Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
VACCINES COMPRISING SYNTHETIC GENES
BACKGROI~NO OF THE INVENTION
5 1. HIV Infection:
~ Iuman Imrnunodeficiency Virus-1 (HIV-1) is the
etiological agent of acquired human immune deficiency syndrome
(AIDS) and related disorders. HIV-1 is an RNA virus of the
Retroviridae family and exhibits the 5'LTR-gag-pol-env-LTR3'
10 org~ni7~ion of all retroviruses. In addition, H~V-1 comprises a handful
of genes with regulatory or unknown functions, including the tat and
rev genes. The env gene encodes the viral envelope glycoprotein that is
translated as a 160-kilodalton (kDa) precursor (gpl60) and then cleaved
by a cellular protease to yield the external 120-kDa envelope
15 glycoprotein (gpl20) and the transmembrane 41-kDa envelope
glycoprotein (gp41). Gpl20 and gp41 remain associated and are
displayed on the viral particles and the surface of HIV-infected cells.
Gpl20 binds to the CD4 receptor present on the surface of helper T-
lymphocytes, macrophages and other target cells. After gpl20 binds to
20 CD4, gp4 l mediates the fusion event responsible for virus entry.
Infection begins when gp 120 on the viral particle binds to
the CD4 receptor on the surface of T4 Iymphocytes or other target cells.
The bound virus merges with the target cell and reverse transcribes its
RNA genome into the double-stranded DNA of the cell. The viral DNA
25 is incorporated into the genetic material in the cell's nucleus, where the
viral DNA directs the production of new viral RNA, viral proteins, and
new virus particles. The new particles bud from the target cell
membrane and infect other cells.
Destruction of T4 Iymphocytes, which are critical to
30 immllne defense, is a major cause of the progressive immune
dysfunction that is the h~llm~rk of HIV infection. The loss of target
cells seriously impairs the body's ability to fight most invaders, but it
has a particularly severe impact on the defenses against viruses, fungi,
parasites and certain bacteria, including mycobacteria.
.. ....
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HIV-1 kills the cells it infects by replicating, budding from
them and ~l~m~ging the cell membrane. HIV-1 may kill target cells
indirectly by means of the viral gpl20 that is displayed on an infected
cell's surface. Since the CD4 receptor on T cells has a strong affinity for
5 gpl20, healthy cells expressing CD4 receptor can bind to gpl20 and
fuse with infected cells to form a syncytium. A syncytium cannot
survlve.
HIV-1 can also elicit normal cellular immune defenses
against infected cells. With or without the help of antibodies, cytotoxic
10 defensive cells can destroy an infected cell that displays viral proteins on
its surface. Finally, free gpl20 may circulate in the blood of individuals
infected with HIV-1. The free protein may bind to the CD4 receptor of
uninfected cells, making them appear to be infected and evoking an
immune response.
Infection with HIV-1 is almost always fatal, and at present
there are no cures for HIV-1 infection. Effective vaccines for
prevention of HIV- 1 infection are not yet available. Because of the
danger of reversion or infection, live attenuated virus probably cannot
be used as a vaccine. Most subunit vaccine approaches have not been
20 successful at preventing HIV infection. Treatments for HIV- 1 infection,
while prolonging the lives of some infected persons, have serious side
effects. There is thus a great need for effective treatments and vaccines
to combat this lethal infection.
25 2. Vaccines
Vaccination is an effective form of disease prevention and
has proven successful against several types of viral infection.
Determining ways to present HIV-1 antigens to the human immune
system in order to evoke protective humoral and cellular immunity, is a
30 difficult task. To date, attempts to generate an effective HIV vaccine
have not been successful. In AIDS patients, free virus is present in low
levels only. Tr~n~mi.~sion of HIV-l is enhanced by cell-to-cell
interaction via fusion and syncytia formation. Hence, antibodies
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generated against free virus or viral subunits are generally ineffective in
elimin~ting virus-infected cells.
Vaccines exploit the body's ability to "remember" an
antigen. After first encounters with a given antigen the immlme system
S generates cells that retain an immunological memory of the antigen for
an individual's lifetime. Subsequent exposure to the antigen stim~ tes
the immune response and results in elimin~tion or inactivation of the
pathogen.
The immune system deals with pathogens in two ways: by
10 humoral and by cell-mediated responses. In the humoral response
Iymphocytes generate specific antibodies that bind to the antigen thus
inactivating the pathogen. The cell-mediated response involves
cytotoxic and helper T Iymphocytes that specifically attack and destroy
infected cells.
Vaccine development with HIV-1 virus presents problems
because HIV-1 infects some of the same cells the vaccine needs to
activate in the immune system (i.e., T4 lymphocytes). It would be
advantageous to develop a vaccine which inactivates HIV before
impairment of the immune system occurs. A particularly suitable type
20 of HIV vaccine would generate an anti-H~V immune response which
recognizes HIV variants and which works in HIV-positive individuals
who are at the beginning of their infection.
A major challenge to the development of vaccines against
viruses, particularly those with a high rate of mutation such as the
25 human immunodeficiency virus, against which elicitation of neutralizing
and protective immllne responses is desirable, is the diversity of the
viral envelope proteins among different viral isolates or strains.
Because cytotoxic T-lymphocytes (CTLs) in both mice and humans are
capable of recognizing epitopes derived from conserved internal viral
30 proteins, and are thought to be important in the immune response
against viruses, efforts have been directed towards the development of
CTL vaccines capable of providing heterologous protection against
different viral strains.
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It is known that CD8+ CTLs kill virally-infected cells when
their T cell receptors recognize viral peptides associated with MHC class
I molecules. The viral peptides are derived from endogenously
synthesized viral proteins, regardless of the protein's location or
function within the virus. Thus, by recognition of epitopes from
conserved viral proteins, CTLs may provide cross-strain protection.
Peptides capable of associating with MHC class I for CTL recognition
originate from proteins that are present in or pass through the
cytoplasm or endoplasmic reticulum. In general, exogenous proteins,
which enter the endosomal processing pathway (as in the case of
antigens presented by MHC class Il molecules), are not effective at
generating CD8+ CTL responses.
Most efforts to generate CTL responses have used
replicating vectors to produce the protein antigen within the cell or they
have focused upon the introduction of peptides into the cytosol. These
approaches have limi~tions that may reduce their utility as vaccines.
Retroviral vectors have restrictions on the size and structure of
polypeptides that can be expressed as fusion proteins while m~int~ininp;
the ability of the recombinant virus to replicate, and the effectiveness of
vectors such as vaccinia for subsequent immlmi7~tions may be
compromised by immune responses against the vectors themselves.
Also, viral vectors and modified pathogens have inherent risks that may
hinder their use in hllm~n~. Furthermore, the selection of peptide
epitopes to be presented is dependent upon the structure of an
individual's MHC antigens and, therefore, peptide vaccines may have
limited effectiveness due to the diversity of MHC haplotypes in outbred
populations.
3. DNA Vaccines
Benvenisty, N., and Reshef, L. [PNAS 83, 9551-9555,
(1986)] showed that CaPO4 precipitated DNA introduced into mice
intraperitoneally (i.p.), intravenously (i.v.) or intramuscularly (i.m.)
could be expressed. The i.m. injection of DNA expression vectors
without CaCl2 treatment in mice resulted in the uptake of DNA by the
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muscle cells and expression of the protein encoded by the DNA . The
plasmids were m:lint~ined episomally and did not replicate.
Subsequently, persistent expression has been observed after i.m.
injection in skeletal muscle of rats, fish and primates, and cardiac
muscle of rats. The technique of using nucleic acids as therapeutic
agents was reported in WO90/11092 (4 October 1990), in which naked
polynucleotides were used to vaccinate vertebrates.
It is not necessary for the success of the method that
immunization be intramuscular. The introduction of gold
microprojectiles coated with DNA encoding bovine growth hormone
(BGH) into the skin of mice resulted in production of anti-BGH
antibodies in the mice. A jet injector has been used to transfect skin,
muscle, fat, and m~mm~ry tissues of living ~nim~l~. Various methods
for introducing nucleic have been reviewed. Intravenous injection of a
DNA:cationic liposome complex in mice was shown by Zhu et al.,
[Science 261:209-211 (9 July 1993) to result in systemic expression of a
cloned transgene. ULrner et al., [Science 259:1745-1749, (1993)]
reported on the heterologous protection against influenza virus infection
by intramuscular injection of DNA encoding influenza virus proteins.
The need for specific therapeutic and prophylactic agents
capable of eliciting desired immune responses against pathogens and
tumor antigens is met by the instant invention. Of particular importance
in this therapeutic approach is the ability to induce T-cell immune
responses which can prevent infections or disease caused even by virus
strains which are heterologous to the strain from which the antigen gene
was obtained. This is of particular concern when dealing with HIV as
this virus has been recognized to mutate rapidly and many virulent
isolates have been identified [see, for example, LaRosa et al., Science
249:932-935 (1990), identifying 245 separate HIV isolates]. In
response to this recognized diversity, researchers have attempted to
generate CTLs based on peptide immllni7~tion. Thus, T~k~h~hi et al.,
[Science 255:333-336 (1992)] reported on the induction of broadly
cross-reactive cytotoxic T cells recogni7ing an HIV envelope (gpl60)
determinant. However, those workers recognized the difficulty in
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achieving a truly cross-reactive CTL response and suggested that there
is a dichotomy between the priming or restimulation of T cells, which is
very stringent, and the elicitation of effector function, including
cytotoxicity, from already stimulated CTLs.
S Wang et al. reported on elicitation of immllne responses in
mice against HIV by intramuscular inoculation with a cloned, genomic
(unspliced) HIV gene. However, the level of immune responses
achieved in these studies was very low. In addition, the Wang et al.,
DNA construct utilized an essentially genomic piece of HIV encoding
contiguous Tat/rev-gpl60-Tat/rev coding sequences. As is described in
detail below, this is a suboptimal system for obtaining high-level
expression of the gpl60. It also is potentially dangerous because
expression of Tat contributes to the progression of Kaposi's Sarcoma.
WO 93/17706 describes a method for vaccinating an ~nim~l
against a virus, wherein carrier particles were coated with a gene
construct and the coated particles are accelerated into cells of an ~nim~l.
ln regard to HIV, essentially the entire genome, minus the long terminal
repeats, was proposed to be used. That method represents substantial
risks for recipients. It is generally believed that constructs of HIV
should contain less than about 50% of the HIV genome to ensure safety
of the vaccine; thi.s ensures that enzymatic moieties and viral regulatory
proteins, many of which have unknown or poorly understood functions
have been elimin~ted. Thus, a number of problems remain if a useful
human HIV vaccine is to emerge from the gene-delivery technology.
The instant invention contemplates any of the known
methods for introducing polynucleotides into living tissue to induce
expression of proteins. However, this invention provides a novel
immunogen for introducing HIV and other proteins into the antigen
processing pathway to efficiently generate HIV-specific CTLs and
antibodies. The ph~rm~ceutical is effective as a vaccine to induce both
cellular and humoral anti-HIV and HIV neutralizing immune responses.
In the instant invention, the problems noted above are addressed and
solved by the provision of polynucleotide immlmogens which, when
introduced into an ~nim~l, direct the efficient expression of HIV
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proteins and epitopes without the attendant risks associated with those
methods. The immune responses thus generated are effective at
recogni7ing HIV, at inhibiting replication of HIV, at identifying and
killing cells infected with HIV, and are cross-reactive against many HIV
5 strains.
4. Codon Usage and Codon Context
The codon pairings of org~ni~m~ are highly nonrandom,
and differ from organism to organism. This information is used to
10 construct and express altered or synthetic genes having desired levels of
translational efficiency, to determine which regions in a genome are
protein coding regions, to introduce translational pause sites into
heterologous genes, and to ascertain relationship or ancestral origin of
nucleotide sequences.
The expression of foreign heterologous genes in
transformed org~ni~ms is now commonplace. A large number of
m~mm~ n genes, including, for example, murine and human genes,
have been successfully inserted into single celled org~ni~m~. Standard
techniques in this regard include introduction of the foreign gene to be
expressed into a vector such as a plasmid or a phage and l-tili7ing that
vector to insert the gene into an org~ni~m. The native promoters for
such genes are commonly replaced with strong promoters compatible
with the host into which the gene is inserted. Protein sequencing
machinery permits elucidation of the amino acid sequences of even
minute quantities of native protein. From these amino acid sequences,
DNA sequences coding for those proteins can be inferred. DNA
synthesis is also a rapidly developing art, and synthetic genes
corresponding to those inferred DNA sequences can be readily
constructed.
Despite the burgeoning knowledge of expression systems
and recombinant DNA, significant obstacles remain when one attempts
to express a foreign or synthetic gene in an organism. Many native,
active proteins, for example, are glycosylated in a manner different
from that which occurs when they are expressed in a foreign host. For
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this reason, eukaryotic hosts such as yeast may be preferred to bacterial
hosts for expressing many m~mm~ n genes. The glycosylation
problem is the subject of continlling research.
Another problem is more poorIy understood. Often
translation of a synthetic gene, even when coupled with a strong
promoter, proceeds much less efficiently than would be expected. The
same is frequently true of exogenous genes foreign to the expression
organism. Even when the gene is transcribed in a sufficiently efficient
manner that recoverable quantities of the translation product are
produced, the protein is often inactive or otherwise different in
properties from the native protein.
It is recognized that the latter problem is commonly due to
differences in protein folding in various org~ni.cm~. The solution to this
problem has been elusive, and the mech~nisms controlling protein
folding are poorly understood.
- The problems related to translational efficiency are
believed to be related to codon context effects. The protein coding
regions of genes in all organisms are subject to a wide variety of
functional constraints, some of which depend on the requirement for
encoding a properly functioning protein, as well as a~ro~liate
translational start and stop signals. However, several features of protein
coding regions have been discerned which are not readily understood in
terms of these constraints. Two important classes of such features are
those involving codon usage and codon context.
It is known that codon utilization is highly biased and varies
considerably between different org~ni~ms. Codon usage patterns have
been shown to be related to the relative abundance of tRNA
isoacceptors. Genes encoding proteins of high versus low abundance
show differences in their codon preferences. The possibility that biases
30 in codon usage alter peptide elongation rates has been widely discussed.
While differences in codon use are associated with differences in
translation rates, direct effects of codon choice on translation have been
difficult to demonstrate. Other proposed constraints on codon usage
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patterns include ma~imi7.in~ the fidelity of translation and optimi7.ing
the kinetic efficiency of protein synthesis.
Apart from the non-random use of codons, considerable
evidence has accumulated that codon/anticodon recognition is influenced
5 by sequences outside the codon itself, a phenomenon termed "codon
context." There exists a strong influence of nearby nucleotides on the
efficiency of suppression of nonsense codons as well as missense codons.
Clearly, the abundance of suppressor activity in natural bacterial
populations, as well as the use of "termination" codons to encode
10 selenocysteine and phosphoserine require that termination be context-
dependent. Similar context effects have been shown to influence the
fidelity of translation, as well as the efficiency of translation initiation.
Statistical analyses of protein coding regions of E. coli have
demonstrate another manifestation of "codon context." The presence of
15 a particular codon at one position strongly influences the frequency of
occurrence of certain nucleotides in neighboring codons, and these
context constraints differ markedly for genes expressed at high versus
low levels. Although the context effect has been recognized, the
predictive value of the statistical rules relating to preferred nucleotides
20 adjacent to codons is relatively low. This has limited the utility of such
nucleotide preference data for selecting codons to effect desired levels
of translational efficiency.
The advent of automated nucleotide sequencing equipment
has made available large quantities of sequence data for a wide variety
25 of or~ni~m~. Understanding those data presents substantial difficulties.
For example, it is important to identify the coding regions of the
genome in order to relate the genetic sequence data to protein sequences.
In addition~ the ancestry of the genome of certain org~ni~m~ is of
substantial interest. It is known that genomes of some org~ni~m~ are of
30 mixed ancestry. Some sequences that are viral in origin are now stably
incorporated into the genome of eukaryotic organisms. The viral
sequences themselves may have originated in another substantially
unrelated species. An understanding of the ancestry of a gene can be
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important in drawing proper analogies between related genes and their
translation products in other org~nism~.
There is a need for a better underst~nclin~; of codon context
effects on translation, and for a method for determining the appropriate
codons for any desired translational effect. There is also a need for a
method for identifying coding regions of the genome from nucleotide
sequence data. There is also a need for a method for controlling protein
folding and for insuring that a foreign gene will fold appropriately
when expressed in a host. Genes altered or constructed in accordance
with desired translational efficiencies would be of significant worth.
Another aspect of the practice of recombinant DNA
techniques for the expression by microorg~ni~m~ of proteins of
industrial and pharmaceutical interest is the phenomenon of "codon
preference". While it was earlier noted that the existing machinery for
gene expression is genetically transformed host cel}s will "operate" to
construct a given desired product, levels of expression attained in a
microorganism can be subject to wide variation, depending in part on
specific alternative forms of the amino acid-specifying genetic code
present in an inserted exogenous gene. A "triplet" codon of four
possible nucleotide bases can exist in 64 variant forms. That these
forms provide the message for only 20 different amino acids (as well as
transcription initiation and termination) means that some amino acids
can be coded for by more than one codon. Indeed, some amino acids
have as many as six "redundant", alternative codons while some others
have a single, required codon. For reasons not completely understood,
alternative codons are not at all uniformly present in the endogenous
DNA of differing types of cells and there appears to exist a variable
natural hierarchy or "preference" for certain codons in certain types of
cells.
As one example, the amino acid leucine is specified by any
of six DNA codons including CTA, CTC, CTG, CTT, TTA, and TTG
(which correspond, respectively, to the mRNA codons, CUA, CUC,
CUG, CUU, UUA and UUG). Exhaustive analysis of genome codon
frequencies for microorg~nism~ has revealed endogenous DNA of E.
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coli most commonly contains the CTG leucine-specifying codon, while
the DNA of yeasts and slime molds most commonly includes a TTA
leucine-specifying codon. In view of this hierarchy, it is generally held
that the likelihood of obt~ining high levels of expression of a leucine-
5 rich polypeptide by an E. coli host will depend to some extent on thei~requency of codon use. For example, a gene rich in TTA codons will
in all probability be poorly expressed in E. coli. whereas a CTG rich
gene will probably highly e~press the polypeptide. Similarly, when yeast
cells are the projected transformation host cells for expression of a
10 leucine-rich polypeptide, a preferred codon for use in an inserted DNA
would be TTA.
The implications of codon preference phenomena on
recombinant DNA techniques are manifest, and the phenomenon may
serve to explain many prior failures to achieve high expression levels of
15 exogenous genes in successfully transformed host org~nism~-a less
"preferred" codon may be repeatedly present in the inserted gene and
the host cell machinery for expression may not operate as efficiently.
This phenomenon suggests that synthetic genes which have been
designed to include a projected host cell's preferred codons provide a
20 preferred form of foreign genetic material for practice of recombinant
DNA techniques.
5. Protein Trafficking
The diversity of function that typifies eukaryote cells
25 depends upon the structural differentiation of their membrane
boundaries. To generate and m~int~in these structures, proteins must be
transported from their site of synthesis in the endoplasmic reticulum to
predetermined destinations throughout the cell. This requires that the
trafficking proteins display sorting signals that are recognized by the
30 molecular machinery responsible for route selection located at the access
points to the main trafficking pathways. Sorting decisions for most
proteins need to be made only once as they traverse their biosynthetic
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pathways since their final destination, the cellular location at which they
perform their function, becomes their permanent residence.
Maintenance of intracellular integrity depends in part on
the selective sorting and accurate transport of proteins to their correct
S destinations. Over the past few years the dissection of the molecular
machinery for targeting and loc~li7~tion of proteins has been studied
vigorously. Defined sequence motifs have been identified on proteins
which can act as 'address labels'. A number of sorting signals have been
found associated with the cytoplasmic domains of membrane proteins.
SUMMARY OF THE INVENTION
Synthetic polynucleotides comprising a DNA sequence
encoding a peptide or protein are provided. The DNA sequence of the
synthetic polynucleotides comprise codons optimized for expression in a
nonhomologous host. The invention is exemplified by synthetic DNA
molecules encoding HIV env as well as modifications of HIV env. The
codons of the synthetic molecules include the projected host cell's
preferred codons. The synthetic molecules provide preferred forms of
foreign genetic material. The synthetic molecules may be used as a
polynucleotide vaccine which provides effective immunoprophylaxis
against HIV infection through neutralizing antibody and cell-mediated
immunity. This invention provides polynucleotides which, when directly
introduced into a vertebrate in vivo, including m~mm~ls such as
primates and hllm~ns, induces the expression of encoded proteins within
the ~nim~l.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows HIV env cassette-based expression
strategies.
Figure 2 shows DNA vaccine mediated anti-gpl20
responses.
Figure 3 shows anti-gpl20 ELISA titers of murine DNA
vaccinee sera.
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Figure 4 shows the relative expression of gpl20 after HIV
env PNV cell culture transfection.
Figure 5 shows the mean anti-gpl20 ELISA responses
following tPA-gpl43/optA vs. opt~ DNA vaccination.
5Figure 6 shows the neutralization of HIV by murine DNA
vaccinee sera.
Figure 7 shows HIV neutralization by sera from murine
HIV env DNA vaccinees.
Figure 8 is an immunoblot analysis of optimized HIV env
DNA constructs.
Figure 9 shows anti-gpl20 ELISA responses in rhesus
monkeys following final vaccination with gpl40 DNA and o-gpl60
protein.
Figure 10 shows SHIV neutralizing antibody responses of
rhesus monkeys following final vaccination.
DETAILED DESCRIPTION OF THE INVENTION
Synthetic polynucleotides comprising a DNA sequence
encoding a peptide or protein are provided. The DNA se~uence of the
synthetic polynucleotides comprise codons optimized for expression in a
nonhomologous host. The invention is exemplified by synthetic DNA
molecules encoding HIV env as well as modifications of HIV env are
provided. The codons of the synthetic molecules include the projected
host cell's preferred codons. The synthetic molecules provide preferred
forms of foreign genetic material. The synthetic molecules may be used
as a polynucleotide vaccine which provides immunoprophylaxis against
HIV infection through neutralizing antibody and cell-mediated
immunity. This invention provides polynucleotides which, when directly
introduced into a vertebrate in vivo, including m~mm~l~ such as
- 30 primates and h~lm~ns, induces the expression of encoded proteins within
the ~nim~l.
Therefore, synthetic DNA molecules encoding HIV env and
synthetic DNA molecules encoding modified forms of HIV env are
provided. The codons of the synthetic molecules are designed so as to
use the codons preferred by the projected host cell. As noted above, the
~ . . .
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synthetic molecules of this portion of the invention may be used as a
polynucleotide vaccine which provides effective immunoprophylaxis
against HIV infection through neutr~li7.in~ antibody and cell-mediated
immlmity. The synthetic molecules may be used as an immunogenic
5 composition. This portion of the invention also provides
polynucleotides which, when directly introduced into a vertebrate in
vivo, including m~mm~ such as primates and humans, induces the
expression of encoded proteins within the ~nim~l.
As used herein, a polynucleotide is a nucleic acid which
10 contains essential regulatory elements such that upon introduction into a
living, vertebrate cell, it is able to direct the cellular machinery to
produce translation products encoded by the genes comprising the
polynucleotide. In one embodiment of the invention, the polynucleotide
is a polydeoxyribonucleic acid comprising at least one HIV gene
15 operatively linked to a transcriptional promoter. In another
embodiment of the invention, the polynucleotide vaccine (PNV)
comprises polyribonucleic acid encoding at least one HIV gene which is
amenable to translation by the eukaryotic cellular machinery
(ribosomes, tRNAs, and other translation factors). Where the protein
20 encoded by the polynucleotide is one which does not normally occur in
that ~nim~l except in pathological conditions, (i.e., a heterologous
protein) such as proteins associated with hllm~n immunodeficiency
virus, (HIV), the etiologic agent of acquired immune deficiency
syndrome, (AIDS), the ~nim~l~' immune system is activated to launch a
25 protective immune response. Because these exogenous proteins are
produced by the ~nim~l~' tissues, the expressed proteins are processed
by the major histocompatibility system, M HC, in a fashion analogous to
when an actual infection with the related org~ni~m (HIV) occurs. The
result, as shown in this disclosure, is induction of immune responses
30 against the cognate pathogen.
Accordingly, the instant inventors have prepared nucleic
acids which, when introduced into the biological system induce the
expression of HIV proteins and epitopes. The induced antibody
response is both specific for the expressed HIV protein, and neutralizes
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HIV. In addition, cytotoxic T-lymphocytes which specifically recognize
and destroy HIV infected cells are induced.
The instant invention provides a method for using a
polynucleotide which, upon introduction into m~mm~ n tissue, induces
5 the expression in a single cell, in vivo, of discrete gene products. The
instant invention provides a different solution which does not require
multiple manipulations of rev dependent HIV genes to obtain rev-
independent genes. The rev-independent expression system described
herein is useful in its own right and is a system for demonstrating the
10 expression in a single cell in vivo of a single desired gene-product.
Because many of the applications of the instant invention
apply to anti-viral vaccination, the polynucleotides are frequently
referred to as a polynucleotide vaccine, or PNV. This is not to say that
additional utilities of these polynucleotides, in immune stimulation and
15 in anti-tumor therapeutics, are considered to be outside the scope of the
invention.
In one embodiment of this invention, a gene encoding an
HIV gene product is incorporated in an expression vector. The vector
contains a transcriptional promoter recognized by an eukaryotic RNA
20 polymerase, and a transcriptional termin~or at the end of the HIV gene
coding sequence. In a preferred embodiment, the promoter is the
cytomegalovirus promoter with the intron A sequence (CMV-intA),
although those skilled in the art will recognize that any of a number of
other known promoters such as the strong immunoglobulin, or other
25 eukaryotic gene promoters may be used. A preferred transcriptional
terminator is the bovine growth hormone terminator. The combination
of CMVintA-BGH terminator is particularly preferred.
To assist in preparation of the polynuc}eotides in
prokaryotic cells, an antibiotic resistance marker is also preferably
30 included in the expression vector under transcriptional control of a
prokaryotic promoter so that expression of the antibiotic does not occur
in eukaryotic cells. Ampicillin resistance genes, neomycin resistance
genes and other pharmaceutically acceptable antibiotic resistance
markers may be used. To aid in the high level production of the
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WO 97148370 PCT/US97/10517
polynucleotide by fermentation in prokaryotic or~ni~ms, it is
advantageous for the vector to contain a prokaryotic origin of
replication and be of high copy number. A number of commercially
available prokaryotic cloning vectors provide these benefits. It is
5 desirable to remove non-essential DNA sequences. It is also desirable
that the vectors not be able to replicate in eukaryotic cells. This
minimi7.es the risk of integration of polynucleotide vaccine sequences
into the recipients' genome. Tissue-specific promoters or enhancers
may be used whenever it is desirable to limit expression of the
10 polynucleotide to a particular tissue type.
In one embodiment, the expression vector pnRSV is used,
wherein the Rous Sarcoma Virus (RSV) long terminal repeat (LTR) is
used as the promoter. In another embodiment, V1, a mutated pBR322
vector into which the CMV promoter and the BGH transcriptional
15 terminator were cloned is used. In another embodiment, the elements of
V1 and pUC19 have been combined to produce an expression vector
named VlJ. Into VlJ or another desirable expression vector is cloned
an HIV gene, such as gpl20, gp41, gpl60, gag, pol, env, or any other
HIV gene which can induce anti-HlV immllne responses. In another
20 embodiment, the ampicillin resistance gene is removed from VlJ and
replaced with a neomycin resistance gene, to generate VlJ-neo into
different HIV genes have been cloned for use according to this
invention. In another embodiment, the vector is VlJns, which is the
same as VlJneo except that a unique Sfil restriction site has been
25 engineered into the single Kpnl site at position 2114 of VlJ-neo. The
incidence of Sfil sites in hllm~n genomic DNA is very low
(approximately 1 site per 100,000 bases). Thus, this vector allows
careful monitoring for expression vector integration into host DNA,
simply by Sfil digestion of extracted genomic DNA. In a further
30 refinement, the vector is VlR. In this vector, as much non-essential
DNA as possible was "trimmed" from the vector to produce a highly
compact vector. This vector is a derivative of VlJns. This vector
allows larger inserts to be used, with less concern that undesirable
sequences are encoded and optimizes uptake by cells.
- 16 -
..
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One embodiment of this invention incorporates genes
encoding HIV gpl60, gpl20, gag and other gene products from
~ Iaboratory adapted strains of HIV such as SF2, IIIB or MN. Those
skilled in the art will recognize that the use of genes from HIV-2 strains
5 having analogous function to the genes from HIV-l would be expected
to generate immllne responses analogous to those described herein for
HIV- 1 constructs. The cloning and manipulation methods for obtaining
these genes are known to those skilled in the art.
It is recognized that elicitation of immune responses against
10 laboratory adapted strains of HIV may not be adequate to provide
neutralization of primary field isolates of HIV. Thus, in another
embodiment of this invention, genes from virulent, primary field
isolates of HIV are incorporated in the polynucleotide immunogen. This
is accomplished by preparing cDNA copies of the viral genes and then
15 subcloning the individual genes into the polynucleotide immunogen.
Sequences for many genes of many HIV strains are now publicly
available on GENBANK and such primary, field isolates of HIV are
available from the National Institute of Allergy and Infectious Diseases
(NIAID) which has contracted with Quality Biological, lnc., [7581
20 Lindbergh Drive, Gaithersburg, Maryland 20879] to make these strains
available. Such strains are also available from the World Health
Organization (W HO) [Network for HIV Isolation and Characterization,
Vaccine Development Unit, Office of Research, Global Programme on
AIDS, CH-1211 Geneva 27, Switzerland]. From this work those skilled
25 in the art will recognize that one of the utilities of the instant invention
is to provide a system for in vivo as well as in vitro testing and analysis
so that a correlation of HIV sequence diversity with serology of HIV
neutralization, as well as other parameters can be made. Incorporation
of genes from primary isolates of HIV strains provides an immunogen
30 which induces immune responses against clinical isolates of the virus and
thus meets a need as yet unmet in the field. Furthermore, as the virulent
isolates change, the immunogen may be modified to reflect new
sequences as necessary.
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To keep the terminology consistent, the following
convention is followed herein for describing polynucleotide immllnogen
constructs: "Vector name-HIV strain-gene-additional elements". Thus,
a construct wherein the gp~60 gene of the MN strain is cloned into the
S expression vector VlJneo, the name it is given herein is: "VlJneo-MN-
gpl60". The additional elements that are added to the construct are
described in further detail below. As the etiologic strain of the virus
changes, the precise gene which is optimal for incorporation in the
ph~ ceutical may be changed. However, as is demonstrated below,
10 because CTL responses are induced which are capable of protecting
against heterologous strains, the strain variability is less critical in the
immunogen and vaccines of this invention, as compared with the whole
virus or subunit polypeptide based vaccines. In addition, because the
ph~ ceutical is easily manipulated to insert a new gene, this is an
15 adjustment which is easily made by the standard techniques of molecular
biology.
The term "promoter" as used herein refers to a recognition
site on a DNA strand to which the RNA polymerase binds. The
promoter forms an initiation complex with RNA polymerase to initiate
20 and drive transcriptional activity. The complex can be modified by
activating sequences termed "enhancers" or inhibiting sequences termed
"silencers."
The term "leader" as used herein refers to a DNA sequence
at the 5' end of a structural gene which is transcribed along with the
25 gene. The leader usually results in the protein having an N-terminal
peptide extension sometimes called a pro-sequence. For proteins
destined for either secretion to the extracellular medium or a
membrane, this signal sequence, which is generally hydrophobic, directs
the protein into endoplasmic reticulum from which it is discharged to
30 the ~yprol~riate destination.
The term "intron" as used herein refers to a section of
DNA occurring in the middle of a gene which does not code for an
amino acid in the gene product. The precursor RNA of the intron is
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- excised and is therefore not transcribed into mRNA nor translated into
protein.
The term "cassette" refers to the sequence of the present
invention which contains the nucleic acid sequence which is to be
expressed. The cassette is similar in concept to a cassette tape. Each
cassette will have its o~,vn sequence. Thus by interch~nging the cassette
the vector will express a different sequence. Because of the restrictions
sites at the 5' and 3' ends, the cassette can be easily inserted, removed or
replaced with another cassette.
The term "3' untranslated region" or "3' UTR" refers to
the sequence at the 3' end of a structural gene which is usually
transcribed with the gene. This 3' UTR region usually contains the poly
A sequence. Although the 3' UTR is transcribed from the DNA it is
excised before translation into the protein.
The term "Non-Coding Region" or "NCR" refers to the
region which is contiguous to the 3' UTR region of the structural gene.
The NCR region contains a transcriptional termination signal.
The term "restriction site" refers to a sequence specific
cleavage site of restriction endonucleases.
The term "vector" refers to some means by which DNA
fragments can be introduced into a host organism or host tissue. There
are various types of vectors including plasmid, bacteriophages and
cosmids.
The term "effective amount" means sufficient PNV is
injected to produce the adequate levels of the polypeptide. One skilled in
the art recognizes that this level may vary.
To provide a description of the instant invention, the
following background on HIV is provided. The human
immunodeficiency virus has a ribonucleic acid (RNA) genome. This
RNA genome must be reverse transcribed according to methods known
in the art in order to produce a cDNA copy for cloning and
manipulation according to the methods taught herein. At each end of
the genome is a long terminal repeat which acts as a promoter. Between
these termini, the genome encodes, in various reading frames, gag-pol-
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env as the major gene products: gag is the group specific antigen; pol is
the reverse transcriptase, or polymerase; also encoded by this region, in
an alternate reading frame, is the viral protease which is responsible for
post-translational processing, for example, of gpl60 into gpl20 and
5 gp41; env is the envelope protein; vif is the virion infectivity factor; rev
is the regulator of virion protein expression; neg is the negative
regulatory factor; vpu is the virion productivity factor "u"; tat is the
trans-activator of transcription; vpr is the viral protein r. The function
of each of these elements has been described.
In one embodiment of this invention, a gene encoding an
HIV or SIV protein is directly linked to a transcriptional promoter.
The env gene encodes a large, membrane bound protein, gpl60, which
is post-translationally modified to gp41 and gpl20. The gpl20 gene
may be placed under the control of the cytomegalovirus promoter for
15 expression. However, gpl20 is not membrane bound and therefore,
upon expression, it may be secreted from the cell. As HIV tends to
remain dormant in infected cells, it is desirable that immune responses
directed at cell-bound HIV epitopes also be generated. Additionally, it
is desirable that a vaccine produce membrane bound, oligomeric ENV
20 antigen similar in structure to that produced by viral infection in order
to generate the most efficacious antibody responses for viral
neutralization. This goal is accomplished herein by expression in vivo
of a secreted gpl40 epitope (gpl40 > gpl20 + ectodomain of gp41) or
the cell-membrane associated epitope, gpl60, to prime the immllne
25 system. However, expression of gpl60 is repressed in the absence of
rev due to non-export from the nucleus of non-spliced genes. For an
understanding of this system, the life cycle of HIV must be described in
further detail.
In the life cycle of HIV, upon infection of a host cell, HIV
30 RNA genome is reverse-transcribed into a proviral DNA which
integrates into host genomic DNA as a single transcriptional unit. The
LTE~ provides the promoter which transcribes HIV genes from the 5' to
3' direction (gag, pol, env), to form an unspliced transcript of the entire
genome. The unspliced transcript functions as the mRNA from which
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WO 97/48370 PCTIUS97/10517
gag and pol are tr~n~l~ted, while limited splicing must occur for
translation of env encoded genes. For the regulatory gene product rev
to be expressed, more than one splicing event must occur because in the
genomic setting, rev and env overlap. In order for transcription of env
5 to occur, rev transcription must stop, and vice versa. In addition, the
presence of rev is required for export of unspliced RNA from the
nucleus. For rev to function in this manner, however, a rev responsive
element (RRE) must be present on the transcript [Malim et al., Nature
338:254-257 (1989)].
In the polynucleotide vaccine of this invention, the
obligatory splicing of certain HIV genes is elimin~ted by providing fully
spliced genes (i.e.: the provision of a complete open reading frame for
the desired gene product without the need for switches in the reading
frame or elimin~tion of noncoding regions; those of ordinary skill in the
art would recognize that when splicing a particular gene, there is some
latitude in the precise sequence that results; however so long as a
functional coding sequence is obtained, this is acceptable). Thus, in one
embodiment, the entire coding sequence for gpl60 is spliced such that
no intermittent expression of each gene product is required.
The dual humoral and cellular immune responses generated
according to this invention are particularly significant to inhibiting HIV
infection, given the propensity of HIV to mutate within the population,
as well as in infected individuals. In order to form~ te an effective
protective vaccine for HIV it is desirable to generate both a multivalent
antibody response for example to gpl60 (env is approximately 80%
conserved across various HIV-1, clade B strains, which are the prevalent
strains in US hllm~n populations), the principal neutralization target on
HIV, as well as cytotoxic T cells reactive to the conserved portions of
gpl60 and, internal viral proteins encoded by gag. We have made an
HIV vaccine comprising gpl60 genes selected from common laboratory
strains; from predomin~nt, primary viral isolates found within the
infected population; from mutated gpl60s designed to nnm~.~k cross-
strain, neutralizing antibody epitopes; and from other representative
CA 022~8~68 1998-12-18
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HIV genes such as the gag and pol genes (~95% conserved across HIV
isolates.
Virtually all HIV seropositive patients who have not
advanced towards an immllnodeficient state harbor anti-gag CTLs while
5 about 60% of these patients show cross-strain, gpl60-specific CTLs.
The amount of HIV specific CTLs found in infected individuals that
have progressed on to the disease state known as AIDS, however, is
much lower, demonstrating the significance of our findings that we can
induce cross-strain CTL responses.
Immune responses induced by our env andgag
polynucleotide vaccine constructs are demonstrated in mice and
primates. Monitoring antibody production to env in mice allows
confirmation that a given construct is suitably immunogenic, i.e., a high
proportion of vaccinated ~nim~ show an antibody response. Mice also
15 provide the most facile ~nim~l model suitable for testing CTL induction
by our constructs and are therefore used to evaluate whether a
particular construct is able to generate such activity. Monkeys (African
green, rhesus, chimpanzees) provide additional species including
primates for antibody evaluation in larger, non-rodent ~nim~l~. These
20 species are also preferred to mice for antisera neutralization assays due
to high levels of endogenous neutralizing activities against retroviruses
observed in mouse sera. These data demonstrate that sufficient
immllnogenicity is engendered by our vaccines to achieve protection in
experiments in a chimpanzee/HIVIIIg challenge model based upon
25 known protective levels of neutralizing antibodies for this system.
However, the currently emerging and increasingly accepted definition of
protection in the scientific commllnity is moving away from so-called
"sterilizing immunity", which indicates complete protection from HIV
infection, to prevention of disease. A number of correlates of this goal
30 include reduced blood viral titer, as measured either by HIV reverse
transcriptase activity, by infectivity of samples of serum, by ELISA
assay of p24 or other HIV antigen concentration in blood, increased
CD4+ T-cell concentration, and by extended survival rates ~see, for
example, Cohen, J., Science 262:1820-1821, 1993, for a discussion of
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the evolving definition of anti-HIV vaccine efficacy]. The immunogens
of the instant invention also generate neutralizing immune responses
against infectious (clinical, primary field) isolates of HIV.
5 Immunolo~y
A. Antibody Responses to env.
1. ~pl60 and gpl20. An ELISA assay is used to
determine whether vaccine vectors expressing either secreted gpl20 or
membrane-bound gpl60 are efficacious for production of env-specific
10 antibodies. Initial in vitro characterization of env expression by our
vaccination vectors is provided by immunoblot analysis of gpl60
transfected cell lysates. These data confirm and quantitate gpl60
expression using anti-gp41 and anti-gpl20 monoclonal antibodies to
visualize transfectant cell gpl60 expression. In one embodiment of this
15 invention, gpl60 is preferred to gpl20 for the following reasons: (1)
an initial gpl20 vector gave inconsistent immllnogenicity in mice and
was very poorly or non-responsive in African green Monkeys; (2)
gpl60 contributes additional neutralizing antibody as well as CTL
epitopes by providing the addition of approximately 190 amino acid
20 residues due to the inclusion of gp41; (3) gpl60 expression is more
similar to viral env with respect to tetramer assembly and overall
conformation, which may provide oligomer-dependent neutralization
epitopes; and (4) we find that, like the success of membrane-bound,
influenza HA constructs for producing neutr~li7ing antibody responses
25 in mice, ferrets, and nonhuman primates [see Ulmer et al., Science
259:1745-1749, 1993; Montgomery, D., et al., DNA and Cell Biol.
12:777 783, 1993] anti-gpl60 antibody generation is superior to anti-
gpl20 antibody generation. Selection of which type of env, or whether
a cocktail of env subfragments, is preferred is determined by the
30 experiments outlined below.
2. Presence and Breadth of Neutralizin~ Activity.
ELISA positive antisera from monkeys is tested and shown to neutralize
both homologous and heterologous HIV strains.
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WO 97/48370 PCT/US97/10517
3. V3 vs. non-V3 Neutr~1i7in~ Antibodies. A major
goal for env PNVs is to generate broadly neutr~li7ing antibodies. It has
now been shown that antibodies directed against V3 loops are very
strain specific, and the serology of this response has been used to define
5 strains.
a. Non-V3 neutr~li7.in~ antibodies appear to
primarily recognize discontinuous, structural epitopes within gpl20
which are responsible for CD4 binding. Antibodies to this domain are
polyclonal and more broadly cross-neutralizing probably due to
10 restraints on mutations imposed by the need for the virus to bind its
cellular ligand. An in vitro assay is used to test for blocking gp l 20
binding to CD4 immobilized on 96 well p}ates by sera from immlmi7ed
~nim~ls. A second in vitro assay detects direct antibody binding to
synthetic peptides representing selected V3 domains immobilized on
15 plastic. These assays are compatible for antisera from any of the ~nim~l
types used in our studies and define the types of neutralizing antibodies
our vaccines have generated as well as provide an in vitro correlate to
virus neutralization.
b. gp41 harbors at least one major neutralization
20 determin~nt, corresponding to the highly conserved linear epitope
recognized by the broadly neutralizing 2F5 monoclonal antibody
(commercially available from Viral Testing Systems Corp., Texas
Commerce Tower, 600 Travis Street, Suite 4750, Houston, TX 77002-
3005(USA), or Waldheim Ph~rm~7eutika GmbH, Boltzmangasse 11, A-
25 1091 Wien, Austria), as well as other potential sites including the well-
conserved "fusion peptide" domain located at the N-terminus of gp41.
Besides the detection of antibodies directed against gp41 by im~nunoblot
as described above, an in vitro assay test is used for antibodies which
bind to synthetic peptides representing these domains immobilized on
30 plastic.
4. Maturation of the Antibody Response. In HIV
seropositive patients, the neutralizing antibody responses progress from
chiefly anti-V3 to include more broadly neutralizing antibodies
comprising the structural gpl20 domain epitopes described above (#3),
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including gp41 epitopes. These types of antibody responses are
monitored over the course of both time and subsequent vaccinations.
B. T Cell Reactivities A~ainst env and gaB.
1. Generation of CTL Responses. Viralproteins which
are synthesized within cells give rise to MHC I-restricted CTL
responses. Each of these proteins elicits CTL in seropositive patients.
Our vaccines also are able to elicit CTL in mice. The immunogenetics
of mouse strains are conducive to such studies, as demonstrated with
influenza NP, [see Ulmer et al., Science 259:1745-1749, 1993]. Several
epitopes have been defined for the HIV proteins env, rev~ nef and gag
in Balb/c mice, thus facilitating in vitro CTL culture and cytotoxicity
assays. It is advantageous to use syngeneic tumor lines, such as the
murine mastocytoma P815, transfected with these genes to provide
targets for CTL as well as for in vitro antigen specific restimulation.
Methods for defining immlmogens capable of eliciting MHC class I-
restricted cytotoxic T Iymphocytes are known [see Calin-Laurens, et al.,
Vaccine 11(9):974-978, 1993; see particularly Eriksson, et al., Vaccine
11(8):859 865, 1993, wherein T-cell activating epitopes on the HIV
gpl20 were mapped in primates and several regions, including gpl20
amino acids 142-192, 296-343, 367-400, and 410-453 were each found
to induce lymphoproliferation; furthermore, discrete regions 248-269
and 270-295 were lymphoproliferative. A peptide encompassing amino
acids 152-176 was also found to induce HIV neutralizing antibodies],
and these methods may be used to identify immllnogenic epitopes for
inclusion in the PNV of this invention. Alternatively, the entire gene
encoding gpl60, gpl20, protease, or gag could be used. For additional
review on this subject, see for example, Shirai et al., J. Immunol
148:1657 1667, 1992; Choppin et al., J. Immunol 147:569 574, 1991;
Choppin et al., J. Immunol 147:575-583, 1991; Berzofsky et al., J.
Clin. Invest. 88:876-884, 1991. As used herein, T-cell effector function
is associated with mature T-cell phenotype, for example, cytotoxicity,
cytokine secretion for B-cell activation, and/or recruitment or
stim~ tion of macrophages and neutrophils.
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2. Measurement of T~ Activities. Spleen cell cultures
derived from vaccinated ~nim~ are tested for recall to specific antigens
by addition of either recombinant protein or peptide epitopes.
Act*ation of T cells by such antigens, presented by accompanying
5 splenic antigen presenting cells, APCs, is monitored by proliferation of
these cultures or by cytol~ine production. The pattern of cytokine
production also allows classification of TH response as type 1 or type 2.
Because domin~nt TH2 responses appear to correlate with the exclusion
of cellular immunity in immunocompromised seropositive patients, it is
10 possible to define the type of response engendered by a given PNV in
patients, permitting manipulation of the resulting immune responses.
3. Delayed Type Hypersensitivity (DTH). DTH to viral
antigen after i.d. injection is indicative of cellular, primarily MHC II-
restricted, immunity. Because of the commercial availability of
15 recombinant HIV proteins and synthetic peptides for known epitopes,
DTH responses are easily determined in vaccinated vertebrates using
these reagents, thus providing an additional in vivo correlate for
inducing cellular immunity.
20 Protection
Based upon the above immunologic studies, it is predictable
that our vaccines are effective in vertebrates against challenge by
virulent HIV. These studies are accomplished in an
HIVIIIg/chimpanzee challenge model after sufficient vaccination of
25 these ~nim~ls with a PNV construct, or a cocktail of PNV constructs
comprised of gpl60IIIg, gagIIIg~ nefIIIB and REVIIIg. The IIIB
strain is useful in this regard as the chimpanzee titer of lethal doses of
this strain has been established. However, the same studies are
envisioned using any strain of HIV and the epitopes specific to or
30 heterologous to the given strain. A second vaccination/challenge model,
in addition to chimpanzees, is the scid-hu PBL mouse. This model
allows testing of the hllm~n lymphocyte immune system and our vaccine
with subsequent HIV challenge in a mouse host. This system is
advantageous as it is easily adapted to use with any HIV strain and it
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provides evidence of protection against multiple strains of primary field
isolates of HIV. A third challenge model utilizes hybrid HIV/SIV
viruses (SHIV), some of which have been shown to infect rhesus
monkeys and lead to immnnodeficiency disease resulting in death [see
S Li, J., et al., J. AIDS 5:639-646, 1992]. Vaccination of rhesus with our
polynucleotide vaccine constructs is protective against subse~uent
challenge with lethal doses of SHIV.
PNV Construct Summary
HIV and other genes are ligated into an expression vector
which has been optimized for polynucleotide vaccinations. Essentially
all extraneous DNA is removed, leaving the essential elements of
transcriptional promoter, immllnogenic epitopes, transcriptional
terminator, bacterial origin of replication and antibiotic resistance gene.
Expression of HIV late genes such as env and gag is rev-
dependent and requires that the rev response element (RRE) be present
on the viral gene transcript. A secreted form of gpl20 can be generated
in the absence of rev by substitution of the gpl20 leader peptide with a
heterologous leader such as from tPA (tissue-type plasminogen
20 activator), and preferably by a leader peptide such as is found in highly
expressed m~mm~ n proteins such as immunoglobulin leader peptides.
We have inserted a tPA-gpl20 chimeric gene into VlJns which
efficiently expresses secreted gpl20 in transfected cells (RD, a human
rhabdomyosarcoma line). Monocistronic gpl60 does not produce any
25 protein upon transfection without the addition of a rev expression
vector.
Representative Construct Components Include (but are not restricted to):
1 . tPA-gp 1 20MN;
- 30 2. gpl6oIIIB;
3. gagIIIB: for anti-gag CTL;
4 . tPA-gp 1 2oIIIB;
5. tPA-gpl40
.. . ., . ~ , ,
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WO 97/48370 PCT/US97/10517
6. tPA-gpl60 with structural mutations: V1, V2, and/or
V3 loop deletions or substitutions
7. Genes encoding antigens expressed by pathogens other
than HIV, such as, but not limited to, influenza virus
nucleoprotein, hemagglutinin, matrix, neuraminidase, and
other antigenic proteins; herpes simplex virus genes; human
papillomavirus genes; tuberculosis antigens; hepatitis A, B,
or C virus antigens.
The protective efficacy of polynucleotide HIV immunogens
against subsequent viral challenge is demonstrated by immllni7.~tion with
the non-replicating plasmid DNA of this invention. This is
advantageous since no infectious agent is involved, assembly of virus
particles is not required, and determin~nt selection is permitted.
lS Furthermore, because the sequence of gag and protease and several of
the other viral gene products is conserved among various strains of
HIV, protection against subsequent challenge by a virulent strain of HIV
that is homologous to, as well as strains heterologous to the strain from
which the cloned gene is obtained, is enabled.
The i.m. injection of a DNA expression vector encoding
gpl60 results in the generation of significant protective immunity
against subsequent viral challenge. In particular, gpl60-specific
antibodies and primary CTLs are produced. Immune responses directed
against conserved proteins can be effective despite the antigenic shift and
drift of the variable envelope proteins. Because each of the HIV gene
products exhibit some degree of conservation, and because CTL are
generated in response to intracellular expression and MHC processing, it
is predictable that many virus genes give rise to responses analogous to
that achieved for gpl60. Thus, many of these genes have been cloned,
as shown by the cloned and sequenced junctions in the expression vector
(see below) such that these constructs are immunogenic agents in
available form.
The invention offers a means to induce cross-strain
protective immunity without the need for self-replicating agents or
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WO 97148370 PCT/US97tlO517
adjuvants. In addition, immllni7~tion with the instant polynucleotides
offers a number of other advantages. This approach to vaccination
- should be applicable to tumors as well as infectious agents, since the
CD8+ CTL response is important for both pathophysiological processes
[K. Tanaka et al., Annu. Rev. Imml-nol. 6, 359 (1988)~. Therefore,
eliciting an immune response against a protein crucial to the
transformation process may be an effective means of cancer protection
or immunotherapy. The generation of high titer antibodies against
expressed proteins after injection of viral protein and human growth
hormone DNA suggests that this is a facile and highly effective means of
making antibody-based vaccines, either separately or in combination
with cytotoxic T-lymphocyte vaccines targeted towards conserved
antigens.
The ease of producing and purifying DNA constructs
compares favorably with traditional methods of protein purification,
thus facilitating the generation of combination vaccines. Accordingly,
multiple constructs, for example encoding gpl60, gpl20, gp41, or any
other HIV gene may be prepared, mixed and co-~(lmini~tered. Because
protein expression is m~int~ined following DNA injection, the
persistence of B- and T-cell memory may be enhanced, thereby
engendering long-lived humoral and cell-mediated immunity.
Standard techniques of molecular biology for preparing and
purifying DNA constructs enable the preparation of the DNA
immllnogens of this invention. While standard techniques of molecular
biology are therefore sufficient for the production of the products of
this invention, the specific constructs disclosed herein provide novel
polynucleotide immunogens which surprisingly produce cross-strain and
primary HIV isolate neutralization, a result heretofore lln~tt~in~ble with
standard inactivated whole virus or subunit protein vaccines.
- 30 The amount of expressible DNA or transcribed RNA to be
introduced into a vaccine recipient will depend on the strength of the
transcriptional and translational promoters used and on the
immunogenicity of the expressed gene product. In general, an
immunologically or prophylactically effective dose of about 1 ng to 100
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WO 97/48370 PCT/US97/10517
mg, and preferably about 10 ~lg to 300 ~lg is ~tlrnini~tered directly into
muscle tissue. Subcutaneous injection, intradermal introduction,
impression through the skin, and other modes of ~lmini~tration such as
intraperitoneal, intravenous, or inh~l~tion delivery are also
S contemplated. It is also contemplated that booster vaccinations are to be
provided. Following vaccination with HIV polynucleotide immunogen,
boosting with HIV protein immunogens such as gpl60, gpl20, and gag
gene products is also contemplated. Parenteral ~lmini~tration, such as
intravenous, intramuscular, subcutaneous or other means of
10 ~mini~tration of interleukin-12 protein or GM-CSF or similar proteins
alone or in combination, concurrently with or subsequent to parenteral
introduction of the PNV of this invention is also advantageous.
The polynucleotide may be naked, that is, unassociated with
any proteins, adjuvants or other agents which impact on the recipients'
15 immllne system. In this case, it is desirable for the polynucleotide to be
in a physiologically acceptable solution, such as, but not limited to,
sterile saline or sterile buffered saline. Alternatively, the DNA may be
associated with liposomes, such as lecithin liposomes or other liposomes
known in the art, as a DNA-liposome mixture, or the DNA may be
20 associated with an adjuvant known in the art to boost immllne responses,
such as a protein or other carrier. Agents which assist in the cellular
uptake of DNA, such as, but not limited to, calcium ions, may also be
used to advantage. These agents are generally referred to herein as
transfection facilitating reagents and pharmaceutically acceptable
25 carriers. Techniques for coating microprojectiles coated with
polynucleotide are known in the art and are also useful in connection
with this invention.
The following examples are offered by way of illustration
and are not intended to limit the invention in any manner.
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WO 97/48370 PCT/US97/10517
EXAMPLE 1
Materials descriptions
Vectors pF411 and pF412: These vectors were subcloned from
vector pSP62 which was constructed in R. Gallo's lab. pSP62 is an
S available reagent from Biotech Research Laboratories, Inc. pSP62 has a
12.5 kb XbaI fragment of the HXB2 genome subcloned from lambda
HXB2. SalI and Xba I digestion of pSP62 yields to HXB2 fragments:
S'-XbaI/SalI, 6.5 kb and 3'- SalI/XbaI, 6 kb. These inserts were
subcloned into pUC 18 at SmaI and SalI sites yielding pF411 (5'-
10 XbaVSalI) and pF412 (3'-XbaI/SalI). pF411 contains gag/pol and pF412
contains tat/rev/env/nef.
Repligen reagents:
recombinant rev (IIIB), ~tRPl024-10
15 rec. gpl20 (IIIB), #RP1001-10
anti-rev monoclonal antibody, #RP1029-10
anti-gpl20 mAB, #lC1, #RPlO10-10
AIDS Research and Reference Reagent Program:
20 anti-gp41 mAB hybridoma, Chessie 8, #526
The strategies are designed to induce both cytotoxic T
lymphocyte (CTL) and neutralizing antibody responses to HIV,
principally directed at the HIV gag (~95% conserved) and env (gpl60
or gpl20; 70-80% conserved) gene products. gpl60 contains the only
2~ kno~vn neutr~li7ing antibody epitopes on the HIV particle while the
importance of anti-env and anti-gag CTL responses are highlighted by
the known association of the onset of these cellular immunities with
clearance of primary viremia following infection, which occurs prior to
the appearance of neutralizing antibodies, as well as a role for CTL in
30 m;lint~ining disease-free status. Because HIV is notorious for its genetic
diversity, we hope to obtain greater breadth of neutralizing antibodies
by including several representative env genes derived from clinical
isolates and gp4l (~90% conserved), while the highly conserved gag
gene should generate broad cross-strain CTL responses.
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EXAMPLE 2
Heterolo~ous Expression of HIV Late Gene Products
HIV structural genes such as env and gag require
expression of the HIV regulatory gene, rev, in order to efficiently
S produce full-length proteins. We have found that rev-dependent
expression of gag yielded low levels of protein and that rev itself may
be toxic to cells. Although we achieved relatively high levels of rev-
dependent expression of gpl60 in vitro this vaccine elicited low levels of
antibodies to gpl60 following in vivo immllni7~tion with rev/gpl60
10 DNA. This may result from known cytotoxic effects of rev as well as
increased difficulty in obt~ining rev function in myotubules cont~ining
hundreds of nuclei (rev protein needs to be in the same nucleus as a rev-
dependent transcript for gag or env protein expression to occur).
However, it has been possible to obtain rev-independent expression
15 using selected modifications of the env gene. Evaluation of these
plasmids for vaccine purposes is under~vay.
In general, our vaccines have utilized primarily HIV (IIIB)
env and gag genes for optimization of expression within our generalized
vaccination vector, VlJns, which is comprised of a CMV immediate-
20 early (IE) promoter, BGH polyadenylation site, and a pUC backbone.Varying efficiencies, depending upon how large a gene segment is used
(e.g., gpl20 vs. gpl60), of rev-independent expression may be achieved
for env by replacing its native secretory leader peptide with that from
the tissue-specific pl~minogen activator (tPA) gene and expressing the
25 resulting chimeric gene behind the CMVIE promoter with the CMV
intron A. tPA-gpl20 is an example of a secreted gpl20 vector
constructed in this fashion which functions well enough to elicit anti-
gpl20 immune responses in vaccinated mice and monkeys.
Because of reports that membrane-anchored proteins may
30 induce much more substantial (and perhaps more specific for HIV
neutralization) antibody responses compared to secreted proteins as well
as to gain additional immllne epitopes, we prepared VlJns-tPA-gpl60
and V13ns-rev/gpl60. The tPA-gpl60 vector produced detectable
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quantities of gpl60 and gpl20, without the addition of rev, as shown by
immunoblot analysis of transfected cells, although levels of expression
were much lower than that obtained for rev/gpl60, a rev-dependent
gpl60-expressing plasmid. This is probably because inhibitory regions
5 (designated INS), which confer rev dependence upon the gpl60
transcript, occur at multiple sites within gpl60 including at the COOH-
terminus of gp41 (see Figure 1 for schematic view of gpl43 construct
strategies). A vector was prepared for a COOH-terminally truncated
form of tPA-gpl60, tPA-gpl43, which was designed to increase the
10 overall expression levels of env by elimin~tion of these inhibitory
sequences. The gpl43 vector also elimin~tes intracellular gp41 regions
cont~ining peptide motifs (such as leu-leu) known to cause diversion of
membrane proteins to the lysosomes rather than the cell surface. Thus,
gpl43 may be expected to increase both expression of the enV protein
15 (by decreasing rev-dependence) and the efficiency of transport of
protein to the cell surface compared to full-length gpl60 where these
proteins may be better able to elicit anti-gpl60 antibodies following
DNA vaccination. tPA-gpl43 was further modified by extensive silent
mutagenesis of the rev response element (RRE) sequence (350 bp) to
20 elimin~te additional inhibitory sequences for expression. This construct,
gpl43/mutRRE, was prepared in two forrns: either elimin~ting (form
A) or retaining (form B) proteolytic cleavage sites for gpl20/41. Both
forrns were prepared because of literature reports that vaccination of
mice using uncleavable gpl60 expressed in vaccinia elicited much higher
25 levels of antibodies to gpl60 than did cleavable folms.
A quantitative ELISA for gpl60/gpl20 expression in cell
transfectants was developed to determine the relative expression
capabilities for these vectors. In vitro transfection of 293 cells followed
by quantification of cell-associated vs. secreted/released gpl20 yielded
30 the following results: (1) tPA-gpl60 expressed 5-lOX less gpl20 than
rev/gpl60 with similar proportions retained intracellularly vs.
trafficked to the cell surface; (2) tPA-gpl43 gave 3-6X greater secretion
of gpl20 than rev/gpl60 with only low levels of cell-associated gpl~3,
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confirming that the cytoplasmic tail of gpl60 causes intracellular
retention of gpl60 which can be overcome by partial deletion of this
sequence; and, (3) tPA-gpl43/mutRRE A and B gave ~lOX greater
expression levels of protein than did parental tPA-gpl43 while
S elimin~tion of proteolytic processing was confirmed for forrn A.
Figure 4 presents representative data supporting points (1) - (3).
Thus, our strategy to increase rev-independent expression
has yielded stepwise increases in overall expression as well as
redirecting membrane-anchored gpl43 to the cell surface away from
10 lysosomes. It is important to note that it should be possible to insert
gpl20 sequences derived from various viral isolates within a vector
cassette containing these modifications which reside either at the NH2-
terminus (tPA leader) or COOH-terminus (gp41), where few antigenic
differences exist between different viral strains. In other words, this is
15 a generic construct which can easily be modified by inserting gpl20
derived from various primary viral isolates to obtain clinically relevant
vaccmes.
To apply these expression strategies to viruses that are
relevant for vaccine purposes and confirm the generality of our
20 approaches, we also prepared a tPA-gpl20 vector derived from a
primary HIV isolate (cont~ining the North American consensus V3
peptide loop; macrophage-tropic and nonsyncytia-inducing phenotypes).
This vector gave high expression/secretion of gpl20 with transfected
293 cells and elicited anti-gpl20 antibodies in mice demonstrating that it
25 was cloned in a functional form. Primary isolate gpl60 genes will also
be used for expression in the same way as for gpl60 derived from
laboratory strains.
EXAMPLE 3
30 Immune Responses to HIV-l env Polynucleotide Vaccines:
African green (AGM) and Rhesus (RHM) monkeys which
received gpl20 DNA vaccines showed low levels of neutralizing
antibodies following 2-3 vaccinations, which could not be increased by
additional vaccination. These results, as well as increasing awareness
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within the HIV vaccine field that oligomeric gpl60 is probably a more
relevant target antigen for eliciting neutr~li7.in~ antibodies than gpl20
monomers (Moore and Ho, J. Virol. 67: 863 (1993)), have led us to
focus upon obtaining effective expression of gpl60-based vectors (see
5 above). Mice and AGM were also vaccinated with the primary isolate
derived tPA-gpl20 vaccine. These ~nim~ exhibited anti-V3 peptide
(using homologous sequence) reciprocal endpoint antibody titers
ranging from 500-5000, demonstrating that this vaccine design is
functional for clinically relevant viral isolates.
The gpl60-based vaccines, rev-gpl60 and tPA-gpl60,
failed to consistently elicit antibody responses in mice and nonhllm~n
primates or yielded low antibody titers. Our initial results with the
tPA-gpl43 plasmid yielded geometric mean titers > 103 in mice and
AGM following two vaccinations. These data indicate that we have
15 significantly improved the immunogenicity of gpl60-like vaccines by
increasing expression levels. This construct, as well as the tPA-
gpl43/mutRRE A and B vectors, will continue to be characterized for
antibody responses, especially for virus neutralization.
Significantly, gpl20 DNA vaccination produced potent
20 helper T cell responses in all lymphatic compartments tested (spleen,
blood, inguinal, mesenteric, and iliac nodes) with TH 1-like cytokine
secretion profiles (i.e., g-interferon and IL-2 production with little or
no IL-4). These cytokines generally promote strong cellular immunity
and have been associated with m~intenance of a disease-free state for
25 HIV-seropositive patients. Lymph nodes have been shown to be
primary sites for HIV replication, harboring large reservoirs of virus
even when virus cannot be readily detected in the blood. A vaccine
which can elicit anti-HIV immune responses at a variety of Iymph sites,
such as we have shown with our DNA vaccine, may help prevent
30 successful colonization of the lymphatics following initial infection.
As stated previously, we consider realization of the
following objectives to be essential to maximize our chances for success
with this program: (1) env-based vectors capable of generating stronger
neutralizing antibody responses in primates; (2) gag and env vectors
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which elicit strong T-lymphocyte responses as characterized by CTL
and helper effector functions in primates; (3) use of env and gag genes
from clinically relevant HIV-l strains in our vaccines and
characterization of the immunologic responses, especially neutralization
5 of primary isolates, they elicit; (4) demonstration of protection in an
~nim~l challenge model such as chimpanzee/HIV (IIIB) or rhesus/SHIV
using appropriate optimized vaccines; and, (5) determination of the
duration of immune responses appropriate to clinical use. Significant
progress has been made on the first three of these objectives and
10 experiments are in progress to determine whether our recent
vaccination constructs for gpl60 and gag will improve upon these initial
results.
EXAMPLE 4
15 Vectors For Vaccine Production
A. VlJneo EXPRESSION VECTOR~ SEO. ID 1:
It was necessary to remove the ampr gene used for
antibiotic selection of bacteria harboring VlJ because ampicillin may
not be used in large-scale fermenters. The ampr gene from the pUC
20 backbone of VlJ was removed by digestion with SspI and Eaml 105I
restriction enzymes. The rem~ining plasmid was purified by agarose
gel electrophoresis, blunt-ended with T4 DNA polymerase, and then
treated with calf intestinal :~lk~line phosphatase. The commercially
available kanr gene, derived from transposon 903 and contained within
25 the pUC4K plasmid, was excised using the PstI restriction enzyme,
purified by agarose gel electrophoresis, and blunt-ended with T4 DNA
polymerase. This fragment was ligated with the VlJ backbone and
plasmids with the kanr gene in either orientation were derived which
were designated as VlJneo #'s 1 and 3. Each of these plasmids was
30 confirmed by restriction enzyme digestion analysis, DNA sequencing of
the junction regions, and was shown to produce similar quantities of
plasmid as VlJ. Expression of heterologous gene products was also
comparable to VlJ for these VlJneo vectors. We arbitrarily selected
VlJneo#3, referred to as VlJneo hereafter (SEQ ID:1), which contains
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the kanr gene in the same orientation as the ampr gene in VlJ as the
expression construct.
B. V13ns Expression Vector:
An Sfi I site was added to VlJneo to facilitate integration
studies. A commercially available 13 base pair Sfi I linker (New
England BioLabs) was added at the Kpn I site within the BGH sequence
of the vector. VlJneo was linearized with Kpn I, gel purified, blunted
by T4 DNA polymerase, and ligated to the blunt Sfi I linker. Clonal
isolates were chosen by restriction mapping and verified by sequencing
through the linker. The new vector was designated VlJns. Expression
of heterologous genes in VlJns (with Sfi I) was comparable to
expression of the same genes in VlJneo (with Kpn I).
1~ C. VlJns-tPA:
In order to provide an heterologous leader peptide sequence
to secreted and/or membrane proteins, VlJn was modified to include the
human tissue-specific pl~minogen activator (tPA) leader. Two
synthetic complementary oligomers were annealed and then ligated into
VlJn which had been BglII digested. The sense and antisense oligomers
were 5'-GATC ACC ATG GAT GCA ATG AAG AGA GGG CTC TGC
TGT GTG CTG CTG CTG TGT GGA GCA GTC l~C GTT TCG CCC
AGC GA-3' (SEQ.ID:2), and 5'-GAT CTC GCT GGG CGA AAC GAA
GAC TGC TCC ACA CAG CAG CAG CAC ACA GCA GAG CCC
TCT CTT CAT TGC ATC CAT GGT-3' (SEQ. ID:3). The Kozak
sequence is underlined in the sense oligomer. These oligomers have
overhanging bases compatible for ligation to BglII-cleaved sequences.
After ligation the upstream BglII site is destroyed while the downstream
BglII is retained for subsequent ligations. Both the junction sites as well
as the entire tPA leader sequence were verified by DNA sequencing.
Additionally, in order to conform with our consensus optimized vector
VlJns (=VlJneo with an SfiI site), an SfiI restriction site was placed at
the KpnI site within the BGH terminator region of VlJn-tPA by
blunting the KpnI site with T4 DNA polymerase followed by ligation
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with an SfiI linker (catalogue #1138, New F.n~l~n~l Biolabs). This
modification was verified by restriction digestion and agarose gel
electrophoresis .
EXAMPLE 5
I. HIV env Vaccine Constructs:
Vaccines Producing Secreted env-derived Anti~en (gp120 and gp140):
Expression of the rev -dependent env gene as gpl20 was
conducted as follows: gpl20 was PCR-cloned from the MN strain of
HIV with either the native leader peptide sequence (VlJns-gpl20), or as
a fusion with the tissue-plasminogen activator (tPA) leader peptide
replacing the native leader peptide (VlJns-tPA-gpl20). tPA-gpl20
expression has been shown to be rev-independent [B.S. Chapman et al.,
Nuc. Acids Res. 19, 3979 (1991); it should be noted that other leader
sequences would provide a similar function in rendering the gpl20 gene
rev independent]. This was accomplished by preparing the following
gpl20 constructs lltili7ing the above described vectors.
EXAMPLE 6
gp 120 Vaccine Constructs:
A. VlJns-tPA-HIV~N ~pl20:
HIVMN gpl20 gene (~edimmune) was PCR amplified
using oligomers designed to remove the first 30 amino acids of the
peptide leader sequence and to facilitate cloning into VlJns-tPA creating
a chimeric protein consisting of the tPA leader peptide followed by the
rem~ining gpl20 sequence following amino acid residue 30. This
design allows for rev -independent gpl20 expression and secretion of
soluble gpl20 from cells harboring this plasmid. The sense and
antisense PCR oligomers used were 5'-CCC CGG ATC CTG ATC ACA
GAA AAA TTG TGGGTC ACA GTC-3' (SEQ. ID:4), and 5'-C CCC
AGG AATC CAC CTG TTA GCG CTT TTC TCT CTG CAC CAC
TCT TCT C-3' (SEQ. ID:5). The translation stop codon is underlined.
These oligomers contain BamHI restriction enzyme sites at either end of
the translation open reading frame with a BclI site located 3' to the
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BamHI of the sense oligomer. The PCR product was sequentially
digested with BclI followed by BamHI and ligated into VlJns-tPA which
had been BglII digested followed by calf intestinal ~Ik~line phosphatase
treatrnent. The resulting vector was sequenced to confirm in-frame
5 fusion between the tPA leader and gpl20 coding sequence, and gpl20
expression and secretion was verified by immllnoblot analysis of
transfected RB cells.
B. VlJns-tpA-HIvnIR ~pl20:
This vector is analogous to I.A. except that the HIV IIIB
strain was used for gpl20 sequence. The sense and antisense PCR
oligomers used were: 5'-GGT ACA TGA TCA CA GAA AAA TTG
TGG GTC ACA GTC-3' (SEQ.ID:6), and 5'-CCA CAT TGA TCA
GAT ATC TTA TCT TTT TTC TCT CTG CAC CAC TCT TC-3'
15 (SEQ.ID:7), respectively. These oligomers provide BclI sites at either
end of the insert as well as an EcoRV just upstream of the BclI site at
the 3'-end. The 5'-terminal BclI site allows ligation into the BglII site
of VlJns-tPA to create a chimeric tPA-gpl20 gene encoding the tPA
leader sequence and gpl20 without its native leader sequence. Ligation
20 products were verified by restriction digestion and DNA sequencing.
EXAMPLE 7
gp l 40 Vaccine Constructs:
These constructs was prepared by PCR similarly as tPA-
25 gpl20 with the tPA leader in place of the native leader, but designed toproduce secreted antigen by termin~ting the gene immediately NH2-
terminal of the transmembrane peptide (projected carboxyterminal
amino acid sequence = NH2-.. TNWLWYIK-COOH) [SEQ.ID:8].
Unlike the gpl20-producing constructs, gpl40 constructs should
30 produce oligomeric antigen and retain known gp4 1 -contained antibody
neutralization epitopes such as ELDKWA (SEQ.ID:53) defined by the
2F5 monoclonal antibody.
Constructs were prepared in two forms (A or B) depending
upon whether the gpl60 proteolytic cleavage sites at the junction of
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gpl20 and gp41 were retained (B) or elimin~ted (A) by appropriate
amino acid substitutions as described by Kieny et al., (Prot. Eng. 2:
219-255 (1988)) (wild type sequence = NH2-
...KAKRRVVQREKR...COOH (SEQ.ID:9) and the mutated sequence =
S NH2-...KAQNHvvQNEHo...cooH (sEQ~ o) withmllt~tedamino
acids underlined).
A. V lJns-tPA-gp 1 40/mutRRE-A/SRV- 1 3'-UTR (based on HIV-
l ~IIB~:
This construct was obtained by PCR using the following
sense and antisense PCR oligomers: S'-CT GAA AGA CCA GCA ACT
CCT AGG GAAT l~G GGG l~G CTC TGG-3' (SEQ.ID: 11 ) :, and 5'-
CGC AGG GGA GGT GGT CTA GAT ATC l~A TTA TTT TAT
ATA CCA CAG CCA ATT TGT TAT G-3' (SEQ ID:12) to obtain an
AvrII/EcoRV segment from vector IVB (con~ining the optimized RRE-
A segment). The 3'-UTR, prepared as a synthetic gene segment, that is
derived from the Simian Retrovirus-l (SRV-1, see below) was inserted
into an SrfT restriction enzyme site introduced immediately 3'- of the
gpl40 open reading frame. This UTR sequence has been described
previously as facilitating rev-independent expression of HIV env and
gag.
B . V 1 Jns-tPA-gp l 40/mutRRE-B/SRV- 1 3'-UTR (based on HIV-
~IIIB):
This construct is similar to TlA except that the env
proteolytic cleavage sites have been retained by using construct IVC as
starting material.
C. VlJns-tPA-gpl40/opt30-A (based on HTV-lnn~):
This construct was derived from IVB by AvrTI and Srfl
restriction enzyme digestion followed by ligation of a synthetic DNA
segment corresponding to gp30 but comprised of optimal codons for
translation (see gp32-opt below). The gp30-opt DNA was obtained
from gp32-opt by PCR amplification using the following sense and anti-
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sense oligomers: 5'-GGT ACA CCT AGG CAT CTG GGG CTG CTC
TGG-3', (SEQ ID:13) and, 5'-CCA CAT GAT ATC G CCC GGG C
TTA TTA l l l GAT GTA CCA CAG CCA GTT GGT GAT G-3',
(SEQ ID:14), respectively. This DNA segment was digested with AvrII
5 and EcoRV restriction enzymes and ligated into VlJns-tPA-
gpl43/opt32-A (IVD) that had been digested with AvrII and SrfI to
remove the corresponding DNA segment. The resulting products were
verified by DNA sequencing of ligation junctions and immunoblot
analysis.
D. VlJns-tPA-g;pl40/opt30-B (based on HIV~
This construct is similar to IIC except that the env
proteolytic cleavage sites have been retained.
15 E. VlJns-tPA-~pl40/opt all-A:
The env gene of this construct is comprised completely of
optimal codons. The constant regions (C1, C5, gp32) are those
described in IVB,D,H with an additional synthetic DNA segment
corresponding to variable regions 1-5 is inserted using a synthetic DNA
20 segment comprised of optimal codons for translation (see example
below based on HIV-1 MN V1-V5).
F. VlJns-tPA-gpl40/opt all-B:
This construct is similar to IE except that the env
25 proteolytic cleavage sites have been retained.
G. VlJns-tPA-gpl40/opt all-A (non-IIIB strains):
This construct is similar to IIE above except that env amino
acid sequences from strains other than IIIB are used to determine
30 optimum codon usage throughout the variable (V1-V5) regions.
H. VlJns-tPA-gpl40/opt all-B (non-IIIB strains):
This construct is similar to IIG except that the env
proteolytic cleavage sites have been retained.
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EXAMPLE 8
~p l 60 Vaccine Constructs:
Constructs were prepared in two forms (A or B) depending
upon whether the gpl60 proteolytic cleavage sites as described above.
A. V lJns-rev/env:
This vector is a variation of the one described in section D
above except that the entire tat coding region in exon l is deleted up to
the beginning of the rev open reading frame. VlJns-gpl60IIIg (see
section A. above) was digested with Pstl and KpnI restriction enzymes
to remove the 5'-region of the gpl60 gene. PCR amplification was used
to obtain a DNA segment encoding the first REV exon up to the KpnI
site in gpl60 from the HXB2 genomic clone. The sense and antisense
PCR oligomers were 5'-GGT ACA CTG CAG TCA CCG TCC T ATG
GCA GGA AGA AGC GGA GAC-3' (SEQ.ID:15) and 5'-CCA CAT
CA GGT ACC CCA TAA TAG ACT GTG ACC-3'(SEQ.ID:16)
respectively. These oligomers provide PstI and KpnI restriction
enzyme sites at the 5'- and 3'- termini of the DNA fragment,
respectively. The resulting DNA was digested with PstI and KpnI,
purified from an agarose electrophoretic gel, and ligated with VlJns-
gpl60(PstI/KpnI). The resulting plasmid was verified by restriction
enzyme digestion.
B. VlJns-~160:
HIVIIIb gpl60 was cloned by PCR amplification from
plasmid pF412 which contains the 3'-terminal half of the ~IVIIIb
genome derived from HIVIIIb clone HXB2. The PCR sense and
antisense oligomers were 5'-GGT ACA TGA TCA ACC ATG AGA
GTG AAG GAG AAA TAT CAG C-3'(SEQ.ID:17), and 5'-CCA CAT
TGA TCA GAT ATC CCC ATC TTA TAG CAA AAT CCT TTC C-3'
(SEQ. ID: 18), respectively. The Kozak sequence and translation stop
codon are underlined. These oligomers provide BclI restriction enzyme
sites outside of the translation open reading frame at both ends of the
env gene. (BclI-digested sites are compatible for ligation with BglII-
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digested sites with subsequent loss of sensitivity to both restriction
enzymes. BclI was chosen for PCR-cloning gpl60 because this gene
contains internal Bglrl and as well as BamHI sites). The antisense
oligomer also inserts an EcoRV site just prior to the BclI site as
described above for other PCR-derived genes. The amplified gpl60
gene was agarose gel-purified, digested with BclI, and ligated to VlJns
which had been digested with BglII and treated with calf intestinal
~lk~line phosphatase. The cloned gene was about 2.6 kb in size and each
junction of gpl60 with VlJns was confirmed by DNA sequencing.
C. VlJns-tPA-gpl60 (based on HIv-lTnR):
This vector is similar to Example l(C) above, except that
the full-length gpl60, without the native leader se~uence, was obtained
by PCR. The sense oligomer was the sarne as used in I.C. and the
antisense oligomer was 5'-CCA CAT TGA TCA GAT ATC CCC ATC
TTA TAG CAA AAT CCT TTC C-3' (SEQ.ID:l9). These oligomers
provide BclI sites at either end of the insert as well as an EcoRV just
upstream of the BclI site at the 3'-end. The 5'-te~ninal BclI site allows
ligation into the BglII site of VlJns-tPA to create a chimeric tPA-gpl60
gene encoding the tPA leader sequence and gpl60 without its native
leader sequence. Ligation products were verified by restriction
digestion and DNA sequencing.
D. VlJns-tPA-gpl60/opt C1/opt41 -A (~oased on HIV-1 lR~:
This construct was based on IVH, having a complete
optimized codon segment for C5 and gp41, rather than gp32, with an
additional optimized codon segment (see below) replacing C1 at the
amino terminus of gpl20 following the tPA leader. The new C1
segment was joined to the rem~ining gpl43 segment via SOE PCR using
the following oligomers for PCR to synthesize the joined C1/143
segment: 5'-CCT GTG TGT GAG TTT AAA C TGC ACT GAT TTG
AAG AAT GAT ACT AAT AC-3' (SEQ ID:20). The resulting gpl43
gene contains optimal codon usage except for Vl-V5 regions and has a
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unique PmeI restriction enzyme site placed at the junction of Cl and V1
for insertion of variable regions from other HIV genes.
E. VlJns-tPA-gp160/opt C1/opt41-B (based on HIV-lmp~:
S This construct is similar to IIID except that the env
proteolytic cleavage sites have been retained.
F VlJns-tPA-~pl60/opt all-A (based on HIV-lmp~):
The env gene of this construct is comprised completely of
optimal codons as described above. The constant regions (Cl, CS,
gp32) are those described in IIID,E which is used as a cassette
(employed for all completely optimized gpl60s) while the variable
regions, V l-V5, are derived from a synthetic DNA segment comprised
of optimal codons.
G. VlJns-tPA-gpl60/opt all-B:
This construct is similar to IIIF except that the env
proteolytic cleavage sites have been retained.
H. VlJns-tPA-gpl60/opt all-A (non-IIIB strains):
This construct is similar to IIIF above except that env
amino acid sequences from strains other than IIIB were used to
determine optimum codon useage throughout the variable (Vl-VS)
regions.
I. VlJns-tPA-gpl60/opt all-B (non-IIIB strains):
This construct is similar to IIIH except that the env
proteolytic cleavage sites have been retained.
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EXAMPLE 9
gpl43 Vaccine Constructs:
These constructs were prepared by PCR similarly as other
tPA-cont~ining constructs described above (tPA-gpl20, tPA-gpl40, and
5 tPA-gpl60), with the tPA leader in place of the native leader, but
designed to produce COOH-termin~ted, membrane-bound env
(projected intracellular amino acid sequence= NH2-NRVRQGYSP-
COOH). This construct was designed with the purpose of combining the
increased expression of env accompanying tPA introduction and
10 minimi7.ing the possibility that a transcript or peptide region
corresponding to the intracellular portion of env might negatively
impact expression or protein stability/transport to the cell surface.
Constructs were prepared in two forms (A or B) depending upon
whether the gpl60 proteo}ytic cleavage sites were removed or retained
15 as described above. The residual gp41 fragment resulting from
truncation to gpl43 is referred to as gp32.
A. VlJns-tPA-~pl43:
This construct was prepared by PCR using plasmid pF412
20 with the following sense and antisense PCR oligomers: 5'-GGT ACA
TGA TCA CA GAA AAA TTG TGG GTC ACA GTC-3' (SEQ.ID:21):,
and 5'- CCA CAT TGA TCA G CCC GGG C TTA GGG TGA ATA
GCC CTG CCT CAC TCT GTT CAC-3' (SEQ.ID:22). The resulting
DNA segment contains BclI restriction sites at either end for cloning
25 into VlJns-tPA/BglII-digested with an Sr~ site located immediately 3'-
to the env open reading frame. Constructs were verified by DNA
sequencing of ligation junctions and immunoblot analysis of transfected
cells (Figure 8).
~ 30 B. VlJns-tPA-gpl43/mutRRE-A:
This construct was based on IVA by excising the DNA
~ segment using the unique MunI restriction enzyme site and the
downstream SrfI site described above. This segment corresponds to a
portion of the gpl20 C5 domain and the entirety of gp32. A synthetic
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DNA segment corresponding to ~350 bp of the rev response element
(RRE A) of gpl60, comprised of optimal codons for translation, was
joined to the rem~ining gp32 segment by splice overlap extension (SOE)
PCR creating an AvrII restriction enzyme site at the junction of the two
5 segments (but no changes in amino acid sequence). These PCR reactions
were performed using the following sense and antisense PCR oligomers
for generating the gp32-cont~ining domain: 5'-CT GAA AGA CCA
GCA ACT CCT AGG GAT TTG GGG TTG CTG TGG-3' (SEQ ID:23)
and 5'-CCA CAT TGA TCA G CCC GGG C TTA GGG TGA ATA
10 GCC CTG CCT CAC TCT GTT CAC-3' [SEQ ID:24] (which was used
as the antisense oligomer for IVA), respectively. The mutated RRE
(mutRRE-A) segment was joined to the wild type sequence of gp32 by
SOE PCR using the following sense oligomer, 5'-GGT ACA CAA TTG
GAG GAG CGA GTT ATA TAA ATA TAA G-3' (SEQ ID:25), and
15 the antisense oligomer used to make the gp32 segment. The resulting
joined DNA segment was digested with MunI and SrfI restriction
enzymes and ligated into the parent gpl43/Munl/SrfI digested plasmid.
The resulting construct was verified by DNA sequencing of ligation and
SOE PCR junctions and immunoblot analysis of transfected cells (Figure
20 8).
C. VlJns-tPA-gp143/mutRRE-B:
This construct is similar to IVB except that the env
proteolytic cleavage sites have been retained by using the mutRRE-B
25 synthetic gene segment in place of mutRRE-A.
D. V lJns-tPA-gpl43/opt32-A:
This construct was derived from IVB by AvrII and SrfI
restriction enzyme digestion followed by ligation of a synthetic DNA
30 segment corresponding to gp32 but comprised of optimal codons for
translation (see gp32 opt below). The resulting products were verified
by DNA sequencing of ligation junctions and immllnoblot analysis.
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E. VlJns-tPA-gp143/opt32-B:
This construct is similar to IVD except that the env
proteolytic cleavage sites have been retained by using IVC as the initial
plasmid.
s
F. VlJns-tPA-gpl43/SRV-1 3'-UTR:
This construct is similar to IVA except that the 3'-UTR
derived from the Simian Retrovirus-l (SRV-l, see below) was inserted
into the SrfI restriction enzyme site introduced immediately 3'- of the
10 gpl43 open reading frame. This UTR sequence has been described
previously as facilit~ting rev-independent expression of HIV env and
gag.
G. VlJns-tPA-~pl43/opt C1/opt32A:
This construct was based on IVD, having a complete
optimized codon segment for C5 and gp32 with an additional optimized
codon segment (see below) replacing C1 at the amino terminus of gpl20
following the tPA leader. The new Cl segment was joined to the
rem~ining gpl43 segment via SOE PCR using the following oligomers
20 for PCR to synthesize the joined C1/143 segment: 5'-CCT GTG TGT
GAG TTT AAA C TGC ACT GAT TTG AAG AAT GAT ACT AAT
AC-3' (SEQ ID:26). The resulting gpl43 gene contains optimal codon
useage except for V1-V5 regions and has a unique PmeI restriction
enzyme site placed at the junction of Cl and V1 for insertion of variable
25 regions from other HIV genes.
H. VlJns-tPA-~pl43/opt C1/opt32B:
This construct is similar to IVH except that the env
proteolytic cleavage sites have been retained.
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I. VlJns-tPA-gpl43/opt all-A:
The env gene of this construct is comprised completely of
optimal codons. The constant regions (Cl, C5, gp32) are those
described in 4B,D,H with an additional synthetic DNA segment
5 corresponding to variable regions V1-V5 is inserted using a synthetic
DNA segment comprised of optimal codons for translation.
J. VlJns-tPA-~pl43/opt all-B:
This construct is similar to IVJ except that the env
10 proteolytic cleavage sites have been retained.
K. VlJns-tPA-gpl43/opt all-A (non-IIIB strains):
This construct is similar to IIIG above except that env
amino acid sequences from strains other than IIIB were used to
15 determine optimum codon useage throughout the variable (Vl-V5)
reglons.
L. VlJns-tPA-gpl43/opt all-B (non-IIIB strains):
This construct is similar to IIIG above except that env
20 amino acid sequences from strains other than IIIB were used to
determine optimum codon useage throughout the variable (Vl-V5)
reglons.
EXAMPLE 10
25 gpl43/~elyB Vaccine Constructs:
These constructs were prepared by PCR similarly as other
tPA-cont~ining constructs described above (tPA-gpl20, tPA-gpl40,
tPA-gpl43 and tPA-gpl60), with the tPA leader in place of the native
leader, but designed to produce COOH-termin~ted, membrane-bound
30 env as with gpl43. However, gpl43/glyB constructs differ from gpl43
in that of the six amino acids projected to comprise the intracellular
peptide domain, the last 4 are the same those at the carboxyl terminus of
human glycophorin B (glyB) protein (projected intracellular amino acid
sequence= NH2-NRLIKA-COOH (SEQ.ID:27) with the underlined
35 residues corresponding to glyB and "R" common to both env and glyB).
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This construct was designed with the purpose gaining additional env
expression and directed targeting to the cell surface by completely
elimin~ting any transcript or peptide region corresponding to the
intracellular portion of env that might negatively impact expression or
protein stability/transport to the cell surface by replacing this region
with a peptide sequence from an abundantly expressed protein (glyB)
having a short cytoplasmic domain (intracellular amino acid sequence=
NH2-RRLIKA-COOH). Constructs were prepared in two forms (A or
B) depending upon whether the gpl60 proteolytic cleavage sites were
removed or retained as described above.
A . V 1 Jns-tPA-gp 1 43/opt32-A/glyB:
This construct is the same as IVD except that the following
antisense PCR oligomer was used to replace the intracellular peptide
domain of gpl43 with that of glycophorin B as described above: 5'-
CCA CAT GAT ATC G CCC GGG C TTA TTA GGC CTT GAT CAG
CCG GTT CAC AAT GGA CAG CAC AGC-3' (SEQ ID:28).
B. VlJns-tPA-gpl43/opt32-B/glyB:
This construct is similar to VA except that the env
proteolytic cleavage sites have been retained.
C. VlJns-tPA-gpl43/opt C1/opt32-A/glyB:
This constmct is the same as VA except that the first
constant region (C1) of gpl20 is replaced by optimal codons for
translation as with IVH.
D. VlJns-tPA-gpl43/opt C1/opt32-B/glyB:
This construct is similar to VC except that the env
proteolytic cleavage sites have been retained.
E. VlJns-tPA-gpl43/opt all-A/glyB:
The env gene of this construct is comprised completely of
optimal codons as described above.
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F. VlJns-tPA-~pl43/opt all-B/glyB:
This construct is similar to VE except that the env
proteolytic cleavage sites have been retained.
5 G. VlJns-tPA-gpl43/opt all-A/glyB (non-IIIB strains):
This construct is similar to IIIG above except that env
amino acid sequences from strains other than IIIB were used to
determine optimum codon useage throughout the variable (V1-V5)
reglons.
H. VlJns-tPA-~pl43/opt all-B/glyB (non-IIIB strains):
This construct is similar to VG except that the env
proteolytic cleavage sites have been retained.
15 HIV env Vaccine Constructs with Variable Loop Deletions:
These constructs may include all env forms listed above
(gpl20, gpl40, gpl43, gpl60, gpl43/glyB) but have had variable loops
within the gpl20 region deleted during preparation (e.g., V1, V2,
and/or V3). The purpose of these modifications is to elimin~te peptide
20 segments which may occlude exposure of conserved neutralization
epitopes such as the CD4 binding site. For exarnple, the following
oligomer was used in a PCR reaction to create a V1/V2 deletion
resulting in adjoining THE C1 and C2 segments: 5'-CTG ACC CCC
CTG TGT GTG GGG GCT GGC AGT TGT AAC ACC TCA GTC
25 ATT ACA CAG-3' (SEQ ID:29).
EXAMPLE 1 l
Design of Synthetic Gene Segments for Increased env Gene Expression:
Gene segments were converted to sequences having
30 identical translated sequences (except where noted) but with alternative
codon usage as defined by R. Lathe in a research article from J. Molec.
Biol. Vol. 183, pp. 1-12 (1985) entitled "Synthetic Oligonucleotide
Probes Deduced from Arnino Acid Sequence Data: Theoretical and
Practical Considerations". The methodology described below to
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increase rev-independent expression of HIV env gene segments was
based on our hypothesis that the known inability to express this gene
- efficiently in m~mm~ n cells is a consequence of the overall transcript
composition. Thus, using alternative codons encoding the same protein
5 sequence may remove the constraints on env expression in the absence
of rev. Inspection of the codon usage within env revealed that a high
percentage of codons were among those infrequently used by highly
expressed human genes. The specific codon replacement method
employed may be described as follows employing data from Lathe et al.:
1. Identify placement of codons for proper open
reading frame.
2. Compare wild type codon for observed frequency of
use by human genes (refer to Table 3 in Lathe et al.).
3. If codon is not the most commonly employed, replace
it with an optimal codon for high expression based on data in Table 5.
4. Inspect the third nucleotide of the new codon and the
first nucleotide of the adjacent codon immediately 3'- of the first. If a
5'-CG-3' pairing has been created by the new codon selection, replace it
20 with the choice indicated in Table 5.
5. Repeat this procedure until the entire gene segment
has been replaced.
6. Inspect new gene sequence for undesired sequences
generated by these codon replacements (e.g., "ATTTA" sequences,
25 inadvertent creation of intron splice recognition sites, unwanted
restriction enzyme sites, etc.) and substitute codons that elimin~te these
sequences.
7. Assemble synthetic gene segments and test for
improved expression.
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These me~ods were used to create the following synthetic gene
segments for HIV env creating a gene comprised entirely of optimal
codon usage for expression: (i) gpl20-C1 (opt); (ii) V1-V5 (opt); (iii)
RRE-A/B (mut or opt); and (iv) gp30 (opt) with percentages of codon
replacements/nucleotide substitutions of 56/19, 73/26, 78/28, and 61/25
obtained for each segment, respectively. Each of these segments has
been described in detail above with actual sequences listed below.
~p120-Cl (o~t)
This is a gpl20 constant region 1 (Cl) gene segment from the mature
N-terminus to the beginning of Vl designed to have optimal codon
usage for expression.
l TGATCACAGA GAAGCTGTGG GTGACAGTGT ATTATGGCGT GCCAGTCTGG
51 AAGGAGGCCA CCACCACCCT(i~ irGCC TCTGATGCCA AGGCCTATGA
101 CACAGAGGTG CACAATGTGT GGGCCACCCA TGCCTGTGTG CCCACAGACC
151 CCAACCCCCA GGAGGTGGTG CTGGTGAATG TGACTGAGAA CTTCAACATG
201 TGGAAGAACA ACATGGTGGA GCAGATGCAT GAGGACATCA TCAGCCTGTG
251 GGACCAGAGC CTGAAGCCCT GTGTGAAGCT GACCCCCCTG TGTGTGAGTT
301 TAAAC(SEQ ID:30)
MN Vl-V5 (opt)
This is a gene segment corresponding to the derived protein sequence
for HIV MN V1-V5 (1066BP) having optimal codon usage for
expression.
1 AG I I I AAACTGCACAGACCTGAGGAACACCACCMCACCAACAACTCCAC
51 AGCG4ACAACMCTCCMCTCCGAGGGCAC CATCAAGGGG G(~3GAGATGA
101 AGMCTGCTC C; l I CMCATC ACCACCTCCATCAGGGACM GATGCAGAAG
151 GAGTATGCCC l (i~ l ci I ACM GCTGGACATT GTGTCCATTG ACMTGACTC
201 CACCTCCTAC AGGCTGATCT CCTGCMCAC C 1~ i l C;ATC ACCCAGGCCT
251 GCCCCAAAAT C I (;C I I I GAG CCCATCCCCA TCCACTACTG TGCCCCTGCT
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~ 301 GG~i I I I dCCATCCTGAAGTG CAATGACAAG M~ ; 1 (i I G GCMGGG~
351 CTGCAAGAAT~ CACAGTGCAGTGCACACATGGCATCAGG~i I ~ iG
401 TGTCCACCCA~; I G(; I (~i I G MT~ I ~;CC l (~;1 GAGGAGGAG(~i I ~i 1~
451 ATCAGGTCTG AGAACTTCAC AGACAATGCC MGACCATCA l (~i l GCACCT
501 GAATGAGTCT GTGCAGATCAACTGCACCAG GCCCAACTAC AACAAGAGGA
551 AGAGGATCCA CA I I (3GcccT GGCAGGG~; I l t~ l ACACCAC CMGMCATC
601 ATTG(3CACCATCAGGCAGGC CCACTGCMC ATCTCCAGGG CCMGTGGAA
l 5 651 TGACACCCTGAGGCAGATTGTGTCCMGCTGMGGAGCAGTTCMGMCA
701 AGACCATTGT GTTCMCCAG TC~ GGG GG,GACCCTGA GATTGTGATG
751 CACTCCTTCAACi l ~ iGGGGGGA(i I I (; I IC TACTGCMCACCTCCCCCCT
801 GTTCMCTCCACCTGGAATG GCMCAACAC CTGGMCMC ACCACAGGCT
851 CCMCMCAA CATCACCCTC CAGTGCMGATCMGCAGAT CATCMCATG
901 T(~CA~G TG~C4AGGC CATGTATGCC CCCO~ TTG Ar~r~l~GAT
951 CA~i I Gl; I (~C TCCAACATCA CAG~CC I (i~; l GCTGACCAGG GAT~'~;~
1001 AGGACACAGA CACCAACGAC ACCGr4MTCT TCAGGCC; 1~ ('~fi{-~C
1051 ATGAGGGACAATTGG (SEQ ID:31)
RRF.Mut (~
This is a DNA segment corresponding to the rev response element
(RRE) of HIV-1 comprised of optimal codon usage for expression. The
"A" form also has removed the known proteolytic cleavage sites at the
gpl20/gp41 junction by using the nucleotides indicated in boldface.
1 GACMTTG,GA GGAGCGAGTT ATATMMTAT MG,GTGGTGA AGATTGAGCC
51 CC I GGGGGTG GCCCCAACM MGCTC~ i I ~i I G CAG~Q~GAGC
101 A~:AGGC~ I G~GCATTG~;G GCCC; I (i I I I ~T(~GC; I I l ~i l t'~; I G(i I
151 G~;CTCCACMTr~C TAGCATGACC CTCACCGTGC AAGCTCGCCA
201 GCTG,CTGAGTGGCATCGTCC AGCAGCAG~A CMC(i l ~; l (~ CGCGCCATCG
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251 AAGCCCAGCA GCAC;C 1~1~; CAGCTGACTG TGTGG,GGGAT CAAACAGC~T
301 CA(~ GGC~il CGA;3CGCrATCTGAAAGACCAGCAACTCCT
351 AGGC (SEQ ID:32)
RRE.Mut (8)
This is a DNA segment corresponding to the rev response element
(RRE) of HIV-1 comprised of optimal codon usage for expression. The
"B" form retains the known proteolytic cleavage sites at the gpl20/gp41
Junctlon.
GACMTTGGA GGAGCGAGTT ATATAAATAT AAGGTGGTGA AGATTGAGCC
51 CCTGGGGGTG GCCCCAACM AAGCTA~ '~r~AGA(i I G~ I G CAG~GAG~
101 AGAGAGCCGTGGGCATTGt3GGC~I(il I IC l~;l I l ~;l t'~r~( I(i~;
151 GGC I CCACAA I ~CGCCGCTAGCATGACC CTCACCGTGC AAG~ I CGCCA
201 G,CTG,CTGAGT GG,CA I (li I (;C AGCAGCAGAA CAAC;CTGCTC CGCGCCATCG
251 MG~CCAG,CA GCAC~ x; l C CAGCTGACTG TGTGGGGGAT CAAACAGC~T
25 301 CAr{~ GCC(il CGAGCGCrATCTGAAAGACCAGCAACTCCT
351 AGGC (SEQ ID:33)
gp32 (opt)
This is a gp32 gene segment from the AvrII site (starting immediately at
the end of the RRE) to the end of gpl43 comprised of optimal codons
for expression.
35 1 CCTAG,GCATCTG,G~GCTG CTCTGGCAAG CTGATCTGCACCACAGCTGT
51 G,CCCTGGAAT GCCTCCTGGT CCAACAAGAG CCTG~;AGCM ATCTGSMCA
101 ACATGACCTG GATGGAGTGG GACAGAGAGA TCMCMCTA CACCTCCCTG
151 ATCCACTCCC TGATTGAGGA GTCCCAGMC CAGCAGGAGA AGMTGAGCA
201 GGAGC; 1~;1 a GAGCTGGACAAGTGGGCCTC CC; I ~:i l ~iMC I ~i I I ~MCA
45 251 TCACCMCTG GC; l ~ i I AC ATCMMTCT TCATCATGAT TGT('~r~
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~ 3û1 CTGGTGGGGCTGCGGATTGTC; I I 1~1 (j I GC;I ~ ;CATTGTGAACOGGGT
351 GAGACAGGGC TACTCCCCCT MTMGCCCG GGCGATATC (SEQ ID~4)
SRV-1 CTE (A)
This is a synthetic gene segment corresponding to a 3'-UTR from the
Simian Retrovirus-1 genome. This DNA is placed in the following
orientation at the 3'-terminus of HIV genes to increase rev-independent
10 expression.
S rf I Ec oRV
5~ ~t~ TATC TA GACCACCTCC CCTGCGAGCT MGCTGGACA
GCCAATGACGGGTA~GAGAGTGACAI ~ CACTAACCTAAGACAGGAGG
GCOt~u (iAGAG CTA~ AATCCAAAGAC GGGTAAAAGT GATAAAAATG
TATCACTCCAACCTA~GACAGGCGCAGCTTCCGAGGGA I I ~ w I
I I IATATATATTTAAAAGGGTGACCTGTCCGGAGCCGTGCTGCCGGGATG
ATGTCTTGGt'-~TATC t~CC~ C-3' (SEQID:35)
E~RV S rf l
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SRV-1 CTE (~)
This synthetic gene segment is identical to SRV-1 CTE (A) shown above
except that a single nucleotide mutation was used (indicated by boldface)
5 to elimin~te an Al-rTA sequence. This sequence has been associated
with increased mRNA turnover.
S rf l EmRV
5~ t~r~ ~r~ t~TATC TA GACCACCTCC CCTGCGAGCT AAGCTGGACA
GCC~ATGACGGGTA~GAGAGTGACATmTcACTA~CCTAA~AGG
G~i I ~iAGAG CTA~; 1~; l A AT~A~ G~;GTAAAAGT GATAAMATG
TATCACTCCAACCTA~GACA(~TCCGAGGG~ l~TCTGT
mATATATA TTL4AAA(3GG TGAC(i ~ C GGa!~rGC ll~CCGGATG
A I G I C; I I GG ~I~ CCC l~r~C -3' (SEQ ID:36)
~oRV Srfl
EXAMPLE l l
In Vitro ~pl20 Vaccine Expression:
In vitro expression was tested in transfected hllm~n
rhabdomyosarcoma (RD) cells for these constructs. Quantitation of
secreted tPA-gpl20 from transfected RD cells showed that VlJns-tPA-
gp 120 vector produced secreted gp 120.
In Vivo gpl20 Vaccination:
VlJns-tPA-gpl20~N PNV-induced Class II MHC-
restricted T Iymphocyte gpl20 specific antigen reactivities. Balb/c mice
which had been vaccinated two times with 200 )lg VlJns-tPA-gpl20MN
were sacrificed and their spleens extracted for in vitro determin~tions of
helper T lymphocyte reactivities to recombinant gpl20. T cell
proliferation assays were performed with PBMC (peripheral blood
mononuclear cells) using recombinant gpl20IIIg (Repligen, catalogue
#RP1016-20) at 5 ~lg/ml with 4 x 105 cells/ml. Basal levels of 3H-
thymidine uptake by these cells were obtained by culturing the cells in
media alone, while maximum proliferation was induced using ConA
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stimulation at 2 ~lg/ml. ConA-induced reactivities peak at ~3 days and
were harvested at that time point with media control samples while
antigen-treated samples were harvested at 5 days with an additional
media control. Vaccinated mice responses were compared with naive,
5 age-matched syngeneic mice. ConA positive controls gave very high
proliferation for both naive and immunized mice as expected. Very
strong helper T cell memory responses were obtained by gpl20
treatment in vaccinated mice while the naive mice did not respond (the
threshold for specific reactivity is an stimulation index (SI) of >3-4; SI
lO is calculated as the ratio of sample cpm/media cpm). SI's of 65 and 14
were obtained for the vaccinated mice which compares with anti-gpl20
ELISA titers of 5643 and 11,900, respectively, for these mice.
Interestingly, for these two mice the higher responder for antibody gave
significantly lower T cell reactivity than the mouse having the lower
15 antibody titer. This experiment demonstrates that the secreted gpl20
vector efficiently activates helper T cells in vivo as well as generates
strong antibody responses. In addition, each of these immune responses
was determined using antigen which was heterologous compared to that
encoded by the inoculation PNV (IIIB vs. MN):
EXAMPLE 12
gpl60 Vaccines
In addition to secreted gpl20 constructs, we have prepared
expression constructs for full-length, membrane-bound gpl60. The
25 rationales for a gpl60 construct, in addition to gpl20, are (1) more
epitopes are available both for both CTL stimulation as well as
neutralizing antibody production including gp41, against which a potent
HIV neutralizing monoclonal antibody (2F5, see above) is directed; (2) a
more native protein structure may be obtained relative to virus-
30 produced gpl60; and, (3) the success of membrane-bound influenza HA
constructs for immllnogenicity [Ulmer et al., Science 259:1745-1749,
1993; Montgomery, D., et al., DNA and Cell Biol.. 12:777 783, 1993].
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gpl60 retains substantial rev dependence even with a heterologous
leader peptide sequence so that further constructs were made to increase
expression in the absence of rev.
EXAMPLE 13
Assay For HIV Cytotoxic T-Lymphocytes:
The methods described in this section illustrate the assay as
used for vaccinated mice. An essentially similar assay can be used with
primates except that autologous B cell lines must be established for use
as target cells for each ~nim~l. This can be accomplished for humans
using the Epstein-Barr virus and for rhesus monkey using the herpes B
virus.
Peripheral blood mononuclear cells (PBMC) are derived
from either freshly drawn blood or spleen using Ficoll-Hypaque
centrifugation to separate erythrocytes from white blood cells. For
mice, lymph nodes may be used as well. Effecter CTLs may be
prepared from the PBMC either by in vitro culture in IL-2 (20 U/ml)
and concanavalin A (2,ug/ml) for 6-12 days or by using specific antigen
using an e~ual number of irradiated antigen presenting cells. Specific
antigen can consist of either synthetic peptides (9-15 amino acids
usually) that are known epitopes for CTL recognition for the MHC
haplotype of the ~nim~ls used, or vaccinia virus constructs engineered to
express appropriate antigen. Target cells may be either syngeneic or
MHC haplotype-matched cell lines which have been treated to present
appropriate antigen as described for in vitro stimulation of the CTLs.
For Balb/c mice the P18 peptide
(ArgIleHisIleGlyProGlyArgAlaPheTyrThrThrLysAsn [SEQ.ID:37~, for
HIV MN strain) can be used at 10 ,uM concentration to restimulate CTL
in vitro using irradiated syngeneic splenocytes and can be used to
sensitize target cells during the cytotoxicity assay at 1-10 }lM by
incubation at 37~C for about two hours prior to the assay. For these H-
2d MHC haplotype mice, the murine mastocytoma cell line, P815,
provides good target cells. Antigen-sensitized target cells are loaded
with Na5 l CrO4, which is released from the interior of the target cells
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upon killing by CTL, by incubation of targets for 1-2 hours at 37~C
(0.2 mCi for ~5 x 106 cells) followed by several washings of the target
cells. CTL populations are mixed with target cells at varying ratios of
effectors to targets such as 100:1, 50:1, 25:1, etc., pelleted together, and
S incubated 4-6 hours at 37~C before harvest of the supern~t~nt~ which
are then assayed for release of radioactivity using a gamma counter.
Cytotoxicity is calculated as a percentage of total releasable counts from
the target cells (obtained using 0.2% Triton X-100 treatment) from
which spontaneous release from target cells has been subtracted.
EXAMPLE 14
Assay For HIV Specific Antibodies:
ELISA were designed to detect antibodies generated against
HIV using either specific recombinant protein or synthetic peptides as
15 substrate antigens. 96 well microtiter plates were coated at 4~C
overnight with recombinant antigen at 2 ,ug/ml in PBS (phosphate
buffered saline) solution using 50 ,ul/well on a rocking platform.
Antigens consisted of either recombinant protein (gpl20, rev: Repligen
~orp.; gpl60, gp41: American Bio-Technologies, Inc.) or synthetic
20 peptide (V3 peptide corresponding to virus isolate sequences from IIIB,
etc.: American Bio-Technologies, lnc.; gp41 epitope for monoclonal
antibody 2F5). Plates were rinsed four times using wash buffer
(PBS/0.05% Tween 20) followed by addition of 200111/well of blocking
buffer (1% Carnation milk solution in PBS/0.05% Tween-20) for 1 hr
25 at room temperature with rocking. Pre-sera and immune sera were
diluted in blocking buffer at the desired range of dilutions and 100 ,ul
added per well. Plates were incubated for 1 hr at room temperature
with rocking and then washed four times with wash buffer. Secondary
antibodies conjugated with horse radish peroxidase, (anti-rhesus Ig,
30 Southern Biotechnology Associates; anti- mouse and anti-rabbit Igs,
Jackson Immuno Research) diluted 1:2000 in blocking buffer, were then
added to each sample at 100 Ill/well and incubated 1 hr at room
temperature with rocking. Plates were washed 4 times with wash buffer
and then developed by addition of 100 ~l/well of an o-phenylenediamine
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(o-PD, Calbiochem) solution at 1 mg/ml in 100 mM citrate buffer at pH
4.5. Plates were read for absorbance at 450 nm both kinetically (first
ten min~ltes of reaction) and at 10 and 30 minute endpoints (Thermo-
max microplate reader, Molecular Devices).
EXAMPLE 15
Assay For HIV Neutralizing Antibodies:
In vitro neutralization of HIV isolates assays using sera
derived from vaccinated ~nim~l~ was performed as follows. Test sera
10 and pre-immune sera were heat inactivated at 56~c for 60 min before
use. A titrated amount of HIV-l was added in 1:2 serial dilutions of test
sera and incubated 60 min at room temperature before addition to 105
MT-4 hllm~n lymphoid cells in 96 well microtiter plates. The virus/cell
mixtures were incubated for 7 days at 37~C and assayed for virus-
15 mediated killing of cells by staining cultures with tetrazolium dye.Neutralization of virus is observed by prevention of virus-mediated cell
death.
EXAMPLE 16
20 Isolation Of Genes From Clinical HIV Isolates:
HIV viral genes were cloned from infected PBMC's which
had been activated by ConA treatment. The preferred method for
obtaining the viral genes was by PCR amplification from infected
cellular genome using specific oligomers fl~nking the desired genes. A
25 second method for obtaining viral genes was by purification of viral
RNA from the supern~t~nts of infected cells and preparing cDNA from
this material with subsequent PCR. This method was very analogous to
that described above for cloning of the murine B7 gene except for the
PCR oligomers used and random hexamers used to make cDNA rather
30 than specific ~lilll-llg oligomers.
Genomic DNA was purified from infected cell pellets by
Iysis in STE solution (10 mM NaCl, 10 mM EDTA, 10 mM Tris-HCl,
pH 8.0) to which Proteinase K and SDS were added to 0.1 mg/ml and
0.5% final concentrations, respectively. This mixture was incubated
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overnight at 56~C and extracted with 0.5 volumes of
phenol:chloroform:isoarnyl alcohol (25:24:1). The aqueous phase was
then precipitated by addition of sodium acetate to 0.3 M final
concentration and two volumes of cold ethanol. After pelleting the
S DNA from solution the DNA was resuspended in O.lX TE solution (lX
TE = 10 mM Tris-HCl, pH 8.0, 1 mM EDTA). At this point SDS was
added to 0.1% with 2 U of RNAse A with incubation for 30 minutes at
37~C. This solution was extracted with phenol/chloroform/isoamyl
alcohol and then precipitated with ethanol as before. DNA was
suspended in 0.1 X TE and quantitated by measuring its ultraviolet
absorbance at 260 nm. Samples were stored at -20~C until used for
PCR.
PCR was performed using the Perkin-Elmer Cetus kit and
procedure using the following sense and antisense oligomers for gpl60:
5'-GA AAG AGC AGA AGA CAG TGG CAA TGA -3' (SEQ.ID:38)
and 5'-GGG Cl-r TGC TAA ATG GGT GGC AAG TGG CCC GGG C
ATG TGG-3' (SEQ.ID:39), respectively. These oligomers add an SrfI
site at the 3'-terminus of the resulting DNA fragment. PCR-derived
segments are cloned into either the VlJns or VlR vaccination vectors
and V3 regions as well as ligation junction sites confirmed by DNA
sequencmg.
EXAMPLE 17
T Cell Proliferation Assays:
PBMCs are obtained and tested for recall responses to
specific antigen as determined by proliferation within the PBMC
population. Proliferation is monitored using 3H-thymidine which is
added to the cell cultures for the last 18-24 hours of incubation ~efore
harvest. Cell harvesters retain isotope-cont~ining DNA on filters if
proliferation has occurred while quiescent cells do not incorporate the
isotope which is not retained on the filter in free form. For either
rodent or primate species 4 X 105 cells are plated in 96 well microtiter
plates in a total of 200 ~1 of complete media (RPMI/10% fetal calf
serum). Background proliferation responses are determined using
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PBMCs and media alone while nonspecific responses are generated by
using lectins such as phytohaemagglutin (PHA) or concanavalin A
(ConA) at 1- 5 ,ug/ml concentrations to serve as a positive control.
Specific antigen consists of either known peptide epitopes, purified
5 protein, or inactivated virus. Antigen concentrations range from 1- 10
~lM for peptides and 1-10 ,ug/ml for protein. Lectin-induced
proliferation peaks at 3-5 days of cell culture incubation while antigen-
specific responses peak at 5-7 days. Specific proliferation occurs when
radiation counts are obtained which are at least three-fold over the
10 media background and is often given as a ratio to background, or
Stimulation Index (SI). HIV gpl60 is known to contain several peptides
known to cause T cell proliferation of gpl60/gpl20 immunized or HIV-
infected individuals. The most commonly used of these are:
Tl (LysGlnIleIleAsnMetTrpGlnGluValGlyLysAlaMetTyrAla
15 [SEQ .ID :40] ) ; T2 (HisGluAspIleIleSerLeuTrpAspGlnSerLeuLys
[SEQ.ID:411; and, TH4 (AspArgValIleGluValValGlnGlyAlaTyrArgAla
IleArg [SEQ.ID:42]). These peptides have been demonstrated to
stim~ te proliferation of PBMC from antigen-sensitized mice,
nonhuman primates, and humans.
EXAMPLE 18
Vector V 1 R Preparation:
In an effort to continue to optimize our basic vaccination
vector, we prepared a derivative of VlJns which was designated as VlR
25 The purpose for this vector construction was to obtain a minimum-sized
vaccine vector, i.e., without unnecessary DNA sequences, which still
retained the overall optimized heterologous gene expression
characteristics and high plasmid yields that VlJ and VlJns afford. We
determined from the literature as well as by experiment that (1) regions
30 within the pUC backbone comprising the E. coli origin of replication
could be removed without affecting plasmid yield from bacteria; (2) the
3'-region of the kanr gene following the k~n~mycin open reading frame
could be removed if a bacterial terminator was inserted in its stead; and,
(3) ~300 bp from the 3'- half of the BGH terminator could be removed
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without affecting its regulatory function (following the original KpnI
restriction enzyme site within the BGH element).
- VlR was constructed by using PCR to synthesize three
segments of DNA from VlJns representing the CMVintA
S promoter/BGH terminator, origin of replication, and k~n~mycin
resistance elements, respectively. Restriction enzymes unique for each
segment were added to each segment end using the PCR oligomers: SspI
and XhoI for CMVintA/BGH; EcoRV and BamHI for the kan r gene;
and, BclI and SalI for the ori r. These enzyme sites were chosen
10 because they allow directional ligation of each of the PCR-derived DNA
segments with subsequent loss of each site: EcoRV and SspI leave blunt-
ended DNAs which are compatible for ligation while BamHI and BclI
leave complementary overhangs as do SalI and XhoI. After obtaining
these segments by PCR each segment was digested with the appropriate
15 restriction enzymes indicated above and then ligated together in a single
reaction mixture cont~ining all three DNA segments. The 5'-end of the
ori r was designed to include the T2 rho independent termin~tor
sequence that is normally found in this region so that it could provide
termination information for the k7~n~mycin resistance gene. The ligated
20 product was confirmed by restriction enzyme digestion (>8 enzymes) as
well as by DNA sequencing of the ligation junctions. DNA plasmid
yields and heterologous expression using viral genes within VlR appear
similar to V lJns. The net reduction in vector size achieved was 1346 bp
(VlJns = 4.86 kb; VlR = 3.52 kb), [SEQ.ID:43 of this specification;
25 also see Figure 11 and SEQ ID:lO0 of WO95/24485; PCT International
Application No. PCT/US95/02633].
PCR oligomer sequences used to synthesize VlR
(restriction enzyme sites are underlined and identified in brackets
30 following sequence):
- (1) 5'-GGT ACA AAT ATT GG CTA TTG GCC ATT GCA TAC G-3' [SspI], (SEQ.ID:44):,
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(2) 5'-CCA CAT CTC GAG GAA CCG GGT CAA l~C lY~C AGC
ACC-3' [XhoI], (SEQ.ID:45):
(for CMVintA/BGH segment)
5 (3) 5'-GGT ACA GAT ATC GGA AAG CCA CGT TGT GTC TCA
AAA TC-3'[EcoRV], (SEQ.ID:46):
(4) 5'-CCA CAT GGA TCC G TAA TGC TCT GCC AGT G~T ACA
ACC-3' [BamHI], (SEQ.ID:47):
(for k~n~mycin resistance gene segment)
(5) 5'-GGT ACA TGA TCA CGT AGA AAA GAT CAA AGG ATC
~YC TTG-3'[BclI], (SEQ.ID:48):,
(6) 5'-CCA CAT GTC GAC CC GTA AAA AGG CCG CGT TGC
TGG-3' [SalI], (SEQ.ID:49):
(for E. coli origin of replication)
Ligation junctions were sequenced for VlR using the
following oligomers:
5'-GAG CCA ATA TAA ATG TAC-3' (SEQ.ID:50):
20 [CMVintA/kanr junction]
5'-CAA TAG CAG GCA TGC-3' (SEQ.ID:Sl): [BGH/ori
junction]
5'-G CAA GCA GCA GAT TAC-3' (SEQ.ID:52): [ori/kanr
junction]
EXAMPLE l9
Heterologous Expression of HIV Late Gene Products
HIV structural genes such as env and gag require
expression of the HIV regulatory gene, rev, in order to efficiently
30 produce full-length proteins. We have found that rev-dependent
expression of gag yielded low levels of protein and that rev itself may
be toxic to cells. Although we achieved relatively high levels of rev-
dependent expression of gpl60 in vitro this vaccine elicited low levels of
antibodies to gpl60 following in vivo imml-ni7.~tion with rev/gpl60
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DNA. This may result from known cytotoxic effects of rev as well as
increased difficulty in obt~ining rev function in myotubules cont~ining
hundreds of nuclei (rev protein needs to be in the same nucleus as a rev-
dependent transcript in order for gag or env protein expression to
5 occur). However, it has been possible to obtain rev-independent
expression using selected modifications of the env gene.
1. rev-independent expression of env:
In general, our vaccines have utilized primarily HIV (IIIB)
10 env and gag genes for optimi7~tion of expression within our generalized
vaccination vector, VlJns, which is comprised of a CMV immediate-
early (IE) promoter, a BGH-derived polyadenylation and transcriptional
termination sequence, and a pUC backbone. Varying efficiencies,
depending upon how large a gene segment is used (e.g., gpl20 vs.
15 gpl60), of rev-independent expression may be achieved for env by
replacing its native secretory leader peptide with that from the tissue-
specific pl~minogen activator (tPA) gene and expressing the resulting
chimeric gene behind the CMVIE promoter with the CMV intron A.
tPA-gpl20 is an example of a secreted gpl20 vector constructed in this
20 fashion which functions well enough to elicit anti-gpl20 immune
responses in vaccinated mice and monkeys.
Because of reports that membrane-anchored proteins may
induce much more substantial (and perhaps more specific for HIV
neutralization) antibody responses compared to secreted proteins as well
25 as to gain additional epitopes, we prepared VlJns-tPA-gpl60 and VlJns-
rev/gpl60. The tPA-gpl60 vector produced detectable quantities of
gpl60 and gpl20, without the addition of rev, as shown by immunoblot
analysis of transfected cells, although levels of expression were much
lower than that obtained for rev/gpl60, a rev-dependent gpl60-
30 expressing plasmid. This is probably because inhibitory regions, whichconfer rev dependence upon the gpl60 transcript, occur at multiple sites
within gpl60 including at the COOH-terminus of gp41. A vector was
prepared for a COOH-terminally truncated form of tPA-gpl60 (tPA-
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gpl43) which was designed to increase the overall expression levels of
env by elimin~tion of these inhibitory sequences. The gpl43 vector also
elimin~tes intracellular gp41 regions cont~ining peptide motifs (such as
Leu-Leu) known to cause diversion of membrane proteins to the
5 lysosomes rather than the cell surface. Thus, gpl43 may be expected to
have increased levels of expression of the env protein (by decreasing
rev-dependence) and greater efficiency of transport of protein to the
cell surface compared to full-length gpl60 where these proteins may be
better able to elicit anti-gpl60 antibodies following DNA vaccination.
10 tPA-gpl43 was further modified by extensive silent mutagenesis of the
rev response element (RRE) sequence (350 bp) to eliminate additional
inhibitory sequences for expression. This construct, gpl43/mutRRE,
was prepared in two forms: either elimin~ling (form A) or re~ining
(form B) proteolytic cleavage sites for gpl20/41. Both forms were
15 prepared because of literature reports that vaccination of mice using
uncleavable gpl60 expressed in vaccinia elicited much higher levels of
antibodies to gpl60 than did cleavable forms.
A quantitative El,ISA for gpl60/gpl20 expression in cell
transfectants was developed to determine the relative expression
20 capabilities for these vectors. In vitro transfection of 293 cells followed
by quantification of cell-associated vs. secreted/released gpl20 yielded
the following results: (l) tPA-gpl60 expressed 5-lOX less gpl20 than
rev/gpl60 with similar proportions retained intracellularly vs. released
from the cell surface; (2) tPA-gpl43 gave 3-6X greater secretion of
25 gpl20 than rev/gpl60 with only low levels of cell-associated gpl43,
confirming that the cytoplasmic tail of gpl60 causes intracellular
retention of gpl60 which can be overcome by partial deletion of this
sequence; and, (3) tPA-gpl43/mutRR~ A and B gave ~lOX greater
expression levels of protein than did parental tPA-gpl43 while
30 elimin~tion of proteolytic processing was confirmed for form A.
Thus, our strategy to increase rev-independent expression
has yielded stepwise increases in overall expression levels as well as
redirecting membrane-anchored gpl43 to the cell surface away from
lysosomes. It is important to note that this is a generic constmct into
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which it should be possible to insert gpl20 sequences derived from
various primary viral isolates within a vector cassette cont~ining these
modifications which reside either at the NH2-terminus (tPA leader) or
COOH-terminus (gp41), where few antigenic differences exist between
5 different viral strains.
Figures 2-7 present data supporting the use of various
constructs, including but not limited to a gpl43-based construct, and
preferably a tPA-gpl43 based construct, as a DNA vaccine against HIV
infection. Figure 2 shows that tPA-143 (opt41) elicits an anti-gpl20
10 antibody response in the in the range of GMT=103. Figure 3 measures
and compares anti-gpl20 antibody titers for several DNA vaccines,
including gpl43-based constructs. Figure 4 shows the relative
expression of tPA-gpl43 and tPA-143/mutRRE in comparison to the
tPA-gpl60 construct. Figure 5 measures generation of anti-gpl20
15 antibodies for both the optA and optB forrns of tPA-gpl43 constructs.
Figure 6 shows the ability of several DNA vaccines, including tPA-
gpl43-optA and tPA-gpl43-optB, to promote generation of neutralizing
antibodies against HIV strains subsequent to murine DNA vaccination.
Figure 7 also shows HIV neutralization data for various DNA vaccine
20 constructions, including tPA-gpl43-optA, tPA-gpl43-optB, tPA-gpl43-
optA-glyB and tPA-gpl43-optB-glyB.
2. Expression of ~pl20 derived from a clinical isolate:
To apply these expression strategies to viruses that are
25 relevant for vaccine purposes and confirm the generality of our
approaches, we also prepared a tPA-gpl20 vector derived from a
primary HIV isolate (cont~ining the North American concensus V3
peptide loop; macrophage-tropic and nonsyncytia-inducing phenotypes).
This vector gave high expression/secretion of gpl20 with transfected
30 293 cells and elicited anti-gpl20 antibodies in mice thus demonstrating
that it was cloned in a functional form. Primary isolate gpl60 genes
will also be used for expression in the same way as for gpl60 derived
from laboratory strains.
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3. ~mmlme Responses to HIV-1 env Polynucleotide Vaccines
Effect of vaccination route on immune responses in mice:
While efforts to improve expression of gpl60 are ongoing, we have
utilized the tPA-gpl20 DNA construct to assess immlme responses and
S ways to augment them. Intramuscular (i.m.) and intradermal (i.d.)
vaccination routes were compared for this vector at 100, 10, and 1 ,ug
doses in mice. Vaccination by either route elicited antibody responses
(GMTs = 103-104) in all recipients following 2-3 vaccinations at all
three dosage levels. Each route elicited similar anti-gpl20 antibody
10 titers with clear dose-dependent responses. However, we observed
greater variability of responses for i.d. vaccination, particularly at the
lower doses following the initial inoculation. Moreover, helper T-cell
responses, as determined by antigen-specific in vitro proliferation and
cytokine secretion, were higher following i.m. vaccination than i.d. We
15 concluded that i.d. vaccination did not offer any advantages compared to
i.m. for this vaccine.
4. ~pl20 DNA vaccine-mediated helper T cell immunity in mice:
gp 120 DNA vaccination produced potent helper T-cell
20 responses in all lymphatic compartments tested (spleen, blood, inguinal,
mesenteric, and iliac nodes) with TH1-like cytokine secretion profiles
(i.e., g-interferon and IL-2 production with little or no IL-4). These
cytokines generally promote strong cellular immunity and have been
associated with maintenance of a disease-free state for HIV-seropositive
25 patients. Lymph nodes have been shown to be primary sites for HIV
replication, harboring large reservoirs of virus even when virus cannot
be readily detected in the blood. A vaccine which can elicit anti-HIV
immllne responses at a variety of Iymph sites, such as we have shown
with our DNA vaccine, may help prevent successful colonization of the
30 lymphatics following initial infection.
5. env DNA vaccine-mediated antibody responses:
African green (AGM) and Rhesus (RHM) monkeys which
received gpl20 DNA vaccines showed low levels of neutralizing
35 antibodies following 2-3 vaccinations, which could not be increased by
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WO 97/48370 PCT/US97/10517
additional vaccination. These results, as well as increasing awareness
within the HIV vaccine field that oligomeric gpl60 is probably a more
relevant target antigen for eliciting neutr~li7.in~ antibodies than gpl20
monomers, have led us to focus upon obt~ining effective expression of
5 gpl60-based vectors (see above). Mice and AGM were also vaccinated
with the primary isolate derived tPA-gpl20 vaccine. These ~nim~l~
exhibited anti-V3 peptide (using homologous sequence) reciprocal
endpoint antibody titers ranging 500-5000, demonstrating that this
vaccine design is functional for clinically relevant viral isolates.
The gpl60-based vaccines, rev-gpl60 and tPA-gpl60,
failed to consistently elicit antibody responses in mice and nonhuman
primates or yielded low antibody titers. Our initial results with the
tPA-gpl43 plasmid yielded geometric mean titers (GMT) > 103 in mice
and AGM following two vaccinations. These data indicate that we have
15 signficantly improved the immunogenicity of gpl60-like vaccines by
increasing expression levels and more efficient intracellular trafficking
of env to the cell surface. This construct, as well as the tPA-
gpl43/mutRRE A and B vectors, will continue to be characterized for
antibody responses, especially for virus neutralization.
6. env DNA vaccine-mediated CTL responses in monkeys:
We continued to characterize CTL responses of RHM that
had been vaccinated with gpl20 and gpl60/IRES/rev DNA. All four
monkeys that received this vaccine showed significant MHC Class I-
25 restricted CTL activities (20-35% specific killing at an effector/target =
20) following two vaccinations. Following a fourth vaccination these
activities increased to 50-60% killing under similar test conditions,
indicating that additional vaccination boosted responses significantly.
The CTL activities have persisted for at least seven months subsequent
30 to the final vaccination at about 50% of their peak levels indicating that
long-term memory had been established.
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EXAMPLE 20
SIV/HIV (SHIV) Chimeras:
A major obstacle for testing the protective efficacy of
candidate HrV-1 vaccines has been the lack of a suitable ~nim~l
5 challenge model for this virus. Although the simian immunodeficiency
virus (SIV), which is closely related to HIV, is infectious and causes
AIDS in rhesus monkeys, the only ~nim~l species which can be infected
with HIV- 1 viral isolates is the chimpanzee. However, the resulting
viremia from this infection is low-level, transient, and no pathogenic
10 effects (e.g., Iymphopenia, immunodeficiency-related opportunistic
infections, etc.) develop. Recently, hybrid viruses comprised of SIV
and HIV genomes have been developed which are also infectious to
rhesus monkeys and which can cause infection-related AIDS. An
example of this type of virus is SHIV-4 (IIIB) (Li et al., J. of Acquired
15 Immune Deficiency Syndrome, Vol. 5, 639-646 (1992)). This virus
contains the SIV (MAC239) genome except for the regulatory genes, tat
and rev, and the structural gene, env. Because the principle component
of candidate HIV vaccines is based upon env this virus allows testing
vaccines developed for human clinical purposes for protective efficacy
20 against infection in an ~nim~l model.
EXAMPLE 21
Plasmid DNA and Recombinant Protein Combination Vaccines.
Vaccines having both a plasmid DNA HIV env component
25 and a recombinant HIV env protein component were tested for their
abilities to induce antibody responses in rhesus monkeys. Figure 9 and
Figure 10 show the resulting anti-gpl20 ELISA antibody and SHIV-4
(IIIB) virus neutralizing antibody titers, respectively, following
vaccination of rhesus with Hrv env gene-cont~ining DNA vaccines and
30 recombinant protein (formulated in an appropriate adjuvant). These
monkeys developed high titers of env-specific antibodies and
neutr~li7ing antibodies. Control monkeys, vaccinated with "blank"
DNA that did not contain a gene and ovalbumin did not develop any
detectable env-specific responses while monkeys vaccinated only with
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the protein component of this vaccine showed low levels of antigen-
specific antibodies detected by ELISA and no neutr~li7in~ antibodies.
When these monkeys were challenged with SHIV-4 (IIIB) virus all
control and protein only monkeys became infected while those receiving
5 both env DNA and protein did not develop a detectable SHIV viremia.
These monkeys are currently being tested periodically for possible
delayed onset of infection.
, . ...... . .. ... . .
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SEQUENCE LISTING
(l) GENERAL INFORMATION:
~i) APPLICANTS: MERCK & CO., INC.
(ii) TITLE OF INVENTION: VACCINES COMPRISING SYNTHETIC GENES
(iii) NUMBER OF SEQUENCES: 53
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: J. MARK HAND - MERCK ~ CO., INC.
~B) STREET: 126 E. LINCOLN AVE., P.O. BOX 2000
(C) CITY: RAHWAY
(D) STATE: NEW JERSEY
(E) COUNTRY: US
(F) ZIP: 07065-0907
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: HAND, J. MARK
(B) REGISTRATION NUMBER: 36,545
(C) REFERENCE/DOCKET NUMBER: l9729Y PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-594-3905
(B) TELEFAX: 908-594-4720
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4864 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
SUeS~ (RUlE~
CA 022~8~68 1998-12-18
W 097/48370 PCT~US97110517
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA 60
CAG~ 'l' GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG 120
TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC 180
ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGATTGG 240
CTATTGGCCA TTGCATACGT TGTATCCATA TCATAATATG TACATTTATA TTGGCTCATG 300
TCCAACATTA CCGCCATGTT GACATTGATT ATTGACTAGT TATTAATAGT AATCAATTAC 360
GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA CGGTAAATGG 420
CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA CGTATGTTCC 480
CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT TACGGTAAAC 540
TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA TTGACGTCAA 600
TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC 660
TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG GTGATGCGGT TTTGGCAGTA 720
CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA 780
CGTCAATGGG AGlll~llll GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA 840
CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT ATATAAGCAG 900
AGCTCGTTTA GTGAACCGTC AGATCGCCTG GAGACGCCAT CCACGCTGTT TTGACCTCCA 960
TAGAAGACAC CGGGACCGAT CCAGCCTCCG CGGCCGGGAA CGGTGCATTG GAACGCGGAT 1020
TCCCCGTGCC AAGAGTGACG TAAGTACCGC CTATAGAGTC TATAGGCCCA CCCCCTTGGC 1080
TTCTTATGCA TGCTATACTG TTTTTGGCTT GGGGTCTATA CACCCCCGCT TCCTCATGTT 1140
ATAGGTGATG GTATAGCTTA GCCTATAGGT GTGGGTTATT GACCATTATT GACCACTCCC 1200
CTATTGGTGA CGATACTTTC CATTACTAAT CCATAACATG G~'l'C'l"l"l'GCC ACAACTCTCT 1260
TTATTGGCTA TATGCCAATA CACTGTCCTT CAGAGACTGA CACGGACTCT GTATTTTTAC 1320
AGGATGGGGT CTCATTTATT ATTTACAAAT TCACATATAC AACACCACCG TCCCCAGTGC 1380
CCGCAGTTTT TATTAAACAT AACGTGGGAT CTCCACGCGA ATCTCGGGTA CGTGTTCCGG 1440
SU~ Sn~
CA 022~8~68 l998-l2-l8
W O 97148370 PCT~US97/10517
ACATGGGCTC TTCTCCGGTA GCGGCGGAGC TTCTACATCC GAGCCCTGCT CCCATGCCTC 1500
CAGCGACTCA TGGTCGCTCG GCAGCTCCTT GCTCCTAACA GTGGAGGCCA GACTTAGGCA 1560
CAGCACGATG CCCACCACCA CCAGTGTGCC GCACAAGGCC GTGGCGGTAG GGTATGTGTC 1620
TGAAAATGAG CTCGGGGAGC GGGCTTGCAC CGCTGACGCA TTTGGAAGAC TTAAGGCAGC 1680
GGCAGAAGAA GATGCAGGCA GCTGAGTTGT 'l~l~ll~lGA TAAGAGTCAG AGGTAACTCC 1740
CGTTGCGGTG CTGTTAACGG TGGAGGGCAG TGTAGTCTGA GCAGTACTCG TTGCTGCCGC 1800
GCGCGCCACC AGACATAATA GCTGACAGAC TAACAGACTG TTCCTTTCCA TGG~l~llll 1860
CTGCAGTCAC CGTCCTTAGA TCTGCTGTGC CTTCTAGTTG CCAGCCATCT ~ll~lllGCC 1920
CCTCCCCCGT GCCTTCCTTG ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA 1980
ATGAGGAAAT TGCATCGCAT TGTCTGAGTA GGTGTCATTC TATTCTGGGG GGTGGGGTGG 2040
GGCAGCACAG CAAGGGGGAG GATTGGGAAG ACAATAGCAG GCATGCTGGG GATGCGGTGG 2100
GCTCTATGGG TACCCAGGTG CTGAAGAATT GACCCGGTTC CTCCTGGGCC AGAAAGAAGC 2160
AGGCACATCC C~ l~l~l GACACACCCT GTCCACGCCC CTGGTTCTTA GTTCCAGCCC 2220
CACTCATAGG ACACTCATAG CTCAGGAGGG CTCCGCCTTC AATCCCACCC GCTAAAGTAC 2280
TTGGAGCGGT CTCTCCCTCC CTCATCAGCC CACCAAACCA AACCTAGCCT CCAAGAGTGG 2340
GAAGAAATTA AAGCAAGATA GGCTATTAAG TGCAGAGGGA GAGAAAATGC CTCCAACATG 2400
TGAGGAAGTA ATGAGAGAAA TCATAGAATT TCTTCCGCTT CCTCGCTCAC TGACTCGCTG 2460
CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA 2520
TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC 2580
AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG 2640
CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC 2700
CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC 2760
GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCAATG CTCACGCTGT 2820
AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC 2880
GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA 2940
CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA 3000
- 74-
S~ (~ULE2i)
CA 022~8~68 l998-l2-l8
W O 97/48370 PCT~US97/10517
GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGGACAGTA 3060
TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA 3120
TCCGGCAAAC AAACCACCGC TGGTAGCGGT G~lllll~llG TTTGCAAGCA GCAGATTACG 3180
CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG 3240
TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC 3300
TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT 3360
TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT 3420
CGTTCATCCA TAGTTGCCTG ACTCCGGGGG GGGGGGGCGC TGAGGTCTGC CTCGTGAAGA 3480
AGGTGTTGCT GACTCATACC AGGCCTGAAT CGCCCCATCA TCCAGCCAGA AAGTGAGGGA 3540
GCCACGGTTG ATGAGAGCTT 'l'~l"l'~'l'AGGT GGACCAGTTG GTGATTTTGA ACTTTTGCTT 3600
TGCCACGGAA CGGTCTGCGT TGTCGGGAAG ATGCGTGATC TGATCCTTCA ACTCAGCAAA 3660
AGTTCGATTT ATTCAACAAA GCCGCCGTCC CGTCAAGTCA GCGTAATGCT CTGCCAGTGT 3720
TACAACCAAT TAACCAATTC TGATTAGAAA AACTCATCGA GCATCAAATG AAACTGCAAT 3780
TTATTCATAT CAGGATTATC AATACCATAT TTTTGAAAAA GCCGl'll~"l'~ TAATGAAGGA 3840
GAAAACTCAC CGAGGCAGTT CCATAGGATG GCAAGATCCT GGTATCGGTC TGCGATTCCG 3900
ACTCGTCCAA CATCAATACA ACCTATTAAT TTCCCCTCGT CAAAAATAAG GTTATCAAGT 3960
GAGAAATCAC CATGAGTGAC GACTGAATCC GGTGAGAATG GCAAAAGCTT ATGCATTTCT 4020
TTCCAGACTT GTTCAACAGG CCAGCCATTA CGCTCGTCAT CAAAATCACT CGCATCAACC 4080
AAACCGTTAT TCATTCGTGA TTGCGCCTGA GCGAGACGAA ATACGCGATC GCTGTTAAAA 4~40
GGACAATTAC AAACAGGAAT CGAATGCAAC CGGCGCAGGA ACACTGCCAG CGCATCAACA 4200
ATATTTTCAC CTGAATCAGG ATA'll~ l' AATACCTGGA ATG~'l'~llll CCCGGGGATC 4260
GCAGTGGTGA GTAACCATGC ATCATCAGGA GTACGGATAA AATGCTTGAT GGTCGGAAGA 4320
GGCATAAATT CCGTCAGCCA GTTTAGTCTG ACCATCTCAT CTGTAACATC ATTGGCAACG 4380
CTACCTTTGC CATGTTTCAG AAACAACTCT GGCGCATCGG GCTTCCCATA CAATCGATAG 4440
ATTGTCGCAC CTGATTGCCC GACATTATCG CGAGCCCATT TATACCCATA TAAATCAGCA 4500
TCCATGTTGG AATTTAATCG CGGCCTCGAG CAAGACGTTT CCCGTTGAAT ATGGCTCATA 4560
- 75 -
SUBS~ S~ (NUIE26)
... ..
CA 022~8~68 1998-12-18
W 097/48370 PCTrUS97/10S17
ACACCCCTTG TATTACTGTT TATGTAAGCA GACAGTTTTA TTGTTCATGA TGATATATTT 4620
TTA~ l'G CAATGTAACA TCAGAGATTT TGAGACACAA CGTGGCTTTC CCCCCCCCCC 4680
CATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT 4740
TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC 4800
TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT 4860
CGTC 4864
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GATCACCATG GATGCAATGA AGAGAGGGCT CTG~ ~ CTGCTGCTGT GTGGAGCAGT 60
CTTCGTTTCG CCCAGCGA 78
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GATCTCGCTG GGCGAAACGA AGACTGCTCC ACACAGCAGC AGCACACAGC AGAGCCCTCT 60
CTTCATTGCA TCCATGGT 78
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
SU~~ 26)
CA 022~8~68 1998-12-18
W O 97/4B370 PCTrUS97/10517
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CCCCGGATCC TGATCACAGA AAAATTGTGG GTCACAGTC 39
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CCCCAGGAAT CCACCTGTTA GCGCTTTTCT CTCTGCACCA CTCTTCTC 48
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GGTACATGAT CACAGAAAAA TTGTGGGTCA CAGTC 35
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
SUBSIllul~Sh~l (RULE26)
CA 022~8~68 1998-12-18
W O 97/48370 PCT~US97/10517
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ~oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCACATTGAT CAGATATCTT A~ lC TCTCTGCACC ACTCTTC 47
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Thr Asn Trp Leu Trp Tyr Ile Lys
l 5
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
- 78 -
SUBSllJU~tSh~l (RULE26)
CA 022~8~68 l998-l2-l8
W 097/48370 PCTrUS97/10517
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Lys Ala Gln Asn His Val Val Gln Asn Glu His Gln
1 5 10
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
CTGAAAGACC AGCAACTCCT AGGGAATTTG GGGTTGCTCT GG 42
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ''oligonucleotideU
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CGCAGGGGAG GTGGTCTAGA TATCTTATTA TTTTATATAC CACAGCCAAT TTGTTATG 58
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GGTACACCTA GGCATCTGGG GCTGCTCTGG 30
SUBSIIIUI~ SIIEr (RUlE26)
CA 022~8~68 l998-l2-l8
W 097/48370 PCTrUS97/10517
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CCACATGATA TCGCCCGGGC TTATTATTTG ATGTACCACA GCCAGTTGGT GATG 54
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GGTACACTGC AGTCACCGTC CTATGGCAGG AAGAAGCGGA GAC 43
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CCACATCAGG TACCCCATAA TAGACTGTGA CC 32
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
- 80 -
Ul~ S~l (RULE 2G)
CA 022~8~68 l998-l2-l8
W O 97/48370 PCTAUS97/10517
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GGTACATGAT CAACCATGAG AGTGAAGGAG AAATATCAGC 40
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CCACATTGAT CAGATATCCC CATCTTATAG CAAAATCCTT TCC 43
(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l9:
CCACATTGAT CAGATATCCC CATCTTATAG CAAAATCCTT TCC 43
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- 81 -
SUBSIIlul~: ShEE~ ~RUL~~)
CA 022~8~68 l998-l2-l8
W O 97/48370 rCT~US97/10517
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
C~ '~'l'~'l'G AGTTTAAACT GCACTGATTT GAAGAATGAT ACTAATAC 48
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GGTACATGAT CACAGAAAAA TTGTGGGTCA CAGTC 35
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CCACATTGAT CAGCCCGGGC TTAGGGTGAA TAGCCCTGCC TCA~~ CAC 53
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
- 82 -
SUI~ SH~ ULE2ii)
CA 022~8~68 1998-12-18
W O 97/48370 PCTAUS97/10517
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CTGAAAGACC AGCAACTCCT AGGGATTTGG GGTTGCTGTG G 4l
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CCACATTGAT CAGCCCGGGC TTAGGGTGAA TAGCCCTGCC TCA~ l CAC 53
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GGTACACAAT TGGAGGAGCG AGTTATATAA ATATAAG 37
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CCTGTGTGTG AGTTTAAACT GCACTGATTT GAAGAATGAT ACTAATAC 48
- 83 -
SUlBS~ Sh~tl (RUIE~;)
CA 022~8~68 1998-12-18
W O 97/48370 PCTrUS97/10517
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
~B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Asn Arg Leu Ile Lys Ala
1 5
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CCACATGATA TCGCCCGGGC TTATTAGGCC TTGATCAGCC GGTTCACAAT GGACAGCACA 60
GC 62
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CTGACCCCCC ~ GGG GGCTGGCAGT TGTAACACCT CAGTCATTAC ACAG 54
(2) INFORMATION FOR SEQ ID NO:30:
- 84 -
u~Sh~ll (RUIE26~
CA 022~8~68 l998-l2-l8
WO 97/48370 PCT/US97/10517
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 305 base pairs
- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
TGATCACAGA GAAGCTGTGG GTGACAGTGT ATTATGGCGT GCCAGTCTGG AAGGAGGCCA 60
CCACCACCCT ~ ~lGCC TCTGATGCCA AGGCCTATGA CACAGAGGTG CACAATGTGT 120
GGGCCACCCA TGCCTGTGTG CCCACAGACC CCAACCCCCA GGAGGTGGTG CTGGTGAATG 180
TGACTGAGAA CTTCAACATG TGGAAGAACA ACATGGTGGA GCAGATGCAT GAGGACATCA 240
TCAGCCTGTG GGACCAGAGC CTGAAGCCCT GTGTGAAGCT GACCCCCCTG TGTGTGAGTT 300
TAAAC 305
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1065 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
AGTTTAAACT GCACAGACCT GAGGAACACC ACCAACACCA ACAACTCCAC AGCCAACAAC 60
AACTCCAACT CCGAGGGCAC CATCAAGGGG GGGGAGATGA AGAACTGCTC CTTCAACATC 120
ACCACCTCCA TCAGGGACAA GATGCAGAAG GAGTATGCCC TGCTGTACAA GCTGGACATT 180
GTGTCCATTG ACAATGACTC CACCTCCTAC AGGCTGATCT CCTGCAACAC CTCTGTCATC 240
ACCCAGGCCT GCCCCAAAAT CTCCTTTGAG CCCATCCCCA TCCACTACTG TGCCCCTGCT 300
GGCTTTGCCA TCCTGAAGTG CAATGACAAG AAGTTCTCTG GCAAGGGCTC CTGCAAGAAT 360
GTGTCCACAG TGCAGTGCAC ACATGGCATC AGGCCTGTGG TGTCCACCCA GCTGCTGCTG 420
AATGGCTCCC TGGCTGAGGA GGAGGTGGTC ATCAGGTCTG AGAACTTCAC AGACAATGCC 480
AAGACCATCA TCGTGCACCT GAATGAGTCT GTGCAGATCA ACTGCACCAG GCCCAACTAC 540
- 85 -
SUBSI~ Sh~l (RULE21i)
.. . . .
CA 022~8~68 1998-12-18
W O 97/48370 PCTAUS97/10517
AACAAGAGGA AGAGGATCCA CATTGGCCCT GGCAGGGCCT TCTACACCAC CAAGAACATC 600
ATTGGCACCA TCAGGCAGGC CCACTGCAAC ATCTCCAGGG CCAAGTGGAA TGACACCCTG 660
AGGCAGATTG TGTCCAAGCT GAAGGAGCAG TTCAAGAACA AGACCATTGT GTTCAACCAG 720
TCCTCTGGGG GGGACCCTGA GATTGTGATG CACTCCTTCA ACTGTGGGGG GGA~ll~ 780
TACTGCAACA CCTCCCCCCT GTTCAACTCC ACCTGGAATG GCAACAACAC CTGGAACAAC 840
ACCACAGGCT CCAACAACAA CATCACCCTC CAGTGCAAGA TCAAGCAGAT CATCAACATG 900
TGGCAGGAGG TGGGCAAGGC CATGTATGCC CCCCCCATTG AGGGCCAGAT CAGGTGCTCC 960
TCCAACATCA CAGGCCTGCT GCTGACCAGG GATGGGGGGA AGGACACAGA CACCAACGAC I020
ACCGAAATCT TCAGGCCTGG GGGGGGGGAC ATGAGGGACA ATTGG l065
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
GACAATTGGA GGAGCGAGTT ATATAAATAT AAGGTGGTGA AGATTGAGCC CCTGGGGGTG 60
GCCCCAACAA AAGCTCAGAA CCACGTGGTG CAGAACGAGC ACCAGGCCGT GGGCATTGGG 120
GCCCTGTTTC TGGGCTTTCT GGGGGCTGCT GGCTCCACAA TGGGCGCCGC TAGCATGACC l80
CTCACCGTGC AAGCTCGCCA GCTGCTGAGT GGCATCGTCC AGCAGCAGAA CAACCTGCTC 240
CGCGCCATCG AAGCCCAGCA GCACCTCCTC CAGCTGACTG TGTGGGGGAT CAAACAGCTT 300
CAGGCCCGGG TGCTGGCCGT CGAGCGCTAT CTGAAAGACC AGCAACTCCT AGGC 354
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- 86-
SU~ Sh~ UIE26)
CA 022~8~68 l998-l2-l8
W O 97/48370 PCTrUS97/10S17
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
GACAATTGGA GGAGCGAGTT ATATAAATAT AAGGTGGTGA AGATTGAGCC CCTGGGGGTG 60
GCCCCAACAA AAGCTAAGAG AAGAGTGGTG CAGAGAGAGA AGAGAGCCGT GGGCATTGGG 120
GCCCTGTTTC TGGGCTTTCT GGGGGCTGCT GGCTCCACAA TGGGCGCCGC TAGCATGACC 180
CTCACCGTGC AAGCTCGCCA GCTGCTGAGT GGCATCGTCC AGCAGCAGAA CAACCTGCTC 240
CGCGCCATCG AAGCCCAGCA GCACCTCCTC CAGCTGACTG TGTGGGGGAT CAAACAGCTT 300
CAGGCCCGGG TGCTGGCCGT CGAGCGCTAT CTGAAAGACC AGCAACTCCT AGGC 354
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 387 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
CCTAGGCATC TGGGGCTGCT CTGGCAAGCT GATCTGCACC ACAGCTGTGC CCTGGAATGC 60
CTCCTGGTCC AACAAGAGCC TGGAGCAAAT CTGGAACAAC ATGACCTGGA TGGAGTGGGA 120
CAGAGAGATC AACAACTACA CCTCCCTGAT CCACTCCCTG ATTGAGGAGT CCCAGAACCA 180
GCAGGAGAAG AATGAGCAGG AGCTGCTGGA GCTGGACAAG TGGGCCTCCC TGTGGAACTG 240
GTTCAACATC ACCAACTGGC TGTGGTACAT CAAAATCTTC ATCATGATTG TGGGGGGCCT 300
GGTGGGGCTG CGGATTGTCT TTGCTGTGCT GTCCATTGTG AACCGGGTGA GACAGGGCTA 360
CTCCCCCTAA TAAGCCCGGG CGATATC 387
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- 87 -
SU~ SM~E~(RULE26)
CA 022~8~68 l998-l2-l8
W 097/48370 PCT~US97/10517
(ii) MOLECULE TYPE: DNA (genomic~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
GCCCGGGCGA TATCTAGACC ACCTCCCCTG CGAGCTAAGC TGGACAGCCA ATGACGGGTA 60
AGAGAGTGAC ATTTTTCACT AACCTAAGAC AGGAGGGCCG TCAGAGCTAC TGCCTAATCC 120
AAAGACGGGT AAAAGTGATA AAAATGTATC ACTCCAACCT AAGACAGGCG CAGCTTCCGA 180
GGGATTTGTC GT~'l'~'l"l"l"l'A TATATATTTA AAAGGGTGAC CTGTCCGGAG CCGTGCTGCC 240
CGGATGATGT CTTGGGATAT CGCCCGGGC 269
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
GCCCGGGCGA TATCTAGACC ACCTCCCCTG CGAGCTAAGC TGGACAGCCA ATGACGGGTA 60
AGAGAGTGAC ATTTTTCACT AACCTAAGAC AGGAGGGCCG TCAGAGCTAC TGCCTAATCC 120
AAAGACGGGT AAAAGTGATA AAAATGTATC ACTCCAACCT AAGACAGGCG CAGCTTCCGA l80
GGGATTTGTC GT~l~llllA TATATATTAA AAAGGGTGAC CTGTCCGGAG CCGTGCTGCC 240
CGGATGATGT CTTGGGATAT CGCCCGGGC 269
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: sing-le
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: pep~ide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn
1 5 10 15
- 88 -
Su~ sht~ (XUlE2~i)
CA 022~8~68 l998-l2-l8
W 097/48370 PCT~US97/10~17
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
GAAAGAGCAG AAGACAGTGG CAATGA 26
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
GGGCTTTGCT AAATGGGTGG CAAGTGGCCC GGGCATGTGG 40
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
- 89 -
Sl~'~ SHI~(~U1~26)
CA 022~8~68 1998-12-18
W097/48370 PCT~US97110517
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
His Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys
l 5 l0
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Asp Arg Val Ile Glu Val Val Gln Gly Ala Tyr Arg Ala Ile Arg
l 5 l0 15
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3547 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
GATATTGGCT ATTGGCCATT GCATACGTTG TATCCATATC ATAATATGTA CATTTATATT 60
GGCTCATGTC CAACATTACC GCCATGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA 120
TCAATTACGG GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG l80
GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG 240
TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA 300
CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT 360
GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC 420
TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT 480
- 90 -
SU~ S~ uaE26)
CA 022~8~68 1998-12-18
W O 97/48370 PCT~US97/10517
TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC 540
CCCATTGACG TCAATGGGAG 'lll'~ll"l"lGG CACCAAAATC AACGGGACTT TCCAAAATGT 600
CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT 660
ATAAGCAGAG ~lC~lllAGT GAACCGTCAG ATCGCCTGGA GACGCCATCC ACG~l~ll'll 720
GACCTCCATA GAAGACACCG GGACCGATCC AGCCTCCGCG GCCGGGAACG GTGCATTGGA 780
ACGCGGATTC CCCGTGCCAA GAGTGACGTA AGTACCGCCT ATAGAGTCTA TAGGCCCACC 840
CCCTTGGCTT CTTATGCATG CTATACTGTT TTTGGCTTGG GGTCTATACA CCCCCGCTTC 900
CTCATGTTAT AGGTGATGGT ATAGCTTAGC CTATAGGTGT GGGTTATTGA CCATTATTGA 960
CCACTCCCCT ATTGGTGACG ATACTTTCCA TTACTAATCC ATAACATGGC TCTTTGCCAC 1020
AA~l~l'~l"l"l' ATTGGCTATA TGCCAATACA CTGTCCTTCA GAGACTGACA CGGACTCTGT 1080
ATTTTTACAG GATGGGGTCT CATTTATTAT TTACAAATTC ACATATACAA CACCACCGTC 1140
CCCAGTGCCC GCAGTTTTTA TTAAACATAA CGTGGGATCT CCACGCGAAT CTCGGGTACG 1200
TGTTCCGGAC ATGGGCTCTT CTCCGGTAGC GGCGGAGCTT CTACATCCGA GCCCTGCTCC 1260
CATGCCTCCA GCGACTCATG GTCGCTCGGC AGCTCCTTGC TCCTAACAGT GGAGGCCAGA 1320
CTTAGGCACA GCACGATGCC CACCACCACC AGTGTGCCGC ACAAGGCCGT GGCGGTAGGG 1380
TATGTGTCTG AAAATGAGCT CGGGGAGCGG GCTTGCACCG CTGACGCATT TGGAAGACTT 1440
AAGGCAGCGG CAGAAGAAGA TGCAGGCAGC TGA~ll~ll~ 'l~l"l'~lGATA AGAGTCAGAG 1500
GTAACTCCCG TTGCGGTGCT GTTAACGGTG GAGGGCAGTG TAGTCTGAGC AGTACTCGTT 1560
GCTGCCGCGC GCGCCACCAG ACATAATAGC TGACAGACTA ACAGACTGTT CCTTTCCATG 1620
GGT~ll"l"l~"l' GCAGTCACCG TCCTTAGATC TGCTGTGCCT TCTAGTTGCC AGCCATCTGT 1680
TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT GCCACTCCCA CTGTCCTTTC 1740
CTAATAAAAT GAGGAAATTG CATCGCATTG TCTGAGTAGG TGTCATTCTA TTCTGGGGGG 1800
TGGGGTGGGG CAGCACAGCA AGGGGGAGGA TTGGGAAGAC AATAGCAGGC ATGCTGGGGA 1860
TGCGGTGGGC TCTATGGGTA CGGCCGCAGC GGCCGTACCC AGGTGCTGAA GAATTGACCC 1920
- 91 -
SUBSIIlul~ SHEr (~ULE 2C)
CA 022~8~68 l998-l2-l8
W O 97/48370 PCTruS97/l0517
GGTTCCTCGA CCCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT 1980
GACGAGCATC ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA 2040
AGATACCAGG CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG 2100
CTTACCGGAT ACCTGTCCGC ~ 'l'CCCT TCGGGAAGCG TGGCGCTTTC TCAATGCTCA 2160
CGCTGTAGGT ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA 2220
CCCCCCGTTC AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG 2280
GTAAGACACG ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG 2340
TATGTAGGCG GTGCTACAGA ~llCll~AAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG 2400
ACAGTATTTG GTATCTGCGC TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC 2460
TCTTGATCCG GCAAACAAAC CACCGCTGGT AGCGGTGGTT 'l"1~l"l"l'~'ll"l'~ CAAGCAGCAG 2520
ATTACGCGCA GAAAAAAAGG ATCTCAAGAA GATCCTTTGA 'l~"l"l"l"l~'l'AC GTGATCCCGT 2580
AATGCTCTGC CAGTGTTACA ACCAATTAAC CAATTCTGAT TAGAAAAACT CATCGAGCAT 2640
CAAATGAAAC TGCAATTTAT TCATATCAGG ATTATCAATA CCATATTTTT GAAAAAGCCG 2700
TTTCTGTAAT GAAGGAGAAA ACTCACCGAG GCAGTTCCAT AGGATGGCAA GATCCTGGTA 2760
TCGGTCTGCG ATTCCGACTC GTCCAACATC AATACAACCT ATTAATTTCC CCTCGTCAAA 2820
AATAAGGTTA TCAAGTGAGA AATCACCATG AGTGACGACT GAATCCGGTG AGAATGGCAA 2880
AAGCTTATGC A'lll~l'll'CC AGA~'l"l~llC AACAGGCCAG CCATTACGCT CGTCATCAAA 2940
ATCACTCGCA TCAACCAAAC CGTTATTCAT TCGTGATTGC GCCTGAGCGA GACGAAATAC 3000
GCGATCGCTG TTAAAAGGAC AATTACAAAC AGGAATCGAA TGCAACCGGC GCAGGAACAC 3060
TGCCAGCGCA TCAACAATAT TTTCACCTGA ATCAGGATAT TCTTCTAATA CCTGGAATGC 3120
TGTTTTCCCG GGGATCGCAG TGGTGAGTAA CCATGCATCA TCAGGAGTAC GGATAAAATG 3180
CTTGATGGTC GGAAGAGGCA TAAATTCCGT CAGCCAGTTT AGTCTGACCA TCTCATCTGT 3240
AACATCATTG GCAACGCTAC CTTTGCCATG TTTCAGAAAC AACTCTGGCG CATCGGGCTT 3300
CCCATACAAT CGATAGATTG TCGCACCTGA TTGCCCGACA TTATCGCGAG CCCATTTATA 3360
CCCATATAAA TCAGCATCCA TGTTGGAATT TAATCGCGGC CTCGAGCAAG ACGTTTCCCG 3420
- 92 -
SUBSIllU~ Sh~ (RUIE26)
CA 022~8~68 1998-12-18
W O 97/48370 PCTrUS97/10517
TTGAATATGG CTCATAACAC CCCTTGTATT ACTGTTTATG TAAGCAGACA GTTTTATTGT 3480
TCATGATGAT ATATTTTTAT CTTGTGCAAT GTAACATCAG AGATTTTGAG ACACAACGTG 3540
GCTTTCC 3547
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "olignucleotide~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
GGTACAAATA TTGGCTATTG GCCATTGCAT ACG 33
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
CCACATCTCG AGGAACCGGG TCAATCCTCC AGCACC 36
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide~
- 93 -
Sl~~ S~ (BUIE26)
CA 022~8~68 1998-12-18
W 097/48370 PCTrUS97/10517
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
GGTACAGATA TCGGAAAGCC ACGll~l~lC TCAAAATC 38
t2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: ~desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
CCACATGGAT CCGTAATGCT CTGCCAGTGT TACAACC 37
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
GGTACATGAT CACGTAGAAA AGATCAAAGG ATCTTCTTG 39
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
CCACATGTCG ACCCGTAAAA AGGCCGCGTT GCTGG35
- 94 -
SUBSIIlul~Sh~ ULE2~)
CA 022~8~68 l998-l2-l8
W 097/48370 PCT~US97/10~17
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
GAGCCAATAT AAATGTAC 18
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
CAATAGCAGG CATGC 15
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
GCAAGCAGCA GATTAC 16
_ 95 _
SUBSIllul~ SI~ (RUL~26)
CA 02258568 1998-12-18
W 097/48370 PCTrUS97/10517
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
Glu Leu Asp Lys Trp Ala
l 5
- 96 -
SUBSIllul~sHE~ (~ULE26)
