U.S. patent application number 12/708940 was filed with the patent office on 2010-08-26 for recombinant viral vectors.
Invention is credited to Ivo Lorenz, Christopher L. PARKS, Sanjay K. Phogat, Timothy J. Zamb.
Application Number | 20100215691 12/708940 |
Document ID | / |
Family ID | 42262391 |
Filed Date | 2010-08-26 |
United States Patent
Application |
20100215691 |
Kind Code |
A1 |
PARKS; Christopher L. ; et
al. |
August 26, 2010 |
RECOMBINANT VIRAL VECTORS
Abstract
The present relation relates to recombinant vesicular stomatitis
virus for use as prophylactic and therapeutic vaccines for
infectious diseases of AIDS. The present invention encompasses the
preparation and purification of immunogenic compositions which are
formulated into the vaccines of the present invention.
Inventors: |
PARKS; Christopher L.;
(Boonton, NJ) ; Lorenz; Ivo; (Brooklyn, NY)
; Phogat; Sanjay K.; (Frederick, MD) ; Zamb;
Timothy J.; (Nyack, NY) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
42262391 |
Appl. No.: |
12/708940 |
Filed: |
February 19, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61154190 |
Feb 20, 2009 |
|
|
|
Current U.S.
Class: |
424/199.1 ;
435/237; 435/320.1 |
Current CPC
Class: |
A61K 2039/5256 20130101;
C12N 2760/20243 20130101; C12N 2740/16051 20130101; A61P 31/18
20180101; C12N 7/00 20130101; G01N 2333/162 20130101; C12N
2760/20251 20130101; A61K 39/21 20130101; A61K 39/00 20130101; C12N
2760/20234 20130101; C07K 14/005 20130101; C12N 2740/16134
20130101; A61K 39/12 20130101; G01N 33/6854 20130101 |
Class at
Publication: |
424/199.1 ;
435/320.1; 435/237 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C12N 15/63 20060101 C12N015/63; C12N 7/08 20060101
C12N007/08; A61P 31/18 20060101 A61P031/18 |
Goverment Interests
FEDERAL FUNDING LEGEND
[0003] This invention was supported, in part, by NIH grant number:
R01-AI084840. The federal government may have certain rights to
this invention.
Claims
1. A recombinant vesicular stomatitis virus (VSV) vector wherein
the gene encoding the VSV surface glycoprotein G (VSV G) is
functionally replaced by HIV Env.
2. The vector of claim 1 wherein the HIV Env is recognized by
antibodies PG9, PG16, 2G12, b12, 2F5, 4E10 or Z13, or other
Env-specific antibodies, including broad potent neutralizing
trimer-specific antibodies.
3. A recombinant vesicular stomatitis virus (VSV) vector encoding a
modified form of VSV G, wherein the modified form of VSV G harbors
natural or modified epitopes from the HIV Env membrane proximal
external region (MPER).
4. The vector of claim 3 wherein the MPER sequence is inserted into
the membrane proximal region of VSV G.
5. The vector of claim 3 wherein a G-MPER protein binds with high
avidity to 2F5 and 4E10 monoclonal antibodies.
6. A recombinant vesicular stomatitis virus (VSV) vector encoding a
an N-terminally truncated form of VSV G (G/Stem), wherein the
G/Stem presents Env epitope sequences on the surface of VSV
particles.
7. The vector of claim 6 wherein G/Stem contains a cytoplasmic tail
(CT) and trans-membrane (TM) spanning domains of G, a 16- to
68-amino acid membrane proximal extracellular polypeptide (the
Stem), wherein HIV Env epitopes are appended to the Stem.
8. The vector of claim 7 wherein the HIV Env epitopes are MPER
epitopes.
9. The vector of claim 8 wherein the G/Stem-MPER molecules bind to
2F5 and 4E10 monoclonal antibodies with high affinity.
10. A method of generating novel chimeric EnvG molecules expressed
and incorporated into VSV comprising: (a) serially passaging
replication-competent chimeric VSV-HIV viruses that lack the
capacity to encode wild-type G and are dependent on Env or EnvG for
infection and propagation on cells to promote emergence of viruses
with greater replicative fitness and (b) identifying novel
mutations that enhance Env or EnvG function.
11. The method of claim 10, wherein the cells are CD4/CCR5.sup.+
cells.
12. The method of claim 10 wherein the novel mutations escalate
trimer abundance on the virus particle and/or increase the
stability of the functional trimeric form of Env or EnvG.
13. The method of claim 10 further comprising determining whether
the Env or EnvG immunogens bind and elicit broadly neutralizing
anti-Env antibodies.
14. The method of claim 10 further comprising applying selective
pressure to generate novel Env or EnvG molecules expressed and
incorporated into VSV, wherein the selective pressure is binding to
broad and potent neutralizing antibodies.
15. The method of claim 14 wherein the antibody is PG9, PG16, b12,
2G12, 2F5 or 4E10 or any other broad potent neutralizing Env trimer
specific antibody.
16. A method of producing an immune response or eliciting an immune
response comprising administering to a mammal the vector of any one
of claim 1, 3 or 6.
Description
INCORPORATION BY REFERENCE
[0001] This application claims priority to U.S. provisional patent
application Ser. No. 61/154,190 filed Feb. 20, 2009.
[0002] The foregoing applications, and all documents cited therein
or during their prosecution ("appln cited documents") and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention.
FIELD OF THE INVENTION
[0004] The present invention relates to recombinant vesicular
stomatitis virus for use as prophylactic and therapeutic vaccines
for infectious diseases of AIDS.
BACKGROUND OF THE INVENTION
[0005] AIDS, or Acquired Immunodeficiency Syndrome, is caused by
human immunodeficiency virus (HIV) and is characterized by several
clinical features including wasting syndromes, central nervous
system degeneration and profound immunosuppression that results in
opportunistic infections and malignancies. HIV is a member of the
lentivirus family of animal retroviruses, which include the visna
virus of sheep and the bovine, feline, and simian immunodeficiency
viruses (SIV). Two closely related types of HIV, designated HIV-1
and HIV-2, have been identified thus far, of which HIV-1 is by far
the most common cause of AIDS. However, HIV-2, which differs in
genomic structure and antigenicity, causes a similar clinical
syndrome.
[0006] An infectious HIV particle consists of two identical strands
of RNA, each approximately 9.2 kb long, packaged within a core of
viral proteins. This core structure is surrounded by a phospholipid
bilayer envelope derived from the host cell membrane that also
includes virally-encoded membrane proteins (Abbas et al., Cellular
and Molecular Immunology, 4th edition, W.B. Saunders Company, 2000,
p. 454). The HIV genome has the characteristic
5'-LTR-Gag-Pol-Env-LTR-3' organization of the retrovirus family.
Long terminal repeats (LTRs) at each end of the viral genome serve
as binding sites for transcriptional regulatory proteins from the
host and regulate viral integration into the host genome, viral
gene expression, and viral replication.
[0007] The HIV genome encodes several structural proteins. The gag
gene encodes structural proteins of the nucleocapsid core and
matrix. The pol gene encodes reverse transcriptase (RT), integrase
(IN), and viral protease (PR) enzymes required for viral
replication. The tat gene encodes a protein that is required for
elongation of viral transcripts. The rev gene encodes a protein
that promotes the nuclear export of incompletely spliced or
unspliced viral RNAs. The vif gene product enhances the infectivity
of viral particles. The vpr gene product promotes the nuclear
import of viral DNA and regulates G2 cell cycle arrest. The vpu and
nef genes encode proteins that down regulate host cell CD4
expression and enhance release of virus from infected cells. The
env gene encodes the viral envelope glycoprotein that is translated
as a 160-kilodalton (kDa) precursor (gp160) and cleaved by a
cellular protease to yield the external 120-kDa envelope
glycoprotein (gp120) and the transmembrane 41-kDa envelope
glycoprotein (gp41), which are required for the infection of cells
(Abbas, pp. 454-456). gp140 is a modified form of the Env
glycoprotein, which contains the external 120-kDa envelope
glycoprotein portion and the extracellular part of the gp41 portion
of Env and has characteristics of both gp120 and gp41. The nef gene
is conserved among primate lentiviruses and is one of the first
viral genes that is transcribed following infection. In vitro,
several functions have been described, including down-regulation of
CD4 and MHC class I surface expression, altered T-cell signaling
and activation, and enhanced viral infectivity.
[0008] HIV infection initiates with gp120 on the viral particle
binding to the CD4 and chemokine receptor molecules (e.g., CXCR4,
CCR5) on the cell membrane of target cells such as CD4.sup.+
T-cells, macrophages and dendritic cells. The bound virus fuses
with the target cell and reverse transcribes the RNA genome. The
resulting viral DNA integrates into the cellular genome, where it
directs the production of new viral RNA, and thereby viral proteins
and new virions. These virions bud from the infected cell membrane
and establish productive infections in other cells. This process
also kills the originally infected cell. HIV can also kill cells
indirectly because the CD4 receptor on uninfected T-cells has a
strong affinity for gp120 expressed on the surface of infected
cells. In this case, the uninfected cells bind, via the CD4
receptor-gp120 interaction, to infected cells and fuse to form a
syncytium, which cannot survive. Destruction of CD4.sup.+
T-lymphocytes, which are critical to immune defense, is a major
cause of the progressive immune dysfunction that is the hallmark of
AIDS disease progression. The loss of CD4.sup.+ T cells seriously
impairs the body's ability to fight most invaders, but it has a
particularly severe impact on the defenses against viruses, fungi,
parasites and certain bacteria, including mycobacteria.
[0009] Research on the Env glycoprotein has shown that the virus
has many effective protective mechanisms with few vulnerabilities
(Wyatt & Sodroski, Science. 1998 Jun. 19; 280(5371):1884-8).
For fusion with its target cells, HIV-1 uses a trimeric Env complex
containing gp120 and gp41 subunits (Burton et al., Nat Immunol.
2004 March; 5(3):233-6). The fusion potential of the Env complex is
triggered by engagement of the CD4 receptor and a coreceptor,
usually CCR5 or CXCR4. Neutralizing antibodies seem to work either
by binding to the mature trimer on the virion surface and
preventing initial receptor engagement events, or by binding after
virion attachment and inhibiting the fusion process (Parren &
Burton, Adv Immunol. 2001; 77:195-262). In the latter case,
neutralizing antibodies may bind to epitopes whose exposure is
enhanced or triggered by receptor binding. However, given the
potential antiviral effects of neutralizing antibodies, it is not
unexpected that HIV-1 has evolved multiple mechanisms to protect it
from antibody binding (Johnson & Desrosiers, Annu Rev Med.
2002; 53:499-518).
[0010] There remains a need to express immunogens that elicit
broadly neutralizing antibodies. Strategies include producing
molecules that mimic the mature trimer on the virion surface,
producing Env molecules engineered to better present neutralizing
antibody epitopes than wild-type molecules, generating stable
intermediates of the entry process to expose conserved epitopes to
which antibodies could gain access during entry and producing
epitope mimics of the broadly neutralizing monoclonal antibodies
determined from structural studies of the antibody-antigen
complexes (Burton et al., Nat Immunol. 2004 March; 5(3):233-6).
However, none of these approaches have yet efficiently elicited
neutralizing antibodies with broad specificity.
[0011] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present application.
SUMMARY OF THE INVENTION
[0012] The current invention is based, in part, on Applicant's
discovery that HIV gp41 epitopes known to elicit broadly
neutralizing antibodies inserted into a viral glycoprotein are
recognized by such broadly neutralizing antibodies in cells
infected with the recombinant virus expressing the viral
glycoprotein.
[0013] Recombinant viruses are viruses generated by introducing
foreign genetic material into the genome of the virus. The genome
of a virus can comprise either DNA or RNA. The genome of an RNA
virus can be further characterized to be either positive-sense
(plus-strand) or negative-sense (minus-strand). A plus-strand (5'
to 3') viral RNA indicates that a particular viral RNA sequence can
be directly translated into the desired viral proteins whereas a
minus-strand (3' to 5') viral RNA must be first converted to a
positive-sense by an RNA polymerase prior to translation.
[0014] In a first embodiment, the invention relates to a
recombinant vesicular stomatitis virus (VSV) vector wherein the
gene encoding the VSV surface glycoprotein G (VSV G) may be
functionally replaced by HIV Env. The HIV Env may be recognized by
antibodies PG9, PG16, 2G12, b12, 2F5, 4E10 or Z13 or other
antibodies, including potent broadly neutralizing trimer-specific
antibodies. VSV is a minus-strand RNA virus that can infect insects
and mammals.
[0015] In a second embodiment, the invention relates to a
recombinant vesicular stomatitis virus (VSV) vector encoding a
modified form of VSV G, wherein the modified form of VSV G may
harbor epitopes from the HIV Env membrane proximal external region
(MPER). The MPER sequence may be inserted into the membrane
proximal region or other domains of VSV G. The G-MPER protein may
bind with high avidity to 2F5, 4E10 or other monoclonal
antibodies.
[0016] In a third embodiment, the invention relates to a
recombinant vesicular stomatitis virus (VSV) vector encoding a an
N-terminally truncated form of VSV G (G/Stem), wherein the G/Stem
may display Env epitope sequences on the surface of VSV particles.
The G/Stem may contain a cytoplasmic tail (CT) and trans-membrane
(TM) spanning domains of G, a 16- to 68-amino acid membrane
proximal extracellular polypeptide (the Stem), wherein HIV Env
epitopes are appended to or inserted into the Stem. The HIV Env
epitopes may be derived from the gp41 MPER or other regions of Env.
The G/Stem-HIV Env epitope molecules may bind to 2F5, 4E10 or other
monoclonal antibodies with high affinity.
[0017] In a fourth embodiment, the invention relates to a method of
generating novel chimeric HIV Env-VSV G (EnvG) molecules expressed
and incorporated into VSV which may comprise:
[0018] (a) serially passaging replication-competent chimeric
VSV-HIV viruses that lack the capacity to encode wild-type G and
are dependent on Env or chimeric EnvG molecules for infection and
propagation on cells to promote emergence of viruses with greater
replicative fitness and
[0019] (b) identifying novel mutations that enhance Env or EnvG
function in VSV-HIV viruses.
[0020] The cells may be CD4/CCR5+ cells. The novel mutations may
escalate trimer abundance on the virus particle and/or increase the
stability of the functional trimeric form of Env or EnvG. The
method may further comprise determining whether the Env or EnvG
immunogens elicit broadly neutralizing anti-Env antibodies.
[0021] In a fifth embodiment, the invention relates to method of
applying selective pressure to generate novel Env, EnvG, or
G/Stem-antigen chimeras molecules expressed and incorporated into
VSV, wherein the selective pressure may be binding to an antibody
of interest, thereby enriching for molecules that may be more
immunogenic. The antibody may be 2F5, 4E10, or other Env-specific
antibodies.
[0022] The present invention also encompasses methods of producing
or eliciting an immune response which may comprise administering to
a mammal any one of the herein disclosed recombinant VSV
vectors.
[0023] The present invention also encompasses other plus and minus
strand viruses which can be used as recombinant viral vectors in
the method of the invention. Such viruses include but are not
limited to: Measles virus, Canine distemper virus, Parainfluenza
viruses, Sendai virus, Newcastle disease virus, Venezuelan equine
encephalitis virus, Sindbis virus, Semliki Forrest virus etc.
[0024] Accordingly, it is an object of the invention to not
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn.112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product.
[0025] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0026] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings, in which:
[0028] FIGS. 1A and 1B depict the HIV-1 envelope protein. A.
Illustration of the gp160 precursor, which is post-translationally
cleaved into the gp120 and gp41 subunits. The locations of the
signal and fusion peptides, the Membrane-Proximal External Region
(MPER) and the transmembrane (TM) segment are indicated. The ruler
denotes amino acid numbering. B. Broadly neutralizing antibodies
directed against Env: PG9 and PG16 interacts with conserved
residues in the V2 and V3 loops and present an accessible target on
gp120; 2G12 binds to oligosaccharides at the tip of gp120; b12
interacts with the CD4 binding site; 2F5 and 4E10 bind adjacent
linear epitopes in the gp41 MPER.
[0029] FIG. 2 depicts vesicular stomatitis virus. The
negative-sense RNA genome (schematically depicted at the top)
encodes five genes in the order 3'-N-P-M-G-L-5'. The surface of the
virus particle (bottom) is decorated with approximately 1,200
copies of the glycoprotein (G), which is arranged as trimers. The
matrix protein (M) lines the inner surface of the virus particle
between the membrane and the nucleocapsid, probably making contact
with G as well as the nucleocapsid (N) protein and giving the virus
particles their characteristic rod- or bullet-shaped morphology.
The polymerase (L) and phosphoprotein (P) are subunits of the
RNA-dependent RNA polymerase complex.
[0030] FIG. 3 depicts the VSV glycoprotein. The model on the left
side is the soluble G ectodomain solved by Roche et al (Roche et
al., Science 2007 315, 843-848), which is composed of a number of
structural elements including an elongated .beta.-sheet that
contains the fusion peptide. In the middle portion of the Figure, a
graphic approximation (in pink) of amino acid residues not included
in the crystal structure was inserted, which includes the
cytoplasmic tail (CT), the transmembrane (TM) domain, and the short
membrane-proximal ectodomain (Stem). Three epitope insertion points
were used for preliminary studies: an .alpha.-helix at the tip (T),
a loop (L) on the side, and a region in the Stem (S). The Stem,
together with the TM and CT domains, but without the remainder of
the ectodomain, forms the G-Stem polypeptide, which is drawn at the
right side of the Figure. The G-Stem protein can be incorporated
into virions and can be used as a presentation platform for foreign
epitopes.
[0031] FIG. 4 depicts HIV Env Immunogens presented on the VSV
vector platform. The different chimeric envelope proteins are
illustrated from top to bottom: i) the native VSV G trimer, ii) a G
trimer with the gp41 MPER inserted into the stem region of G; iii)
the G/Stem displaying MPER epitopes; and iv) the Env ectodomain
including the MPER, which is incorporated into the VSV particle via
the transmembrane segment and cytoplasmic tail of G.
[0032] FIG. 5 depicts insertion of the HIV gp41-derived 2F5 and/or
4E10 epitope into the `stem` region of VSV G, which shares sequence
similarities with the gp41 MPER.
[0033] FIG. 6 depicts HIV-1 Env MPER and VSV G stem sequence
alignment and insertion/substitution strategies. Top, The MPER of
HIV-1 gp41 (JRFL strain) and the Stem region of VSV G (Indiana
strain) share sequence similarities, which guided the selection of
insertion or substitution points in the Stem region for the 2F5 and
4E10 epitopes. The transmembrane domains and the first two residues
of the cytoplasmic tails are depicted on the right. Hydrophobic
residues are shown in blue. Middle, Generation of the VSV G-2F5-Ins
construct by insertion of the 2F5 epitope into the G stem region.
Flanking linker residues are shown in green. Bottom, Substitution
of residues in the G stem region with the 2F5 and/or 4E10 epitopes,
resulting in the VSV G-2F5-Sub, VSV G-4E10-Sub, and VSV
G-2F5-4E10-Sub constructs. Sequences similarities between HIV gp41
and VSV G are shown in red.
[0034] FIG. 7 depicts insertion points for the 2F5 and 4E10
epitopes in the context of full-length VSV G. The leader peptide,
ectodomain, Stem, TM and CT of VSV G are illustrated. The arrow
denotes insertion of the 2F5 epitope, while the orange and blue
boxes indicate substitution of the 2F5 and 4E10 epitopes,
respectively.
[0035] FIG. 8 depicts the expression and antibody detection of the
VSV G constructs. Western blot using VSV-G, 2F5 and 4E10 antibodies
to detect the G protein in lysates from 293T cells transfected with
plasmids coding for unmodified VSV G, VSV G-2F5-Ins, VSV G-2F5-Sub,
VSV G-4E10-Sub, or VSV G-2F5-4E10-Sub. Mock denotes a transfection
with an "empty" plasmid vector. The antibody used for detection is
shown under each panel. Molecular weight standards are indicated on
the right of each gel.
[0036] FIG. 9 depicts the trimerization of the VSV G constructs.
Western blot using VSV-G antibody to detect oligomeric G protein on
the surface of 293T cells transfected with VSV G constructs,
followed by incubation with the chemical crosslinker
3,3'-Dithiobis-[sulfosuccinimidylpropionate] (DTSSP) at various
concentrations as indicated above each lane. Monomeric, dimeric and
trimeric VSV G forms are detected.
[0037] FIG. 10 depicts cell surface expression of VSV G constructs.
293T cells transfected with VSV G constructs were stained with an
antibody specific for the ectodomain of VSV G, or with 2F5 or 4E10
antibodies, followed by analysis of the samples by flow
cytometry.
[0038] FIG. 11 depicts cell-cell fusion mediated by VSV G. 293T
cells transfected with VSV G constructs were exposed briefly to a
medium with pH 5.2. After 6-8 hours, formation of syncitia was
monitored using a light microscope. The inset in the panel for VSV
G-2F5-4E10 at the bottom right shows a small syncitium, which
occurs rarely for this construct.
[0039] FIG. 12 depicts a reporter assay for functional analysis of
modified VSV G proteins. A reporter lentivirus coding for green
fluorescent protein (GFP) or luciferase (Luc) was packaged with
Gag-Pol and pseudotyped with the VSV G variants and subsequently
used to infect naive 293T cells. GFP or luciferase expression was
analyzed 72 hours post-infection.
[0040] FIG. 13 depicts infectivity of lentiviral particles
pseudotyped with VSV G constructs. GFP reporter lentiviruses
pseudotyped with VSV G variants were generated in 293T cells and
used subsequently to infect naive 293T cells. GFP expression was
monitored 72 hours post-infection.
[0041] FIG. 14 depicts quantification of infectivity of lentiviral
particles pseudotyped with VSV G constructs. Naive 293T cells were
infected with luciferase reporter lentiviruses pseudotyped with VSV
G variants, followed by quantification of luciferase expression 48
hours post-infection.
[0042] FIG. 15 depicts neutralization of lentiviral particles
pseudotyped with VSV G constructs with the 2F5 or 4E10 antibodies.
Luciferase reporter lentiviruses pseudotyped with VSV G, VSV
G-2F5-Sub or VSV G-4E10-Sub were incubated with various
concentrations of 2F5 (left panel) or 4E10 antibody (right panel)
prior to infection of naive cells. Luciferase expression was
quantified 48 hours post-infection.
[0043] FIG. 16 depicts growth curves of recombinant VSV in Vero
cells. Recombinant VSV (rVSV) containing the gene for wild-type G,
G-2F5-Sub, G-4E10-Sub or G-2F5-4E10-Sub rescued in 293T cells was
used to infect Vero cells at a multiplicity of infection (m.o.i.)
of 5. Aliquots of the supernatant were taken at various times
post-infection. Subsequently, naive Vero cells were infected with
the samples, followed by a standard plaque assay to determine the
viral titer for each time point.
[0044] FIG. 17 depicts neutralization of recombinant VSV with 2F5
and 4E10 antibodies. Recombinant VSV containing wild-type G,
G-2F5-Sub, G-4E10-Sub or G-2F5-4E10-Sub was incubated with various
concentrations of the broadly neutralizing monoclonal antibodies
VI-10 (which reacts with the ectodomain of G), 2F5 or 4E10 before
addition to naive Vero cells. A standard plaque assay was used to
determine the extent of neutralization for each antibody and
concentration.
[0045] FIGS. 18A and 18B depict a VSV G-Stem platform for
expression of fusion proteins. A. Schematic illustration of the VSV
genome, the G gene, and the primary structures of the G and G-Stem
proteins. B. Foreign gene sequences are fused to the G-Stem via a
NheI restriction site, which facilitates incorporation of immunogen
coding sequences.
[0046] FIGS. 19A-19C depict a schematic illustrating the membrane
topology of G and G-Stem proteins. A. Topology of the full-length G
protein with the extracellular region, the stem, the transmembrane
segment, and the cytoplasmic tail. Four different G-Stem constructs
were generated: no stem, short stem, middle stem, and long stem. B.
The gp41 MPER was fused to the four G-Stem constructs (GS-MPER
fusions) C. Amino acid sequence of the G-Stem. The starting
position for each GS variant (no, short, medium, long) is shown.
The N-terminal signal sequence is shown in purple, whereas the
transmembrane segment is colored red.
[0047] FIG. 20 depicts VSV Vector Design. The gene encoding G-Stem
variants was inserted into the VSV genome upstream of the N protein
near the 3' end. In addition, the full-length G protein is present
in the genome. Upon expression, both the G-Stem and full-length G
will be incorporated into virus particles as illustrated below the
vector genome map.
[0048] FIGS. 21A-21D depict analysis of G-Stem-MPER Expression. A.
Western Blot analysis of rVSV containing the G-Stem-MPER variants
(rVSV-GS-MPER) from the supernatant of infected cells using an
anti-VSV-G antibody that reacts with the cyoplasmic tail. LS, long
stem; MS, medium stem; SS, short stem; NS, no stem. B. Western Blot
analysis of rVSV-GS-MPER from infected cells using an anti-VSV-G
antibody. C. Western Blot analysis of rVSV-GS-MPER with the 2F5
antibody. D. Western Blot analysis of rVSV-GS-MPER with the 4E10
antibody.
[0049] FIG. 22 depicts various VSV G-HIV Env chimeras. The VSV
glycoprotein G is shown at the top with features labeled including
the signal peptide (SP), the soluble extracellular domain, the
Stem, transmembrane (TM) segment and cytoplasmic tail (CT). The
HIV-1 Envelope (Env) protein, illustrated below G, is
proteolytically processed into the extracellular gp120 and the gp41
domains, the latter containing the MPER, TM segment and CT domains.
Various chimeric EnvG proteins are shown at the bottom. Transition
points between HIV gp41 and VSV G are be located i) before the CT,
ii) before the TM domain, iii) before the MPER, or iv) N-terminal
to the complete VSV G-Stem. Translocation of the protein into the
lumen of the endoplasmic reticulum can be driven by either the Env
or the G signal peptide, although the efficiency and destination
vary with the two signals. The ruler at the top denotes the number
of amino acid residues.
[0050] FIG. 23 depicts infectivity of rVSV-EnvG. a, Uninfected
GHOST cells (expressing the HIV co-receptors CD4 and CCR5; Cecilia
D., et al J. Virol. 1998 September; 7:6988-96) near full
confluency. b, GHOST cells infected with rVSV-EnvG virus at 48
hours post-infection. The cytopathic effect (CPE) is clearly
visible.
[0051] FIG. 24 depicts evolution of Env or EnvG proteins expressed
by recombinant VSV. Recombinant VSV encoding a chimeric EnvG
molecule are subjected to serial passage and selective pressure.
Virus particles that bind with high avidity to 2F5 antibody, for
example, are isolated after stringent washing of the antibody
beads. Infectious nucleocapsid is liberated from the antibody beads
and transfected into CD4/CCR5-positive cells, which initiates a new
round of infection. The new generation of recombinant virus
undergoes further rounds of selection with increased stringency,
which enrich new variants of recombinant viruses that may have
improved immunogenic properties.
[0052] FIG. 25 depicts rabbit immunogenicity testing. Vaccination
and blood collection schedules are listed along a timeline (M,
months; W, weeks) at the top. Analysis of antibody reactivity is
illustrated in the flow diagram at the left side. The chart on the
right side outlines a typical rabbit study.
[0053] FIG. 26 depicts a plan for vaccination, sampling, and SHIV
Challenge. rVSV vaccine candidates are administered 3 times at
6-week intervals after which IV SHIV 162P3 challenge is conducted
using a challenge stock obtained from the NIH AIDS Research &
Reference Reagent Program.
[0054] FIGS. 27A-27B depicts the plasmid sequence of pCINeo-VSV-G
that encodes the G protein from the vesicular stomatitis Indiana
virus. Applicants have optimized the gene sequence.
[0055] FIGS. 28A-28B depicts the unique XhoI and NotI sites
(highlighted) added to the 5' and 3' termini respectively of the
VSV G coding sequence as per the Optimization Strategy detailed in
Example 5.
DETAILED DESCRIPTION
[0056] The terms "protein", "peptide", "polypeptide", and "amino
acid sequence" are used interchangeably herein to refer to polymers
of amino acid residues of any length. The polymer may be linear or
branched, it may comprise modified amino acids or amino acid
analogs, and it may be interrupted by chemical moieties other than
amino acids. The terms also encompass an amino acid polymer that
has been modified naturally or by intervention; for example
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling or bioactive component.
[0057] As used herein, the terms "antigen" or "immunogen" are used
interchangeably to refer to a substance, typically a protein, which
is capable of inducing an immune response in a subject. The term
also refers to proteins that are immunologically active in the
sense that once administered to a subject (either directly or by
administering to the subject a nucleotide sequence or vector that
encodes the protein) is able to evoke an immune response of the
humoral and/or cellular type directed against that protein.
[0058] The term "antibody" includes intact molecules as well as
fragments thereof, such as Fab, F(ab').sub.2, Fv and scFv which are
capable of binding the epitope determinant. These antibody
fragments retain some ability to selectively bind with its antigen
or receptor and include, for example:
[0059] (i) Fab, the fragment which contains a monovalent
antigen-binding fragment of an antibody molecule can be produced by
digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain;
[0060] (ii) Fab', the fragment of an antibody molecule can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody
molecule;
[0061] (iii) F(ab').sub.2, the fragment of the antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds;
[0062] (iv) scFv, including a genetically engineered fragment
containing the variable region of a heavy and a light chain as a
fused single chain molecule.
[0063] General methods of making these fragments are known in the
art. (See for example, Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York (1988), which is
incorporated herein by reference).
[0064] It should be understood that the proteins, including the
antibodies and/or antigens of the invention may differ from the
exact sequences illustrated and described herein. Thus, the
invention contemplates deletions, additions and substitutions to
the sequences shown, so long as the sequences function in
accordance with the methods of the invention. In this regard,
particularly preferred substitutions will generally be conservative
in nature, i.e., those substitutions that take place within a
family of amino acids. For example, amino acids are generally
divided into four families: (1) acidic--aspartate and glutamate;
(2) basic--lysine, arginine, histidine; (3) non-polar--alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar--glycine, asparagine,
glutamine, cysteine, serine threonine, tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified as aromatic amino
acids. It is reasonably predictable that an isolated replacement of
leucine with isoleucine or valine, or vice versa; an aspartate with
a glutamate or vice versa; a threonine with a serine or vice versa;
or a similar conservative replacement of an amino acid with a
structurally related amino acid, will not have a major effect on
the biological activity. Proteins having substantially the same
amino acid sequence as the sequences illustrated and described but
possessing minor amino acid substitutions that do not substantially
affect the immunogenicity of the protein are, therefore, within the
scope of the invention.
[0065] As used herein the terms "nucleotide sequences" and "nucleic
acid sequences" refer to deoxyribonucleic acid (DNA) or ribonucleic
acid (RNA) sequences, including, without limitation, messenger RNA
(mRNA), DNA/RNA hybrids, or synthetic nucleic acids. The nucleic
acid can be single-stranded, or partially or completely
double-stranded (duplex). Duplex nucleic acids can be homoduplex or
heteroduplex.
[0066] As used herein the term "transgene" may used to refer to
"recombinant" nucleotide sequences that may be derived from any of
the nucleotide sequences encoding the proteins of the present
invention. The term "recombinant" means a nucleotide sequence that
has been manipulated "by man" and which does not occur in nature,
or is linked to another nucleotide sequence or found in a different
arrangement in nature. It is understood that manipulated "by man"
means manipulated by some artificial means, including by use of
machines, codon optimization, restriction enzymes, etc.
[0067] For example, in one embodiment the nucleotide sequences may
be mutated such that the activity of the encoded proteins in vivo
is abrogated. In another embodiment the nucleotide sequences may be
codon optimized, for example the codons may be optimized for human
use. In preferred embodiments the nucleotide sequences of the
invention are both mutated to abrogate the normal in vivo function
of the encoded proteins, and codon optimized for human use. For
example, each of the Gag, Pol, Env, Nef, RT, and Int sequences of
the invention may be altered in these ways.
[0068] As regards codon optimization, the nucleic acid molecules of
the invention have a nucleotide sequence that encodes the antigens
of the invention and can be designed to employ codons that are used
in the genes of the subject in which the antigen is to be produced.
Many viruses, including HIV and other lentiviruses, use a large
number of rare codons and, by altering these codons to correspond
to codons commonly used in the desired subject, enhanced expression
of the antigens can be achieved. In a preferred embodiment, the
codons used are "humanized" codons, i.e., the codons are those that
appear frequently in highly expressed human genes (Andre et al., J.
Virol. 72:1497-1503, 1998) instead of those codons that are
frequently used by HIV. Such codon usage provides for efficient
expression of the transgenic HIV proteins in human cells. Any
suitable method of codon optimization may be used. Such methods,
and the selection of such methods, are well known to those of skill
in the art. In addition, there are several companies that will
optimize codons of sequences, such as Geneart (geneart.com). Thus,
the nucleotide sequences of the invention can readily be codon
optimized.
[0069] The invention further encompasses nucleotide sequences
encoding functionally and/or antigenically equivalent variants and
derivatives of the antigens of the invention and functionally
equivalent fragments thereof. These functionally equivalent
variants, derivatives, and fragments display the ability to retain
antigenic activity. For instance, changes in a DNA sequence that do
not change the encoded amino acid sequence, as well as those that
result in conservative substitutions of amino acid residues, one or
a few amino acid deletions or additions, and substitution of amino
acid residues by amino acid analogs are those which will not
significantly affect properties of the encoded polypeptide.
Conservative amino acid substitutions are glycine/alanine;
valine/isoleucine/leucine; asparagine/glutamine; aspartic
acid/glutamic acid; serine/threonine/methionine; lysine/arginine;
and phenylalanine/tyrosine/tryptophan. In one embodiment, the
variants have at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98% or at least 99%
homology or identity to the antigen, epitope, immunogen, peptide or
polypeptide of interest.
[0070] For the purposes of the present invention, sequence identity
or homology is determined by comparing the sequences when aligned
so as to maximize overlap and identity while minimizing sequence
gaps. In particular, sequence identity may be determined using any
of a number of mathematical algorithms. A nonlimiting example of a
mathematical algorithm used for comparison of two sequences is the
algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA
1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc.
Natl. Acad. Sci. USA 1993; 90: 5873-5877.
[0071] Another example of a mathematical algorithm used for
comparison of sequences is the algorithm of Myers & Miller,
CABIOS1988; 4: 11-17. Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used. Yet
another useful algorithm for identifying regions of local sequence
similarity and alignment is the FASTA algorithm as described in
Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85:
2444-2448.
[0072] Advantageous for use according to the present invention is
the WU-BLAST (Washington University BLAST) version 2.0 software.
WU-BLAST version 2.0 executable programs for several UNIX platforms
can be downloaded from ftp://blast.wustl.edu/blast/executables.
This program is based on WU-BLAST version 1.4, which in turn is
based on the public domain NCBI-BLAST version 1.4 (Altschul &
Gish, 1996, Local alignment statistics, Doolittle ed., Methods in
Enzymology 266: 460-480; Altschul et al., Journal of Molecular
Biology 1990; 215: 403-410; Gish & States, 1993; Nature
Genetics 3: 266-272; Karlin & Altschul, 1993; Proc. Natl. Acad.
Sci. USA 90: 5873-5877; all of which are incorporated by reference
herein).
[0073] The various recombinant nucleotide sequences and antibodies
and/or antigens of the invention are made using standard
recombinant DNA and cloning techniques. Such techniques are well
known to those of skill in the art. See for example, "Molecular
Cloning: A Laboratory Manual", second edition (Sambrook et al.
1989).
[0074] The nucleotide sequences of the present invention may be
inserted into "vectors." The term "vector" is widely used and
understood by those of skill in the art, and as used herein the
term "vector" is used consistent with its meaning to those of skill
in the art. For example, the term "vector" is commonly used by
those skilled in the art to refer to a vehicle that allows or
facilitates the transfer of nucleic acid molecules from one
environment to another or that allows or facilitates the
manipulation of a nucleic acid molecule.
[0075] Any vector that allows expression of the antibodies and/or
antigens of the present invention may be used in accordance with
the present invention. In certain embodiments, the antigens and/or
antibodies of the present invention may be used in vitro (such as
using cell-free expression systems) and/or in cultured cells grown
in vitro in order to produce the encoded HIV-antigens and/or
antibodies which may then be used for various applications such as
in the production of proteinaceous vaccines. For such applications,
any vector that allows expression of the antigens and/or antibodies
in vitro and/or in cultured cells may be used.
[0076] For applications where it is desired that the antibodies
and/or antigens be expressed in vivo, for example when the
transgenes of the invention are used in DNA or DNA-containing
vaccines, any vector that allows for the expression of the
antibodies and/or antigens of the present invention and is safe for
use in vivo may be used. In preferred embodiments the vectors used
are safe for use in humans, mammals and/or laboratory animals.
[0077] For the antibodies and/or antigens of the present invention
to be expressed, the protein coding sequence should be "operably
linked" to regulatory or nucleic acid control sequences that direct
transcription and translation of the protein. As used herein, a
coding sequence and a nucleic acid control sequence or promoter are
said to be "operably linked" when they are covalently linked in
such a way as to place the expression or transcription and/or
translation of the coding sequence under the influence or control
of the nucleic acid control sequence. The "nucleic acid control
sequence" can be any nucleic acid element, such as, but not limited
to promoters, enhancers, IRES, introns, and other elements
described herein that direct the expression of a nucleic acid
sequence or coding sequence that is operably linked thereto. The
term "promoter" will be used herein to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II and that when operationally
linked to the protein coding sequences of the invention lead to the
expression of the encoded protein. The expression of the transgenes
of the present invention can be under the control of a constitutive
promoter or of an inducible promoter, which initiates transcription
only when exposed to some particular external stimulus, such as,
without limitation, antibiotics such as tetracycline, hormones such
as ecdysone, or heavy metals. The promoter can also be specific to
a particular cell-type, tissue or organ. Many suitable promoters
and enhancers are known in the art, and any such suitable promoter
or enhancer may be used for expression of the transgenes of the
invention. For example, suitable promoters and/or enhancers can be
selected from the Eukaryotic Promoter Database (EPDB).
[0078] The present invention relates to a recombinant vesicular
stomatitis virus (VSV) vector expressing a foreign epitope.
Advantageously, the epitope is an HIV epitope. Any HIV epitope may
be expressed in a VSV vector. Advantageously, the HIV epitope is an
HIV antigen, HIV epitope or an HIV immunogen, such as, but not
limited to, the HIV antigens, HIV epitopes or HIV immunogens of
U.S. Pat. Nos. 7,341,731; 7,335,364; 7,329,807; 7,323,553;
7,320,859; 7,311,920; 7,306,798; 7,285,646; 7,285,289; 7,285,271;
7,282,364; 7,273,695; 7,270,997; 7,262,270; 7,244,819; 7,244,575;
7,232,567; 7,232,566; 7,223,844; 7,223,739; 7,223,534; 7,223,368;
7,220,554; 7,214,530; 7,211,659; 7,211,432; 7,205,159; 7,198,934;
7,195,768; 7,192,555; 7,189,826; 7,189,522; 7,186,507; 7,179,645;
7,175,843; 7,172,761; 7,169,550; 7,157,083; 7,153,509; 7,147,862;
7,141,550; 7,129,219; 7,122,188; 7,118,859; 7,118,855; 7,118,751;
7,118,742; 7,105,655; 7,101,552; 7,097,971 7,097,842; 7,094,405;
7,091,049; 7,090,648; 7,087,377; 7,083,787; 7,070,787; 7,070,781;
7,060,273; 7,056,521; 7,056,519; 7,049,136; 7,048,929; 7,033,593;
7,030,094; 7,022,326; 7,009,037; 7,008,622; 7,001,759; 6,997,863;
6,995,008; 6,979,535; 6,974,574; 6,972,126; 6,969,609; 6,964,769;
6,964,762; 6,958,158; 6,956,059; 6,953,689; 6,951,648; 6,946,075;
6,927,031; 6,919,319; 6,919,318; 6,919,077; 6,913,752; 6,911,315;
6,908,617; 6,908,612; 6,902,743; 6,900,010; 6,893,869; 6,884,785;
6,884,435; 6,875,435; 6,867,005; 6,861,234; 6,855,539; 6,841,381
6,841,345; 6,838,477; 6,821,955; 6,818,392; 6,818,222; 6,815,217;
6,815,201; 6,812,026; 6,812,025; 6,812,024; 6,808,923; 6,806,055;
6,803,231; 6,800,613; 6,800,288; 6,797,811; 6,780,967; 6,780,598;
6,773,920; 6,764,682; 6,761,893; 6,753,015; 6,750,005; 6,737,239;
6,737,067; 6,730,304; 6,720,310; 6,716,823; 6,713,301; 6,713,070;
6,706,859; 6,699,722; 6,699,656; 6,696,291; 6,692,745; 6,670,181;
6,670,115; 6,664,406; 6,657,055; 6,657,050; 6,656,471; 6,653,066;
6,649,409; 6,649,372; 6,645,732; 6,641,816; 6,635,469; 6,613,530;
6,605,427; 6,602,709 6,602,705; 6,600,023; 6,596,477; 6,596,172;
6,593,103; 6,593,079; 6,579,673; 6,576,758; 6,573,245; 6,573,040;
6,569,418; 6,569,340; 6,562,800; 6,558,961; 6,551,828; 6,551,824;
6,548,275; 6,544,780; 6,544,752; 6,544,728; 6,534,482; 6,534,312;
6,534,064; 6,531,572; 6,531,313; 6,525,179; 6,525,028; 6,524,582;
6,521,449; 6,518,030; 6,518,015; 6,514,691; 6,514,503; 6,511,845;
6,511,812; 6,511,801; 6,509,313; 6,506,384; 6,503,882; 6,495,676;
6,495,526; 6,495,347; 6,492,123; 6,489,131; 6,489,129; 6,482,614;
6,479,286; 6,479,284; 6,465,634; 6,461,615 6,458,560; 6,458,527;
6,458,370; 6,451,601; 6,451,592; 6,451,323; 6,436,407; 6,432,633;
6,428,970; 6,428,952; 6,428,790; 6,420,139; 6,416,997; 6,410,318;
6,410,028; 6,410,014; 6,407,221; 6,406,710; 6,403,092; 6,399,295;
6,392,013; 6,391,657; 6,384,198; 6,380,170; 6,376,170; 6,372,426;
6,365,187; 6,358,739; 6,355,248; 6,355,247; 6,348,450; 6,342,372;
6,342,228; 6,338,952; 6,337,179; 6,335,183; 6,335,017; 6,331,404;
6,329,202; 6,329,173; 6,328,976; 6,322,964; 6,319,666; 6,319,665;
6,319,500; 6,319,494; 6,316,205; 6,316,003; 6,309,633; 6,306,625
6,296,807; 6,294,322; 6,291,239; 6,291,157; 6,287,568; 6,284,456;
6,284,194; 6,274,337; 6,270,956; 6,270,769; 6,268,484; 6,265,562;
6,265,149; 6,262,029; 6,261,762; 6,261,571; 6,261,569; 6,258,599;
6,258,358; 6,248,332; 6,245,331; 6,242,461; 6,241,986; 6,235,526;
6,235,466; 6,232,120; 6,228,361; 6,221,579; 6,214,862; 6,214,804;
6,210,963; 6,210,873; 6,207,185; 6,203,974; 6,197,755; 6,197,531;
6,197,496; 6,194,142; 6,190,871; 6,190,666; 6,168,923; 6,156,302;
6,153,408; 6,153,393; 6,153,392; 6,153,378; 6,153,377; 6,146,635;
6,146,614; 6,143,876 6,140,059; 6,140,043; 6,139,746; 6,132,992;
6,124,306; 6,124,132; 6,121,006; 6,120,990; 6,114,507; 6,114,143;
6,110,466; 6,107,020; 6,103,521; 6,100,234; 6,099,848; 6,099,847;
6,096,291; 6,093,405; 6,090,392; 6,087,476; 6,083,903; 6,080,846;
6,080,725; 6,074,650; 6,074,646; 6,070,126; 6,063,905; 6,063,564;
6,060,256; 6,060,064; 6,048,530; 6,045,788; 6,043,347; 6,043,248;
6,042,831; 6,037,165; 6,033,672; 6,030,772; 6,030,770; 6,030,618;
6,025,141; 6,025,125; 6,020,468; 6,019,979; 6,017,543; 6,017,537;
6,015,694; 6,015,661; 6,013,484; 6,013,432 6,007,838; 6,004,811;
6,004,807; 6,004,763; 5,998,132; 5,993,819; 5,989,806; 5,985,926;
5,985,641; 5,985,545; 5,981,537; 5,981,505; 5,981,170; 5,976,551;
5,972,339; 5,965,371; 5,962,428; 5,962,318; 5,961,979; 5,961,970;
5,958,765; 5,958,422; 5,955,647; 5,955,342; 5,951,986; 5,951,975;
5,942,237; 5,939,277; 5,939,074; 5,935,580; 5,928,930; 5,928,913;
5,928,644; 5,928,642; 5,925,513; 5,922,550; 5,922,325; 5,919,458;
5,916,806; 5,916,563; 5,914,395; 5,914,109; 5,912,338; 5,912,176;
5,912,170; 5,906,936; 5,895,650; 5,891,623; 5,888,726; 5,885,580
5,885,578; 5,879,685; 5,876,731; 5,876,716; 5,874,226; 5,872,012;
5,871,747; 5,869,058; 5,866,694; 5,866,341; 5,866,320; 5,866,319;
5,866,137; 5,861,290; 5,858,740; 5,858,647; 5,858,646; 5,858,369;
5,858,368; 5,858,366; 5,856,185; 5,854,400; 5,853,736; 5,853,725;
5,853,724; 5,852,186; 5,851,829; 5,851,529; 5,849,475; 5,849,288;
5,843,728; 5,843,723; 5,843,640; 5,843,635; 5,840,480; 5,837,510;
5,837,250; 5,837,242; 5,834,599; 5,834,441; 5,834,429; 5,834,256;
5,830,876; 5,830,641; 5,830,475; 5,830,458; 5,830,457; 5,827,749;
5,827,723; 5,824,497 5,824,304; 5,821,047; 5,817,767; 5,817,754;
5,817,637; 5,817,470; 5,817,318; 5,814,482; 5,807,707; 5,804,604;
5,804,371; 5,800,822; 5,795,955; 5,795,743; 5,795,572; 5,789,388;
5,780,279; 5,780,038; 5,776,703; 5,773,260; 5,770,572; 5,766,844;
5,766,842; 5,766,625; 5,763,574; 5,763,190; 5,762,965; 5,759,769;
5,756,666; 5,753,258; 5,750,373; 5,747,641; 5,747,526; 5,747,028;
5,736,320; 5,736,146; 5,733,760; 5,731,189; 5,728,385; 5,721,095;
5,716,826; 5,716,637; 5,716,613; 5,714,374; 5,709,879; 5,709,860;
5,709,843; 5,705,331; 5,703,057; 5,702,707 5,698,178; 5,688,914;
5,686,078; 5,681,831; 5,679,784; 5,674,984; 5,672,472; 5,667,964;
5,667,783; 5,665,536; 5,665,355; 5,660,990; 5,658,745; 5,658,569;
5,643,756; 5,641,624; 5,639,854; 5,639,598; 5,637,677; 5,637,455;
5,633,234; 5,629,153; 5,627,025; 5,622,705; 5,614,413; 5,610,035;
5,607,831; 5,606,026; 5,601,819; 5,597,688; 5,593,972; 5,591,829;
5,591,823; 5,589,466; 5,587,285; 5,585,254; 5,585,250; 5,580,773;
5,580,739; 5,580,563; 5,573,916; 5,571,667; 5,569,468; 5,558,865;
5,556,745; 5,550,052; 5,543,328; 5,541,100; 5,541,057; 5,534,406
5,529,765; 5,523,232; 5,516,895; 5,514,541; 5,510,264; 5,500,161;
5,480,967; 5,480,966; 5,470,701; 5,468,606; 5,462,852; 5,459,127;
5,449,601; 5,447,838; 5,447,837; 5,439,809; 5,439,792; 5,418,136;
5,399,501; 5,397,695; 5,391,479; 5,384,240; 5,374,519; 5,374,518;
5,374,516; 5,364,933; 5,359,046; 5,356,772; 5,354,654; 5,344,755;
5,335,673; 5,332,567; 5,320,940; 5,317,009; 5,312,902; 5,304,466;
5,296,347; 5,286,852; 5,268,265; 5,264,356; 5,264,342; 5,260,308;
5,256,767; 5,256,561; 5,252,556; 5,230,998; 5,230,887; 5,227,159;
5,225,347; 5,221,610 5,217,861; 5,208,321; 5,206,136; 5,198,346;
5,185,147; 5,178,865; 5,173,400; 5,173,399; 5,166,050; 5,156,951;
5,135,864; 5,122,446; 5,120,662; 5,103,836; 5,100,777; 5,100,662;
5,093,230; 5,077,284; 5,070,010; 5,068,174; 5,066,782; 5,055,391;
5,043,262; 5,039,604; 5,039,522; 5,030,718; 5,030,555; 5,030,449;
5,019,387; 5,013,556; 5,008,183; 5,004,697; 4,997,772; 4,983,529;
4,983,387; 4,965,069; 4,945,082; 4,921,787; 4,918,166; 4,900,548;
4,888,290; 4,886,742; 4,885,235; 4,870,003; 4,869,903; 4,861,707;
4,853,326; 4,839,288; 4,833,072 and 4,795,739.
[0079] Advantageously, the HIV epitope may be an Env precursor or
gp160 epitope. The Env precursor or gp160 epitope may be recognized
by antibodies PG9, PG16, 2G12, b12, 2F5, 4E10, Z13, or other broad
potent neutralizing antibodies.
[0080] In another embodiment, HIV, or immunogenic fragments
thereof, may be utilized as the HIV epitope. For example, the HIV
nucleotides of U.S. Pat. Nos. 7,393,949, 7,374,877, 7,306,901,
7,303,754, 7,173,014, 7,122,180, 7,078,516, 7,022,814, 6,974,866,
6,958,211, 6,949,337, 6,946,254, 6,896,900, 6,887,977, 6,870,045,
6,803,187, 6,794,129, 6,773,915, 6,768,004, 6,706,268, 6,696,291,
6,692,955, 6,656,706, 6,649,409, 6,627,442, 6,610,476, 6,602,705,
6,582,920, 6,557,296, 6,531,587, 6,531,137, 6,500,623, 6,448,078,
6,429,306, 6,420,545, 6,410,013, 6,407,077, 6,395,891, 6,355,789,
6,335,158, 6,323,185, 6,316,183, 6,303,293, 6,300,056, 6,277,561,
6,270,975, 6,261,564, 6,225,045, 6,222,024, 6,194,391, 6,194,142,
6,162,631, 6,114,167, 6,114,109, 6,090,392, 6,060,587, 6,057,102,
6,054,565, 6,043,081, 6,037,165, 6,034,233, 6,033,902, 6,030,769,
6,020,123, 6,015,661, 6,010,895, 6,001,555, 5,985,661, 5,980,900,
5,972,596, 5,939,538, 5,912,338, 5,869,339, 5,866,701, 5,866,694,
5,866,320, 5,866,137, 5,864,027, 5,861,242, 5,858,785, 5,858,651,
5,849,475, 5,843,638, 5,840,480, 5,821,046, 5,801,056, 5,786,177,
5,786,145, 5,773,247, 5,770,703, 5,756,674, 5,741,706, 5,705,612,
5,693,752, 5,688,637, 5,688,511, 5,684,147, 5,665,577, 5,585,263,
5,578,715, 5,571,712, 5,567,603, 5,554,528, 5,545,726, 5,527,895,
5,527,894, 5,223,423, 5,204,259, 5,144,019, 5,051,496 and 4,942,122
are useful for the present invention.
[0081] Any epitope recognized by an HIV antibody may be used in the
present invention. For example, the anti-HIV antibodies of U.S.
Pat. Nos. 6,949,337, 6,900,010, 6,821,744, 6,768,004, 6,613,743,
6,534,312, 6,511,830, 6,489,131, 6,242,197, 6,114,143, 6,074,646,
6,063,564, 6,060,254, 5,919,457, 5,916,806, 5,871,732, 5,824,304,
5,773,247, 5,736,320, 5,637,455, 5,587,285, 5,514,541, 5,317,009,
4,983,529, 4,886,742, 4,870,003 and 4,795,739 are useful for the
present invention. Furthermore, monoclonal anti-HIV antibodies of
U.S. Pat. Nos. 7,074,556, 7,074,554, 7,070,787, 7,060,273,
7,045,130, 7,033,593, RE39,057, 7,008,622, 6,984,721, 6,972,126,
6,949,337, 6,946,465, 6,919,077, 6,916,475, 6,911,315, 6,905,680,
6,900,010, 6,825,217, 6,824,975, 6,818,392, 6,815,201, 6,812,026,
6,812,024, 6,797,811, 6,768,004, 6,703,019, 6,689,118, 6,657,050,
6,608,179, 6,600,023, 6,596,497, 6,589,748, 6,569,143, 6,548,275,
6,525,179, 6,524,582, 6,506,384, 6,498,006, 6,489,131, 6,465,173,
6,461,612, 6,458,933, 6,432,633, 6,410,318, 6,406,701, 6,395,275,
6,391,657, 6,391,635, 6,384,198, 6,376,170, 6,372,217, 6,344,545,
6,337,181, 6,329,202, 6,319,665, 6,319,500, 6,316,003, 6,312,931,
6,309,880, 6,296,807, 6,291,239, 6,261,558, 6,248,514, 6,245,331,
6,242,197, 6,241,986, 6,228,361, 6,221,580, 6,190,871, 6,177,253,
6,146,635, 6,146,627, 6,146,614, 6,143,876, 6,132,992, 6,124,132,
RE36,866, 6,114,143, 6,103,238, 6,060,254, 6,039,684, 6,030,772,
6,020,468, 6,013,484, 6,008,044, 5,998,132, 5,994,515, 5,993,812,
5,985,545, 5,981,278, 5,958,765, 5,939,277, 5,928,930, 5,922,325,
5,919,457, 5,916,806, 5,914,109, 5,911,989, 5,906,936, 5,889,158,
5,876,716, 5,874,226, 5,872,012, 5,871,732, 5,866,694, 5,854,400,
5,849,583, 5,849,288, 5,840,480, 5,840,305, 5,834,599, 5,831,034,
5,827,723, 5,821,047, 5,817,767, 5,817,458, 5,804,440, 5,795,572,
5,783,670, 5,776,703, 5,773,225, 5,766,944, 5,753,503, 5,750,373,
5,747,641, 5,736,341, 5,731,189, 5,707,814, 5,702,707, 5,698,178,
5,695,927, 5,665,536, 5,658,745, 5,652,138, 5,645,836, 5,635,345,
5,618,922, 5,610,035, 5,607,847, 5,604,092, 5,601,819, 5,597,896,
5,597,688, 5,591,829, 5,558,865, 5,514,541, 5,510,264, 5,478,753,
5,374,518, 5,374,516, 5,344,755, 5,332,567, 5,300,433, 5,296,347,
5,286,852, 5,264,221, 5,260,308, 5,256,561, 5,254,457, 5,230,998,
5,227,159, 5,223,408, 5,217,895, 5,180,660, 5,173,399, 5,169,752,
5,166,050, 5,156,951, 5,140,105, 5,135,864, 5,120,640, 5,108,904,
5,104,790, 5,049,389, 5,030,718, 5,030,555, 5,004,697, 4,983,529,
4,888,290, 4,886,742 and 4,853,326, are also useful for the present
invention.
[0082] The vectors used in accordance with the present invention
should typically be chosen such that they contain a suitable gene
regulatory region, such as a promoter or enhancer, such that the
antigens and/or antibodies of the invention can be expressed.
[0083] For example, when the aim is to express the antibodies
and/or antigens of the invention in vitro, or in cultured cells, or
in any prokaryotic or eukaryotic system for the purpose of
producing the protein(s) encoded by that antibody and/or antigen,
then any suitable vector can be used depending on the application.
For example, plasmids, viral vectors, bacterial vectors, protozoal
vectors, insect vectors, baculovirus expression vectors, yeast
vectors, mammalian cell vectors, and the like, can be used.
Suitable vectors can be selected by the skilled artisan taking into
consideration the characteristics of the vector and the
requirements for expressing the antibodies and/or antigens under
the identified circumstances.
[0084] When the aim is to express the antibodies and/or antigens of
the invention in vivo in a subject, for example in order to
generate an immune response against an HIV-1 antigen and/or
protective immunity against HIV-1, expression vectors that are
suitable for expression on that subject, and that are safe for use
in vivo, should be chosen. For example, in some embodiments it may
be desired to express the antibodies and/or antigens of the
invention in a laboratory animal, such as for pre-clinical testing
of the HIV-1 immunogenic compositions and vaccines of the
invention. In other embodiments, it will be desirable to express
the antibodies and/or antigens of the invention in human subjects,
such as in clinical trials and for actual clinical use of the
immunogenic compositions and vaccine of the invention. Any vectors
that are suitable for such uses can be employed, and it is well
within the capabilities of the skilled artisan to select a suitable
vector. In some embodiments it may be preferred that the vectors
used for these in vivo applications are attenuated to vector from
amplifying in the subject. For example, if plasmid vectors are
used, preferably they will lack an origin of replication that
functions in the subject so as to enhance safety for in vivo use in
the subject. If viral vectors are used, preferably they are
attenuated or replication-defective in the subject, again, so as to
enhance safety for in vivo use in the subject.
[0085] In preferred embodiments of the present invention viral
vectors are used. Viral expression vectors are well known to those
skilled in the art and include, for example, viruses such as
adenoviruses, adeno-associated viruses (AAV), alphaviruses,
herpesviruses, retroviruses and poxviruses, including avipox
viruses, attenuated poxviruses, vaccinia viruses, and particularly,
the modified vaccinia Ankara virus (MVA; ATCC Accession No.
VR-1566). Such viruses, when used as expression vectors are
innately non-pathogenic in the selected subjects such as humans or
have been modified to render them non-pathogenic in the selected
subjects. For example, replication-defective adenoviruses and
alphaviruses are well known and can be used as gene delivery
vectors.
[0086] The present invention relates to recombinant vesicular
stomatitis (VSV) vectors, however, other vectors may be
contemplated in other embodiments of the invention such as, but not
limited to, prime boost administration comprising administration of
a recombinant VSV vector in combination with another recombinant
vector expressing one or more HIV epitopes.
[0087] VSV is a very practical, safe, and immunogenic vector for
conducting animal studies, and an attractive candidate for
developing vaccines for use in humans. VSV is a member of the
Rhabdoviridae family of enveloped viruses containing a
nonsegmented, negative-sense RNA genome. The genome is composed of
5 genes arranged sequentially 3'-N-P-M-G-L-S', each encoding a
polypeptide found in mature virions. Notably, the surface
glycoprotein G is a transmembrane polypeptide that is present in
the viral envelope as a homotrimer, and like Env, it mediates cell
attachment and infection.
[0088] In a first advantageous embodiment, the VSV G is replaced by
HIV Env or fragments thereof. The latter will generate chimeric
EnvG proteins (see, e.g. FIG. 22).
[0089] In a second advantageous embodiment, VSV G is a carrier or
scaffold advantageously for Env MPER epitopes, however, VSV G as a
carrier or scaffold may be extended to any foreign epitope (see,
e.g., FIGS. 5-7).
[0090] In a third advantageous embodiment, Env MPER epitopes are
fused to the VSV G-Stem molecule, however, any foreign epitope may
be fused to the VSV G-Stem molecule (see, e.g, FIGS. 18-19).
[0091] In a fourth embodiment, the invention pertains to the
evolutionary potential of RNA viruses. Such viruses include but are
not limited to: VSV, Measles virus, Canine distemper virus,
Parainfluenza viruses, Sendai virus, Newcastle disease virus,
Venezuelan equine encephalitis virus, Sindbis virus, Semliki
Forrest virus etc. Pertaining to the evolutionary potential of VSV,
in the first step of EnvG construction, a small panel of genes
encoding different forms of EnvG molecules will be produced to
determine which motifs from G will optimize expression.
Replication-competent `chimeric` VSV-HIV viruses that lack the
capacity to encode wild-type G and are dependent on EnvG for
infection and propagation, which are then utilized to direct the
evolution of new EnvG molecules that are expressed and incorporated
into the virus with greater efficiency.
[0092] In a fifth embodiment, the invention pertains to application
of selective pressure to enrich for molecules that are more
immunogenic. The evolution process will occur primarily through
nucleotide substitution, followed by selection using a broadly
neutralizing antibody against HIV Env, e.g. 2F5 or 4E10, or a broad
potent antibody specific for trimeric Env. Due to the nature of
negative-strand virus replication, base changes are far more
frequent than deletions or insertions, consequently the immunogen
will evolve with amino acid substitutions. (see, e.g. FIG. 24)
[0093] The VSVs of U.S. Pat. Nos. 7,468,274; 7,419,829; 7,419,674;
7,344,838; 7,332,316; 7,329,807; 7,323,337; 7,259,015; 7,244,818;
7,226,786; 7,211,247; 7,202,079; 7,198,793; 7,198,784; 7,153,510;
7,070,994; 6,969,598; 6,958,226; RE38,824; PP15,957; 6,890,735;
6,887,377; 6,867,326; 6,867,036; 6,858,205; 6,835,568; 6,830,892;
6,818,209; 6,790,641; 6,787,520; 6,743,620; 6,740,764; 6,740,635;
6,740,320; 6,682,907; 6,673,784; 6,673,572; 6,669,936; 6,653,103;
6,607,912; 6,558,923; 6,555,107; 6,533,855; 6,531,123; 6,506,604;
6,500,623; 6,497,873; 6,489,142; 6,410,316; 6,410,313; 6,365,713;
6,348,312; 6,326,487; 6,312,682; 6,303,331; 6,277,633; 6,207,455;
6,200,811; 6,190,650; 6,171,862; 6,143,290; 6,133,027; 6,121,434;
6,103,462; 6,069,134; 6,054,127; 6,034,073; 5,969,211; 5,935,822;
5,888,727; 5,883,081; 5,876,727; 5,858,740; 5,843,723; 5,834,256;
5,817,491; 5,792,604; 5,789,229; 5,773,003; 5,763,406; 5,760,184;
5,750,396; 5,739,018; 5,698,446; 5,686,279; 5,670,354; 5,540,923;
5,512,421; 5,090,194; 4,939,176; 4,738,846; 4,622,292; 4,556,556
and 4,396,628 may be contemplated by the present invention.
[0094] The nucleotide sequences and vectors of the invention can be
delivered to cells, for example if aim is to express and the HIV-1
antigens in cells in order to produce and isolate the expressed
proteins, such as from cells grown in culture. For expressing the
antibodies and/or antigens in cells any suitable transfection,
transformation, or gene delivery methods can be used. Such methods
are well known by those skilled in the art, and one of skill in the
art would readily be able to select a suitable method depending on
the nature of the nucleotide sequences, vectors, and cell types
used. For example, transfection, transformation, microinjection,
infection, electroporation, lipofection, or liposome-mediated
delivery could be used. Expression of the antibodies and/or
antigens can be carried out in any suitable type of host cells,
such as bacterial cells, yeast, insect cells, and mammalian cells.
The antibodies and/or antigens of the invention can also be
expressed using including in vitro transcription/translation
systems. All of such methods are well known by those skilled in the
art, and one of skill in the art would readily be able to select a
suitable method depending on the nature of the nucleotide
sequences, vectors, and cell types used.
[0095] In preferred embodiments, the nucleotide sequences,
antibodies and/or antigens of the invention are administered in
vivo, for example where the aim is to produce an immunogenic
response in a subject. A "subject" in the context of the present
invention may be any animal. For example, in some embodiments it
may be desired to express the transgenes of the invention in a
laboratory animal, such as for pre-clinical testing of the HIV-1
immunogenic compositions and vaccines of the invention. In other
embodiments, it will be desirable to express the antibodies and/or
antigens of the invention in human subjects, such as in clinical
trials and for actual clinical use of the immunogenic compositions
and vaccine of the invention. In preferred embodiments the subject
is a human, for example a human that is infected with, or is at
risk of infection with, HIV-1.
[0096] For such in vivo applications the nucleotide sequences,
antibodies and/or antigens of the invention_are preferably
administered as a component of an immunogenic composition
comprising the nucleotide sequences and/or antigens of the
invention in admixture with a pharmaceutically acceptable carrier.
The immunogenic compositions of the invention are useful to
stimulate an immune response against HIV-1 and may be used as one
or more components of a prophylactic or therapeutic vaccine against
HIV-1 for the prevention, amelioration or treatment of AIDS. The
nucleic acids and vectors of the invention are particularly useful
for providing genetic vaccines, i.e. vaccines for delivering the
nucleic acids encoding the antibodies and/or antigens of the
invention to a subject, such as a human, such that the antibodies
and/or antigens are then expressed in the subject to elicit an
immune response.
[0097] The compositions of the invention may be injectable
suspensions, solutions, sprays, lyophilized powders, syrups,
elixirs and the like. Any suitable form of composition may be used.
To prepare such a composition, a nucleic acid or vector of the
invention, having the desired degree of purity, is mixed with one
or more pharmaceutically acceptable carriers and/or excipients. The
carriers and excipients must be "acceptable" in the sense of being
compatible with the other ingredients of the composition.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include,
but are not limited to, water, saline, phosphate buffered saline,
dextrose, glycerol, ethanol, or combinations thereof, buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptide;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0098] An immunogenic or immunological composition can also be
formulated in the form of an oil-in-water emulsion. The
oil-in-water emulsion can be based, for example, on light liquid
paraffin oil (European Pharmacopea type); isoprenoid oil such as
squalane, squalene, EICOSANE.TM. or tetratetracontane; oil
resulting from the oligomerization of alkene(s), e.g., isobutene or
decene; esters of acids or of alcohols containing a linear alkyl
group, such as plant oils, ethyl oleate, propylene glycol
di(caprylate/caprate), glyceryl tri(caprylate/caprate) or propylene
glycol dioleate; esters of branched fatty acids or alcohols, e.g.,
isostearic acid esters. The oil advantageously is used in
combination with emulsifiers to form the emulsion. The emulsifiers
can be nonionic surfactants, such as esters of sorbitan, mannide
(e.g., anhydromannitol oleate), glycerol, polyglycerol, propylene
glycol, and oleic, isostearic, ricinoleic, or hydroxystearic acid,
which are optionally ethoxylated, and
polyoxypropylene-polyoxyethylene copolymer blocks, such as the
Pluronic.RTM. products, e.g., L121. The adjuvant can be a mixture
of emulsifier(s), micelle-forming agent, and oil such as that which
is commercially available under the name Provax.RTM. (IDEC
Pharmaceuticals, San Diego, Calif.).
[0099] The immunogenic compositions of the invention can contain
additional substances, such as wetting or emulsifying agents,
buffering agents, or adjuvants to enhance the effectiveness of the
vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack
Publishing Company, (ed.) 1980).
[0100] Adjuvants may also be included. Adjuvants include, but are
not limited to, mineral salts (e.g., AlK(SO.sub.4).sub.2,
AlNa(SO.sub.4).sub.2, AlNH(SO.sub.4).sub.2, silica, alum,
Al(OH).sub.3, Ca.sub.3(PO.sub.4).sub.2, kaolin, or carbon),
polynucleotides with or without immune stimulating complexes
(ISCOMs) (e.g., CpG oligonucleotides, such as those described in
Chuang, T. H. et al, (2002) J. Leuk. Biol. 71(3): 538-44;
Ahmad-Nejad, P. et al (2002) Eur. J. Immunol. 32(7): 1958-68; poly
IC or poly AU acids, polyarginine with or without CpG (also known
in the art as IC31; see Schellack, C. et al (2003) Proceedings of
the 34.sup.th Annual Meeting of the German Society of Immunology;
Lingnau, K. et al (2002) Vaccine 20(29-30): 3498-508), JuvaVax.TM.
(U.S. Pat. No. 6,693,086), certain natural substances (e.g., wax D
from Mycobacterium tuberculosis, substances found in
Cornyebacterium parvum, Bordetella pertussis, or members of the
genus Brucella), flagellin (Toll-like receptor 5 ligand; see
McSorley, S. J. et al (2002) J. Immunol. 169(7): 3914-9), saponins
such as QS21, QS17, and QS7 (U.S. Pat. Nos. 5,057,540; 5,650,398;
6,524,584; 6,645,495), monophosphoryl lipid A, in particular,
3-de-O-acylated monophosphoryl lipid A (3D-MPL), imiquimod (also
known in the art as IQM and commercially available as Aldara.RTM.;
U.S. Pat. Nos. 4,689,338; 5,238,944; Zuber, A. K. et al (2004)
22(13-14): 1791-8), and the CCR5 inhibitor CMPD167 (see Veazey, R.
S. et al (2003) J. Exp. Med. 198: 1551-1562).
[0101] Aluminum hydroxide or phosphate (alum) are commonly used at
0.05 to 0.1% solution in phosphate buffered saline. Other adjuvants
that can be used, especially with DNA vaccines, are cholera toxin,
especially CTA1-DD/ISCOMs (see Mowat, A. M. et al (2001) J.
Immunol. 167(6): 3398-405), polyphosphazenes (Allcock, H.R. (1998)
App. Organometallic Chem. 12(10-11): 659-666; Payne, L. G. et al
(1995) Pharm. Biotechnol. 6: 473-93), cytokines such as, but not
limited to, IL-2, IL-4, GM-CSF, IL-12, IL-15 IGF-1, IFN-.alpha.,
IFN-.beta., and IFN-.gamma. (Boyer et al., (2002) J. Liposome Res.
121:137-142; WO01/095919), immunoregulatory proteins such as CD40L
(ADX40; see, for example, WO03/063899), and the CD1a ligand of
natural killer cells (also known as CRONY or .alpha.-galactosyl
ceramide; see Green, T. D. et al, (2003) J. Virol. 77(3):
2046-2055), immunostimulatory fusion proteins such as IL-2 fused to
the Fc fragment of immunoglobulins (Barouch et al., Science
290:486-492, 2000) and co-stimulatory molecules B7.1 and B7.2
(Boyer), all of which can be administered either as proteins or in
the form of DNA, on the same expression vectors as those encoding
the antigens of the invention or on separate expression
vectors.
[0102] In an advantageous embodiment, the adjuvants may be lecithin
is combined with an acrylic polymer (Adjuplex-LAP), lecithin coated
oil droplets in an oil-in-water emulsion (Adjuplex-LE) or lecithin
and acrylic polymer in an oil-in-water emulsion (Adjuplex-LAO)
(Advanced BioAdjuvants (ABA)).
[0103] The immunogenic compositions can be designed to introduce
the nucleic acids or expression vectors to a desired site of action
and release it at an appropriate and controllable rate. Methods of
preparing controlled-release formulations are known in the art. For
example, controlled release preparations can be produced by the use
of polymers to complex or absorb the immunogen and/or immunogenic
composition. A controlled-release formulations can be prepared
using appropriate macromolecules (for example, polyesters,
polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate,
methylcellulose, carboxymethylcellulose, or protamine sulfate)
known to provide the desired controlled release characteristics or
release profile. Another possible method to control the duration of
action by a controlled-release preparation is to incorporate the
active ingredients into particles of a polymeric material such as,
for example, polyesters, polyamino acids, hydrogels, polylactic
acid, polyglycolic acid, copolymers of these acids, or ethylene
vinylacetate copolymers. Alternatively, instead of incorporating
these active ingredients into polymeric particles, it is possible
to entrap these materials into microcapsules prepared, for example,
by coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsule and
poly-(methylmethacrylate) microcapsule, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in New Trends and
Developments in Vaccines, Voller et al. (eds.), University Park
Press, Baltimore, Md., 1978 and Remington's Pharmaceutical
Sciences, 16th edition.
[0104] Suitable dosages of the nucleic acids and expression vectors
of the invention (collectively, the immunogens) in the immunogenic
composition of the invention can be readily determined by those of
skill in the art. For example, the dosage of the immunogens can
vary depending on the route of administration and the size of the
subject. Suitable doses can be determined by those of skill in the
art, for example by measuring the immune response of a subject,
such as a laboratory animal, using conventional immunological
techniques, and adjusting the dosages as appropriate. Such
techniques for measuring the immune response of the subject include
but are not limited to, chromium release assays, tetramer binding
assays, IFN-.gamma. ELISPOT assays, IL-2 ELISPOT assays,
intracellular cytokine assays, and other immunological detection
assays, e.g., as detailed in the text "Antibodies: A Laboratory
Manual" by Ed Harlow and David Lane.
[0105] When provided prophylactically, the immunogenic compositions
of the invention are ideally administered to a subject in advance
of HIV infection, or evidence of HIV infection, or in advance of
any symptom due to AIDS, especially in high-risk subjects. The
prophylactic administration of the immunogenic compositions can
serve to provide protective immunity of a subject against HIV-1
infection or to prevent or attenuate the progression of AIDS in a
subject already infected with HIV-1. When provided therapeutically,
the immunogenic compositions can serve to ameliorate and treat AIDS
symptoms and are advantageously used as soon after infection as
possible, preferably before appearance of any symptoms of AIDS but
may also be used at (or after) the onset of the disease
symptoms.
[0106] The immunogenic compositions can be administered using any
suitable delivery method including, but not limited to,
intramuscular, intravenous, intradermal, mucosal, and topical
delivery. Such techniques are well known to those of skill in the
art. More specific examples of delivery methods are intramuscular
injection, intradermal injection, and subcutaneous injection.
However, delivery need not be limited to injection methods.
Further, delivery of DNA to animal tissue has been achieved by
cationic liposomes (Watanabe et al., (1994) Mol. Reprod. Dev.
38:268-274; and WO 96/20013), direct injection of naked DNA into
animal muscle tissue (Robinson et al., (1993) Vaccine 11:957-960;
Hoffman et al., (1994) Vaccine 12: 1529-1533; Xiang et al., (1994)
Virology 199: 132-140; Webster et al., (1994) Vaccine 12:
1495-1498; Davis et al., (1994) Vaccine 12: 1503-1509; and Davis et
al., (1993) Hum. Mol. Gen. 2: 1847-1851), or intradermal injection
of DNA using "gene gun" technology (Johnston et al., (1994) Meth.
Cell Biol. 43:353-365). Alternatively, delivery routes can be oral,
intranasal or by any other suitable route. Delivery also be
accomplished via a mucosal surface such as the anal, vaginal or
oral mucosa.
[0107] Immunization schedules (or regimens) are well known for
animals (including humans) and can be readily determined for the
particular subject and immunogenic composition. Hence, the
immunogens can be administered one or more times to the subject.
Preferably, there is a set time interval between separate
administrations of the immunogenic composition. While this interval
varies for every subject, typically it ranges from 10 days to
several weeks, and is often 2, 4, 6 or 8 weeks. For humans, the
interval is typically from 2 to 6 weeks. The immunization regimes
typically have from 1 to 6 administrations of the immunogenic
composition, but may have as few as one or two or four. The methods
of inducing an immune response can also include administration of
an adjuvant with the immunogens. In some instances, annual,
biannual or other long interval (5-10 years) booster immunization
can supplement the initial immunization protocol.
[0108] The present methods also include a variety of prime-boost
regimens, for example DNA prime-Adenovirus boost regimens. In these
methods, one or more priming immunizations are followed by one or
more boosting immunizations. The actual immunogenic composition can
be the same or different for each immunization and the type of
immunogenic composition (e.g., containing protein or expression
vector), the route, and formulation of the immunogens can also be
varied. For example, if an expression vector is used for the
priming and boosting steps, it can either be of the same or
different type (e.g., DNA or bacterial or viral expression vector).
One useful prime-boost regimen provides for two priming
immunizations, four weeks apart, followed by two boosting
immunizations at 4 and 8 weeks after the last priming immunization.
It should also be readily apparent to one of skill in the art that
there are several permutations and combinations that are
encompassed using the DNA, bacterial and viral expression vectors
of the invention to provide priming and boosting regimens.
[0109] The prime-boost regimen can also include VSV vectors that
derive their G protein or G/Stem protein from different serotype
vesicular stomatitis viruses (Rose N F, Roberts A, Buonocore L,
Rose J K. Glycoprotein exchange vectors based on vesicular
stomatitis virus allow effective boosting and generation of
neutralizing antibodies to a primary isolate of human
immunodeficiency virus type 1. J Virol. 2000 December;
74(23):10903-10). The VSV vectors used in these examples contain a
G or G/Stem protein derived from the Indiana serotype of VSV.
Vectors can also be constructed to express epitopes in the context
of G or G/Stem molecules derived from other VSV serotypes (i.e.
vesicular stomatitis New Jersey virus or vesicular stomatitis
Alagoas virus) or other vesiculoviruses (i.e. Chandipura virus,
Cocal virus, Isfahan virus). Thus an epitope like the HIV MPER can
be delivered in a prime in the context of an G or G/Stem molecule
that is from the Indiana serotype and the immune system can be
boosted with a vector that expresses epitopes in the context of
second serotype like New Jersey. This circumvents anti-G immunity
elicited by the prime, and helps focus the boost response against
the foreign epitope.
[0110] A specific embodiment of the invention provides methods of
inducing an immune response against HIV in a subject by
administering an immunogenic composition of the invention,
preferably comprising an adenovirus vector containing DNA encoding
one or more of the epitopes of the invention, one or more times to
a subject wherein the epitopes are expressed at a level sufficient
to induce a specific immune response in the subject. Such
immunizations can be repeated multiple times at time intervals of
at least 2, 4 or 6 weeks (or more) in accordance with a desired
immunization regime.
[0111] The immunogenic compositions of the invention can be
administered alone, or can be co-administered, or sequentially
administered, with other HIV immunogens and/or HIV immunogenic
compositions, e.g., with "other" immunological, antigenic or
vaccine or therapeutic compositions thereby providing multivalent
or "cocktail" or combination compositions of the invention and
methods of employing them. Again, the ingredients and manner
(sequential or co-administration) of administration, as well as
dosages can be determined taking into consideration such factors as
the age, sex, weight, species and condition of the particular
subject, and the route of administration.
[0112] When used in combination, the other HIV immunogens can be
administered at the same time or at different times as part of an
overall immunization regime, e.g., as part of a prime-boost regimen
or other immunization protocol. In an advantageous embodiment, the
other HIV immunogen is env, preferably the HIV env trimer.
[0113] Many other HIV immunogens are known in the art, one such
preferred immunogen is HIVA (described in WO 01/47955), which can
be administered as a protein, on a plasmid (e.g., pTHr.HIVA) or in
a viral vector (e.g., MVA.HIVA). Another such HIV immunogen is
RENTA (described in PCT/US2004/037699), which can also be
administered as a protein, on a plasmid (e.g., pTHr.RENTA) or in a
viral vector (e.g., MVA.RENTA).
[0114] For example, one method of inducing an immune response
against HIV in a human subject comprises administering at least one
priming dose of an HIV immunogen and at least one boosting dose of
an HIV immunogen, wherein the immunogen in each dose can be the
same or different, provided that at least one of the immunogens is
an epitope of the present invention, a nucleic acid encoding an
epitope of the invention or an expression vector, preferably a VSV
vector, encoding an epitope of the invention, and wherein the
immunogens are administered in an amount or expressed at a level
sufficient to induce an HIV-specific immune response in the
subject. The HIV-specific immune response can include an
HIV-specific T-cell immune response or an HIV-specific B-cell
immune response. Such immunizations can be done at intervals,
preferably of at least 2-6 or more weeks.
[0115] It is to be understood and expected that variations in the
principles of invention as described above may be made by one
skilled in the art and it is intended that such modifications,
changes, and substitutions are to be included within the scope of
the present invention.
[0116] The invention will now be further described by way of the
following non-limiting examples.
EXAMPLES
Example 1
Insertion of the HIV-1 gp41 Epitopes 2F5 and 4E10 into the
Membrane-Proximal Region of the Vesicular Stomatitis Virus
Glycoprotein
[0117] The membrane-proximal external region (MPER) of HIV-1 gp41,
which is recognized by the broadly neutralizing monoclonal
antibodies 2F5 and 4E10, is an important target for an HIV vaccine.
However, efforts to mimic the 2F5 and 4E10 epitopes outside the
context of the gp41 MPER have had minimal success so far. In this
study, Applicants used the envelope glycoprotein G of Vesicular
Stomatitis Virus (VSV) as a scaffold. VSV G, which forms
homotrimeric spikes on the viral surface, is responsible for
binding of the virus to cells and promotes fusion of the viral and
cellular membranes. The "stem" region of VSV G, which lies
immediately N-terminal of its single transmembrane segment, shares
sequence similarities with the gp41 MPER. Applicants inserted the
gp41 sequences corresponding to the 2F5 and 4E10 neutralizing
epitopes into the stem region of VSV G and evaluated the function
and antibody reactivity of the chimeric polypeptides. VSV-G-2F5 and
VSV-G-4E10 formed trimers and were transported to the cell surface,
where they were detected by the 2F5 and 4E10 monoclonal antibodies,
respectively. Reporter lentiviruses pseudotyped with VSV G-2F5 or
VSV-G-4E10 were infectious, and they were efficiently neutralized
by the 2F5 or 4E10 monoclonal antibodies. Recombinant VSV
containing G-2F5, G-4E10 or G-2F5-4E10 on the viral surface was
infectious, replication-competent, and sensitive to neutralization
by the 2F5 or 4E10 monoclonal antibodies. Applicants are currently
determining if the recombinant VSVs encoding MPER epitopes elicit
neutralizing antibodies specific for the HIV gp41 epitopes in a
small animal model. Taken together, Applicants' approach represents
a novel strategy to develop a vaccine that induces a humoral immune
response against HIV.
Example 2
Using VSV Vectors to Display and Evolve Novel HIV Envelope
Immunogens
[0118] The goal of this Example is to design and develop novel
HIV-1 envelope protein (Env) immunogens capable of eliciting
broadly protective neutralizing antibody responses for use as
vaccine candidates. Applicants take advantage of the unique
biological properties of vesicular stomatitis virus (VSV) as
vaccine delivery vehicle to present and effectively deliver HIV Env
immunogens. In addition, Applicants use the high evolutionary
potential of VSV to biologically derive unique mutant HIV Envs with
enhanced immunogenicity. Novel candidates are used to vaccinate
rabbits to determine their capacity to elicit antibodies with
enhanced HIV neutralizing activity, and those VSV-vectored vaccines
that evoke responses with increased breadth of neutralization are
tested in macaques. Applicants achieve these goals by completing
the Specific Aims below: [0119] 1. Vaccine Platform 1: Optimize HIV
Env (derived from SHIV.sub.SF162P3) for expression as functional
stable trimers on the surface of VSV particles, and produce
`chimeric viruses`, in which the gene encoding the VSV surface
glycoprotein (G) are functionally replaced by HIV Env. Env
modifications described below are investigated to identify the
optimal form for expressing abundant functional trimers on VSV
particles that specifically direct infection of cells expressing
the CD4 and CCR5 coreceptors (CD4/CCR5+ cells). Additionally,
Applicants take advantage of the innate ability of VSV to rapidly
accrue adaptive mutations to further optimize expression of
functional Env trimers by subjecting replication-competent VSV-Env
chimeric viruses to serial passage on CD4/CCR5+ cell lines to
biologically select for Env mutations that improve replicative
fitness. Moreover, to develop additional novel Env immunogens,
methods to apply selective pressure during serial passage are
developed using the broadly neutralizing antibodies against Env
(e.g. monoclonal antibodies 2F5, 4E10, 2G12, b12, PG9, PG16 and
other antibodies, including broad potent neutralizing
trimer-specific antibodies). [0120] 2. Vaccine Platform 2: Produce
recombinant VSV (rVSV) vectors that encode modified forms of VSV G,
which harbor epitopes from the HIV Env membrane proximal external
region (MPER). This takes advantage of several G protein properties
including: i) it is a glycosylated transmembrane protein abundantly
expressed on the VSV particle; ii) it is a potent immunogen; iii)
it contains a hydrophobic membrane-proximal region that resembles
the Env MPER, and iv) G trimerizes and provides a platform for
multimeric configurations of MPER epitopes. Although several
domains in G are tested as sites for insertion of MPER sequences,
Applicants focus on the membrane proximal region of G, which
provides a similar membrane-associated environment for the most
authentic presentation of MPER epitopes. Env MPER insertions that
do not abolish the function of VSV G are delivered using VSV
vectors and advanced into rabbit immunogenicity studies.
Additionally, VSV encoding G-MPER hybrids are subjected to serial
passage to determine whether virus expressing a fitness advantage
emerges with unique mutations that affect the MPER epitope
configuration. Moreover, serial passage also are conducted using
conditions that select virus expressing G-MPER proteins that bind
with high avidity to the 2F5 and 4E10 mAbs to derive unique
immunogens. [0121] 3. Vaccine Platform 3: An N-terminally truncated
form of VSV G (called G/Stem) are used to present Env epitope
sequences on the surface of VSV particles. The G/Stem molecule
contains the cytoplasmic tail (CT) and trans-membrane (TM) spanning
domains of G as well as a short 16- to 68-amino acid membrane
proximal extracellular polypeptide (the Stem) to which HIV Env
epitopes are appended. Several forms of G/Stem, which vary in
length and amino acid sequence, are investigated to determine the
optimal form for display of MPER epitopes on the surface of VSV
particles and the plasma membrane of infected cells. VSV encoding
G/Stem fusion proteins can be propagated using G
trans-complementation or by generating recombinant virus that
contains a functional G gene in addition to the G/Stem coding
sequence. Novel G/Stem-MPER molecules are evolved by serial passage
under conditions that select for vectors encoding mutant molecules
that bind to the 2F5 and 4E10 mAbs with high affinity. [0122] 4. In
Vivo Studies: After validating their in vitro properties, promising
vaccine candidates developed in Aims 1-3 are evaluated by
vaccinating rabbits. Enzyme-linked immunosorbent assays (ELISAs)
are conducted first to screen for serum antibodies that react with
HIV Env, and those immune sera that contain significant titers are
evaluated in HIV neutralization assays using virus-like particles
pseudotyped with Env from various HIV strains. The top rVSV-Env
vaccine candidates that evoke production of broadly neutralizing
antibodies in vaccinated rabbits are advanced into nonhuman primate
studies. Rhesus macaques are vaccinated to determine whether
immunization protects macaques from subsequent intravenous
challenge with the SIV-HIV chimeric virus SHIV.sub.SF162P3, which
expresses an HIV envelope protein.
Example 3
Optimization of Immunogen Presentation by G-Stem Vectors
[0123] To develop a platform that can be used to display immunogens
on the surface of virus particles or infected cells, Applicants
have engineered vesicular stomatitis virus (VSV) vectors to encode
a truncated form of the viral transmembrane glycoprotein protein
(G) that can be modified to express foreign epitopes anchored to
virus envelop or cell membrane. The truncated form of G, called
G-Stem (FIG. 18A), retains amino acid sequences that are essential
for directing insertion of the molecule into the membrane (the
signal peptide), anchoring the protein in the viral envelop or
cellular lipid bilayer (the transmembrane domain; TM), and
promoting incorporation into the budding viral particle (C-terminal
domain). Additionally, a small membrane proximal region of the
external domain of G (the Stem) is retained in most constructs
because it provides a short stalk on which to append epitopes (FIG.
18B), and importantly, sequences in the Stem are known to promote
efficient assembly of VSV particles [Robison & Whitt, J Virol
2000; 74:2239-2246].
[0124] Because the Stem domain plays at least two significant roles
in Applicants' epitope display vectors--it serves as the platform
on which epitopes are attached and displayed, and it plays a role
in VSV maturation--Applicants anticipated that it might be
necessary to empirically determine the optimal Stem sequence needed
for expression and membrane incorporation of G-Stem-Epitope fusion
proteins. Applicants tested this assumption by constructing 4
different G-Stem fusion proteins that contained the HIV Env
membrane proximal external region (MPER) [Montero et al., Microbiol
Mol Biol Rev 2008; 72:54-84] fused to Stem domains that were 68,
42, 16 or 0 amino acids in length, referred to as long stem (LS),
medium stem (MS), short stem (SS), and no stem (NS), respectively
(FIGS. 19A-C).
[0125] The 4 G-Stem-MPER (GS-MPER) molecules were expressed using a
novel replication-competent VSV vector that retains a functional G
protein and expresses the GS-MPER fusion proteins from an added
transcription unit inserted in the highly-transcribed promoter
proximal position in the viral genome (FIG. 20). Consequently, the
MPER expression vectors express GS-MPER fusion proteins as well as
wild-type G protein. Expression of native G protein confers a
replication-competent phenotype of these recombinant viruses, and
importantly, this also means that infected cells will produce
wild-type G and GS-MPER proteins and that both proteins can be
inserted into cell membrane and viral envelop (right side of FIG.
20B).
[0126] After the recombinant VSV-G-Stem-MPER vectors were
constructed, they were used to infect Vero cells and assess
expression of the GS-MPER fusion proteins and determine their
relative abundance in virus particles (FIG. 21). FIG. 21 shows a
Western blot that was used to analyze G and G-Stem-MPER proteins
found in the medium supernatant of infected cells. The source of G
and GS-MPER fusion proteins in the supernatant primarily should be
virus that has budded out of infected cells; therefore, the
proteins visualized in Panel A provide an estimate of the relative
G and GS-MPER abundance in progeny virus particles. The blot in
Panel A was reacted with antibody that recognizes the C-terminus of
VSV G, which is present on both the native G protein the
G-Stem-MPER molecules. The results indicate that NS-MPER and
SS-MPER are present at higher levels in the virus particle than
MS-MPER or LS-MPER, and that none of the G-Stem-MPERs are as
abundant as the native G protein. It is important to note that a
proteolytic fragment of G comigrates with the NS-MPER at the top of
the gel (Lane 6) making it difficult to estimate its abundance. The
relative amount of the 4 MPER-containing molecules is more clearly
shown in Panels C and D where the GS-MPER proteins are reacted with
MPER-Specific monoclonal antibodies 2F5 and 4E10. In Panel C for
example, the relative amounts of NS-MPER (Lane 6) and SS-MPER (Lane
5) are clearly greater than MS- and LS-MPER (Lanes 3 and 4) in
virus particles found in the supernatant. It is worth noting that
the LS-MPER molecule is expressed at relatively high levels in
infected cells as shown in Panel B (Lane 2) suggesting that this
form of G-Stem-MPER is expressed but not efficiently incorporated
into virus particles. The MS-MPER protein is evident in the
infected cells (Panel B, Lane 3) but at low levels indicating that
it is expressed poorly or it is unstable compared to the other
GS-MPERS. Finally, it is notable that the NS-MPER protein, which
lacks the Stem completely, seems to be incorporated at the highest
levels of all of the G-Stem-MPERs (FIGS. 21C and D, Lanes 5 and 6).
This finding seems to be contrary to the known role of Stem in
virus particle maturation [Robison & Whitt, J Virol 2000;
74:2239-2246], but it is consistent with Applicants' results that
show that the MPER and smaller peptides from the MPER regions can
functionally substitute for the Stem (see, e.g. FIG. 14).
[0127] Taken together, these results show that achieving
significant expression of G-Stem fusion proteins in infected cells
and on virus particles requires optimization of the Stem domain.
Applicants' finding that the NS Stem domain is perhaps optimal for
expression of HIV MPER probably reflects the fact that the MPER has
Stem-like properties. Other antigens expressed as G-Stem-antigen
fusions may require different lengths of Stem to be incorporated
efficiently into cellular or viral membranes.
Example 4
Insertion of the HIV-1 gp41 Epitopes 2F5 and 4E10 into the
Membrane-Proximal Region of the Vesicular Stomatitis Virus
Glycoprotein
[0128] Broadly neutralizing antibodies against the HIV Env protein
may bind epitopes on gp120 and gp41 (see, e.g., FIG. 1B). Such
antibodies include, but are not limited to, PG9 and PG16 (which
bind the base of V1/V2 loops and are trimer-specific), 2G12 (which
binds carbohydrates), b12 (which binds the CD4-binding site) and
2F5, 4E10 and Z13 (which bind the membrane-proximal external region
(MPER)).
[0129] A schematic of VSV is presented in FIG. 2. VSV is an
enveloped, negative-strand RNA virus of the Rhabdoviridae family.
VSV infects human cells, but is not pathogenic and propagates
robustly in vitro and is a safe and immunogenic vector for
conducting animal studies.
[0130] A schematic of the VSV glycoprotein G is presented in FIG.
3. VSV glycoprotein G is a single envelope glycoprotein on the
viral surface that forms trimers (ca. 1,200 molecules arranged as
400 trimers). VSV glycoprotein G mediates attachment, fusion, and
entry of VSV into host cell, accepts insertion of short amino acid
sequences at certain positions and has a membrane-proximal `stem`
region that shares similarities with the MPER of HIV-1 gp41.
[0131] Glycoprotein G is envisioned as an insertion site. In
particular, epitope sequences, in particular HIV epitope sequences,
more preferably HIV gp41 2F5 and 4E10 epitope sequences may be
inserted into the stem region of VSV G. Replication-competent,
recombinant VSV containing the modified G protein may be generated
for use as an immunogen. FIG. 5 presents a schematic of insertion
and substitution of HIV gp41 2F5 and 4E10 epitopes. FIG. 6 depicts
insertion and substitution of the 2F5 and 4E10 epitopes. For an
insertion, the 2F5 epitope and flanking residues was added to the
VSV G stem region. For a substitution, residues in the VSV G stem
region were replaced by the 2F5 and/or 4E10 epitopes. A summary of
the VSV G constructs are presented in FIG. 7. The expression vector
was pCI-Neo (deltaT7).
[0132] A Western blot demonstrating the expression and antibody
recognition of VSV G proteins expressed from plasmid DNA constructs
is presented in FIG. 8. VSV constructs were expressed transiently
in 293T cells and the Western blot was performed with lysates (2%
CHAPS). The Western blot showed that the stem region of VSV G
tolerated the insertion of the 2F5 and/or 4E10 epitope, and that
modified VSV G constructs were detected by the 2F5 and 4E10
antibodies.
[0133] Trimerization of VSV G on the cell surface is presented in
FIG. 9. The VSV G plasmid DNA constructs were expressed in 293T
cells, chemical crosslinking was performed with DTSSP
(3,3'-Dithiobis-[sulfosuccinimidyl-propionate]) on intact cells and
western blot with cell lysates was performed. As shown in FIG. 9,
all VSV G variants form trimers on the surface of 293T cells.
[0134] Cell surface expression of VSV G constructs is presented in
FIG. 10. The VSV G constructs were transiently expressed in 293T
cells, and flow cytometry was performed 24 hours post-transfection.
The modified VSV G constructs were expressed on the cell surface
and detected by the 2F5 and 4E10 antibodies.
[0135] VSV G mediated cell-cell fusion is presented in FIG. 11.
293T cells were transfected with plasmid encoding VSV G, briefly
exposed to pH 5.2 after 24 hours, and syncitia formation was
observed. As shown in FIG. 11, VSV G-2F5-Sub and VSV G-4E10-Sub
both induced cell-cell fusion. In addition, VSV G-2F5-4E10-Sub
showed small areas of cell-cell fusion in rare cases. It was
postulated that the modified G proteins may confer virus entry. To
answer this question, a lentivirus reporter system was
developed.
[0136] A lentivirus reporter system is presented in FIG. 12. 293T
cells were co-transfected with reporter plasmids pV1-GFP or pV1-Luc
(HIV provirus with 5' and 3' LTR), and plasmids coding for Gag-Pol
and VSV-G. Supernatants containing GFP or luciferase-encoding
lentiviruses pseutodypted with VSV G were harvested, followed by
infection of naive 293T cells. If VSV G mediates entry, cells will
express GFP or luciferase.
[0137] Infectivity of lenviruses pseudotyped with VSV G is
presented in FIG. 13. 293T cells were infected with recombinant
GFP-lentiviruses pseudotyped with VSV G variants. As shown in FIG.
13, the infectivity of VSV G-2F5-Sub and VSV G-4E10-Sub was similar
to wild-type G.
[0138] Infectivity of reporter lentiviruses pseudotyped with VSV G
is presented in FIG. 14. 293T cells were infected with recombinant
Luc-lentiviruses pseudotyped with VSV G variants. Lentiviruses
pseudotyped with VSV G-2F5-Sub and VSV G-4E10-Sub retained 33% and
35% of infectivity compared to wild-type VSV G. It was postulated
that these viruses be neutralized with the 2F5 and 4E10
antibodies.
[0139] Neutralization of lentiviruses pseudotyped with VSV G is
depicted in FIG. 15. Luc-lentiviruses pseudotyped with VSV
G-2F5-Sub or VSV G-4E10-Sub were incubated with 2F5 or 4E10
antibody at various concentrations. Subsequently, 293T cells were
infected with the Luc-lentiviruses, followed by assaying luciferase
activity at 3 days post-infection. Luc-lentiviruses pseudotyped
with VSV G-2F5-Sub and VSV G-4E10-Sub were efficiently neutralized
with the 2F5 and 4E10 antibody, respectively. It was then
postulated that modified G proteins could be incorporated into
recombinant VSV.
[0140] Recombinant VSV containing the gene coding for G-2F5-Sub,
G-4E10-Sub and G-2F5-4E10-Sub were rescued. A growth curve analysis
by plaque assay on Vero cells (m.o.i of 5) is shown in FIG. 16. The
growth kinetics of rVSV containing G-2F5-Sub, G-4E10-Sub or
G-2F5-4E10-Sub were similar to wild-type. It was then postulated
that rVSV G-2F5-Sub, rVSV G-4E10-Sub and rVSV G-2F5-4E10-Sub could
be neutralized with the 2F5 and 4E10 antibodies.
[0141] Neutralization of recombinant VSV with various antibodies is
shown in FIG. 17. 5000 pfu rVSV G-2F5-Sub, rVSV G-4E10-Sub or rVSV
G-2F5-4E10-Sub were incubated with VI-10 (control antibody against
the ectodomain of VSV G, i.e. it should neutralize all viruses with
G), 2F5 or 4E10 at various concentrations, followed by a plaque
assay on Vero cells. As shown in FIG. 17, rVSV containing
G-2F5-Sub, G-4E10-Sub or G-2F5-4E10-Sub was efficiently neutralized
by the 2F5 and/or 4E10 antibodies.
[0142] To summarize this Example: (1) the `stem` region of the
Vesicular Stomatitis Virus (VSV) glycoprotein tolerated the
insertion of the HIV-1 gp41 2F5 and 4E10 epitope sequences, (2) the
modified VSV G proteins were expressed on the cell surface and
detected by the respective HIV broadly neutralizing antibodies, (3)
lentiviruses pseudotyped with VSV G-2F5-Sub or VSV G-4E10-Sub were
infectious and could be neutralized with the 2F5 and 4E10 antibody,
respectively and (4) recombinant VSVs with G-2F5-Sub, G-4E10-Sub or
G-2F5-4E10-Sub were infectious, had similar growth kinetics like
wild-type rVSV, and could be efficiently neutralized with the 2F5
and 4E10 antibodies. Applicants conclude that the HIV-1 gp41 2F5
and 4E10 epitope sequences were presented in a native-like
conformation in the `stem` region of the VSV glycoprotein.
Example 5
Optimization Strategy Adopted for Optimization of VSV G Protein
Coding Sequence
[0143] The gene was optimized for expression in eukaryotic cells
using the following steps: 1. Started with amino acid sequence for
VSV G serotype Indiana, strain Orsay (Genbank M11048.1) 2. The
amino acid sequence was reverse-translated using the OPTIMIZER
webtool (available on the OPTIMIZER website associated with
Universitat Rovira i Virgili (URV)) and a human codon frequency
table [Puigb P et al. Nucleic Acids Res. 2007 July; 35 (Web Server
issue):W126-31] 3. The DNA sequence obtained from
reverse-translation was scanned for potential mRNA splice donor and
acceptor sequences using the Splice Site Prediction webtool
available on the fruitfly.org website [Reese M G et al. J Comput
Biol. 1997 Fall; 4(3):311-23]. Potential splicing signals were
disrupted subsequently by introducing one or two synonymous codons,
which altered key elements in the donor or acceptor site.
Synonymous codons were selected based on frequencies found in the
Codon Table published by Zhang et al [Hum Mol. Genet. 1998 May; 7
(5):919-32] for GC-rich transcripts. 4. The reverse-translated
sequence also was scanned for homopolymeric sequences .gtoreq.5
nucleotides. Those that were .gtoreq.5 were interrupted by
substitution of sequence with a synonymous codon as described in
the step above. 5. The sequence was scanned for the presence of
mRNA instability elements [Zubiaga A M et al. 1995, Mol. Cell.
Biol. 15: 2219-2230]. None were found. 6. Optimal translation
initiation (Kozak element [Kozak M. J Biol Chem. 1991 25; 266
(30):19867-70]) and termination signals [Kochetov A V et al. FEBS
Lett. 1998 4; 440(3):351-5] were introduced. 7. Unique XhoI and
NotI sites were added to the 5' and 3' termini, respectively, as
presented in FIGS. 28 A and 28B.
[0144] The invention is further described by the following numbered
paragraphs:
[0145] 1. A recombinant vesicular stomatitis virus (VSV) vector
wherein the gene encoding the VSV surface glycoprotein G (VSV G) is
functionally replaced by HIV Env.
[0146] 2. The vector of paragraph 1 wherein the HIV Env is
recognized by antibodies PG9, PG16, 2G12, b12, 2F5, 4E10 or Z13, or
other Env-specific antibodies, including broad potent neutralizing
trimer-specific antibodies.
[0147] 3. A recombinant vesicular stomatitis virus (VSV) vector
encoding a modified form of VSV G, wherein the modified form of VSV
G harbors epitopes from the HIV Env membrane proximal external
region (MPER).
[0148] 4. The vector of paragraph 3 wherein the MPER sequence is
inserted into the membrane proximal region of VSV G.
[0149] 5. The vector of paragraph 3 or 4 wherein a G-MPER protein
binds with high avidity to 2F5 and 4E10 monoclonal antibodies.
[0150] 6. A recombinant vesicular stomatitis virus (VSV) vector
encoding a an N-terminally truncated form of VSV G (G/Stem),
wherein the G/Stem presents Env epitope sequences on the surface of
VSV particles.
[0151] 7. The vector of paragraph 6 wherein G/Stem contains a
cytoplasmic tail (CT) and trans-membrane (TM) spanning domains of
G, a membrane proximal extracellular polypeptide (the Stem) that
can be 0 to 16 to 68 amino acids in, wherein HIV Env epitopes are
appended to the Stem.
[0152] 8. The vector of paragraph 7 wherein the HIV Env epitopes
are MPER epitopes.
[0153] 9. The vector of paragraph 8 wherein the G/Stem-MPER
molecules bind to 2F5 and 4E10 monoclonal antibodies with high
affinity.
[0154] 10. The vector of any one of paragraphs 1-9 wherein the HIV
Env is a mutant HIV Env.
[0155] 11. A method of generating novel chimeric EnvG molecules
expressed and incorporated into VSV comprising:
[0156] (a) serially passaging replication-competent chimeric
VSV-HIV viruses that lack the capacity to encode wild-type G and
are dependent on EnvG for infection and propagation on cells to
promote emergence of viruses with greater replicative fitness
and
[0157] (b) identifying novel mutations that enhance Env or EnvG
function.
[0158] 12. The method of paragraph 11, wherein the cells are
CD4/CCR5.sup.+ cells.
[0159] 13. The method of paragraph 11 or 12 wherein the novel
mutations escalate trimer abundance on the virus particle and/or
increase the stability of the functional trimeric form of Env or a
chimeric EnvG.
[0160] 14. The method of paragraph 11, 12 or 13 further comprising
determining whether the Env or EnvG immunogens elicit broadly
neutralizing anti-Env antibodies.
[0161] 15. The method of paragraph 11, 12, 13 or 14 further
comprising applying selective pressure to generate novel Env or
EnvG molecules expressed and incorporated into VSV, wherein the
selective pressure is binding to an antibody of interest.
[0162] 16. The method of paragraph 15 wherein the antibody is PG9,
PG16, b12, 2G12, 2F5 or 4E10 or any other broad potent neutralizing
Env trimer specific antibody.
[0163] 17. A method of producing an immune response comprising
administering to a mammal the vector of any one of paragraphs
1-10.
[0164] 18. A method of eliciting an immune response comprising
administering to a mammal the vector of any one of paragraphs
1-10.
[0165] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
Sequence CWU 1
1
15130PRTHuman immunodeficiency virus 1 1Gln Glu Leu Leu Glu Leu Asp
Lys Trp Ala Ser Leu Trp Asn Trp Phe1 5 10 15Asp Ile Thr Asn Trp Leu
Trp Tyr Ile Lys Ile Phe Ile Met 20 25 30230PRTVesicular stomatitis
virus 2Glu Ser Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys Asn Pro Ile
Glu1 5 10 15Leu Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Ser Ile Ala
20 25 30314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Ser Gly Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser
Leu Gly1 5 10419PRTVesicular stomatitis virus 4Glu Ser Leu Phe Phe
Gly Asp Thr Gly Leu Ser Lys Asn Pro Ile Glu1 5 10 15Leu Val
Glu533PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Glu Ser Leu Phe Phe Gly Asp Thr Gly Ser Gly Glu
Leu Leu Glu Leu1 5 10 15Asp Lys Trp Ala Ser Leu Gly Leu Ser Lys Asn
Pro Ile Glu Leu Val 20 25 30Glu619PRTHuman immunodeficiency virus 1
6Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp1 5
10 15Ile Thr Asn716PRTVesicular stomatitis virus 7Ser Lys Asn Pro
Ile Glu Leu Val Glu Gly Trp Phe Ser Ser Trp Lys1 5 10
15824PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Ser Lys Asn Pro Ile Glu Leu Leu Glu Leu Asp Lys
Trp Ala Ser Leu1 5 10 15Trp Asn Trp Phe Ser Ser Trp Lys
20912PRTHuman immunodeficiency virus 1 9Trp Phe Asp Ile Thr Asn Trp
Leu Trp Tyr Ile Lys1 5 101016PRTVesicular stomatitis virus 10Glu
Leu Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Trp Ser Ile Ala1 5 10
151119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Val Glu Gly Trp Phe Asp Ile Thr Asn Trp Leu Trp
Tyr Ile Lys Ser1 5 10 15Ser Ile Ala1234PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Ser
Lys Asn Pro Ile Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu1 5 10
15Trp Asn Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Lys Ser Ser
20 25 30Ile Ala13136PRTVesicular stomatitis virus 13Met Lys Cys Leu
Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys1 5 10 15Lys Ala Ser
Gly Tyr Lys Phe Pro Leu Tyr Met Ile Gly His Gly Met 20 25 30Leu Asp
Ser Asp Leu His Leu Ser Ser Lys Ala Gln Val Phe Glu His 35 40 45Pro
His Ile Gln Asp Ala Ala Ser Gln Leu Pro Asp Asp Glu Ser Leu 50 55
60Phe Phe Gly Asp Thr Gly Leu Ser Lys Asn Pro Ile Glu Leu Val Glu65
70 75 80Gly Trp Phe Ser Ser Trp Lys Ser Ser Ile Ala Ser Phe Phe Phe
Ile 85 90 95Ile Gly Leu Ile Ile Gly Leu Phe Leu Val Leu Arg Val Gly
Ile His 100 105 110Leu Cys Ile Lys Leu Lys His Thr Lys Lys Arg Gln
Ile Tyr Thr Asp 115 120 125Ile Glu Met Asn Arg Leu Gly Lys 130
135146973DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 14tcaatattgg ccattagcca tattattcat
tggttatata gcataaatca atattggcta 60ttggccattg catacgttgt atctatatca
taatatgtac atttatattg gctcatgtcc 120aatatgaccg ccatgttggc
attgattatt gactagttat taatagtaat caattacggg 180gtcattagtt
catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
240gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
atgttcccat 300agtaacgcca atagggactt tccattgacg tcaatgggtg
gagtatttac ggtaaactgc 360ccacttggca gtacatcaag tgtatcatat
gccaagtccg ccccctattg acgtcaatga 420cggtaaatgg cccgcctggc
attatgccca gtacatgacc ttacgggact ttcctacttg 480gcagtacatc
tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
540caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
ccattgacgt 600caatgggagt ttgttttggc accaaaatca acgggacttt
ccaaaatgtc gtaacaactg 660cgatcgcccg ccccgttgac gcaaatgggc
ggtaggcgtg tacggtggga ggtctatata 720agcagagctc gtttagtgaa
ccgtcagatc actagaagct ttattgcggt agtttatcac 780agttaaattg
ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
840gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg
taagtatcaa 900ggttacaaga caggtttaag gagaccaata gaaactgggc
ttgtcgagac agagaagact 960cttgcgtttc tgataggcac ctattggtct
tactgacatc cactttgcct ttctctccac 1020aggtgtccac tcccagttca
attacagctc ttaaggcgag agtactcgta cgctagcctc 1080gagaggagcc
accatgaagt gcctgctgta cctggccttc ctgttcatcg gcgtgaactg
1140caagttcacc atcgtgttcc cccacaacca gaagggcaac tggaagaacg
tgcccagcaa 1200ctaccactac tgccccagca gcagcgacct gaactggcac
aacgacctga tcggcaccgc 1260cctgcaagtc aagatgccca agagccacaa
ggccatccag gccgacggct ggatgtgcca 1320cgccagcaag tgggtgacca
cctgcgactt ccggtggtac ggccccaagt acatcaccca 1380cagcatccgc
agcttcaccc caagcgtgga gcagtgcaag gagagcatcg agcagaccaa
1440gcagggcacc tggctgaacc ccggcttccc tccacaaagc tgcggctacg
ccaccgtgac 1500cgacgccgag gccgccatcg tgcaggtgac ccctcaccac
gtgctggtgg acgagtacac 1560cggcgagtgg gtggacagcc agttcatcaa
cggcaagtgc agcaacgaca tctgccccac 1620cgtgcacaac agcaccacct
ggcacagcga ctacaaagtg aagggcctgt gcgacagcaa 1680cctgatcagc
accgacatca ccttcttctc cgaggacggc gagctgagca gcctgggcaa
1740ggagggcacc ggcttccgca gcaactactt cgcctacgag accggcgaca
aggcctgcaa 1800gatgcagtac tgcaagcact ggggcgtgcg cctgcccagc
ggcgtgtggt tcgagatggc 1860cgacaaggac ctgttcgccg ccgcccgctt
ccccgagtgc cccgagggca gcagcatcag 1920cgccccaagc cagaccagcg
tggacgtgag cctgatccag gacgtggagc gcatcctgga 1980ctacagcctg
tgccaggaga cctggagcaa gatccgcgcc ggcctgccca tcagccccgt
2040ggacctgagc tacctggccc ctaagaaccc cggcaccggc cccgtgttca
ccatcatcaa 2100cggcaccctg aagtacttcg agacccgcta catccgcgtg
gacatcgccg ccccaatcct 2160gagccgcatg gtgggcatga tcagcggcac
caccaccgag cgcgagctgt gggacgactg 2220ggccccttac gaggacgtgg
agatcggccc taacggcgtg ctgcgcacca gcctgggcta 2280caagtttccc
ctgtacatga tcggccacgg catgctggac agcgacctgc acctgagcag
2340caaggcccag gtgttcgagc atccccacat ccaggacgcc gccagccagc
tgcccgacga 2400cgagaccctg ttcttcggcg acaccggcct gagcaagaac
cccatcgagt tcgtggaggg 2460ctggttcagc agctggaaga gcagcatcgc
cagcttcttc ttcatcatcg gcctgatcat 2520cggcctgttc ctggtgctgc
gcgtgggcat ctacctgtgc atcaagctga agcacaccaa 2580gaagcgccag
atctacaccg acatcgagat gaaccgcctg ggcaagtaaa gcggccgctt
2640ccctttagtg agggttaatg cttcgagcag acatgataag atacattgat
gagtttggac 2700aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
tgaaatttgt gatgctattg 2760ctttatttgt aaccattata agctgcaata
aacaagttaa caacaacaat tgcattcatt 2820ttatgtttca ggttcagggg
gagatgtggg aggtttttta aagcaagtaa aacctctaca 2880aatgtggtaa
aatccgataa ggatcgatcc gggctggcgt aatagcgaag aggcccgcac
2940cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc
ctgtagcggc 3000gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
ccgctacact tgccagcgcc 3060ctagcgcccg ctcctttcgc tttcttccct
tcctttctcg ccacgttcgc cggctttccc 3120cgtcaagctc taaatcgggg
gctcccttta gggttccgat ttagtgcttt acggcacctc 3180gaccccaaaa
aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
3240gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
gttccaaact 3300ggaacaacac tcaaccctat ctcggtctat tcttttgatt
tataagggat tttgccgatt 3360tcggcctatt ggttaaaaaa tgagctgatt
taacaaaaat ttaacgcgaa ttttaacaaa 3420atattaacgc ttacaatttc
ctgatgcggt attttctcct tacgcatctg tgcggtattt 3480cacaccgcat
acgcggatct gcgcagcacc atggcctgaa ataacctctg aaagaggaac
3540ttggttaggt accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc
agttagggtg 3600tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
agcatgcatc tcaattagtc 3660agcaaccagg tgtggaaagt ccccaggctc
cccagcaggc agaagtatgc aaagcatgca 3720tctcaattag tcagcaacca
tagtcccgcc cctaactccg cccatcccgc ccctaactcc 3780gcccagttcc
gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
3840cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
ttggaggcct 3900aggcttttgc aaaaagcttg attcttctga cacaacagtc
tcgaacttaa ggctagagcc 3960accatgattg aacaagatgg attgcacgca
ggttctccgg ccgcttgggt ggagaggcta 4020ttcggctatg actgggcaca
acagacaatc ggctgctctg atgccgccgt gttccggctg 4080tcagcgcagg
ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
4140ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
ttgcgcagct 4200gtgctcgacg ttgtcactga agcgggaagg gactggctgc
tattgggcga agtgccgggg 4260caggatctcc tgtcatctca ccttgctcct
gccgagaaag tatccatcat ggctgatgca 4320atgcggcggc tgcatacgct
tgatccggct acctgcccat tcgaccacca agcgaaacat 4380cgcatcgagc
gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
4440gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
gcgcatgccc 4500gacggcgagg atctcgtcgt gacccatggc gatgcctgct
tgccgaatat catggtggaa 4560aatggccgct tttctggatt catcgactgt
ggccggctgg gtgtggcgga ccgctatcag 4620gacatagcgt tggctacccg
tgatattgct gaagagcttg gcggcgaatg ggctgaccgc 4680ttcctcgtgc
tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
4740cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
gcgacgccca 4800acctgccatc acgatggccg caataaaata tctttatttt
cattacatct gtgtgttggt 4860tttttgtgtg aatcgatagc gataaggatc
cgcgtatggt gcactctcag tacaatctgc 4920tctgatgccg catagttaag
ccagccccga cacccgccaa cacccgctga cgcgccctga 4980cgggcttgtc
tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
5040atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
cctcgtgata 5100cgcctatttt tataggttaa tgtcatgata ataatggttt
cttagacgtc aggtggcact 5160tttcggggaa atgtgcgcgg aacccctatt
tgtttatttt tctaaataca ttcaaatatg 5220tatccgctca tgagacaata
accctgataa atgcttcaat aatattgaaa aaggaagagt 5280atgagtattc
aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
5340gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
gttgggtgca 5400cgagtgggtt acatcgaact ggatctcaac agcggtaaga
tccttgagag ttttcgcccc 5460gaagaacgtt ttccaatgat gagcactttt
aaagttctgc tatgtggcgc ggtattatcc 5520cgtattgacg ccgggcaaga
gcaactcggt cgccgcatac actattctca gaatgacttg 5580gttgagtact
caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
5640tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
gacaacgatc 5700ggaggaccga aggagctaac cgcttttttg cacaacatgg
gggatcatgt aactcgcctt 5760gatcgttggg aaccggagct gaatgaagcc
ataccaaacg acgagcgtga caccacgatg 5820cctgtagcaa tggcaacaac
gttgcgcaaa ctattaactg gcgaactact tactctagct 5880tcccggcaac
aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
5940tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
gcgtgggtct 6000cgcggtatca ttgcagcact ggggccagat ggtaagccct
cccgtatcgt agttatctac 6060acgacgggga gtcaggcaac tatggatgaa
cgaaatagac agatcgctga gataggtgcc 6120tcactgatta agcattggta
actgtcagac caagtttact catatatact ttagattgat 6180ttaaaacttc
atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
6240accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
agaaaagatc 6300aaaggatctt cttgagatcc tttttttctg cgcgtaatct
gctgcttgca aacaaaaaaa 6360ccaccgctac cagcggtggt ttgtttgccg
gatcaagagc taccaactct ttttccgaag 6420gtaactggct tcagcagagc
gcagatacca aatactgttc ttctagtgta gccgtagtta 6480ggccaccact
tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
6540ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
aagacgatag 6600ttaccggata aggcgcagcg gtcgggctga acggggggtt
cgtgcacaca gcccagcttg 6660gagcgaacga cctacaccga actgagatac
ctacagcgtg agctatgaga aagcgccacg 6720cttcccgaag ggagaaaggc
ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 6780cgcacgaggg
agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
6840cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
cctatggaaa 6900aacgccagca acgcggcctt tttacggttc ctggcctttt
gctggccttt tgctcacatg 6960gctcgacaga tct 6973151561DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
15ctcgagagga gccaccatga agtgcctgct gtacctggcc ttcctgttca tcggcgtgaa
60ctgcaagttc accatcgtgt tcccccacaa ccagaagggc aactggaaga acgtgcccag
120caactaccac tactgcccca gcagcagcga cctgaactgg cacaacgacc
tgatcggcac 180cgccctgcaa gtcaagatgc ccaagagcca caaggccatc
caggccgacg gctggatgtg 240ccacgccagc aagtgggtga ccacctgcga
cttccggtgg tacggcccca agtacatcac 300ccacagcatc cgcagcttca
ccccaagcgt ggagcagtgc aaggagagca tcgagcagac 360caagcagggc
acctggctga accccggctt ccctccacaa agctgcggct acgccaccgt
420gaccgacgcc gaggccgcca tcgtgcaggt gacccctcac cacgtgctgg
tggacgagta 480caccggcgag tgggtggaca gccagttcat caacggcaag
tgcagcaacg acatctgccc 540caccgtgcac aacagcacca cctggcacag
cgactacaaa gtgaagggcc tgtgcgacag 600caacctgatc agcaccgaca
tcaccttctt ctccgaggac ggcgagctga gcagcctggg 660caaggagggc
accggcttcc gcagcaacta cttcgcctac gagaccggcg acaaggcctg
720caagatgcag tactgcaagc actggggcgt gcgcctgccc agcggcgtgt
ggttcgagat 780ggccgacaag gacctgttcg ccgccgcccg cttccccgag
tgccccgagg gcagcagcat 840cagcgcccca agccagacca gcgtggacgt
gagcctgatc caggacgtgg agcgcatcct 900ggactacagc ctgtgccagg
agacctggag caagatccgc gccggcctgc ccatcagccc 960cgtggacctg
agctacctgg cccctaagaa ccccggcacc ggccccgtgt tcaccatcat
1020caacggcacc ctgaagtact tcgagacccg ctacatccgc gtggacatcg
ccgccccaat 1080cctgagccgc atggtgggca tgatcagcgg caccaccacc
gagcgcgagc tgtgggacga 1140ctgggcccct tacgaggacg tggagatcgg
ccctaacggc gtgctgcgca ccagcctggg 1200ctacaagttt cccctgtaca
tgatcggcca cggcatgctg gacagcgacc tgcacctgag 1260cagcaaggcc
caggtgttcg agcatcccca catccaggac gccgccagcc agctgcccga
1320cgacgagacc ctgttcttcg gcgacaccgg cctgagcaag aaccccatcg
agttcgtgga 1380gggctggttc agcagctgga agagcagcat cgccagcttc
ttcttcatca tcggcctgat 1440catcggcctg ttcctggtgc tgcgcgtggg
catctacctg tgcatcaagc tgaagcacac 1500caagaagcgc cagatctaca
ccgacatcga gatgaaccgc ctgggcaagt aaagcggccg 1560c 1561
* * * * *