U.S. patent application number 12/360615 was filed with the patent office on 2009-10-08 for novel expression vectors and uses thereof.
This patent application is currently assigned to FIT BIOTECH OY. Invention is credited to VESNA BLAZEVIC, KAI KROHN, ANDRES MANNIK, ANNAMARI RANKI, MARJA TAHTINEN, URVE TOOTS, ENE USTAV, MART USTAV.
Application Number | 20090252707 12/360615 |
Document ID | / |
Family ID | 8561113 |
Filed Date | 2009-10-08 |
United States Patent
Application |
20090252707 |
Kind Code |
A1 |
KROHN; KAI ; et al. |
October 8, 2009 |
NOVEL EXPRESSION VECTORS AND USES THEREOF
Abstract
A method for treating an HIV disease in a subject in need of
said treatment, comprising administering to the subject a
therapeutically effective amount of a DNA vaccine comprising an
expression vector and a pharmaceutically acceptable excipient,
where the expression vector comprises: (a) a heterologous promoter
operatively linked to a DNA sequence encoding a nuclear-anchoring
protein, where the nuclear-anchoring protein comprises: (i) a DNA
binding domain which binds to a specific DNA binding sequence, and
(ii) a functional domain of the Bovine Papilloma Virus Type 1 E2
protein, where the functional domain binds to a nuclear component;
(b) a multimerized DNA sequence that forms a binding site for the
nuclear anchoring protein; and (c) at least one expression cassette
comprising a DNA sequence encoding a protein or peptide that
stimulates an immune response specific to the protein or peptide;
where the expression vector lacks an origin of replication
functional in mammalian cells.
Inventors: |
KROHN; KAI; (SALMENTAKA,
FI) ; BLAZEVIC; VESNA; (TAMPERE, FI) ;
TAHTINEN; MARJA; (TAMPERE, FI) ; USTAV; MART;
(TARTU, EE) ; TOOTS; URVE; (TARTU, EE) ;
MANNIK; ANDRES; (TARTU, EE) ; RANKI; ANNAMARI;
(HELSINKI, FI) ; USTAV; ENE; (TARTU, EE) |
Correspondence
Address: |
BUCHANAN, INGERSOLL & ROONEY PC
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
FIT BIOTECH OY
TAMPERE
FI
|
Family ID: |
8561113 |
Appl. No.: |
12/360615 |
Filed: |
January 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10476615 |
Nov 3, 2003 |
7510718 |
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PCT/FI02/00379 |
May 3, 2002 |
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12360615 |
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Current U.S.
Class: |
424/93.2 ;
514/44A; 514/44R |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2840/203 20130101; A61K 48/00 20130101; A61P 31/18 20180101;
C12N 2840/20 20130101; C12N 15/85 20130101; A61P 43/00 20180101;
A61K 2039/53 20130101; A61P 31/00 20180101; C12N 2800/108 20130101;
C12N 2830/42 20130101 |
Class at
Publication: |
424/93.2 ;
514/44.A; 514/44.R |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 35/76 20060101 A61K035/76; A61K 31/713 20060101
A61K031/713; A61P 31/18 20060101 A61P031/18 |
Foreign Application Data
Date |
Code |
Application Number |
May 3, 2001 |
FI |
20010922 |
Claims
1. A method for treating an HIV disease in a subject in need of
said treatment, said method comprising: administering to said
subject a therapeutically effective amount of a DNA vaccine
comprising an expression vector and a pharmaceutically acceptable
excipient, wherein said expression vector comprises: (a) a
heterologous promoter operatively linked to a DNA sequence encoding
a nuclear-anchoring protein, wherein said nuclear-anchoring protein
comprises: (i) a DNA binding domain which binds to a specific DNA
binding sequence, and (ii) a functional domain of the Bovine
Papilloma Virus Type 1 E2 protein, wherein said functional domain
binds to a nuclear component; (b) a multimerized DNA sequence that
forms a binding site for said nuclear anchoring protein; and (c) at
least one expression cassette comprising a DNA sequence encoding a
protein or peptide that stimulates an immune response specific to
the protein or peptide; wherein said expression vector lacks an
origin of replication functional in mammalian cells.
2. The method of claim 1, wherein said nuclear component is
selected from the group consisting of mitotic chromatin, the
nuclear matrix, nuclear domain 10 (ND10), and nuclear domain PML
oncogenic domain (POD).
3. The method of claim 1, wherein said nuclear-anchoring protein is
a chromatin-anchoring protein, and said functional domain binds
mitotic chromatin.
4. The method of claim 1, wherein said nuclear-anchoring protein
comprises a hinge or linker region.
5. The method of claim 1, wherein said nuclear-anchoring protein is
a natural protein of viral origin.
6. The method of claim 1, wherein said nuclear-anchoring protein is
an artificial protein.
7. The method of claim 1, wherein said expression cassette
comprises a DNA sequence of HIV origin.
8. The method of claim 7, wherein said DNA sequence of HIV origin
encodes a non-structural regulatory protein of HIV, or an
immunogenic fragment thereof.
9. The method of claim 8, wherein said nonstructural regulatory
protein of HIV is selected from the group consisting of Nef, Tat
and Rev.
10. The method of claim 9, wherein said nonstructural regulatory
protein of HIV is Nef.
11. The method of claim 7, wherein said DNA sequence of HIV origin
encodes a structural protein of HIV, or an immunogenic fragment
thereof.
12. The method of claim 11, wherein said DNA sequence of HIV origin
is HIV gp120/gp160.
13. The method of claim 1, wherein said vector comprises: (a) a
first expression cassette comprising a DNA sequence encoding Nef,
Tat or Rev; and (b) a second expression cassette comprising a DNA
sequence encoding Nef, Tat or Rev.
14. The method of claim 1, wherein said vector comprises: (a) a
first expression cassette comprising a DNA sequence encoding Nef,
Tat or Rev; and (b) a second expression cassette comprising a DNA
sequence encoding a structural protein of HIV.
15. The method of claim 1, wherein: said DNA binding domain
comprises the DNA binding domain of the Bovine Papilloma Virus Type
1 E2 protein; and said multimerized DNA sequence comprises
multimerized E2 binding sites.
16. The method of claim 1, wherein: said nuclear-anchoring protein
comprises the Bovine Papilloma Virus Type 1 E2 protein, and said
multimerized DNA sequence comprises multimerized E2 binding
sites.
17. The method of claim 1, wherein said expression cassette
comprises a DNA sequence encoding a fusion protein comprising the
following components: (A) Rev, Nef, Tat (RNT); (B) opt 17/24; and
(C) Cytotoxic T cell epitopes (CTL).
18. The method of claim 17, wherein the order of the components
from the 5' end to the 3' end of said fusion protein is A+B+C.
19. The method of claim 17, wherein the components A, B, and C
comprise the sequences of SEQ ID NOS: 5, 13 and 10,
respectively.
20. A DNA vaccine comprising an expression vector and a
pharmaceutically acceptable excipient, wherein said expression
vector comprises: (a) a heterologous promoter operatively linked to
a DNA sequence encoding a nuclear-anchoring protein, wherein said
nuclear-anchoring protein comprises: (i) a DNA binding domain which
binds to a specific DNA binding sequence, and (ii) a functional
domain of the Bovine Papilloma Virus Type 1 E2 protein, wherein
said functional domain binds to a nuclear component; (b) a
multimerized DNA sequence that forms a binding site for said
nuclear anchoring protein; and (c) at least one expression cassette
comprising a DNA sequence encoding a protein or peptide that
stimulates an immune response specific to the protein or peptide;
wherein said expression vector lacks an origin of replication
functional in mammalian cells; and wherein said expression cassette
comprises a DNA sequence encoding a fusion protein comprising the
following components: (A) Rev, Nef, Tat (RNT); (B) opt 17/24; and
(C) Cytotoxic T cell epitopes (CTL).
21. The DNA vaccine of claim 20, wherein the order of the
components from the 5' end to the 3' end of said fusion protein is
A+B+C.
22. The DNA vaccine of claim 20, wherein the components A, B, and C
comprise the sequences of SEQ ID NOS: 5, 13 and 10,
respectively.
23. The DNA vaccine of claim 21, wherein the components A, B, and C
comprise the sequences of SEQ ID NOS: 5, 13 and 10, respectively.
Description
1. FIELD OF THE INVENTION
[0001] The present invention relates to novel vectors, to DNA
vaccines and gene therapeutics containing said vectors, to methods
for the preparation of the vectors and DNA vaccines and gene
therapeutics containing the vectors, and to therapeutic uses of
said vectors. More specifically, the present invention relates to
novel vectors comprising (a) an expression cassette of a gene of a
nuclear-anchoring protein, which contains (i) a DNA binding domain
capable of binding to a specific DNA sequence and (ii) a functional
domain capable of binding to a nuclear component and (b) a
multimerized DNA forming a binding site for the anchoring protein
of a nuclear-anchoring protein, and optionally (c) one or more
expression cassettes of a DNA sequence of interest. In particular
the invention relates to vectors that lack a papilloma virus origin
of replication. The invention also relates to vectors that lack an
origin of replication functional in a mammalian cell. The invention
further relates to methods for expressing a DNA sequence of
interest in a subject.
2. BACKGROUND OF THE INVENTION
[0002] Transfer of autologous or heterologous genes into animal or
human organisms with suitable vectors is emerging as a technique
with immense potential to cure diseases with a genetic background
or to prevent or cure infectious diseases. Several types of viral
and non-viral vectors have been developed and tested in animals and
in human subjects to deliver a gene/genes that are defective by
mutations and therefore non-functional. Examples of such vectors
include Adenovirus vectors, Herpes virus vectors, Retrovirus
vectors, Lentivirus vectors and Adeno-associated vectors.
[0003] Vaccination has proven to be a highly effective and
economical method to prevent a disease caused by infectious agents.
Since the introduction of the Vaccinia virus as an attenuated
vaccine against the smallpox virus (Variola), vaccines against a
multitude of human pathogens have been developed and taken into
routine use. Today small pox has been eradicated by vaccinations
and the same is to be expected shortly for the poliovirus. Several
childhood diseases, such as pertussis, diphtheria and tetanus, can
be effectively prevented by vaccinations.
[0004] In general, the most successful viral vaccines are live
avirulent mutants of the disease-causing viruses. The key to the
success of this approach is the fact that a living virus targets
the same organs, the same type and similar number of cells, and
therefore, by multiplying in the recipient, elicits a long-lasting
immune response without causing the disease or causing only a mild
disease. In effect, a live attenuated vaccine produces a
subclinical infection, the nature's own way of immunizing. As a
result, a full immune response will be induced, including humoral,
cellular and innate responses, providing a long lasting and
sometimes a life-long immune protection against the pathogen.
[0005] Although live attenuated vaccines are most potent, they can
cause harmful side effects. Thus, an attenuated viral vaccine can
revert to a virulent strain or in cases where the attenuated virus
is apathogenic in adults it can still cause a disease in infants or
in disabled persons. This is true in the case of viruses causing
chronic infections, such as Human Immunodeficiency Virus type 1 and
2. Vaccines composed of viral and bacterial proteins or immunogenic
peptides are less likely to cause unwanted side effects but may not
be as potent as the live vaccines. This is especially the case with
vaccines against microbes causing chronic infections, such as
certain viruses and intracellular bacteria.
[0006] The strength and type of immune response is, however, also
dependent on how the viral proteins are processed and how they are
presented to the immune system by antigen presenting cells (APCs),
such as macrophages and dendritic cells. Protein and peptide
antigens are taken up by APCs via endocytosis, processed to small
immunogenic peptides through an endosomal pathway and presented to
T-lymphocytes (T-cells) by MHC (major histocompatibility complex)
class II antigens [in man HLAs (human leukocyte antigens) class
II]. In contrast, proteins synthesized de novo in APCs or in
possible target cells for an immune response, will be processed
through a cytoplasmic pathway and presented to T-cells by MHC class
I antigens (in man HLAs class 1). In general, the presentation of
immunogenic peptides through the class II pathway will lead to the
activation of the helper/inducer T-cells, which in turn will lead
to the activation of B-cells and to antibody response. In contrast,
presentation through class I MHC favors the induction of cytotoxic
T-lymphocytes (CTLs), which are capable of recognition and
destruction of virally infected cells.
[0007] In early 1990's, a method to mimic the antigen processing
and presentation that was normally achieved by live attenuated
vaccines was introduced [Ulmer, J. B. et al Science 259 (1993)
1745-1749]. It was shown that an injection of eukaryotic expression
vectors in the form of circular DNA into the muscle induced take-up
of this DNA by the muscle cells (and probably others) and was able
to induce the expression of the gene of interest, and to raise an
immune response, especially a cellular immune response in the form
of CTLs, to the protein encoded by the inserted gene. Since that
observation, DNA immunization has become a standard method to
induce immune responses to foreign proteins in experimental animals
and human studies with several DNA vaccines are underway.
[0008] Generally, the DNA vectors used in these vaccine studies
contain a cloning site for the gene of interest, a strong viral
promoter, such as the immediate early promoter of the CMV virus, in
order to drive the expression of the gene of interest, a
polyadenylation region, and an antibiotic resistance gene and a
bacterial replication origin for the propagation of the DNA vector
(plasmid) in bacterial cells.
[0009] With the vectors described above it is possible to obtain a
detectable level of expression of the gene of interest after
administering the vector to experimental animals or to humans,
either by a direct injection to muscle or to skin with a particle
bombardment technique or by applying the vector in a solution
directly to mucous membranes. However, the expression obtained by
these vectors is short lived: the vectors tend to disappear from
the transfected cells little by little and are not transferred to
daughter cells in a dividing cell population. The short-term
expression of the gene of interest and limited number of cells
targeted are probably the major reasons, why only temporary immune
responses are observed in subjects immunized with DNA vectors
described above. Thus, for example, Boyer et al. observed only
temporary immune responses to HIV-1 Env and Rev proteins in human
subjects, who were immunized several times with a vector similar to
the those described above [Boyer, J. D., J Infect Dis 181 (2000)
476-483].
[0010] There is a growing interest in developing novel products
useful in gene therapy and DNA vaccination. For instance papilloma
virus vectors carrying the expression cassette for the gene of
interest have been suggested to be useful candidates.
[0011] To date more than 70 subtypes of human papilloma viruses
(HPVs) and many different animal papilloma viruses have been
identified [zur Hausen, H. and de Villiers E., Annu Rev Microbiol
48 (1994) 427-447; Bernard, H., et al., Curr Top Microbiol Immunol
186 (1994) 33-54]. All papilloma viruses share a similar genome
organization and the positioning of all of the translational open
reading frames (ORFs) is highly conserved.
[0012] Papilloma viruses infect squamous epithelial cells of skin
or mucosa at different body sites and induce the formation of
benign tumors, which in some cases can progress to malignancy. The
papilloma virus genomes are replicated and maintained in the
infected cells as multicopy nuclear plasmids. The replication,
episomal maintenance, expression of the late genes and virus
assembly are tightly coupled to the differentiation of the
epithelial tissue: the papilloma virus DNA episomal replication
takes place during the initial amplificational replication and the
second, i.e. latent, and the third, i.e. vegetative, replications
in the differentiating epithelium [Howley, P. M.; Papillomavirinae:
the viruses and their replication. In Virology, Fields, B. C.,
Knipe, D. M., Howley, P. M., Eds., Lippincott-Raven Publishers,
Philadelphia, USA, 1996, 2. Edition, p. 2045-2076].
[0013] Two viral factors encoded by the E1 and E2 open reading
frames have been shown to be necessary and sufficient for the
initiation of the DNA replication from the papilloma virus origin
in the cells [Ustav, M. and Stenlund, A., EMBO J 10 (1991) 449-57;
Ustav, M., et al., EMBO J 10 (1991) 4321-4329; Ustav, E., et al.,
Proc Natl Acad Sci USA 90 (1993) 898-902].
[0014] Functional origins for the initiation of the DNA replication
have been defined for BPV1 [Ustav, M., et al., EMBO J 10 (1991)
4321-4329], HPV1a [Gopalakrishnan, V. and Khan, S., supra], HPV11
[Russell, J., Botchan, M., J Virol 69 (1995) 651-660], HPV18
[Sverdrup, F. and Khan, S., J Virol 69 (1995) 1319-1323: Sverdrup,
F. and Khan, S., J Virol 68 (1994) 505-509] and many others.
Characteristically, all these origin fragments have a high A/T
content, and they contain several overlapping individual E1 protein
recognition sequences, which together constitute the E1 binding
site [Ustav, M., et al., EMBO J 10 (1991) 4321-4329; Holt, S., et
al., J Virol 68 (1994) 1094-1102; Holt, S, and Wilson, V., J Virol
69 (1995) 6525-3652; Sedman, T., et al. J Virol 71 (1997)
2887-2996]. In addition, these functional origin fragments contain
an E2 binding site, which is essential for the initiation of DNA
replication in vivo in most cases (Ustav, E., et al., supra). The
E2 protein facilitates the first step of the origin recognition by
E1. After the initial binding of monomeric E1 to the origin the
multimerization of E1 is initiated. This leads to the formation of
the complex with the ori melting activity. It has been suggested
that E2 has no influence on the following stages of the initiation
of the DNA replication [Lusky, M., et al., Proc Natl Acad Sci USA
91 (1994) 8895-8899].
[0015] The BPV1 E2 ORF encodes three proteins that originate from
selective promoter usage and alternative mRNA splicing [Lambert,
P., et al., Annu Rev Genet. 22 (1988) 235-258]. All these proteins
can form homo- and heterodimers with each other and bind
specifically to a 12 bp interrupted palindromic sequence
5'-ACCNNNNNNGGT-3' [Androphy, E., et al., Nature 325 (1987)
70-739].
[0016] There are 17 E2 binding sites in the BPV1 genome and up to
four sites in the HPV genomes, which play a crucial role in the
initiation of viral DNA replication (Ustav, E., et al., supra) and
in the regulation of viral gene expression (Howley, P. M.,
Papillomavirinae: the viruses and their replication, in Virology,
Fields, B. C., Knipe, D. M., Howley, P. M., Eds., Philadelphia:
Lippincott-Raven Publishers, 1996. 2. edition, p. 2045-2076).
Structural and mutational analyses have revealed three distinct
functional domains in the full size E2 protein. The N-terminal part
(residues 1 to 210) is an activation domain for transcription and
replication. It is followed by the unstructured hinge region
(residues 211 to 324) and the carboxy-terminal DNA
binding-dimerization domain (residues 325 to 410) [Dostatni, N., et
al., EMBO J 7 (1988) 3807-3816; Haugen, T., et al. EMBO J 7 (1988)
4245-4253; McBride, A., et al., EMBO J 7 (1988) 533-539; McBride,
A., et al., Proc Natl Acad Sci USA 86 (1989) 510-514]. On the basis
of X-ray crystallographical data, the DNA binding-dimerization
domain of E2 has a structure of a dyad-symmetric eight-stranded
antiparallel beta barrel, made up of two identical "half-barrel"
subunits [Hegde, R., et al., Nature 359 (1992) 505-512; Hegde, R.,
J Nucl Med 36 (6 Suppl) (1995) 25S-27S]. The functional elements of
the trans-activation domain of E2 have a very high structural
integrity as confirmed by mutational analysis [Abroi, A., et al., J
Virol 70 (1996) 6169-6179; Brokaw, J., et al., J Virol 71 (1996)
23-29; Grossel, M., et al., J Virol 70 (1996) 7264-7269; Ferguson,
M. and Botchan, M., J Virol 70 (1996) 4193-4199] and by X-ray
crystallography [Harris, S., and Botchan, M. R., Science 284 (1999)
1673-1677 and Antson, A. et al., Nature 403 (2000) 805-809]. In
addition, X-ray crystallography shows that the N-terminal domain of
the E2 protein forms a dimeric structure, where Arg 37 has an
important function in dimer formation (Antson, A., et al.,
supra).
[0017] As has been described previously, bovine papillomavirus type
1 E2 protein in trans and its multiple binding sites in cis are
both necessary and sufficient for the chromatin attachment of the
episomal genetic elements. The phenomenon is suggested to provide a
mechanism for partitioning viral genome during viral infection in
the dividing cells [Ilves, I., et al., J Virol. 73 (1999)
4404-4412].
[0018] None of the papilloma vectors or other vectors disclosed so
far fulfills the criteria and requirements set forth for an optimal
vaccine, which are the same for DNA vaccines and for conventional
vaccines. (It should be noted that these requirements are preferred
but not necessary for use as a vaccine.) First, an optimal vaccine
must produce protective immunity with minimal adverse effects. Thus
the vaccine should be devoid of components, which are toxic and/or
cause symptoms of the disease to the recipient. Second, an optimal
vaccine must induce a pathogen-specific immune response, i.e. it
must elicit a strong and measurable immune response to the desired
pathogen without causing an immune response to other components of
the vaccine. These two requirements imply that a vector to be used
as a DNA vaccine should optimally only express the desired gene(s)
and optimally should not replicate in the host or contain any
sequences homologous with those of the recipient, since nucleotide
sequences that are homologous between the vector and the host's
genome may effect the integration of the vector into the host's
genome. Third, an optimal vaccine must induce a right type of
immune response; i.e. it must raise both humoral and cellular
immune responses in order to act on the intracellular and
extracellular pathogen. Finally, an optimal vaccine must be stable,
i.e. it must retain its potency for a sufficiently long time in the
body to raise the immune response in a vaccine formulation for use
in various demanding circumstances during storage and preparation.
Additionally, vaccines should be of reasonable price. Further, the
route and the method of inoculation are important considerations
for optimizing a DNA immunization.
[0019] When developing a DNA vaccine the stability of the
expression of the desired gene is sometimes a major problem. Thus,
the maintenance function or the persistance of the vector in the
recipient cell has been focused on in the prior art, however, often
at the cost of the safety. For example, Ohe, Y., et al.][Hum Gene
Ther 6 (3) (1995) 325-333] disclose a papilloma virus vector
capable of stable, high-level gene expression, which is suggested
for use in gene therapy. Trans-forming early genes E5, E6, and E7
have been deleted from said vector, but it still contains
nucleotide sequences encoding other papilloma viral genes, such as
the E1 and E2 genes, which are involved in the replication of the
virus. Thus, the vector produces several other papilloma proteins,
which may elicit undesired immune responses and which induce a risk
of the vector's integration in the recipient. Also, the vector is
replicable, since it contains the E1 gene. Additionally, it is
large in size and therefore subject to bacterial modification
during preparation.
[0020] International Patent Application PCT/EE96/00004 (WO
97/24451) discloses vectors capable of a long-term maintenance in a
host cell and methods using such vectors for obtaining long-term
production of a gene product of interest in a mammalian host cell,
which expresses E1 and E2. These vectors contain a minimal origin
of replication of a papilloma virus (MO), a Minichromosome
Maintenance Element (MME) of a papilloma virus and a gene encoding
said gene product, the MO and MME consisting of a DNA sequence
different from the natural papilloma virus sequence, and in some
embodiments the E1 gene. Additionally, vectors containing an MME
consisting essentially of ten E2 binding sites are disclosed in
some examples. These vectors require the presence of the E1 protein
either in the host or in the vector for the expression. This
imparts the replication function to the vectors. These vectors also
express the E1 protein in addition to the gene of interest and the
E2 protein and contain sequences, such as rabbit .beta.-globin
sequences, which are partially homologous to human sequences
causing a serious risk of integration to human genome, which
reduces the potential of these vectors as DNA vaccines.
Additionally, the vectors are unstable due to their size (ca 15
kb): at the preparation stage in a bacterial cell, the bacterial
replication machinery tends to modify the vector by random slicing
of the vector, which leads to unsatisfactory expression products
including products totally lacking the gene of interest.
[0021] International Patent Application PCT/EE96/00004 (WO
97/24451) further discloses that E1 and E2 are the only viral
proteins necessary for the episomal long-term replication of the
vectors. Additionally, the maintenance function of the BPV1 genome
is associated with the presence of minimal ori (MO), which is
stated to be necessary, although not sufficient, for the long-term
persistence or the stable maintenance of the vectors the cells. In
addition, the cis-elements, i.e. the Minichromosome Maintenance
Elements of the BPV1, are stated to be required for the stable
replication of BPV1. In particular, multimeric E2 binding sites
(E2BS) are stated to be necessary for the stable maintenance of the
vectors.
[0022] There is a clear need for improved novel vectors, which
would be useful as DNA vaccines.
[0023] An object of the invention is therefore to provide novel
vectors, which are capable of a long-term maintenance in a large
and increasing number of different cells of the host's body and
thereby capable of providing a stable expression of the desired
antigen(s).
[0024] Another object of the invention is to provide novel vectors,
which are maintained for a long period of time in the cells that
originally received the vector and transferred it to the daughter
cells after mitotic cell division.
[0025] Yet another object of the invention is to provide novel
vectors, which express in addition to the gene or genes of interest
preferably only a gene necessary for a long-term maintenance in the
recipient cells and thus are devoid of components that are toxic or
cause symptoms of the disease to the recipient.
[0026] A further object of the invention is to provide novel
vectors, which mimic attenuated live viral vaccines, especially in
their function of multiplying in the body, without inducing any
considerable signs of disease and without expressing undesired
proteins, which may induce adverse reactions in a host injected
with the DNA vaccine.
[0027] Still a further object of the invention is to provide novel
vectors, which do not replicate in the recipient.
[0028] Still another object of the invention is to provide novel
vectors, which induce both humoral and cellular immune responses
when used as DNA vaccines.
[0029] Yet another object of the invention is to provide novel
vectors, which are suitable for a large-scale production in
bacterial cell.
[0030] Yet another object of the invention is to provide novel
vectors, which are not host specific and thus enable the production
in various bacterial cells.
[0031] An additional object of the invention is to provide novel
vectors, which are useful as carrier vectors for a gene or genes of
interest,
[0032] A further object of the invention is to provide novel
vectors, which are useful in gene therapy and as gene therapeutic
agents and for the production of macromolecular drugs in vivo.
3. SUMMARY OF THE INVENTION
[0033] The present invention discloses novel vectors, which meet
the requirements of a carrier vector of a gene or genes of interest
or of an optimal DNA vaccination vector and which are preferably
devoid of drawbacks and side effects of prior art vectors.
[0034] The present invention is based on the surprising finding
that a vector (plasmid) carrying (i) an expression cassette of a
DNA sequence encoding a nuclear-anchoring protein, and (ii)
multiple copies of high affinity binding sites for said
nuclear-anchoring protein spreads in proliferating cells. As a
result, the number of vector-carrying cells increases even without
the replication of the vector. When the vector additionally carries
a gene or genes of interest, the number of such cells that express
a gene or genes of interest similarly increases without the
replication of the vector. Thus, the vector of the invention lacks
a papilloma virus origin of replication. In a preferred embodiment,
the vector of the invention lacks an origin of replication that
functions in a mammalian cell.
[0035] Accordingly, the present invention discloses novel vectors
useful as carrier vectors of a gene or genes of interest, in DNA
vaccination and gene therapy and as gene therapeutic agents. In a
specific embodiment, said vectors are capable of spreading and, if
desired, of expressing a gene or genes of interest in an increasing
number of cells for an extended time. The vectors of the present
invention preferably express only a nuclear-anchoring protein, and,
if desired, the gene or genes of interest, and optionally a
selectable marker. However, they preferably lack any redundant,
oncogenically transforming or potentially toxic sequences, thereby
avoiding a severe drawback of the vectors previously disclosed or
suggested for use as DNA vaccines, i.e. hypersensitivity reactions
against other viral components. In certain embodiments of the
invention, this is achieved by low level of the expressed
nuclear-anchoring protein in the cells. At the same time, the
vectors of the present invention induce both humoral and cellular
immune responses, where the gene or genes of interest is included
in the vector.
[0036] The vectors of the present invention are advantageous for
use both in vitro (e.g., in the production level) and in vivo
(e.g., vaccination).
[0037] The present invention relates to the subject matter of the
invention as set forth in the attached claims.
[0038] The present invention relates to expression vectors
comprising: (a) a DNA sequence encoding a nuclear-anchoring protein
operatively linked to a heterologous promoter, said
nuclear-anchoring protein comprising (i) a DNA binding domain which
binds to a specific DNA sequence, and (ii) a functional domain that
binds to a nuclear component, or a functional equivalent thereof;
and (b) a multimerized DNA sequence forming a binding site for the
nuclear anchoring protein, wherein said vector lacks a papilloma
virus origin of replication. In a preferred embodiment a vector of
the invention lacks an origin of replication functional in a
mammalian cell.
[0039] In certain embodiments, the nuclear component is mitotic
chromatin, the nuclear matrix, nuclear domain 10 (ND10), or nuclear
domain POD.
[0040] In certain specific embodiments, the nuclear
anchoring-protein is a chromatin-anchoring protein, and said
functional domain binds mitotic chromatin.
[0041] In certain embodiments, the nuclear-anchoring protein
contains a hinge or linker region.
[0042] In certain embodiments, the nuclear-anchoring protein is a
natural protein of eukaryotic, prokaryotic, or viral origin. In
certain specific embodiments, the natural protein is of viral
origin.
[0043] In certain embodiments, the nuclear-anchoring protein is a
natural protein of eukaryotic origin.
[0044] In certain embodiments, the nuclear-anchoring protein is
that of a papilloma virus or an Epstein-Barr virus.
[0045] In specific embodiments, the nuclear-anchoring protein is
the E2 protein of Bovine Papilloma Virus type 1 or Epstein-Barr
Virus Nuclear Antigen 1.
[0046] In a specific embodiment, the nuclear-anchoring protein is
the E2 protein of Bovine Papilloma Virus type 1.
[0047] In specific embodiments, the nuclear-anchoring protein is a
High Mobility Group protein.
[0048] In certain embodiments, the nuclear-anchoring protein is a
non-natural protein.
[0049] In certain embodiments, the nuclear-anchoring protein is a
recombinant protein, a fusion protein, or a protein obtained by
molecular modeling techniques.
[0050] In specific embodiments, the recombinant protein, fusion
protein, or protein obtained by molecular modeling techniques
contains any combination of a DNA binding domain which binds to
said specific DNA sequence and a functional domain which binds to a
nuclear component, wherein said functional domain which binds to a
nuclear component is that of a papilloma virus, an
Epstein-Barr-Virus, or a High Mobility Group protein.
[0051] In certain specific embodiments, the recombinant protein,
fusion protein, or protein obtained by molecular modeling
techniques contains any combination of a DNA binding domain which
binds to said specific DNA sequence and a functional domain which
binds to a nuclear component, wherein said functional domain which
binds to a nuclear component is that of E2 protein of Bovine
Papilloma Virus type 1, Epstein-Barr Virus Nuclear Antigen 1, or a
High Mobility Group protein.
[0052] In certain embodiments, the vector further comprises one or
more expression cassettes of a DNA sequence of interest.
[0053] In certain embodiments, the DNA sequence of interest is that
of an infectious pathogen. In certain embodiments, the infectious
pathogen is a virus. In certain specific embodiments, the virus is
selected from the group consisting of Human Immunodeficiency Virus
(HIV), Herpex Simplex Virus (HSV), Hepatitis C Virus, Influenzae
Virus, and Enterovirus.
[0054] In certain embodiments, the DNA sequence of interest is that
of a bacterium. In certain embodiments, the bacterium is selected
from the group consisting of Chlamydia trachomatis, Mycobacterium
tuberculosis, and Mycoplasma pneumonia. In a specific embodiment,
the bacterium is Salmonella.
[0055] In certain embodiments, the DNA sequence of interest is that
of a fungal pathogen. In certain embodiments, the fungal pathogen
is Candida albigans.
[0056] In certain embodiments, the DNA sequence of interest is of
HIV origin.
[0057] In specific embodiments, the DNA sequence of interest
encodes a non-structural regulatory protein of HIV. In more
specific embodiments, the non-structural regulatory protein of HIV
is Nef, Tat and/or Rev. In a specific embodiment, the
non-structural regulatory protein of HIV is Nef.
[0058] In certain embodiments, the DNA sequence of interest encodes
a structural protein of HIV. In a specific embodiment, the DNA
sequence of interest is the gene encoding HIV gp120/gp160.
[0059] In certain embodiments, the vector of the invention
comprises a first expression cassette comprising a DNA sequence of
interest which encodes Nef, Tat and/or Rev, and a second expression
cassette comprising a DNA sequence of interest which encodes Nef,
Tat and/or Rev.
[0060] In certain embodiments, the vector of the invention
comprises a first expression cassette comprising a DNA sequence of
interest which encodes Nef, Tat and/or Rev, and a second expression
cassette comprising a DNA sequence of interest which encodes a
structural protein of HIV.
[0061] In certain embodiments, the DNA sequence of interest encodes
a protein associated with cancer.
[0062] In certain embodiments, the DNA sequence of interest encodes
a protein associated with immune maturation, regulation of immune
responses, or regulation of autoimmune responses. In a specific
embodiment, the protein is APECED.
[0063] In a specific embodiment, the DNA sequence of interest is
the Aire gene.
[0064] In certain embodiments, the DNA sequence of interest encodes
a protein that is defective in any hereditary single gene
disease.
[0065] In certain embodiments, the DNA sequence of interest encodes
a macromolecular drug.
[0066] In certain embodiments, the DNA sequence of interest encodes
a cytokine. In certain specific embodiments, the cytokine is an
interleukin selected from the group consisting of IL1, IL2, IL4,
IL6 and IL12. In certain other specific embodiments, the DNA
sequence of interest encodes an interferon.
[0067] In certain embodiments, the DNA sequence of interest encodes
a biologically active RNA molecule. In certain specific
embodiments, the biologically active RNA molecule is selected from
the group consisting of inhibitory antisense and ribozyme
molecules. In certain specific embodiments, the inhibitory
antisense or ribozyme molecules antagonize the function of an
oncogene.
[0068] A vector of the invention is suitable for the use for the
production of a therapeutic macromolecular agent in vivo.
[0069] In certain embodiments, the invention provides a vector for
use as a medicament.
[0070] In certain embodiments, the invention provides a vector for
use as a carrier vector for a gene, genes, or a DNA sequence or DNA
sequences of interest, such as a gene, genes, or a DNA sequence or
DNA sequences encoding a protein or peptide of an infectious agent,
a therapeutic agent, a macromolecular drug, or any combination
thereof.
[0071] In certain specific embodiments, the invention provides a
vector for use as a medicament for treating inherited or acquired
genetic defects.
[0072] In certain embodiments, the invention provides a vector for
use as a therapeutic DNA vaccine against an infectious agent.
[0073] In certain embodiments, the invention provides a vector for
use as a therapeutic agent.
[0074] The invention further relates to methods for providing a
protein to a subject, said method comprising administering to the
subject a vector of the invention, wherein said vector (i) further
comprises a second DNA sequence encoding the protein to be provided
to the subject, which second DNA sequence is operably linked to a
second promoter, and (ii) does not encode Bovine Papilloma Virus
protein E1, and wherein said subject does not express Bovine
Papilloma Virus protein E1.
[0075] The invention further relates to methods for inducing an
immune response to a protein in a subject, said method comprising
administering to the subject a vector of the invention wherein said
vector (i) further comprises a second DNA sequence encoding said
protein, which second DNA sequence is operably linked to a second
promoter, and (ii) does not encode Bovine Papilloma Virus protein
E1, and wherein said subject does not express Bovine Papilloma
Virus protein E1.
[0076] The invention further relates to methods for treating an
infectious disease in a subject in need of said treatment, said
method comprising administering to said subject a therapeutically
effective amount of a vector of the invention, wherein the DNA
sequence of interest encodes a protein comprising an immunogenic
epitope of an infectious agent.
[0077] The invention further relates to methods for treating an
inherited or acquired genetic defect in a subject in need of said
treatment, said method comprising: administering to said subject a
therapeutically effective amount of a vector of the invention,
wherein said DNA sequence of interest encodes a protein which is
affected by said inherited or acquired genetic defect.
[0078] The invention further relates to methods for expressing a
DNA sequence in a subject, said method comprising administering a
vector of the invention to said subject.
[0079] The invention further relates to methods for expressing a
DNA sequence in a subject, treating an inherited or acquired
genetic defect, treating an infectious disease, inducing an
immune-response to a protein, and providing a protein to a subject,
wherein the vector of the invention does not encode Bovine
Papilloma Virus protein E1, and wherein said subject does not
express Bovine Papilloma Virus protein E1.
[0080] In certain embodiments, a vector of the invention is used
for production of a protein encoded by said DNA sequence of
interest in a cell or an organism.
[0081] The invention further provides a method for the preparation
of a vector of claim 1, 2, or 17 comprising: (a) cultivating a host
cell containing said vector and (b) recovering the vector. In a
specific embodiment, the method for preparing a vector of the
invention further comprises before step (a) a step of transforming
said host cell with said vector. In certain specific embodiments,
the host cell is a prokaryotic cell. In a specific embodiment, the
host cell is an Escherichia coli.
[0082] The invention further relates to a host cell that is
characterized by containing a vector of the invention. In certain
embodiments, the host cell is a bacterial cell. In a certain other
embodiments, the host cell is a mammalian cell.
[0083] The invention further relates to carrier vectors containing
a vector of the invention.
[0084] The invention further relates to a pharmaceutical
composition comprising a vector of the invention and a suitable
pharmaceutical vehicle.
[0085] The invention further relates to a DNA vaccine containing a
vector of the invention.
[0086] The invention further relates to a gene therapeutic agent
containing a vector of the invention.
[0087] The invention further relates to a method for the
preparation of a DNA vaccine, said method comprising combining a
vector of the invention with a suitable pharmaceutical vehicle.
[0088] The invention further relates to a method for the
preparation of an agent for use in gene therapy, said method
comprising combining a vector of the invention with a suitable
pharmaceutical vehicle.
4. DESCRIPTION OF THE FIGURES
[0089] FIG. 1 shows the schematic map of plasmid super6.
[0090] FIG. 2 shows the schematic map of plasmid VI.
[0091] FIG. 3 shows the schematic map of plasmid II.
[0092] FIG. 4 shows the expression of the Nef and E2 proteins from
the vectors super6, super6wt, VI, VIwt, and II in Jurkat cells.
[0093] FIG. 5 shows the schematic map of plasmid product1.
[0094] FIG. 6A shows the schematic map of the plasmids NNV-1 and
NNV-2 and FIG. 6B shows the schematic map of plasmid and
NNV-2wt.
[0095] FIG. 7 shows the expression of the Nef protein from the
plasmids NNV-1, NNV-2, NNV-1wt, NNV-2-wt, super6, and super6wt in
Jurkat cells.
[0096] FIG. 8 shows the expression of the Nef and E2 proteins from
the plasmids NNV-2-wt, NNV-2-wtFS, and product I in Jurkat
cells.
[0097] FIG. 9 shows the expression of the Nef and E2 proteins from
the plasmids NNV-2-wt, NNV-2-wtFS, and product I in P815 cells.
[0098] FIG. 10 shows the expression of the Nef and E2 proteins from
the plasmids NNV-2-wt, NNV-2-wtFS, and product I in CHO cells.
[0099] FIG. 11 shows the expression of the Nef protein from the
plasmids NNV-2-wt, NNV-2-wtFS, and product I in RD cells.
[0100] FIG. 12 shows the expression of the RNA molecules NNV-2wt in
CHO, Jurkat cells, and P815 cells.
[0101] FIG. 13 shows the stability of NNV-2wt in bacterial
cells.
[0102] FIG. 14 shows the Southern blot analysis of stability of the
NNV-2wt as non-replicating episomal element in CHO and Jurkat cell
lines.
[0103] FIG. 15 shows that the vectors NNV2wt, NNV2wtFS and product1
are unable to HPV-11 replication factor-dependent replication.
[0104] FIG. 16 shows the schematic map of the plasmid
2wtd1EGFP.
[0105] FIG. 17 shows the schematic map of the plasmid gf10bse2
[0106] FIG. 18 shows the schematic map of the plasmid
2wtd1EGFPFS.
[0107] FIG. 19 shows the schematic map of the plasmid
NNVd1EGFP.
[0108] FIG. 20 shows the growth curves of the Jurkat cells
transfected with the plasmids 2wtd1EGFP, 2wtd1EGFPFS, NNVd1EGFP or
with carrier DNA only.
[0109] FIG. 21 shows the growth curves of the Jurkat cells
transfected with the plasmids 2wtd1EGFP, 2wtd1EGFPFS, gf10bse2 or
with carrier DNA only.
[0110] FIG. 22 shows the change in the percentage of d1EGFP
positive cells in a population of Jurkat cells transfected with the
vectors 2wtd1EGFP, 2wtd1EGFPFS or NNVd1EGFP.
[0111] FIG. 23 shows the change in percentage of the d1EGFP
positive cells in a population of Jurkat cells transfected with the
vectors 2wtd1EGFP, 2wtd1EGFPFS or gf10bse2.
[0112] FIG. 24 shows the change in the number of d1EGFP expressing
cells in a population of Jurkat cells transfected with the vectors
2wtd1EGFP, 2wtd1EGFPFS or NNVd1EGFP.
[0113] FIG. 25 shows the change in the number of d1EGFP expressing
cells in a population of Jurkat cells transfected with the vectors
2wtd1EGFP, 2wtd1EGFPFS or gf10bse2.
[0114] FIG. 26. T-cell responses towards recombinant Nef proteins
(5 micrograms/well), measured by T-cell proliferation in five
patients immunized with 1 microgram of GTU-Nef.
[0115] FIG. 27. T-cell responses towards recombinant Nef proteins
(5 micrograms/well), measured by T-cell proliferation in five
patients immunized with 20 micrograms of GTU-Nef.
[0116] FIG. 28. T-cell responses towards recombinant Nef proteins
(5 micrograms/well), measured by T-cell proliferation in patient# 1
immunized with 1 microgram of GTU-Nef. The results are given as
stimulation index of the T-cell proliferation assay (Nef SI) and as
IFN-Gamma secretion to the supernatant.
[0117] FIG. 29. (A) plasmid pEBO LPP; (B) plasmid s6E2d1EGFP; (C)
plasmid FRE2d1EGFP
[0118] FIG. 30. Plasmid FREBNAd1EGFP
[0119] FIG. 31. Vectors did not interfere with cell
proliferation
[0120] FIG. 32. Vectors were maintained in the cells with different
kinetics
[0121] FIG. 33. Change of the number of d1EGFP expressing cells in
time in transfected total population of cells
[0122] FIG. 34. Change of the number of d1EGFP expressing cells in
time in transfected total population of cells. (A) human embryonic
cell line 293; (B) mouse cell line 3T6
[0123] FIG. 35. Nef and E2 antibody response
[0124] FIG. 36. Rev and Tat antibody response
[0125] FIG. 37. Gag and CTL response
[0126] FIG. 38. (A) GTU-1; (B) GTU-2Nef; (C) GTU-3Nef; (D) super6
wtd1EGFP; (E) FREBNAd1EGFP; (F) E2BSEBNAd1EGFP; (G) NNV-Rev
[0127] FIG. 39. (A) pNRT; (B) pTRN; (C) pRTN; (D) pTNR; (E) pRNT;
(F) p2TRN; (G) p2RNT; (H) p3RNT; (I) pTRN-iE2-GMCSF; (J)
pTRN-iMG-GMCSF
[0128] FIG. 40. (A) pMV1NTR; (B) pMV2NTR; (C) pMV1N11TR; (D)
pMV2N11TR
[0129] FIG. 41. (A) pCTL; (B) pdgag; (C) psynp17/24; (D)
poptp17/24; (E) p2mCTL; (F) p2optp17/24; (G) p3mCTL; (H)
p3optp17/24
[0130] FIG. 42. (A) pTRN-CTL; (B) pRNT-CTL; (C) pTRN-dgag; (D)
pTRN-CTL-dgag; (E) pRNT-CTL-dgag; (F) pTRN-dgag-CTL; (G)
pRNT-dgag-CTL; (H) pTRN-optp17/24-CTL; (I) pTRN-CTL-optp17/24; (J)
pRNT-CTL-optp17/24; (K) p2TRN-optp17/24-CTL; (L)
p2RNT-optp17/24-CTL; (M) p2TRN-CTL-optp17/24; (N)
p2RNT-CTL-optp17/24; (O) p2TRN-CTL-optp17/24-iE2-mGMCSF; (P)
p2RNT-CTL-optp17/24-iE2-mGMCSF; (O) p3TRN-CTL-optp17/24; (R)
p3RNT-CTL-optp17/24; (S) p3TRN-CTL-optp17/24-iE2-mGMCSF; (T)
p3RNT-CTL-optp17/24-iE2-mGMCSF; (U) FREBNA-RNT-CTL-optp17/24; (V)
super6wt-RNT-CTL-optp17/24; (W) E2BSEBNA-RNT-CTL-optp17/24; (X)
pCMV-RNT-CTL-optp17/24
[0131] FIG. 43. Analysis of expression of the multireg
antigens.
[0132] FIG. 44. Analysis of expression of the multireg antigens
comprised of immunodominant parts of the proteins.
[0133] FIG. 45. Analysis of intracellular localization of multireg
antigens by immunofluorescence.
[0134] FIG. 46. Analysis of expression of the gag coded structural
proteins and the CTL multi-epitope.
[0135] 47. The p17/24 protein localization in membranes of RD
cells.
[0136] FIG. 48. Analysis expression of the dgag and CTL containing
multigenes in Cos-7 cells.
[0137] FIG. 49. Western blot analyses of multiHIV antigens
expressed in Jurkat cells.
[0138] FIG. 50. Analysis of the expression of the TRN-CTL-optp17/24
and RNT-CTL-optp17/24 antigens as well E2 protein from the GTU-1,
GTU-2 and GTU-3 vector.
[0139] FIG. 51. The maintenance of the multiHIV antigen expression
from different vectors.
[0140] FIG. 52. Intracellular localization of the multiHIV antigens
in RD cells.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Vectors of the Invention
[0141] The present invention is based on the unexpected finding
that expression vectors, which carry (A) an expression cassette of
a gene of a nuclear-anchoring protein that binds both to (i) a
specific DNA sequence and (ii) to a suitable nuclear component and
(B) a multimerized DNA binding sequence for said nuclear-anchoring
protein are capable of spreading in a proliferating cell
population. Such nuclear-anchoring proteins include, but are not
limited to, chromatin-anchoring proteins, such as the Bovine
Papilloma Virus type 1 E2 protein (BPV1 E2; SEQ ID NO: 50). The DNA
binding sequences can be, but are not limited to, multimerized E2
binding sites. On the basis of prior art, it could not be expected
that a segregation/partitioning function of, for instance, the
papilloma viruses could be expressed separately and that an
addition of such segregation/partitioning function to the vaccine
vectors would assure the distribution of the vector in the
proliferating cell population. Additionally, on the basis of the
prior art, it could not have been expected that functional vectors
acting independently of the replication origin can be
constructed.
[0142] The term "nuclear-anchoring protein" as used in the present
invention refers to a protein, which binds to a specific DNA
sequence and capable of providing a nuclear compartmentalization
function to the vector, i.e., to a protein, which is capable of
anchoring or attaching the vector to a specific nuclear
compartment. In certain embodiments of the invention, the
nuclear-anchoring protein is a natural protein. Examples of such
nuclear compartments are the mitotic chromatin or mitotic
chromosomes, the nuclear matrix, nuclear domains like ND10 and POD
etc. Examples of nuclear-anchoring proteins are the Bovine
Papilloma Virus type 1 (BPV1) E2 protein, EBNA1 (Epstein-Barr Virus
Nuclear Antigen 1; SEQ ID NO: 52), and High Mobility Group (HMG)
proteins etc. The term "functional equivalent of a
nuclear-anchoring protein" as used in the present invention refers
to a protein or a polypeptide of natural or non-natural origin
having the properties of the nuclear-anchoring protein.
[0143] In certain other embodiments of the invention, the
nuclear-anchoring protein of the invention is a recombinant
protein. In certain specific embodiments of the invention, the
nuclear-anchoring protein is a fusion protein, a chimeric protein,
or a protein obtained by molecular modeling. A fusion protein, or a
protein obtained by molecular modeling in connection with the
present invention is characterized by its ability to bind to a
nuclear component and by its ability to bind sequence-specifically
to DNA. In a preferred embodiment of the invention, such a fusion
protein is encoded by a vector of the invention which also contains
the specific DNA sequence to which the fusion/chimeric protein
binds. Nuclear components include, but are not limited to
chromatin, the nuclear matrix, the ND10 domain and POD. In order to
reduce the risk of interference with the expression of genes
endogenous to the host cell, the DNA binding domain and the
corresponding DNA sequence is preferably non-endogenous to the host
cell/host organism. Such domains include, but are not limited to,
the DNA binding domain of the Bovine Papilloma Virus type 1 (BPV1)
E2 protein (SEQ ID NO: 50), Epstein-Barr Virus Nuclear Antigen 1
(EBNA1; SEQ ID NO: 52), and High Mobility Group (HMG) proteins (HMG
box).
[0144] The vector of the invention can further comprise a "DNA
sequence of interest", that encodes a protein (including a peptide
or polypeptide), e.g., that is an immunogen or a therapeutic. In
certain embodiments of the invention, the DNA sequence of interest
encodes a biologically active RNA molecule, such as an antisense
RNA molecule or a ribozyme.
[0145] The expression vectors of the invention carrying an
expression cassette for a gene of a nuclear-anchoring protein and
multimerized binding sites for said nuclear-anchoring protein
spread in a proliferating host cell population. This means that a
high copy-number of vectors or plasmids are delivered into the
target cells and the use of the segregation/partitioning function
of the nuclear-anchoring protein and its multimerized binding sites
assures the distribution of the vector to the daughter cells during
cell division.
[0146] The vector of the invention lacks a papilloma virus origin
of replication. Further, in a preferred embodiment, the vector of
the invention lacks an origin of replication functional in a
mammalian cell. The omission of a papilloma virus origin of
replication or a mammalian origin of replication constitutes an
improvement over prior art vectors for several reasons. (1)
Omission of the origin of replication reduces the size of the
vector of the invention compared to prior art vectors. Such a
reduction in size increases the stability of the vector and
facilitates uptake by the host cell. (2) Omission of the origin of
replication reduces the risk for recombination with the host cell's
genome, thereby reducing the risk of unwanted side effects. (3) The
omission of the origin of replication allows to control the dosage
simply by adjusting the amount of vector administered. In contrast,
with a functioning origin of replication, replication of the vector
has to be taken into consideration when determining the required
dosage. (4) If the vector is not administered to a host organism
continually, the lack of an origin of replication allows the host
organism to clear itself of the vector, thus providing more control
over the levels of DNA sequences to be expressed in the host
organism. Further, the ability of the organism to clear itself of
the vector will be advantageous if the presence of the vector is
required only during the course of a therapy but is undesirable in
a healthy individual.
[0147] The gene of a nuclear-anchoring protein useful in the
vectors of the present invention can be any suitable DNA sequence
encoding a natural or artificial protein, such as a recombinant
protein, a fusion protein or a protein obtained by molecular
modeling techniques, having the required properties. Thus the gene
of a natural nuclear-anchoring protein, which contains a DNA
binding domain capable of binding to a specific DNA sequence and a
functional domain capable of binding to a nuclear component, can be
that of a viral protein, such as the E2 protein of Bovine Papilloma
Virus or the EBNA1 (Epstein-Barr Virus Nuclear Antigen 1) of the
Epstein-Barr Virus, a eukaryotic protein such a one of the High
Mobility Group (HMG) proteins or a like protein, or a prokaryotic
protein. Alternatively, the gene of a nuclear-anchoring protein,
which contains a DNA binding domain capable of binding to a
specific DNA sequence and a functional domain capable of binding to
a nuclear component, can also be comprised of DNA sequences, which
encode a domain from a cellular protein having the ability to
attach to a suitable nuclear structure, such as to mitotic
chromosomes, the nuclear matrix or nuclear domains like ND10 or
POD.
[0148] Alternatively, the DNA sequence, which encodes a non-natural
or artificial protein, such as a recombinant protein or a fusion
protein or a protein obtained by molecular modeling, which contains
a DNA binding domain capable of binding to a specific DNA sequence
of, e.g., a papilloma virus, such as the DNA binding domain of the
E2 protein of the BPV1, but in which the N-terminus of the
nuclear-anchoring protein, e.g. that of the E2 protein, has been
replaced with domains of any suitable protein of similar capacity,
for example, with the N-terminal domain of Epstein-Barr Virus
Nuclear Antigen 1 sequence, can be used. Similarly, DNA sequences,
which encode a recombinant protein or a fusion protein, which
contains a functional domain capable of binding to a nuclear
component, e.g., the N-terminal functional domain of a papilloma
virus, such as the E2 protein of the BPV1, but in which the
C-terminal DNA-binding dimerization domain of the nuclear-anchoring
protein, e.g., that of the E2 protein, has been replaced with
domains of any protein of a sufficient DNA-binding strength, e.g.,
the DNA binding domain of the BPV-1 E2 protein and the EBNA-1, can
be used.
[0149] In a preferred embodiment of the invention, the
nuclear-anchoring protein is a chromatin-anchoring protein, which
contains a DNA binding domain, which binds to a specific DNA
sequence, and a functional domain capable of binding to mitotic
chromatin. A preferred example of such a chromatin-anchoring
protein and its multimerized binding sites useful in the present
invention are the E2 protein of Bovine Papilloma Virus type 1 and
E2 protein multimerized binding sites. In the case of E2, the
mechanism of the spreading function is due to the dual function of
the E2 protein: the capacity of the E2 protein to attach to mitotic
chromosomes through the N-terminal domain of the protein and the
sequence-specific binding capacity of the C-terminal domain of the
E2 protein, which assures the tethering of vectors, which contain a
multimerized E2 binding site, to mitotic chromosomes. A
segregation/partitioning function is thus provided to the
vectors.
[0150] In another preferred embodiment of the invention, the
expression cassette of a gene of the chromatin-anchoring protein
comprises a gene of any suitable protein of cellular, viral or
recombinant origin having analogous properties to E2 of the BPV1,
i.e., the ability to attach to the mitotic chromatin through one
domain and to cooperatively bind DNA through another domain to
multimerized binding sites specific for this DNA binding
domain.
[0151] In a specific embodiment, sequences obtained from BPV1, are
used in the vectors of the present invention, they are extensively
shortened in size to include just two elements from BPV1. First,
they include the E2 protein coding sequence transcribed from a
heterologous eukaryotic promoter and polyadenylated at the
heterologous polyadenylation site. Second, they include E2 protein
multiple binding sites incorporated into the vector as a cluster,
where the sites can be as head-to-tail structures or can be
included into the vector by spaced positioning. Both of these
elements are necessary and, surprisingly, sufficient for the
function of the vectors to spread in proliferating cells.
Similarly, when DNA sequences based of other suitable sources are
used in the vectors of the present invention, the same principles
are applied.
[0152] According to the present invention, the expression cassette
of a gene of a nuclear-anchoring protein, which contains a DNA
binding domain capable of binding to a specific DNA sequence and a
functional domain capable of binding to a nuclear component, such
as an expression cassette of a gene of a chromatin-anchoring
protein, like BPV1 E2, comprises a heterologous eukaryotic
promoter, the nuclear-anchoring protein coding sequence, such as a
chromatin-anchoring protein coding sequence, for instance the BPV1
E2 protein coding sequence, and a poly A site. Different
heterologous, eukaryotic promoters, which control the expression of
the nuclear-anchoring protein, can be used. Nucleotide sequences of
such heterologous, eukaryotic promoters are well known in the art
and are readily available. Such heterologous eukaryotic promoters
are of different strength and tissue-specificity. In a preferred
embodiment, the nuclear anchoring protein is expressed at low
levels.
[0153] The multimerized DNA binding sequences, i.e., DNA sequences
containing multimeric binding sites, as defined in the context of
the present invention, are the region, to which the DNA binding
dimerization domain binds. The multimerized DNA binding sequences
of the vectors of the present invention can contain any suitable
DNA binding site, provided that it fulfills the above
requirements.
[0154] In a preferred embodiment, the multimerized DNA binding
sequence of a vector of the present invention can contain any one
of known 17 different affinity E2 binding sites as a hexamer or a
higher oligomer, as a octamer or a higher oligomer, as a decamer or
higher oligomer. Oligomers containing different E2 binding sites
are also applicable. Specifically preferred E2 binding sites useful
in the vectors of the present invention are the BPV1 high affinity
sites 9 and 10, affinity site 9 being most preferred. When a higher
oligomer is concerned, its size is limited only by the construction
circumstances and it may contain from 6 to 30 identical binding
sites. Preferred vectors of the invention contain 10 BPV-1 E2
binding sites 9 in tandem. When the multimerized DNA binding
sequences are comprised of different E2 binding sites, their size
and composition is limited only by the method of construction
practice. Thus they may contain two or more different E2 binding
sites attached to a series of 6 to 30, most preferably 10, E2
binding sites.
[0155] The Bovine Papilloma Virus type 1 genome (SEQ ID NO: 49)
contains 17 E2 protein binding sites which differ in their affinity
to E2. The E2 binding sites are described in Li et al. [Genes Dev 3
(4) (1989) 510-526], which is incorporated by reference in its
entirety herein.
[0156] Alternatively, the multimerized DNA binding sequences may be
composed of any suitable multimeric specific sequences capable of
inducing the cooperative binding of the protein to the plasmid,
such as those of the EBNA1 or a suitable HMG protein. 21.times.30
bp repeats of binding sites for EBNA-1 are localized in the region
spanning from nucleotide position 7421 to nucleotide position 8042
of the Epstein-Barr virus genome (SEQ ID NO:51). These EBNA-1
binding sites are described in the following references: Rawlins et
al., Cell 42 (3) (1985) 859-868; Reisman et al., Mol Cell Biol 5
(8) (1985) 1822-1832; and Lupton and Levine, Mol Cell Biol 5 (10)
(1985) 2533-2542, all three of which are incorporated by reference
in their entireties herein.
[0157] The position of the multimerized DNA binding sequences
relative to the expression cassette for the DNA binding
dimerization domain is not critical and can be any position in the
plasmid. Thus the multimerized DNA binding sequences can be
positioned either downstream or upstream relative to the expression
cassette for the gene of interest, a position close to the promoter
of the gene of interest being preferred.
[0158] The vectors of the invention also contain, where
appropriate, a suitable promoter for the transcription of the gene
or genes or the DNA sequences of interest, additional regulatory
sequences, polyadenylation sequences and introns. Preferably the
vectors may also include a bacterial plasmid origin of replication
and one or more genes for selectable markers to facilitate the
preparation of the vector in a bacterial host and a suitable
promoter for the expression the gene for antibiotic selection.
[0159] The selectable marker can be any suitable marker allowable
in DNA vaccines, such a kanamycin or neomycin, and others. In
addition, other positive and negative selection markers can be
included in the vectors of the invention, where applicable.
[0160] The vectors of the present invention only comprise the DNA
sequences, for instance BPV1 DNA sequences, which are necessary and
sufficient for long-term maintenance. All superfluous sequences,
which may induce adverse reactions, such as oncogenic sequences,
have been deleted. Thus in preferred vectors of the invention the
E2 coding sequence is modified by mutational analysis so that this
expresses only E2 protein and overlapping E3, E4 and E5 sequences
have been inactivated by the introduction of mutations, which
inactivate the translation from Open Reading Frames for E3, E4 and
E5. The vector of the invention does not contain a papilloma virus
origin of replication. Preferably, the vector of the invention
further does not contain an origin of replication functional in a
mammalian cell or a mammal.
[0161] Furthermore, the vectors of the present invention are not
host specific, since the expression of the nuclear-anchoring
protein, such as the E2 protein, is controlled by non-native or
heterologous promoters. Depending on the particular promoter
chosen, these promoters may be functional in a broad range of
mammalian cells or they can be cell or tissue specific. Examples of
promoters for the nuclear-anchoring protein, such as for the E2
protein, useful in the vectors of the present invention are
thymidine kinase promoters, Human Cytomegalovirus Immediate Early
Promoter, Rous Sarcoma Virus LTR and like. For the expression of
the gene of interest, preferred promoters are strong promoters
assuring high levels of expression of the gene of interest, an
example for such a promoter is the Human Cytomegalovirus Immediate
Early Promoter.
5.2 The Vectors of the Invention as Vehicles for Expression of a
DNA Sequence of Interest
[0162] A gene, genes or a DNA sequence or DNA sequences to be
expressed via a vector of the invention can be any DNA sequence of
interest, whose expression is desired. Thus the vectors may contain
a gene or genes or a DNA sequence or DNA sequences from infectious
microbial pathogens, such as viruses, against which live attenuated
vaccines or inactivated vaccines cannot be prepared or used. Such
DNA sequences of interest include genes or DNA sequences from
viruses, such as Human Immunodeficiency Virus (HIV), Herpex Simplex
Virus (HSV), Hepatitis C Virus, Influenzae Virus, Enteroviruses
etc.; intracellular bacterial, such as Chlamydia trachomatis,
Mycobacterium tuberculosis, Mycoplasma pneumonia etc.;
extracellular bacteria, such as Salmonella; or fungi, such as
Candida albigans.
[0163] In a preferred embodiment of the invention, the vectors
contain a gene encoding early regulatory proteins of HIV, i.e. the
nonstructural regulatory proteins Nef, Tat or Rev, preferably Nef.
In another preferred embodiment of the invention the vectors of the
invention contain genes encoding structural proteins of the HIV. In
another preferred embodiment the vectors of the present invention
contain two or more genes encoding any combination of early
regulatory proteins and/or structural proteins of HIV. Illustrative
examples of such combinations are a combination of a gene encoding
the Nef protein and a DNA sequence encoding the Tat protein,
possibly together with a DNA sequence encoding outer envelope
glycoprotein of HIV, gp120/gp160 or a combination of any
immunogenic epitopes of the proteins of pathogens incorporated into
artificial recombinant protein.
[0164] Alternatively, the vectors of the invention may contain
genes or DNA sequences for inherited or acquired genetic defects,
such as sequences of differentiation antigens for melanoma, like a
Tyrosinase A coding sequence or a coding sequence of
beta-catenins.
[0165] In a preferred embodiment of the invention, the vectors
contain a gene encoding proteins relating to cancer or other
mutational diseases, preferably diseases related to immune
maturation and regulation of immune response towards self and
nonself, such as the APECED gene.
[0166] In another preferred embodiment of the invention, the
vectors contain any DNA sequence coding for a protein that is
defective in any hereditary single gene hereditary disease.
[0167] In another preferred embodiment of the invention, the
vectors contain any DNA sequence coding for a macromolecular drug
to be delivered and produced in vivo.
[0168] The method of the invention for the preparation of the
vectors of the invention comprises the following steps: (A)
cultivating a host cell containing a vector of the invention, and
(B) recovering the vector. In certain specific embodiments, step
(A) is preceded by transforming a host cell with a vector of the
invention.
[0169] The vectors of the invention are preferably amplified in a
suitable bacterial host cell, such as Escherichia coli. The vectors
of the invention are stable and replicate at high copy numbers in
bacterial cells. If a vector of the invention is to be amplified in
a bacterial hast cell, the vector of the invention contains a
bacterial origin of replication. Nucleotide sequences of bacterial
origins of replication are well known to the skilled artisan and
can readily be obtained.
[0170] Upon transfection into a mammalian host in high copy number,
the vector spreads along with cell divisions and the number of
cells carrying the vector increases without the replication of the
vector, each cell being capable of expressing the protein of
interest.
[0171] The vectors of the invention result in high expression of
the desired protein. For instance, as demonstrated in Examples 4,
7-10: a high expression of the Nef protein of the HIV, green
fluorescent protein (EGFP) and the AIRE protein could be
demonstrated in many different cell lines and the data indicate
that not only the number of positive cells, but the quantity of the
protein encoded by the gene of interest is increasing in time.
[0172] The vectors of the invention also induce both humoral and
cellular response as demonstrated in Examples 9 and 10. The results
indicate that the vectors of the present invention can effectively
be used as DNA vaccines.
[0173] The vaccines of the present invention contain a vector of
the present invention or a mixture of said vectors in a suitable
pharmaceutical carrier. The vaccine may for instance contain a
mixture of vectors containing genes for the three different
regulatory proteins of the HIV and/or structural proteins of the
HIV.
[0174] The vaccines of the invention are formulated using standard
methods of vaccine formulation to produce vaccines to be
administered by any conventional route of administration, i.e.
intramuscularly, intradermally and like.
[0175] The vectors of the invention may contain the ISS stimulatory
sequences in order to activate the immune response of the body.
[0176] The vaccines of the invention can be used in a conventional
preventive manner to protect an individual from infections,
Alternatively, the vaccines of the invention can be used as
therapeutical vaccines, especially in the case of viral infections,
together with a conventional medication.
[0177] As mentioned above, the vectors of the present invention
carrying the mechanism of spreading in the host cell find numerous
applications as vaccines, in gene therapy, in gene transfer and as
therapeutic immunogens. The vectors of the invention can be used to
deliver a normal gene to a host having a gene defect, thus leading
to a cure or therapy of a genetic disease. Furthermore, the vectors
can deliver genes of immunogenic proteins of foreign origin, such
as those from microbes or autologous tumor antigens, to be used in
the development of vaccines against microbes or cancer.
Furthermore, the vectors of the invention can deliver suitable
genes of marker substances to nucleus, to be used in studies of
cellular function or in diagnostics. Finally, the vectors of the
invention can be used to specifically deliver a gene of
macromolecular drug to the nucleus, thus enabling the development
of novel therapeutic principles to treat and cure diseases, where
the expression of the drug in the site of action, the cell nucleus,
is of importance. These drugs can be chemical macromolecules, such
as any proteins or polypeptides with therapeutic or curative
effect, which interfere with any of the nuclear mechanisms, such as
the replication or transcription or the trans-port of substances to
and from the nucleus.
[0178] Specifically, the vectors of the present invention can be
used for the expression of the specific cytokines, like
interleukines (IL1, IL2, IL4, IL6, IL12 and others) or interferon,
with the aim of modulating the specific immune responses of the
organism (immunotherapy) against foreign antigens or boosting of
the activity of the immune system against the mutated
self-antigens. The vectors of the present invention are also useful
in complementing malfunctioning of the brain due to the loss of
specific dopamine-ergic neurons leading to the irreversible
neurodegeneration, which is cause for Parkinson's disease, by
expressing genes involved into synthesis of dopamine, like tyrosine
hydroxylase, as well as other genes deficiency of which would have
the similar effect. The vectors of the present invention are also
useful for the expression of proteins and peptides regulating the
brain activity, like dopamine receptors, CCK-A and CCK-B receptors,
as well as neurotrophic factors, like GDNF, BDNF and other proteins
regulating the brain activity. Further, the vectors of the present
invention are useful for a long-term expression of factor IX in
hepatocytes and alfa1-antitrypsin in muscle cells with the aim of
complementing respective deficiencies of the organism.
5.3 Target Diseases and Disorders
[0179] In certain embodiments, a vector of the invention is used as
a vaccine. In certain embodiments, a vector of the invention
contains a DNA sequence of interest that encodes a protein or a
peptide. Upon administering of such a vector to a subject, the
protein or peptide encoded by the DNA sequence of interest is
expressed and stimulates an immune response specific to the protein
or peptide encoded by the DNA sequence of interest.
[0180] In specific embodiments, the vector of the invention is used
to treat and/or prevent an infectious disease and/or a condition
caused by an infectious agent. Such diseases and conditions
include, but are not limited to, infectious diseases caused by
bacteria, viruses, fungi, protozoa, helminths, and the like. In a
more specific embodiment of the invention, the infectious disease
is Acquired Immunodeficiency Syndrome.
[0181] Preferably, where it is desired to treat or prevent viral
diseases, DNA sequences encoding molecules comprising epitopes of
known viruses are used. For example, such DNA sequences encoding
antigenic epitopes may be prepared from viruses including, but not
limited to, hepatitis type A, hepatitis type B, hepatitis type C,
influenza, varicella, adenovirus, herpes simplex type I (HSV-I),
herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus,
rotavirus, respiratory syncytial virus, papilloma virus, papova
virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,
coxsackie virus, mumps virus, measles virus, rubella virus, polio
virus, human immunodeficiency virus type I (HIV-I), and human
immunodeficiency virus type II (HIV-II).
[0182] Preferably, where it is desired to treat or prevent
bacterial infections, DNA sequences encoding molecules comprising
epitopes of known bacteria are used. For example, such DNA
sequences encoding antigenic epitopes may be prepared from bacteria
including, but not limited to, mycobacteria rickettsia, mycoplasma,
neisseria and legionella.
[0183] Preferably, where it is desired to treat or prevent
protozoal infections, DNA sequences encoding molecules comprising
epitopes of known protozoa are used. For example, such DNA
sequences encoding antigenic epitopes may be prepared from protozoa
including, but not limited to, leishmania, kokzidioa, and
trypanosoma.
[0184] Preferably, where it is desired to treat or prevent
parasitic infections, DNA sequences encoding molecules comprising
epitopes of known parasites are used. For example, such DNA
sequences encoding antigenic epitopes may be prepared from
parasites including, but not limited to, chlamydia and
rickettsia.
[0185] In other specific embodiments, the vector of the invention
is used to treat and/or prevent a neoplastic disease in a subject.
In these embodiments, the DNA sequence of interest encodes a
protein or peptide that is specific to or associated with the
neoplastic disease. By way of non-limiting example, the neoplastic
disease can be a fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,
rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, testicular tumor, lung carcinoma, small
cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and
acute myelocytic leukemia (myeloblastic, promyelocytic,
myelomonocytic, monocytic and erythroleukemia); chronic leukemia
(chronic myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease, etc.
[0186] In certain other embodiments of the invention, the DNA
sequence of interest encodes a protein that is non-functional or
malfunctioning due to an inherited disorder or an acquired mutation
in the gene encoding the protein. Such genetic diseases include,
but are not limited to, metabolic diseases, e.g., Atherosclerosis
(affected gene: APOE); cancer, e.g., Familial Adenomatous Polyposis
Coli (affected gene: APC gene); auto-immune diseases, e.g.,
autoimmune polyendocrinopathy-candidosis-ectodermal dysplasia
(affected gene: APECED); disorders of the muscle, e.g., Duchenne
muscular dystrophyvaccines (affected gene: DMD); diseases of the
nervous system, e.g., Alzheimer's Disease (affected genes: PS1 and
PS2).
[0187] In even other embodiments, the vectors of the invention are
used to treat and/or prevent diseases and disorders caused by
pathologically high activity of a protein. In these embodiments of
the invention, the DNA sequence of interest encodes an antagonist
of the overactive protein. Such antagonists include, but are not
limited to, antisense RNA molecules, ribozymes, antibodies, and
dominant negative proteins. In specific embodiments of the
invention, the DNA sequence of interest encodes an inhibitor of an
oncogene.
[0188] In certain embodiments, the DNA sequence of interest encodes
a molecule that antagonizes neoplastic growth. In specific
embodiments of the invention, the DNA sequence of interest encodes
a tumor suppressor, such as, but not limited to, p53. In other
specific embodiments, the DNA sequence of interest encodes an
activator of apoptosis, such as but not limited to, a Caspase.
[0189] The invention provides methods, whereby a DNA sequence of
interest is expressed in a subject. In certain embodiments, a
vector containing one or more expression cassettes of a DNA
sequence of interest is administered to the subject, wherein the
subject does not express the Bovine Papilloma Virus E1 protein.
5.4 Therapeutic Methods for Use with the Invention
[0190] 5.4.1 Recombinant DNA
[0191] In various embodiments of the invention, the vector of the
invention comprises one or more expression cassettes comprising a
DNA sequence of interest. The DNA sequence of interest can encode a
protein and/or a biologically active RNA molecule. In either case,
the DNA sequence is inserted into the vector of the invention for
expression in recombinant cells or in cells of the host in the case
of gene therapy.
[0192] An expression cassette, as used herein, refers to a DNA
sequence of interest operably linked to one or more regulatory
regions or enhancer/promoter sequences which enables expression of
the protein of the invention in an appropriate host cell.
"Operably-linked" refers to an association in which the regulatory
regions and the DNA sequence to be expressed are joined and
positioned in such a way as to permit transcription, and in the
case of a protein, translation.
[0193] The regulatory regions necessary for transcription of the
DNA sequence of interest can be provided by the vector of the
invention. In a compatible host-construct system, cellular
transcriptional factors, such as RNA polymerase, will bind to the
regulatory regions of the vector to effect transcription of the DNA
sequence of interest in the host organism. The precise nature of
the regulatory regions needed for gene expression may vary from
host cell to host cell. Generally, a promoter is required which is
capable of binding RNA polymerase and promoting the transcription
of an operably-associated DNA sequence. Such regulatory regions may
include those 5'-non-coding sequences involved with initiation of
transcription and translation, such as the TATA box, capping
sequence, CAAT sequence, and the like. The non-coding region 3' to
the coding sequence may contain transcriptional termination
regulatory sequences, such as terminators and polyadenylation
sites.
[0194] Both constitutive and inducible regulatory regions may be
used for expression of the DNA sequence of interest. It may be
desirable to use inducible promoters when the conditions optimal
for growth of the host cells and the conditions for high level
expression of the DNA sequence of interest are different. Examples
of useful regulatory regions are provided below (section
5.4.4).
[0195] In order to attach DNA sequences with regulatory functions,
such as promoters, to the DNA sequence of interest or to insert the
DNA sequence of interest into the cloning site of a vector, linkers
or adapters providing the appropriate compatible restriction sites
may be ligated to the ends of the cDNAs by techniques well known in
the art [Wu et al., Methods in Enzymol 152 (1987) 343-349).
Cleavage with a restriction enzyme can be followed by modification
to create blunt ends by digesting back or filling in
single-stranded DNA termini before ligation. Alternatively, a
desired restriction enzyme site can be introduced into a fragment
of DNA by amplification of the DNA by use of PCR with primers
containing the desired restriction enzyme site.
[0196] The vector comprising a DNA sequence of interest operably
linked to a regulatory region (enhancer/promoter sequences) can be
directly introduced into appropriate host cells for expression of
the DNA sequence of interest without further cloning.
[0197] For expression of the DNA sequence of interest in mammalian
host cells, a variety of regulatory regions can be used, for
example, the SV40 early and late promoters, the cytomegalovirus
(CMV) immediate early promoter, and the Rous sarcoma virus long
terminal repeat (RSV-LTR) promoter. Inducible promoters that may be
useful in mammalian cells include but are not limited to those
associated with the metallothionein II gene, mouse mammary tumor
virus glucocorticoid responsive long terminal repeats (MMTV-LTR),
.beta.-interferon gene, and hsp70 gene [Williams et al., Cancer
Res. 49 (1989) 2735-42; Taylor et al., Mol. Cell. Biol., 10 (1990)
165-75]. It may be advantageous to use heat shock promoters or
stress promoters to drive expression of the DNA sequence of
interest in recombinant host cells.
[0198] In addition, the expression vector may contain a selectable
or screenable marker gene for initially isolating, identifying or
tracking host cells that contain the vector. A number of selection
systems may be used for mammalian cells, including but not limited
to the Herpes simplex virus thymidine kinase [Wigler et al., Cell
11 (1977) 223], hypoxanthine-guanine phosphoribosyltransferase
[Szybalski and Szybalski, Proc. Natl. Acad. Sci. USA 48 (1962)
2026], and adenine phosphoribosyltransferase [Lowy et al., Cell 22
(1980) 817] genes can be employed in tk.sup.-, hgprt.sup.- or
aprt.sup.- cells, respectively. Also, antimetabolite resistance can
be used as the basis of selection for dihydrofolate reductase
(dhfr), which confers resistance to methotrexate [Wigler et al.,
Natl, Acad. Sci. USA 77 (1980) 3567; O'Hare et al., Proc. Natl.
Acad. Sci. USA 78 (1981) 1527]; gpt, which confers resistance to
mycophenolic acid [Mulligan & Berg, Proc. Natl. Acad. Sci. USA
78 (1981) 2072]; neomycin phosphotransferase (neo), which confers
resistance to the aminoglycoside G-418 [Colberre-Garapin et al., J.
Mol. Biol. 150 (1981) 1]; and hygromycin phosphotransferase (hyg),
which confers resistance to hygromycin [Santerre et al., 1984, Gene
30 (1984) 147]. Other selectable markers, such as but not limited
to histidinol and Zeocin.RTM. can also be used.
[0199] 5.4.2 Expression Systems and Host Cells
[0200] For use with the methods of the invention, the host cell
and/or the host organism preferably does not express the Bovine
Papilloma Virus E1 protein. Furthermore, preferably the vector of
the invention does not encode the Bovine Papilloma Virus E1
protein.
[0201] Preferred mammalian host cells include but are not limited
to those derived from humans, monkeys and rodents, (see, for
example, Kriegler M. in "Gene Transfer and Expression: A Laboratory
Manual", New York, Freeman & Co. 1990), such as monkey kidney
cell line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line (293, 293-EBNA, or 293 cells subcloned for
growth in suspension culture, Graham et al., J. Gen. Virol., 36
(1977) 59; baby hamster kidney cells (BHK, ATCC CCL 10); chinese
hamster ovary-cells-DHFR [CHO, Urlaub and Chasin. Proc. Natl. Acad.
Sci. 77 (1980) 4216]; mouse sertoli cells [Mather, Biol. Reprod. 23
(1980) 243-251]; mouse fibroblast cells (NIH-3T3), monkey kidney
cells (CVI ATCC CCL 70); african green monkey kidney cells
(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA,
ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat
liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC
CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary
tumor cells (MMT 060562, ATCC CCL51).
[0202] The vectors of the invention may be synthesized and
assembled from known DNA sequences by well-known techniques in the
art. The regulatory regions and enhancer elements can be of a
variety of origins, both natural and synthetic. Some host cells may
be obtained commercially.
[0203] The vectors of the invention containing a DNA sequence of
interest can be introduced into the host cell by a variety of
techniques known in the art, including but not limited to, for
prokaryotic cells, bacterial transformation (Hanahan, 1985, in DNA
Cloning, A Practical Approach, 1:109-136), and for eukaryotic
cells, calcium phosphate mediated transfection [Wigler et al., Cell
11 (1977) 223-232], liposome-mediated transfection [Schaefer-Ridder
et al., Science 215 (1982) 166-168], electroporation [Wolff et al.,
Proc Natl Acad Sci 84 (1987) 3344], and microinjection [Cappechi,
Cell 22 (1980) 479-4889].
[0204] In a specific embodiment, cell lines that express the DNA
sequence of the invention may be engineered by using a vector that
contains a selectable marker. By way of example but not limitation,
following the introduction of the vector, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the vector
confers resistance to the selection and optimally allows only cells
that contain the vector with the selectable marker to grow in
culture.
[0205] 5.4.3 Vaccine Approaches
[0206] In certain embodiments, a vector of the invention comprising
an expression cassette of a DNA sequence of interest is
administered to a subject to induce an immune response.
Specifically, the DNA sequence of interest encodes a protein (for
example, a peptide or polypeptide), which induces a specific immune
response upon its expression. Examples of such proteins are
discussed in section 5.3.
[0207] For the delivery of a vector of the invention for use as a
vaccine, methods may be selected from among those known in the art
and/or described in section 5.4.6.
[0208] 5.4.4 Gene Therapy Approaches
[0209] In a specific embodiment, a vector of the invention
comprising an expression cassette comprising DNA sequences of
interest is administered to treat, or prevent various diseases. The
DNA sequence of interest may encode a protein and/or a biologically
active RNA molecule. Gene therapy refers to therapy performed by
the administration to a subject of an expressed or expressible DNA
sequence. In this embodiment of the invention, the DNA sequences
produce their encoded protein or RNA molecule that mediates a
therapeutic effect.
[0210] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0211] For general reviews of the method of gene therapy, see,
Goldspiel et al., Clinical Pharmacy 12 (1993) 488-505; Wu and Wu,
Biotherapy 3 (1991) 87-95; Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32 (1993) 573-596; Mulligan, Science 260 (1993) 926-932;
Morgan and Anderson, Ann. Rev. Biochem. 62 (1993) 191-217; May,
TIBTECH 1, I (5) (1993) 155-215. Methods commonly known in the art
of recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0212] The following animal regulatory regions, which exhibit
tissue specificity and have been utilized in transgenic animals,
can be used for expression of the DNA sequence of interest in a
particular tissue type: elastase I gene control region which is
active in pancreatic acinar cells [Swift et al., Cell 38 (1984)
639-646; Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50
(1986) 399-409; MacDonald, Hepatology 7 (1987) 425-515]; insulin
gene control region which is active in pancreatic beta cells
[Hanahan, Nature 315 (1985) 115-122], immunoglobulin gene control
region which is active in lymphoid cells [Grosschedl et al., Cell
38 (1984) 647-658; Adames et al., Nature 318 (1985) 533-538;
Alexander et al., Mol. Cell. Biol. 7 (1987) 1436-1444], mouse
mammary tumor virus control region which is active in testicular,
breast, lymphoid and mast cells [Leder et al., Cell 45 (1986)
485-495], albumin gene control region which is active in the liver
[Pinkert et al., Genes and Devel. 1 (1987) 268-276],
alpha-fetoprotein gene control region which is active in the liver
[Krumlauf et al., Mol. Cell. Biol. 5 (1985) 1639-1648; Hammer et
al., Science 235 (1987) 53-58; alpha 1-antitrypsin gene control
region which is active in the liver [Kelsey et al., Genes and
Devel. 1 (1987) 161-171], beta-globin gene control region which is
active in myeloid cells [Mogram et al., Nature 315 (1985) 338-340;
Kollias et al., Cell 46 (1986) 89-94]; myelin basic protein gene
control region which is active in oligodendrocyte cells in the
brain [Readhead et al., Cell 48 (1987) 703-712]; myosin light
chain-2 gene control region which is active in skeletal muscle
[Sani, Nature 314 (1985) 283-286], and gonadotropic releasing
hormone gene control region which is active in the hypothalamus
[Mason et al., Science 234 (1986) 1372-1378].
[0213] Methods of delivery for gene therapy approaches are well
known in the art and/or described in section 5.4.6.
[0214] 5.4.5 Inhibitory Antisense and Ribozyme
[0215] In certain embodiments of the invention a vector of the
invention contains a DNA sequence of interest that encodes an
antisense or ribozyme RNA molecule. Techniques for the production
and use of such molecules are well known to those of skill in the
art.
[0216] Antisense RNA molecules act to directly block the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. Antisense approaches involve the design of
oligonucleotides that are complementary to a target gene mRNA. The
antisense oligonucleotides will bind to the complementary target
gene mRNA transcripts and prevent translation. Absolute
complementarity, although preferred, is not required.
[0217] A sequence "complementary" to a portion of an RNA, as
referred to herein, means a sequence having sufficient
complementarity to be able to hybridize with at least the non-polyA
portion of an RNA, forming a stable duplex; in the case of
double-stranded antisense nucleic acids, a single strand of the
duplex DNA may thus be tested, or triplex formation may be assayed.
The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid.
Generally, the longer the hybridizing nucleic acid, the more base
mismatches with an RNA it may contain and still form a stable
duplex (or triplex, as the case may be). One skilled in the art can
ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex.
[0218] Antisense nucleic acids should be at least six nucleotides
in length, and are preferably oligonucleotides ranging from 6 to
about 50 nucleotides in length. In specific aspects the
oligonucleotide is at least 10 nucleotides, at least 17
nucleotides, at least 25 nucleotides or at least 50 nucleotides. In
other embodiments of the invention, the antisense nucleic acids are
at least 100, at least 250, at least 500, and at least 1000
nucleotides in length.
[0219] Regardless of the choice of target sequence, it is preferred
that in vitro studies are first performed to quantitate the ability
of the antisense oligonucleotide to inhibit gene expression. It is
preferred that these studies utilize controls that distinguish
between antisense gene inhibition and nonspecific biological
effects of oligonucleotides. It is also preferred that these
studies compare levels of the target RNA or protein with that of an
internal control RNA or protein. Additionally, it is envisioned
that results obtained using the antisense DNA sequence are compared
with those obtained using a control DNA sequence. It is preferred
that the control DNA sequence is of approximately the same length
as the test oligonucleotide and that the DNA sequence of the
oligonucleotide differs from the antisense sequence no more than is
necessary to prevent specific hybridization to the target
sequence.
[0220] While antisense DNA sequences complementary to the target
gene coding region sequence could be used, those complementary to
the transcribed, untranslated region are most preferred.
[0221] For expression of the biologically active RNA, e.g., an
antisense RNA molecule, from the vector of the invention the DNA
sequence encoding the bio logically active RNA molecule is
operatively linked to a strong pol III or pol II promoter. The use
of such a construct to transfect target cells in the patient will
result in the transcription of sufficient amounts of single
stranded RNAs that will form complementary base pairs with the
endogenous target gene transcripts and thereby prevent translation
of the target gene mRNA. For example, a vector of the invention can
be introduced, e.g., such that it is taken up by a cell and directs
the transcription of an antisense RNA. Such vectors can be
constructed by recombinant DNA technology methods standard in the
art. Expression of the sequence encoding the antisense RNA can be
by any promoter known in the art to act in mammalian, preferably
human cells. Such promoters can be inducible or constitutive. Such
promoters include but are not limited to: the SV40 early promoter
region [Bernoist and Chambon, Nature 290 (1981) 304-310], the
promoter contained in the 3 long terminal repeat of Rous sarcoma
virus [Yamamoto, et al., Cell 22 (1980) 787-797], the herpes
thymidine kinase promoter [Wagner, et al., Proc. Natl. Acad. Sci.
U.S.A. 78 (1981) 1441-1445], the regulatory sequences of the
metallothionein gene [Brinster, et al., 1982, Nature 296 (1982)
39-42], etc.
[0222] In certain embodiments of the invention, a vector of the
invention contains a DNA sequence, which encodes a ribozyme.
Ribozyme molecules designed to catalytically cleave target gene
mRNA transcripts can also be used to prevent translation of a
target gene mRNA and, therefore, expression of a target gene
product [see, e.g., PCT International Publication WO90/11364,
published Oct. 4, 1990; Sarver, et al., Science 247 (1990)
1222-1225].
[0223] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. [For a review, see Rossi, Current
Biology 4 (1994) 469-471]. The mechanism of ribozyme action
involves sequence specific hybridization of the ribozyme molecule
to complementary target RNA, followed by an endonucleolytic
cleavage event. The composition of ribozyme molecules must include
one or more sequences complementary to the target gene mRNA, and
must include the well known catalytic sequence responsible for mRNA
cleavage. For this sequence, see, e.g., U.S. Pat. No. 5,093,246,
which is incorporated herein by reference in its entirety.
[0224] While ribozymes that cleave mRNA at site-specific
recognition sequences can be used to destroy target gene mRNAs, the
use of hammerhead ribozymes is preferred. Hammerhead ribozymes
cleave mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes
is well known in the art and is described more fully in Myers,
1995, Molecular Biology and Biotechnology: A Comprehensive Desk
Reference, VCH Publishers, New York, (see especially FIG. 4, page
833) and in Haseloff & Gerlach, Nature, 334 1988) 585-591,
which is incorporated herein by reference in its entirety.
[0225] Preferably the ribozyme is engineered so that the cleavage
recognition site is located near the 5' end of the target gene
mRNA, i.e., to increase efficiency and minimize the intracellular
accumulation of non-functional mRNA transcripts.
[0226] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one that occurs naturally in Tetrahymena thermophila (known as the
IVS, or L-19 IVS RNA) and that has been extensively described by
Thomas Cech and collaborators [Zaug, et al., Science, 224 (1984)
574-578; Zaug and Cech, Science, 231 (1986) 470-475; Zaug, et al.,
Nature, 324 (1986) 429-433; published International patent
application No. WO 88/04300 by University Patents Inc.; Been &
Cech, Cell, 47 (1986) 207-216]. The Cech-type ribozymes have an
eight base pair active site, which hybridizes to a target RNA
sequence whereafter cleavage of the target RNA takes place. The
invention encompasses those Cech-type ribozymes, which target eight
base-pair active site sequences that are present in the target
gene.
[0227] Expression of a ribozyme can be under the control of a
strong constitutive pol III or pol II promoter, so that transfected
cells will produce sufficient quantities of the ribozyme to destroy
endogenous target gene messages and inhibit translation. Because
ribozymes unlike antisense molecules, are catalytic, a lower
intracellular concentration is required for efficiency.
[0228] In instances wherein the antisense and/or ribozyme molecules
described herein are utilized to inhibit mutant gene expression, it
is possible that the technique may so efficiently reduce or inhibit
the translation of mRNA produced by normal target gene alleles that
the possibility may arise wherein the concentration of normal
target gene product present may be lower than is necessary for a
normal phenotype. In such cases, to ensure that substantially
normal levels of target gene activity are maintained, therefore,
nucleic acid molecules that encode and express target gene
polypeptides exhibiting normal target gene activity may, be
introduced into cells via gene therapy methods such as those
described, below, in Section 5.4.4 that do not contain sequences
susceptible to whatever antisense, ribozyme, or triple helix
treatments are being utilized. Alternatively, in instances whereby
the target gene encodes an extracellular protein, it may be
preferable to co-administer normal target gene protein in order to
maintain the requisite level of target gene activity.
[0229] Methods of administering the ribozyme and antisense RNA
molecules are well known in the art and/or described in section
5.4.6.
[0230] 5.4.6 Pharmaceutical Formulations and Modes of
Administration
[0231] In a preferred aspect, a pharmaceutical of the invention
comprises a substantially purified vector of the invention (e.g.,
substantially free from substances that limit its effect or produce
undesired side-effects). The subject to whom the pharmaceutical is
administered in the methods of the invention is preferably an
animal, including but not limited to animals such as cows, pigs,
horses, chickens, cats, dogs, etc., and is preferably a mammal, and
most preferably a human.
[0232] In certain embodiments, the vector of the invention is
directly administered in vivo, where the DNA sequence of interest
is expressed to produce the encoded product. This can be
accomplished by any of numerous methods known in the art. The
vectors of the invention can be administered so that the nucleic
acid sequences become intracellular. The vectors of the invention
can be administered by direct injection of naked DNA; use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont);
coating with lipids or cell-surface receptors or transfecting
agents; encapsulation in microparticles or microcapsules;
administration in linkage to a peptide which is known to enter the
nucleus; administration in linkage to a ligand subject to
receptor-mediated endocytosis [see, e.g., Wu and Wu, J. Biol. Chem.
262 (1987) 4429-4432] (which can be used to target cell types
specifically expressing the receptors); etc. In a specific
embodiment, the compound or composition can be delivered in a
vesicle, in particular a liposome [see Langer, Science 249 (1990)
1527-1533; Treat et al., 1989, in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp.
317-327].
[0233] In certain embodiments, the vector of the invention is
coated with lipids or cell-surface receptors or transfecting
agents, or linked to a homeobox-like peptide which is known to
enter the nucleus [see e.g., Joliot et al., Proc. Natl. Acad. Sci.
USA 88 (1991) 1864-1868], etc.
[0234] In certain other embodiments, nucleic acid-ligand complexes
can be formed in which the ligand comprises a fusogenic viral
peptide to disrupt endosomes, allowing the nucleic acid to avoid
lysosomal degradation.
[0235] In yet other embodiments, the vector of the invention can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO 92/06
180; WO 92/22635; WO92/20316; WO93/14188, and WO 93/20221).
[0236] Methods for use with the invention include, but are not
limited to, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
Methods for use with the invention further include administration
by any convenient route, for example by infusion or bolus
injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). In
a specific embodiment, it may be desirable to administer a vector
of the invention by injection, by means of a catheter, by means of
a suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including a membrane,
such as a sialastic membrane, or a fiber. Care must be taken to use
materials to which the vector does not absorb. Administration can
be systemic or local.
[0237] In certain embodiments, a vector of the invention is
administered together with other biologically active agents such as
chemotherapeutic agents or agents that augment the immune
system.
[0238] In yet another embodiment, methods for use with the
invention include delivery via a controlled release system. In one
embodiment, a pump may be used [see Langer, supra; Sefton, CRC
Crit. Ref. Biomed. Eng. 14 (1989) 201; Buchwald et al., Surgery 88
(1980) 507; Saudek et al., N. Engl. J. Med. 321 (1989) 574]. In
another embodiment, polymeric materials can be used [see Medical
Applications of Controlled Release, 1974, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla.; Controlled Drug Bioavailability, Drug
Product Design and Performance, 1984, Smolen and Ball (eds.),
Wiley, New York; Ranger and Peppas, Macromol. Sci. Rev. Macromol.
Chem. 23 (1983) 61; see also Levy et al., Science 228 (1985) 190;
During et al., Ann. Neurol. 25 (1989) 351; Howard et al., J.
Neurosurg. 71 (1989) 105].
[0239] Other controlled release systems are discussed in the review
by Langer, Science 249 (1990) 1527-1533.
[0240] Pharmaceutical compositions of the invention comprise a
therapeutically effective amount of a vector of the invention, and
a suitable pharmaceutical vehicle. In a specific embodiment, the
term "suitable pharmaceutical vehicle" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. The term
"vehicle" refers to a diluent, adjuvant, excipient, or vehicle with
which the therapeutic is administered. Such suitable pharmaceutical
vehicles can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water is a preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. These compositions can take the
form of solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc Examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Such compositions will
contain a therapeutically effective amount of the nucleic acid or
protein of the invention, preferably in purified form, together
with a suitable amount of carrier so as to provide the form for
proper administration to the patient. The formulation should suit
the mode of administration.
[0241] In a specific embodiment, the pharmaceutical of the
invention is formulated in accordance with routine procedures as a
pharmaceutical composition adapted for intravenous administration
to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer.
Where necessary, the pharmaceutical of the invention may also
include a solubilizing agent and a local anesthetic such as
lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the pharmaceutical of the invention is to be administered by
infusion, it can be dispensed with an infusion bottle containing
sterile pharmaceutical grade water or saline. Where the
pharmaceutical of the invention is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0242] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0243] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0244] The amount of a vector of the invention, which will be
effective in the treatment or prevention of the indicated disease,
can be determined by standard clinical techniques. In addition, in
vitro assays may optionally be employed to help identify optimal
dosage ranges. The precise dose to be employed in the formulation
will also depend on the route of administration, and the stage of
indicated disease, and should be decided according to the judgment
of the practitioner and each patient's circumstances. Effective
doses may be extrapolated from dose-response curves derived from in
vitro or animal model test systems.
[0245] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention. The following examples are presented in
order to more fully illustrate the preferred embodiments of the
invention. They should in no way be construed, however, as limiting
the broad scope of the invention.
6 EXAMPLES
6.1. Example 1
Cloning and Analysis of The Expression Properties of the Vectors
super6 and super6wt
[0246] The vector plasmids super6 (FIG. 1) and super6wt were
prepared from previous generation based gene vaccination vectors VI
(FIG. 2) and VIwt, respectively. Vectors VI and VIwt are
principally synthetic bacterial plasmids that contain a transposon
Tn903 derived kanamycin resistance marker gene [Oka, A., et al., J
Mol Biol 147 (1981) 217-226] and a modified form of pMB1 replicon
[Yanisch-Perron, C., et al., Gene 33 (1985) 103-119] needed for the
propagation in Escherichia coli cells. Vectors VI and VIwt also
contain a Cytomegalovirus Immediately Early Promoter combined with
a HSV1 TK leader sequence and rabbit .beta.-globin gene sequences,
which both are derived from plasmid pCG [Tanaka, M., et al., 60
(1990) Cell 375-386]. The latter elements are needed for expressing
from the nef coding sequence derived from a HAN2 isolate of the
HIV-1 strain [Sauermann, U., et al., AIDS Research. Hum. Retrov. 6
(1990) 813-823]. The expression vectors for the Nef carry clustered
ten high affinity E2 binding sites (derived from plasmid pUC1910BS,
unpublished) just upstream of the CMV promoter.
[0247] The parent vector VI contains a modified E2 coding sequence:
the hinge region of E2 (amino acids 192-311) is replaced with four
glycine-alanine repeats from EBNA1 protein of Epstein-Barr Virus
[Baer, R. J., et al., Nature 310 (1984) 207-211]. The protein
encoded by this sequence was named as E2d192-311+4G, The parent
vector VIwt contains an expression cassette for wild type E2
protein of the bovine papilloma virus type 1 with point mutations
introduced for the elimination E3 and E4 open reading frame (ORF)
coding capacity by two stop codons into both these ORFs. In the
vectors the E2 coding sequences are cloned between a Rous sarcoma
virus proviral 5' LTR [Long, E. O., et al., Hum. Immunol. 31 (1991)
229-235] and bovine growth hormone polyadenylation region [Chesnut,
J. D., et al., J Immunol Methods 193 (1996) 17-27].
[0248] Plasmid vectors super6 and super6wt were constructed by
deleting from the respective parent vectors VI and VIwt all
beta-globin sequences downstream of the nef gene except the second
intron of the rabbit beta-globin gene. The beta-globin sequences
(especially the fragment of the exon) show some homology with
sequences in the human beta-globin gene, whereas the intron lacks
any significant homology to human genomic sequences. The intron was
amplified by PCR from the plasmid pCG [Tanaka, M. et al., Cell 60
(1990) 375-386] using oligonucleotides with some mismatches for
modifying the sequences of splicing donor and acceptor sites of the
intron to the perfect match to consensus motifs. The Herpes Simplex
Virus type 1 thymidine kinase gene polyadenylation region from
pHook [Chesnut, J. D., et al., J Immunol Methods 193 (1996) 17-27]
was then cloned just next to the 3'-end of the intron, because in
parent plasmids the rabbit .beta.-globin polyadenylation signal
were used.
[0249] The expression properties of the Nef and E2 proteins
expressed by the plasmid vectors super6 and super6wt were analyzed
and compared with the expression properties of the Nef and E2
proteins expressed by VI and VIwt by Western blotting [Towbin et
al., Proc Natl Acad Sci USA 76 (1979) 4350-4354] with monoclonal
antibodies against Nef and E2. First, Jurkat cells (a human T-cell
lymphoblast cell line) were transfected by electroporation [Ustav
et al. EMBO J 2 (1991) 449-457] with 1 .mu.g of super6, super6WT or
equimolar amounts of the plasmids VI, VIwt. As a control an
equimolar amount of vector II (FIG. 3), which contains an identical
Nef cassette but no E2 coding sequence, was used. Carrier DNA was
used as a negative control. Briefly, the plasmid and carrier DNA
were mixed with the cell suspension in a 0.4 cm electroporation
cuvette (BioRad Laboratories, Hercules, USA) followed an electric
pulse (200V; 1 mF) using Gene Pulser IITM with capacitance extender
(BioRad Laboratories, Hercules, USA).
[0250] Forty-nine hours post-transfection the cells were lysed by
treating with a sample buffer containing 50 mM Tris-HCl pH 6.8; 2%
SDS, 0.1% bromophenol blue, 100 mM dithiothreitol, and 10% (v/v)
glycerol. The lysates were run on a 10% or 12.5% SDS-polyacrylamide
gel and subsequently transferred onto a 0.45 .mu.m PVDF
nitrocellulose membrane (Millipore). The membrane was first blocked
overnight with a blocking solution containing 5% dry milk
(fat-free), 0.1% Tween 20 in 50 mM Tris-HCl pH 7.5; 150 mM NaCl and
thereafter incubated for 1 h with diluted monoclonal anti-Nef
antisera (1:100) or anti-E2-antisera (1:1000) in the blocking
solution. After each incubation step, unbound proteins were removed
by washing strips three times with TBS-0.1% Tween-20. The binding
of primary immunoglobulins was detected by incubating the strips
with horse raddish peroxidase conjugate anti-mouse IgG (Labas,
Estonia) followed by visualization using a chemoluminesence
detection system (Amersham Pharmacia Biotech, United Kingdom).
[0251] The results are shown in FIG. 4. The expression of the Nef
protein is shown on panel A and the expression of E2 protein on
panel B. The arrows indicate the right molecular sizes of the Nef
and E2 proteins. The expression level of the E2d192-311+4GA is very
low and for this reason cannot be seen on the blot presented in
FIG. 4.
[0252] The amounts of Nef expressed from the plasmids super6,
super6wt, VI and VIwt (lanes 1-4 in FIG. 4A) are quite similar
(FIG. 4, panel A, lanes 1 to 4). Much less protein is produced from
plasmid II (lane 5). The expression levels of the Nef protein are
higher from vectors containing wtE2 (cf. lane 1 compared with lane
2 and lane 3 compared with lane 4). This is in accordance with the
expression levels of E2 and E2d192-311+4GA proteins from these
plasmids (FIG. 4, panel B).
6.2. Example 2
Cloning and Analysis of the Expression Properties of Plasmids in
Series product1 and NNV
[0253] To increase the copy number of the vectors super 6 and
super6wt in Escherichia coli further modifications were made in
these vectors. The Tn903 kanamycin resistance gene, pMB1 replicon
and ten E2 binding sites were removed by HindIII/NheI digestion
followed by replacing with the Hind III/NheI fragment from
retroviral vector pBabe Neo [Morgenstern, J. P. and Land, H.,
Nucleic Acids Research 18 (1990) 3587-3596]. This fragment contains
a modified pMB1 replicon and the Tn5 kanamycin resistance gene that
allow relaxed high copy-number replication of the plasmids in
bacteria. The new plasmids were named as the product1 (FIG. 5), and
product1wt respectively. An unsuccessful attempt to reinsert the
ten E2 binding sites back into the blunted NheI site upstream of
the CMV promoter of the product1 resulted in vector New Vector NNV,
respectively, with only two binding sites integrated in the
plasmid.
[0254] Additional ten E2 binding sites were inserted from plasmid
pUC1910BS into the New Vector in just downstream the E2 expression
cassette. These new vectors were named NNV-1 and NNV-2 (FIG. 6A).
For replacing the E2d192-311+4GA with wt E2 (with deleted E3 and E4
ORFs), the E2d192-311+4GA coding sequence containing Bsp120I
fragment was replaced with wtE2 containing an analogous Bsp120I
fragment from the super6wt. Generated plasmids were named NNV-1wt
and NNV-2wt (FIG. 6B), respectively. The numbers 1 or 2 in vectors
of the NNV series mark the orientation of the 10 E2 binding sites
region relative to the E2 expression cassette.
[0255] The expression properties of the Nef protein from the NNV
plasmids, i.e. NNV-1, NNV-2, NNVwt and NNV-2wt, after the
transfection of Jurkat cells by electroporation at a concentration
of 1 g of the plasmid were analyzed and compared with the
expression properties of the Nef proteins from super6 and super6 wt
by Western blotting essentially as described in Example 1. The
amounts of super6 and super6wt used for the transfection were 0.95
and 1 g, respectively. The results are shown in FIG. 7.
[0256] NNV-1 and NNV-2 vectors have expression potential similar to
plasmid super6 as evident from the comparison of lanes 1 and 2 on
FIG. 8 with lane 5. The same applies to vectors NNV-1wt, NNV-2wt
and super6wt (compare lanes 3 and 4 with lane 6 on FIG. 7). In
accordance with the previous results the plasmids expressing wt E2
produce more Nef protein than E2d192-311+4GA vectors do (compare
lane 1 with lane 3 and lane 2 with lane 4 in FIG. 7). In view of
this and since the Nef expression from NNV-2wt was slightly higher
than that from NNV-1wt, vector NNV-2wt was selected for further
tests.
6.3 Example 3
Analysis of the expression properties of NNV-2wt
[0257] To analyse the expression properties of NNV-2wt, four
different cell lines, i.e. the Jurkat (human T-cell lymphoblasts),
P815 (mouse mastocytoma cells), CHO (Chinese Hamster Ovary cells)
lines and RD (human embryo rhabdomyosarcoma cells), were
transfected by electroporation and analyzed for their expression of
Nef of and E2. To reveal the transcription activation and
maintenance properties mediated by E2 protein and E2 oligomerized
binding sites product1wt, which lacks the E2 binding sites (FIG.
5), was used as a control. An additional control plasmid was
plasmid NNV-2wtFS, which differs from NNV-2wt by containing a
frameshift introduced into E2 coding sequence, whereby it does not
express functional E2 protein.
[0258] Each cell line was transfected with different amounts of the
vector DNA by electroporation essentially as described in Example
1. Time-points were taken approximately two and five days after
transfection. The results of analyses are presented in FIGS. 8 to
10.
[0259] The Jurkat cells were transfected with 0.5 .mu.g or 2 .mu.g
of the NNV-2wt (lanes 1, 2, 8, and 9 in FIG. 8) and equal amounts
of the plasmids NNV-2wtFS (lanes 3, 4, 10, and 11 in FIG. 8) and
product1wt (lanes 5, 6, 12, and 13 in FIG. 8) or carrier only
(lanes 7 and 14 in FIG. 8). Time-points were taken 44 hours (lanes
1-7) and 114 hours (lanes 8-14) after transfection: The expression
of the Nef and E2 proteins was analyzed by Western blotting
essentially as described in Example 1.
[0260] The P815 cells were transfected with 0.5 .mu.g or 2 .mu.g of
the NNV-2wt (lanes 1, 2, 8, and 9 in FIG. 9) and equal amounts of
the plasmids NNV-2wtFS (lanes 3, 4, 10, and 11 in FIG. 9) and
product1wt (lanes 5, 6, 12, and 13 in FIG. 9) or carrier only
(lanes 7 and 14 in FIG. 9). Time-points were taken 45 hours (lanes
1-7) and 119 hours (lanes 8-14) after transfection: The expression
of the Nef proteins was analyzed by Western blotting essentially as
described in Example 1. The blot with anti-E2 antibodies 119 h
post-transfection is not shown, because no special signal could be
detected. Generally, the expression level of the Nef protein
correlated with the expression level of E2 protein in these cells,
which confirms the fact that the function of the E2 protein is to
activate the transcription and to help the plasmid to be maintained
for a longer time in the proliferating cells.
[0261] The CHO cells were transfected with 0.5 .mu.g or 2 .mu.g of
the NNV-2wt (lanes 1, 2, 8, and 9 in FIG. 10) and equal amounts of
the plasmids NNV-2wtFS (lanes 3, 4, 10, and 11 in FIG. 10) and
product1wt (lanes 5, 6, 12, and 13 in FIG. 10) or carrier only
(lanes 7 and 14 in FIG. 10). Time-points were taken 48 hours (lanes
1-7) and 114 hours (lanes 8-14) after transfection. The expression
of the Nef and E2 proteins was analyzed by Western blotting
essentially as described in Example 1.
[0262] The RD cells were transfected with 0.5 .mu.g or 2 .mu.g of
the NNV-2wt (lanes 1, 2, 8, and 9 in FIG. 11) and equal amounts of
the plasmids NNV-2wtFS (lanes 3, 4, 10, and 11 in FIG. 11) and
product1wt (lanes 5, 6, 12, and 13 in FIG. 11) or carrier only
(lanes 7 and 14 in FIG. 11) Time-points were taken 39 hours (lanes
1-7) and 110 hours (lanes 8-14) after transfection. The expression
of the Nef protein was analyzed by Western blotting essentially as
described in Example 1.
[0263] In all four cell lines the expression level of the Nef
protein, taken at earlier time points (lanes 1-7 in FIGS. 8-11) and
at later time points (lanes 8-14 in FIGS. 8-11) hours, from the
NNV-2wt was higher than from control vectors. The superiority of
the NNV-2wt was more obvious at later time-points as evident from
the comparison of lane 8 with lanes 10 and 12 in FIG. 8, and also
from the comparison of lane 9 with lanes 11 and 13 in FIGS. 8, 9
and 10.
[0264] The expression pattern of RNA from these plasmids was also
analyzed using the Northern analysis [Alwine, J. C, et al., Proc
Natl Acad Sci USA 74 (1977) 5350-5354] for the NNV-2wt vector. For
this purpose, Jurkat and CHO cells were transfected with 2 .mu.g of
the NNV-2wt. For the transfection of P815 cells 10 .mu.g of NNV-2wt
were used. The transfections were made essentially as described in
Example 1. Forty-eight hours post-transfection total RNA was
extracted using RNAeasy kit (Qiagen) and samples containing 21
.mu.g (P815), 15 .mu.g (CHO) or 10 .mu.g (Jurkat) of the RNA were
analysed by electrophoresis under the denaturing conditions (1.3%
agarose gel containing 20 mM MOPS pH 7.0; 2 mM NaOAc; 1 mM EDTA pH
8.0; 2.2M formaldehyde). The running buffer contained the same
components except formaldehyde. The samples were loaded in a buffer
containing formamide and formaldehyde. After the electrophoresis
the separated RNAs were blotted onto the HybondN+ membrane
(Amersham Pharmacia Biotech, United Kingdom) and hybridization with
a radio-labeled nef coding sequence, E2 coding sequence or whole
vector probes was carried out. The RNA from cells transfected with
the carrier was used as a control. The results of the Northern blot
analyses are shown in FIG. 12.
[0265] The results indicate that no other RNA species than
complementary mRNAs for E2 and nef are expressed from the vector,
since no additional signals can be detected with the whole vector
probe compared with nef and E2 specific hybridizations (compare
lanes 1-12 with lanes 13-18 in FIG. 12).
6.4 Example 4
Analysis of the Attachment of the NNV-2wt to Mitotic
Chromosomes
[0266] The attachment of the NNV-2wt to mitotic chromosomes in CHO
cells was analyzed by fluoresence in situ hybridisation (FISH)
[Tucker J. D., et al., In: J. E. Celis (ed.), Cell Biology: A
Laboratory Handbook, vol 2, p. 450-458. Academic Press, Inc. New
York, N.Y. 1994.].
[0267] Thirty-six hours post-transfection the CHO cells by
electroporation with 1 .mu.g of NNV-2wt or with equimolar amounts
of the control plasmids NNV-2wtFS and product1wt (performed
essentially as described in Example 1) the cultures were treated
with colchicin (Gibco) for arresting the cells in metaphase of the
mitosis. Briefly, cells were exposed to colchicine added to medium
at final concentration of 0.1 .mu.g/ml for 1-4 h to block the cell
cycle at mitosis. Blocked cells were harvested by a trypsin
treatment and suspended in a 0.075M KCl solution, incubated at room
temperature for 15 min, and fixed in ice-cold methanol-glacial
acetic acid (3:1, vol/vol). The spread-out chromosomes at metaphase
and nuclei at interphase for fluorescence in situ hybridization
analyses were prepared by dropping the cell suspension on wet
slides. Several slides from one culture were prepared.
[0268] Hybridization probes were generated by nick-translation,
using biotin-16-dUTP as a label and plasmid Product1wt as template.
A typical nick-translation reaction mixture contained a
nick-translation buffer, unlabeled dNTPs, biotin-16-dUTP, and E.
coli DNA polymerase.
[0269] Chromosome preparations were denatured at 70.degree. C. in
70% formamide (pH 7.0-7.3) for 5 min, then immediately dehydrated
in a series of washes (70%, 80%, and 96% ice-cold ethanol washes
for 3 min each), and air-dried. The hybridization mixture (18 .mu.l
per slide) was composed of 50% formamide in 2.times.SSC
(1.times.SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 10%
dextran sulfate, 150 ng of biotinylated plasmid probe DNA and 10
.mu.g of herring sperm carrier DNA. After 5 min of denaturation at
70.degree. C., probe DNA was applied to each slide, sealed under a
coverslip, and hybridized for overnight at 37.degree. C. in a moist
chamber. The slides were washed with three changes of 2.times.SSC,
nd 2.times.SSC containing 0.1% IGEPAL CA-630 (Sigma Chemical Co.)
at 45.degree. C. Prior to the immunofluorescence detection, slides
were preincubated for 5 min in PNM a buffer [PN buffer (25.2 g
Na.sub.2HPO.sub.4.7H.sub.2O, 083 g NaH.sub.2PO.sub.4._.quadrature.
H.sub.2O and 0.6 ml of IGEPAL CA-630 in 1 .mu.liter of H.sub.2O]
with 5% nonfat dried milk and 0.02% sodium azide).
[0270] Subsequently, the probe was detected with fluorescein
isothiocyanate (FITC)-conjugated extravidin. The signal was
amplified with biotinylated antiavidin antibody and a second round
of extravidin-FITC treatment. Between each of the steps, the slides
were washed in PN buffer containing 0.05% IGEPAL CA-630 at room
temperature for 2.times.5 min. Chromosomes were counterstained with
propidium iodide and mounted in p-phenylenediamine antifade
mounting medium.
[0271] Slides were analyzed with a Olympus VANOX-S fluorescence
microscope equipped with appropriate filter set.
[0272] The results are shown in Table 1.
TABLE-US-00001 TABLE 1 Chromosomal attachment of the NNV-2wt.
Metaphases with episomal signal Analyzed Culture on chromosomes
metaphases % 0.5 .mu.g 11 158 7 NNV-2wt 0.5 .mu.g 0 100 0 NNV-2wtFS
0.48 .mu.g 0 100 0 product1wt carrier 0 100 0
[0273] The data indicate clearly that the E2 protein and its
binding sites are needed for the chromosomal attachment because
only the NNV-2wt but not two other vectors have this ability.
6.5 Example 5
Stability of NNV-2wt During Propagation in Bacterial Cells
[0274] The stability of NNV-2wt during propagation in bacterial
cells was tested. The plasmid NNV-2wt was mixed with competent
Escherichia coli cells of the DH5alpha strain [prepared as
described in Inoue, H., et al., Gene 96 (1990) 23-28] and incubated
on ice for 30 minutes. Subsequently, the cell suspension was
subjected to a heat-shock for 3 minutes at 37.degree. C. followed
by a rapid cooling on ice. One milliliter of LB medium was added to
the sample and the mixture was incubated for 45 minutes at
37.degree. C. with vigorous shaking. Finally, a portion of the
cells was plated onto dishes containing LB medium with 50 .mu.g/ml
of kanamycin. On the next day, the cells from a single colony were
transferred onto the new dishes containing the same medium. This
procedure was repeated until four generations of bacteria had been
grown, and the plasmid DNA from the colonies of each generation was
analyzed.
[0275] One colony from each generation was used for an inoculation
of 2 ml LB medium containing 50 .mu.g/ml of kanamycin followed by
an overnight incubation at 37.degree. C. with vigorous shaking. The
cells were harvested and the plasmid DNA was extracted from the
cell using classical lysis by boiling. [Sambrook, S., et al.,
Molecular Cloning A Laboratory Manual. Second ed. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. The samples
were digested with restriction endonuclease XbaI (Fermentas,
Lithuania) and analyzed by agarose gel electrophoresis in
comparison with the original DNA used for transformation. The
results are shown in FIG. 13.
[0276] As can be seen in FIG. 13, the vector is stable during the
passage in Escherichia coli cells: no colonies with re-arrangements
were observed when compared with the DNA used for transformation
(lane 9).
6.6 Example 6
Stability of NNV-2wt in Eukaryotic Cells
[0277] The stability of the plasmid NNV-2wt as a non-replicating
episomal element was also analyzed in eukaryotic cells. For this
purpose the CHO and Jurkat cells were transfected with 2 .mu.g of
NNV-2wt. Total DNAs of the cells were extracted at 24, 72 or 96
hours post-transfection. Briefly, the cells were lyzed in 20 mM
Tris-HCl pH 8.0; 10 mM EDTA pH 8.0; 100 mM NaCl; 0.2% SDS; in
presence of 200 .mu.g/ml of proteinase K (Fermentas, Lithuania).
Next, the samples were extracted sequentially with phenol and with
chloroform and precipitated with ethanol. The nucleic acids were
resuspended in 10 mM Tris-HCl pH 8.0; 1 mM EDTA pH 8.0; 20 .mu.g/ml
of RNase A (Fermentas, Lithuania) and incubated for 1 hour at
37.degree. C. Finally the DNA was re-precipitated with ammonium
acetate and ethanol, washed with 70% ethanol and resuspended in 10
mM Tris-HCl pH 8.0; 1 mM EDTA pH 8.0. The samples were digested
with different restriction endonucleases: with Eco81 (Fermentas,
Lithuania) that has two recognition sites on the plasmid, with
HindIII (Fermentas, Lithuania) that does not cut the NNV-2wt DNA
and with DpnI (New England Biolabs, USA) that digest only DNA
synthesized in Escherichia coli cells. Restricted DNAs were
separated on TAE agarose electrophoresis and analyzed by Southern
blotting [Southern, E. M. J. Mol. Biol. 98 (1975) 503-517] with a
vector specific radiolabeled probe. The results are illustrated on
FIG. 14. As obvious from comparison of the fragment sizes of Eco81I
digestion (lanes 1, 2 and 7 in FIG. 14) with respective marker
lanes no arrangements of the vector were detected in the assay.
Neither were signals observed at a position different from the
marker lanes in cases of the Hind III (lanes 3, 4 and 8 in FIG. 14)
or HindIII/DpnI (lanes 5, 6 and 9 in FIG. 14) digestion indicating
that integration and/or replication events were not observed.
6.7 Example 7
Analysis of Replication of the NNV-2wt in the Presence of Human
Papillomaviral Replication Factors
[0278] It has been demonstrated previously that papillomaviral
proteins are able to initiate the replication of heterologous
ori-containing plasmids from many other human and animal
papillomaviruses [Chiang, C. M., et al., Proc Natl Acad Sci USA 89
(1992) 5799-5803]. Although NNV-2wt does not contain an intact
viral origin of replication, it was tested how the replication is
initiated in the presence of human papillomavirus type 1 E1 and E2
proteins. CHO cells were transfected with one microgram of either
plasmids NNV-2wt, NNV-2wtFS or product 1 alone or with 4.5 .mu.g of
the HPV-11 E1 expression vector pMT/E1 HPV11 or with same amount of
pMT/E1 HPV11 and 4.5 .mu.g HPV-11 E2 protein expression vector
pMT/E2 HPV 11 as indicated on the top of the FIG. 15. Transfections
were done essentially as described in Example 1. E1 and E2
expression vectors are described previously (Chiang, C. M. et al.,
supra). An equimolar amount of HPV-11 replication origin containing
plasmid HPV11ORI was transfected with the same expression vectors
as a positive control.
[0279] Low-molecular weight DNA was extracted by modified Hirt
lysis [Ustav, et al., EMBO J 2 (1991) 449-457] at 67 hours
post-transfection. Briefly, the cells washed with PBS were lyzed on
ice at 5 minutes by adding alkaline lysis solutions I (50 mM
glucose; 25 mM Tris-HCl, pH 8.0; 10 mM EDTA, pH 8.0) and II (0.2M
NaOH; 1% SDS) in a ratio of 1:2 onto the dishes. The lysates were
neutralized by 0.5 vol solution III (a mixture of potassium acetate
and acetic acid, 3M with respect to potassium and 5M with respect
to acetate). After centrifugation the supernatant was precipitated
with isopropanol, resuspended and incubated at 55.degree. C. in 20
mM Tris-HCl pH 8.0; 10 mM EDTA pH 8.0; 100 mM NaCl; 0.2% SDS; in
presence of 200 .mu.g/ml of proteinase K (Fermentas, Lithuania).
Next, the samples were extracted sequentially with phenol and with
chloroform followed by precipitation with ethanol. The nucleic
acids were resuspended in 10 mM Tris-HCl pH 8.0; 1 mM EDTA pH 8.0;
20 .mu.g/ml RNase A (Fermentas, Lithuania) and incubated for 30 min
at 65.degree. C. The samples were digested with linearizing
endonuclease (NdeI; Fermentas, Lithuania) in case of the vectors or
HindIII (Fermentas, Lithuania) in case of the HPV11ORI) and DpnI
(New England Biolabs, USA) (breaks non-replicated DNA), followed by
Southern blotting performed essentially as described earlier using
a vector specific radiolabeled probe. For positive control of
hybridization appropriate markers of the linearized vectors and
HPV11ORI were used (lanes marked as M on FIG. 15). As seen from the
results set forth in FIG. 15, no replication signal was detected in
case of any vector plasmids.
6.8 Example 8
Analysis of the E2 and its Binding Sites Dependent Segregation
Function of the Vectors in Dividing Cells
[0280] As has been described previously, bovine papillomavirus type
1 E2 protein in trans and its multiple binding sites in cis are
both necessary and sufficient for the chromatin attachment of the
episomal genetic elements. The phenomenon is suggested to provide a
mechanism for partitioning viral genome during viral infection in
the dividing cells [Ilves, I., et al., J. Virol. 73 (1999)
4404-4412]. Because both functional elements are also included into
our vector system, the aim of this study was analyze the importance
of the E2 protein and oligomerized binding sites for maintenance of
the transcriptionally active vector element in population of
dividing cells.
[0281] For this purpose the Nef coding sequence of the vectors
NNV-2wt and super6wt was replaced with coding sequence of the
destabilized form of green fluorescent protein (d1EGFP) derived
from vector pd1EGFP-N1 (Clontech Laboratories). Because the
half-life of this protein is as short as 1 hour, it does not
accumulate in the cells and the d1EGFP expression detected by flow
cytometer correlates with the presence of transcriptionally active
vector in these cells.
[0282] From NNV-2wt the nef coding sequence was removed and
SmaI-NotI fragment from the pd1EGFP-N1 was inserted instead of it.
New vector was named as 2wtd1EGFP (FIG. 16). Similar replacement
was made in case of super6 wt for generation gf10bse2 (FIG. 17),
respectively. The recognition sequence for restrictional
endonuclease SpeI was introduced into the EcoRI site in the
super6wt just upstream the ten E2BS. The vector gf10bse2 is derived
from this plasmid by replacing the Nef coding sequence containing
NdeI-Bst1107I fragment with d1EGFP coding sequence containing
fragment from 2wtd1EGFP, cut out with same enzymes.
[0283] Negative control plasmids lacking either functional E2
coding sequence or its binding sites were also made: The frameshift
was introduced into the E2 coding sequence in context of the
2wtd1EGFP by replacing E2 coding sequence containing
Bsp120I-Bsp120I with similar fragment from plasmid NNV-2wtFS. The
resulting vector was named as 2wtd1EGFPFS (FIG. 18). For the
construction the control plasmid NNVd1EGFP (FIG. 19) the whole E2
expression cartridge (as well bacterial replicon) from the
2wtd1EGFP was removed by Bst1107 and NheI digestion. The replicon
was reconstituted from plasmid product1 as HindIII (filled in)-NheI
fragment.
[0284] Jurkat cells were transfected by electroporation with 1
.mu.g of the vector 2wtd1EGFP or with equimolar amounts of the
plasmids 2wtd1EGFPFS, NNVd1EGFP, gf10bse2 or with carrier DNA only
as described in Example 1. At different time-points
post-transfection the equal aliquots of the cell suspension were
collected for analysis and the samples were diluted thereafter with
the fresh medium. At every time-point total number of the cells as
well the number of the d1EGFP expressing cells were counted by flow
cytometer (Becton-Dickinson FACSCalibur System). With these data,
the percentages of d1EGFP expressing cells, alterations of total
numbers of cells and numbers of d1EGFP expressing cells in samples
were calculated using the carrier-only transfected cells as a
negative control for background fluorescence. The calculations of
cell numbers were done in consideration of the dilutions made.
Finally, the error values were calculated based on technical data
of the cytometer about fluctuations of speed of the flow.
[0285] Two independent experiments were done. First, the
maintenance of d1EGFP expressed from the plasmids 2wtd1EGFP,
2wtd1EGFPFS and NNVd1EGFP were analyzed during the eight days
post-transfection. In the second experiment the maintenance of
d1EGFP expressed from the plasmids 2wtd1EGFP, 2wtd1EGFPFS and
gf10bse2 were analyzed during the thirteen days
post-transfection.
[0286] As is obvious from FIGS. 20 and 21, there was no difference
of the growth speed of the cells transfected with any vector or
carrier only. It means that differences in the d1EGFP expression
maintenance are not caused by influences of transfected vectors
themselves on the dividing of the cells. Also, during the assay the
logarithmic growth of the cells were detected, except the period
until second time-point in the experiment represented in FIG. 20.
This lag period of the growth is probably caused by the
electroporation shock of the cells, because the first time-point
was taken already 19 hours after the transfection.
[0287] As illustrated in FIGS. 22 and 23, the percentages of green
fluorescent protein expressing cells decrease in all populations
transfected with either plasmid, because the vectors do not
replicate in the cells. However, as is seen on the charts, the
fraction of positive cells declines more rapidly in cases of
control vectors, if compared with the 2wtd1EGFP or gf10bse2. If
compared with each other, the gf10bse2 have clear benefit to
2wtd1EGFP (FIG. 23.). There is also a notable difference of
maintenance between control plasmids 2wtFSd1EGFP and NNVd1EGFP
(FIG. 22).
[0288] These differences between the vectors become much more
obvious, if the data are represented as alterations of the numbers
of the d1EGFP expressing cells in the populations (FIGS. 24 and
25). The numbers of the positive cells in cases of the control
plasmids are not notable changed during the assay. In contrast, in
case of the 2wtd1EGFP the number of d1EGFP expressing cells
increases during the first week after the transfection becoming
approximately five to ten times higher than in control samples
(FIG. 24). After this time-point the number start to decrease (FIG.
25). The difference of maintenance is strongest in the case of the
gf10bse2 vector. The number of positive cells increases
continuously during the analyses period. After two weeks it is 6
times higher than in the sample transfected with 2wtd1EGFP and 45
times higher than in the population transfected with frameshift
mutant (FIG. 25).
[0289] The data demonstrate clearly that the vector system of the
present invention has active mechanism of segregation based on a
nuclear-anchoring protein, i.e. bovine papillomavirus type 1 E2
protein and its binding sites that promotes its maintenance in a
population of proliferating cells as a transcriptionally active
element.
6.9 Example 9
Cloning of the AIRE Gene into super6wt and Expression in an
Epithelial Cell Line
[0290] The AIRE gene coding for the AIRE protein (AIRE=autoimmune
regulator) is mutated in an autosomally heredited syndrome APECED
(Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy).
AIRE is expressed in rare epithelial cells in the medulla of thymus
and in the dendritic cells in peripheral blood and in peripheral
lymphoid organs. APECED could thus be treated by transferring the
non-mutated AIRE gene ex vivo to peripheral blood dendritic cells,
followed by the introduction of the corrected dendritic cells back
to the patient. To test this possibility human AIRE gene and the
homologous murine AIRE gene were transferred to COS-1 cells.
[0291] For cloning of the AIRE gene into Super6wt a
maxi-preparation of the vector was prepared. First a transfection
with Super6wt was done to TOP10-cells (chemically competent
Escherichia coli by Invitrogen) according to manufacturer's
protocol. Briefly, the cells were incubated on ice for 30 minutes,
after which a heat shock was performed in a water bath at
+42.degree. C. for 30 seconds. The cells were then transferred
directly on ice for 2 minutes and grown in 250 .mu.l of SOC-medium
(2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl2, 10 mM MgSO4, 20 mM glucose) at +37.degree. C. with shaking
for 1 hour.
[0292] Plating was done on LB-plates using kanamycin (50 .mu.g/ml)
for selection. For maxiprep, colonies were transferred to 150 ml of
LB-solution containing kanamycin (50 .mu.g/ml) and grown overnight
at +37.degree. C. with shaking. Preparation of maxiprep was done
using Qiagen's Plasmid Maxi Kit according to manufacturer's
protocol.
[0293] A digestion with BamHI and SalI restriction enzymes was used
to check the vector. The reaction mixture contained 500 ng of
Super6w, 5 U of BamHI, 5 U of SalI, 2 .mu.l of 2.times.TANGO buffer
(both the restriction enzymes and buffer from Fermentas) and
sterile water in total volume of 10 .mu.l. The digestion was
carried out at +37.degree. C. for one hour.
[0294] The digested vector was checked with 1% agarose gel
containing ethidium bromide 1 .mu.g/ml in 1.times.TAE-buffer.
[0295] For cloning of the PCR amplified AIRE gene and Aire
fragments into the Super6wt, 4 .mu.g of Super6wt was digested with
10 U NotI restriction enzyme (MBI Fermentas, in 2 .mu.l enzyme
buffer and sterile water added to a final volume of 20 .mu.l. The
digestion was carried at +37.degree. C. for 1.5 hours, after which
1 U of ZIP-enzyme (alkaline phosphatase) was added to the reaction
mixture and incubated further for 30 minutes. The ZIP-enzyme
treatment was done to facilitate the insertion of the AIRE gene
into the vector by preventing the self-ligation of the vector back
to a circular mode. After the digestion the vector was purified
using GFXTM PCR DNA and Gel Band Purification Kit (Amersham
Pharmacia Biotech) and dissolved in to a concentration of 0.2
micrograms/microliter.
[0296] Human and mouse AIRE-gene PCR-products were also digested
with NotI restriction enzyme. To the digestion, 26 .mu.l of PCR
product, 3 .mu.l of an appropriate enzyme buffer and 10 U of NotI
restriction enzyme (the buffer and enzyme from MBI Fermentas) was
used. The digestion was carried out at +37.degree. C. for 2 hours,
after which digested PCR-products were purified and dissolved in
sterile water to a volume of 10 .mu.l.
[0297] The PCR amplified and digested human and mouse AIRE genes
were ligated to Super6wt by a T4 DNA ligase (MBI Fermentas). The
digested insert DNA was taken (a total volume of 10 .mu.l), 1.5
.mu.l of ligase buffer (MBI Fermentas), 5 U of T4 DNA ligase and
sterile water was added to a final concentration of 15 .mu.l. The
ligation was carried out at +17.degree. C. overnight.
[0298] After the ligation 10 .mu.l of ligation reaction mixture was
taken for transfection into TOP10 cells according to manufacturer's
protocol. The cells were incubated on ice for 30 minutes, after
which a heat shock was performed in a water bath at +42.degree. C.
for 30 seconds. The cells were then transferred directly on ice for
2 minutes and grown in 250 .mu.l of SOC-medium (2% tryptone, 0.5%
yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM
MgSO.sub.4, 20 mM glucose) at +37.degree. C. with shaking for 1
hour.
[0299] The transfected bacterial cells were plated onto
LB-kanamycin plates and colonies were picked on the following day
to 2 ml of LB-medium (1% tryptone, 0.5% yeast extract, 170 mM NaCl)
with kanamycin and grown overnight at +37.degree. C.
[0300] Miniprep DNA preparations from selected colonies were
purified using Qiagen's Plasmid Midi Kit and dissolved to a volume
of 50 .mu.l of sterile water. The presence and size of the insert
was checked with NotI and BamHI digestion. 10 .mu.l of miniprep DNA
was taken for digestion, 5 U of NotI and 5 U of BamHI enzymes, 2 ml
of R+enzyme buffer and sterile water was added to a final volume of
20 .mu.l. The digestion was carried out at +37.degree. C. for 1
hour.
[0301] The orientation of the insert was analysed with BamHI
restriction enzyme. Ten .mu.l of minprep DNA was taken, 5 U of
BamHI, 2 .mu.l of BamHI buffer (MBI Fermentas) and sterile water
was added to a final volume of 20 .mu.l. The digestion was carried
out for 1 hour at +37.degree. C. and the products were checked on a
1% agarose gel with EtBr in 1.times.TAE.
[0302] On the basis on these results, a plasmid containing a mouse
AIRE-gene and a plasmid containing a human AIRE-gene were picked
and maxipreps were prepared. Briefly, 0.5 ml of E. coli cell
suspension containing the plasmid of interest or a miniprep culture
was added to a 150 ml LB-medium containing kanamycin (50 .mu.g/ml)
and grown overnight at +37.degree. C. Maxiprep DNAs were prepared
using Qiagen's Plasmid Maxi Kit.
[0303] The plasmid containing the mouse AIRE-gene was designated as
pS6 wtmAIRE and plasmid containing the human AIRE-gene as pS6
wthAIRE.
[0304] The generated vectors were sequenced for approximately 500
bp from both ends to verify the orientation and correctedness of
the insert. The sequencing was performed using the dideoxy method
with PE Biosystem's Big Dye Terminator RR-mix, which contains the
four different terminating dideoxynucletide triphosphates labeled
with different fluorescent labels.
[0305] Plasmids containing the AIRE gene and AIRE gene fragments
were inserted into selected cell lines to check the expression of
the protein with Western blot after the transfection.
[0306] Cos-1 cells were harvested with trypsin-EDTA (Bio Whittaker
Europe) solution and suspended 10.times.106 cells/ml into
Dulbecco's MEM (Life Technologies) medium and 250 .mu.l of cell
suspension was taken for transfection. The transfection of Cos-1
cells was performed using electroporation with 2.5.times.106 cells,
50 .mu.g of salmon sperm DNA as a carrier and 5 .mu.g of
appropriate vector. The transfections were made with pS6 wthAiRE,
pS6 wtmAIRE, Super6wt, pCAIRE, psiAIRE and pCAIRE S1-4. pCAIRE and
psiAIRE are positive human AIRE controls, pCAIRE S1-4 is a positive
mouse AIRE control and Super6wt is a negative control.
[0307] The electroporation was done using Biorad's Gene Pulser with
capacitance 960 .mu.Fd, 240 V and 1 pulse. After the pulse the
cells were kept at room temperature for 10 minutes and 400 .mu.l of
medium was added. The cells were transferred to 5 ml of medium and
centrifuged for 5 minutes with 1000 rpm. Cells were plated and
grown for 3 days at +37.degree. C., 5% CO2.
[0308] The cells were harvested with trypsin-EDTA and centrifuged.
Then Cells were then washed once with 500 ml of 1.times.PBS (0.14
mM NaCl, 2.7 mM KCl, 7.9 .mu.M Na2HPO4, 1.5 .mu.M KH2PO4). 50 .mu.l
of PBS and 100 .mu.l of SDS loading buffer (5% mercaptoethanol, 16
.mu.M Bromphenolblue, 20 .mu.M Xylene Cyanol, 1.6 mM Ficoll 400)
was added and cells were heated at +95.degree. C. for 10
minutes.
[0309] For the western blot analysis SDS-PAGE was prepared with 10%
separation and 5% stacking gels in a SDS running buffer (25 mM
Tris, 250 mM glysin, 0.1% SDS). Cell samples and biotinylated
molecular weigh marker were loaded on the gel and electrophoresis
was performed with 150 V for 1 h 50 minutes. The transfer of
proteins to a nitrocellulose membrane was performed at 100 V for
1.5 hours at room temperature with a cooler in transfer buffer.
[0310] The membrane was blocked in 5% milk in TBS (0.05 M Tris-Cl,
0.15 M NaCl, pH 7.5) for 30 minutes at room temperature. A primary
antibody mixture, anti-AIRE6.1 (human) and anti-AIRE8.1 (mouse)
antibodies at a dilution of 1:100 in 5% milk in TBS, was added onto
membrane and incubated overnight at +4.degree. C. The membrane was
washed two times with 0.1% Tween in TBS for 5 minutes and once with
TBS for 5 minutes. The secondary antibody, biotinylated anti-mouse
IgG at a dilution of 1:500 in 5% milk in TBS was incubated for 1
hour at room temperature. The membrane was washed and horseradish
peroxidase avidin D at a dilution of 1:1000 in 5% milk in TBS was
added. The membrane was incubated at room temperature for 1 hour
and washed. A substrate for the peroxidase was prepared of 5 ml
chloronaphtol, 20 ml TBS and 10 .mu.l hydrogen peroxide and added
onto membrane. After the development of the color the membrane was
washed with TBS and dried.
[0311] The antibody detecting with human AIRE (anti-AIRE6.1)
detected the AIRE protein expression in the preparates transfected
with pS6 wthAIRE, pCAIRE and psiAIRE. The antibody detecting murine
AIRE detected likewise the murine AIRE in cells transfected with
pS6 wtmAIRE and pCAIRE S1-4. The negative control (Super6wt) showed
no AIRE/aire proteins.
6.10 Example 10
Detection of Cellular and Humoral Immune Response Toward HIV.1 Nef
in Mice Immunized with the NNV-Nef Construct DNA Immunizations
[0312] To further study the induction of humoral immunity by the
vectors of the inventions, 5-8 weeks old both male and female
BALB/c (H-2d) mice were used. For the DNA immunizations, the mice
were anaesthetized with 1.2 mg of pentobarbital (i.p) and DNA was
inoculated on shaved abdominal skin using plasmid DNA coated gold
particles. The inoculation was made with Helios Gene Gun (BioRad)
using the pressure of 300 psi. The gold particles were 1 .mu.m in
diameter, .about.1 .mu.g of DNA/cartridge. The mice were immunized
twice (on day 0 and day 7) with a total amount of DNA of 0.4 or 8
.mu.g/mouse. The control mice were immunized with 8 .mu.g of the
plain vector without the nef-gene, i.e. NNV-deltanef.
[0313] A blood sample was taken from the tail of the mice two weeks
after the last immunization. The mice were sacrificed four weeks
after the last immunization and blood samples (100 .mu.l) were
collected to Eppendorf tubes containing 10 .mu.l of 0.5 M blotting
(++vs. +) and in ELISA (higher OD, more mice in higher-dose above
cut-off EDTA. The absolute number of leukocytes/ml of blood was
calculated from these samples for each mouse. The sera were
collected for antibody assays and stored at -20.degree. C. The
spleens were removed aseptically, weighted and then homogenized to
single cell suspensions for use in T, B and NK cell assays and
staining.
[0314] Detection of the Humoral Immunogenicity of the Vectors of
the Invention
[0315] For the detection of Nef-specific antibodies by Western
blotting, serum samples from mice immunized with the vector
constructs of the invention were diluted 1:100 to 5% milk in TBS
and applied on nitrocellulose strips made with recombinant HIV-1
Nef protein. For the preparation of the nitrocellulose strips, the
purified recombinant protein was boiled in a sample buffer
containing 1% SDS and 1% 2-mercaptoethanol, then run on a 10 or
12.5% polyacrylamide gel and subsequently transferred onto a 0.45
.mu.m nitrocellulose paper. The strips were first blocked with 2%
BSA in 5% defatted milk-TBS and thereafter incubated with diluted
sera (1:100) overnight. After incubation, unbound proteins were
removed by washing the strips three times with TBS-0.05% Tween-20
and twice with water. After washings, the strips were probed with a
1:500 dilution of biotinylated anti-mouse IgG (Vector Laboratories,
USA) for 2 hour. After further washings, horseradish
peroxidase-avidin in a dilution of 1:1000 (Vector Laboratories,
USA) was added for 1 h, the strips were washed again and the bound
antibodies were detected with a hydrogen peroxidase substrate,
4-chloro-1-naphtol (Sigma, USA).
[0316] The sera were also tested in ELISA to determine the exact
antibody titers induced by each construct. Nef antibody ELISA was
performed as previously described (Tahtinen et al., 2001). Briefly,
Nunc Maxi Sorp plates were coated with 50 ng of Nef (isolate HAN),
blocked with 2% BSA in phosphate buffered saline (PBS), and the
sera in a dilution of 1:100 to 1:25000 were added in duplicate
wells for an overnight incubation. After extensive washings, the
secondary antibody, peroxidase conjugated anti-mouse IgG or IgM
(DAKO), was added, and the plates were incubated for two hours and
then washed. Color intensity produced from the substrate
(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, ABTS, Sigma)
in a phosphate-citrate buffer was measured at 405 nm using a
Labsystems Multiscan Plus ELISA-plate reader. The optical density
cut-off value for positive antibody reactions was determined as
follows:
cut-off=OD(xl control mice sera)+3 SD.
[0317] Detection of the Cellular Immunogenicity of the Vectors of
the Invention
[0318] To analyze the capacity of the vectors of the invention to
induce cellular immunity, T-cell and B-cell assays as well as cell
surface staining were performed.
[0319] T cell proliferation assay. The spleen cells were suspended
to a final concentration of 1.times.106/ml RPMI-1640 (GibcoBRL)
supplemented with 10% FCS (GibcoBRL), 1% penicillin-streptomycin
(GibcoBRL) and 50 .mu.M betamercaptoethanol (Sigma). Cells were
incubated in microtitre plates at 200 .mu.l/well with media only or
with different stimuli. The final concentrations of stimuli were:
Con A 5 .mu.g/ml, HIV-Nef-protein at a concentration of 1 and 10
.mu.g/ml, and a negative control antigen HIV-gag at a concentration
of 1 and 10 .mu.g/ml. All reactions were made in quadruplicates. On
the sixth day of the incubation 100 .mu.l of supernatant from each
well was collected and stored at -80.degree. C. for cytokine
assays. Six hours before harvesting 1 .mu.Ci of 3H-thymidine
(Amersham Pharmacia Biotech) was added to each well. The cells were
harvested and radioactivity incorporated (cpm) was measured in a
scintillation counter. The stimulation indexes (SI) were calculated
as follows
SI=mean experimental cpm/mean media cpm.
[0320] Lymphocyte activation. T and B cell activation was detected
by double surface staining of fresh splenocytes with anti-CD3-FITC
plus anti-CD69-PE (early activation marker) and anti-CD19-FITC plus
anti-CD69-PE antibodies (all from Pharmingen). Stainings were
analyzed with flow cytometer (FACScan, Becton Dickinson).
[0321] CTL assays. Mouse splenocytes were co-cultured with fixed
antigen presenting cells (P-815 cells infected with MVA-HIV-nef or
control MVA-F6) for five days after which they were tested in a
standard 4 hour 51 chromium release assay [Hiserodt, J., et al., J
Immunol 135 (1995) 53-59; Lagranderie, M., et al., J Virol 71
(1997) 2303-2309) against MVA-HIV-nef infected or control target
cells. In CTL assays the specific lysis of 10% or more was
considered positive.
[0322] Cytokine assay. IFN-gamma and IL-10 were measured from
antigen-stimulated cell culture supernatants in order to analyze,
whether immunized mice develop a Th1 type or Th2 response. The
supernatants were collected from antigen-stimulated cells as
described above. Pro-inflammatory cytokines TNF-alfa and IL-10 were
measured in the sera of the immunized mice. All cytokines were
measured with commercial ELISA kits (Quantikine, R&D
Systems).
[0323] Spontaneous proliferation. Spontaneous splenocyte
proliferation was detected by 3H-thymidine uptake of the cells
cultured in the medium only for 6 days.
[0324] Anti-double strand (ds) DNA antibodies. dsDNA antibodies
were measured in the sera of immunized mice, positive control mice
(mrl/lpr, a generous gift from Dr. Gene Shearer, NIH, USA) and
normal mice. The antibodies were assayed with ELISA on
poly-L-Lysine bounded lambda phage dsDNA. The results are shown in
Tables 2 and 3.
[0325] Table 2 shows complete immunological results of the mice
immunized with HIV-Nef plasmid DNA. Although HIV-1 Nef recombinant
protein, which was used for in vitro T cell stimulation, induced
some non-HIV-specific proliferation of the cells in each immunized
group, there was a significant increase in the mean SI of mice
immunized with 0.4 .mu.g of the plasmid (mean SI=72.2) compared to
others. Furthermore, negative control protein HIV-gag did not
induce any T cell response. Only the T cells of the mice in the
group that had nef-specific proliferation also produced
nef-specific IFN-gamma. None of the immunized mice had cells
producing IL-10, which shows that the T cell response in the
immunized mice was of Th1 type and not of Th2 type. In contrast to
the T cell response, mice immunized with the higher concentration
of nef plasmid DNA (8 .mu.g) had a stronger B cell response
compared to mice immunized with 0.4 .mu.g: the humoral response in
mice immunized with the higher dose was detectable already three
weeks after the last immunization and the response detected was
stronger both in Western-). The antibodies detected belonged to
IgG-class, no IgM response was detected. None of the mice developed
E2 specific antibody.
[0326] The mice immunized with 0.4 .mu.g of HIV-nef plasmid DNA had
an increased number of leukocytes (6.38.times.106/ml) in the
peripheral blood compared to other groups of immunized mice and
normal mice (3.8.times.106/ml) (Table 3). The same mice had twice
as much activated T cells (21%, CD3+CD69+) compared to other mice
(9% and 10%). This finding is in correlation with the positive T
cell response to HIV-Nef (Table 2), since the mice with a positive
T cell response to Nef also had an increased number of activated T
cells in their spleens. The results of Table 3 also show that none
of the immunized mice developed anti-dsDNA anti-bodies as compared
to positive control sera (OD=1,208) indicating that there is no
adverse effect of the immunization.
TABLE-US-00002 TABLE 2 HIV-1 HIV-1 HIV-1 nef gag IFN-g IL-10 nef E2
Mice SI* SI Th1 Th2 Ab Ab NNV-Nef 8 1 6 1 - - ++ - 2 8 1 - - ++ - 3
13 2 - - ++ - 4 15 1 - - ++ - 5 7 1 - - ++ - Mean 9.8 1.2 NNV-NEF
0.4 1 24 1 + - + - 2 112 1 + - + - 3 83 1 + - + - 4 73 1 + - + - 5
69 1 + - + - Mean 72.2 1 NNV-.DELTA.Nef 8 1 6 1 - - - - 2 nt nt - -
- - 3 11 1 - - - - 4 23 2 - - - - 5 12 1 - - - - Mean 13 1.25 SI* =
stimulation index nt = not tested -', negative +', positive +'+',
strong positive
TABLE-US-00003 TABLE 3 CD3 CD3+CD69+ CD19 CD19+CD69+ anti-dsDNA % %
% % ab Mice WBC .times. 10.sup.6/ml spleen spleen spleen spleen
OD(1:10 dil) NM 1 0.355 2 0.255 3 0.231 Mean 0.280 NNV-Nef 8 1 5 nt
nt nt nt 0.387 2 4.3 50 4 11 3 0.457 3 4.9 57 4 15 4 0.514 4 4.3 55
6 15 4 0.367 5 5.1 54 5 7 0 0.478 mean 4.72 54 4.75 (9%) 12 2.75
0.441 NNV-Nef 0.4 1 3.9 nt nt nt nt 0.418 2 8 41 9 18 5 0.263 3 7.5
39 8 25 9 0.375 4 5 46 9 16 6 0.285 5 7.5 43 10 13 7 0.396 mean
6.38 42.25 9 (21%) 18 6.75 0.347 NNV-.DELTA.Nef 8 1 4.5 61 4 9 2
0.413 2 4.6 59 4 15 1 0.353 3 3.8 50 6 17 5 0.382 4 3.1 46 7 25 8
0.448 5 3.5 nt nt nt nt 0.501 mean 3.9 54 5.25 (10%) 16.5 4 0.419
Normal mouse mean WBC = 3.8 .times. 10.sup.6/ml nt, not tested
a-dsDNA positive control sera OD was 1.208 (1:10 dil)
6.11. Example 11
Safety and Immunogenicity of a Prototype HIV Vaccine GTU-nef in HIV
Infected Patients
[0327] Production of the NNV-2-Nef Vaccine (Check Whether NNV-2 or
NNVwt-2 was Used)
[0328] The investigational vaccine NNV-2-Nef was prepared according
to Example 2 with the Manufacturing License No. LLDnro 756/30/2000
(issued by the Finnish National Agency for Medicines on Dec. 21,
2000).
[0329] The manufacturing processes performed fulfilled the current
Good Manufacturing Practices (cGMP) requirements and provided
plasmid DNA preparations suitable for use in clinical phase I and
II studies. The manufacturing process consisted of four steps:
[0330] a) Establishment of Master Cell Banks and Working Cell
Banks
[0331] b) Fermentation
[0332] c) Purification
[0333] d) Aseptic filling of the vaccine
[0334] In detail, NNV-2-Nef was produced in E. coli bacteria. The
Master Cell Banks (MCBs) and Working Cell Banks (WCBs) containing
E. coli DH5 alpha T1 phage resistant cell strain were established
in accordance with the specific Standard Operating Procedure from
pure cultured and released Research Cell Banks.
[0335] a) Establishment of Master Cell Banks and Working Cell
Banks
[0336] The schematic procedure for establishing the cell bank
system is illustrated below:
[0337] Thaw of one vial of Research Cell Bank [E. coli DH5 alpha T1
phage resistant cell strain (Gibco RBL) transformed with the
NNV-2-Nef plasmid.
[0338] Inoculate of the culture on modified Luria Bertani medium
plate (containing 25 .mu.g/ml of kanamycin)
[0339] Incubate overnight (14-16 h) at 37.degree. C.
[0340] Select of a single colony from the plate and inoculation
into 50 ml of modified Luria Bertani medium (containing 25 .mu.g/ml
of kanamycin)
[0341] Incubate overnight (14-16 h) at 37.degree. C.
[0342] Measure optical density of the bacterial culture
(OD.sub.600=2.0-6.0)
[0343] Add glycerol to bacterial culture
[0344] Divide the culture-glycerol mix to aliquots
[0345] Label and store the Master Cell Banks
[0346] Following the same diagram, the Working Cell Bank was
established using one vial of the Master Cell Bank as the starting
material. The routine tests performed on the MCB and WCB were:
microbiological characterization, absence of contamination,
assessment of the plasmid stability by replica plating and the
plasmid identity (restriction enzyme digestion and sequencing).
[0347] b) Fermentation. In the fermentation the DH5 alpha T1 phage
resistant E. coli strain (Gibco RBL, UK) transformed with NNV-2-Nef
(WCB) was first cultured on plate. From the plate a single colony
was inoculated to a 100 ml liquid pre-culture before the actual
fermentation in the fermentation reactor. The fermentation was
carried out in a 5 I fermentor (B. Braun Medical) on a fed-batch
system basis, after which cells were harvested. The culture medium
composition for one litre contained 7 g of yeast extract, 8 g of
peptone from soy meal, 10 g of NaCl, 800 ml of water for injection
(WFI), 1N NaOH, pH 7.0, kanamycin 50 mg/ml (Sigma), silicon
anti-foaming agent (Merck), 1M K.sub.2PO.sub.4 (BioWhittaker).
[0348] In the beginning of the fermentation run, a 1 ml sample was
taken through the harvesting tube to determine the initial cell
density (OD.sub.600). The pre-culture was used to inoculate the
fermentation medium. During the fermentation, fresh culture medium
and 1M potassium phosphate buffer, pH 6.5-7.3, were fed to the
reactor with the pumps. Addition of the medium allows replenishment
of essential nutrients before they run out and phosphate buffer
maintains the pH constant. When the fermentation process had
continued for approximately 5 hours and at the end of the
fermentation run (after approximately 10 hours of fermentation),
samples of 1 ml were taken as above and the cell density was
measured. After the fermentation, the culture medium was
centrifuged (10,000 rpm, 30 minutes, +4.degree. C.) and the
bacterial pellet (50-60 g) was recovered.
[0349] c) Purification. The methodology used for the purification
of DNA was based on the QIAGEN process scale technology (Qiagen
Plasmid Purification Handbook 11/98). The NN2-Nef was purified
using the following steps:
[0350] Resuspend the bacterial pellet in the resuspension buffer
(100-150 ml, RT)
Lyse with the lysis buffer (100-150 ml, 5 minutes, RT)
[0351] Neutralize with the neutralization buffers (100-150 ml,
+4.degree. C.)
[0352] Incubate (30 minutes, +4.degree. C.)
[0353] Centrifugate (10,000 rpm, 30 minutes, +4.degree. C.)
[0354] Filtrate supernatant (0.22 micrometers)
[0355] Remove endotoxins with Endotoxin removal buffer (60-90
ml)
[0356] Equilibrate Ultrapure column with Equilibration buffer (350
ml, flow rate 10 ml/min)
[0357] Load lysate to the column (flow rate 4-6 ml/min)
[0358] Wash the column with Wash buffer (31, overnight, flow rate
4-6 ml/min)
[0359] Elute the plasmid DNA with Elution buffer (400 ml, flow rate
3.1 ml/min)
[0360] Filtrate the eluate (0.22 micrometer)
[0361] Precipitate DNA with isopropanol
[0362] Centrifuge (20000 g, 30 minutes, 4.degree. C.)
[0363] Purified plasmid DNA
[0364] Buffers used within the purification were as follows. The
resuspension buffer contained 50 mM Tris-Cl, pH 8.0, plus RNase A
(50 mg); the lysis buffer was 200 mM NaOH; the neutralization
buffer was 3M potassium acetate, pH 5.5; the endotoxin removal
buffer contained 750 mM NaCl, 10% Triton X-100; 50 mM MOPS, pH7.0;
the equilibration buffer contained 750 mM NaCl, 50 mM MOPS, pH 7.0;
the wash buffer contained 1 M NaCl, 50 mM MOPS, pH 7.0, 15%
isopropanol; and elution buffer contained 1.6 M NaCl, 50 mM MOPS,
pH 7.0, 15% isopropanol.
[0365] d) Aseptic Filling
[0366] The purified DNA representing the final bulk was dissolved
in 0.9% sterile physiological saline to a final concentration of 1
mg/ml and sterile filtered (0.22 micrometer) during the same day.
The purified bulk was filled manually (filling volume 0.5 ml) in
Schott Type 1 plus glass vials using a steam sterilized
Finnpipette.RTM. and sterile endotoxin-free tips. The vials filled
with the NN2-Nef vaccine were closed immediately, labelled and
packed in accordance to the specific Standard Operating Procedure
(SOP).
[0367] 2. Administration of the Test Vaccine to the Patients
[0368] Ten HIV-1 infected patients undergoing Highly Active
Anti-Retroviral therapy (HAART) were immunized with the
experimental DNA vaccine NN2-Nef, expressing the HIV-1 Nef gene
(Clade B). For immunizations, two intramuscular injections in the
gluteal muscle were given two weeks apart. The doses were 1 and 20
micrograms/injection. Blood samples were drawn at -4, 0, 1, 2, 4, 8
and 12 weeks. The samples were analyzed for humoral (ELISA, Western
blot) and cell mediated immune response (T-cell subsets, T-cell
proliferation, ELISPOT, cytokine expression, intracellular
cytokines).
[0369] A clinical examination was performed to each patient
participating the study. The clinical examination included a
patient interview (anamnesis) and weight determination. Cardiac and
pulmonar functions were checked by auscultation and percussion, the
blood pressure and heart rate were recorded. Enlargement of lymph
nodes, liver and thyroid gland were determined by palpation.
[0370] Laboratory tests to evaluate the safety of the vaccine were
performed at each visit. These tests included:
[0371] hematology: red blood cell count, haemoglobin, total and
differential WBC, platelet count, prothrombin time and activated
partial thromboplastin time at baseline; mean erythrocyte
corpuscular volume and hemoglobin content has been calculated.
[0372] Immunology: nuclear and ds-DNA antibodies.
[0373] Serum chemistries: total bilirubin, alkaline phosphatase,
SGOT/SLT or SGPT/ALT, serum creatinine, protein electrophoresis,
total serum cholesterol, triglycerides, glucose (at baseline),
sodium, potassium, and calcium.
[0374] Urine analysis: dipstick protein, glucose, ketones, occult
blood, bile pigments, pH, specific gravity and microscopic
examination of urinary sediment (RBC, WBC, epithelial cells,
bacteria, casts), when dipstick determination showed one or more
abnormal values.
[0375] Viral load: Increases of more than one log 10 should be
followed by a confirmatory viral load estimate after two weeks.
[0376] None of the patients experienced subjective or objective
adverse reactions to the vaccination. No adverse laboratory
abnormalities were observed in the panel of clinical chemistry
tests (see material and methods for details) performed repeatedly
during the vaccination period.
[0377] The following immunological studies were performed:
[0378] Lymphocyte Proliferation Assay (LPA)
[0379] Peripheral blood mononuclear cells (PBMC) were isolated from
heparinized venous blood by Ficoll-Hypaque density-gradient
(Pharmacia) centrifugation and resuspended at 1.times.10.sup.6
cells/ml in RPMI 1640 medium (Gibco) supplemented with 5% pooled,
heat-inactivated AB.sup.+ serum (Sigma), antibiotics (100 U/ml
penicillin and 100 .mu.g/ml streptomycin; Gibco) and L-glutamine
(complete medium, CM). Quadruplicate cultures were then set up in
flat-bottomed micro titer plates (1.times.10.sup.5 PBMC/well) and
the cells were incubated for 6 days in the presence or absence of
the following stimuli: rNef (0.2, 1 and 5 .mu.g/ml), GST (0.2, 1
and 5 .mu.g/ml), purified protein derivative of tuberculin (PPD,
12.5 .mu.g/ml; Statens Seruminstitut), Candida albicans antigen (20
.mu.g/ml; Greer Laboratories) and Phytohaemagglutinin (PHA; 5
.mu.g/ml; Life Technologies). For the last 6 h of the incubation
period .sup.3H-thymidine (1 .mu.Ci/well; Amersham) was added to the
cultures and the cells were harvested onto glass fiber filters and
incorporated radioactivity was measured in a .gamma.-counter.
Results are expresses as delta cpm (cpm in the presence of
antigen-cpm without antigen) or as stimulation index (cpm in the
presence of antigen/cpm without antigen).
[0380] The results are shown in FIGS. 26 and 27. None of the
vaccines showed significant T-cell proliferative response to the
test antigen, HIV-1 Nef before the vaccination. In contrast, 2 out
of 5 vaccines in the group that had received 1 microgram dose of
the test vaccine (patients 1 and 3) (FIG. 26) and 2 out of 5 in the
group receiving 20 micrograms of the test vaccine (patients 9 and
10) (FIG. 27) showed a strong T-cell proliferative response after
the first vaccination. After the second vaccination, one (patient
2) vaccine responded in the 1-microgram group.
[0381] IFN-.gamma. Assays
[0382] The type of immune response (Th1/Th2) induced by the vaccine
was evaluated by measuring interferon-gamma (IFN-.gamma.) released
in 6 days old culture supernatant after antigen (rNef, rGST, PPD)
or mitogen (PHA) stimulation of PBMC. For determinations,
commercial ELISA kits (R&D Quantikine) were used. The assay
employ the quantitative sandwich enzyme immunoassay technique where
a monoclonal antibody specific for IFN-.gamma. has been coated onto
a microplate. Standards and samples are pipetted into the wells and
any IFN-.gamma. present is bound by the immobilized antibody. After
washing away any unbound substances, an enzyme-linked polyclonal
antibody specific for IFN-.gamma. is added to the wells. Following
a wash to remove any unbound anti body-enzyme reagent, a substrate
solution is added to the wells and color develops in proportion to
the amount of the cytokine bound in the initial step. The color
development is stopped and the intensity of the color is
measured.
[0383] IFN-.gamma. response data from patient# 1 is shown in FIG.
28. As can be seen, the vaccine responded to the rNef antigen by
marked IFN-.gamma. response correlated with the T-cell
proliferation, indicating that the response seen in the vaccine is
in fact of the Th1 type.
[0384] HIV-1 infection is characterized by low or totally lacking
cell-mediated immune response towards all HIV proteins. The results
show that it is possible to induce a robust CMI in such patients
with exceptionally low doses of the DNA vaccine NN2-Nef. The doses
used were minimal to what has generally been required with DNA
vaccines. Thus, for instance, Merch announced recently good results
with their experimental HIV vaccine but the doses required were
from 1000 to 5000 micrograms (IAVI report, 2002).
6.12 Example 12
Construction of the Plasmid Expressing Epstein-Barr Virus (EBV)
EBNA-1 Protein and Containing 20 Binding Sites for EBNA-1 (FR
Element)
[0385] To construct a plasmid expressing Epstein-Barr virus (EBV)
EBNA-1 protein and containing 20 binding sites for EBNA-1 (FR
element), BPV-1 E2 binding sites were first replaced by EBV EBNA-1
binding sites (oriP without DS element). Plasmid FRE2d1EGFP (FIG.
29) was constructed by isolating the XmiI(AccI)/Eco32I (EcoRV) DNA
fragment (blunt-ended with Klenow enzyme) of pEBO LPP plasmid (FIG.
29A) (the fragment contains 20 binding sites for EBNA-1) and
inserting it by blunt end ligation into the SpeI/NheI site of
s6E2d1EGFP (FIG. 29B) (blunt-ended with Klenow enzyme). The
constructed plasmid FRE2d1EGFP (FIG. 29) was used as a negative
control in further experiments. It contains binding sites for
EBNA-1 protein instead of the BPV1 E2 10 binding sites, expressing
E2, but not EBNA-1.
[0386] Next, the sequence encoding BPV-1 E2 protein in FRE2d1EGFP
plasmid was replaced by a sequence encoding EBV EBNA-1 protein as
follows. The XmiI(AccI)/EcoRI fragment of pEBO LPP plasmid was
isolated and blunt-ended with Klenow enzyme and inserted into the
XbaI/XbaI site of FRE2d1EGFP plasmid (blunted with Klenow enzyme).
The vector FRE2d1EGFP was previously grown in Escherichia coli
strain DH5.alpha. (lacking Dam.sup.- methylation, because one XbaI
site is sensitive for methylation. The constructed plasmid
FREBNAd1EGFP (FIG. 30) expresses EBNA-1 protein and contains 20
binding sites for EBNA-1.
[0387] For expression, Jurkat, human embryonic kidney cell line 293
(ATCC CRL 1573) and mouse fibroblast cell line 3T6 cells (ATCC CCL
96) were maintained in Iscove's modified Dulbecco's medium (IMDM)
supplemented with 10% fetal calf serum (FCS). Four million cells
(Jurkat), 75% confluent dishes (293) or 1/4 of 75% confluent dishes
(3T6) were used for each transfection, which were carried out by
electroporation as follows. Cells were harvested by centrifugation
(1000 rpm, 5 min, at 20.degree. C., Jouan CR 422), and resuspended
in a complete medium containing 5 mM Na-BES buffer (pH 7.5). 250
.mu.l cell of the cell suspension was mixed with 50 .mu.g of
carrier DNA (salmon sperm DNA) and 1 .mu.g (in the case of Jurkat
and 3T6) or 5 .mu.g (in the case of 293) of plasmid DNA and
electroporated at 200 V and 1000 .mu.F for Jurkat cells, 170 V and
950 .mu.F for 293 cells and 230 V and 975 .mu.F for 3T6 cells. The
transfected Jurkat cells were plated on 6-cm dishes with 5 ml of
medium; 1/3 of transfected 293 and 3T6 cells were plated on a 6-cm
dishes with 5 ml of medium and 2/3 of the cells were plated on a
10-cm dishes with 10 ml of medium.
[0388] The transfected cells were analysed for the expression of
d1EGFP protein (modified enhanced green fluorescent protein). All
of the constructed plasmids expressed d1EGFP protein, which was
detected by measuring the fluorescence using a flow cytometer.
Because of the short half-life of the d1EGFP protein, it does not
accumulate, and the expression of this protein reflects the
presence of transcriptionally active plasmids in the cells.
Becton-Dickinson FACSCalibur system was used. The volume of the
Jurkat cell suspension was measured before each time-point
(approximately after every 24 hour) and if the volume was less than
5 ml, the missing volume of medium was added. Depending on the cell
suspension density the appropriate volume was taken for measuring
(1 or 2 ml) and replaced with the same amount of medium. This was
later taken into consideration when the dilution was
calculated.
[0389] For the first time-point, 293 cells from the 6-cm dish were
suspended in 5 ml of medium for measuring. In every following
time-point half of the cells were taken from the 10-cm dish,
suspended in 5 ml of medium and then measured. An appropriate
volume was added to the rest of the cell suspension. For the first
time-point, 3T6 cells from the 6-cm dish were suspended in 1 ml of
trypsine, which was then inactivated with 100 .mu.l of FCS. For
every following time-point, cells from the 10-cm dish were
suspended in 2 ml of trypsin. 1 ml of this suspension was treated
as described previously. 9 ml of medium was added to the rest of
the suspension. The analyzed cells were taken out of the incubator
immediately before the measurement. The appropriate flow speed
(500-1000 cells/sec) was determined before each time-point using
cells transfected only with carrier DNA as a control. Three
different parameters were used to measure size, surface structure
and fluorescence of the cells.
[0390] The results are presented as graphs in FIG. 31. Cells
transfected only with carrier DNA were used to measure the
auto-fluorescence of the cell-line. 1% of this auto-fluorescence
was considered as background fluorescence and was subtracted later
from the d1EGFP fluorescence. The received data was analyzed using
Microsoft Excel program.
[0391] Percentages of the d1EGFP expressing cells were calculated
using cells transfected with the carrier only as a negative control
for background fluorescence. As shown in FIG. 33, the two vectors
were maintained in the cells with different kinetics.
[0392] The number of the d1EGFP expressing cells was calculated
taking the dilutions into consideration using cells transfected
with the carrier only as a negative control for background
fluorescence. As seen from FIG. 53, the plasmids expressing EBNA-1
and carrying EBNA-1 specific multimeric binding sites are
maintained very efficiently in the transfected cells. At day 1
after transfection approximately 8.times.10.sup.4 cells expressed
EGFP. At day 8, in the case of maintenance vector (FREBNAd1EGFP),
the number of the plasmid positive d1EGFP expressing cells had
increased ten times to 8.times.10.sup.5. With the plasmid lacking
EBNA-1 expression (FRE2d1EGFP) or having no EBNA-1 binding sites,
the number of plasmid positive cells was retained or in many cases
decreased. This fact reflects the mechanism for
segregation/partitioning Epstein-Barr virus. Maintenance and
segregation function by EBNA1 and EBNA-1 binding sites provides
maintenance function to the plasmid if EBNA-1 is expressed and
plasmid carries EBNA-1 binding sites. The same mechanism and the
same components actually provide the segregation function to
Epstein-Barr Virus in the latent phase of life-cycle.
[0393] Similar results were obtained also in human embryonic cell
line 293 and mouse cell line 3T6 (FIG. 34). As a control for the
maintenance for 293 and 3T6 cells, s6HPV11 and 2wtFS, respectively,
were used.
6.53 Example 13
The Immunogenicity of GTU-Multigene Vectors
[0394] The Immunogenicity of GTU-1-Multigene Vectors
[0395] The immunogenicity of six different multi-gene vaccine
constructs prepared in Example 12, i.e. GTU-1-RNT, GTU-1-TRN,
GTU-1-RNT-CTL, GTU-1-TRN-CTL, GTU-1-TRN-optgag-CTL, and
GTU-1-TRN-CTL-optgag vectors were tested in mice. The vectors were
transformed into TOP10 or DH5alpha cells, and the MegaPreps were
prepared using commercial Qiagen columns. Endotoxins were removed
with Pierce Endotoxin Removal Gel.
[0396] The test articles were coated on 1 .mu.m gold particles
according to the instructions given by the manufacturer (Bio-Rad)
with slight modifications. Balb/c mice were immunized with a Helios
Gene Gun using a pressure of 400 psi and 0.5 mg gold/cartridge.
Mice were immunized three times at weeks 0, 1, and 3. Mice were
sacrificed two weeks after the last immunization.
[0397] Mice were divided into six test groups (5 mice/group), which
received 3.times.1 .mu.g DNA as follows:
[0398] Group 1. GTU-1-RNT
[0399] Group 2. GTU-1-TRN
[0400] Group 3. GTU-1-RNT-CTL
[0401] Group 4. GTU-1-TRN-CTL
[0402] Group 5. GTU-1-TRN-optgag-CTL
[0403] Group 6. GTU-1-TRN-CTL-optgag
[0404] Group 7. Control mice immunized with empty gold particles
not loaded with DNA.
[0405] The humoral response was followed from tail-blood samples
from each mouse. First pre-immunization sample was taken from
anesthetized mice before the first immunization was given. Second
sample was taken from anesthetized mice before the third
immunization. At sacrifice, whole blood sample was used for white
blood cell counting, and serum was collected for humoral immunity
tests.
[0406] The blood samples were tested for antibodies with ELISA
using a standard procedure. Nunc Maxi Sorp plates were coated with
100 ng of Nef, Rev, Tat, Gag, CTL or E2 proteins, blocked and sera
at a dilution of 1:100 were added in duplicate wells for an
overnight incubation. After washing, the plates were incubated for
2 hours with a diluted (1:500) secondary antibody, peroxidase
conjugated anti-mouse IgG (DAKO). Color intensity produced from the
substrate (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS) in phosphate-citrate buffer was measured at 405 nm using
Labsystems ELISA-plate reader.
[0407] All vectors induced Nef antibodies in all mice, whereas none
of the mice showed E2, CTL or Rev antibodies (FIGS. 35, 36, and
Table 4) Some of the mice immunized with GTU-1-RNT or GTU-1-RNT-CTL
also developed Tat anti-bodies (FIG. 36 and Table 4). Furthermore,
mice immunized with vectors containing the optgag sequence
developed also Gag antibodies, but the construct
GTU-1-TRN-optgag-CTL was a better antibody inducer that the
construct GTU-1-TRN-CTL-optgag (FIG. 37 and Table 4). The
antibodies induced were mainly of the IgG1 class indicating a Th2
type of response usually seen with gene gun immunization. The
antibody assays shown below were done from the sera collected when
mice were sacrificed.
[0408] The results show that a multigene construct, expressing
several HIV genes as a fusion protein, can induce an immune
response to most of the gene products. The orientation and order of
the genes in the multigene and corresponding proteins in the fusion
proteins affects the results, however, dramatically. Thus, a
response against Tat was seen only when the Tat gene was placed
inside the fusion protein (vectors with RNT motif) and not when Tat
was the amino terminal protein (vectors with the TRN motif).
Response to the Gag proteins was seen only with the vector, where
Gag was placed before the CTL containing a stretch of Th and CTL
epitopes.
TABLE-US-00004 TABLE 4 Immuno III A mice ELISA results (OD mean of
five mice) Group Immunogen number Nef (own prot) Tat Rev Gag CTL
GTU-1-RNT 1 2.194 1.391 0.31 0.155 0.36 GTU-1-TRN 2 1.849 0.197
0.252 0.302 0.38 GTU-1-RNT- 3 1.922 0.555 0.295 0.154 0.439 CTL
GTU-1-TRN- 4 1.677 0.211 0.298 0.14 0.425 CTL GTU-1-TRN- 5 1.722
0.182 0.24 0.667 0.381 optgag-CTL GTU-1-TRN- 6 0.547 0.225 0.322
0.228 0.43 CTL-optgag Controls 7 0.316 0.226 0.282 0.16 0.405
Percent of Nef Tat Rev Gag CTL Immunogen Group response response
response response response GTU-1-RNT 1 100 80 0 0 0 GTU-1-TRN 2 100
0 0 20 0 GTU-1-RNT- 3 100 40 0 0 0 CTL GTU-1-TRN- 4 100 0 0 0 0 CTL
GTU-1-TRN- 5 80 0 0 60 0 optgag-CTL GTU-1-TRN- 6 100 0 0 20 0
CTL-optgag
6.14, Example 14
Expression of Hybrid Protein Expressing Nef, Rev and Tat in
Different Combinations (Multireg)
[0409] For the production of HIV multi-gene vectors, GTU-1 vector
with a multi-cloning site (FIG. 38A) was used as a backbone. Intact
Nef, Rev and Tat coding sequences were amplified by the polymerase
chain reaction (PCR) and attached to each other in various orders
to multi-regulatory (multireg) antigen coding reading frames
(Nef-Tat-Rev, Tat-Rev-Nef, Rev-Tat-Nef, Tat-Nef-Rev and
Rev-Nef-Tat; Sequences Id. No. 1 to 5, respectively). These
sequences were cloned to the Bsp191 and NotI sites of the GTU-1
vector.
[0410] Similarly, Nef protein expressing GTU-2 and GTU-3 vectors
(FIGS. 38B and 38C; see also FIG. 6B for NNV-2wt)) were also used
as backbones for the production of HIV multigene vectors.
Additionally, the vector super6wt expressing destabilized enhanced
green fluorescent protein or d1EGFP (super6wtd1EGFP; FIG. 17 and
FIG. 38D) and plasmid utilizing the EBNA-1 protein and its binding
sites (FREBNAd1EGFP; FIG. 38E) were used as a Gene Transfer Unit
(GTU) platform. For control "non-GTU" vectors, a regular
cytomegalovirus (CMV) vector NNV-Rev expressing Rev and a plasmid
EBNA-1 and E2BS containing d1EGFP plasmid (NNV-Rev and
E2BSEBNAd1EGFP, respectively; FIGS. 38G and F) were used as
backbones.
[0411] For the preparation of different GTU-2 and GTU-3 vectors
(pNRT, pTRN, pRTN, pTNR and pRNT; and p2TRN and p2RNT; and p3RNT,
FIGS. 39A-E, 39F-G and 39H, respectively), the Nef gene in vectors
GTU-2Nef and GTU-3Nef was substituted by the respective multireg
antigen using NdeI and Pag I sites. The sequence of the letters
N(ef), R(ev) and T(at) in the name shows the position of respective
coding sequences of the protein in the multi-gene. Also two
vectors, which contain the IRES element placed into the SalI sites
following either the multi-antigen or E2 coding sequences, were
prepared (pTRN-iE2-GMCSF and pTRN-iMG-GMCSF, respectively; FIGS.
39I and J). The latter sequence, which controls the translation of
the coding sequence of the mouse granulocyte-magrophage colony
stimulating factor (GM-CSF), was cloned into the single BspTI site
introduced with IRES.
[0412] Additionally, a set of the vectors, in which only
immunodominant parts of the regulatory proteins were used for
building up the polyproteins, were cloned into the Bsp119I and NotI
sites of the GTU-1 (pMV1 NTR, pMV2NTR, pMV1N11TR and pMV2N11TR;
FIGS. 40A-D). In case of the pMV2 constructs, linkers that could be
digested by intracellular proteases separate the regions of the
multi-antigene derived from different regulatory proteins.
[0413] Further, GTU-1, GTU-2 and GTU-3 vectors, which express the
structural proteins encoded by the gag gene or an artificial
polyprotein composed by previously described CTL epitopes, were
prepared. The coding sequences were cloned as Bsp119I and Not I
digested PCR fragments into the GTU-1 vector (pCTL=BNmCTL,
pdgag=pBNdgag, psynp17/24=pBNsynp17+24, poptp17/24=pBNoptp17/24;
FIGS. 41A-D), and transferred in a Nde I-Pac I fragment to the
GTU-2 (p2mCTL and p2optp17/24; FIGS. 41E and F) and GTU-3 (p3mCTL
and p3optp17/24; FIGS. 41G and H).
[0414] The coding segment designated as CTL (Sequence Id. No. 10)
contains fragments from pol and env regions involving many
previously identified CTL epitopes. The codon usage is optimized so
that only codons used frequently in human cells are involved. This
coding sequence also contains a well-characterized mouse CTL
epitope used in potency assay and an epitope for recognition by
anti-mouse CD43 antibody. Also, a dominant SIV p27 epitope was
included for use in potency studies in macaques.
[0415] The dgag contains truncated p17 (start at 13 aa) +p24+p2+p7
(p1 and p6 are excluded) (Sequence Id. No. 11) of gag region of the
Han2 isolate. The synp17/24 (Sequence Id. No 12) codes for the
p17+p24 polypeptide of the Han2 HIV-1. The codon usage is modified
to be optimal in human cells. Also, previously identified AU rich
RNA instability elements were removed by this way. The optp17/24
coding (Sequence Id. No. 13) region is very similar to the
synp17/24 with the exception that the two synonymous mutations made
therein do not change the protein composition but remove a
potential splicing donor site.
[0416] Further, a set of the multi-HIV vectors, which contain both
a multireg antigen and structural antigens as a single polyprotein,
were created: pTRN-CTL, pRNT-CTL, pTRN-dgag, pTRN-CTL-dgag,
pRNT-CTL-dgag, pTRN-dgag-CTL, pRNT-dgag-CTL, pTRN-optp17/24-CTL,
pTRN-CTL-optp17/24, and pRNT-CTL-optp17/24; p2TRN-optp17/24-CTL,
p2RNT-optp17/24-CTL, p2TRN-CTL-optp17/24, p2RNT-CTL-optp17/24,
p2TRN-CTL-optp17/24-iE2-mGMCSF, and p2RNT-CTL-optp17/24-iE2-mGMCSF;
and p3TRN-CTL-optp17/24, p3RNT-CTL-optp17/24,
p3TRN-CTL-optp17/24-iE2-mGMCS F, and
p3RNT-CTL-optp17/24-iE2-mGMCSF, FIGS. 42A-T.
[0417] For cloning, as a first step the STOP codon was removed from
the regulatory multi-antigen coding sequences. Then the structural
antigen coding sequences were added by cloning into the NotI site
at the end of the frame so that a NotI site was reconstituted. If
both CTL and gag were added, the first antigen coding sequence was
without the STOP codon. Generally, the clonings were made in
context of GTU-1 and for making the respective GTU-2 (p2 . . . )
and GTU-3 (p3 . . . ) vectors, the Nef gene in the plasmids
GTU-2Nef and GTU-2Nef was replaced using sites for NdeI and Pag I.
However, the RNT-optp17/24-CTL antigen was built up directly in
GTU-2 vector.
[0418] The HIV multi-antigen was cloned to the vectors
super6wtd1EGFP and FREBNAd1EGFP instead of the d1EGFP using sites
for Eco105I and NotI (super6 wt-RNT-CTL-optp17/24 and
FREBNA-RNT-CTL-optp17/24; FIGS. 43V and 42 U, respectively). If
indicated, the IRES and mouse mGM-CSF were cloned into the GTU-2
and GTU-3 vectors behind the E2 coding sequence into the sites
Mph1103I and Eco91I from pTRN-iE2-mGMCSF (cut out using same
restrictases).
[0419] Finally, "non-GTU" control vector E2BSEBNA-RNT-CTL-optp17/24
(FIG. 42W) for the system utilizing EBNA-1 (contains EBNA-1
expression cassette with E2 binding sites) was made in a similar
way as the FREBNA-RNT-CTL-optp17/24. The regular CMV vector
pCMV-RNT-CTL-optp17/24 expressing the multi HIV antigen (FIG. 42D)
was made by cloning the multi-HIV coding fragment from respective
GTU-1 vector using sites for NdeI and Pag I.
[0420] 6.5.2 Expression Properties of the Multireg Antigens
Carrying Only Immunodominant Regions of the Regulatory
Proteins.
1. Intracellular Localization of the MultiREG Antigens
[0421] The intracellular localization of MultiREG antigens
expressed by the vectors of the invention was studied by in situ
immunofluorescence in RD cells using monoclonal antibodies against
Nef, Rev and Tat proteins essentially as described in Example 4.
The results are summarized in Table 5 and illustrated in FIG. 45.
All antigens that are comprised of intact Nef, Rev and Tat proteins
showed exclusive localization in cytoplasm. The aberrant protein
initially designed as N(ef)T(at)R(ev), which has a frame-shift
before the Rev sequence, showed only the nuclear localization.
MultiREG antigens carrying truncated sequences of the regulatory
proteins were localized in cytoplasm. In this cases distinct
structures like "inclusion bodies" were frequently observed. The
same was true for antigens, which carried the protease sites
expressed from pMV2 vectors. However in these cases the proteins in
nucleus were also detected (FIG. 45).
TABLE-US-00005 TABLE 5 Intracellular localization in multireg
antigenes Construct anti-Nef anti-Rev anti-Tat empty negative
negative negative GTU-1 pTRN strong staining in cytoplasm good
staining in cytoplasm positive staining in cytoplasm pNTR strong
staining in negative positive staining in nucleus, nucleolus
nucleus pRNT strong staining in cytoplasm good staining in
cytoplasm good staining in cytoplasm pNRT strong, cytoplasmic good
staining in cytoplasm good staining in cytoplasm pRTN strong,
cytoplasmic good staining in cytoplasm positive staining in
cytoplasm pTNR strong, cytoplasmic good staining in cytoplasm good
staining in cytoplasm pMV1NTR strong, cytoplasmic cytoplasmic +
inclusions cytoplasmic + inclusions pMV1N11TR strong cytoplasmic +
inclusions cytoplasmic + inclusions cytoplasmic + inciusions
pMV2NTR inclusions in nuclei and inclusions in nuclei inclusions in
nuclei in cytopi. and cytoplasm and cytoplasm pMV2N11TR only
inclusions, in nuclei only inclusions in only inclusions in and in
cytopi nuclei and in cytopi nuclei and in cytopi.
[0422] The intracellular localization of dgag and p17+p24 proteins
was also analyzed in RD cells by immunofluorescence with monoclonal
anti p24 antibodies. In accordance with the Western blot results in
Jurkat cells, the dgag could not be detected. However, the p17/24
protein showed localization in plasma membranes (FIG. 45). The
localization of CTL protein was not analyzed, because no suitable
antibody was available.
6.6 Example 15
Analysis of Vectors Encoding Recombinant GAG Antigens and Cytotoxic
T-Cell Epitopes (CTL) from POL
[0423] 6.6.1. Expression
[0424] Analysis of expression of the vectors expressing CTL cds or
proteins from the gag region were performed by western blot. As
seen on FIGS. 46A and 46B, the CTL and dgag expression was clearly
demonstrated in Cos-7 cells as predicted size proteins (25 kD and
47 kD, respectively). The co-transfection of the Nef, Rev and Tat
significantly enhanced the expression of the dgag protein. We
interpret this as a result of REV protein action on the GAG mRNA
expression We also tried to express the dgag protein from GTU-1
vector in Jurkat cells, but we failed to detect any signal (FIG.
46C). The analysis of the codon usage showed that wt GAG sequence
had not optimal codon usage for human cells. When the codon usage
was optimized (constructs psynp17/24 and poptp17/24), strong
p17+p24 (40 kD) protein expression was detected in Jurkat cells
(FIGS. 46C and 46D).
[0425] 6.6.2. Intracellular Localization
[0426] For dgag and p17+p24 proteins, the intracellular
localization was also analyzed in RD cells by immunofluorescence
with anti p24 Mab. Similar to the western blot results in Jurkat
cells, the dgag could not be detected. The p17/24 protein showed
localization in plasma membranes (FIG. 47). The localization of CTL
protein was not analyzed caused by lacking of suitable antibody
6.7 Example 16
Multireg+Structural Proteins as Multi-HIV Antigen Expression
[0427] As next step, the expression of the Multi HIV antigenes
consisting of both, regular multigene together with gag encoded
protein and/or CTL multiepitope as single polypeptide was analysed.
On FIG. 48, the Western blot shows the expression of several
multiHIV-antigenes expressing vectors transfected to the Cos-7
cells. It is clearly seen that the expression levels of all
regulatory+structural multi-antigenes are significantly lower than
of the RNT or TRN proteins. All tested MultiHIV antigenes migrate
in the gel as distinct bands near the position of predicted size
(73 kD for multireg+CTL; 95 kD for multireg+dgag and 120 kD for
multireg+CTL+dgag). Similar to the RNT and TRN, the RNT-CTL
migrates more slowly than TRN-CTL. Also, in cases of both TRN and
RNT constructs, the MultiREG-CTL-dgag combination showed higher
expression level than MultiREG-dgag-CTL.
[0428] More detailed analysis of the multiHIV antigenes was
performed in Jurkat cells. For this reason, most of the constructed
MultiHIV antigenes (multireg+structural), included the
MultiREG+CTL+optp17/24 (with predicted size 113 kD) were analyzed
by Western blotting using antibodies against different parts of the
antigene. The results are presented on FIG. 49 are principally
similar to those were reported in previous section in case of Cos-7
cells. As it was seen in the previous experiments, the dgag
containing multi-antigenes express very low levels of the hybrid
protein in Jurkat cells. The expression from the vector pTRNdgag
was undetectable on all blots. In lanes loaded material from cells
transfected with other dgag containing antigene expression vectors,
very faint signals only on the Nef Mab hybridized blot were
detected at positions of predicted sizes. In contrast, if the dgag
part is replaced with the codon optimized p17/24, the expression
level increase was observed. Because the TRN-CTL-optp17/24 and
RNT-optp17/24 were initially chosen for further analysis, the
expression of the antigenes was analyzed from all GTU vectors
containing these expression cassettes. Also, the E2 protein
expression from these plasmids was analyzed. The results are
illustrated on FIG. 50. There are no big differences between the
vectors in expression levels of both multi-antigene and the E2
protein. The E2 expression level is not significantly influenced by
presence of IRES element followed mouse GM-CSF gene in the plasmid,
translated from the same mRNA as the E2.
6.7.1. Example 17
Maintenance of Expression of Antigen
[0429] The maintenance of the plasmid in a population of dividing
cells was proved using the green fluorescent protein and Nef
protein as markers. The maintenance of the expression of the
RNT-CTL-optp17/24 antigen produced from different GTU or non-GTU
vectors was also analyzed. Specifically, GTU-1 (p
RNT-CTL-optp17/24), GTU-2 (p2 RNT-CTL-optp17/24), GTU-3 (p3
RNT-CTL-optp17/24), super6wt (super6wt-RNT-CTL-optp17/24) vectors
each utilize the E2 protein and its binding sites for the plasmid
maintenance activity. In this experiment, also EBNA-1 and its
binding site utilizing GTU vector FREBNA-RNT-CTL-optp17/24 was
included. As negative controls, "non-GTU" plasmid containing a
mixed pair of the EBNA-1 expression cassette together with E2
binding sites (E2BSEBNA-RNT-CTL-optp17/24) was used. Also, regular
CMV expression vector pCMV-RNT-CTL-optp17/24 was used.
[0430] Jurkat cells were transfected with equimolar amounts of the
plasmids and the antigen expression was studied at 2 and 5 days
post-transfection using a monoclonal anti-Nef antibodies.
Transfection with carrier DNA only was used as a negative control.
The results are presented in FIG. 51.
[0431] As it seen from FIG. 51, the expression is detectable only
from GTU vectors at the second time-point. The antigen expression
from the FREBNA-RNT-CTL-optp17/24 was lower at both time-points,
because, unlike E2, the EBNA-1 does not have transcription
activation ability.
[0432] Also the intracellular localization of the
multireg+structural polyproteins was studied by in situ
immunofluorescence analysis in RD cells essentially as described in
Example 4. The results are presented in FIG. 52.
[0433] In all cases localization only in cytoplasm was detected
using either monoclonal anti-Nef or anti-p24 antibodies. In
accordance with Western blot data, the expression level of
optp17/24 containing proteins was much stronger than dgag fragment
containing antigens.
[0434] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
[0435] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended claims
along with the full scope of equivalents to which such claims are
entitled.
Sequence CWU 1
1
5211260DNAArtificial SequenceHybrid protein comprised of
Nef-Tat-Rev (NTR) 1atggtgggca agtggtcaaa atgtagtgga tggcctactg
taagggaaag aatgaaacaa 60gctgagcctg agccagcagc agatggggtg ggagcagcat
ctcgagacct ggaaaaacat 120ggagcaatca caagtagcaa tacagcaact
aataacgctg cttgtgcctg gctagaagca 180caagaggaag aggaagtggg
ttttccagtc agacctcagg tacctttaag accaatgact 240tacaagggag
ctttagatct tagccacttt ttaaaagaaa aggggggact ggaagggtta
300atttactccc caaaaagaca agagatcctt gatctgtggg tctaccacac
acaaggctac 360ttccctgatt ggcagaacta cacaccaggg ccaggggtca
gatatccact gacctttgga 420tggtgcttca agttagtacc agttgaacca
gatgaagaag agaacagcag cctgttacac 480cctgcgagcc tgcatgggac
agaggacacg gagagagaag tgttaaagtg gaagtttgac 540agccatctag
catttcatca caaggcccga gagctgcatc cggagtacta caaagactgc
600actagtgcag gaagaagcgg agacagcgac gaagagctcc tcaagacagt
cagactcatc 660aagtttctct accaaagcaa ccctcctccc agcaacgagg
ggacccgaca ggcccgaaga 720aatcgaagaa gaaggtggag agagagacag
aggcagatcc gttcgattag tgagcggatt 780cttagcactt ttctgggacg
acctgcggag cctgtgcctc ttcagctacc gccgcttgag 840agacttactc
ttgattgtag cgaagattgt ggaaactctg ggacgcaggg ggtgggaagt
900cctcaagtat tggtggaatc tcctgcagta ttggagccag gaactaaaga
aaagcttgag 960ccagtagatc ctagactaga gccctggaag catccaggaa
gtcagcctag gaccccttgt 1020accaattgct attgtaaaaa gtgttgcctt
cattgccaag tttgtttcac aagaaaaggc 1080ttaggcatct cctatggcag
gaagaagcgg agacagcgac gaagagctcc tcaagacagt 1140cagactcatc
aagtttctct accaaagcaa ccctcctccc agcaacgagg ggacccgaca
1200ggcccgaaga aatcgaagaa gaaggtggag agagagacag aggcagatcc
gttcgattag 126021260DNAArtificial SequenceHybrid protein comprised
of Tat-Rev-Nef (TRN) 2atggagccag tagatcctag actagagccc tggaagcatc
caggaagtca gcctaggacc 60ccttgtacca attgctattg taaaaagtgt tgccttcatt
gccaagtttg tttcacaaga 120aaaggcttag gcatctccta tggcaggaag
aagcggagac agcgacgaag agctcctcaa 180gacagtcaga ctcatcaagt
ttctctacca aagcaaccct cctcccagca acgaggggac 240ccgacaggcc
cgaagaaatc gaagaagaag gtggagagag agacagaggc agatccgttc
300gatactagtg caggaagaag cggagacagc gacgaagagc tcctcaagac
agtcagactc 360atcaagtttc tctaccaaag caaccctcct cccagcaacg
aggggacccg acaggcccga 420agaaatcgaa gaagaaggtg gagagagaga
cagaggcaga tccgttcgat tagtgagcgg 480attcttagca cttttctggg
acgacctgcg gagcctgtgc ctcttcagct accgccgctt 540gagagactta
ctcttgattg tagcgaagat tgtggaaact ctgggacgca gggggtggga
600agtcctcaag tattggtgga atctcctgca gtattggagc caggaactaa
agaaaagctt 660gtgggcaagt ggtcaaaatg tagtggatgg cctactgtaa
gggaaagaat gaaacaagct 720gagcctgagc cagcagcaga tggggtggga
gcagcatctc gagacctgga aaaacatgga 780gcaatcacaa gtagcaatac
agcaactaat aacgctgctt gtgcctggct agaagcacaa 840gaggaagagg
aagtgggttt tccagtcaga cctcaggtac ctttaagacc aatgacttac
900aagggagctt tagatcttag ccacttttta aaagaaaagg ggggactgga
agggttaatt 960tactccccaa aaagacaaga gatccttgat ctgtgggtct
accacacaca aggctacttc 1020cctgattggc agaactacac accagggcca
ggggtcagat atccactgac ctttggatgg 1080tgcttcaagt tagtaccagt
tgaaccagat gaagaagaga acagcagcct gttacaccct 1140gcgagcctgc
atgggacaga ggacacggag agagaagtgt taaagtggaa gtttgacagc
1200catctagcat ttcatcacaa ggcccgagag ctgcatccgg agtactacaa
agactgctga 126031260DNAArtificial SequenceHybrid protein comprised
of Rev-Tat-Nef (RTN) 3atggcaggaa gaagcggaga cagcgacgaa gagctcctca
agacagtcag actcatcaag 60tttctctacc aaagcaaccc tcctcccagc aacgagggga
cccgacaggc ccgaagaaat 120cgaagaagaa ggtggagaga gagacagagg
cagatccgtt cgattagtga gcggattctt 180agcacttttc tgggacgacc
tgcggagcct gtgcctcttc agctaccgcc gcttgagaga 240cttactcttg
attgtagcga agattgtgga aactctggga cgcagggggt gggaagtcct
300caagtattgg tggaatctcc tgcagtattg gagccaggaa ctaaagaaac
tagtgagcca 360gtagatccta gactagagcc ctggaagcat ccaggaagtc
agcctaggac cccttgtacc 420aattgctatt gtaaaaagtg ttgccttcat
tgccaagttt gtttcacaag aaaaggctta 480ggcatctcct atggcaggaa
gaagcggaga cagcgacgaa gagctcctca agacagtcag 540actcatcaag
tttctctacc aaagcaaccc tcctcccagc aacgagggga cccgacaggc
600ccgaagaaat cgaagaagaa ggtggagaga gagacagagg cagatccgtt
cgataagctt 660gtgggcaagt ggtcaaaatg tagtggatgg cctactgtaa
gggaaagaat gaaacaagct 720gagcctgagc cagcagcaga tggggtggga
gcagcatctc gagacctgga aaaacatgga 780gcaatcacaa gtagcaatac
agcaactaat aacgctgctt gtgcctggct agaagcacaa 840gaggaagagg
aagtgggttt tccagtcaga cctcaggtac ctttaagacc aatgacttac
900aagggagctt tagatcttag ccacttttta aaagaaaagg ggggactgga
agggttaatt 960tactccccaa aaagacaaga gatccttgat ctgtgggtct
accacacaca aggctacttc 1020cctgattggc agaactacac accagggcca
ggggtcagat atccactgac ctttggatgg 1080tgcttcaagt tagtaccagt
tgaaccagat gaagaagaga acagcagcct gttacaccct 1140gcgagcctgc
atgggacaga ggacacggag agagaagtgt taaagtggaa gtttgacagc
1200catctagcat ttcatcacaa ggcccgagag ctgcatccgg agtactacaa
agactgctga 126041260DNAArtificial SequenceHybrid protein comprised
of Tat-Nef-Rev (TNR) 4atggagccag tagatcctag actagagccc tggaagcatc
caggaagtca gcctaggacc 60ccttgtacca attgctattg taaaaagtgt tgccttcatt
gccaagtttg tttcacaaga 120aaaggcttag gcatctccta tggcaggaag
aagcggagac agcgacgaag agctcctcaa 180gacagtcaga ctcatcaagt
ttctctacca aagcaaccct cctcccagca acgaggggac 240ccgacaggcc
cgaagaaatc gaagaagaag gtggagagag agacagaggc agatccgttc
300gatactagtg tgggcaagtg gtcaaaatgt agtggatggc ctactgtaag
ggaaagaatg 360aaacaagctg agcctgagcc agcagcagat ggggtgggag
cagcatctcg agacctggaa 420aaacatggag caatcacaag tagcaataca
gcaactaata acgctgcttg tgcctggcta 480gaagcacaag aggaagagga
agtgggtttt ccagtcagac ctcaggtacc tttaagacca 540atgacttaca
agggagcttt agatcttagc cactttttaa aagaaaaggg gggactggaa
600gggttaattt actccccaaa aagacaagag atccttgatc tgtgggtcta
ccacacacaa 660ggctacttcc ctgattggca gaactacaca ccagggccag
gggtcagata tccactgacc 720tttggatggt gcttcaagtt agtaccagtt
gaaccagatg aagaagagaa cagcagcctg 780ttacaccctg cgagcctgca
tgggacagag gacacggaga gagaagtgtt aaagtggaag 840tttgacagcc
atctagcatt tcatcacaag gcccgagagc tgcatccgga gtactacaaa
900gactgcaagc ttgcaggaag aagcggagac agcgacgaag agctcctcaa
gacagtcaga 960ctcatcaagt ttctctacca aagcaaccct cctcccagca
acgaggggac ccgacaggcc 1020cgaagaaatc gaagaagaag gtggagagag
agacagaggc agatccgttc gattagtgag 1080cggattctta gcacttttct
gggacgacct gcggagcctg tgcctcttca gctaccgccg 1140cttgagagac
ttactcttga ttgtagcgaa gattgtggaa actctgggac gcagggggtg
1200ggaagtcctc aagtattggt ggaatctcct gcagtattgg agccaggaac
taaagaatag 126051260DNAArtificial SequenceHybrid protein comprised
of Rev-Nef-Tat (RNT) 5atggcaggaa gaagcggaga cagcgacgaa gagctcctca
agacagtcag actcatcaag 60tttctctacc aaagcaaccc tcctcccagc aacgagggga
cccgacaggc ccgaagaaat 120cgaagaagaa ggtggagaga gagacagagg
cagatccgtt cgattagtga gcggattctt 180agcacttttc tgggacgacc
tgcggagcct gtgcctcttc agctaccgcc gcttgagaga 240cttactcttg
attgtagcga agattgtgga aactctggga cgcagggggt gggaagtcct
300caagtattgg tggaatctcc tgcagtattg gagccaggaa ctaaagaaac
tagtgtgggc 360aagtggtcaa aatgtagtgg atggcctact gtaagggaaa
gaatgaaaca agctgagcct 420gagccagcag cagatggggt gggagcagca
tctcgagacc tggaaaaaca tggagcaatc 480acaagtagca atacagcaac
taataacgct gcttgtgcct ggctagaagc acaagaggaa 540gaggaagtgg
gttttccagt cagacctcag gtacctttaa gaccaatgac ttacaaggga
600gctttagatc ttagccactt tttaaaagaa aaggggggac tggaagggtt
aatttactcc 660ccaaaaagac aagagatcct tgatctgtgg gtctaccaca
cacaaggcta cttccctgat 720tggcagaact acacaccagg gccaggggtc
agatatccac tgacctttgg atggtgcttc 780aagttagtac cagttgaacc
agatgaagaa gagaacagca gcctgttaca ccctgcgagc 840ctgcatggga
cagaggacac ggagagagaa gtgttaaagt ggaagtttga cagccatcta
900gcatttcatc acaaggcccg agagctgcat ccggagtact acaaagactg
caagcttgag 960ccagtagatc ctagactaga gccctggaag catccaggaa
gtcagcctag gaccccttgt 1020accaattgct attgtaaaaa gtgttgcctt
cattgccaag tttgtttcac aagaaaaggc 1080ttaggcatct cctatggcag
gaagaagcgg agacagcgac gaagagctcc tcaagacagt 1140cagactcatc
aagtttctct accaaagcaa ccctcctccc agcaacgagg ggacccgaca
1200ggcccgaaga aatcgaagaa gaaggtggag agagagacag aggcagatcc
gttcgattag 126061164DNAArtificial SequenceProtein comprised of
Immunodominant parts of the Nef-Tat-Rev(NTR) 6atgggatggc ctactgtaag
ggaaagaatg aaacaagctg agcctgagcc agcagcagat 60ggggtgggag cagcatctcg
agacctggaa aaacatggag caatcacaag tagcaataca 120gcaactaata
acgctgcttg tgcctggcta gaagcacaag aggaagagga agtgggtttt
180ccagtcagac ctcaggtacc tttaagacca atgacttaca agggagcttt
agatcttagc 240cactttttaa aagaaaaggg gggactggaa gggttaattt
actccccaaa aagacaagag 300atccttgatc tgtgggtcta ccacacacaa
ggctacttcc ctgattggca gaactacaca 360ccagggccag gggtcagata
tccactgacc tttggatggt gcttcaagtt agtaccagtt 420gaaccagatg
aagaagagaa cagcagcctg ttacaccctg cgagcctgca tgggacagag
480gacacggaga gagaagtgtt aaagtggaag tttgacagcc atctagcatt
tcatcacaag 540gcccgagagc tgcatccgga gtactacaaa gactgcgctc
tggccgccgt tgagccagta 600gatcctagac tagagccctg gaagcatcca
ggaagtcagc ctaggacccc ttgtaccaat 660tgctattgta aaaagtgttg
ccttcattgc caagtttgtt tcacaagaaa aggcttaggc 720atctcctatg
gcaggaagaa gcggagacag cgacgaagag ctcctcaaga cagtcagact
780catcaagttt ctctaccaaa gcaaccctcc tcccagcaac gaggggaccc
gacaggcccg 840aagaaatccg gactggccat cctgctgagc gacgaagagc
tcctcaagac agtcagactc 900atcaagtttc tctaccaaag caaccctcct
cccagcaacg aggggacccg acaggcccga 960agaaatcgaa gaagaaggtg
gagagagaga cagaggcaga tccgttcgat tagtgagcgg 1020attcttagca
cttttctggg acgacctgcg gagcctgtgc ctcttcagct accgccgctt
1080gagagactta ctcttgattg tagcgaagat tgtggaaact ctgggacgca
gggggtggga 1140agtcctcaag tattggtgga atga 116471173DNAArtificial
SequenceProtein comprised of Immunodominant parts of the
Nef-Tat-Rev separated by protease sites(NTR) 7atgggatggc ctactgtaag
ggaaagaatg aaacaagctg agcctgagcc agcagcagat 60ggggtgggag cagcatctcg
agacctggaa aaacatggag caatcacaag tagcaataca 120gcaactaata
acgctgcttg tgcctggcta gaagcacaag aggaagagga agtgggtttt
180ccagtcagac ctcaggtacc tttaagacca atgacttaca agggagcttt
agatcttagc 240cactttttaa aagaaaaggg gggactggaa gggttaattt
actccccaaa aagacaagag 300atccttgatc tgtgggtcta ccacacacaa
ggctacttcc ctgattggca gaactacaca 360ccagggccag gggtcagata
tccactgacc tttggatggt gcttcaagtt agtaccagtt 420gaaccagatg
aagaagagaa cagcagcctg ttacaccctg cgagcctgca tgggacagag
480gacacggaga gagaagtgtt aaagtggaag tttgacagcc atctagcatt
tcatcacaag 540gcccgagagc tgcatccgga gtactacaaa gactgcgctc
tggccttcaa gcgggttgag 600ccagtagatc ctagactaga gccctggaag
catccaggaa gtcagcctag gaccccttgt 660accaattgct attgtaaaaa
gtgttgcctt cattgccaag tttgtttcac aagaaaaggc 720ttaggcatct
cctatggcag gaagaagcgg agacagcgac gaagagctcc tcaagacagt
780cagactcatc aagtttctct accaaagcaa ccctcctccc agcaacgagg
ggacccgaca 840ggcccgaaga aatccgtacg ggagaagcgg ctgctgagcg
acgaagagct cctcaagaca 900gtcagactca tcaagtttct ctaccaaagc
aaccctcctc ccagcaacga ggggacccga 960caggcccgaa gaaatcgaag
aagaaggtgg agagagagac agaggcagat ccgttcgatt 1020agtgagcgga
ttcttagcac ttttctggga cgacctgcgg agcctgtgcc tcttcagcta
1080ccgccgcttg agagacttac tcttgattgt agcgaagatt gtggaaactc
tgggacgcag 1140ggggtgggaa gtcctcaagt attggtggaa tga
117381161DNAArtificial SequenceProtein comprised of Immunodominant
parts of the regulatory proteins Nef-Tat-Rev started from aa1 of
Nef (N11TR) 8atgtggccta ctgtaaggga aagaatgaaa caagctgagc ctgagccagc
agcagatggg 60gtgggagcag catctcgaga cctggaaaaa catggagcaa tcacaagtag
caatacagca 120actaataacg ctgcttgtgc ctggctagaa gcacaagagg
aagaggaagt gggttttcca 180gtcagacctc aggtaccttt aagaccaatg
acttacaagg gagctttaga tcttagccac 240tttttaaaag aaaagggggg
actggaaggg ttaatttact ccccaaaaag acaagagatc 300cttgatctgt
gggtctacca cacacaaggc tacttccctg attggcagaa ctacacacca
360gggccagggg tcagatatcc actgaccttt ggatggtgct tcaagttagt
accagttgaa 420ccagatgaag aagagaacag cagcctgtta caccctgcga
gcctgcatgg gacagaggac 480acggagagag aagtgttaaa gtggaagttt
gacagccatc tagcatttca tcacaaggcc 540cgagagctgc atccggagta
ctacaaagac tgcgctctgg ccgccgttga gccagtagat 600cctagactag
agccctggaa gcatccagga agtcagccta ggaccccttg taccaattgc
660tattgtaaaa agtgttgcct tcattgccaa gtttgtttca caagaaaagg
cttaggcatc 720tcctatggca ggaagaagcg gagacagcga cgaagagctc
ctcaagacag tcagactcat 780caagtttctc taccaaagca accctcctcc
cagcaacgag gggacccgac aggcccgaag 840aaatccggac tggccatcct
gctgagcgac gaagagctcc tcaagacagt cagactcatc 900aagtttctct
accaaagcaa ccctcctccc agcaacgagg ggacccgaca ggcccgaaga
960aatcgaagaa gaaggtggag agagagacag aggcagatcc gttcgattag
tgagcggatt 1020cttagcactt ttctgggacg acctgcggag cctgtgcctc
ttcagctacc gccgcttgag 1080agacttactc ttgattgtag cgaagattgt
ggaaactctg ggacgcaggg ggtgggaagt 1140cctcaagtat tggtggaatg a
116191170DNAArtificial SequenceProtein comprised of Immunodominant
parts of the regulatory proteins Nef-Tat-Rev started from aa1 of
Nef separated by protease sites (N11TR) 9atgtggccta ctgtaaggga
aagaatgaaa caagctgagc ctgagccagc agcagatggg 60gtgggagcag catctcgaga
cctggaaaaa catggagcaa tcacaagtag caatacagca 120actaataacg
ctgcttgtgc ctggctagaa gcacaagagg aagaggaagt gggttttcca
180gtcagacctc aggtaccttt aagaccaatg acttacaagg gagctttaga
tcttagccac 240tttttaaaag aaaagggggg actggaaggg ttaatttact
ccccaaaaag acaagagatc 300cttgatctgt gggtctacca cacacaaggc
tacttccctg attggcagaa ctacacacca 360gggccagggg tcagatatcc
actgaccttt ggatggtgct tcaagttagt accagttgaa 420ccagatgaag
aagagaacag cagcctgtta caccctgcga gcctgcatgg gacagaggac
480acggagagag aagtgttaaa gtggaagttt gacagccatc tagcatttca
tcacaaggcc 540cgagagctgc atccggagta ctacaaagac tgcgctctgg
ccttcaagcg ggttgagcca 600gtagatccta gactagagcc ctggaagcat
ccaggaagtc agcctaggac cccttgtacc 660aattgctatt gtaaaaagtg
ttgccttcat tgccaagttt gtttcacaag aaaaggctta 720ggcatctcct
atggcaggaa gaagcggaga cagcgacgaa gagctcctca agacagtcag
780actcatcaag tttctctacc aaagcaaccc tcctcccagc aacgagggga
cccgacaggc 840ccgaagaaat ccgtacggga gaagcggctg ctgagcgacg
aagagctcct caagacagtc 900agactcatca agtttctcta ccaaagcaac
cctcctccca gcaacgaggg gacccgacag 960gcccgaagaa atcgaagaag
aaggtggaga gagagacaga ggcagatccg ttcgattagt 1020gagcggattc
ttagcacttt tctgggacga cctgcggagc ctgtgcctct tcagctaccg
1080ccgcttgaga gacttactct tgattgtagc gaagattgtg gaaactctgg
gacgcagggg 1140gtgggaagtc ctcaagtatt ggtggaatga
117010663DNAArtificial SequenceProtein comprised of Cytotoxic
T-cell epitopes of Pol and Env genes (CTL) 10atgatcaccc tgtggcagcg
ccccctggtg gccctgatcg agatctgcac cgagatggag 60aaggagggca agatcagcaa
gatcggcccc gccggcctga agaagaagaa gagcgtgacc 120gtgctggacg
tgggcgacgc ctacttcagc gtgcccctgg ataaggactt ccgcaagtac
180accgccttca ccatccccag catctggaag ggcagccccg ccatcttcca
gagcagcatg 240accaagaagc agaaccccga catcgtgatc taccagtaca
tggacgacct gtacgtgccc 300atcgtgctgc ccgagaagga cagctggctg
gtgggcaagc tgaactgggc cagccagatc 360tacgccggca tcaaggtgaa
gcagctgatc ctgaaggagc ccgtgcacgg cgtgtacgag 420cccatcgtgg
gcgccgagac cttctacgtg gacggcgccg ccaaccgcgc cggcaacctg
480tgggtgaccg tgtactacgg cgtgcccgtg tggaaggagg ccaccaccac
cctggtggag 540cgctacctgc gcgaccagca gctgctgggc atctggggct
gcgcctgcac cccctacgac 600atcaaccaga tgctgcgcgg ccctggccgc
gccttcgtga ccatccgcca gggcagcctg 660tag 663111266DNAArtificial
SequenceTruncated Gag protein sequence (dgag) 11atgttagaca
aatgggaaaa aattcggtta aggccagggg gaaagaaaaa atatcaatta 60aaacatatag
tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
120gaaacatcag aaggctgtag acagataatg ggacagctac aaccgtccct
tcagacagga 180tcagaagaac ttagatcatt atataataca gtagcaaccc
tctattgtgt gcatcaaaag 240atagaggtaa aagacaccaa ggaagcttta
gacaaggtag aggaagagca aaacaacagt 300aagaaaaagg cacagcaaga
agcagctgac gcaggaaaca gaaaccaggt cagccaaaat 360taccctatag
tgcaaaacct acagggacaa atggtacatc aggccatatc acctagaact
420ttaaatgcat gggtaaaagt agtggaagag aaggctttca gcccagaagt
aatacccatg 480ttttcagcat tatcagaagg agccacccca caagatttaa
acaccatgct aaacacagtg 540gggggacatc aagcagccat gcaaatgtta
aaagaaacca tcaatgagga agctgcagaa 600tgggatagat tgcacccagt
gcatgcaggg cctattgcac caggccagat gagagaacca 660aggggaagtg
acatagcagg aactactagt acccttcagg aacaaatagg atggatgaca
720aataatccac ctatcccagt aggagaaata tataagagat ggataatcct
gggattaaat 780aaaatagtaa gaatgtatag ccctaccagc attctggata
taaaacaagg accaaaagaa 840ccctttagag attatgtaga ccggttctat
aaaaccctaa gagccgagca agctacacag 900gaagtaaaaa attggatgac
agaaaccttg ttggtccaaa atgcgaatcc agattgtaag 960actattttaa
aagcattagg accagcagct acactagaag aaatgatgac agcatgtcag
1020ggagtggggg gacccggcca taaagcaaga gttttggctg aagcaatgag
ccaagtaaca 1080ggttcagctg ccataatgat gcagagaggc aattttagga
accaaagaaa gactgttaag 1140tgtttcaatt gtggcaaaga agggcacata
gccagaaatt gcagggcccc taggaaaaag 1200ggctgttgga aatgtggaaa
ggaaggacat caaatgaagg attgcacaga aagacaggct 1260aattag
1266121092DNAArtificial SequenceSynthetic coding sequence for
p17/24 protein of Gag gene (syn 17/24) 12atgggcgcaa gagcctccgt
gctgagcggc ggagagctgg acaagtggga gaagatccgc 60ctgcgccccg gcggcaagaa
gaagtaccag ctgaagcaca tcgtgtgggc cagccgcgag 120ctggagcgct
tcgccgtgaa ccccggcctg ctcgagacca gcgaaggctg ccgccagatc
180atgggccagc tccagcccag cctccagacc ggcagcgagg agctgcgcag
cctgtacaac 240accgtggcca ccctgtactg cgtgcaccag aagatcgagg
tgaaggacac caaggaggcc 300ctggacaagg tggaggagga gcagaacaac
agcaagaaga aggcccagca ggaggccgcc 360gacgccggca accgcaacca
ggtgagccag aactacccca tcgtgcagaa cctgcagggc 420cagatggtgc
accaggccat cagcccccgc accctgaacg cctgggtgaa ggtggtggag
480gagaaggcct tcagccccga ggtgatcccc atgttcagcg ccctgagcga
gggcgctacc 540ccccaggacc tgaacaccat gctgaacacc gtgggcggcc
accaggccgc catgcagatg 600ctgaaggaga ccatcaacga ggaggccgcc
gagtgggacc gcctgcaccc cgtgcacgcc 660gggcccatcg cccccggcca
gatgcgcgag ccccgcggca gcgacatcgc cggcaccacc 720agcaccctcc
aggagcagat cggctggatg accaacaacc cccccatccc cgtgggcgag
780atctacaagc gctggatcat cctgggcctg aacaagatcg tccgcatgta
cagccccacc 840agcatcctgg acatcaagca gggccccaag gagcccttcc
gcgactacgt
ggaccgcttc 900tacaagaccc tgcgcgccga gcaggccacc caggaggtga
agaactggat gaccgagacc 960ctgctggtgc agaacgccaa ccccgactgc
aagaccatcc tcaaggccct gggacccgcc 1020gccaccctgg aggagatgat
gaccgcctgc caaggcgtgg gcggccccgg ccacaaggcc 1080cgcgtgctgt ga
1092131092DNAArtificial SequenceSynthetic coding sequence for
p17/24 protein of Gag gene optimized for expression in eukaryotic
cells (optp17/24) 13atgggcgcaa gagcctccgt gctgagcggc ggagagctgg
acaagtggga gaagatccgc 60ctgcgccccg gcggcaagaa gaagtaccag ctgaagcaca
tcgtgtgggc cagccgcgag 120ctggagcgct tcgccgtgaa ccccggcctg
ctcgagacca gcgaaggctg ccgccagatc 180atgggccagc tccagcccag
cctccagacc ggcagcgagg agctgcgcag cctgtacaac 240accgtggcca
ccctgtactg cgtgcaccag aagatcgagg tgaaggacac caaggaggcc
300ctggacaagg tggaggagga gcagaacaac agcaagaaga aggcccagca
ggaggccgcc 360gacgccggca accgcaacca agtcagccag aactacccca
tcgtgcagaa cctgcagggc 420cagatggtgc accaggccat cagcccccgc
accctgaacg cctgggtgaa ggtggtggag 480gagaaggcct tcagccccga
ggtgatcccc atgttcagcg ccctgagcga gggcgctacc 540ccccaggacc
tgaacaccat gctgaacacc gtgggcggcc accaggccgc catgcagatg
600ctgaaggaga ccatcaacga ggaggccgcc gagtgggacc gcctgcaccc
cgtgcacgcc 660gggcccatcg cccccggcca gatgcgcgag ccccgcggca
gcgacatcgc cggcaccacc 720agcaccctcc aggagcagat cggctggatg
accaacaacc cccccatccc cgtgggcgag 780atctacaagc gctggatcat
cctgggcctg aacaagatcg tccgcatgta cagccccacc 840agcatcctgg
acatcaagca gggccccaag gagcccttcc gcgactacgt ggaccgcttc
900tacaagaccc tgcgcgccga gcaggccacc caggaggtga agaactggat
gaccgagacc 960ctgctggtgc agaacgccaa ccccgactgc aagaccatcc
tcaaggccct gggacccgcc 1020gccaccctgg aggagatgat gaccgcctgc
caaggcgtgg gcggccccgg ccacaaggcc 1080cgcgtgctgt ga
1092141926DNAArtificial SequenceHybrid protein cds comprised of
Tat-Rev-Nef and CTL (TRN-CTL) 14atggagccag tagatcctag actagagccc
tggaagcatc caggaagtca gcctaggacc 60ccttgtacca attgctattg taaaaagtgt
tgccttcatt gccaagtttg tttcacaaga 120aaaggcttag gcatctccta
tggcaggaag aagcggagac agcgacgaag agctcctcaa 180gacagtcaga
ctcatcaagt ttctctacca aagcaaccct cctcccagca acgaggggac
240ccgacaggcc cgaagaaatc gaagaagaag gtggagagag agacagaggc
agatccgttc 300gatactagtg caggaagaag cggagacagc gacgaagagc
tcctcaagac agtcagactc 360atcaagtttc tctaccaaag caaccctcct
cccagcaacg aggggacccg acaggcccga 420agaaatcgaa gaagaaggtg
gagagagaga cagaggcaga tccgttcgat tagtgagcgg 480attcttagca
cttttctggg acgacctgcg gagcctgtgc ctcttcagct accgccgctt
540gagagactta ctcttgattg tagcgaagat tgtggaaact ctgggacgca
gggggtggga 600agtcctcaag tattggtgga atctcctgca gtattggagc
caggaactaa agaaaagctt 660gtgggcaagt ggtcaaaatg tagtggatgg
cctactgtaa gggaaagaat gaaacaagct 720gagcctgagc cagcagcaga
tggggtggga gcagcatctc gagacctgga aaaacatgga 780gcaatcacaa
gtagcaatac agcaactaat aacgctgctt gtgcctggct agaagcacaa
840gaggaagagg aagtgggttt tccagtcaga cctcaggtac ctttaagacc
aatgacttac 900aagggagctt tagatcttag ccacttttta aaagaaaagg
ggggactgga agggttaatt 960tactccccaa aaagacaaga gatccttgat
ctgtgggtct accacacaca aggctacttc 1020cctgattggc agaactacac
accagggcca ggggtcagat atccactgac ctttggatgg 1080tgcttcaagt
tagtaccagt tgaaccagat gaagaagaga acagcagcct gttacaccct
1140gcgagcctgc atgggacaga ggacacggag agagaagtgt taaagtggaa
gtttgacagc 1200catctagcat ttcatcacaa ggcccgagag ctgcatccgg
agtactacaa agactgcgcg 1260gccgtcatca ccctgtggca gcgccccctg
gtggccctga tcgagatctg caccgagatg 1320gagaaggagg gcaagatcag
caagatcggc cccgccggcc tgaagaagaa gaagagcgtg 1380accgtgctgg
acgtgggcga cgcctacttc agcgtgcccc tggataagga cttccgcaag
1440tacaccgcct tcaccatccc cagcatctgg aagggcagcc ccgccatctt
ccagagcagc 1500atgaccaaga agcagaaccc cgacatcgtg atctaccagt
acatggacga cctgtacgtg 1560cccatcgtgc tgcccgagaa ggacagctgg
ctggtgggca agctgaactg ggccagccag 1620atctacgccg gcatcaaggt
gaagcagctg atcctgaagg agcccgtgca cggcgtgtac 1680gagcccatcg
tgggcgccga gaccttctac gtggacggcg ccgccaaccg cgccggcaac
1740ctgtgggtga ccgtgtacta cggcgtgccc gtgtggaagg aggccaccac
caccctggtg 1800gagcgctacc tgcgcgacca gcagctgctg ggcatctggg
gctgcgcctg caccccctac 1860gacatcaacc agatgctgcg cggccctggc
cgcgccttcg tgaccatccg ccagggcagc 1920ctgtag 1926151926DNAArtificial
SequenceHybrid protein cds comprised of Rev-Nef-Tat and CTL
(RNT-CTL) 15atggcaggaa gaagcggaga cagcgacgaa gagctcctca agacagtcag
actcatcaag 60tttctctacc aaagcaaccc tcctcccagc aacgagggga cccgacaggc
ccgaagaaat 120cgaagaagaa ggtggagaga gagacagagg cagatccgtt
cgattagtga gcggattctt 180agcacttttc tgggacgacc tgcggagcct
gtgcctcttc agctaccgcc gcttgagaga 240cttactcttg attgtagcga
agattgtgga aactctggga cgcagggggt gggaagtcct 300caagtattgg
tggaatctcc tgcagtattg gagccaggaa ctaaagaaac tagtgtgggc
360aagtggtcaa aatgtagtgg atggcctact gtaagggaaa gaatgaaaca
agctgagcct 420gagccagcag cagatggggt gggagcagca tctcgagacc
tggaaaaaca tggagcaatc 480acaagtagca atacagcaac taataacgct
gcttgtgcct ggctagaagc acaagaggaa 540gaggaagtgg gttttccagt
cagacctcag gtacctttaa gaccaatgac ttacaaggga 600gctttagatc
ttagccactt tttaaaagaa aaggggggac tggaagggtt aatttactcc
660ccaaaaagac aagagatcct tgatctgtgg gtctaccaca cacaaggcta
cttccctgat 720tggcagaact acacaccagg gccaggggtc agatatccac
tgacctttgg atggtgcttc 780aagttagtac cagttgaacc agatgaagaa
gagaacagca gcctgttaca ccctgcgagc 840ctgcatggga cagaggacac
ggagagagaa gtgttaaagt ggaagtttga cagccatcta 900gcatttcatc
acaaggcccg agagctgcat ccggagtact acaaagactg caagcttgag
960ccagtagatc ctagactaga gccctggaag catccaggaa gtcagcctag
gaccccttgt 1020accaattgct attgtaaaaa gtgttgcctt cattgccaag
tttgtttcac aagaaaaggc 1080ttaggcatct cctatggcag gaagaagcgg
agacagcgac gaagagctcc tcaagacagt 1140cagactcatc aagtttctct
accaaagcaa ccctcctccc agcaacgagg ggacccgaca 1200ggcccgaaga
aatcgaagaa gaaggtggag agagagacag aggcagatcc gttcgatgcg
1260gccgtcatca ccctgtggca gcgccccctg gtggccctga tcgagatctg
caccgagatg 1320gagaaggagg gcaagatcag caagatcggc cccgccggcc
tgaagaagaa gaagagcgtg 1380accgtgctgg acgtgggcga cgcctacttc
agcgtgcccc tggataagga cttccgcaag 1440tacaccgcct tcaccatccc
cagcatctgg aagggcagcc ccgccatctt ccagagcagc 1500atgaccaaga
agcagaaccc cgacatcgtg atctaccagt acatggacga cctgtacgtg
1560cccatcgtgc tgcccgagaa ggacagctgg ctggtgggca agctgaactg
ggccagccag 1620atctacgccg gcatcaaggt gaagcagctg atcctgaagg
agcccgtgca cggcgtgtac 1680gagcccatcg tgggcgccga gaccttctac
gtggacggcg ccgccaaccg cgccggcaac 1740ctgtgggtga ccgtgtacta
cggcgtgccc gtgtggaagg aggccaccac caccctggtg 1800gagcgctacc
tgcgcgacca gcagctgctg ggcatctggg gctgcgcctg caccccctac
1860gacatcaacc agatgctgcg cggccctggc cgcgccttcg tgaccatccg
ccagggcagc 1920ctgtag 1926162529DNAArtificial SequenceHybrid
protein cds comprised of Tat-Rev-Nef and truncated Gag protein
(TRN-dgag) 16atggagccag tagatcctag actagagccc tggaagcatc caggaagtca
gcctaggacc 60ccttgtacca attgctattg taaaaagtgt tgccttcatt gccaagtttg
tttcacaaga 120aaaggcttag gcatctccta tggcaggaag aagcggagac
agcgacgaag agctcctcaa 180gacagtcaga ctcatcaagt ttctctacca
aagcaaccct cctcccagca acgaggggac 240ccgacaggcc cgaagaaatc
gaagaagaag gtggagagag agacagaggc agatccgttc 300gatactagtg
caggaagaag cggagacagc gacgaagagc tcctcaagac agtcagactc
360atcaagtttc tctaccaaag caaccctcct cccagcaacg aggggacccg
acaggcccga 420agaaatcgaa gaagaaggtg gagagagaga cagaggcaga
tccgttcgat tagtgagcgg 480attcttagca cttttctggg acgacctgcg
gagcctgtgc ctcttcagct accgccgctt 540gagagactta ctcttgattg
tagcgaagat tgtggaaact ctgggacgca gggggtggga 600agtcctcaag
tattggtgga atctcctgca gtattggagc caggaactaa agaaaagctt
660gtgggcaagt ggtcaaaatg tagtggatgg cctactgtaa gggaaagaat
gaaacaagct 720gagcctgagc cagcagcaga tggggtggga gcagcatctc
gagacctgga aaaacatgga 780gcaatcacaa gtagcaatac agcaactaat
aacgctgctt gtgcctggct agaagcacaa 840gaggaagagg aagtgggttt
tccagtcaga cctcaggtac ctttaagacc aatgacttac 900aagggagctt
tagatcttag ccacttttta aaagaaaagg ggggactgga agggttaatt
960tactccccaa aaagacaaga gatccttgat ctgtgggtct accacacaca
aggctacttc 1020cctgattggc agaactacac accagggcca ggggtcagat
atccactgac ctttggatgg 1080tgcttcaagt tagtaccagt tgaaccagat
gaagaagaga acagcagcct gttacaccct 1140gcgagcctgc atgggacaga
ggacacggag agagaagtgt taaagtggaa gtttgacagc 1200catctagcat
ttcatcacaa ggcccgagag ctgcatccgg agtactacaa agactgcgcg
1260gccgtgttag acaaatggga aaaaattcgg ttaaggccag ggggaaagaa
aaaatatcaa 1320ttaaaacata tagtatgggc aagcagggag ctagaacgat
tcgcagttaa tcctggcctg 1380ttagaaacat cagaaggctg tagacagata
atgggacagc tacaaccgtc ccttcagaca 1440ggatcagaag aacttagatc
attatataat acagtagcaa ccctctattg tgtgcatcaa 1500aagatagagg
taaaagacac caaggaagct ttagacaagg tagaggaaga gcaaaacaac
1560agtaagaaaa aggcacagca agaagcagct gacgcaggaa acagaaacca
ggtcagccaa 1620aattacccta tagtgcaaaa cctacaggga caaatggtac
atcaggccat atcacctaga 1680actttaaatg catgggtaaa agtagtggaa
gagaaggctt tcagcccaga agtaataccc 1740atgttttcag cattatcaga
aggagccacc ccacaagatt taaacaccat gctaaacaca 1800gtggggggac
atcaagcagc catgcaaatg ttaaaagaaa ccatcaatga ggaagctgca
1860gaatgggata gattgcaccc agtgcatgca gggcctattg caccaggcca
gatgagagaa 1920ccaaggggaa gtgacatagc aggaactact agtacccttc
aggaacaaat aggatggatg 1980acaaataatc cacctatccc agtaggagaa
atatataaga gatggataat cctgggatta 2040aataaaatag taagaatgta
tagccctacc agcattctgg atataaaaca aggaccaaaa 2100gaacccttta
gagattatgt agaccggttc tataaaaccc taagagccga gcaagctaca
2160caggaagtaa aaaattggat gacagaaacc ttgttggtcc aaaatgcgaa
tccagattgt 2220aagactattt taaaagcatt aggaccagca gctacactag
aagaaatgat gacagcatgt 2280cagggagtgg ggggacccgg ccataaagca
agagttttgg ctgaagcaat gagccaagta 2340acaggttcag ctgccataat
gatgcagaga ggcaatttta ggaaccaaag aaagactgtt 2400aagtgtttca
attgtggcaa agaagggcac atagccagaa attgcagggc ccctaggaaa
2460aagggctgtt ggaaatgtgg aaaggaagga catcaaatga aggattgcac
agaaagacag 2520gctaattag 2529173195DNAArtificial SequenceHybrid
protein cds comprised of Tat-Rev-Nef, CTL and truncated Gag protein
(TRN-CTL-dgag) 17atggagccag tagatcctag actagagccc tggaagcatc
caggaagtca gcctaggacc 60ccttgtacca attgctattg taaaaagtgt tgccttcatt
gccaagtttg tttcacaaga 120aaaggcttag gcatctccta tggcaggaag
aagcggagac agcgacgaag agctcctcaa 180gacagtcaga ctcatcaagt
ttctctacca aagcaaccct cctcccagca acgaggggac 240ccgacaggcc
cgaagaaatc gaagaagaag gtggagagag agacagaggc agatccgttc
300gatactagtg caggaagaag cggagacagc gacgaagagc tcctcaagac
agtcagactc 360atcaagtttc tctaccaaag caaccctcct cccagcaacg
aggggacccg acaggcccga 420agaaatcgaa gaagaaggtg gagagagaga
cagaggcaga tccgttcgat tagtgagcgg 480attcttagca cttttctggg
acgacctgcg gagcctgtgc ctcttcagct accgccgctt 540gagagactta
ctcttgattg tagcgaagat tgtggaaact ctgggacgca gggggtggga
600agtcctcaag tattggtgga atctcctgca gtattggagc caggaactaa
agaaaagctt 660gtgggcaagt ggtcaaaatg tagtggatgg cctactgtaa
gggaaagaat gaaacaagct 720gagcctgagc cagcagcaga tggggtggga
gcagcatctc gagacctgga aaaacatgga 780gcaatcacaa gtagcaatac
agcaactaat aacgctgctt gtgcctggct agaagcacaa 840gaggaagagg
aagtgggttt tccagtcaga cctcaggtac ctttaagacc aatgacttac
900aagggagctt tagatcttag ccacttttta aaagaaaagg ggggactgga
agggttaatt 960tactccccaa aaagacaaga gatccttgat ctgtgggtct
accacacaca aggctacttc 1020cctgattggc agaactacac accagggcca
ggggtcagat atccactgac ctttggatgg 1080tgcttcaagt tagtaccagt
tgaaccagat gaagaagaga acagcagcct gttacaccct 1140gcgagcctgc
atgggacaga ggacacggag agagaagtgt taaagtggaa gtttgacagc
1200catctagcat ttcatcacaa ggcccgagag ctgcatccgg agtactacaa
agactgcgcg 1260gccgtcatca ccctgtggca gcgccccctg gtggccctga
tcgagatctg caccgagatg 1320gagaaggagg gcaagatcag caagatcggc
cccgccggcc tgaagaagaa gaagagcgtg 1380accgtgctgg acgtgggcga
cgcctacttc agcgtgcccc tggataagga cttccgcaag 1440tacaccgcct
tcaccatccc cagcatctgg aagggcagcc ccgccatctt ccagagcagc
1500atgaccaaga agcagaaccc cgacatcgtg atctaccagt acatggacga
cctgtacgtg 1560cccatcgtgc tgcccgagaa ggacagctgg ctggtgggca
agctgaactg ggccagccag 1620atctacgccg gcatcaaggt gaagcagctg
atcctgaagg agcccgtgca cggcgtgtac 1680gagcccatcg tgggcgccga
gaccttctac gtggacggcg ccgccaaccg cgccggcaac 1740ctgtgggtga
ccgtgtacta cggcgtgccc gtgtggaagg aggccaccac caccctggtg
1800gagcgctacc tgcgcgacca gcagctgctg ggcatctggg gctgcgcctg
caccccctac 1860gacatcaacc agatgctgcg cggccctggc cgcgccttcg
tgaccatccg ccagggcagc 1920ctggcggccg tgttagacaa atgggaaaaa
attcggttaa ggccaggggg aaagaaaaaa 1980tatcaattaa aacatatagt
atgggcaagc agggagctag aacgattcgc agttaatcct 2040ggcctgttag
aaacatcaga aggctgtaga cagataatgg gacagctaca accgtccctt
2100cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
ctattgtgtg 2160catcaaaaga tagaggtaaa agacaccaag gaagctttag
acaaggtaga ggaagagcaa 2220aacaacagta agaaaaaggc acagcaagaa
gcagctgacg caggaaacag aaaccaggtc 2280agccaaaatt accctatagt
gcaaaaccta cagggacaaa tggtacatca ggccatatca 2340cctagaactt
taaatgcatg ggtaaaagta gtggaagaga aggctttcag cccagaagta
2400atacccatgt tttcagcatt atcagaagga gccaccccac aagatttaaa
caccatgcta 2460aacacagtgg ggggacatca agcagccatg caaatgttaa
aagaaaccat caatgaggaa 2520gctgcagaat gggatagatt gcacccagtg
catgcagggc ctattgcacc aggccagatg 2580agagaaccaa ggggaagtga
catagcagga actactagta cccttcagga acaaatagga 2640tggatgacaa
ataatccacc tatcccagta ggagaaatat ataagagatg gataatcctg
2700ggattaaata aaatagtaag aatgtatagc cctaccagca ttctggatat
aaaacaagga 2760ccaaaagaac cctttagaga ttatgtagac cggttctata
aaaccctaag agccgagcaa 2820gctacacagg aagtaaaaaa ttggatgaca
gaaaccttgt tggtccaaaa tgcgaatcca 2880gattgtaaga ctattttaaa
agcattagga ccagcagcta cactagaaga aatgatgaca 2940gcatgtcagg
gagtgggggg acccggccat aaagcaagag ttttggctga agcaatgagc
3000caagtaacag gttcagctgc cataatgatg cagagaggca attttaggaa
ccaaagaaag 3060actgttaagt gtttcaattg tggcaaagaa gggcacatag
ccagaaattg cagggcccct 3120aggaaaaagg gctgttggaa atgtggaaag
gaaggacatc aaatgaagga ttgcacagaa 3180agacaggcta attag
3195183195DNAArtificial SequenceHybrid protein cds comprised of
Rev-Nef-Tat, CTL and truncated Gag protein (RNT-CTL-dgag)
18atggcaggaa gaagcggaga cagcgacgaa gagctcctca agacagtcag actcatcaag
60tttctctacc aaagcaaccc tcctcccagc aacgagggga cccgacaggc ccgaagaaat
120cgaagaagaa ggtggagaga gagacagagg cagatccgtt cgattagtga
gcggattctt 180agcacttttc tgggacgacc tgcggagcct gtgcctcttc
agctaccgcc gcttgagaga 240cttactcttg attgtagcga agattgtgga
aactctggga cgcagggggt gggaagtcct 300caagtattgg tggaatctcc
tgcagtattg gagccaggaa ctaaagaaac tagtgtgggc 360aagtggtcaa
aatgtagtgg atggcctact gtaagggaaa gaatgaaaca agctgagcct
420gagccagcag cagatggggt gggagcagca tctcgagacc tggaaaaaca
tggagcaatc 480acaagtagca atacagcaac taataacgct gcttgtgcct
ggctagaagc acaagaggaa 540gaggaagtgg gttttccagt cagacctcag
gtacctttaa gaccaatgac ttacaaggga 600gctttagatc ttagccactt
tttaaaagaa aaggggggac tggaagggtt aatttactcc 660ccaaaaagac
aagagatcct tgatctgtgg gtctaccaca cacaaggcta cttccctgat
720tggcagaact acacaccagg gccaggggtc agatatccac tgacctttgg
atggtgcttc 780aagttagtac cagttgaacc agatgaagaa gagaacagca
gcctgttaca ccctgcgagc 840ctgcatggga cagaggacac ggagagagaa
gtgttaaagt ggaagtttga cagccatcta 900gcatttcatc acaaggcccg
agagctgcat ccggagtact acaaagactg caagcttgag 960ccagtagatc
ctagactaga gccctggaag catccaggaa gtcagcctag gaccccttgt
1020accaattgct attgtaaaaa gtgttgcctt cattgccaag tttgtttcac
aagaaaaggc 1080ttaggcatct cctatggcag gaagaagcgg agacagcgac
gaagagctcc tcaagacagt 1140cagactcatc aagtttctct accaaagcaa
ccctcctccc agcaacgagg ggacccgaca 1200ggcccgaaga aatcgaagaa
gaaggtggag agagagacag aggcagatcc gttcgatgcg 1260gccgtcatca
ccctgtggca gcgccccctg gtggccctga tcgagatctg caccgagatg
1320gagaaggagg gcaagatcag caagatcggc cccgccggcc tgaagaagaa
gaagagcgtg 1380accgtgctgg acgtgggcga cgcctacttc agcgtgcccc
tggataagga cttccgcaag 1440tacaccgcct tcaccatccc cagcatctgg
aagggcagcc ccgccatctt ccagagcagc 1500atgaccaaga agcagaaccc
cgacatcgtg atctaccagt acatggacga cctgtacgtg 1560cccatcgtgc
tgcccgagaa ggacagctgg ctggtgggca agctgaactg ggccagccag
1620atctacgccg gcatcaaggt gaagcagctg atcctgaagg agcccgtgca
cggcgtgtac 1680gagcccatcg tgggcgccga gaccttctac gtggacggcg
ccgccaaccg cgccggcaac 1740ctgtgggtga ccgtgtacta cggcgtgccc
gtgtggaagg aggccaccac caccctggtg 1800gagcgctacc tgcgcgacca
gcagctgctg ggcatctggg gctgcgcctg caccccctac 1860gacatcaacc
agatgctgcg cggccctggc cgcgccttcg tgaccatccg ccagggcagc
1920ctggcggccg tgttagacaa atgggaaaaa attcggttaa ggccaggggg
aaagaaaaaa 1980tatcaattaa aacatatagt atgggcaagc agggagctag
aacgattcgc agttaatcct 2040ggcctgttag aaacatcaga aggctgtaga
cagataatgg gacagctaca accgtccctt 2100cagacaggat cagaagaact
tagatcatta tataatacag tagcaaccct ctattgtgtg 2160catcaaaaga
tagaggtaaa agacaccaag gaagctttag acaaggtaga ggaagagcaa
2220aacaacagta agaaaaaggc acagcaagaa gcagctgacg caggaaacag
aaaccaggtc 2280agccaaaatt accctatagt gcaaaaccta cagggacaaa
tggtacatca ggccatatca 2340cctagaactt taaatgcatg ggtaaaagta
gtggaagaga aggctttcag cccagaagta 2400atacccatgt tttcagcatt
atcagaagga gccaccccac aagatttaaa caccatgcta 2460aacacagtgg
ggggacatca agcagccatg caaatgttaa aagaaaccat caatgaggaa
2520gctgcagaat gggatagatt gcacccagtg catgcagggc ctattgcacc
aggccagatg 2580agagaaccaa ggggaagtga catagcagga actactagta
cccttcagga acaaatagga 2640tggatgacaa ataatccacc tatcccagta
ggagaaatat ataagagatg gataatcctg 2700ggattaaata aaatagtaag
aatgtatagc cctaccagca ttctggatat aaaacaagga 2760ccaaaagaac
cctttagaga ttatgtagac cggttctata aaaccctaag agccgagcaa
2820gctacacagg aagtaaaaaa ttggatgaca gaaaccttgt tggtccaaaa
tgcgaatcca 2880gattgtaaga ctattttaaa agcattagga ccagcagcta
cactagaaga aatgatgaca 2940gcatgtcagg gagtgggggg acccggccat
aaagcaagag ttttggctga agcaatgagc 3000caagtaacag gttcagctgc
cataatgatg cagagaggca attttaggaa ccaaagaaag 3060actgttaagt
gtttcaattg tggcaaagaa gggcacatag ccagaaattg cagggcccct
3120aggaaaaagg gctgttggaa atgtggaaag gaaggacatc aaatgaagga
ttgcacagaa 3180agacaggcta attag
3195193195DNAArtificial SequenceHybrid protein cds comprised of
Tat-Rev-Nef, truncated Gag protein and CTL (TRN-dgag-CTL)
19atggagccag tagatcctag actagagccc tggaagcatc caggaagtca gcctaggacc
60ccttgtacca attgctattg taaaaagtgt tgccttcatt gccaagtttg tttcacaaga
120aaaggcttag gcatctccta tggcaggaag aagcggagac agcgacgaag
agctcctcaa 180gacagtcaga ctcatcaagt ttctctacca aagcaaccct
cctcccagca acgaggggac 240ccgacaggcc cgaagaaatc gaagaagaag
gtggagagag agacagaggc agatccgttc 300gatactagtg caggaagaag
cggagacagc gacgaagagc tcctcaagac agtcagactc 360atcaagtttc
tctaccaaag caaccctcct cccagcaacg aggggacccg acaggcccga
420agaaatcgaa gaagaaggtg gagagagaga cagaggcaga tccgttcgat
tagtgagcgg 480attcttagca cttttctggg acgacctgcg gagcctgtgc
ctcttcagct accgccgctt 540gagagactta ctcttgattg tagcgaagat
tgtggaaact ctgggacgca gggggtggga 600agtcctcaag tattggtgga
atctcctgca gtattggagc caggaactaa agaaaagctt 660gtgggcaagt
ggtcaaaatg tagtggatgg cctactgtaa gggaaagaat gaaacaagct
720gagcctgagc cagcagcaga tggggtggga gcagcatctc gagacctgga
aaaacatgga 780gcaatcacaa gtagcaatac agcaactaat aacgctgctt
gtgcctggct agaagcacaa 840gaggaagagg aagtgggttt tccagtcaga
cctcaggtac ctttaagacc aatgacttac 900aagggagctt tagatcttag
ccacttttta aaagaaaagg ggggactgga agggttaatt 960tactccccaa
aaagacaaga gatccttgat ctgtgggtct accacacaca aggctacttc
1020cctgattggc agaactacac accagggcca ggggtcagat atccactgac
ctttggatgg 1080tgcttcaagt tagtaccagt tgaaccagat gaagaagaga
acagcagcct gttacaccct 1140gcgagcctgc atgggacaga ggacacggag
agagaagtgt taaagtggaa gtttgacagc 1200catctagcat ttcatcacaa
ggcccgagag ctgcatccgg agtactacaa agactgcgcg 1260gccgtgttag
acaaatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatatcaa
1320ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
tcctggcctg 1380ttagaaacat cagaaggctg tagacagata atgggacagc
tacaaccgtc ccttcagaca 1440ggatcagaag aacttagatc attatataat
acagtagcaa ccctctattg tgtgcatcaa 1500aagatagagg taaaagacac
caaggaagct ttagacaagg tagaggaaga gcaaaacaac 1560agtaagaaaa
aggcacagca agaagcagct gacgcaggaa acagaaacca ggtcagccaa
1620aattacccta tagtgcaaaa cctacaggga caaatggtac atcaggccat
atcacctaga 1680actttaaatg catgggtaaa agtagtggaa gagaaggctt
tcagcccaga agtaataccc 1740atgttttcag cattatcaga aggagccacc
ccacaagatt taaacaccat gctaaacaca 1800gtggggggac atcaagcagc
catgcaaatg ttaaaagaaa ccatcaatga ggaagctgca 1860gaatgggata
gattgcaccc agtgcatgca gggcctattg caccaggcca gatgagagaa
1920ccaaggggaa gtgacatagc aggaactact agtacccttc aggaacaaat
aggatggatg 1980acaaataatc cacctatccc agtaggagaa atatataaga
gatggataat cctgggatta 2040aataaaatag taagaatgta tagccctacc
agcattctgg atataaaaca aggaccaaaa 2100gaacccttta gagattatgt
agaccggttc tataaaaccc taagagccga gcaagctaca 2160caggaagtaa
aaaattggat gacagaaacc ttgttggtcc aaaatgcgaa tccagattgt
2220aagactattt taaaagcatt aggaccagca gctacactag aagaaatgat
gacagcatgt 2280cagggagtgg ggggacccgg ccataaagca agagttttgg
ctgaagcaat gagccaagta 2340acaggttcag ctgccataat gatgcagaga
ggcaatttta ggaaccaaag aaagactgtt 2400aagtgtttca attgtggcaa
agaagggcac atagccagaa attgcagggc ccctaggaaa 2460aagggctgtt
ggaaatgtgg aaaggaagga catcaaatga aggattgcac agaaagacag
2520gctaatgcgg ccgtcatcac cctgtggcag cgccccctgg tggccctgat
cgagatctgc 2580accgagatgg agaaggaggg caagatcagc aagatcggcc
ccgccggcct gaagaagaag 2640aagagcgtga ccgtgctgga cgtgggcgac
gcctacttca gcgtgcccct ggataaggac 2700ttccgcaagt acaccgcctt
caccatcccc agcatctgga agggcagccc cgccatcttc 2760cagagcagca
tgaccaagaa gcagaacccc gacatcgtga tctaccagta catggacgac
2820ctgtacgtgc ccatcgtgct gcccgagaag gacagctggc tggtgggcaa
gctgaactgg 2880gccagccaga tctacgccgg catcaaggtg aagcagctga
tcctgaagga gcccgtgcac 2940ggcgtgtacg agcccatcgt gggcgccgag
accttctacg tggacggcgc cgccaaccgc 3000gccggcaacc tgtgggtgac
cgtgtactac ggcgtgcccg tgtggaagga ggccaccacc 3060accctggtgg
agcgctacct gcgcgaccag cagctgctgg gcatctgggg ctgcgcctgc
3120accccctacg acatcaacca gatgctgcgc ggccctggcc gcgccttcgt
gaccatccgc 3180cagggcagcc tgtag 3195203195DNAArtificial
SequenceHybrid protein cds comprised of Rev-Nef-Tat, truncated Gag
protein and CTL (RNT-dgag-CTL) 20atggcaggaa gaagcggaga cagcgacgaa
gagctcctca agacagtcag actcatcaag 60tttctctacc aaagcaaccc tcctcccagc
aacgagggga cccgacaggc ccgaagaaat 120cgaagaagaa ggtggagaga
gagacagagg cagatccgtt cgattagtga gcggattctt 180agcacttttc
tgggacgacc tgcggagcct gtgcctcttc agctaccgcc gcttgagaga
240cttactcttg attgtagcga agattgtgga aactctggga cgcagggggt
gggaagtcct 300caagtattgg tggaatctcc tgcagtattg gagccaggaa
ctaaagaaac tagtgtgggc 360aagtggtcaa aatgtagtgg atggcctact
gtaagggaaa gaatgaaaca agctgagcct 420gagccagcag cagatggggt
gggagcagca tctcgagacc tggaaaaaca tggagcaatc 480acaagtagca
atacagcaac taataacgct gcttgtgcct ggctagaagc acaagaggaa
540gaggaagtgg gttttccagt cagacctcag gtacctttaa gaccaatgac
ttacaaggga 600gctttagatc ttagccactt tttaaaagaa aaggggggac
tggaagggtt aatttactcc 660ccaaaaagac aagagatcct tgatctgtgg
gtctaccaca cacaaggcta cttccctgat 720tggcagaact acacaccagg
gccaggggtc agatatccac tgacctttgg atggtgcttc 780aagttagtac
cagttgaacc agatgaagaa gagaacagca gcctgttaca ccctgcgagc
840ctgcatggga cagaggacac ggagagagaa gtgttaaagt ggaagtttga
cagccatcta 900gcatttcatc acaaggcccg agagctgcat ccggagtact
acaaagactg caagcttgag 960ccagtagatc ctagactaga gccctggaag
catccaggaa gtcagcctag gaccccttgt 1020accaattgct attgtaaaaa
gtgttgcctt cattgccaag tttgtttcac aagaaaaggc 1080ttaggcatct
cctatggcag gaagaagcgg agacagcgac gaagagctcc tcaagacagt
1140cagactcatc aagtttctct accaaagcaa ccctcctccc agcaacgagg
ggacccgaca 1200ggcccgaaga aatcgaagaa gaaggtggag agagagacag
aggcagatcc gttcgatgcg 1260gccgtgttag acaaatggga aaaaattcgg
ttaaggccag ggggaaagaa aaaatatcaa 1320ttaaaacata tagtatgggc
aagcagggag ctagaacgat tcgcagttaa tcctggcctg 1380ttagaaacat
cagaaggctg tagacagata atgggacagc tacaaccgtc ccttcagaca
1440ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
tgtgcatcaa 1500aagatagagg taaaagacac caaggaagct ttagacaagg
tagaggaaga gcaaaacaac 1560agtaagaaaa aggcacagca agaagcagct
gacgcaggaa acagaaacca ggtcagccaa 1620aattacccta tagtgcaaaa
cctacaggga caaatggtac atcaggccat atcacctaga 1680actttaaatg
catgggtaaa agtagtggaa gagaaggctt tcagcccaga agtaataccc
1740atgttttcag cattatcaga aggagccacc ccacaagatt taaacaccat
gctaaacaca 1800gtggggggac atcaagcagc catgcaaatg ttaaaagaaa
ccatcaatga ggaagctgca 1860gaatgggata gattgcaccc agtgcatgca
gggcctattg caccaggcca gatgagagaa 1920ccaaggggaa gtgacatagc
aggaactact agtacccttc aggaacaaat aggatggatg 1980acaaataatc
cacctatccc agtaggagaa atatataaga gatggataat cctgggatta
2040aataaaatag taagaatgta tagccctacc agcattctgg atataaaaca
aggaccaaaa 2100gaacccttta gagattatgt agaccggttc tataaaaccc
taagagccga gcaagctaca 2160caggaagtaa aaaattggat gacagaaacc
ttgttggtcc aaaatgcgaa tccagattgt 2220aagactattt taaaagcatt
aggaccagca gctacactag aagaaatgat gacagcatgt 2280cagggagtgg
ggggacccgg ccataaagca agagttttgg ctgaagcaat gagccaagta
2340acaggttcag ctgccataat gatgcagaga ggcaatttta ggaaccaaag
aaagactgtt 2400aagtgtttca attgtggcaa agaagggcac atagccagaa
attgcagggc ccctaggaaa 2460aagggctgtt ggaaatgtgg aaaggaagga
catcaaatga aggattgcac agaaagacag 2520gctaatgcgg ccgtcatcac
cctgtggcag cgccccctgg tggccctgat cgagatctgc 2580accgagatgg
agaaggaggg caagatcagc aagatcggcc ccgccggcct gaagaagaag
2640aagagcgtga ccgtgctgga cgtgggcgac gcctacttca gcgtgcccct
ggataaggac 2700ttccgcaagt acaccgcctt caccatcccc agcatctgga
agggcagccc cgccatcttc 2760cagagcagca tgaccaagaa gcagaacccc
gacatcgtga tctaccagta catggacgac 2820ctgtacgtgc ccatcgtgct
gcccgagaag gacagctggc tggtgggcaa gctgaactgg 2880gccagccaga
tctacgccgg catcaaggtg aagcagctga tcctgaagga gcccgtgcac
2940ggcgtgtacg agcccatcgt gggcgccgag accttctacg tggacggcgc
cgccaaccgc 3000gccggcaacc tgtgggtgac cgtgtactac ggcgtgcccg
tgtggaagga ggccaccacc 3060accctggtgg agcgctacct gcgcgaccag
cagctgctgg gcatctgggg ctgcgcctgc 3120accccctacg acatcaacca
gatgctgcgc ggccctggcc gcgccttcgt gaccatccgc 3180cagggcagcc tgtag
3195213020DNAArtificial SequenceHybrid protein cds comprised of
Tat-Rev-Nef, truncated Gag protein and CTL (TRN-optp17/24-CTL)
21atggagccag tagatcctag actagagccc tggaagcatc caggaagtca gcctaggacc
60ccttgtacca attgctattg taaaaagtgt tgccttcatt gccaagtttg tttcacaaga
120aaaggcttag gcatctccta tggcaggaag aagcggagac agcgacgaag
agctcctcaa 180gacagtcaga ctcatcaagt ttctctacca aagcaaccct
cctcccagca acgaggggac 240ccgacaggcc cgaagaaatc gaagaagaag
gtggagagag agacagaggc agatccgttc 300gatactagtg caggaagaag
cggagacagc gacgaagagc tcctcaagac agtcagactc 360atcaagtttc
tctaccaaag caaccctcct cccagcaacg aggggacccg acaggcccga
420agaaatcgaa gaagaaggtg gagagagaga cagaggcaga tccgttcgat
tagtgagcgg 480attcttagca cttttctggg acgacctgcg gagcctgtgc
ctcttcagct accgccgctt 540gagagactta ctcttgattg tagcgaagat
tgtggaaact ctgggacgca gggggtggga 600agtcctcaag tattggtgga
atctcctgca gtattggagc caggaactaa agaaaagctt 660gttggcaagt
ggtcaaaatg tagtggatgg cctactgtaa gggaaagaat gaaacaagct
720gagcctgagc cagcagcaga tggggtggga gcagcatctc gagacctgga
aaaacatgga 780gcaatcacaa gtagcaatac agcaactaat aacgctgctt
gtgcctggct agaagcacaa 840gaggaagagg aagtgggttt tccagtcaga
cctcaggtac ctttaagacc aatgacttac 900aagggagctt tagatcttag
ccacttttta aaagaaaagg ggggactgga agggttaatt 960tactccccaa
aaagacaaga gatccttgat ctgtgggtct accacacaca aggctacttc
1020cctgattggc agaactacac accagggcca ggggtcagat atccactgac
ctttggatgg 1080tgcttcaagt tagtaccagt tgaaccagat gaagaagaga
acagcagcct gttacaccct 1140gcgagcctgc atgggacaga ggacacggag
agagaagtgt taaagtggaa gtttgacagc 1200catctagcat ttcatcacaa
ggcccgagag ctgcatccgg agtactacaa agactgcgcg 1260gccgtgggcg
caagagcctc cgtgctgagc ggcggagagc tggacaagtg ggagaagatc
1320cgcctgcgcc ccggcggcaa gaagaagtac cagctgaagc acatcgtgtg
ggccagccgc 1380gagctggagc gcttcgccgt gaaccccggc ctgctcgaga
ccagcgaagg ctgccgccag 1440atcatgggcc agctccagcc cagcctccag
accggcagcg aggagctgcg cagcctgtac 1500aacaccgtgg ccaccctgta
ctgcgtgcac cagaagatcg aggtgaagga caccaaggag 1560gccctggaca
aggtggagga ggagcagaac aacagcaaga agaaggccca gcaggaggcc
1620gccgacgccg gcaaccgcaa ccaagtcagc cagaactacc ccatcgtgca
gaacctgcag 1680ggccagatgg tgcaccaggc catcagcccc cgcaccctga
acgcctgggt gaaggtggtg 1740gaggagaagg ccttcagccc cgaggtgatc
cccatgttca gcgccctaag cgagggcgct 1800accccccagg acctgaacac
catgctgaac accgtgggcg gccaccaggc cgccatgcag 1860atgctgaagg
agaccatcaa cgaggaggcc gccgagtggg accgcctgca ccccgtgcac
1920gccgggccca tcgcccccgg ccagatgcgc gagccccgcg gcagcgacat
cgccggcacc 1980accagcaccc tccaggagca gatcggctgg atgaccaaca
acccccccat ccccgtgggc 2040gagatctaca agcgctggat catcctgggc
ctgaacaaga tcgtccgcat gtacagcccc 2100accagcatcc tggacatcaa
gcagggcccc aaggagccct tccgcgacta cgtggaccgc 2160ttctacaaga
ccctgcgcgc cgagcaggcc acccaggagg tgaagaactg gatgaccgag
2220accctgctgg tgcagaacgc caaccccgac tgcaagacca tcctcaaggc
cctgggaccc 2280gccgccaccc tggaggagat gatgaccgcc tgccaaggcg
tgggcggccc cggccacaag 2340gcccgcgtgc tggcggccgt catcaccctg
tggcagcgcc ccctggtggc cctgatcgag 2400atctgcaccg agatggagaa
ggagggcaag atcagcaaga tcggccccgc cggcctgaag 2460aagaagaaga
gcgtgaccgt gctggacgtg ggcgacgcct acttcagcgt gcccctggat
2520aaggacttcc gcaagtacac cgccttcacc atccccagca tctggaaggg
cagccccgcc 2580atcttccaga gcagcatgac caagaagcag aaccccgaca
tcgtgatcta ccagtacatg 2640gacgacctgt acgtgcccat cgtgctgccc
gagaaggaca gctggctggt gggcaagctg 2700aactgggcca gccagatcta
cgccggcatc aaggtgaagc agctgatcct gaaggagccc 2760gtgcacggcg
tgtacgagcc catcgtgggc gccgagacct tctacgtgga cggcgccgcc
2820aaccgcgccg gcaacctgtg ggtgaccgtg tactacggcg tgcccgtgtg
gaaggaggcc 2880accaccaccc tggtggagcg ctacctgcgc gaccagcagc
tgctgggcat ctggggctgc 2940gcctgcaccc cctacgacat caaccagatg
ctgcgcggcc ctggccgcgc ctcgtgacca 3000tccgccaggg cagcctgtag
3020223021DNAArtificial SequenceHybrid protein cdscomprised of
Tat-Rev-Nef, CTL and truncated Gag protein (TRN-CTL-optp17/24)
22atggagccag tagatcctag actagagccc tggaagcatc caggaagtca gcctaggacc
60ccttgtacca attgctattg taaaaagtgt tgccttcatt gccaagtttg tttcacaaga
120aaaggcttag gcatctccta tggcaggaag aagcggagac agcgacgaag
agctcctcaa 180gacagtcaga ctcatcaagt ttctctacca aagcaaccct
cctcccagca acgaggggac 240ccgacaggcc cgaagaaatc gaagaagaag
gtggagagag agacagaggc agatccgttc 300gatactagtg caggaagaag
cggagacagc gacgaagagc tcctcaagac agtcagactc 360atcaagtttc
tctaccaaag caaccctcct cccagcaacg aggggacccg acaggcccga
420agaaatcgaa gaagaaggtg gagagagaga cagaggcaga tccgttcgat
tagtgagcgg 480attcttagca cttttctggg acgacctgcg gagcctgtgc
ctcttcagct accgccgctt 540gagagactta ctcttgattg tagcgaagat
tgtggaaact ctgggacgca gggggtggga 600agtcctcaag tattggtgga
atctcctgca gtattggagc caggaactaa agaaaagctt 660gtgggcaagt
ggtcaaaatg tagtggatgg cctactgtaa gggaaagaat gaaacaagct
720gagcctgagc cagcagcaga tggggtggga gcagcatctc gagacctgga
aaaacatgga 780gcaatcacaa gtagcaatac agcaactaat aacgctgctt
gtgcctggct agaagcacaa 840gaggaagagg aagtgggttt tccagtcaga
cctcaggtac ctttaagacc aatgacttac 900aagggagctt tagatcttag
ccacttttta aaagaaaagg ggggactgga agggttaatt 960tactccccaa
aaagacaaga gatccttgat ctgtgggtct accacacaca aggctacttc
1020cctgattggc agaactacac accagggcca ggggtcagat atccactgac
ctttggatgg 1080tgcttcaagt tagtaccagt tgaaccagat gaagaagaga
acagcagcct gttacaccct 1140gcgagcctgc atgggacaga ggacacggag
agagaagtgt taaagtggaa gtttgacagc 1200catctagcat ttcatcacaa
ggcccgagag ctgcatccgg agtactacaa agactgcgcg 1260gccgtcatca
ccctgtggca gcgccccctg gtggccctga tcgagatctg caccgagatg
1320gagaaggagg gcaagatcag caagatcggc cccgccggcc tgaagaagaa
gaagagcgtg 1380accgtgctgg acgtgggcga cgcctacttc agcgtgcccc
tggataagga cttccgcaag 1440tacaccgcct tcaccatccc cagcatctgg
aagggcagcc ccgccatctt ccagagcagc 1500atgaccaaga agcagaaccc
cgacatcgtg atctaccagt acatggacga cctgtacgtg 1560cccatcgtgc
tgcccgagaa ggacagctgg ctggtgggca agctgaactg ggccagccag
1620atctacgccg gcatcaaggt gaagcagctg atcctgaagg agcccgtgca
cggcgtgtac 1680gagcccatcg tgggcgccga gaccttctac gtggacggcg
ccgccaaccg cgccggcaac 1740ctgtgggtga ccgtgtacta cggcgtgccc
gtgtggaagg aggccaccac caccctggtg 1800gagcgctacc tgcgcgacca
gcagctgctg ggcatctggg gctgcgcctg caccccctac 1860gacatcaacc
agatgctgcg cggccctggc cgcgccttcg tgaccatccg ccagggcagc
1920ctggcggccg tgggcgcaag agcctccgtg ctgagcggcg gagagctgga
caagtgggag 1980aagatccgcc tgcgccccgg cggcaagaag aagtaccagc
tgaagcacat cgtgtgggcc 2040agccgcgagc tggagcgctt cgccgtgaac
cccggcctgc tcgagaccag cgaaggctgc 2100cgccagatca tgggccagct
ccagcccagc ctccagaccg gcagcgagga gctgcgcagc 2160ctgtacaaca
ccgtggccac cctgtactgc gtgcaccaga agatcgaggt gaaggacacc
2220aaggaggccc tggacaaggt ggaggaggag cagaacaaca gcaagaagaa
ggcccagcag 2280gaggccgccg acgccggcaa ccgcaaccaa gtcagccaga
actaccccat cgtgcagaac 2340ctgcagggcc agatggtgca ccaggccatc
agcccccgca ccctgaacgc ctgggtgaag 2400gtggtggagg agaaggcctt
cagccccgag gtgatcccca tgttcagcgc cctgagcgag 2460ggcgctaccc
cccaggacct gaacaccatg ctgaacaccg tgggcggcca ccaggccgcc
2520atgcagatgc tgaaggagac catcaacgag gaggccgccg agtgggaccg
cctgcacccc 2580gtgcacgccg ggcccatcgc ccccggccag atgcgcgagc
cccgcggcag cgacatcgcc 2640ggcaccacca gcaccctcca ggagcagatc
ggctggatga ccaacaaccc ccccatcccc 2700gtgggcgaga tctacaagcg
ctggatcatc ctgggcctga acaagatcgt ccgcatgtac 2760agccccacca
gcatcctgga catcaagcag ggccccaagg agcccttccg cgactacgtg
2820gaccgcttct acaagaccct gcgcgccgag caggccaccc aggaggtgaa
gaactggatg 2880accgagaccc tgctggtgca gaacgccaac cccgactgca
agaccatcct caaggccctg 2940ggacccgccg ccaccctgga ggagatgatg
accgcctgcc aaggcgtggg cggccccggc 3000cacaaggccc gcgtgctgtg a
3021233021DNAArtificial SequenceHybrid protein cds comprised of
Rev-Nef-Tat, CTL and truncated Gag protein (RNT-CTL-optp17/24)
23atggcaggaa gaagcggaga cagcgacgaa gagctcctca agacagtcag actcatcaag
60tttctctacc aaagcaaccc tcctcccagc aacgagggga cccgacaggc ccgaagaaat
120cgaagaagaa ggtggagaga gagacagagg cagatccgtt cgattagtga
gcggattctt 180agcacttttc tgggacgacc tgcggagcct gtgcctcttc
agctaccgcc gcttgagaga 240cttactcttg attgtagcga agattgtgga
aactctggga cgcagggggt gggaagtcct 300caagtattgg tggaatctcc
tgcagtattg gagccaggaa ctaaagaaac tagtgtgggc 360aagtggtcaa
aatgtagtgg atggcctact gtaagggaaa gaatgaaaca agctgagcct
420gagccagcag cagatggggt gggagcagca tctcgagacc tggaaaaaca
tggagcaatc 480acaagtagca atacagcaac taataacgct gcttgtgcct
ggctagaagc acaagaggaa 540gaggaagtgg gttttccagt cagacctcag
gtacctttaa gaccaatgac ttacaaggga 600gctttagatc ttagccactt
tttaaaagaa aaggggggac tggaagggtt aatttactcc 660ccaaaaagac
aagagatcct tgatctgtgg gtctaccaca cacaaggcta cttccctgat
720tggcagaact acacaccagg gccaggggtc agatatccac tgacctttgg
atggtgcttc 780aagttagtac cagttgaacc agatgaagaa gagaacagca
gcctgttaca ccctgcgagc 840ctgcatggga cagaggacac ggagagagaa
gtgttaaagt ggaagtttga cagccatcta 900gcatttcatc acaaggcccg
agagctgcat ccggagtact acaaagactg caagcttgag 960ccagtagatc
ctagactaga gccctggaag catccaggaa gtcagcctag gaccccttgt
1020accaattgct attgtaaaaa gtgttgcctt cattgccaag tttgtttcac
aagaaaaggc 1080ttaggcatct cctatggcag gaagaagcgg agacagcgac
gaagagctcc tcaagacagt 1140cagactcatc aagtttctct accaaagcaa
ccctcctccc agcaacgagg ggacccgaca 1200ggcccgaaga aatcgaagaa
gaaggtggag agagagacag aggcagatcc gttcgatgcg 1260gccgtcatca
ccctgtggca gcgccccctg gtggccctga tcgagatctg caccgagatg
1320gagaaggagg gcaagatcag caagatcggc cccgccggcc tgaagaagaa
gaagagcgtg 1380accgtgctgg acgtgggcga cgcctacttc agcgtgcccc
tggataagga cttccgcaag 1440tacaccgcct tcaccatccc cagcatctgg
aagggcagcc ccgccatctt ccagagcagc 1500atgaccaaga agcagaaccc
cgacatcgtg atctaccagt acatggacga cctgtacgtg 1560cccatcgtgc
tgcccgagaa ggacagctgg ctggtgggca agctgaactg ggccagccag
1620atctacgccg gcatcaaggt gaagcagctg atcctgaagg agcccgtgca
cggcgtgtac 1680gagcccatcg tgggcgccga gaccttctac gtggacggcg
ccgccaaccg cgccggcaac 1740ctgtgggtga ccgtgtacta cggcgtgccc
gtgtggaagg aggccaccac caccctggtg 1800gagcgctacc tgcgcgacca
gcagctgctg ggcatctggg gctgcgcctg caccccctac
1860gacatcaacc agatgctgcg cggccctggc cgcgccttcg tgaccatccg
ccagggcagc 1920ctggcggccg tgggcgcaag agcctccgtg ctgagcggcg
gagagctgga caagtgggag 1980aagatccgcc tgcgccccgg cggcaagaag
aagtaccagc tgaagcacat cgtgtgggcc 2040agccgcgagc tggagcgctt
cgccgtgaac cccggcctgc tcgagaccag cgaaggctgc 2100cgccagatca
tgggccagct ccagcccagc ctccagaccg gcagcgagga gctgcgcagc
2160ctgtacaaca ccgtggccac cctgtactgc gtgcaccaga agatcgaggt
gaaggacacc 2220aaggaggccc tggacaaggt ggaggaggag cagaacaaca
gcaagaagaa ggcccagcag 2280gaggccgccg acgccggcaa ccgcaaccaa
gtcagccaga actaccccat cgtgcagaac 2340ctgcagggcc agatggtgca
ccaggccatc agcccccgca ccctgaacgc ctgggtgaag 2400gtggtggagg
agaaggcctt cagccccgag gtgatcccca tgttcagcgc cctgagcgag
2460ggcgctaccc cccaggacct gaacaccatg ctgaacaccg tgggcggcca
ccaggccgcc 2520atgcagatgc tgaaggagac catcaacgag gaggccgccg
agtgggaccg cctgcacccc 2580gtgcacgccg ggcccatcgc ccccggccag
atgcgcgagc cccgcggcag cgacatcgcc 2640ggcaccacca gcaccctcca
ggagcagatc ggctggatga ccaacaaccc ccccatcccc 2700gtgggcgaga
tctacaagcg ctggatcatc ctgggcctga acaagatcgt ccgcatgtac
2760agccccacca gcatcctgga catcaagcag ggccccaagg agcccttccg
cgactacgtg 2820gaccgcttct acaagaccct gcgcgccgag caggccaccc
aggaggtgaa gaactggatg 2880accgagaccc tgctggtgca gaacgccaac
cccgactgca agaccatcct caaggccctg 2940ggacccgccg ccaccctgga
ggagatgatg accgcctgcc aaggcgtggg cggccccggc 3000cacaaggccc
gcgtgctgtg a 3021243021DNAArtificial SequenceHybrid protein cds
comprised of Rev-Nef-Tat, truncated Gag protein and CTL
(RNT-optp17/24-CTL) 24atggcaggaa gaagcggaga cagcgacgaa gagctcctca
agacagtcag actcatcaag 60tttctctacc aaagcaaccc tcctcccagc aacgagggga
cccgacaggc ccgaagaaat 120cgaagaagaa ggtggagaga gagacagagg
cagatccgtt cgattagtga gcggattctt 180agcacttttc tgggacgacc
tgcggagcct gtgcctcttc agctaccgcc gcttgagaga 240cttactcttg
attgtagcga agattgtgga aactctggga cgcagggggt gggaagtcct
300caagtattgg tggaatctcc tgcagtattg gagccaggaa ctaaagaaac
tagtgtgggc 360aagtggtcaa aatgtagtgg atggcctact gtaagggaaa
gaatgaaaca agctgagcct 420gagccagcag cagatggggt gggagcagca
tctcgagacc tggaaaaaca tggagcaatc 480acaagtagca atacagcaac
taataacgct gcttgtgcct ggctagaagc acaagaggaa 540gaggaagtgg
gttttccagt cagacctcag gtacctttaa gaccaatgac ttacaaggga
600gctttagatc ttagccactt tttaaaagaa aaggggggac tggaagggtt
aatttactcc 660ccaaaaagac aagagatcct tgatctgtgg gtctaccaca
cacaaggcta cttccctgat 720tggcagaact acacaccagg gccaggggtc
agatatccac tgacctttgg atggtgcttc 780aagttagtac cagttgaacc
agatgaagaa gagaacagca gcctgttaca ccctgcgagc 840ctgcatggga
cagaggacac ggagagagaa gtgttaaagt ggaagtttga cagccatcta
900gcatttcatc acaaggcccg agagctgcat ccggagtact acaaagactg
caagcttgag 960ccagtagatc ctagactaga gccctggaag catccaggaa
gtcagcctag gaccccttgt 1020accaattgct attgtaaaaa gtgttgcctt
cattgccaag tttgtttcac aagaaaaggc 1080ttaggcatct cctatggcag
gaagaagcgg agacagcgac gaagagctcc tcaagacagt 1140cagactcatc
aagtttctct accaaagcaa ccctcctccc agcaacgagg ggacccgaca
1200ggcccgaaga aatcgaagaa gaaggtggag agagagacag aggcagatcc
gttcgatgcg 1260gccgtgggcg caagagcctc cgtgctgagc ggcggagagc
tggacaagtg ggagaagatc 1320cgcctgcgcc ccggcggcaa gaagaagtac
cagctgaagc acatcgtgtg ggccagccgc 1380gagctggagc gcttcgccgt
gaaccccggc ctgctcgaga ccagcgaagg ctgccgccag 1440atcatgggcc
agctccagcc cagcctccag accggcagcg aggagctgcg cagcctgtac
1500aacaccgtgg ccaccctgta ctgcgtgcac cagaagatcg aggtgaagga
caccaaggag 1560gccctggaca aggtggagga ggagcagaac aacagcaaga
agaaggccca gcaggaggcc 1620gccgacgccg gcaaccgcaa ccaagtcagc
cagaactacc ccatcgtgca gaacctgcag 1680ggccagatgg tgcaccaggc
catcagcccc cgcaccctga acgcctgggt gaaggtggtg 1740gaggagaagg
ccttcagccc cgaggtgatc cccatgttca gcgccctaag cgagggcgct
1800accccccagg acctgaacac catgctgaac accgtgggcg gccaccaggc
cgccatgcag 1860atgctgaagg agaccatcaa cgaggaggcc gccgagtggg
accgcctgca ccccgtgcac 1920gccgggccca tcgcccccgg ccagatgcgc
gagccccgcg gcagcgacat cgccggcacc 1980accagcaccc tccaggagca
gatcggctgg atgaccaaca acccccccat ccccgtgggc 2040gagatctaca
agcgctggat catcctgggc ctgaacaaga tcgtccgcat gtacagcccc
2100accagcatcc tggacatcaa gcagggcccc aaggagccct tccgcgacta
cgtggaccgc 2160ttctacaaga ccctgcgcgc cgagcaggcc acccaggagg
tgaagaactg gatgaccgag 2220accctgctgg tgcagaacgc caaccccgac
tgcaagacca tcctcaaggc cctgggaccc 2280gccgccaccc tggaggagat
gatgaccgcc tgccaaggcg tgggcggccc cggccacaag 2340gcccgcgtgc
tggcggccgt catcaccctg tggcagcgcc ccctggtggc cctgatcgag
2400atctgcaccg agatggagaa ggagggcaag atcagcaaga tcggccccgc
cggcctgaag 2460aagaagaaga gcgtgaccgt gctggacgtg ggcgacgcct
acttcagcgt gcccctggat 2520aaggacttcc gcaagtacac cgccttcacc
atccccagca tctggaaggg cagccccgcc 2580atcttccaga gcagcatgac
caagaagcag aaccccgaca tcgtgatcta ccagtacatg 2640gacgacctgt
acgtgcccat cgtgctgccc gagaaggaca gctggctggt gggcaagctg
2700aactgggcca gccagatcta cgccggcatc aaggtgaagc agctgatcct
gaaggagccc 2760gtgcacggcg tgtacgagcc catcgtgggc gccgagacct
tctacgtgga cggcgccgcc 2820aaccgcgccg gcaacctgtg ggtgaccgtg
tactacggcg tgcccgtgtg gaaggaggcc 2880accaccaccc tggtggagcg
ctacctgcgc gaccagcagc tgctgggcat ctggggctgc 2940gcctgcaccc
cctacgacat caaccagatg ctgcgcggcc ctggccgcgc cttcgtgacc
3000atccgccagg gcagcctgta g 302125419PRTArtificial SequenceHybrid
protein comprised of Nef-Tat-Rev (NTR) 25Met Val Gly Lys Trp Ser
Lys Cys Ser Gly Trp Pro Thr Val Arg Glu1 5 10 15Arg Met Lys Gln Ala
Glu Pro Glu Pro Ala Ala Asp Gly Val Gly Ala 20 25 30Ala Ser Arg Asp
Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr 35 40 45Ala Thr Asn
Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu 50 55 60Glu Val
Gly Phe Pro Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr65 70 75
80Tyr Lys Gly Ala Leu Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly
85 90 95Leu Glu Gly Leu Ile Tyr Ser Pro Lys Arg Gln Glu Ile Leu Asp
Leu 100 105 110Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln
Asn Tyr Thr 115 120 125Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe
Gly Trp Cys Phe Lys 130 135 140Leu Val Pro Val Glu Pro Asp Glu Glu
Glu Asn Ser Ser Leu Leu His145 150 155 160Pro Ala Ser Leu His Gly
Thr Glu Asp Thr Glu Arg Glu Val Leu Lys 165 170 175Trp Lys Phe Asp
Ser His Leu Ala Phe His His Lys Ala Arg Glu Leu 180 185 190His Pro
Glu Tyr Tyr Lys Asp Cys Thr Ser Ala Gly Arg Ser Gly Asp 195 200
205Ser Asp Glu Glu Leu Leu Lys Thr Val Arg Leu Ile Lys Phe Leu Tyr
210 215 220Gln Ser Asn Pro Pro Pro Ser Asn Glu Gly Thr Arg Gln Ala
Arg Arg225 230 235 240Asn Arg Arg Arg Arg Trp Arg Glu Arg Gln Arg
Gln Ile Arg Ser Ile 245 250 255Ser Glu Arg Ile Leu Ser Thr Phe Leu
Gly Arg Pro Ala Glu Pro Val 260 265 270Pro Leu Gln Leu Pro Pro Leu
Glu Arg Leu Thr Leu Asp Cys Ser Glu 275 280 285Asp Cys Gly Asn Ser
Gly Thr Gln Gly Val Gly Ser Pro Gln Val Leu 290 295 300Val Glu Ser
Pro Ala Val Leu Glu Pro Gly Thr Lys Glu Lys Leu Glu305 310 315
320Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro
325 330 335Arg Thr Pro Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Leu
His Cys 340 345 350Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser
Tyr Gly Arg Lys 355 360 365Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln
Asp Ser Gln Thr His Gln 370 375 380Val Ser Leu Pro Lys Gln Pro Ser
Ser Gln Gln Arg Gly Asp Pro Thr385 390 395 400Gly Pro Lys Lys Ser
Lys Lys Lys Val Glu Arg Glu Thr Glu Ala Asp 405 410 415Pro Phe
Asp26419PRTArtificial SequenceHybrid protein comprised of
Tat-Rev-Nef (TRN) 26Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys
His Pro Gly Ser1 5 10 15Gln Pro Arg Thr Pro Cys Thr Asn Cys Tyr Cys
Lys Lys Cys Cys Leu 20 25 30His Cys Gln Val Cys Phe Thr Arg Lys Gly
Leu Gly Ile Ser Tyr Gly 35 40 45Arg Lys Lys Arg Arg Gln Arg Arg Arg
Ala Pro Gln Asp Ser Gln Thr 50 55 60His Gln Val Ser Leu Pro Lys Gln
Pro Ser Ser Gln Gln Arg Gly Asp65 70 75 80Pro Thr Gly Pro Lys Lys
Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 90 95Ala Asp Pro Phe Asp
Thr Ser Ala Gly Arg Ser Gly Asp Ser Asp Glu 100 105 110Glu Leu Leu
Lys Thr Val Arg Leu Ile Lys Phe Leu Tyr Gln Ser Asn 115 120 125Pro
Pro Pro Ser Asn Glu Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg 130 135
140Arg Arg Trp Arg Glu Arg Gln Arg Gln Ile Arg Ser Ile Ser Glu
Arg145 150 155 160Ile Leu Ser Thr Phe Leu Gly Arg Pro Ala Glu Pro
Val Pro Leu Gln 165 170 175Leu Pro Pro Leu Glu Arg Leu Thr Leu Asp
Cys Ser Glu Asp Cys Gly 180 185 190Asn Ser Gly Thr Gln Gly Val Gly
Ser Pro Gln Val Leu Val Glu Ser 195 200 205Pro Ala Val Leu Glu Pro
Gly Thr Lys Glu Lys Leu Val Gly Lys Trp 210 215 220Ser Lys Cys Ser
Gly Trp Pro Thr Val Arg Glu Arg Met Lys Gln Ala225 230 235 240Glu
Pro Glu Pro Ala Ala Asp Gly Val Gly Ala Ala Ser Arg Asp Leu 245 250
255Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Thr Asn Asn Ala
260 265 270Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu Val Gly
Phe Pro 275 280 285Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr
Lys Gly Ala Leu 290 295 300Asp Leu Ser His Phe Leu Lys Glu Lys Gly
Gly Leu Glu Gly Leu Ile305 310 315 320Tyr Ser Pro Lys Arg Gln Glu
Ile Leu Asp Leu Trp Val Tyr His Thr 325 330 335Gln Gly Tyr Phe Pro
Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val 340 345 350Arg Tyr Pro
Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu 355 360 365Pro
Asp Glu Glu Glu Asn Ser Ser Leu Leu His Pro Ala Ser Leu His 370 375
380Gly Thr Glu Asp Thr Glu Arg Glu Val Leu Lys Trp Lys Phe Asp
Ser385 390 395 400His Leu Ala Phe His His Lys Ala Arg Glu Leu His
Pro Glu Tyr Tyr 405 410 415Lys Asp Cys27419PRTArtificial
SequenceHybrid protein comprised of Rev-Tat-Nef (RTN) 27Met Ala Gly
Arg Ser Gly Asp Ser Asp Glu Glu Leu Leu Lys Thr Val1 5 10 15Arg Leu
Ile Lys Phe Leu Tyr Gln Ser Asn Pro Pro Pro Ser Asn Glu 20 25 30Gly
Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg 35 40
45Gln Arg Gln Ile Arg Ser Ile Ser Glu Arg Ile Leu Ser Thr Phe Leu
50 55 60Gly Arg Pro Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu
Arg65 70 75 80Leu Thr Leu Asp Cys Ser Glu Asp Cys Gly Asn Ser Gly
Thr Gln Gly 85 90 95Val Gly Ser Pro Gln Val Leu Val Glu Ser Pro Ala
Val Leu Glu Pro 100 105 110Gly Thr Lys Glu Thr Ser Glu Pro Val Asp
Pro Arg Leu Glu Pro Trp 115 120 125Lys His Pro Gly Ser Gln Pro Arg
Thr Pro Cys Thr Asn Cys Tyr Cys 130 135 140Lys Lys Cys Cys Leu His
Cys Gln Val Cys Phe Thr Arg Lys Gly Leu145 150 155 160Gly Ile Ser
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro 165 170 175Gln
Asp Ser Gln Thr His Gln Val Ser Leu Pro Lys Gln Pro Ser Ser 180 185
190Gln Gln Arg Gly Asp Pro Thr Gly Pro Lys Lys Ser Lys Lys Lys Val
195 200 205Glu Arg Glu Thr Glu Ala Asp Pro Phe Asp Lys Leu Val Gly
Lys Trp 210 215 220Ser Lys Cys Ser Gly Trp Pro Thr Val Arg Glu Arg
Met Lys Gln Ala225 230 235 240Glu Pro Glu Pro Ala Ala Asp Gly Val
Gly Ala Ala Ser Arg Asp Leu 245 250 255Glu Lys His Gly Ala Ile Thr
Ser Ser Asn Thr Ala Thr Asn Asn Ala 260 265 270Ala Cys Ala Trp Leu
Glu Ala Gln Glu Glu Glu Glu Val Gly Phe Pro 275 280 285Val Arg Pro
Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Leu 290 295 300Asp
Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile305 310
315 320Tyr Ser Pro Lys Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His
Thr 325 330 335Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly
Pro Gly Val 340 345 350Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys
Leu Val Pro Val Glu 355 360 365Pro Asp Glu Glu Glu Asn Ser Ser Leu
Leu His Pro Ala Ser Leu His 370 375 380Gly Thr Glu Asp Thr Glu Arg
Glu Val Leu Lys Trp Lys Phe Asp Ser385 390 395 400His Leu Ala Phe
His His Lys Ala Arg Glu Leu His Pro Glu Tyr Tyr 405 410 415Lys Asp
Cys28419PRTArtificial SequenceHybrid protein comprised of
Tat-Nef-Rev (TNR) 28Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys
His Pro Gly Ser1 5 10 15Gln Pro Arg Thr Pro Cys Thr Asn Cys Tyr Cys
Lys Lys Cys Cys Leu 20 25 30His Cys Gln Val Cys Phe Thr Arg Lys Gly
Leu Gly Ile Ser Tyr Gly 35 40 45Arg Lys Lys Arg Arg Gln Arg Arg Arg
Ala Pro Gln Asp Ser Gln Thr 50 55 60His Gln Val Ser Leu Pro Lys Gln
Pro Ser Ser Gln Gln Arg Gly Asp65 70 75 80Pro Thr Gly Pro Lys Lys
Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 90 95Ala Asp Pro Phe Asp
Thr Ser Val Gly Lys Trp Ser Lys Cys Ser Gly 100 105 110Trp Pro Thr
Val Arg Glu Arg Met Lys Gln Ala Glu Pro Glu Pro Ala 115 120 125Ala
Asp Gly Val Gly Ala Ala Ser Arg Asp Leu Glu Lys His Gly Ala 130 135
140Ile Thr Ser Ser Asn Thr Ala Thr Asn Asn Ala Ala Cys Ala Trp
Leu145 150 155 160Glu Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Val
Arg Pro Gln Val 165 170 175Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala
Leu Asp Leu Ser His Phe 180 185 190Leu Lys Glu Lys Gly Gly Leu Glu
Gly Leu Ile Tyr Ser Pro Lys Arg 195 200 205Gln Glu Ile Leu Asp Leu
Trp Val Tyr His Thr Gln Gly Tyr Phe Pro 210 215 220Asp Trp Gln Asn
Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr225 230 235 240Phe
Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Asp Glu Glu Glu 245 250
255Asn Ser Ser Leu Leu His Pro Ala Ser Leu His Gly Thr Glu Asp Thr
260 265 270Glu Arg Glu Val Leu Lys Trp Lys Phe Asp Ser His Leu Ala
Phe His 275 280 285His Lys Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys
Asp Cys Lys Leu 290 295 300Ala Gly Arg Ser Gly Asp Ser Asp Glu Glu
Leu Leu Lys Thr Val Arg305 310 315 320Leu Ile Lys Phe Leu Tyr Gln
Ser Asn Pro Pro Pro Ser Asn Glu Gly 325 330 335Thr Arg Gln Ala Arg
Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg Gln 340 345 350Arg Gln Ile
Arg Ser Ile Ser Glu Arg Ile Leu Ser Thr Phe Leu Gly 355 360 365Arg
Pro Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg Leu 370 375
380Thr Leu Asp Cys Ser Glu Asp Cys Gly Asn Ser Gly Thr Gln Gly
Val385 390 395 400Gly Ser Pro Gln Val Leu Val Glu Ser Pro Ala Val
Leu Glu Pro Gly 405 410 415Thr Lys Glu29419PRTArtificial
SequenceHybrid protein comprised of Rev-Nef-Tat (RNT) 29Met Ala Gly
Arg Ser Gly Asp Ser Asp Glu Glu Leu Leu Lys Thr Val1 5 10 15Arg Leu
Ile Lys Phe Leu Tyr Gln Ser Asn Pro Pro Pro Ser Asn Glu 20
25 30Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu
Arg 35 40 45Gln Arg Gln Ile Arg Ser Ile Ser Glu Arg Ile Leu Ser Thr
Phe Leu 50 55 60Gly Arg Pro Ala Glu Pro Val Pro Leu Gln Leu Pro Pro
Leu Glu Arg65 70 75 80Leu Thr Leu Asp Cys Ser Glu Asp Cys Gly Asn
Ser Gly Thr Gln Gly 85 90 95Val Gly Ser Pro Gln Val Leu Val Glu Ser
Pro Ala Val Leu Glu Pro 100 105 110Gly Thr Lys Glu Thr Ser Val Gly
Lys Trp Ser Lys Cys Ser Gly Trp 115 120 125Pro Thr Val Arg Glu Arg
Met Lys Gln Ala Glu Pro Glu Pro Ala Ala 130 135 140Asp Gly Val Gly
Ala Ala Ser Arg Asp Leu Glu Lys His Gly Ala Ile145 150 155 160Thr
Ser Ser Asn Thr Ala Thr Asn Asn Ala Ala Cys Ala Trp Leu Glu 165 170
175Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro
180 185 190Leu Arg Pro Met Thr Tyr Lys Gly Ala Leu Asp Leu Ser His
Phe Leu 195 200 205Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser
Pro Lys Arg Gln 210 215 220Glu Ile Leu Asp Leu Trp Val Tyr His Thr
Gln Gly Tyr Phe Pro Asp225 230 235 240Trp Gln Asn Tyr Thr Pro Gly
Pro Gly Val Arg Tyr Pro Leu Thr Phe 245 250 255Gly Trp Cys Phe Lys
Leu Val Pro Val Glu Pro Asp Glu Glu Glu Asn 260 265 270Ser Ser Leu
Leu His Pro Ala Ser Leu His Gly Thr Glu Asp Thr Glu 275 280 285Arg
Glu Val Leu Lys Trp Lys Phe Asp Ser His Leu Ala Phe His His 290 295
300Lys Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys Lys Leu
Glu305 310 315 320Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro
Gly Ser Gln Pro 325 330 335Arg Thr Pro Cys Thr Asn Cys Tyr Cys Lys
Lys Cys Cys Leu His Cys 340 345 350Gln Val Cys Phe Thr Arg Lys Gly
Leu Gly Ile Ser Tyr Gly Arg Lys 355 360 365Lys Arg Arg Gln Arg Arg
Arg Ala Pro Gln Asp Ser Gln Thr His Gln 370 375 380Val Ser Leu Pro
Lys Gln Pro Ser Ser Gln Gln Arg Gly Asp Pro Thr385 390 395 400Gly
Pro Lys Lys Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Ala Asp 405 410
415Pro Phe Asp30387PRTArtificial SequenceProtein comprised of
Immunodominant parts of the Nef-Tat-Rev(NTR) 30Met Gly Trp Pro Thr
Val Arg Glu Arg Met Lys Gln Ala Glu Pro Glu1 5 10 15Pro Ala Ala Asp
Gly Val Gly Ala Ala Ser Arg Asp Leu Glu Lys His 20 25 30Gly Ala Ile
Thr Ser Ser Asn Thr Ala Thr Asn Asn Ala Ala Cys Ala 35 40 45Trp Leu
Glu Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro 50 55 60Gln
Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Leu Asp Leu Ser65 70 75
80His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Pro
85 90 95Lys Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly
Tyr 100 105 110Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val
Arg Tyr Pro 115 120 125Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro
Val Glu Pro Asp Glu 130 135 140Glu Glu Asn Ser Ser Leu Leu His Pro
Ala Ser Leu His Gly Thr Glu145 150 155 160Asp Thr Glu Arg Glu Val
Leu Lys Trp Lys Phe Asp Ser His Leu Ala 165 170 175Phe His His Lys
Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys 180 185 190Ala Leu
Ala Ala Val Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys 195 200
205His Pro Gly Ser Gln Pro Arg Thr Pro Cys Thr Asn Cys Tyr Cys Lys
210 215 220Lys Cys Cys Leu His Cys Gln Val Cys Phe Thr Arg Lys Gly
Leu Gly225 230 235 240Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Ala Pro Gln 245 250 255Asp Ser Gln Thr His Gln Val Ser Leu
Pro Lys Gln Pro Ser Ser Gln 260 265 270Gln Arg Gly Asp Pro Thr Gly
Pro Lys Lys Ser Gly Leu Ala Ile Leu 275 280 285Leu Ser Asp Glu Glu
Leu Leu Lys Thr Val Arg Leu Ile Lys Phe Leu 290 295 300Tyr Gln Ser
Asn Pro Pro Pro Ser Asn Glu Gly Thr Arg Gln Ala Arg305 310 315
320Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg Gln Arg Gln Ile Arg Ser
325 330 335Ile Ser Glu Arg Ile Leu Ser Thr Phe Leu Gly Arg Pro Ala
Glu Pro 340 345 350Val Pro Leu Gln Leu Pro Pro Leu Glu Arg Leu Thr
Leu Asp Cys Ser 355 360 365Glu Asp Cys Gly Asn Ser Gly Thr Gln Gly
Val Gly Ser Pro Gln Val 370 375 380Leu Val Glu38531390PRTArtificial
SequenceProtein comprised of Immunodominant parts of the
Nef-Tat-Rev separated by protease sites(NTR) 31Met Gly Trp Pro Thr
Val Arg Glu Arg Met Lys Gln Ala Glu Pro Glu1 5 10 15Pro Ala Ala Asp
Gly Val Gly Ala Ala Ser Arg Asp Leu Glu Lys His 20 25 30Gly Ala Ile
Thr Ser Ser Asn Thr Ala Thr Asn Asn Ala Ala Cys Ala 35 40 45Trp Leu
Glu Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro 50 55 60Gln
Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Leu Asp Leu Ser65 70 75
80His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Pro
85 90 95Lys Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly
Tyr 100 105 110Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val
Arg Tyr Pro 115 120 125Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro
Val Glu Pro Asp Glu 130 135 140Glu Glu Asn Ser Ser Leu Leu His Pro
Ala Ser Leu His Gly Thr Glu145 150 155 160Asp Thr Glu Arg Glu Val
Leu Lys Trp Lys Phe Asp Ser His Leu Ala 165 170 175Phe His His Lys
Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys 180 185 190Ala Leu
Ala Phe Lys Arg Val Glu Pro Val Asp Pro Arg Leu Glu Pro 195 200
205Trp Lys His Pro Gly Ser Gln Pro Arg Thr Pro Cys Thr Asn Cys Tyr
210 215 220Cys Lys Lys Cys Cys Leu His Cys Gln Val Cys Phe Thr Arg
Lys Gly225 230 235 240Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg Ala 245 250 255Pro Gln Asp Ser Gln Thr His Gln Val
Ser Leu Pro Lys Gln Pro Ser 260 265 270Ser Gln Gln Arg Gly Asp Pro
Thr Gly Pro Lys Lys Ser Val Arg Glu 275 280 285Lys Arg Leu Leu Ser
Asp Glu Glu Leu Leu Lys Thr Val Arg Leu Ile 290 295 300Lys Phe Leu
Tyr Gln Ser Asn Pro Pro Pro Ser Asn Glu Gly Thr Arg305 310 315
320Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg Gln Arg Gln
325 330 335Ile Arg Ser Ile Ser Glu Arg Ile Leu Ser Thr Phe Leu Gly
Arg Pro 340 345 350Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu
Arg Leu Thr Leu 355 360 365Asp Cys Ser Glu Asp Cys Gly Asn Ser Gly
Thr Gln Gly Val Gly Ser 370 375 380Pro Gln Val Leu Val Glu385
39032386PRTArtificial SequenceProtein comprised of Immunodominant
parts of the regulatory proteins Nef-Tat-Rev started from aa1 of
Nef(N11TR) 32Met Trp Pro Thr Val Arg Glu Arg Met Lys Gln Ala Glu
Pro Glu Pro1 5 10 15Ala Ala Asp Gly Val Gly Ala Ala Ser Arg Asp Leu
Glu Lys His Gly 20 25 30Ala Ile Thr Ser Ser Asn Thr Ala Thr Asn Asn
Ala Ala Cys Ala Trp 35 40 45Leu Glu Ala Gln Glu Glu Glu Glu Val Gly
Phe Pro Val Arg Pro Gln 50 55 60Val Pro Leu Arg Pro Met Thr Tyr Lys
Gly Ala Leu Asp Leu Ser His65 70 75 80Phe Leu Lys Glu Lys Gly Gly
Leu Glu Gly Leu Ile Tyr Ser Pro Lys 85 90 95Arg Gln Glu Ile Leu Asp
Leu Trp Val Tyr His Thr Gln Gly Tyr Phe 100 105 110Pro Asp Trp Gln
Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu 115 120 125Thr Phe
Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Asp Glu Glu 130 135
140Glu Asn Ser Ser Leu Leu His Pro Ala Ser Leu His Gly Thr Glu
Asp145 150 155 160Thr Glu Arg Glu Val Leu Lys Trp Lys Phe Asp Ser
His Leu Ala Phe 165 170 175His His Lys Ala Arg Glu Leu His Pro Glu
Tyr Tyr Lys Asp Cys Ala 180 185 190Leu Ala Ala Val Glu Pro Val Asp
Pro Arg Leu Glu Pro Trp Lys His 195 200 205Pro Gly Ser Gln Pro Arg
Thr Pro Cys Thr Asn Cys Tyr Cys Lys Lys 210 215 220Cys Cys Leu His
Cys Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile225 230 235 240Ser
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp 245 250
255Ser Gln Thr His Gln Val Ser Leu Pro Lys Gln Pro Ser Ser Gln Gln
260 265 270Arg Gly Asp Pro Thr Gly Pro Lys Lys Ser Gly Leu Ala Ile
Leu Leu 275 280 285Ser Asp Glu Glu Leu Leu Lys Thr Val Arg Leu Ile
Lys Phe Leu Tyr 290 295 300Gln Ser Asn Pro Pro Pro Ser Asn Glu Gly
Thr Arg Gln Ala Arg Arg305 310 315 320Asn Arg Arg Arg Arg Trp Arg
Glu Arg Gln Arg Gln Ile Arg Ser Ile 325 330 335Ser Glu Arg Ile Leu
Ser Thr Phe Leu Gly Arg Pro Ala Glu Pro Val 340 345 350Pro Leu Gln
Leu Pro Pro Leu Glu Arg Leu Thr Leu Asp Cys Ser Glu 355 360 365Asp
Cys Gly Asn Ser Gly Thr Gln Gly Val Gly Ser Pro Gln Val Leu 370 375
380Val Glu38533389PRTArtificial SequenceProtein comprised of
Immunodominant parts of the regulatory proteins Nef-Tat-Rev started
from aa1 of Nef separated by protease sites(N11TR) 33Met Trp Pro
Thr Val Arg Glu Arg Met Lys Gln Ala Glu Pro Glu Pro1 5 10 15Ala Ala
Asp Gly Val Gly Ala Ala Ser Arg Asp Leu Glu Lys His Gly 20 25 30Ala
Ile Thr Ser Ser Asn Thr Ala Thr Asn Asn Ala Ala Cys Ala Trp 35 40
45Leu Glu Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln
50 55 60Val Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Leu Asp Leu Ser
His65 70 75 80Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr
Ser Pro Lys 85 90 95Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr
Gln Gly Tyr Phe 100 105 110Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro
Gly Val Arg Tyr Pro Leu 115 120 125Thr Phe Gly Trp Cys Phe Lys Leu
Val Pro Val Glu Pro Asp Glu Glu 130 135 140Glu Asn Ser Ser Leu Leu
His Pro Ala Ser Leu His Gly Thr Glu Asp145 150 155 160Thr Glu Arg
Glu Val Leu Lys Trp Lys Phe Asp Ser His Leu Ala Phe 165 170 175His
His Lys Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys Ala 180 185
190Leu Ala Phe Lys Arg Val Glu Pro Val Asp Pro Arg Leu Glu Pro Trp
195 200 205Lys His Pro Gly Ser Gln Pro Arg Thr Pro Cys Thr Asn Cys
Tyr Cys 210 215 220Lys Lys Cys Cys Leu His Cys Gln Val Cys Phe Thr
Arg Lys Gly Leu225 230 235 240Gly Ile Ser Tyr Gly Arg Lys Lys Arg
Arg Gln Arg Arg Arg Ala Pro 245 250 255Gln Asp Ser Gln Thr His Gln
Val Ser Leu Pro Lys Gln Pro Ser Ser 260 265 270Gln Gln Arg Gly Asp
Pro Thr Gly Pro Lys Lys Ser Val Arg Glu Lys 275 280 285Arg Leu Leu
Ser Asp Glu Glu Leu Leu Lys Thr Val Arg Leu Ile Lys 290 295 300Phe
Leu Tyr Gln Ser Asn Pro Pro Pro Ser Asn Glu Gly Thr Arg Gln305 310
315 320Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg Gln Arg Gln
Ile 325 330 335Arg Ser Ile Ser Glu Arg Ile Leu Ser Thr Phe Leu Gly
Arg Pro Ala 340 345 350Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu
Arg Leu Thr Leu Asp 355 360 365Cys Ser Glu Asp Cys Gly Asn Ser Gly
Thr Gln Gly Val Gly Ser Pro 370 375 380Gln Val Leu Val
Glu38534220PRTArtificial SequenceProtein comprised of Cytotoxic
T-cell epitopes of Pol and Env genes(CTL) 34Met Ile Thr Leu Trp Gln
Arg Pro Leu Val Ala Leu Ile Glu Ile Cys1 5 10 15Thr Glu Met Glu Lys
Glu Gly Lys Ile Ser Lys Ile Gly Pro Ala Gly 20 25 30Leu Lys Lys Lys
Lys Ser Val Thr Val Leu Asp Val Gly Asp Ala Tyr 35 40 45Phe Ser Val
Pro Leu Asp Lys Asp Phe Arg Lys Tyr Thr Ala Phe Thr 50 55 60Ile Pro
Ser Ile Trp Lys Gly Ser Pro Ala Ile Phe Gln Ser Ser Met65 70 75
80Thr Lys Lys Gln Asn Pro Asp Ile Val Ile Tyr Gln Tyr Met Asp Asp
85 90 95Leu Tyr Val Pro Ile Val Leu Pro Glu Lys Asp Ser Trp Leu Val
Gly 100 105 110Lys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly Ile Lys
Val Lys Gln 115 120 125Leu Ile Leu Lys Glu Pro Val His Gly Val Tyr
Glu Pro Ile Val Gly 130 135 140Ala Glu Thr Phe Tyr Val Asp Gly Ala
Ala Asn Arg Ala Gly Asn Leu145 150 155 160Trp Val Thr Val Tyr Tyr
Gly Val Pro Val Trp Lys Glu Ala Thr Thr 165 170 175Thr Leu Val Glu
Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly Ile Trp 180 185 190Gly Cys
Ala Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu Arg Gly Pro 195 200
205Gly Arg Ala Phe Val Thr Ile Arg Gln Gly Ser Leu 210 215
22035421PRTArtificial SequenceTruncated Gag protein sequence(dgag)
35Met Leu Asp Lys Trp Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys1
5 10 15Lys Tyr Gln Leu Lys His Ile Val Trp Ala Ser Arg Glu Leu Glu
Arg 20 25 30Phe Ala Val Asn Pro Gly Leu Leu Glu Thr Ser Glu Gly Cys
Arg Gln 35 40 45Ile Met Gly Gln Leu Gln Pro Ser Leu Gln Thr Gly Ser
Glu Glu Leu 50 55 60Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr Cys
Val His Gln Lys65 70 75 80Ile Glu Val Lys Asp Thr Lys Glu Ala Leu
Asp Lys Val Glu Glu Glu 85 90 95Gln Asn Asn Ser Lys Lys Lys Ala Gln
Gln Glu Ala Ala Asp Ala Gly 100 105 110Asn Arg Asn Gln Val Ser Gln
Asn Tyr Pro Ile Val Gln Asn Leu Gln 115 120 125Gly Gln Met Val His
Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp 130 135 140Val Lys Val
Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met145 150 155
160Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met
165 170 175Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu
Lys Glu 180 185 190Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu
His Pro Val His 195 200 205Ala Gly Pro Ile Ala Pro Gly Gln Met Arg
Glu Pro Arg Gly Ser Asp 210
215 220Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp Met
Thr225 230 235 240Asn Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys
Arg Trp Ile Ile 245 250 255Leu Gly Leu Asn Lys Ile Val Arg Met Tyr
Ser Pro Thr Ser Ile Leu 260 265 270Asp Ile Lys Gln Gly Pro Lys Glu
Pro Phe Arg Asp Tyr Val Asp Arg 275 280 285Phe Tyr Lys Thr Leu Arg
Ala Glu Gln Ala Thr Gln Glu Val Lys Asn 290 295 300Trp Met Thr Glu
Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys305 310 315 320Thr
Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met 325 330
335Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala Arg Val Leu
340 345 350Ala Glu Ala Met Ser Gln Val Thr Gly Ser Ala Ala Ile Met
Met Gln 355 360 365Arg Gly Asn Phe Arg Asn Gln Arg Lys Thr Val Lys
Cys Phe Asn Cys 370 375 380Gly Lys Glu Gly His Ile Ala Arg Asn Cys
Arg Ala Pro Arg Lys Lys385 390 395 400Gly Cys Trp Lys Cys Gly Lys
Glu Gly His Gln Met Lys Asp Cys Thr 405 410 415Glu Arg Gln Ala Asn
42036363PRTArtificial SequenceSynthetic p17/24 protein of Gag
gene(Syn 17/24) 36Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu
Leu Asp Lys Trp1 5 10 15Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys
Lys Tyr Gln Leu Lys 20 25 30His Ile Val Trp Ala Ser Arg Glu Leu Glu
Arg Phe Ala Val Asn Pro 35 40 45Gly Leu Leu Glu Thr Ser Glu Gly Cys
Arg Gln Ile Met Gly Gln Leu 50 55 60Gln Pro Ser Leu Gln Thr Gly Ser
Glu Glu Leu Arg Ser Leu Tyr Asn65 70 75 80Thr Val Ala Thr Leu Tyr
Cys Val His Gln Lys Ile Glu Val Lys Asp 85 90 95Thr Lys Glu Ala Leu
Asp Lys Val Glu Glu Glu Gln Asn Asn Ser Lys 100 105 110Lys Lys Ala
Gln Gln Glu Ala Ala Asp Ala Gly Asn Arg Asn Gln Val 115 120 125Ser
Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His 130 135
140Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val
Glu145 150 155 160Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe
Ser Ala Leu Ser 165 170 175Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr
Met Leu Asn Thr Val Gly 180 185 190Gly His Gln Ala Ala Met Gln Met
Leu Lys Glu Thr Ile Asn Glu Glu 195 200 205Ala Ala Glu Trp Asp Arg
Leu His Pro Val His Ala Gly Pro Ile Ala 210 215 220Pro Gly Gln Met
Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr225 230 235 240Ser
Thr Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile 245 250
255Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys
260 265 270Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Lys
Gln Gly 275 280 285Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe
Tyr Lys Thr Leu 290 295 300Arg Ala Glu Gln Ala Thr Gln Glu Val Lys
Asn Trp Met Thr Glu Thr305 310 315 320Leu Leu Val Gln Asn Ala Asn
Pro Asp Cys Lys Thr Ile Leu Lys Ala 325 330 335Leu Gly Pro Ala Ala
Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly 340 345 350Val Gly Gly
Pro Gly His Lys Ala Arg Val Leu 355 36037363PRTArtificial
SequenceSynthetic p17/24 protein of Gag gene optimized for
expression in eukaryotic cells(optp 17/24) 37Met Gly Ala Arg Ala
Ser Val Leu Ser Gly Gly Glu Leu Asp Lys Trp1 5 10 15Glu Lys Ile Arg
Leu Arg Pro Gly Gly Lys Lys Lys Tyr Gln Leu Lys 20 25 30His Ile Val
Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45Gly Leu
Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Met Gly Gln Leu 50 55 60Gln
Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn65 70 75
80Thr Val Ala Thr Leu Tyr Cys Val His Gln Lys Ile Glu Val Lys Asp
85 90 95Thr Lys Glu Ala Leu Asp Lys Val Glu Glu Glu Gln Asn Asn Ser
Lys 100 105 110Lys Lys Ala Gln Gln Glu Ala Ala Asp Ala Gly Asn Arg
Asn Gln Val 115 120 125Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln
Gly Gln Met Val His 130 135 140Gln Ala Ile Ser Pro Arg Thr Leu Asn
Ala Trp Val Lys Val Val Glu145 150 155 160Glu Lys Ala Phe Ser Pro
Glu Val Ile Pro Met Phe Ser Ala Leu Ser 165 170 175Glu Gly Ala Thr
Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly 180 185 190Gly His
Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu 195 200
205Ala Ala Glu Trp Asp Arg Leu His Pro Val His Ala Gly Pro Ile Ala
210 215 220Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly
Thr Thr225 230 235 240Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr
Asn Asn Pro Pro Ile 245 250 255Pro Val Gly Glu Ile Tyr Lys Arg Trp
Ile Ile Leu Gly Leu Asn Lys 260 265 270Ile Val Arg Met Tyr Ser Pro
Thr Ser Ile Leu Asp Ile Lys Gln Gly 275 280 285Pro Lys Glu Pro Phe
Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu 290 295 300Arg Ala Glu
Gln Ala Thr Gln Glu Val Lys Asn Trp Met Thr Glu Thr305 310 315
320Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala
325 330 335Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys
Gln Gly 340 345 350Val Gly Gly Pro Gly His Lys Ala Arg Val Leu 355
36038641PRTArtificial SequenceHybrid protein comprised of
Tat-Rev-Nef and CTL (TRN-CTL) 38Met Glu Pro Val Asp Pro Arg Leu Glu
Pro Trp Lys His Pro Gly Ser1 5 10 15Gln Pro Arg Thr Pro Cys Thr Asn
Cys Tyr Cys Lys Lys Cys Cys Leu 20 25 30His Cys Gln Val Cys Phe Thr
Arg Lys Gly Leu Gly Ile Ser Tyr Gly 35 40 45Arg Lys Lys Arg Arg Gln
Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr 50 55 60His Gln Val Ser Leu
Pro Lys Gln Pro Ser Ser Gln Gln Arg Gly Asp65 70 75 80Pro Thr Gly
Pro Lys Lys Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 90 95Ala Asp
Pro Phe Asp Thr Ser Ala Gly Arg Ser Gly Asp Ser Asp Glu 100 105
110Glu Leu Leu Lys Thr Val Arg Leu Ile Lys Phe Leu Tyr Gln Ser Asn
115 120 125Pro Pro Pro Ser Asn Glu Gly Thr Arg Gln Ala Arg Arg Asn
Arg Arg 130 135 140Arg Arg Trp Arg Glu Arg Gln Arg Gln Ile Arg Ser
Ile Ser Glu Arg145 150 155 160Ile Leu Ser Thr Phe Leu Gly Arg Pro
Ala Glu Pro Val Pro Leu Gln 165 170 175Leu Pro Pro Leu Glu Arg Leu
Thr Leu Asp Cys Ser Glu Asp Cys Gly 180 185 190Asn Ser Gly Thr Gln
Gly Val Gly Ser Pro Gln Val Leu Val Glu Ser 195 200 205Pro Ala Val
Leu Glu Pro Gly Thr Lys Glu Lys Leu Val Gly Lys Trp 210 215 220Ser
Lys Cys Ser Gly Trp Pro Thr Val Arg Glu Arg Met Lys Gln Ala225 230
235 240Glu Pro Glu Pro Ala Ala Asp Gly Val Gly Ala Ala Ser Arg Asp
Leu 245 250 255Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Thr
Asn Asn Ala 260 265 270Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu
Glu Val Gly Phe Pro 275 280 285Val Arg Pro Gln Val Pro Leu Arg Pro
Met Thr Tyr Lys Gly Ala Leu 290 295 300Asp Leu Ser His Phe Leu Lys
Glu Lys Gly Gly Leu Glu Gly Leu Ile305 310 315 320Tyr Ser Pro Lys
Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr 325 330 335Gln Gly
Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val 340 345
350Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu
355 360 365Pro Asp Glu Glu Glu Asn Ser Ser Leu Leu His Pro Ala Ser
Leu His 370 375 380Gly Thr Glu Asp Thr Glu Arg Glu Val Leu Lys Trp
Lys Phe Asp Ser385 390 395 400His Leu Ala Phe His His Lys Ala Arg
Glu Leu His Pro Glu Tyr Tyr 405 410 415Lys Asp Cys Ala Ala Val Ile
Thr Leu Trp Gln Arg Pro Leu Val Ala 420 425 430Leu Ile Glu Ile Cys
Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys 435 440 445Ile Gly Pro
Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp 450 455 460Val
Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp Lys Asp Phe Arg Lys465 470
475 480Tyr Thr Ala Phe Thr Ile Pro Ser Ile Trp Lys Gly Ser Pro Ala
Ile 485 490 495Phe Gln Ser Ser Met Thr Lys Lys Gln Asn Pro Asp Ile
Val Ile Tyr 500 505 510Gln Tyr Met Asp Asp Leu Tyr Val Pro Ile Val
Leu Pro Glu Lys Asp 515 520 525Ser Trp Leu Val Gly Lys Leu Asn Trp
Ala Ser Gln Ile Tyr Ala Gly 530 535 540Ile Lys Val Lys Gln Leu Ile
Leu Lys Glu Pro Val His Gly Val Tyr545 550 555 560Glu Pro Ile Val
Gly Ala Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn 565 570 575Arg Ala
Gly Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp 580 585
590Lys Glu Ala Thr Thr Thr Leu Val Glu Arg Tyr Leu Arg Asp Gln Gln
595 600 605Leu Leu Gly Ile Trp Gly Cys Ala Cys Thr Pro Tyr Asp Ile
Asn Gln 610 615 620Met Leu Arg Gly Pro Gly Arg Ala Phe Val Thr Ile
Arg Gln Gly Ser625 630 635 640Leu39641PRTArtificial SequenceHybrid
protein comprised of Rev-Nef-Tat and CTL(RNT-CTL) 39Met Ala Gly Arg
Ser Gly Asp Ser Asp Glu Glu Leu Leu Lys Thr Val1 5 10 15Arg Leu Ile
Lys Phe Leu Tyr Gln Ser Asn Pro Pro Pro Ser Asn Glu 20 25 30Gly Thr
Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg 35 40 45Gln
Arg Gln Ile Arg Ser Ile Ser Glu Arg Ile Leu Ser Thr Phe Leu 50 55
60Gly Arg Pro Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg65
70 75 80Leu Thr Leu Asp Cys Ser Glu Asp Cys Gly Asn Ser Gly Thr Gln
Gly 85 90 95Val Gly Ser Pro Gln Val Leu Val Glu Ser Pro Ala Val Leu
Glu Pro 100 105 110Gly Thr Lys Glu Thr Ser Val Gly Lys Trp Ser Lys
Cys Ser Gly Trp 115 120 125Pro Thr Val Arg Glu Arg Met Lys Gln Ala
Glu Pro Glu Pro Ala Ala 130 135 140Asp Gly Val Gly Ala Ala Ser Arg
Asp Leu Glu Lys His Gly Ala Ile145 150 155 160Thr Ser Ser Asn Thr
Ala Thr Asn Asn Ala Ala Cys Ala Trp Leu Glu 165 170 175Ala Gln Glu
Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln Val Pro 180 185 190Leu
Arg Pro Met Thr Tyr Lys Gly Ala Leu Asp Leu Ser His Phe Leu 195 200
205Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Pro Lys Arg Gln
210 215 220Glu Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe
Pro Asp225 230 235 240Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val Arg
Tyr Pro Leu Thr Phe 245 250 255Gly Trp Cys Phe Lys Leu Val Pro Val
Glu Pro Asp Glu Glu Glu Asn 260 265 270Ser Ser Leu Leu His Pro Ala
Ser Leu His Gly Thr Glu Asp Thr Glu 275 280 285Arg Glu Val Leu Lys
Trp Lys Phe Asp Ser His Leu Ala Phe His His 290 295 300Lys Ala Arg
Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys Lys Leu Glu305 310 315
320Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro
325 330 335Arg Thr Pro Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Leu
His Cys 340 345 350Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser
Tyr Gly Arg Lys 355 360 365Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln
Asp Ser Gln Thr His Gln 370 375 380Val Ser Leu Pro Lys Gln Pro Ser
Ser Gln Gln Arg Gly Asp Pro Thr385 390 395 400Gly Pro Lys Lys Ser
Lys Lys Lys Val Glu Arg Glu Thr Glu Ala Asp 405 410 415Pro Phe Asp
Ala Ala Val Ile Thr Leu Trp Gln Arg Pro Leu Val Ala 420 425 430Leu
Ile Glu Ile Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys 435 440
445Ile Gly Pro Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp
450 455 460Val Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp Lys Asp Phe
Arg Lys465 470 475 480Tyr Thr Ala Phe Thr Ile Pro Ser Ile Trp Lys
Gly Ser Pro Ala Ile 485 490 495Phe Gln Ser Ser Met Thr Lys Lys Gln
Asn Pro Asp Ile Val Ile Tyr 500 505 510Gln Tyr Met Asp Asp Leu Tyr
Val Pro Ile Val Leu Pro Glu Lys Asp 515 520 525Ser Trp Leu Val Gly
Lys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly 530 535 540Ile Lys Val
Lys Gln Leu Ile Leu Lys Glu Pro Val His Gly Val Tyr545 550 555
560Glu Pro Ile Val Gly Ala Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn
565 570 575Arg Ala Gly Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro
Val Trp 580 585 590Lys Glu Ala Thr Thr Thr Leu Val Glu Arg Tyr Leu
Arg Asp Gln Gln 595 600 605Leu Leu Gly Ile Trp Gly Cys Ala Cys Thr
Pro Tyr Asp Ile Asn Gln 610 615 620Met Leu Arg Gly Pro Gly Arg Ala
Phe Val Thr Ile Arg Gln Gly Ser625 630 635 640Leu40842PRTArtificial
SequenceHybrid protein cds comprised of Tat-Rev-Nef and truncated
Gag protein(TRN-dgag) 40Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp
Lys His Pro Gly Ser1 5 10 15Gln Pro Arg Thr Pro Cys Thr Asn Cys Tyr
Cys Lys Lys Cys Cys Leu 20 25 30His Cys Gln Val Cys Phe Thr Arg Lys
Gly Leu Gly Ile Ser Tyr Gly 35 40 45Arg Lys Lys Arg Arg Gln Arg Arg
Arg Ala Pro Gln Asp Ser Gln Thr 50 55 60His Gln Val Ser Leu Pro Lys
Gln Pro Ser Ser Gln Gln Arg Gly Asp65 70 75 80Pro Thr Gly Pro Lys
Lys Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 90 95Ala Asp Pro Phe
Asp Thr Ser Ala Gly Arg Ser Gly Asp Ser Asp Glu 100 105 110Glu Leu
Leu Lys Thr Val Arg Leu Ile Lys Phe Leu Tyr Gln Ser Asn 115 120
125Pro Pro Pro Ser Asn Glu Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg
130 135 140Arg Arg Trp Arg Glu Arg Gln Arg Gln Ile Arg Ser Ile Ser
Glu Arg145 150 155 160Ile Leu Ser Thr Phe Leu Gly Arg Pro Ala Glu
Pro Val Pro Leu Gln 165 170 175Leu Pro Pro Leu Glu Arg Leu Thr Leu
Asp Cys Ser Glu Asp Cys Gly 180 185 190Asn Ser Gly Thr Gln Gly Val
Gly Ser Pro Gln
Val Leu Val Glu Ser 195 200 205Pro Ala Val Leu Glu Pro Gly Thr Lys
Glu Lys Leu Val Gly Lys Trp 210 215 220Ser Lys Cys Ser Gly Trp Pro
Thr Val Arg Glu Arg Met Lys Gln Ala225 230 235 240Glu Pro Glu Pro
Ala Ala Asp Gly Val Gly Ala Ala Ser Arg Asp Leu 245 250 255Glu Lys
His Gly Ala Ile Thr Ser Ser Asn Thr Ala Thr Asn Asn Ala 260 265
270Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu Val Gly Phe Pro
275 280 285Val Arg Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys Gly
Ala Leu 290 295 300Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu
Glu Gly Leu Ile305 310 315 320Tyr Ser Pro Lys Arg Gln Glu Ile Leu
Asp Leu Trp Val Tyr His Thr 325 330 335Gln Gly Tyr Phe Pro Asp Trp
Gln Asn Tyr Thr Pro Gly Pro Gly Val 340 345 350Arg Tyr Pro Leu Thr
Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu 355 360 365Pro Asp Glu
Glu Glu Asn Ser Ser Leu Leu His Pro Ala Ser Leu His 370 375 380Gly
Thr Glu Asp Thr Glu Arg Glu Val Leu Lys Trp Lys Phe Asp Ser385 390
395 400His Leu Ala Phe His His Lys Ala Arg Glu Leu His Pro Glu Tyr
Tyr 405 410 415Lys Asp Cys Ala Ala Val Leu Asp Lys Trp Glu Lys Ile
Arg Leu Arg 420 425 430Pro Gly Gly Lys Lys Lys Tyr Gln Leu Lys His
Ile Val Trp Ala Ser 435 440 445Arg Glu Leu Glu Arg Phe Ala Val Asn
Pro Gly Leu Leu Glu Thr Ser 450 455 460Glu Gly Cys Arg Gln Ile Met
Gly Gln Leu Gln Pro Ser Leu Gln Thr465 470 475 480Gly Ser Glu Glu
Leu Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr 485 490 495Cys Val
His Gln Lys Ile Glu Val Lys Asp Thr Lys Glu Ala Leu Asp 500 505
510Lys Val Glu Glu Glu Gln Asn Asn Ser Lys Lys Lys Ala Gln Gln Glu
515 520 525Ala Ala Asp Ala Gly Asn Arg Asn Gln Val Ser Gln Asn Tyr
Pro Ile 530 535 540Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala
Ile Ser Pro Arg545 550 555 560Thr Leu Asn Ala Trp Val Lys Val Val
Glu Glu Lys Ala Phe Ser Pro 565 570 575Glu Val Ile Pro Met Phe Ser
Ala Leu Ser Glu Gly Ala Thr Pro Gln 580 585 590Asp Leu Asn Thr Met
Leu Asn Thr Val Gly Gly His Gln Ala Ala Met 595 600 605Gln Met Leu
Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg 610 615 620Leu
His Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu625 630
635 640Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu
Gln 645 650 655Ile Gly Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly
Glu Ile Tyr 660 665 670Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile
Val Arg Met Tyr Ser 675 680 685Pro Thr Ser Ile Leu Asp Ile Lys Gln
Gly Pro Lys Glu Pro Phe Arg 690 695 700Asp Tyr Val Asp Arg Phe Tyr
Lys Thr Leu Arg Ala Glu Gln Ala Thr705 710 715 720Gln Glu Val Lys
Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala 725 730 735Asn Pro
Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr 740 745
750Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His
755 760 765Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr Gly
Ser Ala 770 775 780Ala Ile Met Met Gln Arg Gly Asn Phe Arg Asn Gln
Arg Lys Thr Val785 790 795 800Lys Cys Phe Asn Cys Gly Lys Glu Gly
His Ile Ala Arg Asn Cys Arg 805 810 815Ala Pro Arg Lys Lys Gly Cys
Trp Lys Cys Gly Lys Glu Gly His Gln 820 825 830Met Lys Asp Cys Thr
Glu Arg Gln Ala Asn 835 840411064PRTArtificial SequenceHybrid
protein cds comprised of Tat-Rev-Nef, CTL and truncated Gag
protein(TRN-TCL-dgag) 41Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp
Lys His Pro Gly Ser1 5 10 15Gln Pro Arg Thr Pro Cys Thr Asn Cys Tyr
Cys Lys Lys Cys Cys Leu 20 25 30His Cys Gln Val Cys Phe Thr Arg Lys
Gly Leu Gly Ile Ser Tyr Gly 35 40 45Arg Lys Lys Arg Arg Gln Arg Arg
Arg Ala Pro Gln Asp Ser Gln Thr 50 55 60His Gln Val Ser Leu Pro Lys
Gln Pro Ser Ser Gln Gln Arg Gly Asp65 70 75 80Pro Thr Gly Pro Lys
Lys Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 90 95Ala Asp Pro Phe
Asp Thr Ser Ala Gly Arg Ser Gly Asp Ser Asp Glu 100 105 110Glu Leu
Leu Lys Thr Val Arg Leu Ile Lys Phe Leu Tyr Gln Ser Asn 115 120
125Pro Pro Pro Ser Asn Glu Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg
130 135 140Arg Arg Trp Arg Glu Arg Gln Arg Gln Ile Arg Ser Ile Ser
Glu Arg145 150 155 160Ile Leu Ser Thr Phe Leu Gly Arg Pro Ala Glu
Pro Val Pro Leu Gln 165 170 175Leu Pro Pro Leu Glu Arg Leu Thr Leu
Asp Cys Ser Glu Asp Cys Gly 180 185 190Asn Ser Gly Thr Gln Gly Val
Gly Ser Pro Gln Val Leu Val Glu Ser 195 200 205Pro Ala Val Leu Glu
Pro Gly Thr Lys Glu Lys Leu Val Gly Lys Trp 210 215 220Ser Lys Cys
Ser Gly Trp Pro Thr Val Arg Glu Arg Met Lys Gln Ala225 230 235
240Glu Pro Glu Pro Ala Ala Asp Gly Val Gly Ala Ala Ser Arg Asp Leu
245 250 255Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Thr Asn
Asn Ala 260 265 270Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu
Val Gly Phe Pro 275 280 285Val Arg Pro Gln Val Pro Leu Arg Pro Met
Thr Tyr Lys Gly Ala Leu 290 295 300Asp Leu Ser His Phe Leu Lys Glu
Lys Gly Gly Leu Glu Gly Leu Ile305 310 315 320Tyr Ser Pro Lys Arg
Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr 325 330 335Gln Gly Tyr
Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val 340 345 350Arg
Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu 355 360
365Pro Asp Glu Glu Glu Asn Ser Ser Leu Leu His Pro Ala Ser Leu His
370 375 380Gly Thr Glu Asp Thr Glu Arg Glu Val Leu Lys Trp Lys Phe
Asp Ser385 390 395 400His Leu Ala Phe His His Lys Ala Arg Glu Leu
His Pro Glu Tyr Tyr 405 410 415Lys Asp Cys Ala Ala Val Ile Thr Leu
Trp Gln Arg Pro Leu Val Ala 420 425 430Leu Ile Glu Ile Cys Thr Glu
Met Glu Lys Glu Gly Lys Ile Ser Lys 435 440 445Ile Gly Pro Ala Gly
Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp 450 455 460Val Gly Asp
Ala Tyr Phe Ser Val Pro Leu Asp Lys Asp Phe Arg Lys465 470 475
480Tyr Thr Ala Phe Thr Ile Pro Ser Ile Trp Lys Gly Ser Pro Ala Ile
485 490 495Phe Gln Ser Ser Met Thr Lys Lys Gln Asn Pro Asp Ile Val
Ile Tyr 500 505 510Gln Tyr Met Asp Asp Leu Tyr Val Pro Ile Val Leu
Pro Glu Lys Asp 515 520 525Ser Trp Leu Val Gly Lys Leu Asn Trp Ala
Ser Gln Ile Tyr Ala Gly 530 535 540Ile Lys Val Lys Gln Leu Ile Leu
Lys Glu Pro Val His Gly Val Tyr545 550 555 560Glu Pro Ile Val Gly
Ala Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn 565 570 575Arg Ala Gly
Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp 580 585 590Lys
Glu Ala Thr Thr Thr Leu Val Glu Arg Tyr Leu Arg Asp Gln Gln 595 600
605Leu Leu Gly Ile Trp Gly Cys Ala Cys Thr Pro Tyr Asp Ile Asn Gln
610 615 620Met Leu Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Arg Gln
Gly Ser625 630 635 640Leu Ala Ala Val Leu Asp Lys Trp Glu Lys Ile
Arg Leu Arg Pro Gly 645 650 655Gly Lys Lys Lys Tyr Gln Leu Lys His
Ile Val Trp Ala Ser Arg Glu 660 665 670Leu Glu Arg Phe Ala Val Asn
Pro Gly Leu Leu Glu Thr Ser Glu Gly 675 680 685Cys Arg Gln Ile Met
Gly Gln Leu Gln Pro Ser Leu Gln Thr Gly Ser 690 695 700Glu Glu Leu
Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr Cys Val705 710 715
720His Gln Lys Ile Glu Val Lys Asp Thr Lys Glu Ala Leu Asp Lys Val
725 730 735Glu Glu Glu Gln Asn Asn Ser Lys Lys Lys Ala Gln Gln Glu
Ala Ala 740 745 750Asp Ala Gly Asn Arg Asn Gln Val Ser Gln Asn Tyr
Pro Ile Val Gln 755 760 765Asn Leu Gln Gly Gln Met Val His Gln Ala
Ile Ser Pro Arg Thr Leu 770 775 780Asn Ala Trp Val Lys Val Val Glu
Glu Lys Ala Phe Ser Pro Glu Val785 790 795 800Ile Pro Met Phe Ser
Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu 805 810 815Asn Thr Met
Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met 820 825 830Leu
Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu His 835 840
845Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg
850 855 860Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln
Ile Gly865 870 875 880Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly
Glu Ile Tyr Lys Arg 885 890 895Trp Ile Ile Leu Gly Leu Asn Lys Ile
Val Arg Met Tyr Ser Pro Thr 900 905 910Ser Ile Leu Asp Ile Lys Gln
Gly Pro Lys Glu Pro Phe Arg Asp Tyr 915 920 925Val Asp Arg Phe Tyr
Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln Glu 930 935 940Val Lys Asn
Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro945 950 955
960Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr Leu Glu
965 970 975Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His
Lys Ala 980 985 990Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr Gly
Ser Ala Ala Ile 995 1000 1005Met Met Gln Arg Gly Asn Phe Arg Asn
Gln Arg Lys Thr Val Lys 1010 1015 1020Cys Phe Asn Cys Gly Lys Glu
Gly His Ile Ala Arg Asn Cys Arg 1025 1030 1035Ala Pro Arg Lys Lys
Gly Cys Trp Lys Cys Gly Lys Glu Gly His 1040 1045 1050Gln Met Lys
Asp Cys Thr Glu Arg Gln Ala Asn 1055 1060421064PRTArtificial
SequenceHybrid protein comprised of Tat-Rev-Nef, CTL and truncated
Gag protein(TRN-CTL-dgag) 42Met Ala Gly Arg Ser Gly Asp Ser Asp Glu
Glu Leu Leu Lys Thr Val1 5 10 15Arg Leu Ile Lys Phe Leu Tyr Gln Ser
Asn Pro Pro Pro Ser Asn Glu 20 25 30Gly Thr Arg Gln Ala Arg Arg Asn
Arg Arg Arg Arg Trp Arg Glu Arg 35 40 45Gln Arg Gln Ile Arg Ser Ile
Ser Glu Arg Ile Leu Ser Thr Phe Leu 50 55 60Gly Arg Pro Ala Glu Pro
Val Pro Leu Gln Leu Pro Pro Leu Glu Arg65 70 75 80Leu Thr Leu Asp
Cys Ser Glu Asp Cys Gly Asn Ser Gly Thr Gln Gly 85 90 95Val Gly Ser
Pro Gln Val Leu Val Glu Ser Pro Ala Val Leu Glu Pro 100 105 110Gly
Thr Lys Glu Thr Ser Val Gly Lys Trp Ser Lys Cys Ser Gly Trp 115 120
125Pro Thr Val Arg Glu Arg Met Lys Gln Ala Glu Pro Glu Pro Ala Ala
130 135 140Asp Gly Val Gly Ala Ala Ser Arg Asp Leu Glu Lys His Gly
Ala Ile145 150 155 160Thr Ser Ser Asn Thr Ala Thr Asn Asn Ala Ala
Cys Ala Trp Leu Glu 165 170 175Ala Gln Glu Glu Glu Glu Val Gly Phe
Pro Val Arg Pro Gln Val Pro 180 185 190Leu Arg Pro Met Thr Tyr Lys
Gly Ala Leu Asp Leu Ser His Phe Leu 195 200 205Lys Glu Lys Gly Gly
Leu Glu Gly Leu Ile Tyr Ser Pro Lys Arg Gln 210 215 220Glu Ile Leu
Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp225 230 235
240Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe
245 250 255Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Asp Glu Glu
Glu Asn 260 265 270Ser Ser Leu Leu His Pro Ala Ser Leu His Gly Thr
Glu Asp Thr Glu 275 280 285Arg Glu Val Leu Lys Trp Lys Phe Asp Ser
His Leu Ala Phe His His 290 295 300Lys Ala Arg Glu Leu His Pro Glu
Tyr Tyr Lys Asp Cys Lys Leu Glu305 310 315 320Pro Val Asp Pro Arg
Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro 325 330 335Arg Thr Pro
Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Leu His Cys 340 345 350Gln
Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys 355 360
365Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln
370 375 380Val Ser Leu Pro Lys Gln Pro Ser Ser Gln Gln Arg Gly Asp
Pro Thr385 390 395 400Gly Pro Lys Lys Ser Lys Lys Lys Val Glu Arg
Glu Thr Glu Ala Asp 405 410 415Pro Phe Asp Ala Ala Val Ile Thr Leu
Trp Gln Arg Pro Leu Val Ala 420 425 430Leu Ile Glu Ile Cys Thr Glu
Met Glu Lys Glu Gly Lys Ile Ser Lys 435 440 445Ile Gly Pro Ala Gly
Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp 450 455 460Val Gly Asp
Ala Tyr Phe Ser Val Pro Leu Asp Lys Asp Phe Arg Lys465 470 475
480Tyr Thr Ala Phe Thr Ile Pro Ser Ile Trp Lys Gly Ser Pro Ala Ile
485 490 495Phe Gln Ser Ser Met Thr Lys Lys Gln Asn Pro Asp Ile Val
Ile Tyr 500 505 510Gln Tyr Met Asp Asp Leu Tyr Val Pro Ile Val Leu
Pro Glu Lys Asp 515 520 525Ser Trp Leu Val Gly Lys Leu Asn Trp Ala
Ser Gln Ile Tyr Ala Gly 530 535 540Ile Lys Val Lys Gln Leu Ile Leu
Lys Glu Pro Val His Gly Val Tyr545 550 555 560Glu Pro Ile Val Gly
Ala Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn 565 570 575Arg Ala Gly
Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp 580 585 590Lys
Glu Ala Thr Thr Thr Leu Val Glu Arg Tyr Leu Arg Asp Gln Gln 595 600
605Leu Leu Gly Ile Trp Gly Cys Ala Cys Thr Pro Tyr Asp Ile Asn Gln
610 615 620Met Leu Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Arg Gln
Gly Ser625 630 635 640Leu Ala Ala Val Leu Asp Lys Trp Glu Lys Ile
Arg Leu Arg Pro Gly 645 650 655Gly Lys Lys Lys Tyr Gln Leu Lys His
Ile Val Trp Ala Ser Arg Glu 660 665 670Leu Glu Arg Phe Ala Val Asn
Pro Gly Leu Leu Glu Thr Ser Glu Gly 675 680 685Cys Arg Gln Ile Met
Gly Gln Leu Gln Pro Ser Leu Gln Thr Gly Ser 690 695 700Glu Glu Leu
Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu Tyr Cys Val705 710 715
720His Gln Lys Ile Glu Val Lys Asp Thr Lys Glu Ala Leu Asp Lys
Val
725 730 735Glu Glu Glu Gln Asn Asn Ser Lys Lys Lys Ala Gln Gln Glu
Ala Ala 740 745 750Asp Ala Gly Asn Arg Asn Gln Val Ser Gln Asn Tyr
Pro Ile Val Gln 755 760 765Asn Leu Gln Gly Gln Met Val His Gln Ala
Ile Ser Pro Arg Thr Leu 770 775 780Asn Ala Trp Val Lys Val Val Glu
Glu Lys Ala Phe Ser Pro Glu Val785 790 795 800Ile Pro Met Phe Ser
Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu 805 810 815Asn Thr Met
Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met 820 825 830Leu
Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu His 835 840
845Pro Val His Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg
850 855 860Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln
Ile Gly865 870 875 880Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly
Glu Ile Tyr Lys Arg 885 890 895Trp Ile Ile Leu Gly Leu Asn Lys Ile
Val Arg Met Tyr Ser Pro Thr 900 905 910Ser Ile Leu Asp Ile Lys Gln
Gly Pro Lys Glu Pro Phe Arg Asp Tyr 915 920 925Val Asp Arg Phe Tyr
Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln Glu 930 935 940Val Lys Asn
Trp Met Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro945 950 955
960Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr Leu Glu
965 970 975Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His
Lys Ala 980 985 990Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr Gly
Ser Ala Ala Ile 995 1000 1005Met Met Gln Arg Gly Asn Phe Arg Asn
Gln Arg Lys Thr Val Lys 1010 1015 1020Cys Phe Asn Cys Gly Lys Glu
Gly His Ile Ala Arg Asn Cys Arg 1025 1030 1035Ala Pro Arg Lys Lys
Gly Cys Trp Lys Cys Gly Lys Glu Gly His 1040 1045 1050Gln Met Lys
Asp Cys Thr Glu Arg Gln Ala Asn 1055 1060431064PRTArtificial
SequenceHybrid protein comprised of Tat-Rev-Nef, truncated Gag
protein and CTL (TRN-dgag-CTL) 43Met Glu Pro Val Asp Pro Arg Leu
Glu Pro Trp Lys His Pro Gly Ser1 5 10 15Gln Pro Arg Thr Pro Cys Thr
Asn Cys Tyr Cys Lys Lys Cys Cys Leu 20 25 30His Cys Gln Val Cys Phe
Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly 35 40 45Arg Lys Lys Arg Arg
Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr 50 55 60His Gln Val Ser
Leu Pro Lys Gln Pro Ser Ser Gln Gln Arg Gly Asp65 70 75 80Pro Thr
Gly Pro Lys Lys Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 90 95Ala
Asp Pro Phe Asp Thr Ser Ala Gly Arg Ser Gly Asp Ser Asp Glu 100 105
110Glu Leu Leu Lys Thr Val Arg Leu Ile Lys Phe Leu Tyr Gln Ser Asn
115 120 125Pro Pro Pro Ser Asn Glu Gly Thr Arg Gln Ala Arg Arg Asn
Arg Arg 130 135 140Arg Arg Trp Arg Glu Arg Gln Arg Gln Ile Arg Ser
Ile Ser Glu Arg145 150 155 160Ile Leu Ser Thr Phe Leu Gly Arg Pro
Ala Glu Pro Val Pro Leu Gln 165 170 175Leu Pro Pro Leu Glu Arg Leu
Thr Leu Asp Cys Ser Glu Asp Cys Gly 180 185 190Asn Ser Gly Thr Gln
Gly Val Gly Ser Pro Gln Val Leu Val Glu Ser 195 200 205Pro Ala Val
Leu Glu Pro Gly Thr Lys Glu Lys Leu Val Gly Lys Trp 210 215 220Ser
Lys Cys Ser Gly Trp Pro Thr Val Arg Glu Arg Met Lys Gln Ala225 230
235 240Glu Pro Glu Pro Ala Ala Asp Gly Val Gly Ala Ala Ser Arg Asp
Leu 245 250 255Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Thr
Asn Asn Ala 260 265 270Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu
Glu Val Gly Phe Pro 275 280 285Val Arg Pro Gln Val Pro Leu Arg Pro
Met Thr Tyr Lys Gly Ala Leu 290 295 300Asp Leu Ser His Phe Leu Lys
Glu Lys Gly Gly Leu Glu Gly Leu Ile305 310 315 320Tyr Ser Pro Lys
Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr 325 330 335Gln Gly
Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val 340 345
350Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Glu
355 360 365Pro Asp Glu Glu Glu Asn Ser Ser Leu Leu His Pro Ala Ser
Leu His 370 375 380Gly Thr Glu Asp Thr Glu Arg Glu Val Leu Lys Trp
Lys Phe Asp Ser385 390 395 400His Leu Ala Phe His His Lys Ala Arg
Glu Leu His Pro Glu Tyr Tyr 405 410 415Lys Asp Cys Ala Ala Val Leu
Asp Lys Trp Glu Lys Ile Arg Leu Arg 420 425 430Pro Gly Gly Lys Lys
Lys Tyr Gln Leu Lys His Ile Val Trp Ala Ser 435 440 445Arg Glu Leu
Glu Arg Phe Ala Val Asn Pro Gly Leu Leu Glu Thr Ser 450 455 460Glu
Gly Cys Arg Gln Ile Met Gly Gln Leu Gln Pro Ser Leu Gln Thr465 470
475 480Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu
Tyr 485 490 495Cys Val His Gln Lys Ile Glu Val Lys Asp Thr Lys Glu
Ala Leu Asp 500 505 510Lys Val Glu Glu Glu Gln Asn Asn Ser Lys Lys
Lys Ala Gln Gln Glu 515 520 525Ala Ala Asp Ala Gly Asn Arg Asn Gln
Val Ser Gln Asn Tyr Pro Ile 530 535 540Val Gln Asn Leu Gln Gly Gln
Met Val His Gln Ala Ile Ser Pro Arg545 550 555 560Thr Leu Asn Ala
Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro 565 570 575Glu Val
Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln 580 585
590Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Ala Met
595 600 605Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp
Asp Arg 610 615 620Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly
Gln Met Arg Glu625 630 635 640Pro Arg Gly Ser Asp Ile Ala Gly Thr
Thr Ser Thr Leu Gln Glu Gln 645 650 655Ile Gly Trp Met Thr Asn Asn
Pro Pro Ile Pro Val Gly Glu Ile Tyr 660 665 670Lys Arg Trp Ile Ile
Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser 675 680 685Pro Thr Ser
Ile Leu Asp Ile Lys Gln Gly Pro Lys Glu Pro Phe Arg 690 695 700Asp
Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Thr705 710
715 720Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn
Ala 725 730 735Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro
Ala Ala Thr 740 745 750Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val
Gly Gly Pro Gly His 755 760 765Lys Ala Arg Val Leu Ala Glu Ala Met
Ser Gln Val Thr Gly Ser Ala 770 775 780Ala Ile Met Met Gln Arg Gly
Asn Phe Arg Asn Gln Arg Lys Thr Val785 790 795 800Lys Cys Phe Asn
Cys Gly Lys Glu Gly His Ile Ala Arg Asn Cys Arg 805 810 815Ala Pro
Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly His Gln 820 825
830Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Ala Ala Val Ile Thr Leu
835 840 845Trp Gln Arg Pro Leu Val Ala Leu Ile Glu Ile Cys Thr Glu
Met Glu 850 855 860Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Ala Gly
Leu Lys Lys Lys865 870 875 880Lys Ser Val Thr Val Leu Asp Val Gly
Asp Ala Tyr Phe Ser Val Pro 885 890 895Leu Asp Lys Asp Phe Arg Lys
Tyr Thr Ala Phe Thr Ile Pro Ser Ile 900 905 910Trp Lys Gly Ser Pro
Ala Ile Phe Gln Ser Ser Met Thr Lys Lys Gln 915 920 925Asn Pro Asp
Ile Val Ile Tyr Gln Tyr Met Asp Asp Leu Tyr Val Pro 930 935 940Ile
Val Leu Pro Glu Lys Asp Ser Trp Leu Val Gly Lys Leu Asn Trp945 950
955 960Ala Ser Gln Ile Tyr Ala Gly Ile Lys Val Lys Gln Leu Ile Leu
Lys 965 970 975Glu Pro Val His Gly Val Tyr Glu Pro Ile Val Gly Ala
Glu Thr Phe 980 985 990Tyr Val Asp Gly Ala Ala Asn Arg Ala Gly Asn
Leu Trp Val Thr Val 995 1000 1005Tyr Tyr Gly Val Pro Val Trp Lys
Glu Ala Thr Thr Thr Leu Val 1010 1015 1020Glu Arg Tyr Leu Arg Asp
Gln Gln Leu Leu Gly Ile Trp Gly Cys 1025 1030 1035Ala Cys Thr Pro
Tyr Asp Ile Asn Gln Met Leu Arg Gly Pro Gly 1040 1045 1050Arg Ala
Phe Val Thr Ile Arg Gln Gly Ser Leu 1055 1060441064PRTArtificial
SequenceHybrid protein comprised of Rev-Nef-Tat, truncated Gag
protein and CTL(RNT-dgag-CTL) 44Met Ala Gly Arg Ser Gly Asp Ser Asp
Glu Glu Leu Leu Lys Thr Val1 5 10 15Arg Leu Ile Lys Phe Leu Tyr Gln
Ser Asn Pro Pro Pro Ser Asn Glu 20 25 30Gly Thr Arg Gln Ala Arg Arg
Asn Arg Arg Arg Arg Trp Arg Glu Arg 35 40 45Gln Arg Gln Ile Arg Ser
Ile Ser Glu Arg Ile Leu Ser Thr Phe Leu 50 55 60Gly Arg Pro Ala Glu
Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg65 70 75 80Leu Thr Leu
Asp Cys Ser Glu Asp Cys Gly Asn Ser Gly Thr Gln Gly 85 90 95Val Gly
Ser Pro Gln Val Leu Val Glu Ser Pro Ala Val Leu Glu Pro 100 105
110Gly Thr Lys Glu Thr Ser Val Gly Lys Trp Ser Lys Cys Ser Gly Trp
115 120 125Pro Thr Val Arg Glu Arg Met Lys Gln Ala Glu Pro Glu Pro
Ala Ala 130 135 140Asp Gly Val Gly Ala Ala Ser Arg Asp Leu Glu Lys
His Gly Ala Ile145 150 155 160Thr Ser Ser Asn Thr Ala Thr Asn Asn
Ala Ala Cys Ala Trp Leu Glu 165 170 175Ala Gln Glu Glu Glu Glu Val
Gly Phe Pro Val Arg Pro Gln Val Pro 180 185 190Leu Arg Pro Met Thr
Tyr Lys Gly Ala Leu Asp Leu Ser His Phe Leu 195 200 205Lys Glu Lys
Gly Gly Leu Glu Gly Leu Ile Tyr Ser Pro Lys Arg Gln 210 215 220Glu
Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp225 230
235 240Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr
Phe 245 250 255Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Asp Glu
Glu Glu Asn 260 265 270Ser Ser Leu Leu His Pro Ala Ser Leu His Gly
Thr Glu Asp Thr Glu 275 280 285Arg Glu Val Leu Lys Trp Lys Phe Asp
Ser His Leu Ala Phe His His 290 295 300Lys Ala Arg Glu Leu His Pro
Glu Tyr Tyr Lys Asp Cys Lys Leu Glu305 310 315 320Pro Val Asp Pro
Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro 325 330 335Arg Thr
Pro Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Leu His Cys 340 345
350Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys
355 360 365Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr
His Gln 370 375 380Val Ser Leu Pro Lys Gln Pro Ser Ser Gln Gln Arg
Gly Asp Pro Thr385 390 395 400Gly Pro Lys Lys Ser Lys Lys Lys Val
Glu Arg Glu Thr Glu Ala Asp 405 410 415Pro Phe Asp Ala Ala Val Leu
Asp Lys Trp Glu Lys Ile Arg Leu Arg 420 425 430Pro Gly Gly Lys Lys
Lys Tyr Gln Leu Lys His Ile Val Trp Ala Ser 435 440 445Arg Glu Leu
Glu Arg Phe Ala Val Asn Pro Gly Leu Leu Glu Thr Ser 450 455 460Glu
Gly Cys Arg Gln Ile Met Gly Gln Leu Gln Pro Ser Leu Gln Thr465 470
475 480Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu
Tyr 485 490 495Cys Val His Gln Lys Ile Glu Val Lys Asp Thr Lys Glu
Ala Leu Asp 500 505 510Lys Val Glu Glu Glu Gln Asn Asn Ser Lys Lys
Lys Ala Gln Gln Glu 515 520 525Ala Ala Asp Ala Gly Asn Arg Asn Gln
Val Ser Gln Asn Tyr Pro Ile 530 535 540Val Gln Asn Leu Gln Gly Gln
Met Val His Gln Ala Ile Ser Pro Arg545 550 555 560Thr Leu Asn Ala
Trp Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro 565 570 575Glu Val
Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln 580 585
590Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His Gln Ala Ala Met
595 600 605Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp
Asp Arg 610 615 620Leu His Pro Val His Ala Gly Pro Ile Ala Pro Gly
Gln Met Arg Glu625 630 635 640Pro Arg Gly Ser Asp Ile Ala Gly Thr
Thr Ser Thr Leu Gln Glu Gln 645 650 655Ile Gly Trp Met Thr Asn Asn
Pro Pro Ile Pro Val Gly Glu Ile Tyr 660 665 670Lys Arg Trp Ile Ile
Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser 675 680 685Pro Thr Ser
Ile Leu Asp Ile Lys Gln Gly Pro Lys Glu Pro Phe Arg 690 695 700Asp
Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Thr705 710
715 720Gln Glu Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val Gln Asn
Ala 725 730 735Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro
Ala Ala Thr 740 745 750Leu Glu Glu Met Met Thr Ala Cys Gln Gly Val
Gly Gly Pro Gly His 755 760 765Lys Ala Arg Val Leu Ala Glu Ala Met
Ser Gln Val Thr Gly Ser Ala 770 775 780Ala Ile Met Met Gln Arg Gly
Asn Phe Arg Asn Gln Arg Lys Thr Val785 790 795 800Lys Cys Phe Asn
Cys Gly Lys Glu Gly His Ile Ala Arg Asn Cys Arg 805 810 815Ala Pro
Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys Glu Gly His Gln 820 825
830Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Ala Ala Val Ile Thr Leu
835 840 845Trp Gln Arg Pro Leu Val Ala Leu Ile Glu Ile Cys Thr Glu
Met Glu 850 855 860Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Ala Gly
Leu Lys Lys Lys865 870 875 880Lys Ser Val Thr Val Leu Asp Val Gly
Asp Ala Tyr Phe Ser Val Pro 885 890 895Leu Asp Lys Asp Phe Arg Lys
Tyr Thr Ala Phe Thr Ile Pro Ser Ile 900 905 910Trp Lys Gly Ser Pro
Ala Ile Phe Gln Ser Ser Met Thr Lys Lys Gln 915 920 925Asn Pro Asp
Ile Val Ile Tyr Gln Tyr Met Asp Asp Leu Tyr Val Pro 930 935 940Ile
Val Leu Pro Glu Lys Asp Ser Trp Leu Val Gly Lys Leu Asn Trp945 950
955 960Ala Ser Gln Ile Tyr Ala Gly Ile Lys Val Lys Gln Leu Ile Leu
Lys 965 970 975Glu Pro Val His Gly Val Tyr Glu Pro Ile Val Gly Ala
Glu Thr Phe 980 985 990Tyr Val Asp Gly Ala Ala Asn Arg Ala Gly Asn
Leu Trp Val Thr Val 995 1000 1005Tyr Tyr Gly Val Pro Val Trp Lys
Glu Ala Thr Thr Thr Leu Val 1010 1015 1020Glu Arg Tyr Leu Arg Asp
Gln Gln Leu Leu Gly Ile Trp Gly Cys 1025 1030 1035Ala Cys Thr Pro
Tyr Asp
Ile Asn Gln Met Leu Arg Gly Pro Gly 1040 1045 1050Arg Ala Phe Val
Thr Ile Arg Gln Gly Ser Leu 1055 1060451006PRTArtificial
SequenceHybrid protein cds comprised of Tat-Rev-Nef, truncated Gag
protein and CTL(TRN-optp17/24-CTL ) 45Met Glu Pro Val Asp Pro Arg
Leu Glu Pro Trp Lys His Pro Gly Ser1 5 10 15Gln Pro Arg Thr Pro Cys
Thr Asn Cys Tyr Cys Lys Lys Cys Cys Leu 20 25 30His Cys Gln Val Cys
Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly 35 40 45Arg Lys Lys Arg
Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr 50 55 60His Gln Val
Ser Leu Pro Lys Gln Pro Ser Ser Gln Gln Arg Gly Asp65 70 75 80Pro
Thr Gly Pro Lys Lys Ser Lys Lys Lys Val Glu Arg Glu Thr Glu 85 90
95Ala Asp Pro Phe Asp Thr Ser Ala Gly Arg Ser Gly Asp Ser Asp Glu
100 105 110Glu Leu Leu Lys Thr Val Arg Leu Ile Lys Phe Leu Tyr Gln
Ser Asn 115 120 125Pro Pro Pro Ser Asn Glu Gly Thr Arg Gln Ala Arg
Arg Asn Arg Arg 130 135 140Arg Arg Trp Arg Glu Arg Gln Arg Gln Ile
Arg Ser Ile Ser Glu Arg145 150 155 160Ile Leu Ser Thr Phe Leu Gly
Arg Pro Ala Glu Pro Val Pro Leu Gln 165 170 175Leu Pro Pro Leu Glu
Arg Leu Thr Leu Asp Cys Ser Glu Asp Cys Gly 180 185 190Asn Ser Gly
Thr Gln Gly Val Gly Ser Pro Gln Val Leu Val Glu Ser 195 200 205Pro
Ala Val Leu Glu Pro Gly Thr Lys Glu Lys Leu Val Gly Lys Trp 210 215
220Ser Lys Cys Ser Gly Trp Pro Thr Val Arg Glu Arg Met Lys Gln
Ala225 230 235 240Glu Pro Glu Pro Ala Ala Asp Gly Val Gly Ala Ala
Ser Arg Asp Leu 245 250 255Glu Lys His Gly Ala Ile Thr Ser Ser Asn
Thr Ala Thr Asn Asn Ala 260 265 270Ala Cys Ala Trp Leu Glu Ala Gln
Glu Glu Glu Glu Val Gly Phe Pro 275 280 285Val Arg Pro Gln Val Pro
Leu Arg Pro Met Thr Tyr Lys Gly Ala Leu 290 295 300Asp Leu Ser His
Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile305 310 315 320Tyr
Ser Pro Lys Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr 325 330
335Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val
340 345 350Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu Val Pro
Val Glu 355 360 365Pro Asp Glu Glu Glu Asn Ser Ser Leu Leu His Pro
Ala Ser Leu His 370 375 380Gly Thr Glu Asp Thr Glu Arg Glu Val Leu
Lys Trp Lys Phe Asp Ser385 390 395 400His Leu Ala Phe His His Lys
Ala Arg Glu Leu His Pro Glu Tyr Tyr 405 410 415Lys Asp Cys Ala Ala
Val Gly Ala Arg Ala Ser Val Leu Ser Gly Gly 420 425 430Glu Leu Asp
Lys Trp Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys 435 440 445Lys
Tyr Gln Leu Lys His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg 450 455
460Phe Ala Val Asn Pro Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg
Gln465 470 475 480Ile Met Gly Gln Leu Gln Pro Ser Leu Gln Thr Gly
Ser Glu Glu Leu 485 490 495Arg Ser Leu Tyr Asn Thr Val Ala Thr Leu
Tyr Cys Val His Gln Lys 500 505 510Ile Glu Val Lys Asp Thr Lys Glu
Ala Leu Asp Lys Val Glu Glu Glu 515 520 525Gln Asn Asn Ser Lys Lys
Lys Ala Gln Gln Glu Ala Ala Asp Ala Gly 530 535 540Asn Arg Asn Gln
Val Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln545 550 555 560Gly
Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp 565 570
575Val Lys Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met
580 585 590Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn
Thr Met 595 600 605Leu Asn Thr Val Gly Gly His Gln Ala Ala Met Gln
Met Leu Lys Glu 610 615 620Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp
Arg Leu His Pro Val His625 630 635 640Ala Gly Pro Ile Ala Pro Gly
Gln Met Arg Glu Pro Arg Gly Ser Asp 645 650 655Ile Ala Gly Thr Thr
Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr 660 665 670Asn Asn Pro
Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile 675 680 685Leu
Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu 690 695
700Asp Ile Lys Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp
Arg705 710 715 720Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln
Glu Val Lys Asn 725 730 735Trp Met Thr Glu Thr Leu Leu Val Gln Asn
Ala Asn Pro Asp Cys Lys 740 745 750Thr Ile Leu Lys Ala Leu Gly Pro
Ala Ala Thr Leu Glu Glu Met Met 755 760 765Thr Ala Cys Gln Gly Val
Gly Gly Pro Gly His Lys Ala Arg Val Leu 770 775 780Ala Ala Val Ile
Thr Leu Trp Gln Arg Pro Leu Val Ala Leu Ile Glu785 790 795 800Ile
Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro 805 810
815Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp Val Gly Asp
820 825 830Ala Tyr Phe Ser Val Pro Leu Asp Lys Asp Phe Arg Lys Tyr
Thr Ala 835 840 845Phe Thr Ile Pro Ser Ile Trp Lys Gly Ser Pro Ala
Ile Phe Gln Ser 850 855 860Ser Met Thr Lys Lys Gln Asn Pro Asp Ile
Val Ile Tyr Gln Tyr Met865 870 875 880Asp Asp Leu Tyr Val Pro Ile
Val Leu Pro Glu Lys Asp Ser Trp Leu 885 890 895Val Gly Lys Leu Asn
Trp Ala Ser Gln Ile Tyr Ala Gly Ile Lys Val 900 905 910Lys Gln Leu
Ile Leu Lys Glu Pro Val His Gly Val Tyr Glu Pro Ile 915 920 925Val
Gly Ala Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn Arg Ala Gly 930 935
940Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu
Ala945 950 955 960Thr Thr Thr Leu Val Glu Arg Tyr Leu Arg Asp Gln
Gln Leu Leu Gly 965 970 975Ile Trp Gly Cys Ala Cys Thr Pro Tyr Asp
Ile Asn Gln Met Leu Arg 980 985 990Gly Pro Gly Arg Ala Phe Val Thr
Ile Arg Gln Gly Ser Leu 995 1000 1005461006PRTArtificial
SequenceHybrid protein cdscomprised of Tat-Rev-Nef, CTL and
truncated Gag protein (TRN-CTL-optp17/24) 46Met Glu Pro Val Asp Pro
Arg Leu Glu Pro Trp Lys His Pro Gly Ser1 5 10 15Gln Pro Arg Thr Pro
Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Leu 20 25 30His Cys Gln Val
Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly 35 40 45Arg Lys Lys
Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr 50 55 60His Gln
Val Ser Leu Pro Lys Gln Pro Ser Ser Gln Gln Arg Gly Asp65 70 75
80Pro Thr Gly Pro Lys Lys Ser Lys Lys Lys Val Glu Arg Glu Thr Glu
85 90 95Ala Asp Pro Phe Asp Thr Ser Ala Gly Arg Ser Gly Asp Ser Asp
Glu 100 105 110Glu Leu Leu Lys Thr Val Arg Leu Ile Lys Phe Leu Tyr
Gln Ser Asn 115 120 125Pro Pro Pro Ser Asn Glu Gly Thr Arg Gln Ala
Arg Arg Asn Arg Arg 130 135 140Arg Arg Trp Arg Glu Arg Gln Arg Gln
Ile Arg Ser Ile Ser Glu Arg145 150 155 160Ile Leu Ser Thr Phe Leu
Gly Arg Pro Ala Glu Pro Val Pro Leu Gln 165 170 175Leu Pro Pro Leu
Glu Arg Leu Thr Leu Asp Cys Ser Glu Asp Cys Gly 180 185 190Asn Ser
Gly Thr Gln Gly Val Gly Ser Pro Gln Val Leu Val Glu Ser 195 200
205Pro Ala Val Leu Glu Pro Gly Thr Lys Glu Lys Leu Val Gly Lys Trp
210 215 220Ser Lys Cys Ser Gly Trp Pro Thr Val Arg Glu Arg Met Lys
Gln Ala225 230 235 240Glu Pro Glu Pro Ala Ala Asp Gly Val Gly Ala
Ala Ser Arg Asp Leu 245 250 255Glu Lys His Gly Ala Ile Thr Ser Ser
Asn Thr Ala Thr Asn Asn Ala 260 265 270Ala Cys Ala Trp Leu Glu Ala
Gln Glu Glu Glu Glu Val Gly Phe Pro 275 280 285Val Arg Pro Gln Val
Pro Leu Arg Pro Met Thr Tyr Lys Gly Ala Leu 290 295 300Asp Leu Ser
His Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile305 310 315
320Tyr Ser Pro Lys Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr
325 330 335Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro
Gly Val 340 345 350Arg Tyr Pro Leu Thr Phe Gly Trp Cys Phe Lys Leu
Val Pro Val Glu 355 360 365Pro Asp Glu Glu Glu Asn Ser Ser Leu Leu
His Pro Ala Ser Leu His 370 375 380Gly Thr Glu Asp Thr Glu Arg Glu
Val Leu Lys Trp Lys Phe Asp Ser385 390 395 400His Leu Ala Phe His
His Lys Ala Arg Glu Leu His Pro Glu Tyr Tyr 405 410 415Lys Asp Cys
Ala Ala Val Ile Thr Leu Trp Gln Arg Pro Leu Val Ala 420 425 430Leu
Ile Glu Ile Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys 435 440
445Ile Gly Pro Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp
450 455 460Val Gly Asp Ala Tyr Phe Ser Val Pro Leu Asp Lys Asp Phe
Arg Lys465 470 475 480Tyr Thr Ala Phe Thr Ile Pro Ser Ile Trp Lys
Gly Ser Pro Ala Ile 485 490 495Phe Gln Ser Ser Met Thr Lys Lys Gln
Asn Pro Asp Ile Val Ile Tyr 500 505 510Gln Tyr Met Asp Asp Leu Tyr
Val Pro Ile Val Leu Pro Glu Lys Asp 515 520 525Ser Trp Leu Val Gly
Lys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly 530 535 540Ile Lys Val
Lys Gln Leu Ile Leu Lys Glu Pro Val His Gly Val Tyr545 550 555
560Glu Pro Ile Val Gly Ala Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn
565 570 575Arg Ala Gly Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro
Val Trp 580 585 590Lys Glu Ala Thr Thr Thr Leu Val Glu Arg Tyr Leu
Arg Asp Gln Gln 595 600 605Leu Leu Gly Ile Trp Gly Cys Ala Cys Thr
Pro Tyr Asp Ile Asn Gln 610 615 620Met Leu Arg Gly Pro Gly Arg Ala
Phe Val Thr Ile Arg Gln Gly Ser625 630 635 640Leu Ala Ala Val Gly
Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu 645 650 655Asp Lys Trp
Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr 660 665 670Gln
Leu Lys His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala 675 680
685Val Asn Pro Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Met
690 695 700Gly Gln Leu Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu
Arg Ser705 710 715 720Leu Tyr Asn Thr Val Ala Thr Leu Tyr Cys Val
His Gln Lys Ile Glu 725 730 735Val Lys Asp Thr Lys Glu Ala Leu Asp
Lys Val Glu Glu Glu Gln Asn 740 745 750Asn Ser Lys Lys Lys Ala Gln
Gln Glu Ala Ala Asp Ala Gly Asn Arg 755 760 765Asn Gln Val Ser Gln
Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln 770 775 780Met Val His
Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys785 790 795
800Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser
805 810 815Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met
Leu Asn 820 825 830Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu
Lys Glu Thr Ile 835 840 845Asn Glu Glu Ala Ala Glu Trp Asp Arg Leu
His Pro Val His Ala Gly 850 855 860Pro Ile Ala Pro Gly Gln Met Arg
Glu Pro Arg Gly Ser Asp Ile Ala865 870 875 880Gly Thr Thr Ser Thr
Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn 885 890 895Pro Pro Ile
Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly 900 905 910Leu
Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile 915 920
925Lys Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr
930 935 940Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln Glu Val Lys Asn
Trp Met945 950 955 960Thr Glu Thr Leu Leu Val Gln Asn Ala Asn Pro
Asp Cys Lys Thr Ile 965 970 975Leu Lys Ala Leu Gly Pro Ala Ala Thr
Leu Glu Glu Met Met Thr Ala 980 985 990Cys Gln Gly Val Gly Gly Pro
Gly His Lys Ala Arg Val Leu 995 1000 1005471006PRTArtificial
SequenceHybrid protein comprised of Rev-Nef-Tat, CTL and truncated
Gag protein (RNT-CTL-optp17/24) 47Met Ala Gly Arg Ser Gly Asp Ser
Asp Glu Glu Leu Leu Lys Thr Val1 5 10 15Arg Leu Ile Lys Phe Leu Tyr
Gln Ser Asn Pro Pro Pro Ser Asn Glu 20 25 30Gly Thr Arg Gln Ala Arg
Arg Asn Arg Arg Arg Arg Trp Arg Glu Arg 35 40 45Gln Arg Gln Ile Arg
Ser Ile Ser Glu Arg Ile Leu Ser Thr Phe Leu 50 55 60Gly Arg Pro Ala
Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg65 70 75 80Leu Thr
Leu Asp Cys Ser Glu Asp Cys Gly Asn Ser Gly Thr Gln Gly 85 90 95Val
Gly Ser Pro Gln Val Leu Val Glu Ser Pro Ala Val Leu Glu Pro 100 105
110Gly Thr Lys Glu Thr Ser Val Gly Lys Trp Ser Lys Cys Ser Gly Trp
115 120 125Pro Thr Val Arg Glu Arg Met Lys Gln Ala Glu Pro Glu Pro
Ala Ala 130 135 140Asp Gly Val Gly Ala Ala Ser Arg Asp Leu Glu Lys
His Gly Ala Ile145 150 155 160Thr Ser Ser Asn Thr Ala Thr Asn Asn
Ala Ala Cys Ala Trp Leu Glu 165 170 175Ala Gln Glu Glu Glu Glu Val
Gly Phe Pro Val Arg Pro Gln Val Pro 180 185 190Leu Arg Pro Met Thr
Tyr Lys Gly Ala Leu Asp Leu Ser His Phe Leu 195 200 205Lys Glu Lys
Gly Gly Leu Glu Gly Leu Ile Tyr Ser Pro Lys Arg Gln 210 215 220Glu
Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe Pro Asp225 230
235 240Trp Gln Asn Tyr Thr Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr
Phe 245 250 255Gly Trp Cys Phe Lys Leu Val Pro Val Glu Pro Asp Glu
Glu Glu Asn 260 265 270Ser Ser Leu Leu His Pro Ala Ser Leu His Gly
Thr Glu Asp Thr Glu 275 280 285Arg Glu Val Leu Lys Trp Lys Phe Asp
Ser His Leu Ala Phe His His 290 295 300Lys Ala Arg Glu Leu His Pro
Glu Tyr Tyr Lys Asp Cys Lys Leu Glu305 310 315 320Pro Val Asp Pro
Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro 325 330 335Arg Thr
Pro Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Leu His Cys 340 345
350Gln Val Cys Phe Thr Arg Lys Gly Leu Gly Ile Ser Tyr Gly Arg Lys
355 360 365Lys Arg Arg Gln Arg Arg Arg Ala Pro Gln Asp Ser Gln Thr
His Gln 370 375 380Val Ser Leu Pro Lys
Gln Pro Ser Ser Gln Gln Arg Gly Asp Pro Thr385 390 395 400Gly Pro
Lys Lys Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Ala Asp 405 410
415Pro Phe Asp Ala Ala Val Ile Thr Leu Trp Gln Arg Pro Leu Val Ala
420 425 430Leu Ile Glu Ile Cys Thr Glu Met Glu Lys Glu Gly Lys Ile
Ser Lys 435 440 445Ile Gly Pro Ala Gly Leu Lys Lys Lys Lys Ser Val
Thr Val Leu Asp 450 455 460Val Gly Asp Ala Tyr Phe Ser Val Pro Leu
Asp Lys Asp Phe Arg Lys465 470 475 480Tyr Thr Ala Phe Thr Ile Pro
Ser Ile Trp Lys Gly Ser Pro Ala Ile 485 490 495Phe Gln Ser Ser Met
Thr Lys Lys Gln Asn Pro Asp Ile Val Ile Tyr 500 505 510Gln Tyr Met
Asp Asp Leu Tyr Val Pro Ile Val Leu Pro Glu Lys Asp 515 520 525Ser
Trp Leu Val Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly 530 535
540Ile Lys Val Lys Gln Leu Ile Leu Lys Glu Pro Val His Gly Val
Tyr545 550 555 560Glu Pro Ile Val Gly Ala Glu Thr Phe Tyr Val Asp
Gly Ala Ala Asn 565 570 575Arg Ala Gly Asn Leu Trp Val Thr Val Tyr
Tyr Gly Val Pro Val Trp 580 585 590Lys Glu Ala Thr Thr Thr Leu Val
Glu Arg Tyr Leu Arg Asp Gln Gln 595 600 605Leu Leu Gly Ile Trp Gly
Cys Ala Cys Thr Pro Tyr Asp Ile Asn Gln 610 615 620Met Leu Arg Gly
Pro Gly Arg Ala Phe Val Thr Ile Arg Gln Gly Ser625 630 635 640Leu
Ala Ala Val Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu 645 650
655Asp Lys Trp Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr
660 665 670Gln Leu Lys His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg
Phe Ala 675 680 685Val Asn Pro Gly Leu Leu Glu Thr Ser Glu Gly Cys
Arg Gln Ile Met 690 695 700Gly Gln Leu Gln Pro Ser Leu Gln Thr Gly
Ser Glu Glu Leu Arg Ser705 710 715 720Leu Tyr Asn Thr Val Ala Thr
Leu Tyr Cys Val His Gln Lys Ile Glu 725 730 735Val Lys Asp Thr Lys
Glu Ala Leu Asp Lys Val Glu Glu Glu Gln Asn 740 745 750Asn Ser Lys
Lys Lys Ala Gln Gln Glu Ala Ala Asp Ala Gly Asn Arg 755 760 765Asn
Gln Val Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln 770 775
780Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val
Lys785 790 795 800Val Val Glu Glu Lys Ala Phe Ser Pro Glu Val Ile
Pro Met Phe Ser 805 810 815Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp
Leu Asn Thr Met Leu Asn 820 825 830Thr Val Gly Gly His Gln Ala Ala
Met Gln Met Leu Lys Glu Thr Ile 835 840 845Asn Glu Glu Ala Ala Glu
Trp Asp Arg Leu His Pro Val His Ala Gly 850 855 860Pro Ile Ala Pro
Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala865 870 875 880Gly
Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn 885 890
895Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly
900 905 910Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu
Asp Ile 915 920 925Lys Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val
Asp Arg Phe Tyr 930 935 940Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln
Glu Val Lys Asn Trp Met945 950 955 960Thr Glu Thr Leu Leu Val Gln
Asn Ala Asn Pro Asp Cys Lys Thr Ile 965 970 975Leu Lys Ala Leu Gly
Pro Ala Ala Thr Leu Glu Glu Met Met Thr Ala 980 985 990Cys Gln Gly
Val Gly Gly Pro Gly His Lys Ala Arg Val Leu 995 1000
1005481006PRTArtificial SequenceHybrid protein comprised of
Rev-Nef-Tat, truncated Gag protein and CTL (RNT-optp17/24-CTL)
48Met Ala Gly Arg Ser Gly Asp Ser Asp Glu Glu Leu Leu Lys Thr Val1
5 10 15Arg Leu Ile Lys Phe Leu Tyr Gln Ser Asn Pro Pro Pro Ser Asn
Glu 20 25 30Gly Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg
Glu Arg 35 40 45Gln Arg Gln Ile Arg Ser Ile Ser Glu Arg Ile Leu Ser
Thr Phe Leu 50 55 60Gly Arg Pro Ala Glu Pro Val Pro Leu Gln Leu Pro
Pro Leu Glu Arg65 70 75 80Leu Thr Leu Asp Cys Ser Glu Asp Cys Gly
Asn Ser Gly Thr Gln Gly 85 90 95Val Gly Ser Pro Gln Val Leu Val Glu
Ser Pro Ala Val Leu Glu Pro 100 105 110Gly Thr Lys Glu Thr Ser Val
Gly Lys Trp Ser Lys Cys Ser Gly Trp 115 120 125Pro Thr Val Arg Glu
Arg Met Lys Gln Ala Glu Pro Glu Pro Ala Ala 130 135 140Asp Gly Val
Gly Ala Ala Ser Arg Asp Leu Glu Lys His Gly Ala Ile145 150 155
160Thr Ser Ser Asn Thr Ala Thr Asn Asn Ala Ala Cys Ala Trp Leu Glu
165 170 175Ala Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln
Val Pro 180 185 190Leu Arg Pro Met Thr Tyr Lys Gly Ala Leu Asp Leu
Ser His Phe Leu 195 200 205Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile
Tyr Ser Pro Lys Arg Gln 210 215 220Glu Ile Leu Asp Leu Trp Val Tyr
His Thr Gln Gly Tyr Phe Pro Asp225 230 235 240Trp Gln Asn Tyr Thr
Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe 245 250 255Gly Trp Cys
Phe Lys Leu Val Pro Val Glu Pro Asp Glu Glu Glu Asn 260 265 270Ser
Ser Leu Leu His Pro Ala Ser Leu His Gly Thr Glu Asp Thr Glu 275 280
285Arg Glu Val Leu Lys Trp Lys Phe Asp Ser His Leu Ala Phe His His
290 295 300Lys Ala Arg Glu Leu His Pro Glu Tyr Tyr Lys Asp Cys Lys
Leu Glu305 310 315 320Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His
Pro Gly Ser Gln Pro 325 330 335Arg Thr Pro Cys Thr Asn Cys Tyr Cys
Lys Lys Cys Cys Leu His Cys 340 345 350Gln Val Cys Phe Thr Arg Lys
Gly Leu Gly Ile Ser Tyr Gly Arg Lys 355 360 365Lys Arg Arg Gln Arg
Arg Arg Ala Pro Gln Asp Ser Gln Thr His Gln 370 375 380Val Ser Leu
Pro Lys Gln Pro Ser Ser Gln Gln Arg Gly Asp Pro Thr385 390 395
400Gly Pro Lys Lys Ser Lys Lys Lys Val Glu Arg Glu Thr Glu Ala Asp
405 410 415Pro Phe Asp Ala Ala Val Gly Ala Arg Ala Ser Val Leu Ser
Gly Gly 420 425 430Glu Leu Asp Lys Trp Glu Lys Ile Arg Leu Arg Pro
Gly Gly Lys Lys 435 440 445Lys Tyr Gln Leu Lys His Ile Val Trp Ala
Ser Arg Glu Leu Glu Arg 450 455 460Phe Ala Val Asn Pro Gly Leu Leu
Glu Thr Ser Glu Gly Cys Arg Gln465 470 475 480Ile Met Gly Gln Leu
Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu 485 490 495Arg Ser Leu
Tyr Asn Thr Val Ala Thr Leu Tyr Cys Val His Gln Lys 500 505 510Ile
Glu Val Lys Asp Thr Lys Glu Ala Leu Asp Lys Val Glu Glu Glu 515 520
525Gln Asn Asn Ser Lys Lys Lys Ala Gln Gln Glu Ala Ala Asp Ala Gly
530 535 540Asn Arg Asn Gln Val Ser Gln Asn Tyr Pro Ile Val Gln Asn
Leu Gln545 550 555 560Gly Gln Met Val His Gln Ala Ile Ser Pro Arg
Thr Leu Asn Ala Trp 565 570 575Val Lys Val Val Glu Glu Lys Ala Phe
Ser Pro Glu Val Ile Pro Met 580 585 590Phe Ser Ala Leu Ser Glu Gly
Ala Thr Pro Gln Asp Leu Asn Thr Met 595 600 605Leu Asn Thr Val Gly
Gly His Gln Ala Ala Met Gln Met Leu Lys Glu 610 615 620Thr Ile Asn
Glu Glu Ala Ala Glu Trp Asp Arg Leu His Pro Val His625 630 635
640Ala Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp
645 650 655Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp
Met Thr 660 665 670Asn Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys
Arg Trp Ile Ile 675 680 685Leu Gly Leu Asn Lys Ile Val Arg Met Tyr
Ser Pro Thr Ser Ile Leu 690 695 700Asp Ile Lys Gln Gly Pro Lys Glu
Pro Phe Arg Asp Tyr Val Asp Arg705 710 715 720Phe Tyr Lys Thr Leu
Arg Ala Glu Gln Ala Thr Gln Glu Val Lys Asn 725 730 735Trp Met Thr
Glu Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys 740 745 750Thr
Ile Leu Lys Ala Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met 755 760
765Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala Arg Val Leu
770 775 780Ala Ala Val Ile Thr Leu Trp Gln Arg Pro Leu Val Ala Leu
Ile Glu785 790 795 800Ile Cys Thr Glu Met Glu Lys Glu Gly Lys Ile
Ser Lys Ile Gly Pro 805 810 815Ala Gly Leu Lys Lys Lys Lys Ser Val
Thr Val Leu Asp Val Gly Asp 820 825 830Ala Tyr Phe Ser Val Pro Leu
Asp Lys Asp Phe Arg Lys Tyr Thr Ala 835 840 845Phe Thr Ile Pro Ser
Ile Trp Lys Gly Ser Pro Ala Ile Phe Gln Ser 850 855 860Ser Met Thr
Lys Lys Gln Asn Pro Asp Ile Val Ile Tyr Gln Tyr Met865 870 875
880Asp Asp Leu Tyr Val Pro Ile Val Leu Pro Glu Lys Asp Ser Trp Leu
885 890 895Val Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly Ile
Lys Val 900 905 910Lys Gln Leu Ile Leu Lys Glu Pro Val His Gly Val
Tyr Glu Pro Ile 915 920 925Val Gly Ala Glu Thr Phe Tyr Val Asp Gly
Ala Ala Asn Arg Ala Gly 930 935 940Asn Leu Trp Val Thr Val Tyr Tyr
Gly Val Pro Val Trp Lys Glu Ala945 950 955 960Thr Thr Thr Leu Val
Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly 965 970 975Ile Trp Gly
Cys Ala Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu Arg 980 985 990Gly
Pro Gly Arg Ala Phe Val Thr Ile Arg Gln Gly Ser Leu 995 1000
1005497945DNABovine papillomavirusmisc_feature1205n = A,T,C or G
49gttaacaata atcacaccat caccgttttt tcaagcggga aaaaatagcc agctaactat
60aaaaagctgc tgacagaccc cggttttcac atggacctga aaccttttgc aagaaccaat
120ccattctcag ggttggattg tctgtggtgc agagagcctc ttacagaagt
tgatgctttt 180aggtgcatgg tcaaagactt tcatgttgta attcgggaag
gctgtagata tggtgcatgt 240accatttgtc ttgaaaactg tttagctact
gaaagaagac tttggcaagg tgttccagta 300acaggtgagg aagctgaatt
attgcatggc aaaacacttg ataggctttg cataagatgc 360tgctactgtg
ggggcaaact aacaaaaaat gaaaaacatc ggcatgtgct ttttaatgag
420cctttctgca aaaccagagc taacataatt agaggacgct gctacgactg
ctgcagacat 480ggttcaaggt ccaaataccc atagaaactt ggatgattca
cctgcaggac cgttgctgat 540tttaagtcca tgtgcaggca cacctaccag
gtctcctgca gcacctgatg cacctgattt 600cagacttccg tgccatttcg
gccgtcctac taggaagcga ggtcccacta cccctccgct 660ttcctctccc
ggaaaactgt gtgcaacagg gccacgtcga gtgtattctg tgactgtctg
720ctgtggaaac tgcggaaaag agctgacttt tgctgtgaag accagctcga
cgtccctgct 780tggatttgaa caccttttaa actcagattt agacctcttg
tgtccacgtt gtgaatctcg 840cgagcgtcat ggcaaacgat aaaggtagca
attgggattc gggcttggga tgctcatatc 900tgctgactga ggcagaatgt
gaaagtgaca aagagaatga ggaacccggg gcaggtgtag 960aactgtctgt
ggaatctgat cggtatgata gccaggatga ggattttgtt gacaatgcat
1020cagtctttca gggaaatcac ctggaggtct tccaggcatt agagaaaaag
gcgggtgagg 1080agcagatttt aaatttgaaa agaaaagtat tggggagttc
gcaaaacagc agcggttccg 1140aagcatctga aactccagtt aaaagacgga
aatcaggagc aaagcgaaga ttatttgctg 1200aaaangaagc taaccgtgtt
cttacgcccc tccaggtaca gggggagggg gaggggaggc 1260aagaacttaa
tgaggagcag gcaattagtc atctacatct gcagcttgtt aaatctaaaa
1320atgctacagt ttttaagctg gggctcttta aatctttgtt cctttgtagc
ttccatgata 1380ttacgaggtt gtttaagaat gataagacca ctaatcagca
atgggtgctg gctgtgtttg 1440gccttgcaga ggtgtttttt gaggcgagtt
tcgaactcct aaagaagcag tgtagttttc 1500tgcagatgca aaaaagatct
catgaaggag gaacttgtgc agtttactta atctgcttta 1560acacagctaa
aagcagagaa acagtccgga atctgatggc aaacacgcta aatgtaagag
1620aagagtgttt gatgctgcag ccagctaaaa ttcgaggact cagcgcagct
ctattctggt 1680ttaaaagtag tttgtcaccc gctacactta aacatggtgc
tttacctgag tggatacggg 1740cgcaaactac tctgaacgag agcttgcaga
ccgagaaatt cgacttcgga actatggtgc 1800aatgggccta tgatcacaaa
tatgctgagg agtctaaaat agcctatgaa tatgctttgg 1860ctgcaggatc
tgatagcaat gcacgggctt ttttagcaac taacagccaa gctaagcatg
1920tgaaggactg tgcaactatg gtaagacact atctaagagc tgaaacacaa
gcattaagca 1980tgcctgcata tattaaagct aggtgcaagc tggcaactgg
ggaaggaagc tggaagtcta 2040tcctaacttt ttttaactat cagaatattg
aattaattac ctttattaat gctttaaagc 2100tctggctaaa aggaattcca
aaaaaaaact gtttagcatt tattggccct ccaaacacag 2160gcaagtctat
gctctgcaac tcattaattc attttttggg tggtagtgtt ttatcttttg
2220ccaaccataa aagtcacttt tggcttgctt ccctagcaga tactagagct
gctttagtag 2280atgatgctac tcatgcttgc tggaggtact ttgacacata
cctcagaaat gcattggatg 2340gctaccctgt cagtattgat agaaaacaca
aagcagcggt tcaaattaaa gctccacccc 2400tcctggtaac cagtaatatt
gatgtgcagg cagaggacag atatttgtac ttgcatagtc 2460gggtgcaaac
ctttcgcttt gagcagccat gcacagatga atcgggtgag caacctttta
2520atattactga tgcagattgg aaatcttttt ttgtaaggtt atgggggcgt
ttagacctga 2580ttgacgagga ggaggatagt gaagaggatg gagacagcat
gcgaacgttt acatgtagcg 2640caagaaacac aaatgcagtt gattgagaaa
agtagtgata agttgcaaga tcatatactg 2700tactggactg ctgttagaac
tgagaacaca ctgctttatg ctgcaaggaa aaaaggggtg 2760actgtcctag
gacactgcag agtaccacac tctgtagttt gtcaagagag agccaagcag
2820gccattgaaa tgcagttgtc tttgcaggag ttaagcaaaa ctgagtttgg
ggatgaacca 2880tggtctttgc ttgacacaag ctgggaccga tatatgtcag
aacctaaacg gtgctttaag 2940aaaggcgcca gggtggtaga ggtggagttt
gatggaaatg caagcaatac aaactggtac 3000actgtctaca gcaatttgta
catgcgcaca gaggacggct ggcagcttgc gaaggctggg 3060gctgacggaa
ctgggctcta ctactgcacc atggccggtg ctggacgcat ttactattct
3120cgctttggtg acgaggcagc cagatttagt acaacagggc attactctgt
aagagatcag 3180gacagagtgt atgctggtgt ctcatccacc tcttctgatt
ttagagatcg cccagacgga 3240gtctgggtcg catccgaagg acctgaagga
gaccctgcag gaaaagaagc cgagccagcc 3300cagcctgtct cttctttgct
cggctccccc gcctgcggtc ccatcagagc aggcctcggt 3360tgggtacggg
acggtcctcg ctcgcacccc tacaattttc ctgcaggctc ggggggctct
3420attctccgct cttcctccac cccgtgcagg gcacggtacc ggtggacttg
gcatcaaggc 3480aggaagaaga ggagcagtcg cccgactcca cagaggaaga
accagtgact ctcccaaggc 3540gcaccaccaa tgatggattc cacctgttaa
aggcaggagg gtcatgcttt gctctaattt 3600caggaactgc taaccaggta
aagtgctatc gctttcgggt gaaaaagaac catagacatc 3660gctacgagaa
ctgcaccacc acctggttca cagttgctga caacggtgct gaaagacaag
3720gacaagcaca aatactgatc acctttggat cgccaagtca aaggcaagac
tttctgaaac 3780atgtaccact acctcctgga atgaacattt ccggctttac
agccagcttg gacttctgat 3840cactgccatt gccttttctt catctgactg
gtgtactatg ccaaatctat ggtttctatt 3900gttcttggga ctagttgctg
caatgcaact gctgctatta ctgttcttac tcttgttttt 3960tcttgtatac
tgggatcatt ttgagtgctc ctgtacaggt ctgccctttt aatgccttta
4020catcactggc tattggctgt gtttttactg ttgtgtggat ttgatttgtt
ttatatactg 4080tatgaagttt tttcatttgt gcttgtattg ctgtttgtaa
gttttttact agagtttgta 4140ttccccctgc tcagatttta tatggtttaa
gctgcagcaa taaaaatgag tgcacgaaaa 4200agagtaaaac gtgccagtgc
ctatgacctg tacaggacat gcaagcaagc gggcacatgt 4260ccaccagatg
tgataccaaa ggtagaagga gatactatag cagataaaat tttgaaattt
4320gggggtcttg caatctactt aggagggcta ggaataggaa catggtctac
tggaagggtt 4380gctgcaggtg gatcaccaag gtacacacca ctccgaacag
cagggtccac atcatcgctt 4440gcatcaatag gatccagagc tgtaacagca
gggacccgcc ccagtatagg tgcgggcatt 4500cctttagaca cccttgaaac
tcttggggcc ttgcgtccag gggtgtatga ggacactgtg 4560ctaccagagg
cccctgcaat agtcactcct gatgctgttc ctgcagattc agggcttgat
4620gccctgtcca taggtacaga ctcgtccacg gagaccctca ttactctgct
agagcctgag 4680ggtcccgagg acatagcggt tcttgagctg caacccctgg
accgtccaac ttggcaagta 4740agcaatgctg ttcatcagtc ctctgcatac
cacgcccctc tgcagctgca atcgtccatt 4800gcagaaacat ctggtttaga
aaatattttt gtaggaggct cgggtttagg ggatacagga 4860ggagaaaaca
ttgaactgac atacttcggg tccccacgaa caagcacgcc ccgcagtatt
4920gcctctaaat cacgtggcat tttaaactgg ttcagtaaac ggtactacac
acaggtgccc
4980acggaagatc ctgaagtgtt ttcatcccaa acatttgcaa acccactgta
tgaagcagaa 5040ccagctgtgc ttaagggacc tagtggacgt gttggactca
gtcaggttta taaacctgat 5100acacttacaa cacgtagcgg gacagaggtg
ggaccacagc tacatgtcag gtactcattg 5160agtactatac atgaagatgt
agaagcaatc ccctacacag ttgatgaaaa tacacaggga 5220cttgcattcg
tacccttgca tgaagagcaa gcaggttttg aggagataga attagatgat
5280tttagtgaga cacatagact gctacctcag aacacctctt ctacacctgt
tggtagtggt 5340gtacgaagaa gcctcattcc aactcaggaa tttagtgcaa
cacggcctac aggtgttgta 5400acctatggct cacctgacac ttactctgct
agcccagtta ctgaccctga ttctacctct 5460cctagtctag ttatcgatga
cactactact acaccaatca ttataattga tgggcacaca 5520gttgatttgt
acagcagtaa ctacaccttg catccctcct tgttgaggaa acgaaaaaaa
5580cggaaacatg cctaattttt tttgcagatg gcgttgtggc aacaaggcca
gaagctgtat 5640ctccctccaa cccctgtaag caaggtgctt tgcagtgaaa
cctatgtgca aagaaaaagc 5700attttttatc atgcagaaac ggagcgcctg
ctaactatag gacatccata ttacccagtg 5760tctatcgggg ccaaaactgt
tcctaaggtc tctgcaaatc agtatagggt atttaaaata 5820caactacctg
atcccaatca atttgcacta cctgacagga ctgttcacaa cccaagtaaa
5880gagcggctgg tgtgggcagt cataggtgtg caggtgtcca gagggcagcc
tcttggaggt 5940actgtaactg ggcaccccac ttttaatgct ttgcttgatg
cagaaaatgt gaatagaaaa 6000gtcaccaccc aaacaacaga tgacaggaaa
caaacaggcc tagatgctaa gcaacaacag 6060attctgttgc taggctgtac
ccctgctgaa ggggaatatt ggacaacagc ccgtccatgt 6120gttactgatc
gtctagaaaa tggcgcctgc cctcctcttg aattaaaaaa caagcacata
6180gaagatgggg atatgatgga aattgggttt ggtgcagcca acttcaaaga
aattaatgca 6240agtaaatcag atctacctct tgacattcaa aatgagatct
gcttgtaccc agactacctc 6300aaaatggctg aggacgctgc tggtaatagc
atgttctttt ttgcaaggaa agaacaggtg 6360tatgttagac acatctggac
cagagggggc tcggagaaag aagcccctac cacagatttt 6420tatttaaaga
ataataaagg ggatgccacc cttaaaatac ccagtgtgca ttttggtagt
6480cccagtggct cactagtctc aactgataat caaattttta atcggcccta
ctggctattc 6540cgtgcccagg gcatgaacaa tggaattgca tggaataatt
tattgttttt aacagtgggg 6600gacaatacac gtggtactaa tcttaccata
agtgtagcct cagatggaac cccactaaca 6660gagtatgata gctcaaaatt
caatgtatac catagacata tggaagaata taagctagcc 6720tttatattag
agctatgctc tgtggaaatc acagctcaaa ctgtgtcaca tctgcaagga
6780cttatgccct ctgtgcttga aaattgggaa ataggtgtgc agcctcctac
ctcatcgata 6840ttagaggaca cctatcgcta tatagagtct cctgcaacta
aatgtgcaag caatgtaatt 6900cctgcaaaag aagaccctta tgcagggttt
aagttttgga acatagatct taaagaaaag 6960ctttctttgg acttagatca
atttcccttg ggaagaagat ttttagcaca gcaaggggca 7020ggatgttcaa
ctgtgagaaa acgaagaatt agccaaaaaa cttccagtaa gcctgcaaaa
7080aaaaaaaaaa aataaaagct aagtttctat aaatgttctg taaatgtaaa
acagaaggta 7140agtcaactgc acctaataaa aatcacttaa tagcaatgtg
ctgtgtcagt tgtttattgg 7200aaccacaccc ggtacacatc ctgtccagca
tttgcagtgc gtgcattgaa ttattgtgct 7260ggctagactt catggcgcct
ggcaccgaat cctgccttct cagcgaaaat gaataattgc 7320tttgttggca
agaaactaag catcaatggg acgcgtgcaa agcaccggcg gcggtagatg
7380cggggtaagt actgaatttt aattcgacct atcccggtaa agcgaaagcg
acacgctttt 7440ttttcacaca tagcgggacc gaacacgtta taagtatcga
ttaggtctat ttttgtctct 7500ctgtcggaac cagaactggt aaaagtttcc
attgcgtctg ggcttgtcta tcattgcgtc 7560tctatggttt ttggaggatt
agacggggcc accagtaatg gtgcatagcg gatgtctgta 7620ccgccatcgg
tgcaccgata taggtttggg gctccccaag ggactgctgg gatgacagct
7680tcatattata ttgaatgggc gcataatcag cttaattggt gaggacaagc
tacaagttgt 7740aacctgatct ccacaaagta cgttgccggt cggggtcaaa
ccgtcttcgg tgctcgaaac 7800cgccttaaac tacagacagg tcccagccaa
gtaggcggat caaaacctca aaaaggcggg 7860agccaatcaa aatgcagcat
tatattttaa gctcaccgaa accggtaagt aaagactatg 7920tattttttcc
cagtgaataa ttgtt 794550306PRTbovine papillomavirus type 1 50Met Glu
Thr Ala Cys Glu Arg Leu His Val Ala Gln Glu Thr Gln Met 1 5 10
15Gln Leu Ile Glu Lys Ser Ser Asp Lys Leu Gln Asp His Ile Leu Tyr
20 25 30Trp Thr Ala Val Arg Thr Glu Asn Thr Leu Leu Tyr Ala Ala Arg
Lys 35 40 45Lys Gly Val Thr Val Leu Gly His Cys Arg Val Pro His Ser
Val Val 50 55 60Cys Gln Glu Arg Ala Lys Gln Ala Ile Glu Met Gln Leu
Ser Leu Gln65 70 75 80Glu Leu Ser Lys Thr Glu Phe Gly Asp Glu Pro
Trp Ser Leu Leu Asp 85 90 95Thr Ser Trp Asp Arg Tyr Met Ser Glu Pro
Lys Arg Cys Phe Lys Lys 100 105 110Gly Ala Arg Val Val Glu Val Glu
Phe Asp Gly Asn Ala Ser Asn Thr 115 120 125Asn Trp Tyr Thr Val Tyr
Ser Asn Leu Tyr Met Arg Thr Glu Asp Gly 130 135 140Trp Gln Leu Ala
Lys Ala Gly Ala Asp Gly Thr Gly Leu Tyr Tyr Cys145 150 155 160Thr
Met Ala Gly Ala Gly Arg Ile Tyr Tyr Ser Arg Phe Gly Asp Glu 165 170
175Ala Ala Arg Phe Ser Thr Thr Gly His Tyr Ser Val Arg Asp Gln Asp
180 185 190Arg Val Tyr Ala Gly Val Ser Ser Thr Ser Ser Asp Phe Arg
Asp Arg 195 200 205Pro Asp Gly Val Trp Val Ala Ser Glu Gly Pro Glu
Gly Asp Pro Ala 210 215 220Gly Lys Glu Ala Glu Pro Ala Gln Pro Val
Ser Ser Leu Leu Gly Ser225 230 235 240Pro Ala Cys Gly Pro Ile Arg
Ala Gly Leu Gly Trp Val Arg Asp Gly 245 250 255Pro Arg Ser His Pro
Tyr Asn Phe Pro Ala Gly Ser Gly Gly Ser Ile 260 265 270Leu Arg Ser
Ser Ser Thr Pro Cys Arg Ala Arg Tyr Arg Trp Thr Trp 275 280 285His
Gln Gly Arg Lys Lys Arg Ser Ser Arg Pro Thr Pro Gln Arg Lys 290 295
300Asn Gln30551622DNAHuman herpesvirus 4 51gggtatcata tgctgactgt
atatgcatga ggatagcata tgctacccgg atacagatta 60ggatagcata tactacccag
atatagatta ggatagcata tgctacccag atatagatta 120ggatagccta
tgctacccag atataaatta ggatagcata tactacccag atatagatta
180ggatagcata tgctacccag atatagatta ggatagccta tgctacccag
atatagatta 240ggatagcata tgctacccag atatagatta ggatagcata
tgctatccag atatttgggt 300agtatatgct acccagatat aaattaggat
agcatatact accctaatct ctattaggat 360agcatatgct acccggatac
agattaggat agcatatact acccagatat agattaggat 420agcatatgct
acccagatat agattaggat agcctatgct acccagatat aaattaggat
480agcatatact acccagatat agattaggat agcatatgct acccagatat
agattaggat 540agcctatgct acccagatat agattaggat agcatatgct
atccagatat ttgggtagta 600tatgctaccc atggcaacat ta 62252641PRTHuman
herpesvirus 4 52Met Ser Asp Glu Gly Pro Gly Thr Gly Pro Gly Asn Gly
Leu Gly Glu 1 5 10 15Lys Gly Asp Thr Ser Gly Pro Glu Gly Ser Gly
Gly Ser Gly Pro Gln 20 25 30Arg Arg Gly Gly Asp Asn His Gly Arg Gly
Arg Gly Arg Gly Arg Gly 35 40 45Arg Gly Gly Gly Arg Pro Gly Ala Pro
Gly Gly Ser Gly Ser Gly Pro 50 55 60Arg His Arg Asp Gly Val Arg Arg
Pro Gln Lys Arg Pro Ser Cys Ile65 70 75 80Gly Cys Lys Gly Thr His
Gly Gly Thr Gly Ala Gly Ala Gly Ala Gly 85 90 95Gly Ala Gly Ala Gly
Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly 100 105 110Gly Gly Ala
Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly 115 120 125Gly
Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala 130 135
140Gly Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala
Gly145 150 155 160Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly
Gly Gly Ala Gly 165 170 175Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala
Gly Gly Gly Ala Gly Gly 180 185 190Ala Gly Gly Ala Gly Ala Gly Gly
Gly Ala Gly Ala Gly Gly Ala Gly 195 200 205Gly Ala Gly Gly Ala Gly
Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala 210 215 220Gly Gly Ala Gly
Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala225 230 235 240Gly
Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly 245 250
255Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly
260 265 270Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly
Ala Gly 275 280 285Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala
Gly Gly Ala Gly 290 295 300Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly
Gly Gly Ala Gly Ala Gly305 310 315 320Gly Ala Gly Ala Gly Gly Gly
Gly Arg Gly Arg Gly Gly Ser Gly Gly 325 330 335Arg Gly Arg Gly Gly
Ser Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly 340 345 350Arg Arg Gly
Arg Gly Arg Glu Arg Ala Arg Gly Gly Ser Arg Glu Arg 355 360 365Ala
Arg Gly Arg Gly Arg Gly Arg Gly Glu Lys Arg Pro Arg Ser Pro 370 375
380Ser Ser Gln Ser Ser Ser Ser Gly Ser Pro Pro Arg Arg Pro Pro
Pro385 390 395 400Gly Arg Arg Pro Phe Phe His Pro Val Gly Glu Ala
Asp Tyr Phe Glu 405 410 415Tyr His Gln Glu Gly Gly Pro Asp Gly Glu
Pro Asp Val Pro Pro Gly 420 425 430Ala Ile Glu Gln Gly Pro Ala Asp
Asp Pro Gly Glu Gly Pro Ser Thr 435 440 445Gly Pro Arg Gly Gln Gly
Asp Gly Gly Arg Arg Lys Lys Gly Gly Trp 450 455 460Phe Gly Lys His
Arg Gly Gln Gly Gly Ser Asn Pro Lys Phe Glu Asn465 470 475 480Ile
Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val Glu Arg 485 490
495Thr Thr Asp Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr Gly Gly
500 505 510Ser Lys Thr Ser Leu Tyr Asn Leu Arg Arg Gly Thr Ala Leu
Ala Ile 515 520 525Pro Gln Cys Arg Leu Thr Pro Leu Ser Arg Leu Pro
Phe Gly Met Ala 530 535 540Pro Gly Pro Gly Pro Gln Pro Gly Pro Leu
Arg Glu Ser Ile Val Cys545 550 555 560Tyr Phe Met Val Phe Leu Gln
Thr His Ile Phe Ala Glu Val Leu Lys 565 570 575Asp Ala Ile Lys Asp
Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn 580 585 590Ile Arg Val
Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu Pro Pro 595 600 605Trp
Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp Asp Gly 610 615
620Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly
Gln625 630 635 640Glu
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