U.S. patent application number 14/019601 was filed with the patent office on 2014-05-01 for compositions and methods for exosome targeted expression.
The applicant listed for this patent is Morehouse School of Medicine. Invention is credited to Syed ALI, Vincent Craig BOND, MingBo HUANG, Michael POWELL.
Application Number | 20140120156 14/019601 |
Document ID | / |
Family ID | 48610356 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140120156 |
Kind Code |
A2 |
BOND; Vincent Craig ; et
al. |
May 1, 2014 |
COMPOSITIONS AND METHODS FOR EXOSOME TARGETED EXPRESSION
Abstract
The present application relates to methods of producing
exosomes. The application also provides a method for preparing a
protein composition comprising culturing an exosome-producing cell
expressing a Nef-fusion protein comprising a Nef-derived peptide
fused to a protein of interest; isolating exosomes from the
exosome-producing cell culture; and purifying the protein of
interest from the isolated exosomes. The application further
discloses compositions that comprise exosomes containing the
Nef-fusion protein, as well as methods of using the Nef-fusion
protein and exosomes containing the Nef-fusion protein.
Inventors: |
BOND; Vincent Craig; (Stone
Mountain, GA) ; POWELL; Michael; (Douglasville,
GA) ; HUANG; MingBo; (Atlanta, GA) ; ALI;
Syed; (Kepala Batas, Pulau Pinang, MY) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Morehouse School of Medicine |
Atlanta |
GA |
US |
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20140004181 A1 |
January 2, 2014 |
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Family ID: |
48610356 |
Appl. No.: |
14/019601 |
Filed: |
September 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13327244 |
Dec 15, 2011 |
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14019601 |
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Current U.S.
Class: |
424/450 ;
424/188.1; 424/192.1; 424/94.3; 435/258.3; 435/357; 435/358;
435/366; 435/369; 435/7.92; 436/506; 514/21.2; 514/21.3; 530/324;
530/350 |
Current CPC
Class: |
C07K 14/163 20130101;
C07K 2319/055 20130101; A61K 45/06 20130101; C07K 2319/00 20130101;
A61K 2039/55555 20130101; A61K 9/127 20130101; C12P 21/02 20130101;
A61P 37/04 20180101; G01N 33/566 20130101; C07K 14/005 20130101;
A61K 39/39 20130101; C07K 2319/60 20130101; A61K 38/162 20130101;
C12N 15/00 20130101; C12N 2740/16322 20130101; G01N 33/543
20130101; A61K 38/00 20130101; C07K 2319/50 20130101 |
Class at
Publication: |
424/450 ;
514/21.3; 514/21.2; 424/192.1; 424/188.1; 436/506; 435/7.92;
435/366; 435/369; 435/358; 435/357; 435/258.3; 530/350; 424/94.3;
530/324 |
International
Class: |
C07K 14/005 20060101
C07K014/005; G01N 33/566 20060101 G01N033/566; A61K 39/39 20060101
A61K039/39; A61K 45/06 20060101 A61K045/06; A61K 9/127 20060101
A61K009/127; A61K 38/16 20060101 A61K038/16 |
Claims
1. A method for preparing a protein composition, comprising: a)
culturing an exosome-producing cell expressing a Nef-fusion protein
comprising a Nef-derived peptide fused to a protein of interest;
(b) isolating exosomes from cultured exosome-producing cell; and
(c) purifying said protein of interest from isolated exosomes.
2. The method of claim 1, wherein said Nef-derived peptide
comprises a Nef fragment comprising SEQ ID NO:3, or a variant of
said Nef fragment.
3. The method of claim 1, wherein said Nef-derived peptide
comprises a Nef fragment comprising SEQ ID NO:4 or a variant of
said Nef fragment.
4. The method of claim 1, wherein said Nef-derived peptide
comprises a Nef fragment comprising SEQ ID NO:5, or a variant of
said Nef fragment.
5. The method of claim 1, wherein said Nef-derived peptide
comprises a Nef fragment comprising SEQ ID NO:6 or a variant of
said Nef fragment.
6. The method of claim 1, wherein said Nef fusion protein further
comprises an amino acid sequence encoding a functional domain
selected from the group consisting of affinity tag, protease
cleavage site, targeting domain, reporter, enzyme and combination
thereof.
7. The method of claim 1, wherein said exosome-producing cell is
stably transformed with a vector expressing said Nef-fusion
protein.
8. The method of claim 1, wherein said exosome-producing cell is
transiently transfected with a vector expressing said Nef-fusion
protein.
9. A cell line that produces exosomes containing a Nef-fusion
protein comprising a Nef-derived peptide fused to a protein of
interest, wherein said Nef-derived peptide comprises SEQ ID NO:3,
4, 5 or 6.
10. A method for delivering a protein of interest to a target cell
in a mammal, comprising administering to the mammal an exosome
comprising a Nef-fusion protein, wherein said Nef-fusion protein
comprises a Nef-derived peptide fused to said protein of interest
and wherein said Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or
6.
11. The method of claim 10, wherein said Nef-derived peptide
comprises SEQ ID NO:3.
12. The method of claim 10, wherein said Nef-derived peptide
comprises SEQ ID NO:4.
13. The method of claim 10, wherein said Nef-derived peptide
comprises SEQ ID NO:5.
14. The method of claim 10, wherein said Nef-derived peptide
comprises SEQ ID NO:6.
15. The method of claim 10, wherein said exosome is further loaded
with a member from the group consisting of antigen, peptide, small
molecule drug, and siRNA.
16. A method for inducing an immune response in a mammal,
comprising administering to a mammal an exosome comprising a
Nef-fusion protein comprising a Nef-derived peptide fused to an
immunogenic protein of interest, wherein said Nef-derived peptide
comprises SEQ ID NO:3, 4, 5 or 6.
17. The method of claim 16, wherein said immunogenic protein
comprises at least a portion of a tumor antigen.
18. The method of claim 16, wherein said exosome is isolated from
an antigen-presenting cell.
19. A method for detecting a target molecule in a sample,
comprising: contacting a sample from a subject with a Nef-fusion
protein that binds specifically to said target molecule, wherein
said Nef-fusion protein comprises SEQ ID NO:3, 4, 5 or 6. detecting
a binding of said target molecule in the sample to said Nef-fusion
protein, and determining a level of said target molecule in said
sample, wherein a medical condition is indicated if a level of said
target molecule is outside a reference range.
20. A pharmaceutical composition, comprising: an exosome comprising
a Nef-fusion protein containing a Nef-derived peptide fused to a
protein of interest; and a pharmaceutically acceptable carrier.
21. The pharmaceutical composition of claim 20, wherein said
Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6.
22. A Nef-fusion protein, produced by culturing cells that produce
exosomes containing said Nef-fusion protein; isolating exosomes
from said cells; and purifying said Nef-fusion protein from
isolated exosomes, wherein said Nef-fusion protein comprises a
Nef-derived peptide fused to a protein of interest and wherein said
Nef-derived peptide comprises SEQ ID NO:3, 4, 5 or 6.
Description
[0001] This application is a Continuation of U.S. application Ser.
No. 13/327,244, filed Dec. 15, 2011. The entirety of the
aforementioned applications is incorporated herein by
reference.
FIELD
[0002] The present application relates to methods of producing
exosomes, and exosome targeted expression of fusion proteins with
predefined sequences of interest for the therapeutic and diagnostic
uses.
BACKGROUND
[0003] Exosomes are small vesicles 40-100 nm in diameter, that are
secreted by a number of different cell types for communicating with
other cells via the proteins and ribonucleic acids they carry.
Depending on their cellular origin, exosomes carry a uniquely
distinct profile of proteins, which can trigger signalling pathways
in other cells and/or transfer exosomal products into other cells
by exosomal fusion with cellular plasma membranes. The protein
composition of exosomes is distinct from that of other organelles,
including early endosomes and plasma membranes, more closely
resembling that of late endosomes or multivesicular bodies,
(MVBs).
[0004] Exosome release has been demonstrated from different cell
types in varied physiological contexts. For example, it has been
demonstrated that B lymphocytes release exosomes carrying class II
major histocompatibility complex molecules, which play a role in
antigenic presentation (Raposo et al., J. Exp. Med., 183:1161,
1996). Similarly, it has been demonstrated that dendritic cells
produce exosomes (i.e., dexosomes, Dex), which play a role in
immune response mediation, particularly in cytotoxic T lymphocyte
stimulation (Zitvogel et al., Nature Medicine, 4:594, 1998).
Further, it has also been demonstrated that tumor cells secrete
specific exosomes (i.e., texosomes, Tex) carrying tumor antigens in
a regulated manner, which can present these antigens to antigen
presenting cells. The application of exosomes for use as cancer
vaccines has been reviewed by Tan et al., Int. J. Nanomed.,
5:889-900, 2010.
[0005] Nef is a protein expressed by primate lentiviruses, such as
HIV and SIV. Nef is known to be secreted in association with
exosomes and has been also shown to be present on the surface of
HIV-infected cells. Nef-expressing cells have a dramatically
altered subcellular morphology and have been shown to induce the
intracellular accumulation of multivesicular bodies and the
extracellular accumulation of exosomes. Exosomes have been
postulated to play a role in the production of HIV-1 virions. The
so called "Trojan Exosome" hypothesis suggests that HIV-1 particles
can "piggyback" on the process of exosome biogenesis to provide a
means of transfer of infectious particles from one cell to another
(Izquierdo-Useros et al., PLoS pathogens, 6(3):1-9, 2010). Although
some of the aspects of this theory have been questioned, the
research has established a precedent for HIV-1 proteins being
carried out of the cell and from one cell to another via the
exosome network.
[0006] There is great interest in exploiting the properties of
exosomes for diagnostic, vaccination, and therapeutic applications,
including new and effective methods for preparing recombinant
proteins at an industrial scale, for vaccine preparation, and for
immunotherapy. The present invention provides compositions and
methods for exosomal expression of recombinant proteins.
SUMMARY
[0007] One aspect of the present application relates to a method
for preparing a protein composition. The method comprises the steps
of culturing an exosome-producing cell expressing a Nef-fusion
protein comprising a Nef-derived peptide fused to a protein of
interest; isolating exosomes from the exosome-producing cell
culture; and purifying the protein of interest from the isolated
exosomes.
[0008] Another aspect of the present application relates to a
method for delivering a protein of interest to a target cell in a
mammal. The method comprises administering to the mammal an exosome
comprising a Nef-fusion protein comprising a Nef-derived peptide
fused to the protein of interest.
[0009] Another aspect of the present application relates to a
method for inducing an immune response in a mammal. The method
comprises administering to a mammal an exosome comprising a
Nef-fusion protein comprising a Nef-derived peptide fused to an
immunogenic protein of interest.
[0010] Another aspect of the present application relates to a
method for detecting a target molecule in a sample. The method
comprises contacting a sample from a subject with a Nef-fusion
protein that binds specifically to the target molecule, detecting a
binding of the target molecule in the sample to the Nef-fusion
protein, and determining a level of the target molecule in the
sample, wherein a medical condition is indicated if the level of
the target molecule is outside a reference range.
[0011] Another aspect of the present application relates to a
pharmaceutical composition, comprising an exosome comprising a
Nef-fusion protein containing a Nef-derived peptide fused to a
protein of interest, and a pharmaceutically acceptable carrier.
[0012] Another aspect of the present application relates to a
Nef-fusion protein produced by culturing cells that produce
exosomes containing the Nef-fusion protein; isolating exosomes from
the exosome-producing cell culture; and purifying the Nef-fusion
protein from the isolated exosomes, wherein the Nef-fusion protein
comprises a Nef-derived peptide fused to a protein of interest.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1A-C show co-expression of Nef with exosomal markers
acetylcholinesterase (AChE) and CD45 release from untransfected and
Nef-GFP-transfected Jurkat cells. In FIG. 1A, 1.times.10.sup.6
Jurkat cells were mock-transfected or HIV-1 wtNef-GFP for 48 h at
37.degree. C. Cell lysates prepared therefrom were examined for
HIV-1 Nef, AChE, CD45, and tubulin expression by Western analysis.
Columns: UT, untransfected Jurkat cell lysates; Nef, HIV-1
wtNef-GFP-transfected cell lysates. Rows: Probed with: Nef, HIV-1
Nef monoclonal antibody; AChE, AChE antibody; CD45, CD45 antibody;
tubulin, tubulin antibody. In FIG. 1B, differential centrifugal
high-molecular-weight pellets were examined for HIV-1 Nef, AChE,
and CD45 by Western analysis. Columns: lane 1, 10,000.times.g
pellet; lane 2, 50,000.times.g pellet; lane 3, 100,000.times.g
pellet; lane 4, 400,000.times.g pellet; lane 5, 400,000.times.g
spent supernatant. Rows: top two panel set: untransfected Jurkat
culture pellet examined with AChE or CD45 antibodies; lower three
panel set: HIV-1 Nef-GFP-transfected Jurkat culture pellet examined
with HIV-1 Nef monoclonal, AChE, or CD45 antibodies. FIG. 1C shows
densitometric analysis of the Western data (NIH Image J software
analysis) in FIGS. 1A and 1B. AChE and CD45 band densities from
untransfected cells and AChE, CD45, and Nef band densities from
HIV-1 Nef-transfected cells were normalized against intracellular
tubulin, and to report combined 100,000.times.g plus
400,000.times.g band density units per 1.times.10.sup.6 cells. This
is the combined data from multiple experiments and the data were
analyzed using Student's t-test comparing untransfected values and
wtNef-GFP-transfected values and displaying the p-values where
p<0.05 is significant. Transfection efficiencies for these
experiments were 85%.+-.2%.
[0014] FIGS. 2A-B show an analysis of the vesicular nature of
secreted Nef protein. Cells and conditioned media were collected
from untransfected or wtNef-GFP-transfected Jurkat cultures.
Culture media were processed via differential centrifugation, with
spins at 1200.times.g, 10,000.times.g, 50,000.times.g,
200,000.times.g, and 400,000.times.g. Both 200,000.times.g and
400,000.times.g pellets were subjected to sucrose gradient
flotation. Cell lysates, culture medium, 50,000.times.g pellets and
400,000.times.g pellets and flotation gradient fractions were
examined by Western blotting for Nef, GFP, and Alix. FIG. 2A shows
representative images from one experiment: Cell lysate (lane 1);
culture media (supernatant; lane 2); 50,000.times.g pellet (lane
3); 400,000.times.g pellet (Diff. Cent.; lane 4); Gradient
fractions 4-11 (lanes 5-12). Gradient fractions 1, 2, 3, and 12,
which had no protein in them, are not shown. FIG. 2B shows data
collated from multiple experiments. Bands visualized on Western
blots were measured by densitometry. Data were analyzed using
Student's t-test comparing Alix from untransfected cell cultures
and wtNef-GFP-transfected cell cultures, with p values <0.01
being scored as significant.
[0015] FIG. 3 shows a transient transfection strategy for
identifying the Nef structural requirements for exosome
secretion.
[0016] FIGS. 4A-B show a schematic representation of HIV NL4-3 Nef
mutants generated for testing. FIG. 4A shows various Nef deletion
mutants, including Nef: Nef.DELTA.31-206 containing aa residues
1-30; Nef.DELTA.1-12 containing aa 13-70 but lacking the
myristoylation site G2 and the K4K7 basic region; Nef.DELTA.1-40
containing aa 41-70; Nef.DELTA.51-206 containing aa 1-50;
Nef.DELTA.65-206 containing aa 1-65; Nef.DELTA.71-206 containing aa
1-70; Nef.DELTA.91-206 containing aa 1-90; and Nef.DELTA.150-206
containing aa 1-150; Nef.DELTA.200-206 containing aa 1-200. FIG. 4B
shows various amino acid replacement mutants, including
Nef4R(17-22)/4A, in which R17, R19, R21, and R22 are replaced with
four alanines; NefK39P, in which K39 is replaced with a proline to
disrupt the helix; SS45,46AA, in which S45 and S46 are replaced
with two alanines; P25A, in which P25 is replaced with an alanine;
29GVG31/3A, in which G29, V30, and G31 are replaceed with three
alanines; T44A, in which T44 is replaced with an alanine;
Nef.sup.62EEEE.sup.65/4A (PACS), in which E62-65 are replaced with
five alanines; NefSMR/.sup.66VGFPV.sup.70/5A, in which V66, G67,
F68, P69, and V70 are replaced with five alanines (in wt as well as
Nef13-70 background); SMR/.sup.66AGFPV.sup.70, in which V66 is
replaced with an alanine; SMR/.sup.66VAFPV.sup.70, in which F68 is
replaced with an alanine; SMR/.sup.66VGFAV.sup.70, in which P69 is
replaced with an alanine; and SMR.sup.66VGFPA.sup.70, in which V70
is replaced with an alanine.
[0017] FIG. 5A shows the sequence of HIV-1 Nef showing structural
domains required for cellular interactions, including the basic
amino acid 1 and 2 motifs (BAA-1, BAA-2), helix-1 and helix-2,
membrane targeting domain, PACS, and SMR motifs. FIG. 5B shows the
sequence of SIV Nef showing structural domains required for
cellular interactions.
[0018] FIG. 6A shows truncation mutagenesis to determine Nef
secretion sequences. The relative fluorescence of carboxy-terminal
deletion mutants of Nef compared to the wtNef-GFP is shown. Media
were collected and assayed from the 48-h-old cultures of HEK293
cells transfected with the wt, Nef.DELTA.31-206 (1-30),
Nef.DELTA.51-206 (1-50), Nef.DELTA.66-206 (1-65), Nef.DELTA.71-206
(1-70), Nef.DELTA.91-206 (1-90), Nef.DELTA.151-206 (1-150),
Nef.DELTA.201-206 (1-200), Nef.DELTA.1-12 (13-206), Nef.DELTA.1-12
and .DELTA.71-206 (13-70), Nef.DELTA.1-40 and .DELTA.71-206
(41-70), and untransfected HEK293 cells (bar 12). FIG. 6B shows
replacement mutagenesis to fine map Nef secretion sequences. The
relative fluorescence of N-terminal replacement mutants of Nef in
the 1-70 aa region and compared to the wtNef-GFP is shown. Media
were collected and assayed from the 48-h cultures of HEK293 cells
transfected with wtNef, Nef.DELTA.71-206 (1-70), NefG2A, NefK4K7,
Nef K39P, Nef.sup.39K/P, Nef.sup.45,46S/2A, NefP25A,
Nef.sup.29GVG.sup.31/3A, NefT44A, Nef.sup.66VGFPV.sup.70/5A,
Nef.sup.62EEEE.sup.65/4A (PACS), Nef.sup.17,19,21,22R/4A, and
untransfected HEK293 cells. FIG. 6C shows a newly identified domain
on HIV-1 Nef. The relative fluorescence of N-terminal deletion and
replacement mutants of Nef in the 66-70 aa region compared to the
wtNef is shown. Media were collected and assayed from the 48-h
cultures of HEK293 cells transfected with wtNef, GFP,
Nef.sup.66AGFPV.sup.70, Nef.sup.66VAFPV.sup.70,
Nef.sup.66VGAPV.sup.70, Nef.sup.66VGFAV.sup.70,
Nef.sup.66VGFPA.sup.70, and untransfected HEK293 cells. FIGS. 6D-F
show the Nef-induced secretion domains function similarly in
multiple cell types. Jurkat cells (1.times.10.sup.6) transfected
with HIV-1 wtNef (bar 1), Nef.DELTA.71-206 (bar 2), Nef.DELTA.1-12
and .DELTA.71-206 (bar 3), Nef.DELTA.1-12 (bar 4),
Nef.sup.17,19,21,22 R/4A (bar 5), Nef.sup.62EEE.sup.65/4A (PACS,
bar 6), Nef.sup.66AGFPV.sup.70 (SMR, bar 7), GFP (bar 8), and
untransfected cells (bar 9) (FIG. 6D), THP-1 (FIG. 6E), and U937
(FIG. 6F) monocytes by Gene Pulser Xcell Electroporation System
(Bio-Rad Laboratories, Inc., CA). Cells were incubated in RPMI 1640
medium for 48 h at 37.degree. C. and removed from the culture
supernatant by centrifugation at 2000.times.g for 5 min. In all
experiments, the error bars show the standard errors of the
measurements. Transfection efficiencies for Jurkat cells
(80-86.67%), for THP-1 cells (60-65%), and for U937 cells (55-60%).
These results are a compilation of at least three independent
experiments.
[0019] FIG. 7 shows an alignment of the PACS/SMR regions of HIV-1
Nef. Amino acid consensus sequences for 13 HIV-1 subtypes were
determined as described in Materials and Methods. The PACS-SMR
consensus sequences were then aligned to illustrate the degree of
homology in these required secretion domains of Nef. Dashes (-)
indicate gaps inserted to facilitate the alignment.
[0020] FIGS. 8A-B shows that HIV Nef expression in cells is not
toxic or apoptotic to transfected cells. HIV-1 Nef-GFP mutants were
transfected into HEK293 cells at 37.degree. C. for 48 h.
Subsequently, the cultures were stained with propidium iodide (PI)
to visualize the nucleus. Finally, a comparative morphological
examination of the individual cells in these cultures was performed
to determine whether and how much cytotoxicity or apoptosis was
observed in the transfected cells. In FIG. 8A, HEK293 cells were
transfected with HIV-1 Nef-GFP mutants and stained by PI. Columns:
bar 1, mock, untransfected HEK293 cells; bar 2, pQBI-GFP,
transfected pQBI-GFP; bar 3, wtNef-GFP(1-206), transfected HIV-1
wtNef-GFP; bar 4, wtNef-GFP (1-70), transfected Nef.DELTA.71-206;
bar 5, wtNef-GFP (13-70), transfected Nef.DELTA.1-12 and
.DELTA.71-206; bar 6, Nef-4R4A-GFP (1-206), transfected
Nef.sup.17,19,21,22R/4A; bar 7, Nef-PACS-GFP (1-206), transfected
Nef.sup.62EEEE.sup.65/4A; bar 8, Nef-AGFPV-GFP (1-206), transfected
Nef.sup.66AGFPV.sup.70. In FIG. 8B, HEK293 cells were transfected
with HIV-1 Nef at 37.degree. C. for 48 h and then cells were
assayed by TUNEL. Columns: bar 1, pQBI-RFP, transfected pQBI-RFP in
HEK293 cells; bar 2, HIV-1 wtNef-RFP, transfected HIV-1
wtNef-RFP.
[0021] FIGS. 9A-B demonstrate that Nef-induced vesicles do not
display attributes of apoptotic vesicles. HIV-1 wtNef-GFP and
Nef-GFP mutants were transfected into HEK293 cells. In FIG. 9A,
cell lysates and vesicles collected from each condition were
examined for histones through Coomassie brilliant blue staining of
PAGE gels. Lanes 1, 3, 5, and 7 are cell lysates from each
condition; lanes 2, 4, 6, and 8 are pellets from cell lysates spun
at 130,000.times.g. Lanes 1 and 2 are from cells treated with 10
.mu.M camptothecin; lanes 3 and 4 are from cells transfected with
HIV-1 wtNef; lanes 5 and 6 are from cells transfected with
Nef.sup.66AGFPV.sup.70; lanes 7 and 8 are from untransfected cells.
His denotes the region of the gel containing the histone bands. In
FIG. 9B, cell lysates and vesicles were analyzed by Western
analysis for the presence of histones (top panel set, histone
antibody), GFP (middle panel set, GFP antibody), and HIV-1 Nef
(bottom panel set, Nef polyclonal antibody). Lane 1, cell lysates;
lane 2, 300.times.g pellet; lane 3, 1200.times.g pellet; lane 4,
10,000.times.g pellet; lane 5, 130,000.times.g pellet. Individual
panels of each panel set: top panel, Camp, cells were treated with
10 .mu.M camptothecin; second panel, Nef, HIV-1
wtNef-GFP-transfected cells; third panel, Nef-SMR (HIV-1
Nef-.sup.66VGFPV.sup.70/5A-GFP)-transfected cells; bottom panel,
UT, untransfected cells.
[0022] FIGS. 10A-D show that the effects of Nef mutants is not due
to variable transfection/expression efficiencies. In FIGS. 10A-10C,
1.times.10.sup.6 HEK293 cells were transfected with 1 mg of HIV-1
wtNef-GFP for 48 h and then followed by Western analysis of HEK293
cell lysates from wtNef or mutant transfections. Cell cultures were
transfected with pQBI-Nef-GFP (NefGFP; lane 1), pQBI-GFP (GFP; lane
2), pQBI-Nef.sup.62EEEE.sup.65/4AGFP (PACS replacement; lane 3),
pQBI-Nef.sup.66AGFPV.sup.70GFP (SMR replacement lane 4),
pQBI-Nef.sup.17,19,21,22R/4AGFP (BAA-2 replacement; lane 5), or
untransfected HEK293 cells (UT; lane 6). Cell lysates were
collected and analyzed by SDS-PAGE followed by Western analysis
probing with anti-GFP antiserum. Representative images of several
independent experiments are shown. The relative positions of
Nef-GFP and Nef-GFP deletion mutants' cellular protein that
hybridizes to the anti-GFP antiserum are indicated. FIG. 10D shows
densitometry was performed and the readings from multiple
independent analyses are displayed as the average densitometric
units for any particular assay with standard error of measurement
displayed.
DETAILED DESCRIPTION
[0023] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed method and compositions
belong. It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a peptide" includes a plurality of such
peptides, reference to "the peptide" is a reference to one or more
peptides and equivalents thereof known to those skilled in the art,
and so forth.
Method of Producing Nef-Fusion Protein
[0024] One aspect of the present application relates to a method
for producing a protein composition comprising culturing an
exosome-producing cell expressing a Nef-fusion protein comprising a
Nef-derived peptide fused to a protein of interest; isolating
exosomes from the exosome-producing cell culture; and purifying the
Nef-fusion protein or the protein of interest from the isolated
exosomes.
[0025] As used herein, the term "Nef-derived peptide" refers to the
full length HIV Nef peptide (SEQ ID NO:1), the full length SIV Nef
peptide (SEQ ID NO:2), a fragment of the full length HIV Nef
peptide that comprises amino acid residues 13-41 of SEQ ID NO:1
(i.e., SEQ ID NO:3), a fragment of the full length SIV Nef peptide
that comprises amino acid residues 1-102 of SEQ ID NO:2 (i.e., SEQ
ID NO:4), or variants thereof. A variant of the full length Nef
peptide or the Nef fragment includes peptides that share at least
95%, 96%, 97%, 98% or 99% homology to the full length Nef peptide
or the Nef fragment, as well as peptides that contain one or more
substitutions, additions and/or deletions that do not significantly
alter the bioactivity of the full length Nef peptide or the Nef
fragment. In some embodiments, the Nef-derived peptide is a Nef
fragment comprising SEQ ID NO:3 or a variant the HIV Nef fragment.
In some other embodiments, the Nef-derived peptide is a Nef
fragment comprising amino acid residues 13-70 of SEQ ID NO:1 (SEQ
ID NO:5) or a variant the Nef fragment. In some other embodiments,
the Nef-derived peptide is a Nef fragment comprising amino acid
residues 1-70 of SEQ ID NO:1 (SEQ ID NO:6) or a variant the Nef
fragment. In some embodiments, the Nef-derived peptide is a Nef
fragment comprising SEQ ID NO:4 or a variant the Nef fragment. In
certain embodiments, the Nef-derived peptide has a length of 30-70,
60-70, 70-150, 150-206, 30-102, 102-180 and 180-263 amino
acids.
The Nef-Fusion Protein
[0026] The Nef-fusion protein comprises a Nef-derived peptide fused
to a protein of interest. In some embodiments, the Nef-fusion
protein further comprises one or more additional amino acid
sequences encoding one or more functional domains. Exemplary
functional domains include, but are not limited to, affinity tags,
protease cleavage sites, targeting domains, reporters, enzymes, or
combination thereof.
[0027] In certain embodiments, an affinity tag may be included to
facilitate purification of the Nef-fusion protein and/or protein of
interest by affinity chromatography. The affinity tag may include
affinity tag known to those of skill in the art, including, but not
limited to, glutathione S-transferase (GST), Histidine tag (e.g.,
6.times.His), maltose binding protein (MBP), Protein A,
thioredoxin, ubiquitin, biotin, calmodulin binding peptide (CBP),
streptavidin tag, and various immunogenic peptide tags, including
FLAG octapeptide tag, hemaglutinin A (HA) tag, myc tag, and the
like.
[0028] In some embodiments, proteolytic cleavage sites may be
engineered into the Nef-fusion protein to promote the release of
the protein of interest from Nef and/or other peptide functional
domains, including affinity tags, in conjunction with fusion
protein synthesis or purification. Exemplary protease cleavage
sites include, but are not limited to, cleavage sites sensitive to
thrombin, furin, factor Xa, metalloproteases, enterokinases, and
cathepsin.
[0029] The targeting domain may comprise amino acid sequences
conferring cell-type specific or cell differentiation-specific
targeting. The targeting domain may be incorporated into the
Nef-fusion protein or it can be fused to a coexpressed
membrane-bound exosomal marker protein. Preferably the targeting
domain is fused to an extracellular domain in the membrane-bound
protein. The targeting domain may comprise an antibody or antibody
derivative, a peptide ligand, a receptor ligand, a receptor
fragment, a hormone, etc. Exemplary membrane-bound exosomal marker
proteins include, but are not limited to tetraspanins, such as CD9,
CD63, CD81, CD82, and CD151, and a variety of GPI
(glycerol-phosphatidyl inositol)-anchored proteins, among
others.
[0030] Exemplary antibody or antibody derived targeting domains may
include any member of the group consisting of: IgG, antibody
variable region; isolated CDR region; single chain Fv molecule
(scFv) comprising a VH and VL domain linked by a peptide linker
allowing for association between the two domains to form an antigen
binding site; bispecific scFv dimer; minibody comprising a scFv
joined to a CH3 domain, single chain diabody fragment, dAb
fragment, which consists of a VH or a VL domain; Fab fragment
consisting of VL, VH, CL and CH1 domains; Fab' fragment, which
differs from a Fab fragment by the addition of a few residues at
the carboxyl terminus of the heavy chain CH1 domain, including one
or more cysteines from the antibody hinge region; Fab'-SH fragment,
which is a Fab' fragment in which the cysteine residue(s) of the
constant domains bear a free thiol group; F(ab').sub.2, bivalent
fragment comprising two linked Fab fragments; Fd fragment
consisting of VH and CH1 domains; derivatives thereof, and any
other antibody fragment(s) retaining antigen-binding function. Fv,
scFv, or diabody molecules may be stabilized by the incorporation
of disulphide bridges linking the VH and VL domains. When using
antibody-derived targeting agents, any or all of the targeting
domains therein and/or Fc regions may be "humanized" using
methodologies well known to those of skill in the art.
[0031] In some embodiments, the targeting domain comprises an
antibody-derived or peptide-derived targeting domain from a phage
display library. Phage display libraries engineered for binding
cell surface molecules or receptors are well known to those of
skill in the art.
[0032] Functional domains in the Nef-fusion proteins of the present
invention may be separated from one another by a spacer or linker
to facilitate the independent folding of each peptide portion
relative to one another and ensure that the individual peptide
portions in a fusion protein do not interfere with one another. The
spacer may include any amino acid or mixtures thereof. In one
embodiment, the spacer comprises between 1 to 50 amino acids,
preferably 3 to 15 amino acids in length. Preferably, a chosen
spacer will increase the flexibility of the protein and facilitate
adoption of an extended conformation. Preferred peptide spacers are
comprised of the amino acids proline, lysine, glycine, alanine, and
serine, and combinations thereof. In one embodiment, the linker is
a glycine rich linker. In a particular embodiment, the spacer
having the formula [(Gly).sub.n-Ser/Ala].sub.m (SEQ ID NO:7) where
n is from 1 to 4, inclusive, and m is from 1 to 4, inclusive.
The Exosome-Producing Cell
[0033] The exosome-producing cell can be any cell capable of
producing exosomes. In some embodiments, the exosome-producing cell
is a cell of mammalian origin. In other embodiments, the
exosome-producing cell is a human cell. The exosome-producing cell
produces and secretes membrane vesicles of endosomal origin by
fusion of late endosomal multivesicular bodies with the plasma
membrane. Cells from various tissue types have been shown to
secrete exosomes, such as dendritic cells, B lymphocytes, tumor
cells, T lymphocytes and mast cells, for instance. Preferred
exosome-producing cells include mammalian tumor cells, mammalian B
and T lymphocytes, and mammalian dendritic cells, typically of
murine or human origin. In this regard, the cells are preferably
immortalized dendritic cells, immature dendritic cells or tumor
cells. Furthermore, for the production of antibody, it may be
advantageous to use B lymphocytes as exosome-producing cells, since
the resulting exosomes comprise accessory functions and molecules
such as MHC class II molecules that facilitate antibody production.
Furthermore, it has been shown that B cells-derived exosomes are
able to bind to follicular dendritic cells, which is another
important feature for antibody induction.
[0034] In some embodiments, the exosome-producing cell is stably
transformed with a vector expressing the Nef-fusion protein. In
other embodiments, the exosome-producing cell is transiently
transfected with a vector expressing the fusion protein.
[0035] Any suitable expression vector may be used to introduce and
express Nef-fusion proteins. As used herein, the term "expression
vector" includes any nucleic acid capable of expressing the fusion
protein in vivo. Expression vectors may be delivered to cells using
two primary delivery schemes: viral-based delivery systems using
viral vectors and non-viral based delivery systems using, for
example, plasmid vectors. Such methods are well known in the art
and readily adaptable for use with the compositions and methods
described herein. In certain cases, these methods can be used to
target certain diseases and cell populations by using the targeting
characteristics inherent to the carrier or engineered into the
carrier.
[0036] The expression vector contains one or more transcriptional
regulatory elements, including promoters and/or enhancers, for
directing the expression of Nef-fusion proteins. A promoter
comprises a DNA sequence that functions to initiate transcription
from a relatively fixed location in regard to the transcription
start site. A promoter contains core elements required for basic
interaction of RNA polymerase and transcription factors, and may
operate in conjunction with other upstream elements and response
elements.
[0037] As used herein, the term "promoter" is to be taken in its
broadest context and includes transcriptional regulatory elements
(TREs) from genomic genes or chimeric TREs therefrom, including the
TATA box or initiator element for accurate transcription
initiation, with or without additional TREs (i.e., upstream
activating sequences, transcription factor binding sites,
enhancers, and silencers) which regulate activation or repression
of genes operably linked thereto in response to developmental
and/or external stimuli, and trans-acting regulatory proteins or
nucleic acids. The promoter may be constitutively active or it may
be active in one or more tissues or cell types in a developmentally
regulated manner. A promoter may contain a genomic fragment or it
may contain a chimera of one or more TREs combined together.
[0038] Preferred promoters are those capable of directing
expression in a target cell of interest. The promoters may include
constitutive promoters (e.g., HCMV, SV40, elongation
factor-1.alpha. (EF-1.alpha.)) or those exhibiting preferential
expression in a particular cell type of interest. Enhancers
generally refer to DNA sequences that function away from the
transcription start site and can be either 5' or 3' to the
transcription unit. Furthermore, enhancers can be within an intron
as well as within the coding sequence. They are usually between 10
and 300 bp in length, and they function in cis. Enhancers function
to increase and/or regulate transcription from nearby promoters.
Preferred enhancers are those directing high-level expression in
the exosome expressing cell.
[0039] The promoter and/or enhancer may be specifically activated
either by light or specific chemical inducing agents. In some
embodiments, inducible expression systems regulated by
administration of tetracycline or dexamethasone, for example, may
be used. In other embodiments, gene expression may be enhanced by
exposure to radiation, including gamma irradiation and external
beam radiotherapy (EBRT), or alkylating chemotherapeutic drugs.
[0040] Cell or tissue-specific transcriptional regulatory elements
(TREs) can be incorporated into expression vectors to allow for
transcriptional targeting of expression to desired cell types.
Expression vectors generally contain sequences for transcriptional
termination, and may additionally contain one or more elements
positively affecting mRNA stability. An expression vector may
further include an internal ribosome entry site (IRES) between
adjacent protein coding regions to facilitate expression two or
more proteins from a common mRNA in an infected or transfected
cell. Additionally, the expression vectors may further include
nucleic acid sequence encoding a marker product. This marker
product is used to determine if the gene has been delivered to the
cell and once delivered is being expressed. Preferred marker genes
are the E. coli lacZ gene, which encodes .beta.-galactosidase, and
green fluorescent protein.
[0041] The expression vector can be introduced into the
exosome-producing cells by any conventional method, such as by
naked DNA technique, cationic lipid-mediated transfection,
polymer-mediated transfection, peptide-mediated transfection,
virus-mediated infection, physical or chemical agents or
treatments, electroporation, etc. In one embodiment, cells
transfected with the vector may be used directly as a source of
exosomes (transient transfection). Alternatively, cells may be
transfected with a vector expressing a Nef-fusion protein along
with a selectable marker facilitating selection of stably
transformed clones expressing the fusion protein. The exosomes
produced by such cells may be collected and/or purified according
to techniques known in the art, such as by centrifugation,
chromatography, etc. as further described in the cited references
and Examples herein.
[0042] Examples of suitable selectable markers for mammalian cells
include dihydrofolate reductase (DHFR), thymidine kinase, neomycin,
neomycin analog G418, hydromycin, and puromycin. When such
selectable markers are successfully transferred into a mammalian
host cell, the transformed mammalian host cell can survive if
placed under selective pressure. There are two widely used distinct
categories of selective regimes. The first category is based on a
cell's metabolism and the use of a mutant cell line which lacks the
ability to grow independent of a supplemented media. Two examples
are: CHO DHFR-cells and mouse LTK-cells. These cells lack the
ability to grow without the addition of such nutrients as thymidine
or hypoxanthine. Because these cells lack certain genes necessary
for a complete nucleotide synthesis pathway, they cannot survive
unless the missing nucleotides are provided in a supplemented
media. An alternative to supplementing the media is to introduce an
intact DHFR or TK gene into cells lacking the respective genes,
thus altering their growth requirements. Individual cells which
were not transformed with the DHFR or TK gene will not be capable
of survival in non-supplemented media.
[0043] The second category is dominant selection which refers to a
selection scheme used in any cell type and does not require the use
of a mutant cell line. These schemes typically use a drug to arrest
growth of a host cell. Those cells which have a novel gene would
express a protein conveying drug resistance and would survive the
selection. Examples of such dominant selection use the drugs
neomycin, mycophenolic acid, or hygromycin. The three examples
employ bacterial genes under eukaryotic control to convey
resistance to the appropriate drug G418 or neomycin (geneticin),
xgpt (mycophenolic acid) or hygromycin, respectively. Others
include the neomycin analog G418 and puromycin.
[0044] In some embodiments, the Nef-fusion proteins are delivered
from viral-derived expression vectors. Exemplary viral vectors may
include or be derived from adenovirus, adeno-associated virus,
herpesvirus, vaccinia virus, poliovirus, poxvirus, HIV virus,
lentivirus, retrovirus, Sindbis and other RNA viruses, and the
like. Also preferred are any viral families which share the
properties of these viruses which make them suitable for use as
vectors. Retroviruses include Murine Moloney Leukemia virus (MMLV),
HIV and other lentivirus vectors. Adenovirus vectors are relatively
stable and easy to work with, have high titers, and can be
delivered in aerosol formulation, and can transfect non-dividing
cells. Poxyiral vectors are large and have several sites for
inserting genes, they are thermostable and can be stored at room
temperature. Viral delivery systems typically utilize viral vectors
having one or more genes removed and with and an exogenous gene
and/or gene/promoter cassette being inserted into the viral genome
in place of the removed viral DNA. The necessary functions of the
removed gene(s) may be supplied by cell lines which have been
engineered to express the gene products of the early genes in
trans.
[0045] Exemplary exosome-producing cells include human Jurkat,
human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO)
cells, mouse WEHI fibrosarcoma cells, and unicellular protozoan
species, such as Leishmania tarentolae. In addition, stably
transformed, exosome-producing cell lines may be produced using
primary cells immortalized with c-myc or other immortalizing
agents. In some embodiments, the cell lines expresses at least 1
mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg,
at least 50 mg, or at least 100 mg of the Nef-fusion protein/liter
of culture.
[0046] In one embodiment, the cell line comprises a stably
transformed Leishmania cell line, such as Leishmania tarentolae.
Leishmania are known to secrete exosomes and are known to provide a
robust, fast-growing unicellular host for high level expression of
eukaryotic proteins exhibiting mammalian-type glycosylation
patterns. A commercially available Leishmania eukaryotic expression
kit is available (Jena Bioscience GmbH, Jena, Germany).
Isolation of Exosomes and Purification of Nef-Fusion Protein
[0047] Exosomes are isolated from exosome-producing cells.
Exosome-producing cells are cultured and maintained in any
appropriate culture medium, such as RPMI, DMEM, and AIM V.RTM.. The
culture medium is preferably a protein-free medium so as to avoid
contamination of exosomes by media-derived proteins. In some
embodiments, exosomes are isolated from the culture supernatants by
sequential centrifugation. The Nef-fusion proteins are then
purified using conventional protein purification methodologies
(e.g., affinity purification, chromatography, etc) known to those
of skill in the art. In certain embodiments, the purified
Nef-fusion protein is treated to release the protein of interest
from the Nef-derived peptide. The protein of interest is then
purified from the treated Nef-fusion protein using conventional
protein purification methodologies.
[0048] In some other embodiments, the isolated exosomes are treated
to release the protein of interest from the Nef-derived peptide.
The protein of interest is then purified from the treated exosomes
using conventional protein purification methodologies. Therefore,
one aspect of the present application relates to a Nef-fusion
protein produced by culturing cells that produce exosomes
containing the Nef-fusion protein; isolating exosomes from the
exosome-producing cell culture; and purifying the Nef-fusion
protein from the isolated exosomes, wherein the Nef-fusion protein
comprises a Nef-derived peptide fused to a protein of interest.
Methods of Using the Nef-Fusion Protein and Exosomes Containing the
Nef-Fusion Protein
[0049] Another aspect of the present application relates to a
method of treating cancer in a subject. In certain embodiments, the
method includes the step of administering to a subject in need of
such treatment, an effective amount of an exosome comprising the
Nef-fusion protein described above, wherein the protein of interest
is a cancer-specific antigen and wherein the exosome is isolated
from a professional antigen presenting cell, such a B lymphocyte or
a dendritic cell.
[0050] In other embodiments, exosomes containing the Nef-fusion
protein are further loaded with one or more immunogenic agents,
including antigens, peptides, small molecule drugs and/or nucleic
acids, such as siRNAs. Such agents may be loaded into exosomes
using conventional delivery methodologies, employing, for example,
transfection agents, including liposomal and peptide-based
transfection agents, electroporation, microinjection and the
like.
[0051] In certain embodiments, exosomes containing the Nef-fusion
protein are loaded with an siRNA targeting a cancer marker that is
over-expressed in cancer cells. In one embodiment, purified
exosomes are loaded with exogenous siRNA by electroporation. The
exosomes may be further modified to target specific organ, tissue
or cells.
[0052] Another aspect of the present application relates to a
method for inducing an immune response in a mammal. The method
comprises administering to a mammal an exosome containing a
Nef-fusion protein comprising an immunogenic protein of interest,
wherein the exosome composition is sufficient to induce an immune
response in the mammal. The exosome may be introduced into the
mammal as a vaccine, an immunotherapeutic composition for treating
a disease, or an immunogen for raising antibodies in an animal.
[0053] In one embodiment, the exosome is administered as a vaccine.
In another embodiment, the exosome is administered as an
immunotherapeutic composition, such as an immunosuppressive
exosome. In another embodiment, the Nef-fusion protein comprises a
Nef-derived fragment fused to an immunogenic protein from a
bacterium, virus, fungus, or protozoan. In a further embodiment,
the exosome is isolated from an antigen presenting cell, such as a
dendritic cell, B lymphocyte, or macrophage.
[0054] Another aspect of the present application relates to
immunoassay methods, compositions or devices using the Nef-fusion
protein produced by the method of the present application. In some
embodiments, the method is a detection method comprising the steps
of contacting a sample from a subject with a Nef-fusion protein
that binds specifically to a target molecule, detecting a binding
of the target molecule in the sample to the Nef-fusion protein, and
determining a level of the target molecule in the sample, wherein a
medical condition is indicated if the level of the target molecule
is outside a reference range.
[0055] The sample can be a cell sample, tissue sample or body fluid
sample, such as a blood sample or a urine sample.
[0056] In some embodiments, the Nef-fusion protein is attached to a
solid support to capture an antibody of interest or an antigen of
interest from a sample. By "solid support" is meant a non-aqueous
matrix to which the Nef-fusion protein of the present invention can
adhere or attach. Examples of solid phases encompassed herein
include those formed partially or entirely of glass (e.g.,
controlled pore glass), polysaccharides (e.g., agarose),
polyacrylamides, silicones, and plastics such as polystyrene,
polypropylene and polyvinyl alcohol. The solid support can be in
the form of tubes, microtiter plates, beads, or cells.
[0057] Examples of immunoassays include enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), flow cytometry, protein
array, microbead assay, magnetic capture, and combinations thereof.
The medical condition can comprise any disease state in which the
presence of a target antigen and/or an antibody against the target
antigen in the subject is indicative of the medical condition, such
as a cancerous conditions, a microbial infection etc.
[0058] In one embodiment, the exosome is conjugated to a solid
support. In some embodiments, exosome coated assay plates or wells
are contacted with serum from a patient and tested for the presence
or absence of antibodies binding to the Nef-fusion protein
comprising a target antigen or marker diagnostic for a medical
condition. As the antigen concentration increases in the plates or
wells the amount of antibody increases leading to a higher measured
response. Typically an enzyme is attached to the secondary antibody
which must be generated in a different species than primary
antibodies (i.e., if the primary antibody is a rabbit antibody than
the secondary antibody would be an anti-rabbit from goat, chicken,
etc., but not rabbit). The substrate for the enzyme is added to the
reaction that forms a colorimetric readout as the detection signal.
The signal generated is proportional to the amount of target
antigen present in the sample.
[0059] The antibody linked reporter used to measure the binding
event determines the detection mode. A spectrophotometric plate
reader may be used for colorimetric detection. Several types of
reporters have been recently developed in order to increase
sensitivity in an immunoassay. For example, chemiluminescent
substrates have been developed which further amplify the signal and
can be read on a luminescent plate reader. Also, a fluorescent
readout where the enzyme step of the assay is replaced with a
fluorophor tagged antibody is becoming quite popular. This readout
is then measured using a fluorescent plate reader.
[0060] In some embodiments, a competitive binding assay based on
the competition of labeled and unlabeled ligand for a limited
number of antibody binding sites may be used. Competitive
inhibition assays are often used to measure small analytes. Only
one antibody is used in a competitive binding ELISA. This is due to
the steric hindrance that occurs if two antibodies would attempt to
bind to a very small molecule. A fixed amount of labeled ligand
(tracer) and a variable amount of unlabeled ligand are incubated
with the antibody. According to law of mass action, the amount of
labeled ligand is a function of the total concentration of labeled
and unlabeled ligand. As the concentration of unlabeled ligand is
increased, less labeled ligand can bind to the antibody and the
measured response decreases. Thus the lower the signal, the more
unlabeled analyte there is in the sample. The standard curve of a
competitive binding assay has a negative slope.
[0061] In certain other embodiments, a detection marker may be
detected using exosome or Nef-fusion protein coated microbeads. In
some embodiments, the microbeads are magnetic beads. In other
embodiments, the beads are internally color-coded with fluorescent
dyes and the surface of the bead is tagged with an exosome
expressing a fusion protein of interest that can bind an antibody
in a test sample. Antibody-bound exosomes may be directly labeled
with a fluorescent tag or indirectly labeled with an anti-marker
antibody conjugated to a fluorescent tag and may contain two
sources of color, one from the bead and the other from the
fluorescent tag. The beads can then pass through a laser and, on
the basis of their color (and/or size), either get sorted or
measured for color intensity, which is processed into quantitative
data for each reaction.
Compositions Containing the Nef-Fusion Protein
[0062] A further aspect of the present application relates to
compositions for treating a disease condition in accordance with
the methods described herein. In one embodiment, the composition
comprises a Nef-fusion protein containing a Nef-derived peptide
fused to a protein of interest and a pharmaceutically acceptable
carrier. In another embodiment, the composition comprises an
exosome comprising a Nef-fusion protein containing a Nef-derived
peptide fused to a protein of interest as described above and a
pharmaceutically acceptable carrier.
[0063] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be administered to a subject, along with the nucleic acid or
vector, without causing any undesirable biological effects or
interacting in a deleterious manner with any of the other
components of the pharmaceutical composition in which it is
contained. The carrier would naturally be selected to minimize any
degradation of the active ingredient and to minimize any adverse
side effects in the subject, as would be well known to one of skill
in the art.
[0064] In another embodiments, the composition comprises a
Nef-fusion protein containing exosome further loaded with one or
more immunogenic agents, including antigens, peptides, small
molecule drugs, and nucleic acids, such as siRNAs. Such agents may
be loaded into exosomes as described above.
[0065] In other embodiments, the composition comprises an
expression vector configured to express the fusion protein so as to
redirect its localization to secreted exosomes.
[0066] Pharmaceutical carriers are known to those skilled in the
art. These most typically would be standard carriers for
administration of drugs to humans, including solutions such as
sterile water, saline, Ringer's solution, dextrose solution, and
buffered solutions at physiological pH. Typically, an appropriate
amount of a pharmaceutically-acceptable salt is used in the
formulation to render the formulation isotonic. The pH of the
solution is preferably from about 5 to about 8, and more preferably
from about 7 to about 7.5. It will be apparent to those skilled in
the art that certain carriers may be more preferable depending
upon, for instance, the route of administration and exosome
concentration being administered.
[0067] Pharmaceutical compositions may include carriers,
thickeners, diluents, buffers, preservatives, surface active agents
and the like in addition to the molecule of choice. Pharmaceutical
compositions may also include one or more active ingredients such
as antimicrobial agents, anti-inflammatory agents, anesthetics, and
the like.
[0068] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0069] Compositions for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0070] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0071] Some of the compositions may potentially be administered as
a pharmaceutically acceptable acid- or base-addition salt, formed
by reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
[0072] The exosome materials may be targeted to a particular cell
type via targeting domains as described above. The targeting domain
may be incorporated into the Nef-fusion protein or in another
coexpressed exosome protein as described above.
[0073] The pharmaceutical compositions described herein can be
packaged together in a suitable combination as a kit useful for
performing, or aiding in the performance of, the disclosed
method.
[0074] The pharmaceutical composition disclosed herein may be
administered in a number of ways depending on whether local or
systemic treatment is desired, and on the area to be treated. For
example, the compositions may be administered orally, parenterally
(e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular
injection), by inhalation, extracorporeally, topically (including
transdermally, ophthalmically, vaginally, rectally, intranasally)
or the like.
[0075] As used herein, "topical intranasal administration" means
delivery of the pharmaceutical composition into the nose and nasal
passages through one or both of the nares and can comprise delivery
by a spraying mechanism or droplet mechanism, or through
aerosolization of the pharmaceutical composition. Administration of
the composition by inhalant can be through the nose or mouth via
delivery by a spraying or droplet mechanism. Delivery can also be
directly to any area of the respiratory system (e.g., lungs) via
intubation.
[0076] Parenteral administration of the composition, if used, is
generally characterized by injection. Injectables can be prepared
in conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution of suspension in liquid prior to
injection, or as emulsions. A more recently revised approach for
parenteral administration involves use of a slow release or
sustained release system such that a constant dosage is
maintained.
[0077] The exact amount of the compositions required will vary from
subject to subject, depending on the species, age, weight and
general condition of the subject, the particular nucleic acid or
vector used, its mode of administration and the like. An
appropriate amount can be determined by one of ordinary skill in
the art using only routine experimentation given the teachings
herein. Thus, effective dosages and schedules for administering the
compositions may be determined empirically, and making such
determinations is within the skill in the art. The dosage ranges
for the administration of the compositions are those large enough
to produce the desired effect in which the symptoms of the
disorders are affected. The dosage should not be so large as to
cause adverse side effects, such as unwanted cross-reactions,
anaphylactic reactions, and the like. Generally, the dosage will
vary with the age, condition, sex and extent of the disease in the
patient, route of administration, or whether other drugs are
included in the regimen, and can be determined by one of skill in
the art. The dosage can be adjusted by the individual physician in
the event of any counter indications. Dosage can vary, and can be
administered in one or more dose administrations daily, for one or
several days. Guidance can be found in the literature for
appropriate dosages for given classes of pharmaceutical
products.
[0078] For example, a typical daily dosage of the disclosed
composition used alone might range from about 1 mg/kg to up to 100
mg/kg of body weight or more per day, depending on the factors
mentioned above.
[0079] The present invention is further illustrated by the
following examples which should not be construed as limiting. The
contents of all references, patents and published patent
applications cited throughout this application, as well as the
Figures and Tables are incorporated herein by reference.
Example 1
Characterization of HIV Type 1 Nef-Induced Exosome Secretion
[0080] Within the N-terminal 70 amino acids of HIV-1 Nef several
domains were identified as important for Nef-induced vesicle
secretion, including: (i) four arginine residues (aa 17-22)
comprising the basic region; (ii) a phosphofurin acidic cluster
sequence (PACS; Glu61-64); and (iii) a secretion modification
region (SMR) spanning amino acid residues 65-70 (VGFPV). Additional
amino acids associated with Nef secretion include P.sub.25,
29GVG.sub.31, and T44. The portion of HIV-1 Nef containing the
amino acids 1-70 was found to be sufficient to drive Nef-induced
vesicle secretion in all cell types tested.
[0081] SMS Allows Other Proteins to be Released into the
Supernatant.
[0082] The green fluorescent protein (GFP) gene was cloned
downstream of the HIV-1 Nef sequences such that a Nef-GFP fusion
protein would be expressed. Nef sequences were able to drive
secretion of GFP into the extracellular supernatant in vesicles.
The conditioned supernatant was assayed for GFP expression by a
fluorescent plate reader assay. The GFP clone alone is not secreted
into the extracellular supernatant. HIV-1 Nef .DELTA.71-206-GFP,
containing only the N-terminal 70 amino acids of HIV-1 Nef protein,
secretes GFP into the conditioned supernatant in vesicles as well
as the full wtNef-GFP construct. Red fluorescent protein (RFP)
fused to these same Nef sequences can also be secreted into the
conditioned supernatant in vesicular format. Thus, Nef N-terminal
sequences are useful for redirecting exogenous proteins into
vesicles, which are released from the cell they are expressed
in.
Materials and Methods
[0083] Cells and Reagents.
[0084] Escherichia coli STBL-2 cells (Invitrogen, Palo Alto,
Calif.) were maintained in LB broth or LB agar (Becton, Dickinson
and Company, Sparks, Md.) plates at 30.degree. C. and
plasmid-containing transformants were selected on LB agar plates
containing ampicillin (100 .mu.g/ml). Jurkat CD4+ T cell lines
derived from human T cell leukemia and human cutaneous T cell
lymphoma cells, respectively, were obtained from the NIH AIDS
Research and Reference Reagent Program (ARRRP). THP-1 and U-937
monocytic leukemia cell lines were obtained from the American Type
Culture Collection (Manassas, Va.). Cells were maintained in RPMI
1640 medium (Invitrogen) supplemented with streptomycin (100 U/ml),
penicillin (100 U/ml), L-glutamine (2.0 mM), and HEPES-buffered
saline solution (10 .mu.M). HEK293 cells derived from a human
primary embryonic kidney transformed by adenovirus type 5 were
obtained from the NIH ARRRP. The cells were maintained in 5% fetal
bovine serum HEK293 medium (Invitrogen) supplemented with
streptomycin (100 U/ml) and penicillin (100 U/ml). FRhK-4 (rhesus
monkey epithelial cells) cells were maintained in DMEM with
penicillin (100 U/ml)/streptomycin (100 U/ml), 4.0 mM L-glutamine,
4500 mg/liter glucose, 1.0 mM sodium pyruvate, 1500 mg/liter sodium
bicarbonate, and 10% fetal bovine serum. The cells were incubated
at 37.degree. C. for 2-4 days and were harvested when they reached
80-90% confluence.
[0085] The following antibodies were used: (1) rabbit polyclonal
anti-GFP antibody (Abcam, Inc., Cambridge, Mass.), (2) rabbit
polyclonal anti-Nef antibody (NIH ARRRP) and murine monoclonal
anti-Nef HIV-1 antibody (ImmunoDiagnostic, Inc., Woburn, Mass.),
(3) monoclonal anti-CD45 antibody (Abcam Inc., Cambridge, Mass.);
(4) monoclonal anti-AChE antibody (Chemicon, Temecula, Calif.), (5)
rabbit monoclonal anti-GFP antibody (Abcam Inc., Cambridge, Mass.),
(6) goat anti-Alix polyclonal antibody (Santa Cruz, Inc., Santa
Cruz, Calif.), (7) monoclonal antitubulin antibody (Sigma, St.
Louis, Mo.), (8) goat antirabbit IgG (H+L) labeled with horseradish
peroxidase (HRP; Pierce, Rockford, Ill.), (9) camptothecin (Sigma,
St. Louis, Mo.), and (10) donkey antigoat IgG-HRP (Santa Cruz,
Inc., Santa Cruz, Calif.).
[0086] Construction of the Nef mutants. The HIV-1 NL4-3 nef
construct in expression vector pQBI-Nef-GFP (Quantum
Biotechnologies, Montreal, Canada) was used as a template for
amplifying various Nef amplicons as well as for the subcloning of
the Nef mutants to create Nef-GFP fusion constructs (FIG. 1).
Nef-GFP was expressed under the control of the CMV promoter in pQBI
in the various cell types tested (HEK-293, FRhK-4, Jurkat T cells
and monocytes, THP-1/U937).
[0087] Deletion mutants of the C-terminus of HIV-1 Nef (FIG. 4A)
.DELTA.31-206, .DELTA.51-206, .DELTA.66-206, .DELTA.71-206,
.DELTA.91-206, .DELTA.151-206, and .DELTA.201-206 were constructed
by polymerase chain reaction (PCR) amplification using primers
Nef-R-5798-NheI-F, Nef-R-5735-NheI-F, Nef-R-5690-NheI-F,
Nef-R-5675-NheI-F, Nef-R-5615-NheI-F, Nef-R-5435-NheI-F, and
Nef-R-5285-NheI-F, respectively, in combination with
Nef-R-541-PvuI-R1 for PCR (see Table 1). The resulting amplicons
had NheI and PvuI restriction enzyme sites on each flank. These
amplicons were subsequently cloned into the NheI PvuI sites in the
pQBI vector. N-terminal deletion mutants of HIV-1 Nef .DELTA.1-12
and Nef .DELTA.1-40 were constructed with primers Nef13-F-SacII and
Nef41-F-SacII, respectively, in combination with GFP-R-EcoRI for
PCR amplification (see Table 1). The resulting amplicons had SacII
and EcoRI restriction enzyme sites on each flank for subcloning
into the pQBI-GFP SacII EcoRI sites. To obtain the
.DELTA.1-12/.DELTA.1-40 deletion mutants in the context of a
full-length Nef gene, the pQBI-Nef-GFP was used as a DNA template
whereas to obtain the .DELTA.1-12/.DELTA.1-40 in the context of the
first HIV Nef 70 aa, pQBI-Nef 1-70-GFP was used as a DNA
template.
[0088] For the construction of HIV-1 substitution mutants (FIG. 4B)
Nef-EEEE/4A-GFP, NefR/4A-GFP, NefK/P-GFP, NefS/A-GFP, and
NefVGFPV-GFP, primers PACS-F/PACS-R, RXRXRR-F/RXRXRR-R,
XKX-F/XKX-R, XSSX-F/XSSX-R, and VGFPV-F/VGFPV-R (Table 1),
respectively, were used for site-directed mutagenesis in
combination with the QuikChange Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, Calif.). A GFP expression plasmid (pQBI-GFP)
was constructed by amplifying GFP using GFP-1-F-SacII/GFP-R-EcoRI
primers (Table 1). The amplicon had SacII and EcoRI restriction
enzyme sites on each flank for subcloning into SacII/EcoRI sites of
the pQBI vector to yield pQBI-GFP.
[0089] All of the HIV Nef-GFP constructs used in this study were
confirmed by sequencing of both DNA strands using CMV-846-F and
GFP-1855-R primers, respectively (Table 1).
TABLE-US-00001 TABLE 1 PCR and Site-Directed Mutagenesis Primers
Used in Example 1 Primer Sequence CMV-846-F
CGTGTACGGTGGGAGGTCTATATAAGC (SEQ ID NO: 8) GFP-1855-R
CATAACCTTCGGGCATGGCACTC (SEQ ID NO: 9) Nef-R-5798-NheI-F
CATTGCTAGCCCCATCTGCTGCTGGCTCAGC (SEQ ID NO: 10) Nef-R-5735-NheI-F
CATTGCTAGCAGCTGCTGTATTGCTACTTGTGATTGC (SEQ ID NO: 11)
Nef-R-5690-NheI-F CATTGCTAGCCTCTTCCTCCTCTTGTGCTTCTAGC (SEQ ID NO:
12) Nef-R-5675-NheI-F CATTGCTAGCGACTGGAAAACCCACCTCTTCCTC (SEQ ID
NO: 13) Nef-R-5615-Nhel-F CATTGCTAGCAAAGTGGCTAAGATCTACAGCTGCCTT
(SEQ ID NO: 14) Nef-R-5435-NheI-F
CATTGCTAGCTGGCTCAACTGGTACTAGCTTGTAGCA (SEQ ID NO: 15)
Nef-R-5285-NheI-F CATTGCTAGCCGGATGCAGCTCTCGGGCCA (SEQ ID NO: 16)
Nef-R-541-PvuI-R1 GGTCCTCCGATCGTTGTCAGAAGT (SEQ ID NO: 17)
Nef13-F-SacII CAGTCCGCGGATG TGGCCTGCTGTAAGGGAAAGAATG (SEQ ID NO:
18) Nef41-F-SacII CAGTCCGCGGATG GGAGCAATCACAAGTAGCAATACAGCA (SEQ ID
NO: 19) PACS-F CTAGAAGCACAAGCGGCGGCAGCGGTGGGTTTTCCA (SEQ ID NO: 20)
PACS-R TGGAAAACCCACCGCTGCCGCCGCTTGTGCTTCTAG (SEQ ID NO: 21)
RXRXRR-F ATGTGGCCTGCTGTAGCGGAAGCAATGGCAGCAGCTGAGCCAGCA (SEQ ID NO:
22) RXRXRR-R TGCTGGCTCAGCTGCTGCCATTGCTTCCGCTACAGCAGGCCACAT (SEQ ID
NO: 23) XKX-F GCAGTATCTCGAGACCTAGAACCGCATGGAGCAATCACAAGTAGC (SEQ ID
NO: 24) XKX-R GCTACTTGTGATTGCTCCATGCGGTTCTAGGTCTCGAGATACTGC (SEQ ID
NO: 25) XSSX-F CATGGAGCAATCACAGCCGCGAATACAGCAGCTAAC (SEQ ID NO: 26)
XSSX-R GTTAGCTGCTGTATTCGCGGCTGTGATTGCTCCATG (SEQ ID NO: 27) XEEEX-F
TGGCTAGAAGCACAAGACGACGACGACGTGGGTTTTCCAGTC (SEQ ID NO: 28) XEEEE-R
GACTGGAAAACCCACGTCGTCGTCGTCTTGTGCTTCTAGCCA (SEQ ID NO: 29) VGFPV-F
CAAGAGGAGGAAGAGGCGGCTGCTGCAGCCGCTAGCAAAGGAGAA (SEQ ID NO: 30)
VGFPV-R TTCTCCTTTGCTAGCGGCTGCAGCAGCCGCCTCTTCCTCCTCTTG (SEQ ID NO:
31) GFP-1-F-SacII CAGTCCGCGGATGGCTAGCAAAGGAGAAGAACTCTTCACT (SEQ ID
NO: 32) GFP-R-EcoRI TGCAGAATTCCAGCACACTGG (SEQ ID NO: 33)
GFP-1-F-SacII CAGTCCGCGGATG GCTAGCAAAGGAGAAGAACTCTTCACT (SEQ ID NO:
34) GFP-R-EcoRI TGCAGAATTCCAGCACACTGG (SEQ ID NO: 35) Cherry-F-NheI
CGCG GCTAGC TCATCT GTGAGCAAGGGCGAGGAGGAT (SEQ ID NO: 36)
Cherry-R-BamHI CGCG GGATCC TCA CTTGTACAGCTCGTCCATGCC (SEQ ID NO:
37) Cherry-F-HindIII CGCG AAGCTT ATG GTGAGCAAGGGCGAGGAGGAT (SEQ ID
NO: 38) .sup.aAll primers are from 5' to 3' orientation.
[0090] Cell Transfection.
[0091] HEK293cells were grown in serum-free medium (GIBCO 293
Freestyle, Invitrogen) at 37.degree. C. to a confluence of 75-80%.
Cells were trypsinized, washed, and counted before transfection
with wtNef-GFP and Nef mutants using electroporation (Bio-Rad Model
1652108). Jurkat, FRhK-4, THP-1, and U937 monocytes were grown in
serum-free RPMI 1640 medium and then diluted to a final
concentration of 1.times.10.sup.6 cells/100 .mu.l of medium and
mixed with 1 .mu.g of plasmid DNA. The cells were transferred to
electroporation cuvettes (2 mm, Bio-Rad), pulsed at 140V (Jurkat),
130V (FRhK-4), and 140V (THP-1 and U937 monocytes) using a Bio-Rad
Model Gene Pulser Xcell system, following the manual to select
conditions. The cell/DNA solution was then centrifuged at
600.times.g for 5 min, the floating dead cells were removed, and
the pellet was resuspended in 1 ml of fresh media containing 5%
fetal bovine serum (FBS). The cells were put in culture plates and
incubated for 48 h at 37.degree. C. Cells were collected by
centrifuging at 600.times.g for 5 min. The cells were mounted on a
slide and the transfection efficiency was calculated by counting
the green fluorescent cells using a fluorescent microscope.
[0092] Propidium Iodide (PI) Assay.
[0093] HEK293 cells were transfected with pQBI/HIV-1 Nef mutant
plasmid DNA for 48 h as described above. The cells were washed in
PBS after which freshly prepared PI solution (1.25 .mu.g/ml) was
added. The cells were incubated at room temperature for 2 min and
examined immediately under a microscope, with dead cells staining
red.
[0094] TUNEL Assay.
[0095] The HEK293 cells were transfected with pQBI/HIV-1 wtNef-GFP
or wtNef-RFP plasmid DNA for 48 h as described above. The cell
cultures were assayed for apoptosis by TUNEL assay, by
epifluorescence detection, on a computer-controlled fluorescence
microscope system (Carl Zeiss, Thornwood, N.Y.). Cells transfected
with wtNef-RFP were visualized as red, whereas the TUNEL-labeled
apoptotic cells were green.
[0096] Exosome Isolation and Purification from the Transfected
Cells.
[0097] Cells transfected with HIV-1 wtNef-GFP (10.sup.6 cells/ml,
as described above) were harvested at 48 h posttransfection. The
cells were removed from the culture media by centrifugation at
600.times.g for 5 min. The cell-free supernatant was subjected to a
second spin at 10,000.times.g for 30 min to pellet the cell debris.
Exosomes were collected by sequential centrifugations of this
cleared supernatant at 50,000.times.g for 45 min, 100,000.times.g
for 1 h, and 400,000.times.g for 2 h at 4.degree. C. As a negative
control, culture media from a similar volume of untransfected cells
were also subjected to sequential centrifugations. It was further
determined that exosome-like vesicles could be isolated from
untransfected Jurkat cells by starting with conditioned media from
a larger number (2.5.times.10.sup.7 cells) of cells using the same
procedure.
[0098] Exosome Flotation on Continuous Sucrose Gradients.
[0099] Jurkat cell cultures were transfected and distributed in
35-mm dishes (1 ml/dish) as described. For the preparation of
exosomes on flotation gradients, 28 ml of untransfected Jurkat cell
cultures and 14 ml of HIV-1 wtNef-GFP-transfected Jurkat cell
cultures were centrifuged for 5 min at 600.times.g to remove the
cells. The cell pellets (see FIG. 2, lane 1) were set aside for
processing (SDS-PAGE and Western blot, described below). The
cell-free supernatants were then centrifuged for 10 min at
1200.times.g and an aliquot (4 ml untransfected and 1 ml
wtNef-GFP-transfected) of this 1200.times.g clarified supernatant
(see FIG. 2, lane 2) was also processed for Western blotting. The
remaining clarified supernatants (24 ml untransfected and 12 ml
wtNef-GFP-transfected) were subjected to sequential centrifugation
for 30 min at 10,000.times.g, 45 min at 50,000.times.g, 60 min at
200,000.times.g, and 60 min at 400,000.times.g, using a Type 42.1
ultracentrifuge rotor (Beckman Instruments, Inc., Fullerton,
Calif.). The 50,000.times.g pellets were saved for Western blotting
(see FIG. 2, lane, 3). The 200,000.times.g and 400,000.times.g
pellets were resuspended in 1 ml of 2.5 M sucrose, 20 mM
HEPES/NaOH, pH 7.2. An aliquot (250 .mu.l) of each sucrose
suspension was centrifuged at 400,000.times.g for 60 min. These
samples (see FIG. 2, lane 4) were set aside for Western analysis. A
10-ml linear sucrose gradient (2.0-0.25 M sucrose, 20 mM
HEPES/NaOH, pH 7.2) was layered on top of the remaining 750 .mu.l
of sucrose suspension in a Beckman Ultra-Clear 14.times.95-mm tube
and centrifuged at 100,000.times.g for 16 h using a Type 40-Ti
rotor (Beckman Instruments, Inc.). Gradient fractions (12 fractions
of 750 .mu.l) were collected, subsequently diluted 1:3 with
phosphate-buffered saline (PBS), and centrifuged for 60 min at
400,000.times.g using a TLA 100.4 rotor (Beckman Instruments,
Inc.). The resultant pellets (gradient fractions; (see FIG. 2,
lanes 5-12) were set aside for Western blot analysis.
[0100] Processing of Fractions for SDS-PAGE.
[0101] Aliquots of the 1200.times.g clarified supernatants from
untransfected and wtNef-GFP-transfected cultures and the fractions
from the other steps were centrifuged at 400,000.times.g. The
pellets were collected and lysed in 2.times.SDS-PAGE sample buffer
and heated at 95-100.degree. C. for 5 min. The 400,000.times.g
spent supernatants after differential centrifugation were processed
by trichloracetic acid (TCA) and acetone precipitation. TCA was
added to each supernatant to a final concentration of 15% and the
precipitates were allowed to form at 4.degree. C. overnight.
Precipitated proteins were collected by centrifugation at
16,000.times.g for 30 min and the pellets were washed twice with
ice-cold acetone and finally resuspended in 2.times.SDS sample
buffer for analysis.
[0102] Fluorescent Plate Reader Assay.
[0103] One hundred microliters of cell-free conditioned media was
transferred to each well of a 96-well black microtiter plate
(Corning Incorporated, NY). These were assayed for fluorescence on
a Tecan GENEios fluorimeter (Tecan Group, Switzerland) with
excitation wavelength 485 nm and emission wavelength 515 nm.
Conditioned media from pQBI-GFP-transfected and untransfected cells
were used as positive and negative control, respectively.
[0104] Immunoblot Analysis.
[0105] Cells and vesicle proteins were analyzed by Western blot
analysis. The cell or vesicles protein samples were separated by
SDS-PAGE on a 4-20% Tris-HCl-Criterion precast gel (Bio-Rad
Laboratories, Hercules, Calif.) and electrophoretically transferred
to the nitrocellulose membrane. The membrane was washed in
Tris-buffered saline (TBS) for 5 min, blocked with 5% nonfat milk
in TTBS (TBS with 0.1% Tween 20) for 1 h by shaking at room
temperature, processed for immunoblotting using a specific first
primary antibody with shaking at 4.degree. C. overnight, followed
by a secondary HRP-conjugated IgG (H+L) antibody. Protein bands
were detected by Western Blotting Luminol Reagent (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.) followed by an exposure to
photographic film (BioMax film; Fisher Scientific, Pittsburgh,
Pa.). In some experiments, the membrane was stripped using a
stripping reagent (Pierce, Rockford, Ill.) and used to hybridize
with a different primary and secondary antibody. The X-ray films
were scanned into Adobe Photoshop 5.0.2 and arranged for
publication in Adobe Illustrator 10 (Adobe Systems, San Jose,
Calif.).
[0106] Nef protein sequence alignment. The consensus Nef amino acid
sequence for each HIV-1 clade (A through O) was determined by
alignment of individual Nef variant sequences downloaded from the
HIV Sequence Database (Los Alamos National Laboratory) using the
algorithms in GENEious Pro 4.0.2 (Biomatters Ltd., Auckland, NZ).
Specifically, alignments were generated using a Blosum62 Cost
Matrix, with a gap opening penalty=12 and gap extension penalty=3.
The 13 HIV-1 clade consensus sequences thus determined were then
submitted for alignment in GENEious Pro, using the same
parameters.
[0107] Data Analysis.
[0108] The numerical and graphic analyses of all data obtained were
obtained through analysis using at least three repetitions of each
experiment. Data were calculated and graphs were generated using
SigmaPlot 10 (Systat, San Jose, Calif.). One-sided Student's t-test
analysis was used to compare data conditions.
[0109] Exosome Secretion.
[0110] As shown in FIG. 1, when Nef-GFP is transfected in Jurkat
cells, AChE and CD45 (exosomal marker proteins) are released also.
This suggests that more of these two proteins were secreted from
Nef-GFP-transfected cells (FIG. 1B, lower panel set; FIG. 1C) than
from untransfected cells (FIG. 1B, top panel set; FIG. 1C).
Nef-GFP-transfected cells also display an increase in intracellular
AChE and CD45 concomitant with AChE release (FIG. 1A, UT vs. Nef,
AChE; FIG. 1C) or CD45 release (FIG. 1A, UT vs. Nef, CD45; FIG. 1C)
while no change in intracellular tubulin is observed (FIG. 1A, UT
vs. Nef, tubulin). This clearly establishes a Nef protein-induced
increase in intracellular AChE and CD45 concomitant with release of
Nef, AChE, and CD45 in high-molecular-weight format. This is
consistent with intracellularly expressed Nef-inducing secretion of
vesicles containing Nef, AChE, and CD45.
[0111] Nef Protein is Found in Vesicular Form and not in Soluble
Form.
[0112] If Nef is associated with vesicles, some fraction of the
secreted material should be membrane associated. This can be
demonstrated by subjecting the pelleted material from the cell
supernatants to membrane flotation. Thus, Jurkat cell cultures were
transfected with pQBI-Nef-GFP expressing full-length HIV-1 NL4-3
Nef, and the conditioned media from these and from untransfected
cells were collected, lysed, assayed for total protein, and stored
for Western analysis. Conditioned cell media were spun at
1200.times.g for 10 min, the supernatant was collected, and an
aliquot of this was set aside for Western analysis. The bulk of the
material was subjected to differential centrifugation at
10,000.times.g, 50,000.times.g, 200,000.times.g, and
400,000.times.g and the pellets from each spin were collected. The
50,000.times.g pellet was set aside to be assayed, aliquots of the
200,000.times.g and 400,000.times.g pellets were set aside for
assay, and the bulk of these two pellets was loaded onto sucrose
gradients and subjected to flotation centrifugation. Fractions from
each gradient were collected and were assayed. Finally, the spent
supernatant from the 400,000.times.g differential centrifugation
step was TCA precipitated and the pellet was resuspended in a small
volume to be assayed. Each of these collected samples was assayed
by SDS-PAGE and Western analysis probing for Nef, GFP, and Alix, an
exosomal marker. Representative Western blot images for
untransfected cultures and NefGFP-expressing cultures are shown in
FIG. 2A with collated densitometric measurements of multiple
gradients shown in FIG. 2B.
[0113] As shown in FIG. 2B, all of the Nef protein in the
conditioned cell media was pelleted in the differential centrifuge
steps and was found in the floated fractions of the flotation
gradients. In contrast, no (soluble) Nef protein or GFP was
detected in the 400,000.times.g spent supernatant fraction (data
not shown). Second, in the flotation gradients of pelleted
vesicles, the peak band densities for Nef, GFP, and Alix were
detected in gradient fractions 6-8 (FIG. 2B, lanes 7-9). The
vesicle preparations floated at a sucrose density of 1.11-1.17,
which is similar to flotation data reported for exosomes from
B-lymphocytes (Raposo et al., J. Exp. Med., 183(3):1161-1172,
1996). Third, the amount of Alix (as measured by band densities) in
all four fractions assayed was larger in the Nef-GFP-expressing
cultures than in the untransfected cultures (FIG. 2B; all p values
were less than 0.01). Furthermore, the difference in amount of Alix
in Nef-GFP expressing vs. untransfected cell lysates and
supernatants was smaller than that observed for untransfected cell
lysates vs. supernatants (FIG. 2B). Finally, Nef, GFP, and Alix
densitometric measurements in the differential centrifugations and
the sucrose flotation gradient were found to be approximately
equivalent. All this suggests that Nef increases intracellular
expression of at least some specific proteins, and is released from
transfected cells in vesicular form and in vesicles containing the
exosomal marker Alix.
[0114] The Genetics of Exosome Secretion.
[0115] The N-terminal 70 amino acids of Nef are sufficient to
induce secretion. As shown above, Nef-GFP transfected into cells
appears to induce release (secretion) of itself in
high-molecular-weight form along with AChE and CD45. This suggests
that sequences or motif(s) on Nef protein actively induce and
regulate this release/secretion function. Truncation mutants
deleting various lengths of the C-terminal
region--Nef.DELTA.31-206GFP, Nef.DELTA.51-206GFP,
Nef.DELTA.71-206GFP, Nef.DELTA.91-206GFP, Nef.DELTA.151-206GFP, and
Nef.DELTA.201-206GFP (FIG. 4A)--were developed to examine their
ability to induce secretion of Nef-GFP into the conditioned media
using transient transfection of HEK293 cells (FIG. 6A). The clone
pQBI-Nef-GFP (wt in FIG. 6A), containing the full-length HIV-I
NL4-3 Nef, was used as a positive control, while pQBI-GFP,
containing only the GFP sequence, was used as a negative control in
some experiments. Media collected from the cells transfected with
pQBI-Nef.DELTA.71-206GFP (1-70 in FIG. 6A),
pQBI-Nef.DELTA.91-206GFP (1-90 in FIG. 6A),
pQBI-Nef.DELTA.151-206GFP (1-150 in FIG. 6A), and
pQBI-Nef.DELTA.201-206GFP (1-200 in FIG. 6A) displayed fluorescence
comparable to the cells transfected with full-length nef-containing
plasmid. Alternatively, conditioned media from cells transfected
with pQBI-Nef.DELTA.31-206GFP (1-30 in FIG. 6A) and
pQBI-Nef.DELTA.51-206GFP (1-50 in FIG. 6A) displayed only
background levels of fluorescence comparable to the negative
control. These results showed that the N-terminal 70 aa of HIV-1
Nef were sufficient to induce secretion of the Nef-GFP protein into
the conditioned media.
[0116] The PACS Motif (.sup.62-65E) was Required for Nef-Induced
Vesicle Secretion.
[0117] Because the first 70 amino acids of Nef were sufficient for
the secretion of Nef-GFP but the first 50 amino acids were not, it
was anticipated that a secretion regulatory motif was within amino
acids 50-70. There were two known motifs within this 20-amino acid
region: (1) amino acids 51-61 are the apoptotic motif (James et
al., J. Virol., 78(6):3099-3109, 2004) and (2) amino acids 62-65
are the phosphofurin acidic cluster sequence (PACS) motif (Piguet
et al., Nat. Cell Biol., 2(3):163-167, 2000). The PACS replacement
mutant clone pQBI-Nef.sup.62EEEE.sup.65/4AGFP (PACS in FIG. 6B) was
constructed by replacing the four glutamic acid residues with four
alanine residues as described in Materials and Methods (FIG. 4B).
As shown in FIG. 6B, conditioned media collected from cells
transfected with pQBI-Nef.sup.62EEEE.sup.65/4AGFP had only
background fluorescence whereas pQBI-Nef.DELTA.71-206GFP (1-70 in
FIG. 6B) had fluorescence comparable to that of pQBI-NefGFP (wt in
FIG. 6B). This result suggested that the PACS region of HIV-1 Nef
is a secretion regulatory motif.
[0118] The Helix-1 Domain but not the Myristoylation Domain is
Required for Nef Secretion.
[0119] Within the N-terminal 70 amino acids, five distinct motifs
have been identified as being involved in membrane interactions
(FIG. 5A). These include the myristoylation region (amino acid 2),
basic amino acid region 1 (BAA-1; Lys4 and Lys7), basic amino acid
region 2 (BAA-2; Arg17, 19, 21, 22), which overlaps with helix-1
(Trp13-Arg21), the helix-2 (Ser34-Gly41; Geyer et al., J. Mol.
Biol., 289(1):123-138, 1999) and the plasma membrane targeting
domain (PMTD, Gly41-Ala60). Similar domains are also found in
SIV-Nef (FIG. 5B). It was possible that these or other as yet
unidentified domains were also required for Nef-induced secretion.
Several truncation mutants with N-terminal amino acids deleted
(pQBI-Nef .DELTA.1-12GFP and pQBI-Nef .DELTA.1-40GFP) were
constructed by deleting 1-12 aa (myristoylation region and BAA-1
were deleted) and 1-40 aa (BAA-2/helix-1 and helix-2 were deleted),
respectively. No fluorescence was observed in the conditioned media
collected from cultures transfected with pQBI-Nef .DELTA.1-40GFP
(41-70 in FIG. 6A), but conditioned media from pQBI-Nef
.DELTA.1-12GFP (13-206 in FIG. 6A), pQBID1-12/D71-206GFP (13-70 in
FIG. 6A), and pQBI-Nef.DELTA.71-206GFP (1-70 in FIG. 6A) exhibited
fluorescence intensity comparable to that of pQBI-NefGFP (wt in
FIG. 6A). Cultures transfected with the mutant pQBI-NefG2A (G2A in
FIG. 6B) and pQBI-NefK4K7/2A (NefK4K7 in FIG. 6B) also displayed
fluorescence levels comparable to the wild-type construct,
confirming the data obtained with deletion constructs. This
indicated that the myristoylation domain and basic region 1 were
not involved in Nef-induced secretion, whereas either the helix-1
or -2 regions, or another, as yet, unidentified domain between 13
and 41 aa was required for the secretion.
[0120] The Basic Amino Acid Motif in Helix-1 is Required for
Secretion.
[0121] To determine what domain(s) between 13 and 41 aa was
required for the secretion, several mutant clones were constructed
(FIG. 4B). These were pQBI-Nef.sup.17,19,21,22R/4AGFP, in which the
four basic arginines of BAA-2/helix-1 were replaced with four
alanines; pQBI-NefK/PGFP, in which a proline was inserted in place
of .sup.39K as a helix breaker in helix-2; and
pQBI-Nef.sup.45,46S/AGFP, in which the PMTD was mutated replacing
the two serines at positions 45 and 46 with two alanines. The
mutations in pQBI-Nef.sup.39K/PGFP (FIG. 6B, K39P) and
pQBI-Nef.sup.45,46S/AGFP (FIG. 6B, SS4546AA) had no effect on
secretion of fluorescence in the conditioned media from transfected
cultures comparable to that of the pQBI-Nef.DELTA.71-206GFP (FIG.
6B, 1-70) or pQBI-NefGFP (FIG. 6B, wt). Cultures transfected with
pQBI-Nef.sup.17,19,21,22R/4AGFP (FIG. 6B, 4R4A) had significantly
decreased fluorescence in the conditioned media suggesting that
basic region 2 in helix-1 is important for Nef secretion.
[0122] Other Previously Unexplored Sequences on Nef are Required
for Secretion.
[0123] To determine the minimum N-terminal sequence required for
secretion we constructed a C-terminal truncation removing all amino
acids after the PACS motif (pQBI-Nef.DELTA.66-206GFP; FIG. 4A). A
significant decrease in the fluorescence in the conditioned media
from cells transfected with this construct was observed (FIG. 6A,
1-65). This suggested that a third secretion regulatory motif lay
within the amino acids 66-70 (VGFPV; see FIG. 5A). Using an alanine
replacement mutant clone, pQBI-Nef.sup.66VGFPV.sup.70GFP, with
amino acids .sup.66VGFPV.sup.70 replaced with five alanines,
significantly decreased fluorescence was observed in conditioned
media collected from these cultures (FIG. 6B, VGFPV/5A). Thus, this
region, a domain not previously described in the literature that we
named the secretion modification region (SMR), is a third region
important for Nef secretion.
[0124] A phylogenetic analysis of HIV-1 Nef amino acids 1-70
intra-B-clade and across all HIV-1 clades found that the secretion
domains are highly conserved within the SMR region. with the newly
identified. The SMR was 100% conserved across all HIV-1 clades.
This evidence indicates the relevance of these domains,
particularly in a virus that displays high sequence variability.
Further, domain conservation was also found to apply when the
N-terminal sequences of HIV-1 and SIV were compared (data not
shown). Although most of the Nef secretion regulatory sequences
were found in the Nterminal 102 amino acids of SIV Nef, the three
functional motifs in association with Nef secretion in
high-molecularweight form are very similar to HIV and comprise two
BAA regions, a PACS domain and an SMR-like region located
immediately downstream of the PACS.
[0125] To characterize the SMR more fully, an individual alanine
replacement analysis was performed. Five clones were developed
containing the full-length nef gene with nucleotides coding for one
of the five amino acids of the SMR replaced with nucleotides for
alanine (see FIG. 4B; lanes 5-9). Alanine replacement mutants V66A,
G67A, and V70A each displayed only background levels (FIG. 6C,
AGFPV, VAFPV, VGFPA; 1.8%, 2%, 1.9%, respectively), similar to the
ones measured by the pQBI-GFP-negative control (FIG. 6C, pQBI-GFP;
.about.1.7%), of extracellular fluorescence in the conditioned
media collected from the transfected cultures. Alanine replacement
mutant P69A displayed a small but reproducible amount of
extracellular fluorescence (FIG. 6C, VGFAV; .about.6%) compared to
the positive control. Alanine replacement mutant F68A displayed a
reduced but significant amount of extracellular fluorescence (FIG.
6C, VGAPV; .about.30%) in the conditioned media as compared to the
positive control. Thus, three of the five amino acids are critical
for secretion, with single mutations in any one of those three
leading to complete elimination of the ability of Nef to induce
secretion of itself in vesicles.
[0126] The amino acids between R22, the C-terminal amino acid in
the BAA-2 motif in helix-1 and E62, the N-terminal amino acid in
the PACS domain, were also screened using alanine replacement
identifying several amino acids that influence secretion. These
clones were developed in the full-length nef background. The
pQBI-NefP25A-GFP clone (FIG. 4B) displayed background amounts of
extracellular fluorescence (FIG. 6B, P25A; .about.4%) in the
conditioned media as compared to the positive control.
pQBI-Nef.sup.29GVG.sup.313A (FIG. 4B) and pQBI-NefT44A-GFP clone
(FIG. 4B) also displayed background amounts of extracellular
fluorescence (FIG. 6B, 29GVG31/3A, 4%; T44A, 4% respectively).
[0127] These Domains are Relevant in Other Cell Lineages.
[0128] The initial secretion analysis described above was performed
in HEK293 cells. These cells are easily transfectable and do not
normally secret vesicles. Thus, they are optimal for viewing
secretion and identifying changes in the secretion ability although
not a normal target for viral infection. More appropriate would be
Nef secretion analysis of these constructs in either lymphocytic or
monocytic cell lines as these lineages are targets of HIV
infection. Specific Nef mutants described above were analyzed in a
lymphocytic cell line (Jurkat cells) and in two monocytic lines
(THP-1 and U937 cells; FIG. 6). The pQBI-Nef.sup.17,19,21,22R/4AGFP
mutant clone (BAA-2 region knockdown), the
pQBI-Nef.sup.62EEEE.sup.65/4AGFP mutant clone (PACS region
knockdown), and the pQBI-NefV.sup.65AGFP mutant clone (SMR region
substitution mutation knockdown) all displayed extracellular
fluorescence levels in lymphocytic and monocytic cells similar to
those observed in HEK293 cells. There was some variation in the
extracellular fluorescence levels of the truncation mutant's
transfected in lymphocytic (FIG. 6D, 1-70, 13-70, 13-206) and
monocytic cell lines (FIG. 6E or 6F, 1-70, 13-70, 13-206) relative
to each other or to HEK293 cells (FIG. 6A, 1-70, 13-70, 13-206).
However, the variations observed were not significant and the trend
for each of these truncation mutants was for them to display
wild-type or close to wild-type levels of fluorescence.
[0129] Phylogenetic analysis across HIV clades. The genetic
analysis of Nef secretion was performed using HIV-1 NL4-3 Nef. A
logical next step was to determine the conservation of the
identified secretion domains across HIV B clade viruses and across
the other HIV-1 clades uncovering the relative importance of these
domains. An analysis of that region of Nef involved in secretion
(amino acids 1-70) demonstrates significant sequence conservation
within the secretion domains across all HIV-1 clades (FIG. 7).
Interestingly, the SMR domain, which was always found contiguous to
and C-terminal of the PACS domain, displayed 100% sequence
conservation across all the HIV clades suggesting the importance of
these sequences.
[0130] HIV Nef Expressed in Cells is not Toxic/Apoptotic to
Transfected Cells.
[0131] One alternative explanation of the effects being observed is
that endogenous Nef protein causes toxicity to the cells in which
it is expressed, leading to those cells releasing Nef protein in
apoptotic microvesicles or microparticles. Prior studies of cells
releasing putative exosomes have shown that cells in the early
stages of apoptosis release membrane vesicles that are very similar
to vesicles released by healthy cells (e.g., exosomes; Thery et
al., J. Immunol., 166(12):7309-7318, 2001; Aupeix et al., J. Clin.
Invest., 99(7):1546-1554, 1997). However, the protein composition
of the apoptotic vesicles was different from that of the exosomal
vesicles. For example, the apoptotic vesicles contained large
amounts of histones as opposed to little or no histone protein
found in the exosomal vesicles.
[0132] It was previously shown that soluble recombinant Nef (rNef)
protein and the conditioned supernatant from Nef-transfected cells
are apoptotic to naive cells expressing CXCR4 (Huang et al., J.
Virol., 78(20):11084-11096, 2004). Thus, it is possible that these
Nef-containing vesicles represent apoptotic vesicles. To evaluate
this possibility, cells were transfected with the various Nef-GFP
constructs for cell death and apoptosis (FIG. 8) and the
supernatant/vesicles released from the Nef-transfected cells were
examined for histone content in the vesicles, a marker of apoptotic
vesicles (FIG. 9).
[0133] HEK293 cells were transfected with specific Nef constructs
described above, and the cell populations were stained with PI.
These cells were analyzed for GFP fluorescence (NefGFP expression),
PI fluorescence (necrotic cells hallmark of cell death), and
coincidence of PI and GFP (dying cells expressing Nef) in the cells
(FIG. 8A). Endogenously expressed GFP fluorescence, a measure of
Nef expression, for all treatments ranged between 70% and 80% and
did not vary significantly. PI fluorescence, a measure of cell
death, varied from 3% (pQBI-GFP; FIG. 8A, PI measure) in the
negative control and the Nef mutants to .about.12% (pQBI-NefGFP;
FIG. 8A, PI measure) in the transfections with wtNef-GFP. Thus,
wtNef-GFP protein expressed within the cells does increase the
amount of cell death by about 4-fold with about half of that cell
death occurring in the transfected cells (see FIG. 8A, wtNef-GFP,
GFP/PI overlay measure). However, the total amount of cell death
remained modest.
[0134] HEK293 cells were transfected with wild-type pQBI-Nef-RFP
and then TUNEL labeled for detection or earlier signs of apoptosis
in the form of DNA fragmentation. These cells were analyzed for RFP
fluorescence (Nef-RFP expression), TUNEL (apoptosis), and the
coincidence of RFP and TUNEL (apoptotic cells expressing Nef) in
the cells (FIG. 8B). Endogenously expressed RFP fluorescence, a
measure of Nef expression, for all treatments ranged between 75%
and 80% and did not vary significantly. FITC fluorescence, in
TUNEL-labeled apoptotic cells, ranged from 2% (pQBI-RFP, FIG. 8B,
RFP measure) in the negative control to .about.12% (pQBI-Nef-RFP,
TUNEL measure) in the transfections with wtNef. Again, wtNef
protein expressed within cells increased the amount of apoptosis by
about 6-fold with half of that apoptosis occurring in the
transfected cells (see FIG. 8B, wtNef-RFP, RFP/TUNEL overlay
measure). Again, the total amount of cell death in the population
was very modest.
[0135] Thus, evidence for direct and indirect induction of
apoptosis was present but minimal. Next, to see whether transfected
cells released histone-containing apoptotic vesicles into the
conditioned supernatant, HEK293 cells were either treated with
camptothecin, an apoptosis-inducing factor (FIG. 9A, lanes 1 and
2), or transfected with pQBI-NefGFP (FIG. 9A, lanes 3 and 4),
pQBI-Nef.sup.66VGFPV.sup.70GFP (FIG. 9A, lanes 5 and 6), or
pQBI-GFP (FIG. 9A, lanes 7 and 8). The 48 h cultures were harvested
for the conditioned media and the cell lysates. The conditioned
media from each treatment were subjected to differential
centrifugation with four sequential centrifugation steps of
300.times.g, 1200.times.g, 10,000.times.g, and finally
130,000.times.g. A silver-stained SDS-PAGE analysis of the cell
lysates (FIG. 9A, lanes 1, 3, 5, and 7) and 130,000.times.g pellets
(FIG. 9A, lanes 2, 4, 6, and 8) was examined for the protein
composition of those two fractions. The banding pattern in the
camptothecin-treated cells (FIG. 9A, lanes 1 and 2) was distinct
from the other three transfection treatments (FIG. 9A, lanes
3-8).
[0136] To specifically look at the histones in these treatment
conditions, SDS-PAGE analyses of the cell lysate of each treatment
and the pellets from each centrifugation step were screened by
Western analysis (FIG. 9A). This was done with (1) a histone
polyclonal antibody (FIG. 9B, first panel set) to screen and
quantify histones, (2) a GFP antibody (FIG. 9B, second panel set),
and (3) an HIV-1 Nef antibody (FIG. 9B, third panel set). The
camptothecin-treated cells (FIG. 9B, histone set, panel one)
displayed a histone band in both the cell lysate as well as in all
four differential centrifugation-generated pellets as expected
following camptothecin-induced apoptosis: histones were detected in
both the cell lysates and vesicles released in the supernatants. In
comparison, HEK293 transfected with pQBI expressing wtNef, SMR
mutated Nef, or untransfected control, histone bands are detected
only in the cell lysates and in the low-speed centrifugations
(300.times.g and 1200.times.g) in which cellular debris is normally
pelleted. This suggests that Nef transfection does not result in
significant release of apoptotic histone-containing vesicles. The
transfected and wtNef-GFP-expressing cultures analyzed by GFP
antibodies (FIG. 9B, GFP set, panel two) or by Nef antibodies (FIG.
9B, Nef set, panel two) display Nef-GFP protein in both the cell
lysate as well as in all four differential centrifugation
conditions. This indicates that Nef is there in a
high-molecular-weight format indicative of Nef-containing
vesicles.
[0137] The evidence suggests that despite finding an increased (but
small total) amount of cell death/apoptosis in the Nef-transfected
cells, the vesicles released from these cultures have very little
if any histones in them, suggesting a morphology distinct from
apoptotic vesicles. Alternatively, they do have Nef-GFP in them,
suggesting that the Nef-containing vesicles may be exosomes.
[0138] The Effect of Nef Mutants was not Due to Variable
Expression.
[0139] The effects observed in the various mutants could be due to
variation in the ability of each clone to express the resultant
fusion protein and not due to differences in their ability to
secrete the fusion protein. This issue was addressed by examining
the expression pattern of untransfected and transfected HEK293
cells by Western analysis of whole cell extracts probed with
anti-Nef antibody (FIG. 10A) or anti-GFP antibody (FIG. 10B).
Cultures were transfected with pQBI-Nef-GFP (Nef-GFP; FIG. 10, lane
1), pQBI-GFP (GFP; FIG. 10, lane 2),
pQBI-Nef.sup.62EEEE.sup.65/4AGFP (PACS; FIG. 10, lane 3),
pQBI-NefV66/A (SMR AGFPV, FIG. 10, lane 4),
pQBI-Nef.sup.17,19,21,22R14AGFP (Basic Region 2, FIG. 10, lane 5),
or untransfected (FIG. 10, lane 6). FIG. 10D is the densitometric
analysis of FIGS. 10A-C. The Nef and GFP band densities in the
mutant expressing cultures are similar (FIG. 10D). Alternatively, a
significant difference was observed in the band densities of
wtNef-GFP-expressing cells vs. the Nef mutant-expressing cells
(FIG. 10D, wtNef-GFP vs. all others). This suggests that NefGFP
protein made and released in the wtNef-GFP-expressing cells
accumulates within the mutant Nef-GFP-expressing cells.
[0140] The above description is for the purpose of teaching the
person of ordinary skill in the art how to practice the present
invention, and it is not intended to detail all those obvious
modifications and variations of it which will become apparent to
the skilled worker upon reading the description. It is intended,
however, that all such obvious modifications and variations be
included within the scope of the present invention, which is
defined by the following claims. The claims are intended to cover
the claimed components and steps in any sequence which is effective
to meet the objectives there intended, unless the context
specifically indicates the contrary.
Sequence CWU 1
1
521206PRTHuman immunodeficiency virus type 1 1Met Gly Gly Lys Trp
Ser Lys Ser Ser Val Ile Gly Trp Pro Ala Val 1 5 10 15 Arg Glu Arg
Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala 20 25 30 Val
Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr 35 40
45 Ala Ala Asn Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu
50 55 60 Glu Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro
Met Thr 65 70 75 80 Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys
Glu Lys Gly Gly 85 90 95 Leu Glu Gly Leu Ile His Ser Gln Arg Arg
Gln Asp Ile Leu Asp Leu 100 105 110 Trp Ile Tyr His Thr Gln Gly Tyr
Phe Pro Asp Trp Gln Asn Tyr Thr 115 120 125 Pro Gly Pro Gly Val Arg
Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys 130 135 140 Leu Val Pro Val
Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu 145 150 155 160 Asn
Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro 165 170
175 Glu Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His
180 185 190 His Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys
195 200 205 2263PRTSimian immunodeficiency virus 2Met Gly Gly Ala
Ile Ser Met Arg Arg Ser Arg Pro Ser Gly Asp Leu 1 5 10 15 Arg Gln
Arg Leu Leu Arg Ala Arg Gly Glu Thr Tyr Gly Arg Leu Leu 20 25 30
Gly Glu Val Glu Asp Gly Tyr Ser Gln Ser Pro Gly Gly Leu Asp Lys 35
40 45 Gly Leu Ser Ser Leu Ser Cys Glu Gly Gln Lys Tyr Asn Gln Gly
Gln 50 55 60 Tyr Met Asn Thr Pro Trp Arg Asn Pro Ala Glu Lys Arg
Glu Lys Leu 65 70 75 80 Ala Tyr Arg Lys Gln Asn Met Asp Asp Ile Asp
Glu Glu Asp Asn Asp 85 90 95 Leu Val Gly Val Ser Val Trp Pro Arg
Val Pro Leu Arg Thr Met Ser 100 105 110 Tyr Lys Leu Ala Ile Asp Met
Ser His Phe Ile Lys Glu Lys Gly Gly 115 120 125 Leu Glu Gly Ile Tyr
Tyr Ser Glu Arg Arg His Arg Ile Leu Asp Ile 130 135 140 Tyr Leu Glu
Lys Glu Glu Gly Ile Ile Pro Asp Trp Gln Asp Tyr Thr 145 150 155 160
Ser Gly Pro Gly Ile Arg Tyr Pro Lys Thr Phe Gly Trp Leu Trp Lys 165
170 175 Leu Val Pro Val Asn Val Ser Asp Glu Ala Gln Glu Asp Glu Glu
His 180 185 190 Cys Leu Ile His Pro Ala Gln Thr Ser Gln Trp Asp Asp
Pro Trp Gly 195 200 205 Glu Val Leu Ala Trp Lys Phe Asp Pro Thr Leu
Ala Tyr Thr His Glu 210 215 220 Ala Tyr Val Arg Tyr Pro Glu Glu Phe
Gly Ser Lys Ser Gly Leu Ser 225 230 235 240 Glu Glu Glu Val Arg Arg
Arg Leu Thr Ala Arg Gly Leu Leu Asn Met 245 250 255 Ala Asp Lys Lys
Glu Thr Arg 260 329PRTHuman immunodeficiency virus type 1 3Trp Pro
Ala Val Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp 1 5 10 15
Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly 20 25
4102PRTSimian immunodeficiency virus 4Met Gly Gly Ala Ile Ser Met
Arg Arg Ser Arg Pro Ser Gly Asp Leu 1 5 10 15 Arg Gln Arg Leu Leu
Arg Ala Arg Gly Glu Thr Tyr Gly Arg Leu Leu 20 25 30 Gly Glu Val
Glu Asp Gly Tyr Ser Gln Ser Pro Gly Gly Leu Asp Lys 35 40 45 Gly
Leu Ser Ser Leu Ser Cys Glu Gly Gln Lys Tyr Asn Gln Gly Gln 50 55
60 Tyr Met Asn Thr Pro Trp Arg Asn Pro Ala Glu Lys Arg Glu Lys Leu
65 70 75 80 Ala Tyr Arg Lys Gln Asn Met Asp Asp Ile Asp Glu Glu Asp
Asn Asp 85 90 95 Leu Val Gly Val Ser Val 100 558PRTHuman
immunodeficiency virus type 1 5Trp Pro Ala Val Arg Glu Arg Met Arg
Arg Ala Glu Pro Ala Ala Asp 1 5 10 15 Gly Val Gly Ala Val Ser Arg
Asp Leu Glu Lys His Gly Ala Ile Thr 20 25 30 Ser Ser Asn Thr Ala
Ala Asn Asn Ala Ala Cys Ala Trp Leu Glu Ala 35 40 45 Gln Glu Glu
Glu Glu Val Gly Phe Pro Val 50 55 670PRTHuman immunodeficiency
virus type 1 6Met Gly Gly Lys Trp Ser Lys Ser Ser Val Ile Gly Trp
Pro Ala Val 1 5 10 15 Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala
Asp Gly Val Gly Ala 20 25 30 Val Ser Arg Asp Leu Glu Lys His Gly
Ala Ile Thr Ser Ser Asn Thr 35 40 45 Ala Ala Asn Asn Ala Ala Cys
Ala Trp Leu Glu Ala Gln Glu Glu Glu 50 55 60 Glu Val Gly Phe Pro
Val 65 70 720PRTArtificial Sequencesynthesized 7Gly Gly Gly Gly Xaa
Gly Gly Gly Gly Xaa Gly Gly Gly Gly Xaa Gly 1 5 10 15 Gly Gly Gly
Xaa 20 827DNAArtificial Sequencesynthesized 8cgtgtacggt gggaggtcta
tataagc 27923DNAArtificial Sequencesynthesized 9cataaccttc
gggcatggca ctc 231031DNAArtificial Sequencesynthesized 10cattgctagc
cccatctgct gctggctcag c 311137DNAArtificial Sequencesynthesized
11cattgctagc agctgctgta ttgctacttg tgattgc 371235DNAArtificial
Sequencesynthesized 12cattgctagc ctcttcctcc tcttgtgctt ctagc
351334DNAArtificial Sequencesynthesized 13cattgctagc gactggaaaa
cccacctctt cctc 341437DNAArtificial Sequencesynthesized
14cattgctagc aaagtggcta agatctacag ctgcctt 371537DNAArtificial
Sequencesynthesized 15cattgctagc tggctcaact ggtactagct tgtagca
371630DNAArtificial Sequencesynthesized 16cattgctagc cggatgcagc
tctcgggcca 301724DNAArtificial Sequencesynthesized 17ggtcctccga
tcgttgtcag aagt 241837DNAArtificial Sequencesynthesized
18cagtccgcgg atgtggcctg ctgtaaggga aagaatg 371940DNAArtificial
Sequencesynthesized 19cagtccgcgg atgggagcaa tcacaagtag caatacagca
402036DNAArtificial Sequencesynthesized 20ctagaagcac aagcggcggc
agcggtgggt tttcca 362136DNAArtificial Sequencesynthesized
21tggaaaaccc accgctgccg ccgcttgtgc ttctag 362245DNAArtificial
Sequencesynthesized 22atgtggcctg ctgtagcgga agcaatggca gcagctgagc
cagca 452345DNAArtificial Sequencesynthesized 23tgctggctca
gctgctgcca ttgcttccgc tacagcaggc cacat 452445DNAArtificial
Sequencesynthesized 24gcagtatctc gagacctaga accgcatgga gcaatcacaa
gtagc 452545DNAArtificial Sequencesynthesized 25gctacttgtg
attgctccat gcggttctag gtctcgagat actgc 452636DNAArtificial
Sequencesynthesized 26catggagcaa tcacagccgc gaatacagca gctaac
362736DNAArtificial Sequencesynthesized 27gttagctgct gtattcgcgg
ctgtgattgc tccatg 362842DNAArtificial Sequencesynthesized
28tggctagaag cacaagacga cgacgacgtg ggttttccag tc
422942DNAArtificial Sequencesynthesized 29gactggaaaa cccacgtcgt
cgtcgtcttg tgcttctagc ca 423045DNAArtificial Sequencesynthesized
30caagaggagg aagaggcggc tgctgcagcc gctagcaaag gagaa
453145DNAArtificial Sequencesynthesized 31ttctcctttg ctagcggctg
cagcagccgc ctcttcctcc tcttg 453240DNAArtificial Sequencesynthesized
32cagtccgcgg atggctagca aaggagaaga actcttcact 403321DNAArtificial
Sequencesynthesized 33tgcagaattc cagcacactg g 213440DNAArtificial
Sequencesynthesized 34cagtccgcgg atggctagca aaggagaaga actcttcact
403521DNAArtificial Sequencesynthesized 35tgcagaattc cagcacactg g
213637DNAArtificial Sequencesynthesized 36cgcggctagc tcatctgtga
gcaagggcga ggaggat 373734DNAArtificial Sequencesynthesized
37cgcgggatcc tcacttgtac agctcgtcca tgcc 343834DNAArtificial
Sequencesynthesized 38cgcgaagctt atggtgagca agggcgagga ggat
3439209PRTHuman immunodeficiency virus type 1 39Met Gly Gly Lys Trp
Ser Lys Ser Ser Val Ile Gly Trp Pro Ala Val 1 5 10 15 Arg Glu Arg
Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala 20 25 30 Val
Ser Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr 35 40
45 Ala Ala Asn Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu
50 55 60 Glu Val Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro
Met Thr 65 70 75 80 Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys
Glu Lys Gly Gly 85 90 95 Leu Glu Gly Leu Ile His Ser Gln Arg Arg
Gln Asp Ile Leu Asp Leu 100 105 110 Trp Ile Tyr His Thr Gln Gly Tyr
Phe Pro Asp Trp Gln Asn Tyr Thr 115 120 125 Pro Gly Pro Gly Val Arg
Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys 130 135 140 Leu Val Pro Val
Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu 145 150 155 160 Asn
Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro 165 170
175 Glu Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His
180 185 190 His Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys
Gly Ala 195 200 205 Gly 4010PRTHuman immunodeficiency virus type 1
40Gln Glu Glu Glu Glu Val Gly Phe Pro Val 1 5 10 4111PRTHuman
immunodeficiency virus type 1 41Gln Glu Glu Glu Glu Glu Val Gly Phe
Pro Val 1 5 10 4210PRTHuman immunodeficiency virus type 1 42Gln Glu
Glu Glu Glu Val Gly Phe Pro Val 1 5 10 4311PRTHuman
immunodeficiency virus type 1 43Gln Glu Glu Glu Glu Glu Val Gly Phe
Pro Val 1 5 10 4411PRTHuman immunodeficiency virus type 1 44Gln Glu
Glu Glu Glu Glu Val Gly Phe Pro Val 1 5 10 4510PRTHuman
immunodeficiency virus type 1 45Gln Glu Glu Glu Glu Val Gly Phe Pro
Val 1 5 10 4610PRTHuman immunodeficiency virus type 1 46Gln Glu Asp
Glu Glu Val Gly Phe Pro Val 1 5 10 4711PRTHuman immunodeficiency
virus type 1 47Gln Gln Glu Asp Ser Glu Val Gly Phe Pro Val 1 5 10
4811PRTHuman immunodeficiency virus type 1 48Gln Glu Glu Glu Glu
Glu Val Gly Phe Pro Val 1 5 10 4910PRTHuman immunodeficiency virus
type 1 49Gln Thr Glu Glu Glu Val Gly Phe Pro Val 1 5 10
5010PRTHuman immunodeficiency virus type 1 50Gln Glu Glu Glu Glu
Val Gly Phe Pro Val 1 5 10 5111PRTHuman immunodeficiency virus type
1 51Gln Glu Glu Glu Glu Glu Val Gly Phe Pro Val 1 5 10 5210PRTHuman
immunodeficiency virus type 1 52His Gln Asp Glu Glu Val Gly Phe Pro
Val 1 5 10
* * * * *