticulin
secretory signal peptide, an apple pectinase secretory signal
peptide, and a barley alpha-amylase secretory signal peptide. In
some embodiments, the one or more modifications comprise a
secretory signal peptide having an amino acid sequence selected
from the group consisting of SEQ ID NOS: 18, 20, 22, and 24. In
some embodiments, the secretory signal polypeptide is a
rice-alpha-amylase secretory signal peptide. In some embodiments,
the cholera toxin B subunit variant polypeptide comprises an amino
acid sequence selected from the group consisting of SEQ ID NOS:
26-29.
[0014] With further regard to the polypeptides of the
presently-disclosed subject matter, in some embodiments, the one or
more modifications made to the cholera toxin B subunit variant
polypeptides comprise the addition of an endoplasmic reticulum
retention signal having, in some embodiments, the amino acid
sequence KDEL (SEQ ID NO: 31). In some embodiments, the cholera
toxin B subunit variant polypeptide comprises two or more N-linked
glycosylation sequons, such as, in some embodiments, the
polypeptides of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ
ID NO: 14.
[0015] Further provided, in some embodiments of the
presently-disclosed subject matter, are pharmaceutical
compositions. In some embodiments, a pharmaceutical composition is
provided that comprises a polypeptide of the presently-disclosed
subject matter and a pharmaceutically-acceptable vehicle, carrier,
or excipient. In some embodiments, the pharmaceutical composition
further comprises an adjuvant.
[0016] Still further provided by the presently-disclosed subject
matter are isolated nucleic acid molecules. In some embodiments, an
isolated nucleic acid molecule is provided that comprises a nucleic
acid sequence encoding a polypeptide of the presently-disclosed
subject matter. In some embodiments, the nucleic acids are
incorporated into an appropriate expression vector for expressing
the polypeptides of the presently-disclosed subject matter in a
desired cell, such as, in some embodiments, a plant cell
transfected with the vectors, or a progeny of the plant cell, where
the cell or the progeny of the cell expresses the polypeptide. In
some embodiments, the nucleic acids incorporated into the vectors
are operably linked to an expression cassette.
[0017] In yet further embodiments of the presently-disclosed
subject matter are methods for producing a cholera toxin B subunit
variant polypeptide, such as those described herein. In some
embodiments, a method of producing a cholera toxin B subunit
variant polypeptide is provided that includes the steps of:
transforming a plant cell with a nucleic acid encoding a cholera
toxin B subunit variant polypeptide having one or more
modifications to increase the expression of the variant polypeptide
in a plant cell; expressing the cholera toxin B subunit variant
polypeptide in the plant cell; and purifying the cholera toxin B
subunit variant polypeptide. In some embodiments, the plant cell
comprises a Nicotiana plant cell, such as, in some embodiments, a
Nicotiana benthamiana plant cell. In some embodiments, the nucleic
acid encoding the cholera toxin B subunit variant polypeptide
expressed by the plant cell comprises the sequence of SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID
NO: 13.
[0018] In still further embodiments of the presently-disclosed
subject matter are methods for isolating a cholera toxin B subunit
variant polypeptide from a plant tissue. In some embodiments, an
isolation method is provided that comprises: obtaining a plant cell
expressing a cholera toxin B subunit variant polypeptide having one
or more modifications to increase the expression of the polypeptide
in the plant cell; extracting the cholera toxin B subunit variant
polypeptide from the plant cell; and purifying the cholera toxin B
subunit variant polypeptide from the plant cell. In some
embodiments, the step of extracting the cholera toxin B subunit
variant polypeptide from the plant cell comprises homogenizing the
plant tissue in an aqueous buffer having an acidic pH. In some
embodiments, the pH of the aqueous buffer is about 5. In some
embodiments, the step of purifying the cholera toxin B subunit
variant polypeptide from the plant cell comprises purifying the
variant polypeptide using chromatography.
[0019] Additionally provided, in some embodiments of the
presently-disclosed subject matter, are methods for eliciting an
immune response. In some embodiments, a method for eliciting an
immune response in a subject is provided that comprises
administering to a subject in need thereof an effective amount of a
cholera toxin B subunit variant polypeptide of the
presently-disclosed subject matter. In some embodiments,
administering an effective amount of the cholera toxin B subunit
variant polypeptide increases an amount of IgG, IgA, IgM, effector
T cells, regulatory T cells, or combinations thereof in a subject.
In some embodiments, administering an effective amount of the
cholera toxin B subunit variant polypeptide comprises orally
administering the cholera toxin B subunit variant polypeptide.
[0020] Further advantages of the presently-disclosed subject matter
will become evident to those of ordinary skill in the art after a
study of the description, Figures, and non-limiting Examples in
this document.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A and 1B are an image and a graph showing the
expression of various cholera toxin B subunit variant polypeptides
in N. benthamiana, including an image of a SDS-PAGE analysis of
leaf extracts containing the various cholera toxin B subunit
variant polypeptides (FIG. 1A); and a graph showing the amounts of
the various cholera toxin B subunit variant polypeptides present in
the leaf extracts based on a GM1-ELISA (FIG. 1B);
[0022] FIG. 2 is an image of a gel used for SDS-PAGE analysis of
the expression of an aglycosylated cholera toxin B subunit variant
polypeptide in N. benthamiana;
[0023] FIG. 3 is a schematic diagram showing an exemplary method
for isolating a cholera toxin B subunit variant polypeptide from
plant tissue in accordance with the presently-disclosed subject
matter;
[0024] FIGS. 4A-4B are an image and a graph showing the results of
a SDS-PAGE analysis of a wild-type (native) cholera toxin B subunit
polypeptide, a plant-produced aglycosylated cholera toxin B subunit
variant polypeptide, and a plant-produced N-glycosylated cholera
toxin B subunit variant polypeptide, including an image of a
denaturing and non-denaturing gel used for the SDS-PAGE analysis
(FIG. 4A), and a graph showing the ability of the polypeptides to
bind to GM1 ganglioside (FIG. 4B);
[0025] FIG. 5 is a graph showing the ability of a goat polyclonal
anti-cholera toxin B antibody to bind to a plant-produced
N-glycosylated cholera toxin B subunit variant polypeptide
(N-glyc-plant) and to a wild-type cholera toxin B subunit
polypeptide produced in E. coli (native);
[0026] FIGS. 6A and 6B are graphs showing a comparison of a
biochemical characterization of a wild-type (native) cholera toxin
B subunit polypeptide, a plant-produced aglycosylated cholera toxin
B subunit variant polypeptide (Plant CTB, agly), and a
plant-produced N-glycosylated cholera toxin B subunit variant
polypeptide (Plant CTB, N-gly), including a graph showing the
results of a thermal shift assay used to determine the melting
points of the three polypeptides (FIG. 6A) and a graph showing the
results of a size exclusion chromatography-high performance liquid
chromatography experiment used to determine the purity of the three
polypeptides as produced (FIG. 6B);
[0027] FIGS. 7A-7B are graphs showing the oral immunogenicity of a
wild-type cholera toxin B subunit polypeptide produced in E. coli
(eCTB) and a plant-produced aglycosylated cholera toxin B subunit
variant polypeptide (pCTB), including a graph showing the endpoint
titers of serum anti-cholera toxin B subunit IgG titer (FIG. 7A),
and a graph showing the endpoint titers of intestinal anti-cholera
toxin B subunit IgA titer (FIG. 7B);
[0028] FIG. 8 is a graph showing the duration of intestinal
anti-cholera toxin B subunit IgA titers in mice orally immunized
with either a vehicle control (PBS), a wild-type cholera toxin B
subunit polypeptide produced in E. coli (eCTB), or a plant-produced
aglycosylated cholera toxin B subunit variant polypeptide
(pCTB);
[0029] FIG. 9 is an image of gels showing an SDS-PAGE and lectin
blot analysis of a mono-N-glycosylated cholera toxin B subunit
variant polypeptide (lanes 1, 3, and 5) and a di-N-glycosylated
cholera toxin B subunit variant polypeptide (lanes 2, 4, and 6)
including an image of Coomassie stained gels under non-denaturing
(lanes 1 and 2) and denaturing (lanes 3 and 4) conditions, and an
image of a concanavalin A blot used to detect the glycosylated
polypeptides (lanes 5 and 6);
[0030] FIG. 10 is an image of gels showing an SDS-PAGE analysis of
a tri-N-glycosylated cholera toxin B subunit variant polypeptide,
including an image of a Coomassie-stained gel under denaturing
conditions (crude extract, lane 1; purified product, lane 2) and an
image of a Coomassie-stained gel under non-denaturing conditions
(purified product, lane 3);
[0031] FIG. 11 includes images of a gel used for SDS-PAGE analysis
and Western blot analysis of Endoglycosidase H (Endo H) and
Peptide: N-Glycosidase F (PNGase F) digestion of a
mono-N-glycoslyated cholera toxin B subunit variant polypeptide
obtained from N. benthamiana and grown with and without a chemical
inhibitor (CI) of class I .alpha.-mannosidases, including an
analysis of mono-N-glycosylated cholera toxin B subunit variant
polypeptide (+CI) digested with PNGase F (lanes 1),
mono-N-glycosylated cholera toxin B subunit variant polypeptide
(+CI) digested with Endo H (lanes 2), undigested
mono-N-glycosylated cholera toxin B subunit variant polypeptide
(+CI) (lanes 3), mono-N-glycosylated cholera toxin B subunit
variant polypeptide digested with PNGase F (lanes 4),
mono-glycosylated cholera toxin B subunit variant polypeptide
digested with Endo H (lanes 5), and undigested mono-N-glycosylated
cholera toxin B subunit variant polypeptide (lanes 6);
[0032] FIG. 12 is a graph showing the extent of recognition of an
aglycosylated cholera toxin B subunit variant polypeptide, a
mono-N-glycosylated cholera toxin B subunit variant polypeptide, a
mono-N-glycosylated cholera toxin B subunit variant polypeptide
grown in a plant exposed to a chemical inhibitor (CI) of class I a
mannosidases, and a tri-N-glycosylated cholera toxin B subunit
variant polypeptide, where the recognition is occurring by the
pattern recognition C-type lectin receptor DC-SIGN (Dendritic
Cell-Specific Intercellular adhesion molecule-3-Grabbing
Non-integrin); and
[0033] FIG. 13 includes images of gels showing SDS-PAGE and Western
blot analysis of two mutations (S26C and A102C) introduced into a
mono-N-glycosylated cholera toxin B subunit variant polypeptide,
including images of a Coomassie-stained gel (lanes 1-3) and a
Western blot (lanes 4-6) under denaturing conditions, where a
mono-N-glycosylated cholera toxin B subunit variant polypeptide was
loaded onto lanes 1 and 4, an aglycosylated cholera toxin B subunit
variant polypeptide was loaded onto lanes 2 and 5, and a
mono-glycosylated cholera toxin B subunit variant polypeptide with
the S26C and A102C mutations was loaded onto lanes 3 and 6.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0034] SEQ ID NO: 1 is nucleic acid sequence of a wild-type cholera
toxin B subunit from Vibrio cholerae;
[0035] SEQ ID NO: 2 is an amino acid sequence of a wild-type
cholera toxin B subunit from Vibrio cholerae;
[0036] SEQ ID NO: 3 is nucleic acid sequence encoding a cholera
toxin B subunit variant polypeptide modified to include a
C-terminal endoplasmic reticulum signal and to include no N-linked
glycosylation sequons at Asn4;
[0037] SEQ ID NO: 4 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide modified to include a C-terminal
endoplasmic reticulum signal and to include no N-linked
glycosylation sequons at Asn4;
[0038] SEQ ID NO: 5 is a nucleic acid sequence encoding a cholera
toxin B subunit variant polypeptide modified to include a
C-terminal endoplasmic reticulum retention signal and having one
N-linked glycosylation sequon at Asn4;
[0039] SEQ ID NO: 6 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide modified to include a C-terminal
endoplasmic reticulum retention signal and having one N-linked
glycosylation sequon at Asn4;
[0040] SEQ ID NO: 7 is a nucleic acid sequence encoding a cholera
toxin B subunit variant polypeptide modified to include a
C-terminal endoplasmic reticulum retention signal and to include
two N-linked glycosylation sequons at Asn4 and Asn103;
[0041] SEQ ID NO: 8 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide modified to include a C-terminal
endoplasmic reticulum retention signal and to include two N-linked
glycosylation sequons at Asn4 and Asn103;
[0042] SEQ ID NO: 9 is a nucleic acid sequence encoding another
cholera toxin B subunit variant polypeptide modified to include a
C-terminal endoplasmic reticulum retention signal and to include
two N-linked glycosylation sequons at Asn4 and Asn21;
[0043] SEQ ID NO: 10 is an amino acid sequence of another cholera
toxin B subunit variant polypeptide modified to include a
C-terminal endoplasmic reticulum retention signal and to include
two N-linked glycosylation sequons at Asn4 and Asn21;
[0044] SEQ ID NO: 11 is a nucleic acid sequence encoding a cholera
toxin B subunit variant polypeptide modified to include a
C-terminal endoplasmic reticulum retention signal and to include
three N-linked glycosylation sequons at Asn4, Asn21, and
Asn103;
[0045] SEQ ID NO: 12 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide modified to include a C-terminal
endoplasmic reticulum retention signal and to include three
N-linked glycosylation sequons at Asn4, Asn21, and Asn103;
[0046] SEQ ID NO: 13 is a nucleic acid sequence encoding another
cholera toxin B subunit variant polypeptide modified to include a
C-terminal endoplasmic reticulum retention signal and to include
three N-linked glycosylation sequons at Asn4, Asn21, and
Asn103;
[0047] SEQ ID NO: 14 is an amino acid sequence of another cholera
toxin B subunit variant polypeptide modified to include a
C-terminal endoplasmic reticulum retention signal and to include
three N-linked glycosylation sequons at Asn4, Asn21, and
Asn103;
[0048] SEQ ID NO: 15 is a nucleic acid sequence encoding a cholera
toxin B subunit variant polypeptide with an N-terminal secretory
signal from Vibrio cholerae and a C-terminal endoplasmic reticulum
retention signal;
[0049] SEQ ID NO: 16 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide including an N-terminal secretory
signal from Vibrio cholerae and a C-terminal endoplasmic reticulum
retention signal;
[0050] SEQ ID NO: 17 is a nucleic acid sequence encoding a rice
alpha-amylase secretory signal peptide;
[0051] SEQ ID NO: 18 is an amino acid sequence of a rice
alpha-amylase secretory signal peptide;
[0052] SEQ ID NO: 19 is nucleic acid sequence encoding a Nicotiana
plumbagenifolia calreticulin secretory signal peptide;
[0053] SEQ ID NO: 20 is an amino acid sequence of a Nicotiana
plumbagenifolia calreticulin secretory signal peptide;
[0054] SEQ ID NO: 21 is a nucleic acid sequence encoding an apple
pectinase secretory signal peptide;
[0055] SEQ ID NO: 22 is an amino acid sequence of an apple
pectinase secretory signal peptide;
[0056] SEQ ID NO: 23 is a nucleic acid sequence encoding a barley
alpha-amylase secretory signal peptide;
[0057] SEQ ID NO: 24 is an amino acid sequence encoding a barley
alpha-amylase secretory signal peptide;
[0058] SEQ ID NO: 25 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide including a Ser26.fwdarw.Cys and an
Ala102.fwdarw.Cys mutation;
[0059] SEQ ID NO: 26 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide including a rice alpha-amylase
N-terminal secretory signal peptide and a C-terminal endoplasmic
reticulum retention signal peptide;
[0060] SEQ ID NO: 27 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide including a Nicotiana plumbagenifolia
calreticulin N-terminal secretory signal peptide and a C-terminal
endoplasmic reticulum retention signal peptide;
[0061] SEQ ID NO: 28 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide including an apple pectinase N-terminal
secretory signal peptide and a C-terminal endoplasmic reticulum
retention signal peptide;
[0062] SEQ ID NO: 29 is an amino acid sequence of a cholera toxin B
subunit variant polypeptide including a barley alpha-amylase
N-terminal secretory signal peptide and a C-terminal endoplasmic
reticulum retention signal peptide;
[0063] SEQ ID NO: 30 is an amino acid sequence of an exemplary
endoplasmic reticulum retention signal peptide, KDEL (SEQ ID NO:
31), including a two amino acid linker, SE, preceding the KDEL
sequence (SEQ ID NO: 31); and
[0064] SEQ ID NO: 31 is an amino acid of the exemplary endoplasmic
reticulum retention signal peptide, KDEL.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0065] The details of one or more embodiments of the
presently-disclosed subject matter are set forth in this document.
Modifications to embodiments described in this document, and other
embodiments, will be evident to those of ordinary skill in the art
after a study of the information provided in this document. The
information provided in this document, and particularly the
specific details of the described exemplary embodiments, is
provided primarily for clearness of understanding and no
unnecessary limitations are to be understood therefrom. In case of
conflict, the specification of this document, including
definitions, will control.
[0066] Some of the polynucleotide and polypeptide sequences
disclosed herein are cross-referenced to GENBANK.RTM./GENPEPT.RTM.
accession numbers. The sequences cross-referenced in the
GENBANK.RTM./GENPEPT.RTM. database are expressly incorporated by
reference as are equivalent and related sequences present in
GENBANK.RTM./GENPEPT.RTM. or other public databases. Also expressly
incorporated herein by reference are all annotations present in the
GENBANK.RTM./GENPEPT.RTM. database associated with the sequences
disclosed herein. Unless otherwise indicated or apparent, the
references to the GENBANK.RTM./GENPEPT.RTM. database are references
to the most recent version of the database as of the filing date of
this Application.
[0067] While the following terms are believed to be well understood
by one of ordinary skill in the art, definitions are set forth to
facilitate explanation of the presently-disclosed subject matter.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the presently-disclosed subject
matter belongs. Although any methods, devices, and materials
similar or equivalent to those described herein can be used in the
practice or testing of the presently-disclosed subject matter,
representative methods, devices, and materials are now
described.
[0068] Following long-standing patent law convention, the terms
"a," "an," and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a cell" includes a plurality of such cells, and so forth.
[0069] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as reaction conditions,
and so forth used in the specification and claims are to be
understood as being modified in all instances by the term "about."
Accordingly, unless indicated to the contrary, the numerical
parameters set forth in this specification and claims are
approximations that can vary depending upon the desired properties
sought to be obtained by the presently-disclosed subject
matter.
[0070] As used herein, the term "about," when referring to a value
or to an amount of mass, weight, time, volume, concentration or
percentage is meant to encompass variations of in some embodiments
.+-.20%, in some embodiments .+-.10%, in some embodiments .+-.5%,
in some embodiments .+-.1%, in some embodiments .+-.0.5%, and in
some embodiments .+-.0.1% from the specified amount, as such
variations are appropriate to perform the disclosed method.
[0071] As used herein, ranges can be expressed as from "about" one
particular value, and/or to "about" another particular value. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0072] Glycans are polysaccharides or oligosaccharides that are
commonly attached to proteins in the endoplasmic reticulum of
cells. This attachment occurs via the nitrogen atom in the side
chain of the asparagine residue (i.e., amino acid) of the three
amino acid sequence Asn-X-Ser or Asn-X-Thr, which are also referred
to as N-linked glycosylation sequons, where X can be any amino acid
except proline. In other words, the presence of the N-linked
glycosylation sequon in proteins leads to the formation of
glycoproteins and proteoglycans, which are, generally, found on the
exterior surface of eukaryotic cells and, to a certain degree, in
prokaryotes.
[0073] Glycans are also widely found on the surface of the
enveloped viruses that constitute a large group of viral pathogens.
Mannose (Man) represents a major fraction of these envelope
carbohydrates, often comprising a cluster of N-linked high
(H)-Man-type glycans. In this regard, and because such a structure
is not commonly found in host glycoproteins, targeting the sugar
cluster of N-linked high (H)-Man-type glycans on the envelop of
viruses is believed to be a strategy to block the transmission and
infection of enveloped viruses, including a number of human
immunodeficiency virus (HIV) strains, as well as hepatitis C,
influenza, Ebola, and Marburg viruses that have been shown to be
neutralized by Man-specific lectins. Despite this possible
strategy, however, due to the poor antigenicity and immunogenicity
of sugar molecules, induction of carbohydrate-specific antibodies
has generally been a major challenge in modern vaccinology.
[0074] To that end, the presently-disclosed subject matter is
based, at least in part, on the discovery of polypeptides that are
capable of inducing H-Man-specific antiviral antibodies by:
presenting multiple H-Man glycans in a dense cluster as found on
viruses; conjugating H-Man glycans with a highly immunogenic
protein; and/or inducing high-avidity immunoglobulins (Igs) that
can tightly bind to flexible carbohydrate epitopes through multiple
antigen-binding sites. In particular, it has been determined that
variant polypeptides derived from an enteric bacterial cholera
toxin B subunit can be produced and used to display multiple
N-linked H-Man glycans that mimic a virus-like carbohydrate
cluster, such that, upon immunization of a subject, the immunogen
efficiently induces secretory IgA and IgG along with other mucosal
and systemic antibodies to provide a mechanism of protection
against the transmission and infection of enveloped viruses.
Furthermore, it has been determined that H-Man glycan-displaying
cholera toxin B subunit variant polypeptides can be developed as a
vaccine scaffold to carry various antigens and efficiently
stimulate mucosal and systemic immune systems. It has also been
determined that the N-glycosylated cholera toxin B subunit variant
polypeptides can exhibit higher vaccine efficacy against cholera.
Additionally, it has been discovered that these variant
polypeptides can be configured to be effectively produced in a
plant-based platform, thus making these immunogens capable of being
produced in an economical manner and on a large-scale.
[0075] The presently-disclosed subject matter includes polypeptides
having broad immunoactivating activity, including the induction of
H-Man glycan-specific antibodies, as well as methods for producing
and purifying such polypeptides. In some embodiments of the
presently-disclosed subject matter, isolated variant polypeptides
are provided. In some embodiments, an isolated variant polypeptide
is provided that comprises a cholera toxin B subunit variant having
one or more modifications to increase the expression of the variant
polypeptide in a plant cell.
[0076] As would be recognized by those of ordinary skill in the
art, cholera toxin is an oligomeric protein complex, which is
secreted by the bacterium Vibrio cholerae and is thought to be
responsible for the enteric symptoms characteristic of a cholera
infection. The cholera toxin itself is generally composed of six
protein subunits, namely a single copy of the A subunit, which is
thought to be the toxic portion of the molecule responsible for its
enzymatic action; and five copies of the B subunit, which form a
pentameric ring and are thought to comprise the non-toxic portions
of the molecule responsible for binding to receptors, such as the
GM1 ganglioside receptor, which contains a glycosphingolipid (e.g.,
a ceramide and oligosaccharide) with one sialic acid and which is
attached to the surface of a host cell. As such, the term "cholera
toxin B subunit" is used herein to refer to a single B subunit of
the cholera toxin as well as to B subunits of the cholera toxic in
the form of multimers (e.g., in a pentameric form). Exemplary
nucleic acid and amino acid sequence of a native cholera toxin B
subunit polypeptide from wild-type Vibrio cholerae are provided
herein in SEQ ID NOS: 1 and 2.
[0077] The terms "polypeptide," "protein," and "peptide," which are
used interchangeably herein, refer to a polymer of the 20 protein
amino acids, or amino acid analogs, regardless of its size or
function. Although "protein" is often used in reference to
relatively large polypeptides, and "peptide" is often used in
reference to small polypeptides, usage of these terms in the art
overlaps and varies. The term "polypeptide" as used herein refers
to peptides, polypeptides, and proteins, unless otherwise noted.
The terms "protein," "polypeptide," and "peptide" are used
interchangeably herein when referring to a gene product. Thus,
exemplary polypeptides include gene products, naturally occurring
or native proteins, homologs, orthologs, paralogs, fragments and
other equivalents, variants, and analogs of the foregoing. The term
"native," when used with reference to a polypeptide, refers to a
polypeptide that is encoded by a gene that is naturally present in
the genome of an untransformed cell.
[0078] The terms "polypeptide fragment" or "fragment," when used in
reference to a reference polypeptide, refers to a polypeptide in
which amino acid residues are deleted as compared to the reference
polypeptide itself, but where the remaining amino acid sequence is
usually identical to the corresponding positions in the reference
polypeptide. Such deletions can occur at the amino-terminus or
carboxy-terminus of the reference polypeptide, or alternatively
both.
[0079] A fragment can also be a "functional fragment," in which
case the fragment retains some or all of the activity of the
reference polypeptide as described herein. For example, in some
embodiments, a functional fragment of a cholera toxin B subunit
polypeptide can refer to a polypeptide in which amino acid residues
have been deleted as compared to the full-length cholera toxin B
subunit polypeptide, but which retains some or all of the ability
of the full-length cholera toxin B subunit polypeptide to bind to a
GM1 ganglioside and/or some or all of the ability of the
full-length cholera toxin B subunit polypeptide to attach to a
glycan.
[0080] The terms "modified amino acid," "modified polypeptide," and
"variant" are used herein to refer to an amino acid sequence that
is different from the reference polypeptide by one or more amino
acids, e.g., one or more amino acid substitutions or additions. A
variant of a reference polypeptide also refers to a variant of a
fragment of the reference polypeptide, for example, a fragment
wherein one or more amino acid substitutions have been made
relative to the reference polypeptide. A variant can also be a
"functional variant," in which the variant retains some or all of
the activity of the reference protein as described herein. For
example, in some embodiments, the cholera toxin B subunit variant
polypeptides described herein include amino acid sequences in which
one or more amino acids have been added and/or replaced, but which
nonetheless retain and/or enhance some or all of the ability of the
full-length cholera toxin B subunit polypeptide to bind to a GM1
ganglioside and/or some or all of the ability of the full-length
cholera toxin B subunit polypeptide to attach to a glycan.
[0081] As noted, in some embodiments of the presently-disclosed
subject matter, an isolated polypeptide is provided that comprises
a cholera toxin B subunit variant polypeptide having one or more
modifications to increase the expression of the polypeptide in a
plant cell. In some embodiments, the one or more modifications to
the cholera toxin B subunit variant polypeptide include an
endoplasmic reticulum retention signal having the amino acid
sequence KDEL (SEQ ID NO: 31). In some embodiments, the KDEL
sequence is linked to the cholera toxin by a two amino acid linker
to comprise, in some embodiments, the signal: SEKDEL (SEQ ID NO:
30). In some embodiments, the cholera toxin B subunit variant
polypeptide comprises the amino acid sequence of SEQ ID NO: 4, SEQ
ID NO: 6, or SEQ ID NO: 25.
[0082] In some embodiments of the presently-disclosed polypeptides,
the one or more modifications to the cholera toxin B subunit
variant polypeptide include the addition (e.g., an addition at the
N-terminal of the cholera toxin B subunit variant polypeptide) of a
secretory signal peptide capable of transferring or translocating
the cholera toxin B subunit peptide such that the cholera toxin B
subunit variant polypeptides is accumulated in a particular
location in a plant tissue, such as in the apoplasts of plant
cells. In some embodiments, the secretor signal peptide is selected
from the group consisting of a rice (e.g., Oryza sativa)
alpha-amylase secretory signal peptide (e.g., SEQ ID NO: 18), a
Nicotiana plumbagenifolia calreticulin secretory signal peptide
(e.g., SEQ ID NO: 20), an apple (e.g., Malus domestica) pectinase
secretory signal peptide (e.g., SEQ ID NO: 22), and a barley
(Hordeum vulgare) alpha-amylase secretory signal peptide (e.g., SEQ
ID NO: 24). In some embodiments, the secretory signal peptide has
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 18, 20, 22, and 24. In some embodiments, the secretory signal
peptide comprises a rice alpha-amylase secretory signal peptide,
such as the rice alpha-amylase secretory signal peptide of SEQ ID
NO: 18.
[0083] In some embodiments, an isolated cholera toxin B subunit
variant polypeptide is provided that comprises a cholera toxin B
subunit variant linked to a secretory signal peptide, such as those
described herein above, and an endoplasmic reticulum retention
signal. In some embodiments, the variant polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 26-29.
[0084] With further regard to the polypeptides of the
presently-disclosed subject matter, in some embodiments, a cholera
toxin B subunit variant polypeptide includes one or more mutations
so as to include a plurality of N-linked glycosylation sequons
(i.e., Asn-X-Ser or Asn-X-Thr sequences) in the variant polypeptide
sequences and thereby provide a mechanism to display multiple
N-linked H-Man glycans and mimic a virus-like carbohydrate cluster.
In some embodiments, about 1, about 2, about 3, about 4, about, 5,
about 6, about 7, about 8, about 9, or about 10 N-linked
glycosylation sequons are included in an exemplary cholera toxin B
subunit variant polypeptide of the presently-disclosed subject
matter. In some embodiments, a cholera toxin B subunit variant
polypeptide is provided that comprises 2 N-linked glycosylation
sequons, such as, in some embodiments, a cholera toxin B subunit
variant polypeptide having the amino acid sequence of SEQ ID NO: 8
or SEQ ID NO: 10. In other embodiments, a cholera toxin B subunit
variant polypeptide is provided that comprises 3 N-linked
glycosylation sequons, such as, in some embodiments, a cholera
toxin B subunit variant polypeptide having the amino acid sequence
of SEQ ID NO: 12 or SEQ ID NO: 14. In some embodiments, the
polypeptide comprises two or more N-linked glycosylation sequons,
such as, in some embodiments, the polypeptides of SEQ ID NO: 8, SEQ
ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.
[0085] Further provided, in some embodiments of the
presently-disclosed subject matter are pharmaceutical compositions.
In some embodiments, a pharmaceutical composition is provided that
comprises a cholera toxin B subunit variant polypeptide of the
presently-disclosed subject matter and a
pharmaceutically-acceptable vehicle, carrier, or excipient.
[0086] With regard to the pharmaceutically-acceptable vehicle,
carrier, or excipient suitable for use with the polypeptides of the
presently-disclosed subject matter, solid formulations of the
compositions for oral administration can contain suitable carriers
or excipients, such as corn starch, gelatin, lactose, acacia,
sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium
phosphate, calcium carbonate, sodium chloride, or alginic acid.
Disintegrators that can be used include, but are not limited to,
microcrystalline cellulose, corn starch, sodium starch glycolate,
and alginic acid. Tablet binders that can be used include acacia,
methylcellulose, sodium carboxymethylcellulose,
polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose,
starch, and ethylcellulose. Lubricants that can be used include
magnesium stearates, stearic acid, silicone fluid, talc, waxes,
oils, and colloidal silica. Further, the solid formulations can be
uncoated or they can be coated by known techniques to delay
disintegration and absorption in the gastrointestinal tract and
thereby provide a sustained/extended action over a longer period of
time. For example, glyceryl monostearate or glyceryl distearate can
be employed to provide a sustained-/extended-release formulation.
Numerous techniques for formulating sustained release preparations
are known to those of ordinary skill in the art and can be used in
accordance with the present invention, including the techniques
described in the following references: U.S. Pat. Nos. 4,891,223;
6,004,582; 5,397,574; 5,419,917; 5,458,005; 5,458,887; 5,458,888;
5,472,708; 6,106,862; 6,103,263; 6,099,862; 6,099,859; 6,096,340;
6,077,541; 5,916,595; 5,837,379; 5,834,023; 5,885,616; 5,456,921;
5,603,956; 5,512,297; 5,399,362; 5,399,359; 5,399,358; 5,725,883;
5,773,025; 6,110,498; 5,952,004; 5,912,013; 5,897,876; 5,824,638;
5,464,633; 5,422,123; and 4,839,177; and WO 98/47491, each of which
is incorporated herein by this reference.
[0087] Furthermore, liquid formulations of the compositions for
oral administration can be prepared in water or other aqueous
vehicles, and can contain various suspending agents such as
methylcellulose, alginates, tragacanth, pectin, kelgin,
carrageenan, acacia, polyvinylpyrrolidone, and include solutions,
emulsions, syrups, and elixirs containing, together with the active
components of the composition, wetting agents, sweeteners, and
coloring and flavoring agents.
[0088] Various liquid and powder formulations can also be prepared
by conventional methods for inhalation into the lungs of the
subject to be treated. For example, the compositions can be
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
Capsules and cartridges of, for example, gelatin for use in an
inhaler or insufflator may be formulated containing a powder mix of
the desired compound and a suitable powder base such as lactose or
starch.
[0089] Injectable formulations of the compositions can contain
various carriers such as vegetable oils, dimethylacetamide,
dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl
myristate, ethanol, polyols (glycerol, propylene glycol, liquid
polyethylene glycol), and the like. For intravenous injections,
water soluble versions of the compositions can be administered by
the drip method, whereby a formulation including a pharmaceutical
composition of the presently-disclosed subject matter and a
physiologically-acceptable excipient is infused.
Physiologically-acceptable excipients can include, for example, 5%
dextrose, 0.9% saline, Ringer's solution or other suitable
excipients. Intramuscular preparations, e.g., a sterile formulation
of a suitable soluble salt form of the compositions, can be
dissolved and administered in a pharmaceutical excipient such as
Water-for-Injection, 0.9% saline, or 5% glucose solution. A
suitable insoluble form of the compositions can be prepared and
administered as a suspension in an aqueous base or a
pharmaceutically-acceptable oil base, such as an ester of a long
chain fatty acid, (e.g., ethyl oleate).
[0090] In addition to the formulations described above, the
compositions of the presently-disclosed subject matter can also be
formulated as rectal compositions, such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides. Further, the compositions
can also be formulated as a depot preparation by combining the
compositions with suitable polymeric or hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0091] In some embodiments of the present invention, the
compositions of the present invention may be incorporated as part
of a nanoparticle. A "nanoparticle" within the scope of the
presently-disclosed subject matter is meant to include particles at
the single molecule level as well as those aggregates of particles
that exhibit microscopic properties. Methods of using and making a
nanoparticle that incorporates a compound of interest are known to
those of ordinary skill in the art and can be found following
references: U.S. Pat. Nos. 6,395,253, 6,387,329, 6,383,500,
6,361,944, 6,350,515, 6,333,051, 6,323,989, 6,316,029, 6,312,731,
6,306,610, 6,288,040, 6,272,262, 6,268,222, 6,265,546, 6,262,129,
6,262,032, 6,248,724, 6,217,912, 6,217,901, 6,217,864 , 6,214,560,
6,187,559, 6,180,415, 6,159,445, 6,149,868, 6,121,005, 6,086,881,
6,007,845, 6,002,817, 5,985,353, 5,981,467, 5,962,566, 5,925,564,
5,904,936, 5,856,435, 5,792,751, 5,789,375, 5,770,580, 5,756,264,
5,705,585, 5,702,727, and 5,686,113, each of which is incorporated
herein by this reference.
[0092] A topical formulation (e.g., a semi-solid ointment
formulation) can also be provided and can contain a desired
concentration of the active ingredient (e.g., a polypeptide of the
presently-disclosed subject matter) in a carrier such as a
pharmaceutical cream base. Various formulations for topical use
include drops, tinctures, lotions, creams, solutions, and ointments
containing the active ingredient and various supports and vehicles.
The optimal percentage of the therapeutic agent in each
pharmaceutical formulation varies according to the formulation
itself and the therapeutic effect desired in the specific
pathologies and correlated therapeutic.
[0093] In some embodiments, the pharmaceutical compositions of the
presently-disclosed subject matter are in the form of a vaccine. In
some embodiments, such immunogenic compositions and vaccines
according to the presently disclosed subject matter can comprise or
consist essentially of one or more adjuvants. Suitable adjuvants
for use in the practice of the presently-disclosed subject matter
include, but are not limited to: (1) polymers of acrylic or
methacrylic acid, maleic anhydride and alkenyl derivative polymers,
(2) immunostimulating sequences (ISS), such as
oligodeoxyribonucleotide sequences having one ore more
non-methylated CpG units (Klinman et al., Proc. Natl. Acad. Sci.,
USA, 1996, 93, 2879-2883; WO98/16247), (3) an oil in water
emulsion, such as the SPT emulsion described on p. 147 of "Vaccine
Design, The Subunit and Adjuvant Approach" published by M. Powell,
M. Newman, Plenum Press 1995, and the emulsion MF59 described on p
183 of the same work, (4) cation lipids containing a quaternary
ammonium salt, (5) cytokines, (6) aluminum hydroxide or aluminum
phosphate or (7) other adjuvants such as toll-like receptor ligands
or those discussed in any document cited and incorporated by
reference into the instant application, or (8) any combinations or
mixtures thereof.
[0094] The oil in water emulsion (3), which can be particularly
appropriate for viral vaccines, can be based on: light liquid
paraffin oil (European pharmacopoeia type), isoprenoid oil such as
squalane, squalene, oil resulting from the oligomerization of
alkenes, e.g. isobutene or decene, esters of acids or alcohols
having a straight-chain alkyl group, such as vegetable oils, ethyl
oleate, propylene glycol, di(caprylate/caprate), glycerol
tri(caprylate/caprate) and propylene glycol dioleate, or esters of
branched, fatty alcohols or acids, especially isostearic acid
esters. The oil can be used in combination with emulsifiers to form
an emulsion. The emulsifiers can be nonionic surfactants, such as:
esters of, on the one hand, sorbitan, mannide (e.g. anhydromannitol
oleate), glycerol, polyglycerol or propylene glycol and, on the
other hand, oleic, isostearic, ricinoleic or hydroxystearic acids,
the esters being optionally ethoxylated, or
polyoxypropylene-polyoxyethylene copolymer blocks, such as
Pluronic.RTM. (BASF Corporation, NJ), e.g., L121.
[0095] Among the type (1) adjuvant polymers, in some embodiments,
the polymers are polymers of crosslinked acrylic or methacrylic
acid, including those crosslinked by polyalkenyl ethers of sugars
or polyalcohols. These compounds are known under the name carbomer
(Pharmeuropa, vol. 8, no. 2, June 1996). One skilled in the art can
also refer to U.S. Pat. No. 2,909,462, which provides such acrylic
polymers crosslinked by a polyhydroxyl compound having at least
three hydroxyl groups, preferably no more than eight such groups,
the hydrogen atoms of at least three hydroxyl groups being replaced
by unsaturated, aliphatic radicals having at least two carbon
atoms. The preferred radicals are those containing 2 to 4 carbon
atoms, e.g. vinyls, allyls and other ethylenically unsaturated
groups. The unsaturated radicals can also contain other
substituents, such as methyl. Products sold under the name
CARBOPOL.TM. (BF Goodrich, Ohio, USA) are, in some embodiments,
especially suitable, as such products are crosslinked by allyl
saccharose or by allyl pentaerythritol. Among them, reference is
made to CARBOPOL.TM. 974P, 934P and 971 P. As to the maleic
anhydride-alkenyl derivative copolymers, in some embodiments, the
derivative copolymers are EMA polymers, which are straight-chain or
crosslinked ethylene-maleic anhydride copolymers that are, for
example, crosslinked by divinyl ether. Reference is also made to J.
Fields et al., Nature 186: 778-780, Jun. 4, 1960.
[0096] Still further provided, in some embodiments of the
presently-disclosed subject matter, are isolated nucleic acids. In
some embodiments, isolated nucleic acid sequences are provided that
encode the cholera toxin subunit B variant polypeptides of the
presently-disclosed subject matter. In some embodiments, a nucleic
acid is provided that comprises the nucleic acid sequence of SEQ ID
NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or
SEQ ID NO: 13. In some embodiments, a nucleic acid sequence is
provided that comprises the nucleic acid sequence of SEQ ID NO: 15.
In some embodiments, additional nucleic acid sequences are provided
wherein the nucleic acid sequences are derived from a Vibrio
cholerae cholera toxin B subunit gene and used to produce a cholera
toxin B subunit variant polypeptide of the presently-disclosed
subject matter.
[0097] The term "gene" is used broadly to refer to any segment of
DNA associated with a biological function. Thus, genes include, but
are not limited to, coding sequences and/or the regulatory
sequences required for their expression. Genes can also include
non-expressed DNA segments that, for example, form recognition
sequences for a polypeptide. Genes can be obtained from a variety
of sources, including cloning from a source of interest or
synthesizing from known or predicted sequence information, and can
include sequences designed to have desired parameters.
[0098] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or double-
stranded form. Unless specifically limited, the term encompasses
nucleic acids containing known analogues of natural nucleotides
that have similar binding properties as the reference nucleic acid
and are metabolized in a manner similar to naturally-occurring
nucleotides. Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences and as well as the sequence explicitly
indicated.
[0099] The term "isolated", when used in the context of an isolated
nucleic acid molecule or an isolated polypeptide, is a nucleic acid
molecule or polypeptide that, by the hand of man, exists apart from
its native environment and is therefore not a product of nature. An
isolated nucleic acid molecule or polypeptide can exist in a
purified form or can exist in a non-native environment such as, for
example, in a transgenic host cell.
[0100] The term "degenerate variant" refers to a nucleic acid
having a residue sequence that differs from a reference nucleic
acid by one or more degenerate codon substitutions. Degenerate
codon substitutions can be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed base and/or deoxyinosine residues (Batzer et
al. (1991) Nucleic Acid Res 19:5081; Ohtsuka et al. (1985) J Biol
Chem 260:2605 2608; Rossolini et al. (1994) Mol Cell Probes 8:91
98).
[0101] In some embodiments, an isolated nucleic acid sequence is
provided that selectively hybridizes to the nucleic acid sequence
of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. The term "selectively
hybridize" as used herein refers to the ability of a nucleic acid
sequence to hybridize to a target polynucleotide (e.g., a
polynucleotide of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15) with
specificity. Thus, the nucleic acid sequence comprises a
polynucleotide sequence that is complementary, or essentially
complementary, to at least a portion of the target polynucleotide
sequence. For example, in some embodiments, the nucleic acid
sequence that selectively hybridizes to the sequence of SEQ ID NO:
3 is complementary to the sequence of SEQ ID NO: 3. Nucleic acid
sequences which are "complementary" are those which are
base-pairing according to the standard Watson-Crick complementarity
rules. As used herein, the term "complementary sequences" means
nucleic acid sequences which are substantially complementary, as
can be assessed by the same nucleotide comparison set forth above,
or as defined as being capable of hybridizing to the nucleic acid
segment in question under relatively stringent conditions such as
those described herein. A particular example of a contemplated
complementary nucleic acid segment is an antisense oligonucleotide.
With regard to the nucleic acid sequences disclosed herein as
selectively hybridizing to the sequence of SEQ ID NO: 3, SEQ ID NO:
5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ
ID NO: 15, the hybridizing nucleic acid sequence need not
necessarily be completely complementary to the nucleic acid
sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 along the entire
length of the target polynucleotide so long as the hybridizing
nucleic acid sequence can bind the nucleic acid of SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, or SEQ ID NO: 15 with specificity. In some embodiments, the
nucleic acid sequences that selectively hybridize to the sequence
of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15 are about 80%, about 85%,
about 90%, about 95%, about 98%, or about 100% complementary to the
sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, respectively.
[0102] Nucleic acid hybridization will be affected by such
conditions as salt concentration, temperature, or organic solvents,
in addition to the base composition, length of the complementary
strands, and the number of nucleotide base mismatches between the
hybridizing nucleic acids, as will be readily appreciated by those
skilled in the art. Stringent temperature conditions will generally
include temperatures in excess of 30.degree. C., typically in
excess of 37.degree. C., and preferably in excess of 45.degree. C.
Stringent salt conditions will ordinarily be less than 1,000 mM,
typically less than 500 mM, and preferably less than 200 mM. For
example, in some embodiments, nucleic acid hybridization can be
performed at 60.degree. C. with 0.1.times. sodium citrate-sodium
chloride (SSC) and 0.1% sodium dodecyl sulfate (SDS). However, the
combination of parameters is much more important than the measure
of any single parameter. (See, e.g., Wetmur & Davidson, 1968).
Determining appropriate hybridization conditions to identify and/or
isolate sequences containing high levels of homology is well known
in the art. (See, e.g., Sambrook, et al., 1989).
[0103] Further provided, in some embodiments, are expression
vectors comprising the nucleic acid molecules of the
presently-disclosed subject matter operably linked to an expression
cassette. The term "vector" is used herein to refer to any vehicle
that is capable of transferring a nucleic acid sequence into
another cell. For example, vectors which can be used in accordance
with the presently-disclosed subject matter include, but are not
limited to, plasmids, cosmids, bacteriophages, or viruses, which
can be transformed by the introduction of a nucleic acid sequence
of the presently-disclosed subject matter. Such vectors are well
known to those of ordinary skill in the art.
[0104] In some embodiments, the nucleic acids of the
presently-disclosed subject matter are operably linked to an
expression cassette. The terms "associated with", "operably
linked", and "operatively linked" refer to two sequences that are
related physically or functionally. For example, a promoter or
regulatory DNA sequence is said to be "associated with" a DNA
sequence that encodes an RNA or a polypeptide if the two sequences
are operatively linked, or situated such that the regulator DNA
sequence will affect the expression level of the coding or
structural DNA sequence.
[0105] The term "expression cassette" refers to a nucleic acid
molecule capable of directing expression of a particular nucleotide
sequence in an appropriate host cell, comprising a promoter
operatively linked to the nucleotide sequence of interest which is
operatively linked to termination signals. It also typically
comprises sequences required for proper translation of the
nucleotide sequence. The coding region usually encodes a
polypeptide of interest but can also encode a functional RNA of
interest, for example antisense RNA or a non-translated RNA, in the
sense or antisense direction. The expression cassette comprising
the nucleotide sequence of interest can be chimeric, meaning that
at least one of its components is heterologous with respect to at
least one of its other components. The expression cassette can also
be one that is naturally occurring but has been obtained in a
recombinant form useful for heterologous expression.
[0106] The presently-disclosed subject matter also provides
transgenic plant cells or plants that have been transformed with
one or more of the vectors disclosed herein (i.e., a vector
including a nucleic acid molecule encoding for a cholera toxin B
subunit polypeptide or variant thereof). In some embodiments, a
plant cell, or a progeny of the plant cell, is provided wherein the
plant cell and/or its progeny is transfected with a vector of the
presently-disclosed subject matter such that the cell and/or its
progeny expresses the polypeptide. As used herein, the term "plant
cell" is understood to mean any cell derived from a
monocotyledonous or a dicotyledonous plant and capable of
constituting undifferentiated tissues such as calli, differentiated
tissues such as embryos, portions of monocotyledonous plants,
monocotyledonous plants or seed. The term "plant" is understood to
mean any differentiated multi-cellular organism capable of
photosynthesis, including monocotyledons and dicotyledons. In some
embodiments, the plant cell is a Nicotiana or tobacco plant cell,
such as a Nicotiana benthamiana plant cell that has been
transformed with a vector of the presently-disclosed subject
matter.
[0107] The terms "transformed," "transgenic," and "recombinant" are
used herein to refer to a cell of a host organism, such as a plant,
into which a heterologous nucleic acid molecule has been
introduced. The nucleic acid molecule can be stably integrated into
the genome of the cell or the nucleic acid molecule can also be
present as an extrachromosomal molecule. Such an extrachromosomal
molecule can be auto-replicating. Transformed cells, tissues, or
subjects are understood to encompass not only the end product of a
transformation process, but also transgenic progeny thereof.
[0108] The terms "heterologous," "recombinant," and "exogenous,"
when used herein to refer to a nucleic acid sequence (e.g., a DNA
sequence) or a gene, refer to a sequence that originates from a
source foreign to the particular host cell or, if from the same
source, is modified from its original form. Thus, a heterologous
gene in a host cell includes a gene that is endogenous to the
particular host cell but has been modified through, for example,
the use of site-directed mutagenesis or other recombinant
techniques. The terms also include non-naturally occurring multiple
copies of a naturally occurring DNA sequence. Thus, the terms refer
to a DNA segment that is foreign or heterologous to the cell, or
homologous to the cell but in a position or form within the host
cell in which the element is not ordinarily found. Similarly, when
used in the context of a polypeptide or amino acid sequence, an
exogenous polypeptide or amino acid sequence is a polypeptide or
amino acid sequence that originates from a source foreign to the
particular host cell or, if from the same source, is modified from
its original form. Thus, exogenous DNA segments can be expressed to
yield exogenous polypeptides.
[0109] Introduction of a nucleic acid (e.g., a nucleic acid
incorporated into an appropriate vector) of the presently-disclosed
subject matter into a plant cell can be performed by a variety of
methods known to those of ordinary skill in the art including, but
not limited to, insertion of a nucleic acid sequence of interest
into an Agrobacterium rhizogenes Ri or Agrobacterium tumefaciens Ti
plasmid, microinjection, electroporation, or direct precipitation.
By way of providing an example, in some embodiments, transient
expression of a nucleic acid sequence or gene of interest can be
performed by agro-infiltration methods. In this regard, a
suspension of Agrobacterium tumefaciens containing a nucleic acid
sequence or gene of interest can be grown in culture and then
vacuum-infiltrated into a plant. Once inside the tissues of the
plant (e.g., the leaves of the plant), the Agrobacterium transforms
the gene of interest to a portion of the plant cells where the gene
is then transiently expressed.
[0110] As another example, transformation of a plasmid or nucleic
acid of interest into a plant cell can be performed by particle gun
bombardment techniques. In this regard, a suspension of plant
embryos can be grown in liquid culture and then bombarded with
plasmids or nucleic acids that are attached to gold particles,
wherein the gold particles bound to the plasmid or nucleic acid of
interest can be propelled through the membranes of the plant
tissues, such as embryonic tissue. Following bombardment, the
transformed embryos can then be selected using an appropriate
antibiotic to generate new, clonally propagated, transformed
embryogenic suspension cultures.
[0111] For additional guidance regarding methods of transforming
and producing transgenic plant cells, see U.S. Pat. Nos. 4,459,355;
4,536,475; 5,464,763; 5,177,010; 5,187,073; 4,945,050; 5,036,006;
5,100,792; 5,371,014; 5,478,744; 5,179,022; 5,565,346; 5,484,956;
5,508,468; 5,538,877; 5,554,798; 5,489,520; 5,510,318; 5,204,253;
5,405,765; EP Nos. 267,159; 604,662; 672,752; 442,174; 486,233;
486,234; 539,563; 674,725; and, International Patent Application
Publication Nos. WO 91/02071 and WO 95/06128, each of which is
incorporated herein by this reference.
[0112] In yet further embodiments of the presently-disclosed
subject matter, methods of producing a cholera toxin B subunit
polypeptide are provided. In some embodiments, a method of
producing a cholera toxin B subunit polypeptide is provided that
comprises: transforming a plant cell with a nucleic acid encoding a
cholera toxin B subunit variant polypeptide of the
presently-disclosed subject matter (i.e., a cholera toxin B subunit
variant polypeptide having one or more modifications to increase
the expression of the polypeptide in a plant cell and/or to display
one or more H-Man glycans); expressing the cholera toxin B subunit
variant polypeptide in the plant cell; and purifying the cholera
toxin B subunit variant polypeptide. In some embodiments, the plant
cell comprises a plant cell from the genus Nicotiana, such as, in
some embodiments, a Nicotiana benthamiana plant cell.
[0113] The term "purifying" as used herein in reference to the
production of the cholera toxin B subunit variant polypeptide
refers to methods by which the cholera toxin B subunit variant
polypeptide can be isolated from unwanted materials, including
contaminants, that may be found or be otherwise present in plant
tissue expressing an exemplary cholera toxin B subunit variant
polypeptide of the presently-disclosed subject matter. Such
purification methods include, but are not limited, to: protein
precipitation including immunoprecipitation, ultracentrifugation,
and chromatography including size-exclusion chromatography,
hydrophobic interaction chromatography, ion exchange
chromatography, and affinity chromatography, immunoaffinity
chromatography, high-performance liquid chromatography, and the
like.
[0114] As one example of the purification of a cholera toxin B
subunit variant polypeptide of the presently-disclosed subject
matter, in some embodiments, the purification of a cholera toxin B
subunit variant polypeptide is accomplished by first homogenizing
transgenic plant tissue (e.g., leaf tissue) of the
presently-disclosed subject matter to obtain plant tissue extracts.
These tissue extracts are then clarified and the pH of the extracts
is adjusted to about 5 to about 8 before performing liquid
chromatography to obtain the variant polypeptides. In some
embodiments, after the initial chromatography steps are performed,
an additional chromatography step is performed (e.g., using a
hydroxyapatite column) followed by a phase separation step to
remove endotoxins to obtain the purified protein. It has been
determined, however, that in some embodiments, a variant
polypeptide of the presently-disclosed subject matter that is
produced in plants can be highly-purified to, in some embodiments,
a purification level of about 80%, about 85%, about 90%, about 95%,
about 96%, about 97%, about 98%, or about 99% without the use of a
second chromatographic step and/or without the use of an endotoxin
removal step.
[0115] In some embodiments of the presently-disclosed subject
matter, a method of isolating a cholera toxin B subunit polypeptide
or variant thereof from a plant tissue is provided that comprises:
obtaining a plant cell expressing a cholera toxin B subunit variant
polypeptide of the presently-disclosed subject matter; extracting
the cholera toxin B subunit variant polypeptide from the plant
cell; and purifying the cholera toxin B subunit variant from the
plant cell. In some embodiments, the step of extracting the cholera
toxin B subunit polypeptide or variant thereof from the plant cell
comprises homogenizing the plant tissue in an aqueous buffer having
an acidic pH of about 4, about 5, or about 6. In some embodiments,
the pH of the buffer is about 5. In some embodiments, the buffer
has a basic pH, such as a pH of about 8. In some embodiments, the
step of purifying the cholera toxin B subunit polypeptide or
variant thereof from the plant cell comprises purifying the
polypeptide or variant thereof using chromatography.
[0116] In further embodiments of the presently-disclosed subject
matter, methods for eliciting an immune response in a subject are
provided. In some embodiments, a method for eliciting an immune
response in a subject is provided that comprises administering to a
subject an effective amount of a cholera toxin B subunit variant
polypeptide of the presently-disclosed subject matter. In some
embodiments, administering an effective amount of the cholera toxin
B subunit variant polypeptide increases an amount of IgG, IgA, IgM,
and/or other immunglobulins, and effector or regulatory T cells in
a subject. In some embodiments, administering an effective amount
of the cholera toxin B subunit variant polypeptide increases an
amount of IgG, IgA, IgM, effector T cells, regulatory T cells, or
combinations thereof in a subject.
[0117] Various methods known to those skilled in the art can be
used to determine an increase in the amount of IgG, IgA, IgM, other
immunoglobulins, and/or T cells in a subject. For example, in
certain embodiments, the amounts of expression of the
immunoglobulins and the activation of the T cells in a subject can
be determined by probing for mRNA of the gene encoding the
immunoglobulin in a biological sample obtained from the subject
(e.g., a tissue sample, a urine sample, a saliva sample, a blood
sample, a serum sample, a plasma sample, or sub-fractions thereof)
using any RNA identification assay known to those skilled in the
art. Briefly, RNA can be extracted from the sample, amplified,
converted to cDNA, labeled, and allowed to hybridize with probes of
a known sequence, such as known RNA hybridization probes
immobilized on a substrate, e.g., array, or microarray, or
quantitated by real time PCR (e.g., quantitative real-time PCR,
such as available from Bio-Rad Laboratories, Hercules, Calif.).
Because the probes to which the nucleic acid molecules of the
sample are bound are known, the molecules in the sample can be
identified. In this regard, DNA probes for one or more of the mRNAs
encoded by the immunoglobulins or T cell activation marker
molecules can be immobilized on a substrate and provided for use in
practicing a method in accordance with the present invention.
[0118] With further regard to determining levels of the
immunoglobulins in samples, mass spectrometry and/or immunoassay
devices and methods can be used to measure the immunoglobulins in
samples, although other methods can also be used and are well known
to those skilled in the art. See, e.g., U.S. Pat. Nos. 6,143,576;
6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615;
5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and
5,480,792, each of which is hereby incorporated by reference in its
entirety. Immunoassay devices and methods can utilize labeled
molecules in various sandwich, competitive, or non-competitive
assay formats, to generate a signal that is related to the presence
or amount of an analyte of interest. Additionally, certain methods
and devices, such as biosensors and optical immunoassays, can be
employed to determine the presence or amount of analytes without
the need for a labeled molecule. See, e.g., U.S. Pat. Nos.
5,631,171; and 5,955,377, each of which is hereby incorporated by
reference in its entirety.
[0119] Any suitable immunoassay can be utilized, for example,
enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs),
competitive binding assays, and the like. Specific immunological
binding of the immunoglobulins can be detected directly or
indirectly. Direct labels include fluorescent or luminescent tags,
metals, dyes, radionucleotides, and the like, attached to the
antibody. Indirect labels include various enzymes well known in the
art, such as alkaline phosphatase, horseradish peroxidase and the
like.
[0120] The use of immobilized antibodies or fragments thereof
specific for the immunoglobulins is also contemplated by the
presently-disclosed subject matter. The antibodies can be
immobilized onto a variety of solid supports, such as magnetic or
chromatographic matrix particles, the surface of an assay plate
(such as microtiter wells), pieces of a solid substrate material
(such as plastic, nylon, paper), and the like. An assay strip can
be prepared by coating the antibody or a plurality of antibodies in
an array on a solid support. This strip can then be dipped into the
test biological sample and then processed quickly through washes
and detection steps to generate a measurable signal, such as for
example a colored spot.
[0121] Mass spectrometry (MS) analysis can be used, either alone or
in combination with other methods (e.g., immunoassays), to
determine the presence and/or quantity of an inflammatory molecule
in a subject. Exemplary MS analyses that can be used in accordance
with the present invention include, but are not limited to: liquid
chromatography-mass spectrometry (LC-MS); matrix-assisted laser
desorption/ionization time-of-flight MS analysis (MALDI-TOF-MS),
such as for example direct-spot MALDI-TOF or liquid chromatography
MALDI-TOF mass spectrometry analysis; electrospray ionization MS
(ESI-MS), such as for example liquid chromatography (LC) ESI-MS;
and surface enhanced laser desorption/ionization time-of-flight
mass spectrometry analysis (SELDI-TOF-MS). Each of these types of
MS analysis can be accomplished using commercially-available
spectrometers, such as, for example, triple quadropole mass
spectrometers. Methods for utilizing MS analysis to detect the
presence and quantity of peptides, such as immunoglobulins, in
biological samples are known in the art. See, e.g., U.S. Pat. Nos.
6,925,389; 6,989,100; and 6,890,763 for further guidance, each of
which are incorporated herein by this reference.
[0122] With regard to the various methods of eliciting an immune
response described herein, although certain embodiments of the
methods only call for a qualitative assessment (e.g., the presence
or absence of the expression of an immunoglobulin), other
embodiments of the methods call for a quantitative assessment
(e.g., an amount of increase in a level of immunoglobulins, T
cells, or both in a subject). Such quantitative assessments can be
made, for example, using one of the above-mentioned methods, as
will be understood by those skilled in the art.
[0123] The skilled artisan will also understand that measuring an
increase in the amount of a certain feature (e.g., IgA levels) in a
subject is a statistical analysis. For example, a reduction in an
amount of IgA levels in a subject can be compared to control level
of IgA, and an amount of IgA of more than the control level can be
indicative of an increase in the amount of IgA, as evidenced by a
level of statistical significance. Statistical significance is
often determined by comparing two or more populations, and
determining a confidence interval and/or a p value. See, e.g.,
Dowdy and Wearden, Statistics for Research, John Wiley & Sons,
New York, 1983, incorporated herein by reference in its entirety.
Preferred confidence intervals of the present subject matter are
90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred
p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and
0.0001.
[0124] For administration of a therapeutic composition as disclosed
herein (e.g., a composition comprising a cholera toxin B subunit
variant polypeptide of the presently-disclosed subject matter and a
pharmaceutically-acceptable vehicle, carrier, or excipient),
conventional methods of extrapolating human dosage based on doses
administered to a murine animal model can be carried out using the
conversion factor for converting the mouse dosage to human dosage:
Dose Human per kg=Dose Mouse per kg.times.12 (Freireich, et al.,
(1966) Cancer Chemother Rep. 50:219-244). Drug doses can also be
given in milligrams per square meter of body surface area because
this method rather than body weight achieves a good correlation to
certain metabolic and excretionary functions. Moreover, body
surface area can be used as a common denominator for drug dosage in
adults and children as well as in different animal species as
described by Freireich, et al. (Freireich et al., (1966) Cancer
Chemother Rep. 50:219-244). Briefly, to express a mg/kg dose in any
given species as the equivalent mg/sq m dose, multiply the dose by
the appropriate km factor. In an adult human, 100 mg/kg is
equivalent to 100 mg/kg.times.37 kg/sq m=3700 mg/m2.
[0125] Suitable methods for administering a therapeutic composition
in accordance with the methods of the presently-disclosed subject
matter include, but are not limited to, systemic administration,
parenteral administration (including intravascular, intramuscular,
intraarterial administration), oral delivery, topical
administration, buccal delivery, rectal delivery, vaginal delivery,
subcutaneous administration, intraperitoneal administration,
inhalation, intratracheal installation, surgical implantation,
transdermal delivery, local injection, and hyper-velocity
injection/bombardment. Where applicable, continuous infusion can
enhance drug accumulation at a target site (see, e.g., U.S. Pat.
No. 6,180,082). In some embodiments, such as those which include a
pharmaceutical composition comprising a cholera toxin B subunit
variant polypeptide of the presently-disclosed subject matter, the
pharmaceutical composition can be administered orally to thereby
elicit an immune response.
[0126] Regardless of the route of administration, the compounds of
the presently-disclosed subject matter are typically administered
in amount effective to achieve the desired response. As used
herein, the terms "effective amount" and "therapeutically effective
amount" refer to an amount of the therapeutic composition (e.g., a
composition comprising a cholera toxin B subunit variant
polypeptide of the presently-disclosed subject matter, and a
pharmaceutically-acceptable vehicle, carrier, or excipient)
sufficient to produce a measurable biological response (e.g., an
increase in levels of IgA). Actual dosage levels of active
ingredients in a therapeutic composition of the presently-disclosed
subject matter can be varied so as to administer an amount of the
active polypeptide(s) that is effective to achieve the desired
therapeutic response for a particular subject and/or application.
Of course, the effective amount in any particular case will depend
upon a variety of factors including the activity of the therapeutic
composition, formulation, the route of administration, combination
with other drugs or treatments, severity of the condition being
treated, and the physical condition and prior medical history of
the subject being treated. Preferably, a minimal dose is
administered, and the dose is escalated in the absence of
dose-limiting toxicity to a minimally effective amount.
Determination and adjustment of a therapeutically effective dose,
as well as evaluation of when and how to make such adjustments, are
known to those of ordinary skill in the art.
[0127] For additional guidance regarding formulation and dose, see
U.S. Pat. Nos. 5,326,902 and 5,234,933; PCT International
Publication No. WO 93/25521; Berkow, et al., (1997) The Merck
Manual of Medical Information, Home ed. Merck Research
Laboratories, Whitehouse Station, N.J.; Goodman, et al., (2006)
Goodman & Gilman's the Pharmacological Basis of Therapeutics,
11th ed. McGraw-Hill Health Professions Division, New York; Ebadi.
(1998) CRC Desk Reference of Clinical Pharmacology. CRC Press, Boca
Raton, Fla.; Katzung, (2007) Basic & Clinical Pharmacology,
10th ed. Lange Medical Books/McGraw-Hill Medical Pub. Division, New
York; Remington, et al., (1990) Remington's Pharmaceutical
Sciences, 18th ed. Mack Pub. Co., Easton, Pa.; Speight, et al.,
(1997) Avery's Drug Treatment: A Guide to the Properties, Choice,
Therapeutic Use and Economic Value of Drugs in Disease Management,
4th ed. Adis International, Auckland/Philadelphia; and Duch, et
al., (1998) Toxicol. Lett. 100-101:255-263, each of which are
incorporated herein by reference.
[0128] As used herein, the term "subject" includes both human and
animal subjects. Thus, veterinary therapeutic uses are provided in
accordance with the presently-disclosed subject matter. As such,
the presently-disclosed subject matter provides for the treatment
of mammals such as humans, as well as those mammals of importance
due to being endangered, such as Siberian tigers; of economic
importance, such as animals raised on farms for consumption by
humans; and/or animals of social importance to humans, such as
animals kept as pets or in zoos. Examples of such animals include
but are not limited to: carnivores such as cats and dogs; swine,
including pigs, hogs, and wild boars; ruminants and/or ungulates
such as cattle, oxen, sheep, giraffes, deer, goats, bison, and
camels; and horses. Also provided is the treatment of birds,
including the treatment of those kinds of birds that are endangered
and/or kept in zoos, as well as fowl, and more particularly
domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks,
geese, guinea fowl, and the like, as they are also of economic
importance to humans. Thus, also provided is the treatment of
livestock, including, but not limited to, domesticated swine,
ruminants, ungulates, horses (including race horses), poultry, and
the like.
[0129] The practice of the presently-disclosed subject matter can
employ, unless otherwise indicated, conventional techniques of cell
biology, cell culture, molecular biology, transgenic biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Molecular Cloning A Laboratory Manual
(1989), 2nd Ed., ed. by Sambrook, Fritsch and Maniatis, eds., Cold
Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Pat. No.
4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985;
Polynucleotide Synthesis, M. J. Gait, ed., 1984; Nucleic Acid
Hybridization, D. Hames & S. J. Higgins, eds., 1984;
Transcription and Translation, B. D. Hames & S. J. Higgins,
eds., 1984; Culture Of Animal Cells, R. I. Freshney, Alan R. Liss,
Inc., 1987; Immobilized Cells And Enzymes, IRL Press, 1986; Perbal
(1984), A Practical Guide To Molecular Cloning; See Methods In
Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For
Mammalian Cells, J. H. Miller and M. P. Calos, eds., Cold Spring
Harbor Laboratory, 1987; Methods In Enzymology, Vols. 154 and 155,
Wu et al., eds., Academic Press Inc., N.Y.; Immunochemical Methods
In Cell And Molecular Biology (Mayer and Walker, eds., Academic
Press, London, 1987; Handbook Of Experimental Immunology, Volumes
I-IV, D. M. Weir and C. C. Blackwell, eds., 1986.
[0130] The presently-disclosed subject matter is further
illustrated by the following specific but non-limiting
examples.
EXAMPLES
Example 1
Expression of Cholera Toxin B Subunit Variants in Nicotiana
benthamiana
[0131] To evaluate the expression of cholera toxin B subunit
variants in plant cells in an effort to develop a cholera toxin B
subunit variant production platform, deconstructed virus vectors
were designed to allow for the transient expression of a
recombinant (r) CTB in Nicotiana benthamiana. Briefly, a
"deconstructed" tobamovirus replicon system [1, 2] (magnICON.RTM.;
Icon Genetics GmbH, Halle/Saale, Germany) was used to express a
cholera toxin B subunit variant polypeptide (SEQ ID NO: 16) in N.
benthamiana, which included the plant-expression-optimized
synthetic CTB gene (corresponding to nucleotides 64 to 372 of
GENBANK.RTM. accession no. AY475128) containing a C-terminal
endoplasmic reticulum (ER) retention signal attached to a two amino
acid linker sequence (SEKDEL; SEQ ID NO: 30) [3] that was
sub-cloned into the magnICON vector pICH115991 using NcoI and SacI
endonuclease restriction sites to generate pNM134. For expression
of the cholera toxin B subunit variant polypeptide with the
original Vibrio cholerae N-terminal secretory signal peptide, the
nucleic acid sequence (SEQ ID NO: 15) encoding the above-mentioned
cholera toxin B subunit variant polypeptide (SEQ ID NO: 16) was
used as a template for PCR. A 5' oligonucleotide corresponding to
the V. cholerae secretory signal sequence (GENBANK.RTM. accession
no. U25679, nucleotide 1 to 63) and the 5' nucleotide region of the
CTB gene and a 3' oligonucleotide corresponding to the 3'
nucleotide region of the CTB gene were used to amplify the V.
cholerae secretory signal+CTB+SEKDEL-coding sequence (SEQ ID NO:
15). The resulting PCR product was sub-cloned into pIHC11599 using
NcoI and SacI endonuclease restriction sites to generate pNM47. The
in-frame DNA sequences were then confirmed at the University of
Louisville Microarray Core Facility. For expression of further
cholera toxin B subunit variant polypeptides including secretory
signals other than the original V. cholerae peptide, as described
further below, the 5' provectors pICH20155, pICH20188, pICH20388,
and pICH20999 containing the secretory signal peptides of rice
alpha-amylase (GENBANK.RTM. accession no. P27932; SEQ ID NO: 18),
N. plumbagenifolia calreticulin (GENBANK.RTM. accession no. Z71395;
SEQ ID NO: 20), apple pectinase (GENBANK.RTM. accession no. P48978;
SEQ ID NO: 22), barley alpha-amylase (GENBANK.RTM. accession no.
CAX51374; SEQ ID NO: 24), respectively were used.
[0132] Once the vectors were assembled, plant expression of the
cholera toxin B subunit variants was then performed using the
magnICON.RTM. system. For expression of the cholera toxin B subunit
variant polypeptide with the V. cholerae secretory signal (SEQ ID
NO: 16), the three component plasmids, pNM47, pICH20111, and
pICH14011, were used. For the cholera toxin B subunit variant
polypeptides with the other secretory signals, the appropriate 5'
provector (see above) was used in combination with pNM134 and
pICH14011. The vectors were delivered via Agrobacterium tumefaciens
into N. benthamiana leaves using vacuum infiltration methods.
Briefly, midlogarithmic cultures of A. tumefaciens GV3101 harboring
the plasmids were harvested, and bacteria were resuspended to
OD.sub.600 of 0.03 in 10 mM 2-(N-morpholino)ethanesulfonic acid
(IVIES) buffer containing 10 mM MgSO.sub.4, pH 5.5. The suspension
was vacuum infiltrated into N. benthamiana leaves by using a vacuum
pump.
[0133] After 4-7 days, leaf proteins were subsequently analyzed for
cholera toxin B subunit variant polypeptide expression. Leaf
material was homogenized by a Waring blender in extraction buffer
(20 mM Tris-Cl, pH 5.0, 500 mM NaCl, 20 mM Ascorbic Acid, 10 mM
Sodium Metabisulfate) and the extracts were filtered through four
layers of cheese cloth followed by a single layer of Miracloth. The
extracts were then heated at 50.degree. C. for 25 minutes to
precipitate plant endogenous proteins and starch and centrifuged at
22,100.times.g at 4.degree. C. for 15 minutes followed by
filtration through a 0.22 .mu.m filter. The clarified extracts were
then analyzed for cholera toxin B subunit variant polypeptide
expression by SDS-PAGE and GM1-ganglioside-capture enzyme-linked
immunosorbent assay (GM1-ELISA), as described previously [3,4]
(FIGS. 1A-1B, respectively). Briefly, to perform the SDS-PAGE
analysis, an aliquot of 5 .mu.L of 4.times. Loading Dye (40% v/v
glycerol, 8% w/v SDS. 4% v/v (3-mercaptoethanol, 0.08% w/v
bromophenol blue) was added to 20 .mu.L of the clarified leaf
extracts and heated at 95.degree. C. for 10 minutes. Samples were
resolved using Lonza 15% Tris Glycine gels (Catalog No. 58510) in
Bio-Rad gel boxes with lx SDS running buffer (25 mM Tris, 192 mM
glycine, 0.1% SDS, pH 8.3), and the gels were subsequently stained
with Commassie Blue stain for 20 minutes at room temperature and
then destained overnight at room temperature in Commassie
destaining solution.
[0134] To perform the GM1-ELISA experiments, plates were coated
with 50 .mu.L/well of 2 .mu.g/mL monosialoganglioside-GM1
(Sigma-Aldrich # G-7641) in coating buffer (15 mM Na.sub.2CO.sub.3,
35 mM NaHCO.sub.3, pH 9.6) by incubating at 37.degree. C. for 1
hour. Plates were blocked with 150 .mu.L per well of 5% PBSTM
(Phosphate Buffered Saline, pH 7.4, 0.05% [v/v] Tween 20, 5%
Non-fat dry milk) for 1 hour at room temperature. Fifty .mu.L per
well of the clarified leaf extracts or a CTB standard (100 ng/mL to
1.56 ng/ml; Sigma-Aldrich, # C9903), serially diluted 2-fold in 1%
PBSTM, were incubated for 1 hour at 37.degree. C. The plate-bound
CTB was detected by a goat anti-CTB antiserum (List Biological
Laboratories #703) diluted at 1:5,000 in 1% PBSTM with a rabbit
anti-goat IgG peroxidase-conjugated secondary antibody (Sigma,
Catalog No. A5420) diluted 1:10,000 in 1% PBSTM and a
chemiluminescence substrate (TMB Super Sensitive HRP Substrate,
BioFX Laboratories # TMB S-1000-01). Plates were incubated with the
primary and secondary antibodies for 1 hour at 37.degree. C. The
reaction was stopped with stop solution (0.6 N H.sub.250.sub.4, 1N
HCl). Absorbance values at 450 nm were read on a Beckman Coulter
DTX880 Multimode Detector. A standard curve was also generated,
which was used to estimate the cholera toxin B subunit variant
polypeptide concentrations in the clarified extracts.
[0135] To accomplish the foregoing experiments, a non-viral vector
was initially constructed for the stable expression of the cholera
toxin B subunit variant polypeptides in transgenic plants, where
the secretory signal used in the construct was the native secretory
signal derived from V. cholerae. In spite of the attachment of the
ER retention signal at the C-terminus of that cholera toxin B
subunit variant polypeptide (SEQ ID NOS: 15 and 16), however, the
cholera toxin B subunit variant polypeptide expression level was
relatively low in those experiments, with only approximately 0.5 mg
of the protein being obtained per kg of leaf material (FIG. 1A, Tg
lane), as determined by GM1-ELISA. As such, to investigate whether
transient expression based on a viral vector could provide higher
expression of the same cholera toxin B subunit variant polypeptide
(SEQ ID NOS: 15 and 16), the magnICON.RTM. vector was used due to
its alleged ability to produce high-level protein expression in N.
benthamiana [5]. However, the magnICON.RTM. vector also exhibited a
low level of cholera toxin B subunit polypeptide expression, i.e.,
0.2 mg/kg (FIG. 1A, lane 1; FIG. 1B, Original). In this regard, and
without wishing to be bound by any particular theory, it was next
hypothesized that the native V. cholerae-derived secretory signal
may not function well in a plant cell, and that changing the signal
could lead to an increase in cholera toxin B subunit variant
polypeptide accumulation. As such, the V. cholerae secretory signal
(corresponding to amino acids 1 to 21 of GENBANK.RTM. Accession no.
AY475128) was replaced with the various other secretory signals of
plant origin mentioned herein above and the additional cholera
toxin B subunit variant polypeptides were analyzed using SDS-PAGE,
these additional cholera toxin B subunit variant polypeptides
included cholera toxin B subunit variant polypeptides having the
secretory signal peptides of rice .alpha.-amylase (SEQ ID NO: 26,
lane 2), N. plumbagenifolia calreticulin (SEQ ID NO: 27, lane 3),
apple pectinase (SEQ ID NO: 28, lane 4), and barley .alpha.-amylase
(SEQ ID NO: 29, lane 5). GM1-ELISA was also employed to screen
different cholera toxin B subunit variant polypeptides having the
various secretory signals for polypeptide expression in leaf
extracts (FIG. 1B). Upon analysis of the results from these
experiments, it was found that a secretory signal derived from rice
.alpha.-amylase (SEQ ID NO: 18) provided the highest expression of
cholera toxin B subunit variant polypeptides at levels up to 3
g/kg. Therefore, to perform the vaccine and immunological studies
described further below, a cholera toxin B subunit variant
polypeptide containing the rice .alpha.-amylase and the SEKDEL
signal (SEQ ID NOS: 26) was produced.
[0136] One unique feature of the plant-produced cholera toxin B
subunit variant polypeptides is the presence of an Asn (N)-linked
glycan (NLG) at position 4 (SEQ ID NO: 6). The SDS-PAGE analysis
shown in FIG. 1A, however, revealed that the glycosylation was not
a uniform event, and, in particular, revealed that there was a
heterogeneous population with 1 or 0 glycan per cholera toxin B
subunit, represented by a band at around 14.5 kDa and 12.5 kDa,
respectively. To eliminate the NLG and produce an aglycosylated
cholera toxin B subunit variant polypeptides (SEQ ID NO: 4),
site-directed mutagenesis was then preformed to mutate the AAC
codon (Asn4) to AGC (Ser) as it was though that such a mutation
would not affect CTB's structure or function (see, e.g., the E.
coli heat-liable enterotoxin B subunit having a Ser amino acid at a
corresponding position; GENBANK.RTM. Accession No. AAC60441).
Example 2
Expression of Aglycosylated Cholera Toxin B Subunit Variant in
Nicotiana benthamiana
[0137] To characterize the aglycosylated cholera toxin B subunit
variant polypeptide (SEQ ID NO: 4), an SDS-PAGE analysis of the
Asn4.fwdarw.Ser cholera toxin B subunit variant expressed in N.
benthamiana was conducted at 0, 4, 5, 6 and 7 days post
magnICON.RTM. vector inoculation (dpi). Briefly, an aliquot of 10
.mu.L of 2.times. native sample buffer (Bio-Rad No. 161-0738) was
added to 20 .mu.L of clarified leaf extracts. Samples were resolved
using Lonza 15% Tris Glycine gels (Catalog No. 58510) in Bio-Rad
gel boxes with 1.times.SDS running buffer (25 mM Tris, 192 mM
glycine, 0.1% SDS, pH 8.3). The gels were stained with Commassie
Blue stain for 20 minutes at room temperature and destained
overnight at room temperature in Commassie destaining solution.
[0138] Upon analyzing the SDS-PAGE results, it was found that the
Asn4.fwdarw.Ser mutation successfully eliminated the
N-glycosylation, as shown by a single band at around 12 kDa under
denaturing conditions, while retaining a pentameric form that is
necessary for GM1-ganglioside binding activity, as shown by the
single band at around 60 kDa on a non-reducing SDS-PAGE gel (FIG.
2). The expression of the aglycosylated cholera toxin B subunit
variant polypeptide appeared to peak at 5 dpi, after which plants
became very necrotic and expression levels began to decrease.
GM1-ELISA showed that the aglycosylated cholera toxin B subunit
variant polypeptide was expressed at 0.5-1.5 g/kg of leaf material,
which was among the highest for the plant-based recombinant protein
expression [5], even though it was not as high as its
N-glycosylated counterpart.
Example 3
Purification of Cholera Toxin B Subunit Variants
[0139] To purify the various cholera toxin B subunit variants
produced by the foregoing methods, an immobilized metal affinity
chromatography (IMAC) procedure was used as depicted in FIG. 3.
Briefly, clarified leaf extracts were adjusted to pH 8 with a Tris,
pH 9.0 buffer followed by filtration with a 0.22 .mu.m filter.
Chromatography was performed using an AKTA Purifier (GE
Healthcare). Talon Superflow Metal Affinity Resin (Clontech No.
635670), packed in an XK-26 column (GE Healthcare), was
equilibrated with 10 column volume (CV) of Talon equilibration
buffer (20 mM Tris-Cl, pH 8.0, 500 mM NaCl). Samples were loaded at
2.5 ml/min. The column was washed with 8 CV of Talon equilibration
buffer. The cholera toxin B subunit variant polypeptides were
eluted with a step gradient using 100% Talon elution buffer (20 mM
Tris-Cl, pH 8.0, 500 mM NaCl, 150 mM Imidazole) and collected by
monitoring absorbance at 280 nm. SDS-PAGE was employed to verify
the collected fraction for the presence of the cholera toxin B
subunit variant polypeptides. The variant polypeptide fraction was
further purified using a Bio-Rad CHT Hydroxyapatite Fast Flow 5 mL
pre-packed column (Catalog No. 732-4324). The column was
equilibrated with 10 CV of CHT equilibration buffer (10 mM Tris-Cl,
pH 8.0, 5 mM sodium phosphate). The sample was loaded at a flow
rate of 2.5 ml/min followed by a 10 CV wash with CHT equilibration
buffer. The proteins were eluted using a gradient from 0 to 100%
CHT elution buffer (10 mM Tris-Cl, pH 8.0, 500 mM sodium phosphate)
over 20 CV. Five mL fractions were collected. The cholera toxin B
subunit variant polypeptide-containing fractions, after verified by
SDS-PAGE, were combined and endotoxin was removed using a Triton
X-114 phase separation method. [5] After endotoxin removal, the
cholera toxin B subunit variant polypeptides were ultrafiltrated
and diafiltrated into sterile Dulbecco's PBS (Gibco No. 14190)
using Amicon Ultra-15 3000 MWCO centrifugal devices (Millipore No.
UFC900324) according to the manufacturer's instructions. Endotoxin
levels were checked with a Charles River PTS Endotoxin test system,
using 10-0.1 EU/mL cartridges (Charles River No. PTS201). Purity
was determined to be greater than 95% via overloaded SDS-PAGE and
size-exclusion HPLC.
[0140] In certain of the purification procedures, the purification
procedure described above was shortened by eliminating the second
CHT chromatography step and endotoxin removal, as it was found that
the cholera toxin B subunit variant polypeptide purity was greater
than 98% after the first Talon affinity step.
Example 4
Comparison of Production of Native, Aglycosylated-Plant and
N-Glycosylated-Plant Cholera Toxin B Subunit Variants
[0141] To further evaluate the cholera toxin B subunit variant
polypeptides produced above, experiments were performed to assess
whether the Nicotiana-produced cholera toxin B subunit variant
polypeptides were comparable to their native bacterial
counterparts. Briefly, to perform these experiments, native cholera
toxin B subunit polypeptides were first produced in E. coli as
described previously [3], and the SDS-PAGE analysis was conducted
using the E. coli-produced cholera toxin B subunit polypeptide and
certain of the plant-made cholera toxin B subunit variant
polypeptides produced above. As shown in FIG. 4A, the denaturing
SDS-PAGE (left) showed each polypeptide in a monomeric form, with
the native (lane 1) and the plant-made aglycosylated cholera toxin
B subunit variant polypeptides (lane 2) both having a single band,
whereas the plant-made N-glycosylated cholera toxin B subunit
variant polypeptides (lane 3) showed two bands corresponding to
approximately 78% N-glycosylated and 22% aglycosylated forms (based
on a densitometric analysis). The non-denaturing SDS-PAGE (FIG. 4A,
right) showed that native, plant-made aglycosylated, and plant-made
N-glycosylated cholera toxin B subunits all retained pentamer
formation. For both denaturing and non-denaturing SDS-PAGE
analysis, 2 .mu.g of purified proteins were loaded on the gels.
[0142] To then test whether the Nicotiana-produced N-glycosylated
and aglycosylated cholera toxin B subunit variant polypeptides
retained affinity for GM1-ganglioside, a competitive GM1-ELISA was
performed. Briefly, to perform the competitive GM1 ELISA, a 96-well
plate was coated and blocked as described above. In 1.5 mL tubes,
serial dilutions of each cholera toxin B subunit variant
polypeptides sample (SEQ ID NOS: 4 and 26) were then prepared in 1%
PBSTM (1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 2.0, 0.2
and 0 nM). To each cholera toxin B subunit variant polypeptide at
each concentration, an equal volume of 2 .mu.g/mL HRP-CTB
(Molecular Probes # C34780) was then added and mixed. Then, 100
.mu.L/well of each mixture (triplicates for each dilution) was
added to the plate and incubated for 1 hour at 37.degree. C. The
plate-bound HRP-CTB was detected by adding a chemiluminescence
substrate (TMB Super Sensitive HRP Substrate, BioFX Laboratories #
TMB S-1000-01). The reaction was then stopped with stop solution
(0.6 N H.sub.2SO.sub.4, 1 N HCl) and absorbance values at 450 nm
were read on a Beckman Coulter DTX880 Multimode Detector. Fifty
percent inhibitory concentrations (IC50) were determined by the
GraphPad Prism 5.0 software.
[0143] As shown in FIG. 4B, it was observed that there was no
significant difference between their apparent affinities to the
receptor; the native, plant-made aglycosylated and plant-made
N-glycosylated cholera toxin B subunits each showed 50% inhibitory
concentrations (IC50) of 8.97 nM, 5.65 nM, and 11.52 nM,
respectively. The result demonstrated that the modifications made
on plant-produced cholera toxin B subunit variant polypeptides,
i.e., glycosylation, Asn4 Ser mutation, change of the secretory
signal, and attachment of a C-terminal ER retention signal, did not
affect the molecular binding properties of the cholera toxin B
subunits.
Example 5
Antibody Responses to Cholera Toxin B Subunit Variants
[0144] To evaluate antibody responses to the cholera toxin B
subunit variants of the presently-disclosed subject matter, a
further GM1-ELISA was conducted using the procedures described
above, where the concentration of native cholera toxin B subunits
and plant-made, N-glycosylated cholera toxin B subunit variant
polypeptides was determined using theoretical extinction
coefficients at 280 nm of 0.8181 (mg/mL).sup.-1 cm.sup.-1 and
0.7660 (mg/mL).sup.-1 cm.sup.-1, respectively. As shown in FIG. 5,
this additional GM1-ELISA showed that the goat polyclonal anti-CTB
antisera bound less to plant-derived N-glycosylated cholera toxin B
subunit variant polypeptide (SEQ ID NO: 6) than native cholera
toxin B subunit produced in E. coli (as indicated by the arrow on
the graph). The result indicated that the NLG prevented the access
of some anti-CTB antibodies to their epitopes. Based on these
results, it was thus believed that immunization with N-glycosylated
cholera toxin B subunit variant polypeptide can direct an antibody
response toward the more accessible GM1-binding site, which would
in turn provide better neutralization of cholera toxin, i.e.,
higher vaccine efficacy against cholera.
Example 6
Biochemical Characterization of Native, Aglycosylated-Plant, and
N-Glycosylated-Plant Cholera Toxin B Subunit Variants
[0145] To further evaluate the native cholera toxin B subunit (SEQ
ID NO: 2), plant-made aglycosylated cholera toxin B subunit variant
polypeptides (SEQ ID NO: 4), and plant-made N-glycosylated cholera
toxin B subunit variant polypeptides (SEQ ID NO: 6), biochemical
characterizations of the polypeptides were conducted using
size-exclusion chromatography-high performance liquid
chromatography (SEC-HPLC) and a thermal shift assay. Briefly, the
chromatography was performed on a Beckman Coulter System Gold HPLC.
An aliquot of 17 .mu.L of the native, plant-made aglycosylated, and
plant-made N-glycosylated cholera toxin B subunits at 1 mg/ml were
applied, at 1.0 mL/min, to an SEC column (YMC-Pack Diol-200,
500.times.8.0 mm I.D., S--5 .mu.m, 20 nm) equilibrated with 100 mM
sodium phosphate, pH 7.0, 200 mM NaCl. After injection, 100 mM
sodium phosphate, pH 7.0, 200 mM NaCl was applied to the column at
flow rate of 1.0 mL/min for 35 minutes. Before and after
polypeptide analysis, an aliquot of 17 .mu.L of gel filtration
standards (Bio-Rad No. 151-1901) were applied to the column to
confirm integrity of the SEC results. The gel filtration standards
are a mixture of five proteins with molecular sizes of 660 kDa, 140
kDa, 45 kDa, 18 kDa, and 1.3 kDa. Polypeptide elution was monitored
by absorbance at 280 nm.
[0146] For the thermal shift assay, the melting temperature of
cholera toxin B subunit polypeptides were determined by using a
fluorescence-based thermal shift assay performed on a Bio-Rad iQ5
multicolor real-time PCR system. Each polypeptide, at a final
concentration of 60.0 .mu.M in PBS, was mixed with a final
concentration of 50.times.Sypro orange (Molecular Probes No.
5-6650) in a total volume of 20 .mu.L in a 96 well plate (USA
Scientific No. 1402-9200). Blank controls were set up for protein
alone, and samples and blanks were analyzed in triplicate. The
plate was set to be heated from 20.degree. C. to 95.degree. C. in
0.2.degree. C. increments at interval of 15 seconds. Data were then
plotted using the GraphPad Prism 5 software.
[0147] The biochemical characterizations of the native cholera
toxin B subunit polypeptide, plant-made aglycosylated cholera toxin
B subunit variant polypeptides, and plant-made N-glycosylated
cholera toxin B subunit variant polypeptides demonstrated that all
of the polypeptides have similar purity and thermal stabilities. As
shown in FIG. 6A, the fluorescence-based thermal shift assay, which
was utilized to determine the melting temperatures of the
polypeptide variants, revealed that the native cholera toxin B
subunit polypeptides, plant-made aglycosylated cholera toxin B
subunit variant polypeptides, and plant-made N-glycosylated cholera
toxin B subunit variant polypeptides had melting temperatures of
75.degree. C., 72.2.degree. C., and 72.0.degree. C., respectively,
indicating that the modifications introduced into the sequence to
generate the plant-made polypeptide variants did not compromise the
thermal stability of the proteins.
[0148] As shown in FIG. 6B, the SEC-HPLC was used to determine the
purity of the polypeptides, and revealed that all of the
polypeptides showed one large peak with greater than 95% of the
peak area and a small peak at a shorter retention time. The elution
time of the large peak roughly corresponded to the size of a
pentamer form (50-60 kDa), such that the purity of the pentameric
form was estimated to be greater than 95% for all of the
polypeptides.
Example 7
Oral Immunization with Bacterial-Produced Cholera Toxin B Subunit
Variants and with Plant-Made Cholera Toxin B Subunit Variants
[0149] To further assess the cholera toxin B subunit variant
polypeptides of the presently-disclosed subject matter, an oral
immunization experiment was performed in a mouse model to determine
if there was a difference in an antibody response between the
native (SEQ ID NO: 2) and plant-made aglycosylated polypeptides
(SEQ ID NO: 4). C57b1/6 mice (four mice per group) were immunized
twice by gavage with 30 or 300 .mu.g of native cholera toxin B
subunit polypeptide (produced in E. coli, referred to as "eCTB") or
plant-made aglycosylated cholera toxin B subunit variant
polypeptide ("pCTB") at Week 0 and 2. A control group was immunized
with PBS vehicle. One week after the second immunization, serum and
fecal pellets were collected, the endpoint titers of serum anti-CTB
immunoglobulin (Ig)G and intestinal anti-CTB IgA were determined by
ELISA, as described previously. [3,6]. Endpoint titers were defined
as the reciprocal of sample dilutions giving a positive OD value
after subtracting background in ELISA. As shown in FIGS. 7A and 7B,
both eCTB and pCTB induced equivalent levels of serum anti-CTB IgG
and intestinal anti-CTB IgA that was sustained for greater than 3.5
months (FIG. 8), indicating that the plant-produced aglycosylated
cholera toxin B subunit polypeptide variants are immunologically
equivalent to their native counterparts, and further indicating
that the plant-produced aglycosylated variants can serve as a
viable alternative to their bacterially-produced counterparts that
are currently used in the oral cholera vaccine Dukoral.
Example 8
Cholera Toxin B Subunit Variants Having Increased
N-Glycosylation
[0150] As described above, the initially produced cholera toxin B
subunit variant polypeptides were N-glycosylated when expressed in
plant cells, with one NLG being attached to Asn4 of the amino acid
sequence to thereby comprise up to 5 NLGs per cholera toxin
molecule given that cholera toxin B subunit is a homo-pentameric
protein. Due to the C-terminal ER retention signal on the
plant-expressed cholera toxin B subunits, the CTB-attached NLGs
consist mainly of oligomannose sugars that are commonly known as
H-Man glycans. [7] H-Man glycans are often abundantly displayed on
the envelope glycoproteins of many enveloped viruses such as HIV,
hepatitis C, Ebola, and influenza viruses. [8] However, it has been
observed that such glycans are rare on host glycoproteins,
particular in humans, as the human immune system makes use of
mechanisms to sense and capture mannose-rich substances via the
mannose-binding lectin (MBL) and C-type lectin receptors (CLR) on
antigen presenting cells, such as the mannose receptor and
Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing
Non-integrin (DC-SIGN). In this regard, it was thought that the
"mannosylation" of immunogens may be one strategy to enhance the
efficacy of vaccines, and it was hypothesized that N-glycosylated
cholera toxin B subunit variant polypeptides could potentially be:
(1) developed as a vaccine that would induce H-Man glycan-specific
antibodies (Abs) exhibiting broad antiviral activity against
enveloped viruses; and (2) developed as a vaccine scaffold to carry
various antigens and efficiently stimulate mucosal and systemic
immune systems. Additionally, from the results shown in FIG. 5 and
as described above, it was believed that the N-glycosylated cholera
toxin B subunit variant polypeptides would exhibit higher vaccine
efficacy against cholera.
[0151] To assess the ability of N-glycosylated cholera toxin B
subunit variant polypeptides to efficiently induce H-Man
glycan-specific Abs and/or to assess whether the cholera toxin B
subunit variant polypeptides can effectively be recognized by the
immune system via CLR, experiments were undertaken to attempt to
increase the number of NLGs on plant-produced cholera toxin B
subunit variant polypeptides. In eukaryotic cells including plants,
N-glycosylation of a newly synthesized polypeptide requires a
tripeptide sequence composed of Asn-X-Ser/Thr ("sequon"; X is any
amino acid but Pro) in its primary structure. In this regard, the
amino acid sequence of the cholera toxin B subunit variant
polypeptides was modified by means of site directed mutagenesis to
have more than one sequon, besides the existing one at Asn4, and,
more specifically, cholera toxin B subunit variant polypeptides
were designed that had two and three sequons. For a two-sequon
variant, the C-terminal extension peptide containing the ER
retention signal was modified to Val-Thr-Lys-Asp-Glu-Leu
(originally Ser-Glu-Lys-Asp-Glu-Leu) to create a sequon at
Asn103-Val-Thr (SEQ ID NOS: 8 and 10). For a three-sequon variant,
Lys23 was mutated to Thr to create a new sequon at
Asn21-Asp22-Thr23 (SEQ ID NOS: 12 and 14). Without wishing to be
bound by any particular theory, it was believed that the attachment
of NLGs to those particular locations would not interfere with GM1
receptor-binding of CTB.
[0152] Once the additional cholera toxin B subunit variant
polypeptides were designed and expressed using the magnICON.RTM.
system described above, SDS-PAGE and lectin blot analysis using
concanavalin A (ConA) were then performed to prove that CTB-VTKDEL
(i.e., the construct containing 2 glycosylation sites) was
successfully N-glycosylated upon expression in Nicotiana
benthamiana. To perform these further analyses, the SDS-PAGE
analysis was performed as described above. The lectin blot was
performed by first using an SDS-PAGE gel to resolve mono- and
di-N-glycosylated cholera toxin B subunit variant polypeptides
under denaturing conditions, as described above. The cholera toxin
B subunit variant polypeptides were then transferred to a PVDF
membrane (Millipore No. IPSN07852) for 1 hour at 100 volts. The
membrane was subsequently blocked by PBS containing 2% (v/v) TWEEN
20 for 2 minutes at room temperature and incubated with 2 .mu.g/mL
of ConA-HRP (Sigma No. L6397) in BS containing 0.05% (v/v) TWEEN 20
with 1 mM CaCl.sub.2, 1 mM MnCl.sub.2, 1 mM MgCl.sub.2 for 16 hours
at room temperature. The membrane-bound ConA was then detected
using the Amersham ECL Western Blotting Analysis System (GE
Healthcare No. RPN2108) according to the manufacturer's
instructions.
[0153] Upon analysis of the results from this experiment, it was
observed that both mono- and di-N-glycosylated cholera toxin B
subunit variant polypeptides retained pentamer formation as shown
by a band at approximately 60 kDa on the non-denaturing SDS-PAGE
(FIG. 9, Lanes 1 and 2, respectively). The denaturing SDS-PAGE
further demonstrated that the mono-N-glycosylated cholera toxin B
subunit variant polypeptides had two different forms of the monomer
subunit, with 1 and 0 NLG (FIG. 9, Lane 3). Densitometry analysis
showed that the ratio of 1 and 0 NLG forms was 7.8: 2.2. The
di-N-glycosylated cholera toxin B subunit variant polypeptides had
three different forms of the monomer subunit with 2, 1, and 0 NLGs
(FIG. 9, Lane 3). Densitometry analysis revealed that the ratio of
2, 1, and 0 N-glycosylated forms was 3.9: 4.3: 1.7. The lectin blot
using ConA, a lectin having high affinity for Man residues of NLGs,
showed that both 1- and 2-NLGs-attached cholera toxin B subunit
variant polypeptides were recognized by ConA (FIG. 9, Lanes 5 and
6), indicating that both the mono- and di-N-glycosylated cholera
toxin B subunit variant polypeptides contained Man residues.
Similar experiments were also performed with the Nicotiana-produced
[Thr.sup.23]-CTB-VTKDEL (tri-N-glycosylated cholera toxin B subunit
variant polypeptide). The denaturing SDS-PAGE (FIG. 10, Lanes 1 and
2) showed that four different forms (with 3, 2, 1, and 0 NLGs) of
the monomer subunits were expressed, providing evidence that the
Lys23.fwdarw.Thr mutation resulted in a successful addition of a
third NLG. As shown by the non-denaturing SDS-PAGE (FIG. 10, Lane
3), tri-N-glycosylated cholera toxin B subunit variant polypeptides
were purified to greater than 98% purity and retained pentamer
formation (band at 60-70 kDa). Furthermore, GM1-ELISA has confirmed
that tri-N-glycosylated cholera toxin B subunit variant
polypeptides bind to GM1-ganglioside.
Example 9
Analysis of N-Glycosylation of Cholera Toxin B Subunit Variants
[0154] To further analyze the N-gycosylation of the cholera toxin B
subunit variant polypeptides of the presently-disclosed subject
matter, Peptide: N-Glycosidase F (PNGase F), an amidase that
cleaves between the innermost N-acetylglucosamine and Asn residues
of H-Man, hybrid, and complex NLGs (except for those with
.alpha.-1,3-linked core fucose found in plants) from glycoproteins
was used along with endoglycosidase H (Endo H), a glycosidase that
cleaves within the chitobiose core of H-Man Glycans and some hybrid
oligosaccharides from N-glycosylated glycoproteins. [7] Briefly,
the N-glycosylated cholera toxin B subunit variant polypeptides
(SEQ ID NO: 26) were first expressed in N. benthamiana grown with
and without the chemical inhibitor of class 1 a mannosidases,
kifunensine. For expression of mono-N-glycosylated cholera toxin B
subunit variant polypeptides with kifunensine, the plants were
vector-inoculated as outlined above, except that roots were removed
from the soil and placed in water containing 580 ng/ml kifunensine
(Cayman Chemical Company No. 10009437) after vector inoculation.
The plants were re-treated with freshly prepared kifunensine at 2
and 4 dpi and harvested at 5 dpi.
[0155] For Endo H Digestion, Endo H and buffers were purchased from
New England BioLabs (Catalog No. P0703 S), and two .mu.g of cholera
toxin B subunit variant polypeptides, 1 of 10.times. Glycoprotein
Denaturing Buffer and H.sub.2O were mixed to make a 10 .mu.l total
reaction volume. N-glycosylated cholera toxin B subunit variant
polypeptide was denatured by heating at 100.degree. C. for 10
minutes. The total reaction volume was adjusted to 20 .mu.L by
adding 2 .mu.L of 10.times.G5 Reaction Buffer, 4 .mu.L of Endo H
and H.sub.2O. The reaction was performed at 37.degree. C.
overnight.
[0156] For PNGase F Digestion, PNGase F and buffers were purchased
from New England BioLabs (Catalog No. P0704S). Two .mu.g of cholera
toxin B subunit variant polypeptides, 1 .mu.L of 10.times.
Glycoprotein Denaturing Buffer and H.sub.2O were mixed to make a 10
.mu.L total reaction volume. N-glycosylated cholera toxin B subunit
variant polypeptide was denatured by heating at 100.degree. C. for
10 minutes. The total reaction volume was adjusted to 20 .mu.L by
adding 2 .mu.L 10.times. G7 Reaction Buffer, 2 .mu.L 10% NP40, 2
.mu.L PNGase F and H.sub.2O. The reaction was performed at
37.degree. C. overnight.
[0157] After digestions, SDS-PAGE and western blot analysis, using
a goat anti-CTB antiserum as described above, showed that
mono-N-glycosylated cholera toxin B subunit variant polypeptides
produced in N. benthamiana was only partially cleaved by PNGase F
and Endo H (FIG. 11, Lanes 4-6), indicating that NLGs attached to
the cholera toxin B subunit variant polypeptides were not uniform
and contain fewer H-Man glycans than expected. To obtain more
uniform H-Man glycan-displaying cholera toxin B subunit variant
polypeptides, mono-N-glycosylated cholera toxin B subunit variant
polypeptides were expressed in N. benthamiana treated with
kifunensine. SDS-PAGE and western blot showed that NLGs of
mono-N-glycosylated cholera toxin B subunit variant polypeptides
produced in the presence of the kifunensine were completely cleaved
by PNGase F and Endo H, which indicated that NLGs attached to
cholera toxin B subunit variant polypeptides can be restricted to
uniform HMGs by inhibiting the .alpha.-mannosidases of the
host.
Example 10
Recognition of Cholera Toxin B Subunit Variants by DC-SIGN
Receptor
[0158] To evaluate whether the cholera toxin B subunit variants of
the presently-disclosed subject matter were capable of being
recognized the DC-SIGN receptor, the mono- and tri-N-glycosylated
cholera toxin B subunit variant polypeptides produced above were
tested for DC-SIGN binding using ELISA. Briefly, a 96-well plate
was coated with monosialoganglioside-GM1 as described above. Plates
were then blocked with 150 .mu.L per well with 5% BSA in TCN buffer
(10 mM Tris, 50 mM CaCl.sub.2, 150 mM NaCl pH 7.4), overnight at
4.degree. C. Fifty .mu.L/well of 2-fold serially diluted cholera
toxin B subunit variant polypeptides samples (starting from 10
.mu.g/mL) in 1% BSA in TCN were incubated for 1 hour at 37.degree.
C. Next, 50 .mu.L/well of recombinant DC-SIGN (rhDC-SIGN/Fc
chimera, R&D Systems No. 161-DC) diluted at 0.5 .mu.g/mL in 1%
BSA in TCN was added and incubated for 2 hours at room temperature.
Bound DC-SIGN was detected by mouse anti-human IgG
(Fc-specific)-HRP conjugate (diluted at 1:10,000 in 1% BSA in TCN;
Southern Biotech No. 9040-05) and chemiluminescence (TMB Super
Sensitive HRP Substrate, BioFX Laboratories No. TMBS-1000-01). The
reaction was stopped with stop solution (0.6N H.sub.2SO.sub.4, 1N
HCL) and absorbance values at 450 nm were read on a Beckman Coulter
DTX880 Multimode Detector. Data were analyzed using GraphPad Prism
5.
[0159] Upon analysis of the results from this experiment, it was
found that the N-glycosylated cholera toxin B subunit variant
polypeptides, but not the aglycosylated counterpart, were
recognized by DC-SIGN. The CLR bound tri-N-glycosylated cholera
toxin B subunit variant polypeptides better than the
mono-N-glycosylated protein, indicating that additional NLGs
attached to cholera toxin B subunit variant polypeptides enhanced
DC-SIGN recognition. This in turn indicated that N-glycosylated
cholera toxin B subunit variant polypeptides can be used as a
scaffold to target antigens to DCs. Furthermore, as shown in FIG.
12, it was found that mono-N-glycosylated cholera toxin B subunit
variant polypeptides (SEQ ID NO: 6) produced in kifunensine-treated
plants (i.e., displaying only H-Man glycans) was significantly more
captured by DC-SIGN than the protein produced under the normal
conditions.
Example 11
Mutagenesis of Cholera Toxin B Subunit Variants for Binding of
Homogenous Population N-linked Glycans
[0160] As described above and shown in FIG. 13 (Lanes 1 and 4),
Nicotiana-produced mono-N-glycosylated cholera toxin B subunit
variant polypeptides displayed a heterogeneous population of NLGs
with 1 and 0 glycan per monomer. In conducting these experiments,
however, it was also found that Ser26.fwdarw.Cys and
Ala102.fwdarw.Cys mutations of the cholera toxin B subunit (SEQ ID
NO: 25), which were produced via site-directed mutagenesis and were
originally intended for inter-subunit disulfide bond formation,
resulted in a homogeneous population of N-glycosylated cholera
toxin B subunit monomer with one NLG (FIG. 13, Lanes 3 and 6) that
is desirable for the use of N-glycosylated cholera toxin B subunit
variant polypeptides.
[0161] Throughout this document, various references are mentioned,
including publications, patents, and patent applications. All such
references, including the references set forth in the following
list, are incorporated herein by reference to the same extent as if
each individual publication, patent, or patent application was
specifically and individually indicated to be incorporated by
reference.
REFERENCES
[0162] 1. Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V,
Gleba Y. In planta engineering of viral RNA replicons: efficient
assembly by recombination of DNA modules delivered by
Agrobacterium. Proc Natl Acad Sci U S A. 2004; 101(18): 6852-7.
[0163] 2. Gleba Y, Klimyuk V, Marillonnet S. Magnifection--a new
platform for expressing recombinant vaccines in plants. Vaccine.
2005; 23(17-18): 2042-8. [0164] Matoba N, Magerus A, Geyer B C,
Zhang Y, Muralidharan M, Alfsen A, et al. A mucosally targeted
subunit vaccine candidate eliciting HIV-1 transcytosis-blocking
Abs. Proc Natl Acad Sci U S A. 2004; 101(37): 13584-9. [0165] 4.
Matoba N, Griffin T A, Mittman M, Doran J D, Hanson C V, Montefiori
D, et al. Transcytosis-blocking Abs elicited by an oligomeric
immunogen based on the membrane proximal region of HIV-1 gp41
target non-neutralizing epitopes. Curr HIV Res. 2008; 6(3): 218-29.
[0166] 5. Matoba N, Davis K R, Palmer K E. Recombinant Protein
Expression in Nicotiana. Methods Mol Biol. 2011; 701: 199-219.
[0167] 6. Matoba N, Geyer B C, Kilbourne J, Alfsen A, Bomsel M, Mor
T S. Humoral immune responses by prime-boost heterologous route
immunizations with CTB-MPR(649-684), a mucosal subunit HIV/AIDS
vaccine candidate. Vaccine. 2006; 24(23): 5047-55. Matoba N,
Kajiura H, Cherni I, Doran J D, Bomsel M, Fujiyama K, et al.
Biochemical and immunological characterization of the plant-derived
candidate human immunodeficiency virus type 1 mucosal vaccine
CTB-MPR(649-684). Plant Biotechnol J. 2009; 7(2): 129-45. [0168] 8.
Balzarini J. Targeting the glycans of glycoproteins: a novel
paradigm for antiviral therapy. Nat Rev Microbiol. 2007; 5(8):
583-97. [0169] 9. Irache J M, Salman H H, Gamazo C, Espuelas S.
Mannose-targeted systems for the delivery of therapeutics. Expert
Opin Drug Deliv. 2008; 5(6): 703-24. [0170] 10. Keler T,
Ramakrishna V, Fanger M W. Mannose receptor-targeted vaccines.
Expert opinion on biological therapy. 2004; 4(12): 1953-62. [0171]
11. Sheng K C, Kalkanidis M, Pouniotis D S, Esparon S, Tang C K,
Apostolopoulos V, et al. Delivery of antigen using a novel
mannosylated dendrimer potentiates immunogenicity in vitro and in
vivo. Eur J Immunol. 2008; 38(2): 424-36.
[0172] It will be understood that various details of the presently
disclosed subject matter can be changed without departing from the
scope of the subject matter disclosed herein. Furthermore, the
foregoing description is for the purpose of illustration only, and
not for the purpose of limitation.
Sequence CWU 1
1
311309DNAVibrio cholerae 1accccacaaa acatcactga cttgtgtgct
gagtaccaca acacccaaat ccacaccctc 60aatgacaaga tctttagcta caccgagagc
cttgctggca agagggagat ggctatcatc 120accttcaaga atggtgctac
cttccaagtg gaggtgcctg gaagccaaca cattgatagc 180caaaagaagg
ccattgagag gatgaaggac acacttagga tagcttacct cactgaggct
240aaggtggaga agctttgtgt gtggaacaac aagacccccc atgctattgc
tgccatcagc 300atggccaac 3092103PRTVibrio cholerae 2Thr Pro Gln Asn
Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln1 5 10 15Ile His Thr
Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30Gly Lys
Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe 35 40 45Gln
Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55
60Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala65
70 75 80Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala
Ile 85 90 95Ala Ala Ile Ser Met Ala Asn 1003327DNAArtificial
SequenceCholera Toxin B Subunit Polypeptide Variant Polypeptide
3accccacaaa gcatcactga cttgtgtgct gagtaccaca acacccaaat ccacaccctc
60aatgacaaga tctttagcta caccgagagc cttgctggca agagggagat ggctatcatc
120accttcaaga atggtgctac cttccaagtg gaggtgcctg gaagccaaca
cattgatagc 180caaaagaagg ccattgagag gatgaaggac acacttagga
tagcttacct cactgaggct 240aaggtggaga agctttgtgt gtggaacaac
aagacccccc atgctattgc tgccatcagc 300atggccaact ccgagaagga tgaactc
3274109PRTArtificial SequenceCholera Toxin B Subunit Polypeptide
Variant Polypeptide 4Thr Pro Gln Ser Ile Thr Asp Leu Cys Ala Glu
Tyr His Asn Thr Gln1 5 10 15Ile His Thr Leu Asn Asp Lys Ile Phe Ser
Tyr Thr Glu Ser Leu Ala 20 25 30Gly Lys Arg Glu Met Ala Ile Ile Thr
Phe Lys Asn Gly Ala Thr Phe 35 40 45Gln Val Glu Val Pro Gly Ser Gln
His Ile Asp Ser Gln Lys Lys Ala 50 55 60Ile Glu Arg Met Lys Asp Thr
Leu Arg Ile Ala Tyr Leu Thr Glu Ala65 70 75 80Lys Val Glu Lys Leu
Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95Ala Ala Ile Ser
Met Ala Asn Ser Glu Lys Asp Glu Leu 100 1055327DNAArtificial
SequenceCholera Toxin B Subunit Polypeptide Variant Polypeptide
5accccacaaa acatcactga cttgtgtgct gagtaccaca acacccaaat ccacaccctc
60aatgacaaga tctttagcta caccgagagc cttgctggca agagggagat ggctatcatc
120accttcaaga atggtgctac cttccaagtg gaggtgcctg gaagccaaca
cattgatagc 180caaaagaagg ccattgagag gatgaaggac acacttagga
tagcttacct cactgaggct 240aaggtggaga agctttgtgt gtggaacaac
aagacccccc atgctattgc tgccatcagc 300atggccaact ccgagaagga tgaactc
3276109PRTArtificial SequenceCholera Toxin B Subunit Polypeptide
Variant Polypeptide 6Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu
Tyr His Asn Thr Gln1 5 10 15Ile His Thr Leu Asn Asp Lys Ile Phe Ser
Tyr Thr Glu Ser Leu Ala 20 25 30Gly Lys Arg Glu Met Ala Ile Ile Thr
Phe Lys Asn Gly Ala Thr Phe 35 40 45Gln Val Glu Val Pro Gly Ser Gln
His Ile Asp Ser Gln Lys Lys Ala 50 55 60Ile Glu Arg Met Lys Asp Thr
Leu Arg Ile Ala Tyr Leu Thr Glu Ala65 70 75 80Lys Val Glu Lys Leu
Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95Ala Ala Ile Ser
Met Ala Asn Ser Glu Lys Asp Glu Leu 100 1057327DNAArtificial
SequenceCholera Toxin B Subunit Polypeptide Variant Polypeptide
7accccacaaa acatcactga cttgtgtgct gagtaccaca acacccaaat ccacaccctc
60aatgacaaga tctttagcta caccgagagc cttgctggca agagggagat ggctatcatc
120accttcaaga atggtgctac cttccaagtg gaggtgcctg gaagccaaca
cattgatagc 180caaaagaagg ccattgagag gatgaaggac acacttagga
tagcttacct cactgaggct 240aaggtggaga agctttgtgt gtggaacaac
aagacccccc atgctattgc tgccatcagc 300atggccaacg ttactaagga tgaactc
3278109PRTArtificial SequenceCholera Toxin B Subunit Polypeptide
Variant Polypeptide 8Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu
Tyr His Asn Thr Gln1 5 10 15Ile His Thr Leu Asn Asp Lys Ile Phe Ser
Tyr Thr Glu Ser Leu Ala 20 25 30Gly Lys Arg Glu Met Ala Ile Ile Thr
Phe Lys Asn Gly Ala Thr Phe 35 40 45Gln Val Glu Val Pro Gly Ser Gln
His Ile Asp Ser Gln Lys Lys Ala 50 55 60Ile Glu Arg Met Lys Asp Thr
Leu Arg Ile Ala Tyr Leu Thr Glu Ala65 70 75 80Lys Val Glu Lys Leu
Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95Ala Ala Ile Ser
Met Ala Asn Val Thr Lys Asp Glu Leu 100 1059327DNAArtificial
SequenceCholera Toxin B Subunit Polypeptide Variant Polypeptide
9accccacaaa acatcactga cttgtgtgct gagtaccaca acacccaaat ccacaccctc
60aatgacacta tctttagcta caccgagagc cttgctggca agagggagat ggctatcatc
120accttcaaga atggtgctac cttccaagtg gaggtgcctg gaagccaaca
cattgatagc 180caaaagaagg ccattgagag gatgaaggac acacttagga
tagcttacct cactgaggct 240aaggtggaga agctttgtgt gtggaacaac
aagacccccc atgctattgc tgccatcagc 300atggccaact ccgagaagga tgaactc
32710109PRTArtificial SequenceCholera Toxin B Subunit Polypeptide
Variant Polypeptide 10Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu
Tyr His Asn Thr Gln1 5 10 15Ile His Thr Leu Asn Asp Thr Ile Phe Ser
Tyr Thr Glu Ser Leu Ala 20 25 30Gly Lys Arg Glu Met Ala Ile Ile Thr
Phe Lys Asn Gly Ala Thr Phe 35 40 45Gln Val Glu Val Pro Gly Ser Gln
His Ile Asp Ser Gln Lys Lys Ala 50 55 60Ile Glu Arg Met Lys Asp Thr
Leu Arg Ile Ala Tyr Leu Thr Glu Ala65 70 75 80Lys Val Glu Lys Leu
Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95Ala Ala Ile Ser
Met Ala Asn Ser Glu Lys Asp Glu Leu 100 10511327DNAArtificial
SequenceCholera Toxin B Subunit Polypeptide Variant Polypeptide
11accccacaaa acatcactga cttgtgtgct gagtaccaca acacccaaat ccacaccctc
60aatgacacta tctttagcta caccgagagc cttgctggca agagggagat ggctatcatc
120accttcaaga atggtgctac cttccaagtg gaggtgcctg gaagccaaca
cattgatagc 180caaaagaagg ccattgagag gatgaaggac acacttagga
tagcttacct cactgaggct 240aaggtggaga agctttgtgt gtggaacaac
aagacccccc atgctattgc tgccatcagc 300atggccaacg ttactaagga tgaactc
32712109PRTArtificial SequenceCholera Toxin B Subunit Polypeptide
Variant Polypeptide 12Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu
Tyr His Asn Thr Gln1 5 10 15Ile His Thr Leu Asn Asp Thr Ile Phe Ser
Tyr Thr Glu Ser Leu Ala 20 25 30Gly Lys Arg Glu Met Ala Ile Ile Thr
Phe Lys Asn Gly Ala Thr Phe 35 40 45Gln Val Glu Val Pro Gly Ser Gln
His Ile Asp Ser Gln Lys Lys Ala 50 55 60Ile Glu Arg Met Lys Asp Thr
Leu Arg Ile Ala Tyr Leu Thr Glu Ala65 70 75 80Lys Val Glu Lys Leu
Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95Ala Ala Ile Ser
Met Ala Asn Val Thr Lys Asp Glu Leu 100 10513345DNAArtificial
SequenceCholera Toxin B Subunit Polypeptide Variant Polypeptide
13accccacaaa acatcactga cttgtgtgct gagtaccaca acacccaaat ccacaccctc
60aatgacacta tctttagcta caccgagagc cttgctggca agagggagat ggctatcatc
120accttcaaga atggtgctac cttccaagtg gaggtgcctg gaagccaaca
cattgatagc 180caaaagaagg ccattgagag gatgaaggac acacttagga
tagcttacct cactgaggct 240aaggtggaga agctttgtgt gtggaacaac
aagacccccc atgctattgc tgccatcagc 300atggccaacg ttactggtgg
tggaggatcc gagaaggatg aactc 34514115PRTArtificial SequenceCholera
Toxin B Subunit Polypeptide Variant Polypeptide 14Thr Pro Gln Asn
Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln1 5 10 15Ile His Thr
Leu Asn Asp Thr Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30Gly Lys
Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe 35 40 45Gln
Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55
60Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala65
70 75 80Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala
Ile 85 90 95Ala Ala Ile Ser Met Ala Asn Val Thr Gly Gly Gly Gly Ser
Glu Lys 100 105 110Asp Glu Leu 11515393DNAArtificial
SequenceCholera Toxin B Subunit Polypeptide Variant Polypeptide
15atggctatca agctcaagtt tggagtgttc ttcactgtgc tccttagctc tgcctatgca
60catggcaccc cacaaaacat cactgacttg tgtgctgagt accacaacac ccaaatccac
120accctcaatg acaagatctt tagctacacc gagagccttg ctggcaagag
ggagatggct 180atcatcacct tcaagaatgg tgctaccttc caagtggagg
tgcctggaag ccaacacatt 240gatagccaaa agaaggccat tgagaggatg
aaggacacac ttaggatagc ttacctcact 300gaggctaagg tggagaagct
ttgtgtgtgg aacaacaaga ccccccatgc tattgctgcc 360atcagcatgg
ccaactccga gaaggatgaa ctc 39316131PRTArtificial SequenceCholera
Toxin B Subunit Polypeptide Variant Polypeptide 16Met Ala Ile Lys
Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser1 5 10 15Ser Ala Tyr
Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala 20 25 30Glu Tyr
His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser 35 40 45Tyr
Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe 50 55
60Lys Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile65
70 75 80Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg
Ile 85 90 95Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp
Asn Asn 100 105 110Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala
Asn Ser Glu Lys 115 120 125Asp Glu Leu 1301778DNAOryza sativa
17atggggaagc aaatggccgc cctgtgtggc tttctcctcg tggcgttgct ctggctcacg
60cccgacgtcg cgcatggt 781826PRTOryza sativa 18Met Gly Lys Gln Met
Ala Ala Leu Cys Gly Phe Leu Leu Val Ala Leu1 5 10 15Leu Trp Leu Thr
Pro Asp Val Ala His Gly 20 251981DNANicotiana plumbaginifolia
19atggctactc aacgaagggc aaaccctagc tctctccatc taattactgt attctctctg
60ctcgtcgctg tcgtctcagg t 812027PRTNicotiana plumbaginifolia 20Met
Ala Thr Gln Arg Arg Ala Asn Pro Ser Ser Leu His Leu Ile Thr1 5 10
15Val Phe Ser Leu Leu Val Ala Val Val Ser Gly 20 252178DNAMalus
domestica 21atggcattga agacacagtt gttgtggtca ttcgtggttg tgttcgttgt
gtccttcagt 60acaacttcat gctcaggt 782226PRTMalus domestica 22Met Ala
Leu Lys Thr Gln Leu Leu Trp Ser Phe Val Val Val Phe Val1 5 10 15Val
Ser Phe Ser Thr Thr Ser Cys Ser Gly 20 252372DNAHordeum vulgare
23atggcgaaca aacacttgtc cctctccctc ttcctcgtcc tccttggcct gtcggccagc
60ttggcctcag gt 722424PRTHordeum vulgare 24Met Ala Asn Lys His Leu
Ser Leu Ser Leu Phe Leu Val Leu Leu Gly1 5 10 15Leu Ser Ala Ser Leu
Ala Ser Gly 2025109PRTArtificial SequenceCholera Toxin B Subunit
Polypeptide Variant Polypeptide 25Thr Pro Gln Asn Ile Thr Asp Leu
Cys Ala Glu Tyr His Asn Thr Gln1 5 10 15Ile His Thr Leu Asn Asp Lys
Ile Phe Cys Tyr Thr Glu Ser Leu Ala 20 25 30Gly Lys Arg Glu Met Ala
Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe 35 40 45Gln Val Glu Val Pro
Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55 60Ile Glu Arg Met
Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala65 70 75 80Lys Val
Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95Ala
Ala Ile Ser Met Cys Asn Ser Glu Lys Asp Glu Leu 100
10526135PRTArtificial SequenceCholera Toxin B Subunit Polypeptide
Variant Polypeptide 26Met Gly Lys Gln Met Ala Ala Leu Cys Gly Phe
Leu Leu Val Ala Leu1 5 10 15Leu Trp Leu Thr Pro Asp Val Ala His Gly
Thr Pro Gln Asn Ile Thr 20 25 30Asp Leu Cys Ala Glu Tyr His Asn Thr
Gln Ile His Thr Leu Asn Asp 35 40 45Lys Ile Phe Ser Tyr Thr Glu Ser
Leu Ala Gly Lys Arg Glu Met Ala 50 55 60Ile Ile Thr Phe Lys Asn Gly
Ala Thr Phe Gln Val Glu Val Pro Gly65 70 75 80Ser Gln His Ile Asp
Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp 85 90 95Thr Leu Arg Ile
Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys 100 105 110Val Trp
Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala 115 120
125Asn Ser Glu Lys Asp Glu Leu 130 13527136PRTArtificial
SequenceCholera Toxin B Subunit Polypeptide Variant Polypeptide
27Met Ala Thr Gln Arg Arg Ala Asn Pro Ser Ser Leu His Leu Ile Thr1
5 10 15Val Phe Ser Leu Leu Val Ala Val Val Ser Gly Thr Pro Gln Asn
Ile 20 25 30Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln Ile His Thr
Leu Asn 35 40 45Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys
Arg Glu Met 50 55 60Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe Gln
Val Glu Val Pro65 70 75 80Gly Ser Gln His Ile Asp Ser Gln Lys Lys
Ala Ile Glu Arg Met Lys 85 90 95Asp Thr Leu Arg Ile Ala Tyr Leu Thr
Glu Ala Lys Val Glu Lys Leu 100 105 110Cys Val Trp Asn Asn Lys Thr
Pro His Ala Ile Ala Ala Ile Ser Met 115 120 125Ala Asn Ser Glu Lys
Asp Glu Leu 130 13528135PRTArtificial SequenceCholera Toxin B
Subunit Polypeptide Variant Polypeptide 28Met Ala Leu Lys Thr Gln
Leu Leu Trp Ser Phe Val Val Val Phe Val1 5 10 15Val Ser Phe Ser Thr
Thr Ser Cys Ser Gly Thr Pro Gln Asn Ile Thr 20 25 30Asp Leu Cys Ala
Glu Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp 35 40 45Lys Ile Phe
Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala 50 55 60Ile Ile
Thr Phe Lys Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly65 70 75
80Ser Gln His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp
85 90 95Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu
Cys 100 105 110Val Trp Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile
Ser Met Ala 115 120 125Asn Ser Glu Lys Asp Glu Leu 130
13529133PRTArtificial SequenceCholera Toxin B Subunit Polypeptide
Variant Polypeptide 29Met Ala Asn Lys His Leu Ser Leu Ser Leu Phe
Leu Val Leu Leu Gly1 5 10 15Leu Ser Ala Ser Leu Ala Ser Gly Thr Pro
Gln Asn Ile Thr Asp Leu 20 25 30Cys Ala Glu Tyr His Asn Thr Gln Ile
His Thr Leu Asn Asp Lys Ile 35 40 45Phe Ser Tyr Thr Glu Ser Leu Ala
Gly Lys Arg Glu Met Ala Ile Ile 50 55 60Thr Phe Lys Asn Gly Ala Thr
Phe Gln Val Glu Val Pro Gly Ser Gln65 70 75 80His Ile Asp Ser Gln
Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu 85 90 95Arg Ile Ala Tyr
Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp 100 105 110Asn Asn
Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn Ser 115 120
125Glu Lys Asp Glu Leu 130306PRTArtificial
SequenceEndoplasmic Reticulum Retention Signal 30Ser Glu Lys Asp
Glu Leu1 5314PRTArtificial SequenceEndoplasmic Reticulum Retention
Signal 31Lys Asp Glu Leu1
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