U.S. patent application number 10/371069 was filed with the patent office on 2003-11-20 for expression vectors for stimulating an immune response and methods of using the same.
Invention is credited to Fikes, John D., Hermanson, Gary G., Sette, Alessandro.
Application Number | 20030216342 10/371069 |
Document ID | / |
Family ID | 26761083 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030216342 |
Kind Code |
A1 |
Fikes, John D. ; et
al. |
November 20, 2003 |
Expression vectors for stimulating an immune response and methods
of using the same
Abstract
The present invention relates to nucleic acid vaccines encoding
multiple CTL and HTL epitopes and MHC targeting sequences.
Inventors: |
Fikes, John D.; (San Diego,
CA) ; Hermanson, Gary G.; (Encinitas, CA) ;
Sette, Alessandro; (La Jolla, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
26761083 |
Appl. No.: |
10/371069 |
Filed: |
February 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10371069 |
Feb 21, 2003 |
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09311784 |
May 13, 1999 |
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6534482 |
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09311784 |
May 13, 1999 |
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09078904 |
May 13, 1998 |
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60085751 |
May 15, 1998 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61P 31/16 20180101;
A61K 39/385 20130101; C07K 2319/00 20130101; C07K 2319/02 20130101;
Y02A 50/30 20180101; C12N 2770/24222 20130101; C12N 2740/16322
20130101; A61K 38/00 20130101; A61P 31/20 20180101; A61P 37/02
20180101; C07K 14/005 20130101; C12N 2740/16222 20130101; A01K
2217/05 20130101; A61P 31/14 20180101; C07K 14/445 20130101; C07K
14/70539 20130101; C12N 2730/10122 20130101; A61K 2039/605
20130101; Y02A 50/412 20180101; C12N 2740/16122 20130101 |
Class at
Publication: |
514/44 |
International
Class: |
A61K 048/00 |
Goverment Interests
[0002] This invention was made with government support under NIH
Grant No. AI-42699-01, NIH Grant No. A138584-03, and NIH Contract
No. N01-AI-45241. The Government has certain rights in this
invention.
Claims
What is claimed:
1. An expression system which comprises a promoter operably linked
to a nucleotide sequence which encodes a peptide comprising a first
amino acid sequence which is a MHC targeting sequence fused to a
second amino acid sequence encoding a class I MHC restricted CTL
peptide epitope and HIV HTL peptide epitope, wherein the HIV HTL
peptide epitope is selected from the group consisting of HIV HTL
peptide epitopes set forth as SEQ ID NOs: 295, 298-303, 305-311,
313, 316, and 318-323, and an analog of each of the foregoing HTL
peptide epitopes.
2. The expression system of claim 1, wherein the CTL peptide
epitope is an HIV CTL peptide epitope.
3. The expression system of claim 2, wherein the CTL peptide
epitope is an A2 peptide epitope.
4. The expression system of claim 2, wherein the CTL peptide
epitope is an A3 peptide epitope.
5. The expression system of claim 2, wherein the CTL peptide
epitope is a B7 peptide epitope.
6. The expression system of claim 2, wherein the CTL peptide
epitope is selected from the group consisting of CTL peptide
epitopes set forth as SEQ ID NOs: 101, 105, 324-412, and 463, and
an analog of each of the foregoing CTL peptide epitopes.
7. The method of claim 1, wherein the MHC targeting sequence is the
signal sequence from Ig kappa.
8. The method of claim 1, wherein the MHC targeting sequence is the
signal sequence from insulin.
9. The method of claim 1, wherein the MHC targeting sequence is the
signal sequence from tissue plasminogen.
10. The method of claim 1, wherein the MHC targeting sequence is
the signal sequence from LAMP-1.
11. A method for inducing an immune response in a subject which
comprises administering to a mammalian subject the expression
system comprising a promoter operably linked to a nucleotide
sequence which encodes a peptide comprising a first amino acid
sequence which is a MHC targeting sequence fused to a second amino
acid sequence encoding a class I MHC restricted CTL peptide epitope
and HIV HTL peptide epitope, wherein the HIV HTL peptide epitope is
selected from the group consisting of HIV HTL peptide epitopes set
forth as SEQ ID NOs: 295, 298-303, 305-311, 313, 316, and 318-323,
and an analog of each of the foregoing HTL peptide epitopes.
12. The method of claim 11, wherein the CTL peptide epitope is an
HIV CTL peptide epitope.
13. The method of claim 12, wherein the CTL peptide epitope is an
A2 peptide epitope.
14. The method of claim 12, wherein the CTL peptide epitope is an
A3 peptide epitope.
15. The method of claim 12, wherein the CTL peptide epitope is a B7
peptide epitope.
16. The method of claim 12, wherein the CTL peptide epitope is
selected from the group consisting of CTL peptide epitopes set
forth as SEQ ID NOs: 101, 105, 324-412, and 463, and an analog of
each of the foregoing CTL peptide epitopes.
17. The method of claim 11, wherein the MHC targeting sequence is
the signal sequence from Ig kappa.
18. The method of claim 11, wherein the MHC targeting sequence is
the signal sequence from insulin.
19. The method of claim 11, wherein the MHC targeting sequence is
the signal sequence from tissue plasminogen.
20. The method of claim 11, wherein the MHC targeting sequence is
the signal sequence from LAMP-1.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 09/311,784, filed May 13, 1999, which is a
continuation of U.S. patent application Ser. No. 09/078,904, filed
May 13, 1998. This patent application also claims the benefit of
U.S. Patent Application No. 60/085,751, filed May 15, 1998. Each of
these documents is herein incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to nucleic acid vaccines
encoding multiple CTL and HTL epitopes and MHC targeting
sequences.
BACKGROUND OF THE INVENTION
[0004] Vaccines are of fundamental importance in modem medicine and
have been highly effective in combating certain human diseases.
However, despite the successful implementation of vaccination
programs that have greatly limited or virtually eliminated several
debilitating human diseases, there are a number of diseases that
affect millions worldwide for which effective vaccines have not
been developed.
[0005] Major advances in the field of immunology have led to a
greater understanding of the mechanisms involved in the immune
response and have provided insights into developing new vaccine
strategies (Kuby, Immunology, 443-457 (3rd ed., 1997), which is
incorporated herein by reference). These new vaccine strategies
have taken advantage of knowledge gained regarding the mechanisms
by which foreign material, termed antigen, is recognized by the
immune system and eliminated from the organism. An effective
vaccine is one that elicits an immune response to an antigen of
interest.
[0006] Specialized cells of the immune system are responsible for
the protective activity required to combat diseases. An immune
response involves two major groups of cells, lymphocytes, or white
blood cells, and antigen-presenting cells. The purpose of these
immune response cells is to recognize foreign material, such as an
infectious organism or a cancer cell, and remove that foreign
material from the organism.
[0007] Two major types of lymphocytes mediate different aspects of
the immune response. B cells display on their cell surface
specialized proteins, called antibodies, that bind specifically to
foreign material, called antigens. Effector B cells produce soluble
forms of the antibody, which circulate throughout the body and
function to eliminate antigen from the organism. This branch of the
immune system is known as the humoral branch. Memory B cells
function to recognize the antigen in future encounters by
continuing to express the membrane-bound form of the antibody.
[0008] A second major type of lymphocyte is the T cell. T cells
also have on their cell surface specialized proteins that recognize
antigen but, in contrast to B cells, require that the antigen be
bound to a specialized membrane protein complex, the major
histocompatibility complex (MHC), on the surface of an
antigen-presenting cell. Two major classes of T cells, termed
helper T lymphocytes ("HTL") and cytotoxic T lymphocytes ("CTL"),
are often distinguished based on the presence of either CD4 or CD8
protein, respectively, on the cell surface. This branch of the
immune system is known as the cell-mediated branch.
[0009] The second major class of immune response cells are cells
that function in antigen presentation by processing antigen for
binding to MHC molecules expressed in the antigen presenting cells.
The processed antigen bound to MHC molecules is transferred to the
surface of the cell, where the antigen-MHC complex is available to
bind to T cells.
[0010] MHC molecules can be divided into MHC class I and class II
molecules and are recognized by the two classes of T cells. Nearly
all cells express MHC class I molecules, which function to present
antigen to cytotoxic T lymphocytes. Cytotoxic T lymphocytes
typically recognize antigen bound to MHC class I. A subset of cells
called antigen-presenting cells express MHC class II molecules.
Helper T lymphocytes typically recognize antigen bound to MHC class
II molecules. Antigen-presenting cells include dendritic cells,
macrophages, B cells, fibroblasts, glial cells, pancreatic beta
cells, thymic epithelial cells, thyroid epithelial cells and
vascular endothelial cells. These antigen-presenting cells
generally express both MHC class I and class II molecules. Also, B
cells function as both antibody-producing and antigen-presenting
cells.
[0011] Once a helper T lymphocyte recognizes an antigen-MHC class
II complex on the surface of an antigen-presenting cell, the helper
T lymphocyte becomes activated and produces growth factors that
activate a variety of cells involved in the immune response,
including B cells and cytotoxic T lymphocytes. For example, under
the influence of growth factors expressed by activated helper T
lymphocytes, a cytotoxic T lymphocyte that recognizes an
antigen-MHC class I complex becomes activated. CTLs monitor and
eliminate cells that display antigen specifically recognized by the
CTL, such as infected cells or tumor cells. Thus, activation of
helper T lymphocytes stimulates the activation of both the humoral
and cell-mediated branches of the immune system.
[0012] An important aspect of the immune response, in particular as
it relates to vaccine efficacy, is the manner in which antigen is
processed so that it can be recognized by the specialized cells of
the immune system. Distinct antigen processing and presentation
pathways are utilized. The one is a cytosolic pathway, which
results in the antigen being bound to MHC class I molecules. An
alternative pathway is an endoplasmic reticulum pathway, which
bypasses the cytosol. Another is an endocytic pathway, which
results in the antigen being bound to MHC class II molecules. Thus,
the cell surface presentation of a particular antigen by a MHC
class II or class I molecule to a helper T lymphocyte or a
cytotoxic T lymphocyte, respectively, is dependent on the
processing pathway for that antigen.
[0013] The cytosolic pathway processes endogenous antigens that are
expressed inside the cell. The antigen is degraded by a specialized
protease complex in the cytosol of the cell, and the resulting
antigen peptides are transported into the endoplasmic reticulum, an
organelle that processes cell surface molecules. In the endoplasmic
reticulum, the antigen peptides bind to MHC class I molecules,
which are then transported to the cell surface for presentation to
cytotoxic T lymphocytes of the immune system.
[0014] Antigens that exist outside the cell are processed by the
endocytic pathway. Such antigens are taken into the cell by
endocytosis, which brings the antigens into specialized vesicles
called endosomes and subsequently to specialized vesicles called
lysosomes, where the antigen is degraded by proteases into antigen
peptides that bind to MHC class II molecules. The antigen
peptide-MHC class II molecule complex is then transported to the
cell surface for presentation to helper T lymphocytes of the immune
system.
[0015] A variety of factors must be considered in the development
of an effective vaccine. For example, the extent of activation of
either the humoral or cell-mediated branch of the immune system can
determine the effectiveness of a vaccine against a particular
disease. Furthermore, the development of immunologic memory by
inducing memory-cell formation can be important for an effective
vaccine against a particular disease (Kuby, supra). For example,
protection from infectious diseases caused by pathogens with short
incubation periods, such as influenza virus, requires high levels
of neutralizing antibody generated by the humoral branch because
disease symptoms are already underway before memory cells are
activated. Alternatively, protection from infectious diseases
caused by pathogens with long incubation periods, such as polio
virus, does not require neutralizing antibodies at the time of
infection but instead requires memory B cells that can generate
neutralizing antibodies to combat the pathogen before it is able to
infect target tissues. Therefore, the effectiveness of a vaccine at
preventing or ameliorating the symptoms of a particular disease
depends on the type of immune response generated by the
vaccine.
[0016] Many traditional vaccines have relied on intact pathogens
such as attenuated or inactivated viruses or bacteria to elicit an
immune response. However, these traditional vaccines have
advantages and disadvantages, including reversion of an attenuated
pathogen to a virulent form. The problem of reversion of an
attenuated vaccine has been addressed by the use of molecules of
the pathogen rather than the whole pathogen. For example,
immunization approaches have begun to incorporate recombinant
vector vaccines and synthetic peptide vaccines (Kuby, supra).
Recently, DNA vaccines have also been used (Donnelly et al, Annu.
Rev. Immunol. 15:617-648 (1997), which is incorporated herein by
reference). The use of molecules of a pathogen provides safe
vaccines that circumvent the potential for reversion to a virulent
form of the vaccine.
[0017] The targeting of antigens to MHC class II molecules to
activate helper T lymphocytes has been described using lysosomal
targeting sequences, which direct antigens to lysosomes, where the
antigen is digested by lysosomal proteases into antigen peptides
that bind to MHC class II molecules (U.S. Pat. No. 5,633,234;
Thomson et al., J. Virol. 72:2246-2252 (1998)). It would be
advantageous to develop vaccines that deliver multiple antigens
while exploiting the safety provided by administering individual
epitopes of a pathogen rather than a whole organism. In particular,
it would be advantageous to develop vaccines that effectively
target antigens to MHC class II molecules for activation of helper
T lymphocytes.
[0018] Several studies also point to the crucial role of cytotoxic
T cells in both production and eradication of infectious diseases
and cancer by the immune system (Byrne et al., J. Immunol. 51:682
(1984); McMichael et al., N. Engl. J. Med. 309:13 (1983)).
Recombinant protein vaccines do not reliably induce CTL responses,
and the use of otherwise immunogenic vaccines consisting of
attenuated pathogens in humans is hampered, in the case of several
important diseases, by overriding safety concerns. In the case of
diseases such as HIV, HBV, HCV, and malaria, it appears desirable
not only to induce a vigorous CTL response, but also to focus the
response against highly conserved epitopes in order to prevent
escape by mutation and overcome variable vaccine efficacy against
different isolates of the target pathogen.
[0019] Induction of a broad response directed simultaneously
against multiple epitopes also appears to be crucial for
development of efficacious vaccines. HIV infection is perhaps the
best example where an infected host may benefit from a
multispecific response. Rapid progression of HIV infection has been
reported in cases where a narrowly focused CTL response is induced
whereas nonprogressors tend to show a broader specificity of CTLs
(Goulder et al., Nat. Med. 3:212 (1997); Borrow et al., Nat. Med.
3:205 (1997)). The highly variable nature of HIV CTL epitopes
resulting from a highly mutating genome and selection by CTL
responses directed against only a single or few epitopes also
supports the need for broad epitope CTL responses (McMichael et
al., Annu. Rev. Immunol. 15:271 (1997)).
[0020] One potential approach to induce multispecific responses
against conserved epitopes is immunization with a minigene plasmid
encoding the epitopes in a string-of-beads fashion. Induction of
CTL, HTL, and B cell responses in mice by minigene plasmids have
been described by several laboratories using constructs encoding as
many as 11 epitopes (An et al., J. Virol. 71:2292 (1997); Thomson
et al, J. Immunol. 157:822 (1996); Whitton et al., J. Virol. 67:348
(1993); Hanke et al, Vaccine 16:426 (1998); Vitiello et al, Eur. J.
Immunol. 27:671-678 (1997)). Minigenes have been delivered in vivo
by infection with recombinant adenovirus or vaccinia, or by
injection of purified DNA via the intramuscular or intradermal
route (Thomson et al., J. Immunol. 160:1717 (1998); Toes et al.,
Proc. Natl. Acad. Sci. USA 94:14660 (1997)).
[0021] Successful development of minigene DNA vaccines for human
use will require addressing certain fundamental questions dealing
with epitope MHC affinity, optimization of constructs for maximum
in vivo immunogenicity, and development of assays for testing in
vivo potency of multi-epitope minigene constructs. Regarding MHC
binding affinity of epitopes, it is not currently known whether
both high and low affinity epitopes can be included within a single
minigene construct, and what ranges of peptide affinity are
permissible for CTL induction in vivo. This is especially important
because dominant epitopes can vary in their affinity and because it
might be important to be able to deliver mixtures of dominant and
subdominant epitopes that are characterized by high and low MHC
binding affinities.
[0022] With respect to minigene construct optimization for maximum
immunogenicity in vivo, conflicting data exists regarding whether
the exact position of the epitopes in a given construct or the
presence of flanking regions, helper T cell epitopes, and signal
sequences might be crucial for CTL induction (Del Val et al., Cell
66:1145 (1991); Bergmann et al., J. Virol. 68:5306 (1994); Thomson
et al., Proc. Natl. Acad. Sci. USA 92:5845 (1995); Shirai et al.,
J. Infect. Dis. 173:24 (1996); Rahemtulla et al., Nature 353:180
(1991); Jennings et al., Cell. Immunol. 133:234 (1991); Anderson et
al., J. Exp. Med. 174:489 (1991); Uger et al., J. Immunol. 158:685
(1997)). Finally, regarding development of assays that allow
testing of human vaccine candidates, it should be noted that, to
date, all in vivo immunogenicity data of multi-epitope minigene
plasmids have been performed with murine class I MHC-restricted
epitopes. It would be advantageous to be able to test the in vivo
immunogenicity of minigenes containing human CTL epitopes in a
convenient animal model system.
[0023] Thus, there exists a need to develop methods to effectively
deliver a variety of HTL (helper T lymphocyte) and CTL (cytotoxic T
lymphocyte) antigens to stimulate an immune response. The present
invention satisfies this need and provides related advantages as
well.
SUMMARY OF THE INVENTION
[0024] The invention therefore provides expression vectors encoding
two or more HTL epitopes fused to a MHC class II targeting
sequence, as well as expression vectors encoding a CTL epitope and
a universal HTL epitope fused to an MHC class I targeting sequence.
The HTL epitope can be a universal HTL epitope (also referred to as
a universal MHC class II epitope). The invention also provides
expression vectors encoding two or more HTL epitopes fused to a MHC
class II targeting sequence and encoding one or more CTL epitopes.
The invention additionally provides methods of stimulating an
immune response by administering an expression vector of the
invention in vivo, as well as methods of assaying the human
immunogenicity of a human T cell peptide epitope in vivo in a
non-human mammal.
[0025] In one aspect, the present invention provides an expression
vector comprising a promoter operably linked to a first nucleotide
sequence encoding a major histocompatibility (MHC) targeting
sequence fused to a second nucleotide sequence encoding two or more
heterologous peptide epitopes, wherein the heterologous peptide
epitopes comprise two HTL peptide epitopes or a CTL peptide epitope
and a universal HTL peptide epitope.
[0026] In another aspect, the present invention provides a method
of inducing an immune response in vivo comprising administering to
a mammalian subject an expression vector comprising a promoter
operably linked to a first nucleotide sequence encoding a major
histocompatibility (MHC) targeting sequence fused to a second
nucleotide sequence encoding two or more heterologous peptide
epitopes, wherein the heterologous peptide epitopes comprise two
HTL peptide epitopes or a CTL peptide epitope and a universal HTL
peptide epitope.
[0027] In another aspect, the present invention provides a method
of inducing an immune response in vivo comprising administering to
a mammalian subject an expression vector comprising a promoter
operably linked to a first nucleotide sequence encoding a major
histocompatibility (MHC) targeting sequence fused to a second
nucleotide sequence encoding a heterologous human HTL peptide
epitope.
[0028] In another aspect, the present invention provides a method
of assaying the human immunogenicity of a human T cell peptide
epitope in vivo in a non-human mammal, comprising the step of
administering to the non-human mammal an expression vector
comprising a promoter operably linked to a first nucleotide
sequence encoding a heterologous human CTL or HTL peptide
epitope.
[0029] In one embodiment, the heterologous peptide epitopes
comprise two or more heterologous HTL peptide epitopes. In another
embodiment, the heterologous peptide epitopes comprise a CTL
peptide epitope and a universal HTL peptide epitope. In another
embodiment, the heterologous peptide epitopes further comprise one
to two or more heterologous CTL peptide epitopes. In another
embodiment, the expression vector comprises both HTL and CTL
peptide epitopes.
[0030] In one embodiment, one of the HTL peptide epitopes is a
universal HTL epitope. In another embodiment, the universal HTL
epitope is a pan DR epitope. In another embodiment, the pan DR
epitope has the sequence AlaLysPheValAlaAlaTrpThrLeuLysAlaAlaAla
(SEQ ID NO:52).
[0031] In one embodiment, the peptide epitopes are hepatitis B
virus epitopes, hepatitis C virus epitopes, human immunodeficiency
virus epitopes, human papilloma virus epitopes, MAGE epitopes, PSA
epitopes, PSM epitopes, PAP epitopes, p53 epitopes, CEA epitopes,
Her2/neu epitopes, or Plasmodium epitopes. In another embodiment,
the peptide epitopes each have a sequence selected from the group
consisting of the peptides depicted in Tables 1-8. In another
embodiment, at least one of the peptide epitopes is an analog of a
peptide depicted in Tables 1-8.
[0032] In one embodiment, the MHC targeting sequence comprises a
region of a polypeptide selected from the group consisting of the
Ii protein, LAMP-1, HLS-DM, HLA-DO, H2-DO, influenza matrix
protein, hepatitis B surface antigen, hepatitis B virus core
antigen, Ty particle, Ig-.alpha. protein, Ig-.beta. protein, and Ig
kappa chain signal sequence.
[0033] In one embodiment, the expression vector further comprises a
second promoter sequence operably linked to a third nucleotide
sequence encoding one or more heterologous HtL or CTL peptide
epitopes. In another embodiment, the CTL peptide epitope comprises
a structural motif for an HLA supertype, whereby the peptide CTL
epitope binds to two or more members of the supertype with an
affinity of greater that 500 nM. In another embodiment, the CTL
peptide epitopes have structural motifs that provide binding
affinity for more than one HLA allele supertype.
[0034] In one embodiment, the non-human mammal is a transgenic
mouse that expresses a human HLA allele. In another embodiment, the
human HLA allele is selected from the group consisting of A11 and
A2.1. In another embodiment, the non-human mammal is a macaque that
expresses a human HLA allele.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows the nucleotide and amino acid sequences (SEQ ID
NOS: 1 and 2, respectively) of the IiPADRE construct encoding a
fusion of the murine Ii gene with a pan DR epitope sequence
substituted for the CLIP sequence of the Ii protein.
[0036] FIG. 2 shows the nucleotide and amino acid sequences (SEQ ID
NOS:3 and 4, respectively) of the 180T construct encoding a fusion
of the cytoplasmic domain, the transmembrane domain and part of the
luminal domain of the Ii protein fused to multiple MHC class II
epitopes.
[0037] FIG. 3 shows the nucleotide and amino acid sequences (SEQ ID
NOS:5 and 6, respectively) of the IiThfull construct encoding a
fusion of the cytoplasmic domain, transmembrane domain and a
portion of the luminal domain of the Ii protein fused to multiple T
helper epitopes and amino acid residues 101 to 215 of the Ii
protein, which encodes the trimerization region of the Ii
protein.
[0038] FIG. 4 shows the nucleotide and amino acid sequences (SEQ ID
NOS:7 and 8, respectively) of the KappaLAMP-Th construct encoding a
fusion of the murine immunoglobulin kappa signal sequence fused to
multiple T helper epitopes and the transmembrane and cytoplasmic
domains of LAMP-1.
[0039] FIG. 5 shows the nucleotide and amino acid sequences (SEQ ID
NOS:9 and 10, respectively) of the H2M-Th construct encoding a
fusion of the signal sequence of H2-M fused to multiple MHC class
II epitopes and the transmembrane and cytoplasmic domains of
H2-M.
[0040] FIG. 6 shows the nucleotide and amino acid sequences (SEQ ID
NOS: 11 and 12, respectively) of the H.sub.2O-Th construct encoding
a fusion of the signal sequence of H2-DO fused to multiple MHC
class II epitopes and the transmembrane and cytoplasmic domains of
H2-DO.
[0041] FIG. 7 shows the nucleotide and amino acid sequences (SEQ ID
NOS:13 and 14, respectively) of the PADRE-Influenza matrix
construct encoding a fusion of a pan DR epitope sequence fused to
the amino-terminus of influenza matrix protein.
[0042] FIG. 8 shows the nucleotide and amino acid sequences (SEQ ID
NOS:15 and 16, respectively) of the PADRE-HBV-s construct encoding
a fusion of a pan DR epitope sequence fused to the amino-terminus
of hepatitis B virus surface antigen.
[0043] FIG. 9 shows the nucleotide and amino acid sequences (SEQ ID
NOS: 17 and 18, respectively) of the Ig-alphaTh construct encoding
a fusion of the signal sequence of the Ig-.alpha. protein fused to
multiple MHC class II epitopes and the transmembrane and
cytoplasmic domains of the Ig-.alpha. protein.
[0044] FIG. 10 shows the nucleotide and amino acid sequences (SEQ
ID NOS:19 and 20, respectively) of the Ig-betaTh construct encoding
a fusion of the signal sequence of the Ig-.beta. protein fused to
multiple MHC class II epitopes and the transmembrane and
cytoplasmic domains of the Ig-.beta. protein.
[0045] FIG. 11 shows the nucleotide and amino acid sequences (SEQ
ID NOS:21 and 22, respectively) of the SigTh construct encoding a
fusion of the signal sequence of the kappa immunoglobulin fused to
multiple MHC class II epitopes.
[0046] FIG. 12 shows the nucleotide and amino acid sequences (SEQ
ID NOS:23 and 24, respectively) of human HLA-DR, the invariant
chain (Ii) protein.
[0047] FIG. 13 shows the nucleotide and amino acid sequences (SEQ
ID NOS:25 and 26, respectively) of human lysosomal membrane
glycoprotein-1 (LAMP-1).
[0048] FIG. 14 shows the nucleotide and amino acid sequences (SEQ
ID NOS:27 and 28, respectively) of human HLA-DMB. [0049] FIG. 15
shows the nucleotide and amino acid sequences (SEQ ID NOS:29 and
30, respectively) of human HLA-DO beta.
[0049] FIG. 16 shows the nucleotide and amino acid sequences (SEQ
ID NOS:31 and 32, respectively) of the human MB-1 Ig-.alpha..
[0050] FIG. 17 shows the nucleotide and amino acid sequences (SEQ
ID NOS:33 and 34, respectively) of human Ig-.alpha. protein.
[0051] FIG. 18 shows a schematic diagram depicting the method of
generating some of the constructs encoding a MHC class II targeting
sequence fused to multiple MHC class II epitopes.
[0052] FIG. 19 shows the nucleotide sequence of the vector pEP2
(SEQ ID NO:35).
[0053] FIG. 20 shows the nucleotide and amino acid sequences of the
vector pMIN.0 (SEQ ID NOS:36 and 37, respectively).
[0054] FIG. 21 shows the nucleotide and amino acid sequences of the
vector pMIN.1 (SEQ ID NOS:38 and 39, respectively).
[0055] FIG. 22. Representative CTL responses in
HLA-A2.1/K.sup.b-H-2.sup.b- xs mice immunized with pMin.1 DNA.
Splenocytes from primed animals were cultured in triplicate flasks
and stimulated twice in vitro with each peptide epitope.
Cytotoxicity of each culture was assayed in a .sup.51Cr release
assay against Jurkat-A2.1/K.sup.b target cells in the presence
(filled symbols, solid lines) or absence (open symbols, dotted
lines) of peptide. Each symbol represents the response of a single
culture.
[0056] FIG. 23. Presentation of viral epitopes to specific CTLs by
Jurkat-A2.1/K.sup.b tumor cells transfected with DNA minigene. Two
constructs were used for transfection, pMin.1 and pMin.2-GFP.
pMin.2-GFP-transfected targets cells were sorted by FACS and the
population used in this experiment contained 60% fluorescent cells.
CTL stimulation was measured by quantitating the amount of
IFN-.gamma. release (A, B) or by lysis of .sup.51Cr-labeled target
cells (C, D, hatched bars). CTLs were stimulated with transfected
cells (A, C) or with parental Jurkat-A2.1/K.sup.b cells in the
presence of 1 .mu.g/ml peptide (B, D). Levels of IFN-.gamma.
release and cytotoxicity for the different CTL lines in the absence
of epitope ranged from 72-126 pg/ml and 2-6% respectively.
[0057] FIG. 24. Summary of modified minigene constructs used to
address variables critical for in vivo immunogenicity. The
following modifications were incorporated into the prototype pMin.1
construct; A, deletion of PADRE HTL epitope; B, incorporation of
the native HBV Pol 551 epitope that contains an alanine in position
9; C, deletion of the Ig kappa signal sequence; and D, switching
position of the HBV Env 335 and HBV Pol 455 epitopes.
[0058] FIG. 25. Examination of variables that may influence pMin.1
immunogenicity. In vivo CTL-inducing activity of pMin.1 is compared
to modified constructs. For ease of comparison, the CTL response
induced by each of the modified DNA minigene constructs (shaded
bars) is compared separately in each of the four panels to the
response induced by the prototype pMin.1 construct (solid bars).
The geometric mean response of CTL-positive cultures from two to
five independent experiments are shown. Numbers shown with each bar
indicate the number of positive cultures/total number tested for
that particular epitope. The ratio of positive cultures/total
tested for the pMin.1 group is shown in panel A and is the same for
the remaining Figure panels (see Example V, Materials and Methods,
in vitro CTL cultures, for the definition of a positive CTL
culture). Theradigm responses were obtained by immunizing animals
with the lipopeptide and stimulating and testing splenocyte
cultures with the HBV Core 18-27 peptide.
[0059] Definitions
[0060] An "HTL" peptide epitopeor an "MHC II epitope" is an MHC
class II restricted epitope, i.e., one that is bound by an MHC
class II molecule.
[0061] A "CTL" peptide epitope or an "MHC I epitope" is an MHC
class I restricted epitope, i.e., one that is bound by an MHC class
I molecule.
[0062] An "MHC targeting sequence" refers to a peptide sequence
that targets a polypeptide, e.g., comprising a peptide epitope, to
a cytosolic pathway (e.g., an MHC class I antigen processing
pathway), en endoplasmic reticulum pathways, or an endocytic
pathway (e.g., an MHC class II antigen processing pathway).
[0063] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more sequences that are not found in the same
relationship to each other in nature, e.g., a fusion polypeptide
comprising subsequence from different polypeptides, peptide
epitopes from fusion polypeptide that are not naturally in an
adjacent position, or repeats of a single peptide epitope.
[0064] As used herein, the term "universal MHC class II epitope" or
a "universal HTL epitope" refers to a MHC class II peptide epitope
that binds to gene products of multiple MHC class II alleles. For
example, the DR, DP and DQ alleles are human MHC II alleles.
Generally, a unique set of peptides binds to a particular gene
product of a MHC class II allele. In contrast, a universal MHC
class II epitope is able to bind to gene products of multiple MHC
class II alleles. A universal MHC class II epitope binds to 2 or
more MHC class II alleles, generally 3 or more MHC class II
alleles, and particularly 5 or more MHC class II alleles. Thus, the
a universal MHC class II epitope in an expression vector is
advantageous in that it o increase the number of allelic MHC class
II molecules that can bind to the peptide and, consequently, the
number of Helper T lymphocytes that are activated.
[0065] Universal MHC class II epitopes are well known in the art
and include, for example, epitopes such as the "pan DR epitopes,"
also referred to as "PADRE" (Alexander et al., Immunity 1:751-761
(1994); WO 95/07707, U.S. S No. 60/036,713, U.S. S No. 60/037,432,
PCT/US98/01373, Ser. No. 09/009,953, and U.S. S No. 60/087,192 each
of which is incorporated herein by reference). A "pan DR binding
peptide" or a "PADRE" peptide of the invention is a peptide capable
of binding at least about 7 different DR molecules, preferably 7 of
the 12 most common DR molecules, most preferably 9 of the 12 most
common DR molecules (DR1, 2w2b, 2w2a, 3, 4w4, 4w14, 5, 7, 52a, 52b,
52c, and 53), or alternatively, 50% of a panel of DR molecules
representative of greater than or equal to 75% of the human
population, preferably greater than or equal to 80% of the human
population. Pan DR epitopes can bind to a number of DR alleles and
are strongly immunogenic for T cells. For example, pan DR epitopes
were found to be more effective at inducing an immune response than
natural MHC class II epitopes (Alexander, supra). An example of a
PADRE epitope is the peptide
AlaLysPheValAlaAlaTrpThrLeuLysAlaAlaAla (SEQ ID NO:52).
[0066] With regard to a particular amino acid sequence, an
"epitope" is a set of amino acid residues which is involved in
recognition by a particular immunoglobulin, or in the context of T
cells, those residues necessary for recognition by T cell receptor
proteins and/or Major Histocompatibility Complex (MHC) receptors.
In an immune system setting, in vivo or in vitro, an epitope is the
collective features of a molecule, such as primary, secondary and
tertiary peptide structure, and charge, that together form a site
recognized by an immunoglobulin, T cell receptor or HLA molecule.
Throughout this disclosure epitope and peptide are often used
interchangeably. It is to be appreciated, however, that isolated or
purified protein or peptide molecules larger than and comprising an
epitope of the invention are still within the bounds of the
invention.
[0067] As used herein, "high affinity" with respect to HLA class I
molecules is defined as binding with an IC50 (or K.sub.D) of less
than 50 nM. "Intermediate affinity" is binding with an IC50 (or
K.sub.D) of between about 50 and about 500 nM. "High affinity" with
respect to binding to HLA class II molecules is defined as binding
with an K.sub.D of less than 100 nM. "Intermediate affinity" is
binding with a K.sub.D of between about 100 and about 1000 nM.
Assays for determining binding are described in detail, e.g., in
PCT publications WO 94/20127 and WO 94/03205. Alternatively,
binding is expressed relative to a reference peptide. As a
particular assay becomes more, or less, sensitive, the IC50s of the
peptides tested may change somewhat. However, the binding relative
to the reference peptide will not significantly change. For
example, in an assay run under conditions such that the IC50 of the
reference peptide increases 10-fold, the IC50 values of the test
peptides will also shift approximately 10-fold. Therefore, to avoid
ambiguities, the assessment of whether a peptide is a good,
intermediate, weak, or negative binder is generally based on its
IC50, relative to the IC50 of a standard peptide.
[0068] Throughout this disclosure, results are expressed in terms
of "IC50s." IC50 is the concentration of peptide in a binding assay
at which 50% inhibition of binding of a reference peptide is
observed. Given the conditions in which the assays are run (i.e.,
limiting HLA proteins and labeled peptide concentrations), these
values approximate K.sub.D values. It should be noted that IC50
values can change, often dramatically, if the assay conditions are
varied, and depending on the particular reagents used (e.g., HLA
preparation, etc.). For example, excessive concentrations of HLA
molecules will increase the apparent measured IC50 of a given
ligand.
[0069] The terms "identical" or percent "identity," in the context
of two or more peptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues that are the same, when compared and aligned
for maximum correspondence over a comparison window, as measured
using a sequence comparison algorithms using default program
parameters or by manual alignment and visual inspection.
[0070] The phrases "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany the material as it is found in its native
state. Thus, isolated peptides in accordance with the invention
preferably do not contain materials normally associated with the
peptides in their in situ environment.
[0071] "Major histocompatibility complex" or "MHC" is a cluster of
genes that plays a role in control of the cellular interactions
responsible for physiologic immune responses. In humans, the MHC
complex is also known as the HLA complex. For a detailed
description of the MHC and HLA complexes, see Paul, Fundamental
Immunology (3rd ed. 1993).
[0072] "Human leukocyte antigen" or "HLA" is a human class I or
class II major histocompatibility complex (MHC) protein (see, e.g.,
Stites, et al., Immunology, (8th Ed., 1994).
[0073] An "HLA supertype or family", as used herein, describes sets
of HLA molecules grouped on the basis of shared peptide-binding
specificities. HLA class I molecules that share somewhat similar
binding affinity for peptides bearing certain amino acid motifs are
grouped into HLA supertypes. The terms HLA superfamily, HLA
supertype family, HLA family, and HLA xx-like supertype molecules
(where xx denotes a particular HLA type), are synonyms.
[0074] The term "motif" refers to the pattern of residues in a
peptide of defined length, usually a peptide of from about 8 to
about 13 amino acids for a class I HLA motif and from about 6 to
about 25 amino acids for a class II HLA motif, which is recognized
by a particular HLA molecule. Peptide motifs are typically
different for each protein encoded by each human HLA allele and
differ in the pattern of the primary and secondary anchor
residues.
[0075] A "supermotif" is a peptide binding specificity shared by
HLA molecules encoded by two or more HLA alleles. Thus, a
preferably is recognized with high or intermediate affinity (as
defined herein) by two or more HLA antigens.
[0076] "Cross-reactive binding" indicates that a peptide is bound
by more than one HLA molecule; a synonym is degenerate binding.
[0077] The term "peptide" is used interchangeably with
"oligopeptide" in the present specification to designate a series
of residues, typically L-amino acids, connected one to the other,
typically by peptide bonds between the .alpha.-amino and carboxyl
groups of adjacent amino acids. The preferred CTL-inducing
oligopeptides of the invention are 13 residues or less in length
and usually consist of between about 8 and about 11 residues,
preferably 9 or 10 residues. The preferred HTL-inducing
oligopeptides are less than about 50 residues in length and usually
consist of between about 6 and about 30 residues, more usually
between about 12 and 25, and often between about 15 and 20
residues.
[0078] An "immunogenic peptide" or "peptide epitope" is a peptide
which comprises an allele-specific motif or supernotif such that
the peptide will bind an HLA molecule and induce a CTL and/or HTL
response. Thus, immunogenic peptides of the invention are capable
of binding to an appropriate HLA molecule and thereafter inducing a
cytotoxic T cell response, or a helper T cell response, to the
antigen from which the immunogenic peptide is derived.
[0079] A "protective immune response" refers to a CTL and/or an HTL
response to an antigen derived from an infectious agent or a tumor
antigen, which prevents or at least partially arrests disease
symptoms or progression. The immune response may also include an
antibody response which has been facilitated by the stimulation of
helper T cells.
[0080] The term "residue" refers to an amino acid or amino acid
mimetic incorporated into an oligopeptide by an amide bond or amide
bond mimetic.
[0081] "Synthetic peptide" refers to a peptide that is not
naturally occurring, but is man-made using such methods as chemical
synthesis or recombinant DNA technology.
[0082] The nomenclature used to describe peptide compounds follows
the conventional practice wherein the amino group is presented to
the left (the N-terminus) and the carboxyl group to the right (the
C-terminus) of each amino acid residue. When amino acid residue
positions are referred to in a peptide epitope they are numbered in
an amino to carboxyl direction with position one being the position
closest to the amino terminal end of the epitope, or the peptide or
protein of which it may be a part. In the formulae representing
selected specific embodiments of the present invention, the amino-
and carboxyl-terminal groups, although not specifically shown, are
in the form they would assume at physiologic pH values, unless
otherwise specified. In the amino acid structure formulae, each
residue is generally represented by standard three letter or single
letter designations. The L-form of an amino acid residue is
represented by a capital single letter or a capital first letter of
a three-letter symbol, and the D-form for those amino acids having
D-forms is represented by a lower case single letter or a lower
case three letter symbol. Glycine has no asymmetric carbon atom and
is simply referred to as "Gly" or G.
[0083] As used herein, the term "expression vector" is intended to
refer to a nucleic acid molecule capable of expressing an antigen
of interest such as a MHC class I or class II epitope in an
appropriate target cell. An expression vector can be, for example,
a plasmid or virus, including DNA or RNA viruses. The expression
vector contains such a promoter element to express an antigen of
interest in the appropriate cell or tissue in order to stimulate a
desired immune response.
DETAILED DESCRIPTION OF THE INVENTION
[0084] Cytotoxic T lymphocytes (CTLs) and helper T lymphocytes
(HTLs) are critical for immunity against infectious pathogens; such
as viruses, bacteria, and protozoa; tumor cells; autoimmune
diseases and the like. The present invention provides minigenes
that encode peptide epitopes which induce a CTL and/or HTL
response. The minigenes of the invention also include an MHC
targeting sequence. A variety of minigenes encoding different
epitopes can be tested for immunogenicity using an HLA transgenic
mouse. The epitopes are typically a combination of at least two or
more HTL epitopes, or a CTL epitope plus a universal HTL epitope,
and optimally include additional HTL and/or CTL epitopes. Two,
three, four, five, six, seven, eight, nine, ten, twenty, thirty,
forty or about fifty different epitopes, either HTL and/or CTL, can
be included in the minigene, along with the MHC targeting sequence.
The epitopes can have different HLA restriction. Epitopes to be
tested include those derived from viruses such as HIV, HBV, HCV,
HSV, CMV, HPV, and HTLV; cancer antigens such as p53, Her2/Neu,
MAGE, PSA, human papilloma virus, and CEA; parasites such as
Trypanosoma, Plasmodium, Leishmania, Giardia, Entamoeba; autoimmune
diseases such as rheumatoid arthritis, myesthenia gravis, and lupus
erythematosus; fungi such as Aspergillus and Candida; and bacteria
such as Escherichia coli, Staphylococci, Chlamydia, Mycobacteria,
Streptococci, and Pseudomonas. The epitopes to be encoded by the
minigene are selected and tested using the methods described in
published PCT applications WO 93/07421, WO 94/02353, WO 95/01000,
WO 97/04451, and WO 97/05348, herein incorporated by reference.
[0085] HTL and CTL Epitopes
[0086] The expression vectors of the invention encode one or more
MHC class II and/or class I epitopes and an MHC targeting sequence.
Multiple MHC class II or class I epitopes present in an expression
vector can be derived from the same antigen, or the MHC epitopes
can be derived from different antigens. For example, an expression
vector can contain one or more MHC epitopes that can be derived
from two different antigens of the same virus or from two different
antigens of different viruses. Furthermore, any MHC epitope can be
used in the expression vectors of the invention. For example, any
single MHC epitope or a combination of the MHC epitopes shown in
Tables 1 to 8 can be used in the expression vectors of the
invention. Other peptide epitopes can be selected by one of skill
in the art, e.g., by using a computer to select epitopes that
contain HLA allele-specific motifs or supermotifs. The expression
vectors of the invention can also encode one or more universal MHC
class II epitopes, e.g., PADRE (see, e.g., SEQ ID NO:52).
[0087] Universal MHC class II epitopes can be advantageously
combined with other MHC class I and class II epitopes to increase
the number of cells that are activated in response to a given
antigen and provide broader population coverage of MHC-reactive
alleles. Thus, the expression vectors of the invention can encode
MHC epitopes specific for an antigen, universal MHC class II
epitopes, or a combination of specific MHC epitopes and at least
one universal MHC class II epitope.
[0088] MHC class I epitopes are generally about 5 to 15 amino acids
in length, in particular about 8 to 11 amino acids in length. MHC
class II epitopes are generally about 10 to 25 amino acids in
length, in particular about 13 to 21 amino acids in length. A MHC
class I or II epitope can be derived from any desired antigen of
interest. The antigen of interest can be a viral antigen, surface
receptor, tumor antigen, oncogene, enzyme, or any pathogen, cell or
molecule for which an immune response is desired. Epitopes can be
selected based on their ability to bind one or multiple HLA
alleles, and can also be selected using the "analog" technique
described below.
[0089] Targeting Sequences
[0090] The expression vectors of the invention encode one or more
MHC epitopes operably linked to a MHC targeting sequence. The use
of a MHC targeting sequence enhances the immune response to an
antigen, relative to delivery of antigen alone, by directing the
peptide epitope to the site of MHC molecule assembly and transport
to the cell surface, thereby providing an increased number of MHC
molecule-peptide epitope complexes available for binding to and
activation of T cells.
[0091] MHC class I targeting sequences are used in the present
invention, e.g., those sequences that target an MHC class I epitope
peptide to a cytosolic pathway or to the endoplasmic reticulum
(see, e.g., Rammensee et al., Immunogenetics 41:178-228 (1995)).
For example, the cytosolic pathway processes endogenous antigens
that are expressed inside the cell. Although not wishing to be
bound by any particular theory, cytosolic proteins are thought to
be at least partially degraded by an endopeptidase activity of a
proteasome and then transported to the endoplasmic reticulum by the
TAP molecule (transporter associated with processing). In the
endoplasmic reticulum, the antigen binds to MHC class I molecules.
Endoplasmic reticulum signal sequences bypass the cytosolic
processing pathway and directly target endogenous antigens to the
endoplasmic reticulum, where proteolytic degradation into peptide
fragments occurs. Such MHC class I targeting sequences are well
known in the art, and include, e.g., signal sequences such as those
from Ig kappa ,tissue plasminogen activator or insulin. A preferred
signal peptide is the human Ig kappa chain sequence. Endoplasmic
reticulum signal sequences can also be used to target MHC class II
epitopes to the endoplasmic reticulum, the site of MHC class I
molecule assembly.
[0092] MHC class II targeting sequences are also used in the
invention, e.g., those that target a peptide to the endocytic
pathway. These targeting sequences typically direct extracellular
antigens to enter the endocytic pathway, which results in the
antigen being transferred to the lysosomal compartment where the
antigen is proteolytically cleaved into antigen peptides for
binding to MHC class II molecules. As with the normal processing of
exogenous antigen, a sequence that directs a MHC class II epitope
to the endosomes of the endocytic pathway and/or subsequently to
lysosomes, where the MHC class II epitope can bind to a MHC class
II molecule, is a MHC class II targeting sequence. For example,
group of MHC class II targeting sequences useful in the invention
are lysosomal targeting sequences, which localize polypeptides to
lysosomes. Since MHC class II molecules typically bind to antigen
peptides derived from proteolytic processing of endocytosed
antigens in lysosomes, a lysosomal targeting sequence can function
as a MHC class II targeting sequence. Lysosomal targeting sequences
are well known in the art and include sequences found in the
lysosomal proteins LAMP-1 and LAMP-2 as described by August et al.
(U.S. Pat. No. 5,633,234, issued May 27, 1997), which is
incorporated herein by reference.
[0093] Other lysosomal proteins that contain lysosomal targeting
sequences include HLA-DM. HLA-DM is an endosomal/lysosomal protein
that functions in facilitating binding of antigen peptides to MHC
class II molecules. Since it is located in the lysosome, HLA-DM has
a lysosomal targeting sequence that can function as a MHC class II
molecule targeting sequence (Copier et al., J. Immunol.
157:1017-1027 (1996), which is incorporated herein by
reference).
[0094] The resident lysosomal protein HLA-DO can also function as a
lysosomal targeting sequence. In contrast to the above described
resident lysosomal proteins LAMP-1 and HLA-DM, which encode
specific Tyr-containing motifs that target proteins to lysosomes,
HLA-DO is targeted to lysosomes by association with HLA-DM
(Liljedahl et al., EMBO J. 15:4817-4824 (1996)), which is
incorporated herein by reference. Therefore, the sequences of
HLA-DO that cause association with HLA-DM and, consequently,
translocation of HLA-DO to lysosomes can be used as MHC class II
targeting sequences. Similarly, the murine homolog of HLA-DO,
H2-DO, can be used to derive a MHC class II targeting sequence. A
MHC class II epitope can be fused to HLA-DO or H2-DO and targeted
to lysosomes.
[0095] In another example, the cytoplasmic domains of B cell
receptor subunits Ig-.alpha. and Ig-.beta. mediate antigen
internalization and increase the efficiency of antigen presentation
(Bonnerot et al., Immunity 3:335-347 (1995)), which is incorporated
herein by reference. Therefore, the cytoplasmic domains of the
Ig-.alpha. and Ig-.beta. proteins can function as MHC class II
targeting sequences that target a MHC class II epitope to the
endocytic pathway for processing and binding to MHC class II
molecules.
[0096] Another example of a MHC class II targeting sequence that
directs MHC class II epitopes to the endocytic pathway is a
sequence that directs polypeptides to be secreted, where the
polypeptide can enter the endosomal pathway. These MHC class II
targeting sequences that direct polypeptides to be secreted mimic
the normal pathway by which exogenous, extracellular antigens are
processed into peptides that bind to MHC class II molecules. Any
signal sequence that functions to direct a polypeptide through the
endoplasmic reticulum and ultimately to be secreted can function as
a MHC class II targeting sequence so long as the secreted
polypeptide can enter the endosomal/lysosomal pathway and be
cleaved into peptides that can bind to MHC class II molecules. An
example of such a fusion is shown in FIG. 11, where the signal
sequence of kappa immunoglobulin is fused to multiple MHC class II
epitopes.
[0097] In another example, the Ii protein binds to MHC class II
molecules in the endoplasmic reticulum, where it functions to
prevent peptides present in the endoplasmic reticulum from binding
to the MHC class II molecules. Therefore, fusion of a MHC class II
epitope to the Ii protein targets the MHC class II epitope to the
endoplasmic reticulum and a MHC class II molecule. For example, the
CLIP sequence of the Ii protein can be removed and replaced with a
MHC class II epitope sequence so that the MHC class II epitope is
directed to the endoplasmic reticulum, where the epitope binds to a
MHC class II molecule.
[0098] In some cases, antigens themselves can serve as MHC class II
or I targeting sequences and can be fused to a universal MHC class
II epitope to stimulate an immune response. Although cytoplasmic
viral antigens are generally processed and presented as complexes
with MHC class I molecules, long-lived cytoplasmic proteins such as
the influenza matrix protein can enter the MHC class II molecule
processing pathway (Gueguen & Long, Proc. Natl. Acad. Sci. USA
93:14692-14697 (1996)), which is incorporated herein by reference.
Therefore, long-lived cytoplasmic proteins can function as a MHC
class II targeting sequence. For example, an expression vector
encoding influenza matrix protein fused to a universal MHC class II
epitope can be advantageously used to target influenza antigen and
the universal MHC class II epitope to the MHC class II pathway for
stimulating an immune response to influenza.
[0099] Other examples of antigens functioning as MHC class II
targeting sequences include polypeptides that spontaneously form
particles. The polypeptides are secreted from the cell that
produces them and spontaneously form particles, which are taken up
into an antigen-presenting cell by endocytosis such as
receptor-mediated endocytosis or are engulfed by phagocytosis. The
particles are proteolytically cleaved into antigen peptides after
entering the endosomal/lysosomal pathway.
[0100] One such polypeptide that spontaneously forms particles is
HBV surface antigen (HBV-S) (Diminsky et al., Vaccine 15:637-647
(1997); Le Borgne et al., Virology 240:304-315 (1998)), each of
which is incorporated herein by reference. Another polypeptide that
spontaneously forms particles is HBV core antigen (Kuhrober et al.,
International Immunol. 9:1203-1212 (1997)), which is incorporated
herein by reference. Still another polypeptide that spontaneously
forms particles is the yeast Ty protein (Weber et al., Vaccine
13:831-834 (1995)), which is incorporated herein by reference. For
example, an expression vector containing HBV-S antigen fused to a
universal MHC class II epitope can be advantageously used to target
HBV-S antigen and the universal MHC class II epitope to the MHC
class II pathway for stimulating an immune response to HBV.
[0101] Binding Affinity of Peptide Epitopes for HLA Molecules
[0102] The large degree of HLA polymorphism is an important factor
to be taken into account with the epitope-based approach to vaccine
development. To address this factor, epitope selection encompassing
identification of peptides capable of binding at high or
intermediate affinity to multiple HLA molecules is preferably
utilized, most preferably these epitopes bind at high or
intermediate affinity to two or more allele specific HLA
molecules.
[0103] CTL-inducing peptides of interest for vaccine compositions
preferably include those that have a binding affinity for class I
HLA molecules of less than 500 nM. HTL-inducing peptides preferably
include those that have a binding affinity for class II HLA
molecules of less than 1000 nM. For example, peptide binding is
assessed by testing the capacity of a candidate peptide to bind to
a purified HLA molecule in vitro. Peptides exhibiting high or
intermediate affinity are then considered for further analysis.
Selected peptides are tested on other members of the supertype
family. In preferred embodiments, peptides that exhibit
cross-reactive binding are then used in vaccines or in cellular
screening analyses.
[0104] Higher HLA binding affinity is typically correlated with
greater immunogenicity. Greater immunogenicity can be manifested in
several different ways. Immunogenicity corresponds to whether an
immune response is elicited at all, and to the vigor of any
particular response, as well as to the extent of a population in
which a response is elicited. For example, a peptide might elicit
an immune response in a diverse array of the population, yet in no
instance produce a vigorous response. In accordance with these
principles, close to 90% of high binding peptides have been found
to be immunogenic, as contrasted with about 50% of the peptides
which bind with intermediate affinity. Moreover, higher binding
affinity peptides leads to more vigorous immunogenic responses. As
a result, less peptide is required to elicit a similar biological
effect if a high affinity binding peptide is used. Thus, in
preferred embodiments of the invention, high binding epitopes are
particularly useful.
[0105] The relationship between binding affinity for HLA class I
molecules and immunogenicity of discrete peptide epitopes on bound
antigens has been determined for the first time in the art by the
present inventors. The correlation between binding affinity and
immunogenicity was analyzed in two different experimental
approaches (Sette et al., J. Immunol. 153:5586-5592 (1994)). In the
first approach, the immunogenicity of potential epitopes ranging in
HLA binding affinity over a 10,000-fold range was analyzed in
HLA-A*0201 transgenic mice. In the second approach, the
antigenicity of approximately 100 different hepatitis B virus
(HBV)-derived potential epitopes, all carrying A*0201 binding
motifs, was assessed by using PBL (peripheral blood lymphocytes)
from acute hepatitis patients. Pursuant to these approaches, it was
determined that an affinity threshold of approximately 500 nM
(preferably 50 nM or less) determines the capacity of a peptide
epitope to elicit a CTL response. These data are true for class I
binding affinity measurements for naturally processed peptides and
for synthesized T cell epitopes. These data also indicate the
important role of determinant selection in the shaping of T cell
responses (see, e.g., Schaeffer et al. Proc. Natl. Acad. Sci. USA
86:4649-4653, 1989).
[0106] An affinity threshold associated with immunogenicity in the
context of HLA class II DR molecules has also been delineated (see,
e.g., Southwood et al. J. Immunology 160:3363-3373 (1998), and U.S.
S No. 60/087,192, filed May 29, 1998). In order to define a
biologically significant threshold of DR binding affinity, a
database of the binding affinities of 32 DR-restricted epitopes for
their restricting element (i.e., the HLA molecule that binds the
motif) was compiled. In approximately half of the cases (15 of 32
epitopes), DR restriction was associated with high binding
affinities, i.e. binding affinities of less than 100 nM. In the
other half of the cases (16 of 32), DR restriction was associated
with intermediate affinity (binding affinities in the 100-1000 nM
range). In only one of 32 cases was DR restriction associated with
an IC50 of 1000 nM or greater. Thus, 1000 nM can be defined as an
affinity threshold associated with immunogenicity in the context of
DR molecules.
[0107] Peptide Epitope Binding Motifs and Supermotifs
[0108] In the past few years evidence has accumulated to
demonstrate that a large fraction of HLA class I and class II
molecules can be classified into a relatively few supertypes, each
characterized by largely overlapping peptide binding repertoires,
and consensus structures of the main peptide binding pockets.
[0109] For HLA molecule pocket analyses, the residues comprising
the B and F pockets of HLA class I molecules as described in
crystallographic studies were analyzed (Guo et al., Nature 360:364
(1992); Saper et al., J. Mol. Biol. 219:277 (1991); Madden et al.,
Cell 75:693 (1993); Parham et al., Immunol. Rev. 143:141 (1995)).
In these analyses, residues 9, 45, 63, 66, 67, 70, and 99 were
considered to make up the B pocket; and the B pocket was deemed to
determine the specificity for the amino acid residue in the second
position of peptide ligands. Similarly, residues 77, 80, 81, and
116 were considered to determine the specificity of the F pocket;
the F pocket was deemed to determine the specificity for the
C-terminal residue of a peptide ligand bound by the HLA class I
molecule.
[0110] Through the study of single amino acid substituted antigen
analogs and the sequencing of endogenously bound, naturally
processed peptides, critical residues required for allele-specific
binding to HLA molecules have been identified. The presence of
these residues correlates with binding affinity for HLA molecules.
The identification of motifs and/or supermotifs that correlate with
high and intermediate affinity binding is an important issue with
respect to the identification of immunogenic peptide epitopes for
the inclusion in a vaccine. Kast et al. (J. Immunol. 152:3904-3912
(1994)) have shown that motif-bearing peptides account for 90% of
the epitopes that bind to allele-specific HLA class I molecules. In
this study all possible peptides of 9 amino acids in length and
overlapping by eight amino acids (240 peptides), which cover the
entire sequence of the E6 and E7 proteins of human papillomavirus
type 16, were evaluated for binding to five allele-specific HLA
molecules that are expressed at high frequency among different
ethnic groups. This unbiased set of peptides allowed an evaluation
of the predictive value of HLA class I motifs. From the set of 240
peptides, 22 peptides were identified that bound to an
allele-specific HLA molecules with high or intermediate affinity.
Of these 22 peptides, 20, (i.e., 91%), were motif-bearing. Thus,
this study demonstrates the value of motifs for the identification
of peptide epitopes for inclusion in a vaccine: application of
motif-based identification techniques eliminates screening of 90%
of the potential epitopes in a target antigen protein sequence.
[0111] Peptides of the present invention may also include epitopes
that bind to MHC class II DR molecules. There is a significant
difference between class I and class II HLA molecules. This
difference corresponds to the fact that, although a stringent size
restriction and motif position relative to the binding pocket
exists for peptides that bind to class I molecules, a greater
degree of heterogeneity in both size and binding frame position of
the motif, relative to the N and C termini of the peptide, exists
for class II peptide ligands.
[0112] This increased heterogeneity of HLA class II peptide ligands
is due to the structure of the binding groove of the HLA class II
molecule which, unlike its class I counterpart, is open at both
ends. Crystallographic analysis of HLA class II DRB*0101-peptide
complexes showed that the residues occupying position 1 and
position 6 of peptides complexed with DRB*0101 engage two
complementary pockets on the DRBa*0101 molecules, with the P1
position corresponding to the most crucial anchor residue and the
deepest hydrophobic pocket (see, e.g., Madden, Ann. Rev. Immunol.
13:587 (1995)). Other studies have also pointed to the P6 position
as a crucial anchor residue for binding to various other DR
molecules.
[0113] Thus, peptides of the present invention are identified by
any one of several HLA class I or II-specific amino acid motifs
(see, e.g., Tables I-III of U.S. Ser. Nos. 09/226,775, and
09/239,043, herein incorporated by reference in their entirety). If
the presence of the motif corresponds to the ability to bind
several allele-specific HLA antigens it is referred to as a
supermotif. The allele-specific HLA molecules that bind to peptides
that possess a particular amino acid supermotif are collectively
referred to as an HLA "supertype."
[0114] Immune Response-Stimulating Peptide Analogs
[0115] In general, CTL and HTL responses are not directed against
all possible epitopes. Rather, they are restricted to a few
"immunodominant" determinants (Zinkemagel et al., Adv. Immunol.
27:5159 (1979); Bennink et al., J. Exp. Med. 168:1935-1939 (1988);
Rawle et al., J. Immunol. 146:3977-3984 (1991)). It has been
recognized that immunodominance (Benacerraf et al., Science
175:273-279 (1972)) could be explained by either the ability of a
given epitope to selectively bind a particular HLA protein
(determinant selection theory) (Vitiello et al., J. Immunol.
131:1635 (1983)); Rosenthal et al., Nature 267:156-158 (1977)), or
being selectively recognized by the existing TCR (T cell receptor)
specificity (repertoire theory) (Klein, Immunology, The Science of
Self on self Discrimination, pp. 270-310 (1982)). It has been
demonstrated that additional factors, mostly linked to processing
events, can also play a key role in dictating, beyond strict
immunogenicity, which of the many potential determinants will be
presented as immunodominant (Sercarz et al., Annu. Rev. Immunol.
11:729-766 (1993)).
[0116] The concept of dominance and subdominance is relevant to
immunotherapy of both infectious diseases and cancer. For example,
in the course of chronic viral disease, recruitment of subdominant
epitopes can be important for successful clearance of the
infection, especially if dominant CTL or HTL specificities have
been inactivated by functional tolerance, suppression, mutation of
viruses and other mechanisms (Franco et al., Curr. Opin. Immunol.
7:524-531 (1995)). In the case of cancer and tumor antigens, CTLs
recognizing at least some of the highest binding affinity peptides
might be functionally inactivated. Lower binding affinity peptides
are preferentially recognized at these times, and may therefore be
preferred in therapeutic or prophylactic anti-cancer vaccines.
[0117] In particular, it has been noted that a significant number
of epitopes derived from known non-viral tumor associated antigens
(TAA) bind HLA class I with intermediate affinity (IC50 in the
50-500 nM range). For example, it has been found that 8 of 15 known
TAA peptides recognized by tumor infiltrating lymphocytes (TIL) or
CTL bound in the 50-500 nM range. (These data are in contrast with
estimates that 90% of known viral antigens were bound by HLA class
I molecules with IC50 of 50 nM or less, while only approximately
10% bound in the 50-500 nM range (Sette et al., J. Immunol.,
153:558-5592 (1994)). In the cancer setting this phenomenon is
probably due to elimination, or functional inhibition of the CTL
recognizing several of the highest binding peptides, presumably
because of T cell tolerization events.
[0118] Without intending to be bound by theory, it is believed that
because T cells to dominant epitopes may have been clonally
deleted, selecting subdominant epitopes may allow extant T cells to
be recruited, which will then lead to a therapeutic or prophylactic
response. However, the binding of HLA molecules to subdominant
epitopes is often less vigorous than to dominant ones. Accordingly,
there is a need to be able to modulate the binding affinity of
particular immunogenic epitopes for one or more HLA molecules, and
thereby to modulate the immune response elicited by the peptide,
for example to prepare analog peptides which elicit a more vigorous
response. This ability would greatly enhance the usefulness of
peptide-based vaccines and therapeutic agents.
[0119] Thus, although peptides with suitable cross-reactivity among
all alleles of a superfamily are identified by the screening
procedures described above, cross-reactivity is not always as
complete as possible, and in certain cases procedures to further
increase cross-reactivity of peptides can be useful; moreover, such
procedures can also be used to modify other properties of the
peptides such as binding affinity or peptide stability. Having
established the general rules that govern cross-reactivity of
peptides for HLA alleles within a given motif or supermotif,
modification (i.e., analoging) of the structure of peptides of
particular interest in order to achieve broader (or otherwise
modified) HLA binding capacity can be performed. More specifically,
peptides which exhibit the broadest cross-reactivity patterns, can
be produced in accordance with the teachings herein. The present
concepts related to analog generation are set forth in greater
detail in co-pending U.S. Ser. No. 09/226,775.
[0120] In brief, the strategy employed utilizes the motifs or
supermotifs which correlate with binding to certain HLA class I and
II molecules. The motifs or supermotifs are defined by having
primary anchors, and in many cases secondary anchors (see Tables
I-III of U.S. Ser. No. 09/226,775). Analog peptides can be created
by substituting amino acids residues at primary anchor, secondary
anchor, or at primary and secondary anchor positions. Generally,
analogs are made for peptides that already bear a motif or
supermotif. Preferred secondary anchor residues of supernotifs and
motifs that have been defined for HLA class I and class II binding
peptides are shown in Tables II and III, respectively, of U.S. Ser.
No. 09/226,775.
[0121] For a number of the motifs or supermotifs in accordance with
the invention, residues are defined which are deleterious to
binding to allele-specific HLA molecules or members of HLA
supertypes that bind to the respective motif or supermotif (see
Tables II and III of U.S. Ser. No. 09/226,775). Accordingly,
removal of such residues that are detrimental to binding can be
performed in accordance with the methods described therein. For
example, in the case of the A3 supertype, when all peptides that
have such deleterious residues are removed from the population of
analyzed peptides, the incidence of cross-reactivity increases from
22% to 37% (I., Sidney et al., Hu. Immunol. 45:79 (1996)). Thus,
one strategy to improve the cross-reactivity of peptides within a
given supermotif is simply to delete one or more of the deleterious
residues present within a peptide and substitute a small "neutral"
residue such as Ala (that may not influence T cell recognition of
the peptide). An enhanced likelihood of cross-reactivity is
expected if, together with elimination of detrimental residues
within a peptide, "preferred" residues associated with high
affinity binding to an allele-specific HLA molecule or to multiple
HLA molecules within a superfamily are inserted.
[0122] To ensure that an analog peptide, when used as a vaccine,
actually elicits a CTL response to the native epitope in vivo (or,
in the case of class II epitopes, a failure to elicit helper T
cells that cross-react with the wild type peptides), the analog
peptide may be used to immunize T cells in vitro from individuals
of the appropriate HLA allele. Thereafter, the immunized cells'
capacity to induce lysis of wild type peptide sensitized target
cells is evaluated. In both class I and class II systems it will be
desirable to use as targets, cells that have been either infected
or transfected with the appropriate genes to establish whether
endogenously produced antigen is also recognized by the relevant T
cells.
[0123] Another embodiment of the invention is to create analogs of
weak binding peptides, to thereby ensure adequate numbers of
cross-reactive cellular binders. Class I peptides exhibiting
binding affinities of 500-50000 nM, and carrying an acceptable but
suboptimal primary anchor residue at one or both positions can be
"fixed" by substituting preferred anchor residues in accordance
with the respective supertype. The analog peptides can then be
tested for crossbinding activity.
[0124] Another embodiment for generating effective peptide analogs
involves the substitution of residues that have an adverse impact
on peptide stability or solubility in, e.g., a liquid environment.
This substitution may occur at any position of the peptide epitope.
For example, a cysteine (C) can be substituted out in favor of
gamma-amino butyric acid. Due to its chemical nature, cysteine has
the propensity to form disulfide bridges and sufficiently alter the
peptide structurally so as to reduce binding capacity. Substituting
gamma-amino butyric acid for C not only alleviates this problem,
but actually improves binding and crossbinding capability in
certain instances (Sette et al, In: Persistent Viral Infections
(Ahmed & Chen, eds., 1998)). Substitution of cysteine with
gamma-amino butyric acid may occur at any residue of a peptide
epitope, i.e., at either anchor or non-anchor positions.
[0125] Expression Vectors and Construction of a Minigene
[0126] The expression vectors of the invention contain at least one
promoter element that is capable of expressing a transcription unit
encoding the antigen of interest, for example, a MHC class I
epitope or a MHC class II epitope and an MHC targeting sequence in
the appropriate cells of an organism so that the antigen is
expressed and targeted to the appropriate MHC molecule. For
example, if the expression vector is administered to a mammal such
as a human, a promoter element that functions in a human cell is
incorporated into the expression vector. An example of an
expression vector useful for expressing the MHC class II epitopes
fused to MHC class II targeting sequences and the MHC class I
epitopes described herein is the pEP2 vector described in Example
IV.
[0127] This invention relies on routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of
use in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994); Oligonucleotide
Synthesis: A Practical Approach (Gait, ed., 1984); Kuijpers,
Nucleic Acids Research 18(17):5197 (1994); Dueholm, J. Org. Chem.
59:5767-5773 (1994); Methods in Molecular Biology, volume 20
(Agrawal, ed.); and Tijssen, Laboratory Techniques in Biochemistry
and Molecular Biology--Hybridization with Nucleic Acid Probes,
e.g., Part I, chapter 2 "Overview of principles of hybridization
and the strategy of nucleic acid probe assays" (1993)).
[0128] The minigenes are comprised of two or many different
epitopes (see, e.g., Tables 1-8). The nucleic acid encoding the
epitopes are assembled in a minigene according to standard
techniques. In general, the nucleic acid sequences encoding
minigene epitopes are isolated using amplification techniques with
oligonucleotide primers, or are chemically synthesized. Recombinant
cloning techniques can also be used when appropriate.
Oligonucleotide sequences are selected which either amplify (when
using PCR to assemble the minigene) or encode (when using synthetic
oligonucleotides to assemble the minigene) the desired
epitopes.
[0129] Amplification techniques using primers are typically used to
amplify and isolate sequences encoding the epitopes of choice from
DNA or RNA (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR
Protocols: A Guide to Methods and Applications (Innis et al., eds,
1990)). Methods such as polymerase chain reaction (PCR) and ligase
chain reaction (LCR) can be used to amplify epitope nucleic acid
sequences directly from mRNA, from cDNA, from genomic libraries or
cDNA libraries. Restriction endonuclease sites can be incorporated
into the primers. Minigenes amplified by the PCR reaction can be
purified from agarose gels and cloned into an appropriate
vector.
[0130] Synthetic oligonucleotides can also be used to construct
minigenes. This method is performed using a series of overlapping
oligonucleotides, representing both the sense and non-sense strands
of the gene. These DNA fragments are then annealed, ligated and
cloned. Oligonucleotides that are not commercially available can be
chemically synthesized according to the solid phase phosphoramidite
trimester method first described by Beaucage & Caruthers,
Tetrahedron Letts. 22:1859-1862 (1981), using an automated
synthesizer, as described in Van Devanter et. al., Nucleic Acids
Res. 12:6159-6168 (1984). Purification of oligonucleotides is by
either native acrylamide gel electrophoresis or by anion-exchange
HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149
(1983).
[0131] The epitopes of the minigene are typically subcloned into an
expression vector that contains a strong promoter to direct
transcription, as well as other regulatory sequences such as
enhancers and polyadenylation sites. Suitable promoters are well
known in the art and described, e.g., in Sambrook et al. and
Ausubel et al. Eukaryotic expression systems for mammalian cells
are well known in the art and are commercially available. Such
promoter elements include, for example, cytomegalovirus (CMV), Rous
sarcoma virus LTR and SV40.
[0132] The expression vector typically contains a transcription
unit or expression cassette that contains all the additional
elements required for the expression of the minigene in host cells.
A typical expression cassette thus contains a promoter operably
linked to the minigene and signals required for efficient
polyadenylation of the transcript. Additional elements of the
cassette may include enhancers and introns with functional splice
donor and acceptor sites.
[0133] In addition to a promoter sequence, the expression cassette
can also contain a transcription termination region downstream of
the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes.
[0134] The particular expression vector used to transport the
genetic information into the cell is not particularly critical. Any
of the conventional vectors used for expression in eukaryotic cells
may be used. Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors
derived from Epstein Bar virus. Other exemplary eukaryotic vectors
include pMSG, pAV009/A+, pMTOIO/A+, pMAMneo-5, baculovirus pDSVE,
and any other vector allowing expression of proteins under the
direction of the SV40 early promoter, SV40 later promoter,
metallothionein promoter, murine mammary tumor virus promoter, Rous
sarcoma virus promoter, polyhedrin promoter, or other promoters
shown effective for expression in eukaryotic cells. In one
embodiment, the vector pEP2 is used in the present invention.
[0135] Other elements that are typically included in expression
vectors also include a replicon that functions in E. coli, a gene
encoding antibiotic resistance to permit selection of bacteria that
harbor recombinant plasmids, and unique restriction sites in
nonessential regions of the plasmid to allow insertion of
eukaryotic sequences. The particular antibiotic resistance gene
chosen is not critical, any of the many resistance genes known in
the art are suitable. The prokaryotic sequences are preferably
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells, if necessary.
[0136] Administration In Vivo
[0137] The invention also provides methods for stimulating an
immune response by administering an expression vector of the
invention to an individual. Administration of an expression vector
of the invention for stimulating an immune response is advantageous
because the expression vectors of the invention target MHC epitopes
to MHC molecules, thus increasing the number of CTL and HTL
activated by the antigens encoded by the expression vector.
[0138] Initially, the expression vectors of the invention are
screened in mouse to determine the expression vectors having
optimal activity in stimulating a desired immune response. Initial
studies are therefore carried out, where possible, with mouse genes
of the MHC targeting sequences. Methods of determining the activity
of the expression vectors of the invention are well known in the
art and include, for example, the uptake of .sup.3H-thymidine to
measure T cell activation and the release of .sup.51Cr to measure
CTL activity as described below in Examples II and III. Experiments
similar to those described in Example IV are performed to determine
the expression vectors having activity at stimulating an immune
response. The expression vectors having activity are further tested
in human. To circumvent potential adverse immunological responses
to encoded mouse sequences, the expression vectors having activity
are modified so that the MHC class II targeting sequences are
derived from human genes. For example, substitution of the
analogous regions of the human homologs of genes containing various
MHC class II targeting sequences are substituted into the
expression vectors of the invention. Examples of such human
homologs of genes containing MHC class II targeting sequences are
shown in FIGS. 12 to 17. Expression vectors containing human MHC
class II targeting sequences, such as those described in Example I
below, are tested for activity at stimulating an immune response in
human.
[0139] The invention also relates to pharmaceutical compositions
comprising a pharmaceutically acceptable carrier and an expression
vector of the invention. Pharmaceutically acceptable carriers are
well known in the art and include aqueous or non-aqueous solutions,
suspensions and emulsions, including physiologically buffered
saline, alcohol/aqueous solutions or other solvents or vehicles
such as glycols, glycerol, oils such as olive oil or injectable
organic esters.
[0140] A pharmaceutically acceptable carrier can contain
physiologically acceptable compounds that act, for example, to
stabilize the expression vector or increase the absorption of the
expression vector. Such physiologically acceptable compounds
include, for example, carbohydrates, such as glucose, sucrose or
dextrans, antioxidants such as ascorbic acid or glutathione,
chelating agents, low molecular weight polypeptides, antimicrobial
agents, inert gases or other stabilizers or excipients. Expression
vectors can additionally be complexed with other components such as
peptides, polypeptides and carbohydrates. Expression vectors can
also be complexed to particles or beads that can be administered to
an individual, for example, using a vaccine gun. One skilled in the
art would know that the choice of a pharmaceutically acceptable
carrier, including a physiologically acceptable compound, depends,
for example, on the route of administration of the expression
vector.
[0141] The invention further relates to methods of administering a
pharmaceutical composition comprising an expression vector of the
invention to stimulate an immune response. The expression vectors
are administered by methods well known in the art as described in
Donnelly et al. (Ann. Rev., Immunol. 15:617-648 (1997)); Felgner et
al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Felgner (U.S.
Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S.
Pat. No. 5,679,647, issued Oct. 21, 1997), each of which is
incorporated herein by reference. In one embodiment, the minigene
is administered as naked nucleic acid.
[0142] A pharmaceutical composition comprising an expression vector
of the invention can be administered to stimulate an immune
response in a subject by various routes including, for example,
orally, intravaginally, rectally, or parenterally, such as
intravenously, intramuscularly, subcutaneously, intraorbitally,
intracapsularly, intraperitoneally, intracisternally or by passive
or facilitated absorption through the skin using, for example, a
skin patch or transdermal iontophoresis, respectively. Furthermore,
the composition can be administered by injection, intubation or
topically, the latter of which can be passive, for example, by
direct application of an ointment or powder, or active, for
example, using a nasal spray or inhalant. An expression vector also
can be administered as a topical spray, in which case one component
of the composition is an appropriate propellant. The pharmaceutical
composition also can be incorporated, if desired, into liposomes,
microspheres or other polymer matrices (Felgner et al., U.S. Pat.
No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III
(2nd ed. 1993), each of which is incorporated herein by reference).
Liposomes, for example, which consist of phospholipids or other
lipids, are nontoxic, physiologically acceptable and metabolizable
carriers that are relatively simple to make and administer.
[0143] The expression vectors of the invention can be delivered to
the interstitial spaces of tissues of an animal body (Felgner et
al., U.S. Pat. Nos. 5,580,859 and 5,703,055). Administration of
expression vectors of the invention to muscle is a particularly
effective method of administration, including intradermal and
subcutaneous injections and transdermal administration. Transdermal
administration, such as by iontophoresis, is also an effective
method to deliver expression vectors of the invention to muscle.
Epidermal administration of expression vectors of the invention can
also be employed. Epidermal administration involves mechanically or
chemically irritating the outermost layer of epidermis to stimulate
an immune response to the irritant (Carson et al., U.S. Pat. No.
5,679,647).
[0144] Other effective methods of administering an expression
vector of the invention to stimulate an immune response include
mucosal administration (Carson et al., U.S. Pat. No. 5,679,647).
For mucosal administration, the most effective method of
administration includes intranasal administration of an appropriate
aerosol containing the expression vector and a pharmaceutical
composition. Suppositories and topical preparations are also
effective for delivery of expression vectors to mucosal tissues of
genital, vaginal and ocular sites. Additionally, expression vectors
can be complexed to particles and administered by a vaccine
gun.
[0145] The dosage to be administered is dependent on the method of
administration and will generally be between about 0.1 .mu.g up to
about 200 .mu.g. For example, the dosage can be from about 0.05
.mu.g/kg to about 50 mg/kg, in particular about 0.005-5 mg/kg. An
effective dose can be determined, for example, by measuring the
immune response after administration of an expression vector. For
example, the production of antibodies specific for the MHC class II
epitopes or MHC class I epitopes encoded by the expression vector
can be measured by methods well known in the art, including ELISA
or other immunological assays. In addition, the activation of T
helper cells or a CTL response can be measured by methods well
known in the art including, for example, the uptake of
.sup.3H-thymidine to measure T cell activation and the release of
.sup.51Cr to measure CTL activity (see Examples II and III
below).
[0146] The pharmaceutical compositions comprising an expression
vector of the invention can be administered to mammals,
particularly humans, for prophylactic or therapeutic purposes.
Examples of diseases that can be treated or prevented using the
expression vectors of the invention include infection with HBV,
HCV, HIV and CMV as well as prostate cancer, renal carcinoma,
cervical carcinoma, lymphoma, condyloma acuminatum and acquired
immunodeficiency syndrome (AIDS).
[0147] In therapeutic applications, the expression vectors of the
invention are administered to an individual already suffering from
cancer, autoimmune disease or infected with a virus. Those in the
incubation phase or acute phase of the disease can be treated with
expression vectors of the invention, including those expressing all
universal MHC class II epitopes, separately or in conjunction with
other treatments, as appropriate.
[0148] In therapeutic and prophylactic applications, pharmaceutical
compositions comprising expression vectors of the invention are
administered to a patient in an amount sufficient to elicit an
effective immune response to an antigen and to ameliorate the signs
or symptoms of a disease. The amount of expression vector to
administer that is sufficient to ameliorate the signs or symptoms
of a disease is termed a therapeutically effective dose. The amount
of expression vector sufficient to achieve a therapeutically
effective dose will depend on the pharmaceutical composition
comprising an expression vector of the invention, the manner of
administration, the state and severity of the disease being
treated, the weight and general state of health of the patient and
the judgment of the prescribing physician.
[0149] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0150] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
EXAMPLES
[0151] The following example is provided by way of illustration
only and not by way of limitation. Those of skill in the art will
readily recognize a variety of noncritical parameters that could be
changed or modified to yield essentially similar results.
Example I
Construction of Expression Vectors Containing MHC Class II
Epitopes
[0152] This example shows construction of expression vectors
containing MHC class II epitopes that can be used to target
antigens to MHC class II molecules.
[0153] Expression vectors comprising DNA constructs were prepared
using overlapping oligonucleotides, polymerase chain reaction (PCR)
and standard molecular biology techniques (Dieffenbach &
Dveksler, PCR Primer: A Laboratory Manual (1995); Sambrook et al.,
Molecular Cloning: A Laboratory Manual (2nd ed., 1989), each of
which is incorporated herein by reference).
[0154] To generate full length wild type Ii, the full length
invariant chain was amplified, cloned, and sequenced and used in
the construction of the three invariant chain constructs. Except
where noted, the source of cDNA for all the constructs listed below
was Mouse Spleen Marathon-Ready cDNA made from Balb/c males
(Clontech; Palo Alto Calif.). The primer pairs were the
oligonucleotide GCTAGCGCCGCCACCATGGATGACCAACGCG- ACCTC (SEQ ID
NO:40), which is designated murli-F and contains an NheI site
followed by the consensus Kozak sequence and the 5' end of the Ii
cDNA; and the oligonucleotide GGTACCTCACAGGGTGACTTGACCCAG (SEQ ID
NO:41), which is designated murli-R and contains a KpnI site and
the 3' end of the Ii coding sequence.
[0155] For the PCR reaction, 5 .mu.l of spleen cDNA and 250 nM of
each primer were combined in a 100 .mu.l reaction with 0.25 mM each
dNTP and 2.5 units of Pfu polymerase in Pfu polymerase buffer
containing 10 mM KCl, 10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM
Tris-chloride, pH 8.75, 2 mM MgSO.sub.4, 0.1% TRITON X-100 and 100
.mu.g/ml bovine serum albumin (BSA). A Perkin/Elmer 9600 PCR
machine (Perkin Elmer; Foster City Calif.) was used and the cycling
conditions were: 1 cycle of 95.degree. C. for 5 minutes, followed
by 30 cycles of 95.degree. C. for 15 seconds, 52.degree. C. for 30
seconds, and 72.degree. C. for 1 minute. The PCR reaction was run
on a 1% agarose gel, and the 670 base pair product was cut out,
purified by spinning through a Millipore Ultrafree-MC filter
(Millipore; Bedford Mass.) and cloned into pCR-Blunt from
Invitrogen (San Diego, Calif.). Individual clones were screened by
sequencing, and a correct clone (named bli#3) was used as a
template for the helper constructs.
[0156] DNA constructs containing pan DR epitope sequences and MHC
II targeting sequences derived from the li protein were prepared.
The Ii murine protein has been previously described (Zhu &
Jones, Nucleic Acids Res. 17:447-448 (1989)), which is incorporated
herein by reference. Briefly, the 11PADRE construct contains the
full length Ii sequence with PADRE precisely replacing the CLIP
region. The DNA construct encodes amino acids 1 through 87 of
invariant chain, followed with the 13 amino acid PADRE sequence
(SEQ ID NO:52) and the rest of the invariant chain DNA sequence
(amino acids 101-215). The construct was amplified in 2 overlapping
halves that were joined to produce the final construct. The two
primers used to amplify the 5' half were murli-F and the
oligonucleotide CAGGGTCCAGGCAGCCACGAACTTGGCCACAGGTTTGGCAGA (SEQ ID
NO:42), which is designated IiPADRE-R. The IiPADRE-R primer
includes nucleotides 303-262 of IiPADRE. The 3' half was amplified
with the primer GGCTGCCTGGACCCTGAAGGCTGCCGCTATGTCCATGGATAAC (SEQ ID
NO:43), which is designated IiPADRE-F and includes nucleotides
288-330 of IiPADRE; and murli-R. The PCR conditions were the same
as described above, and the two halves were isolated by agarose gel
electrophoresis as described above.
[0157] Ten microliters of each PCR product was combined in a 100
.mu.l PCR reaction with an annealing temperature of 50.degree. C.
for five cycles to generate a full length template. Primers murli-F
and murli-R were added and 25 more cycles carried out. The full
length IiPADRE product was isolated, cloned, and sequenced as
described above. This construct contains the murine Ii gene with a
pan DR epitope sequence substituted for the CLIP sequence of Ii
(FIG. 1).
[0158] A DNA construct, designated 180T, containing the cytoplasmic
domain, the transmembrane domain and part of the luminal domain of
Ii fused to a string of multiple MHC class II epitopes was
constructed (FIG. 2). Briefly, the string of multiple MHC class II
epitopes was constructed with three overlapping oligonucleotides
(oligos). Each oligo overlapped its neighbor by 15 nucleotides and
the final MHC class II epitope string was assembled by extending
the overlapping oligonucleotides in three sets of reactions using
PCR. The three oligonucleotides were: oligo 1, nucleotides
241-310,
1 oligo 1, nucleotides 241-310, CTTCGCATGAAGCTTATCAGCCAGGC-
TGTGCACGCCGCTCACGCCGAAATCAACGA (SEQ ID NO:44) AGCTGGAAGAACCC; oligo
2, nucleotides 364-295, TTCTGGTCAGCAGAAAGAACAGGATA-
GGAGCGTTTGGAGGGCGATAAGCTGGAGGG (SEQ ID NO:45) GTTCTTCCAGCTTC; and
oligo 3, nucleotides 350-42, TTCTGCTGACCAGAATCCTGACA-
ATCCCCCAGTCCCTGGACGCCAAGTTCGTGGCTG (SEQ ID NO:46)
CCTGGACCCTGAAG.
[0159] For the first PCR reaction, 5 .mu.g of oligos 1 and 2 were
combined in a 100 .mu.l reaction containing Pfu polymerase. A
Perkin/Elmer 9600 PCR machine was used and the annealing
temperature used was 45.degree. C. The PCR product was
gel-purified, and a second reaction containing the PCR product of
oligos 1 and 2 with oligo 3 was annealed and extended for 10 cycles
before gel purification of the full length product to be used as a
"mega-primer."
[0160] The 180T construct was made by amplifying bli#3 with murli-F
and the mega-primer. The cycling conditions were: 1 cycle of
95.degree. C. for 5 minutes, followed by 5 cycles of 95.degree. C.
for 15 seconds, 37.degree. C. for 30 seconds, and 72.degree. C. for
1 minute. Primer Help-epR was added and an additional 25 cycles
were carried out with the annealing temperature raised to
47.degree. C. The Help-epR primer
GGTACCTCAAGCGGCAGCCTTCAGGGTCCAGGCA (SEQ ID NO:47) corresponds to
nucleotides 438-405. The full length 180T product was isolated,
cloned, and sequenced as above.
[0161] The 180T construct (FIG. 2) encodes amino acid residues 1
through 80 of Ii, containing the cytoplasmic domain, the
transmembrane domain and part of the luminal domain, fused to a
string of multiple MHC class II epitopes corresponding to: amino
acid residues 323-339 of ovalbumin
(IleSerGlnAlaValHisAlaAlaHisAlaGluIleAsnGluAlaGlyArg; SEQ ID
NO:48); amino acid residues 128 to 141 of HBV core antigen (amino
acids ThrProProAlaTyrArgProProAsnAlaProlleLeu; SEQ ID NO:49); amino
acid residues 182 to 196 of HBV env (amino acids
PhePheLeuLeuThrArglIeLeuThrIl- eProGlnSerLeuAsp; SEQ ID NO:50); and
the pan DR sequence designated SEQ ID NO:52.
[0162] A DNA construct containing the cytoplasmic domain,
transmembrane domain and a portion of the luminal domain of Ii
fused to the MHC class II epitope string shown in FIG. 2 and amino
acid residues 101 to 215 of Ii encoding the trimerization region of
Ii was generated (FIG. 3). This construct, designated IiThfull,
encodes the first 80 amino acids of invariant chain followed by the
MHC class II epitope string (replacing CLIP) and the rest of the
invariant chain (amino acids 101-215). Briefly, the construct was
generated as two overlapping halves that were annealed and extended
by PCR to yield the final product.
[0163] The 5' end of IiThfull was made by amplifying 180T with
murli-F (SEQ ID NO:40) and Th-Pad-R. The Th-Pad-R primer
AGCGGCAGCCTTCAGGGTC (SEQ ID NO:51) corresponds to nucleotides
429-411. The 3' half was made by amplifying bli#3 with IiPADRE-F
and murli-R (SEQ ID NO:41). The IiPADRE-F primer
GGCTGCCTGGACCCTGAAGGCTGCCGCTATGTCCATGGATAAC (SEQ ID NO:43)
corresponds to nucleotides 402-444. Each PCR product was gel
purified and mixed, then denatured, annealed, and extended by five
cycles of PCR. Primers murli-F (SEQ ID NO:40) and murli-R (SEQ ID
NO:41) were added and another 25 cycles performed. The full length
product was gel purified, cloned, and sequenced.
[0164] All of the remaining constructs described below were made
essentially according to the scheme shown in FIG. 18. Briefly,
primer pairs IF plus 1R, designated below for each specific
construct, were used to amplify the specific signal sequence and
contained an overlapping 15 base pair tail identical to the 5' end
of the MHC class II epitope string. Primer pair Th-ova-F,
ATCAGCCAGGCTGTGCACGC (SEQ ID NO:53), plus Th-Pad-R (SEQ ID NO:51)
were used to amplify the MHC class II epitope string. A 15 base
pair overlap and the specific transmembrane and cytoplasmic tail
containing the targeting signals were amplified with primer pairs
2F plus 2R.
[0165] All three pieces of each cDNA were amplified using the
following conditions: 1 cycle of 95.degree. C. for 5 minutes,
followed by 30 cycles of 95.degree. C. for 15 seconds, 52.degree.
C. for 30 seconds, and 72.degree. C. for 1 minute. Each of the
three fragments was agrose-gel purified, and the signal sequence
and MHC class II string fragments were combined and joined by five
cycles in a second PCR. After five cycles, primers IF and Th-Pad-R
were added for 25 additional cycles and the PCR product was gel
purified. This signal sequence plus MHC class II epitope string
fragment was combined with the transmembrane plus cytoplasmic tail
fragment for the final PCR. After five cycles, primers IF plus 2R
were added for 25 additional cycles and the product was gel
purified, cloned and sequenced.
[0166] A DNA construct containing the murine immunoglobulin kappa
signal sequence fused to the T helper epitope string shown in FIG.
2 and the transmembrane and cytoplasmic domains of LAMP-1 was
generated (FIG. 4) (Granger et al., J. Biol. Chem. 265:12036-12043
(1990)), which is incorporated by reference (mouse LAMP-1 GenBank
accession No. M32015). This construct, designated kappaLAMP-Th,
contains the consensus mouse immunoglobulin kappa signal sequence
and was amplified from a plasmid containing full length
immunoglobulin kappa as depicted in FIG. 18. The primer IF used was
the oligonucleotide designated KappaSig-F,
GCTAGCGCCGCCACCATGGGAATGCAG (SEQ ID NO:54). The primer IR used was
the oligonucleotide designated Kappa-Th-R,
CACAGCCTGGCTGATTCCTCTGGACCC (SEQ ID NO:55). The primer 2F used was
the oligonucleotide designated PAD/LAMP-F,
CTGAAGGCTGCCGCTAACAACATGTTGATCCCC (SEQ ID NO:56). The primer 2R
used was the oligonucleotide designated LAMP-CYTOR,
GGTACCCTAGATGGTCTGATAGCC (SEQ ID NO:57).
[0167] A DNA construct containing the signal sequence of H2-M fused
to the MHC class II epitope string shown in FIG. 2 and the
transmembrane and cytoplasmic domains of H2-M was generated (FIG.
5). The mouse H2-M gene has been described previously, Peleraux et
al., Immunogenetics 43:204-214 (1996)), which is incorporated
herein by reference. This construct was designated H2M-Th and was
constructed as depicted in FIG. 18. The primer IF used was 1the
oligonucleotide designated H2-Mb-1F, GCC GCT AGC GCC GCC ACC ATG
GCT GCA CTC TGG (SEQ ID NO:58). The primer IR used was the
oligonucleotide designated H2-Mb-IR, CAC AGC CTG GCT GAT CCC CAT
ACA GTG CAG (SEQ ID NO:59). The primer 2F used was the
oligonucleotide designated H2-Mb-2F, CTG AAG GCT GCC GCT AAG GTC
TCT GTG TCT (SEQ ID NO:60). The primer 2R used was the
oligonucleotide designated H2-Mb-2R, GCG GGT ACC CTA ATG CCG TCC
TTC (SEQ ID NO:61).
[0168] A DNA construct containing the signal sequence of H2-DO
fused to the MHC class II epitope string shown in FIG. 2 and the
transmembrane and cytoplasmic domains of H2-DO was generated (FIG.
6). The mouse H2-DO gene has been described previously (Larhammar
et al., J. Biol. Chem. 260:14111-14119 (1985)), which is
incorporated herein by reference (GenBank accession No. M19423).
This construct, designated H.sub.2O-Th, was constructed as depicted
in FIG. 18. The primer IF used was the oligonucleotide designated
H2-Ob-lF, GCG GCT AGC GCC GCC ACC ATG GGC GCT GGG AGG (SEQ ID
NO:62). The primer 1 R used was the oligonucleotide designated
H2-Ob-1R, TGC ACA GCC TGG CTG ATG GAA TCC AGC CTC (SEQ ID NO:63).
The primer 2F used was the oligonucleotide designated H2-Ob-2F, CTG
AAG GCT GCC GCT ATA CTG AGT GGA GCT (SEQ ID NO:64). The primer 2R
used was the oligonucleotide designated H2-Ob-2R, GCC GGT ACC TCA
TGT GAC ATG TCC CG (SEQ ID NO:65).
[0169] A DNA construct containing a pan DR epitope sequence (SEQ ID
NO:52) fused to the amino-terminus of influenza matrix protein is
generated (FIG. 7). This construct, designated PADRE-Influenza
matrix, contains the universal MHC class II epitope PADRE attached
to the amino terminus of the influenza matrix coding sequence. The
construct is made using a long primer on the 5' end primer. The 5'
primer is the oligonucleotide
GCTAGCGCCGCCACCATGGCCAAGTTCGTGGCTGCCTGGACCCTGAAGGCTGCCGCT
ATGAGTCTTCTAACCGAGGTCGA (SEQ ID NO:66). The 3' primer is the
oligonucleotide TCACTTGAATCGCTGCATCTGCACCCCCAT (SEQ ID NO:67).
Influenza virus from the America Type Tissue Collection (ATCC) is
used as a source for the matrix coding region (Perdue et al.
Science 279:393-396 (1998)), which is incorporated herein by
reference (GenBank accession No. AF036358).
[0170] A DNA construct containing a pan DR epitope sequence (SEQ ID
NO:52) fused to the amino-terminus of HBV-S antigen was generated
(FIG. 8). This construct is designated PADRE-HBV-s and was
generated by annealing two overlapping oligonucleotides to add
PADRE onto the amino terminus of hepatitis B surface antigen
(Michel et al., Proc. Natl. Acad. Sci. USA 81:7708-7712 (1984);
Michel et al., Proc. Natl. Acad. Sci. USA 92:5307-5311 (1995)),
each of which is incorporated herein by reference. One
oligonucleotide was
GCTAGCGCCGCCACCATGGCCAAGTTCGTGGCTGCCTGGACCCTGAAGG- CTGCCGCT C (SEQ
ID NO:68). The second oligonucleotide was
CTCGAGAGCGGCAGCCTTCAGGGTCCAGGCAGCCACGAACTTGGCCATGGTGGCGG CG (SEQ ID
NO:69). When annealed, the oligos have NheI and XhoI cohesive ends.
The oligos were heated to 100.degree. C. and slowly cooled to room
temperature to anneal. A three part ligation joined PADRE with an
XhoI-KpnI fragment containing HBV-s antigen into the NheI plus KpnI
sites of the expression vector.
[0171] A DNA construct containing the signal sequence of Ig-.alpha.
fused to the MHC class II epitope string shown in FIG. 2 and the
transmembrane and cytoplasmic domains of Ig-.alpha. was generated
(FIG. 9). The mouse Ig-.alpha. gene has been described previously
(Kashiwamura et al., J. Immunol. 145:337-343 (1990)), which is
incorporated herein by reference (GenBank accession No. M31773).
This construct, designated Ig-alphaTh, was constructed as depicted
in FIG. 18. The primer 1 F used was the oligonucleotide designated
Ig alpha-1 F, GCG GCT AGC GCC GCC ACC ATG CCA GGG GGT CTA (SEQ ID
NO:70). The primer IR used was the oligonucleotide designated
Igalpha-1R, GCA CAG CCT GGC TGA TGG CCT GGC ATC CGG (SEQ ID NO:71).
The primer 2F used was the oligonucleotide designated Igalpha-2F,
CTG AAG GCT GCC GCT GGG ATC ATC TTG CTG (SEQ ID NO:72). The primer
2R used was the oligonucleotide designated Igalpha-2R, GCG GGT ACC
TCA TGG CTT TTC CAG CTG (SEQ ID NO:73).
[0172] A DNA construct containing the signal sequence of Ig-.beta.
fused to the MHC class II string shown in FIG. 2 and the
transmembrane and cytoplasmic domains of Ig.beta. was generated
(FIG. 10). The Ig-.beta. sequence is the B29 gene of mouse and has
been described previously (Hermanson et al., Proc. Natl. Acad. Sci.
USA 85:6890-6894 (1988)), which is incorporated herein by reference
(GenBank accession No. J03857). This construct, designated
Ig-betaTh, was constructed as depicted in FIG. 18. The primer IF
used was the oligonucleotide designated B29-1F (33mer) GCG GCT AGC
GCC GCC ACC ATG GCC ACA CTG GTG (SEQ ID NO:74). The primer IR used
was the oligonucleotide designated B29-1R (30mer) CAC AGC CTG GCT
GAT CGG CTC ACC TGA GAA (SEQ ID NO:75). The primer 2F used was the
oligonucleotide designated B292F (30mer) CTG AAG GCT GCC GCT ATT
ATC TTG ATC CAG (SEQ ID NO: 76). The primer 2R used was the
oligonucleotide designated B29-2R (27mer), GCC GGT ACC TCA TTC CTG
GCC TGG ATG (SEQ ID NO:77).
[0173] A DNA construct containing the signal sequence of the kappa
immunoglobulin signal sequence fused to the MHC class II epitope
string shown in FIG. 2 was constructed (FIG. 11). This construct is
designated SigTh and was generated by using the kappaLAMP-Th
construct (shown in FIG. 4) and amplifying with the primer pair
KappaSig-F (SEQ ID NO:54) plus Help-epR (SEQ ID NO:47) to create
SigTh. SigTh contains the kappa immunoglobulin signal sequence
fused to the T helper epitope string and terminated with a
translational stop codon.
[0174] Constructs encoding human sequences corresponding to the
above described constructs having mouse sequences are prepared by
substituting human sequences for the mouse sequences. Briefly, for
the IiPADRE construct, corresponding to FIG. 1, amino acid residues
1-80 from the human Ii gene HLA-DR sequence (FIG. 12) (GenBank
accession No. X00497 Ml 4765) is substituted for the mouse Ii
sequences, which is fused to PADRE, followed by human invariant
chain HLA-DR amino acid residues 114-223. For the 180T construct,
corresponding to FIG. 2, amino acid residues 1-80 from the human
sequence of Ii is followed by a MHC class II epitope string. For
the IiThfull construct, corresponding to FIG. 3, amino acid
residues 1-80 from the human sequence of Ii, which is fused to a
MHC class II epitope string, is followed by human invariant chain
amino acid residues 114-223.
[0175] For the LAMP-Th construct, similar to FIG. 4, the signal
sequence encoded by amino acid residues 1-19 (nucleotides 11-67) of
human LAMP-1 (FIG. 13) (GenBank accession No. J04182), which is
fused to the MHC class II epitope string, is followed by the
transmembrane (nucleotides 1163-1213) and cytoplasmic tail
(nucleotides 1214-1258) region encoded by amino acid residues
380-416 of human LAMP-1.
[0176] For the HLA-DM-Th construct, corresponding to FIG. 5, the
signal sequence encoded by amino acid residues 1-17 (nucleotides
1-51) of human HLA-DMB (FIG. 14) (GenBank accession No. UI 5085),
which is fused to the MHC class II epitope string, is followed by
the transmembrane (nucleotides 646-720) and cytoplasmic tail
(nucleotides 721-792) region encoded by amino acid residues 216-263
of human HLA-DMB.
[0177] For the HLA-DO-Th construct, corresponding to FIG. 6, the
signal sequence encoded by amino acid residues 1-21 (nucleotides
1-63) of human HLA-DO (FIG. 15) (GenBank accession No. L29472
J02736 N00052), which is fused to the MHC class II epitope string,
is followed by the transmembrane (nucleotides 685-735) and
cytoplasmic tail (nucleotides 736-819) region encoded by amino acid
residues 223-273 of human HLA-DO.
[0178] For the Ig-alphaTh construct, corresponding to FIG. 9, the
signal sequence encoded by amino acid residues 1-29 (nucleotides
1-87) of human Ig-.alpha. MB-1 (FIG. 16) (GenBank accession No.
U05259), which is fused to the MHC class II epitope string, is
followed by the transmembrane (nucleotides 424-498) and cytoplasmic
tail (nucleotides 499-678) region encoded by amino acid residues
142-226 of human Ig-.alpha. MB-1.
[0179] For the Ig-betaTh construct, corresponding to FIG. 10, the
signal sequence encoded by amino acid residues 1-28 (nucleotides
17-100) of human Ig-.beta. B29 (FIG. 17) (GenBank accession No.
M80461), which is fused to the MHC class II epitope string, is
followed by the transmembrane (nucleotides 500-547) and cytoplasmic
tail (nucleotides 548-703) region encoded by amino acid residues
156-229 of human Ig-.beta..
[0180] The SigTh construct shown in FIG. 11 can be used in mouse
and human. Alternatively, a signal sequence derived from an
appropriate human gene containing a signal sequence can be
substituted for the mouse kappa immunoglobulin sequence in the Sig
Th construct.
[0181] The PADRE-Influenza matrix construct shown in FIG. 7 and the
PADRE-HBVs construct shown in FIG. 8 can be used in mouse and
human.
[0182] Some of the DNA constructs described above were cloned into
the vector pEP2 (FIG. 19; SEQ ID NO:35). The pEP2 vector was
constructed to contain dual CMV promoters. The pEP2 vector used the
backbone of pcDNA3.1 (-)Myc-His A from Invitrogen and pIRES 1 hyg
from Clontech. Changes were made to both vectors before the CMV
transcription unit from pIRES1hyg was moved into the modified pcDNA
vector.
[0183] The pcDNA3.1(-)Myc-HisAvector (http://www.invitrogen.com)
was modified. Briefly, the PvuII fragment (nucleotides 1342-3508)
was deleted. A BspHI fragment that contains the Ampicillin
resistance gene (nucleotides 4404-5412) was cut out. The Ampicillin
resistance gene was replaced with the kanamycin resistance gene
from pUC4K (GenBank Accession #X06404). pUC4K was amplified with
the primer set: TCTGATGTTACATTGCACAAG (SEQ ID NO:78) (nucleotides
1621-1601) and GCGCACTCATGATGCTCTGCCAGTGTTACA- ACC (SEQ ID NO:79)
(nucleotides 682-702 plus the addition of a BspHI restriction site
on the 5' end). The PCR product was digested with BspHI and ligated
into the vector digested with BspHI. The region between the PmeI
site at nucleotide 905 and the EcoRV site at nucleotide 947 was
deleted. The vector was then digested with PmeI (cuts at nucleotide
1076) and ApaI (cuts at nucleotide 1004), Klenow filled in at the
cohesive ends and ligated. The KpnI site at nucleotide 994 was
deleted by digesting with KpnI and filling in the ends with Klenow
DNA polymerase, and ligating. The intron A sequence from CMV
(GenBank accession M21295, nucleotides 635-1461) was added by
amplifying CMV DNA with the primer set:
GCGTCTAGAGTAAGTACCGCCTATAGACTC (SEQ ID NO:80) (nucleotides 635-655
plus an XbaI site on the 5' end) and CCGGCTAGCCTGCAGAAAAGACCCATGGAA
(SEQ ID NO:81) (nucleotides 1461-1441 plus an NheI site on the 3'
end). The PCR product was digested with XbaI and NheI and ligated
into the NheI site of the vector (nucleotide 895 of the original
pcDNA vector) so that the NheI site was on the 3' end of the
intron.
[0184] To modify the pIRES1hyg vector (GenBank Accession U89672,
Clontech), the KpnI site (nucleotide 911) was deleted by cutting
and filling in with Klenow. The plasmid was cut with NotI
(nucleotide 1254) and XbaI (nucleotide 3196) and a polylinker oligo
was inserted into the site. The polylinker was formed by annealing
the following two oligos:
GGCCGCAAGGAAAAAATCTAGAGTCGGCCATAGACTAATGCCGGTACCG (SEQ ID NO:82)
and CTAGCGGTACCGGCATTAGTCTATGGCCCGACTCTAGATTTTTTCCTTGC (SEQ ID
NO:83). The resulting plasmid was cut with HincII and the fragment
between HincII sites 234 and 3538 was isolated and ligated into the
modified pcDNA vector. This fragment contains a CMV promoter,
intron, polylinker, and polyadenylation signal.
[0185] The pIREShyg piece and the pcDNA piece were combined to form
pEP2. The modified pcDNA3.1 (-)Myc-His A vector was partially
digested with PvuII to isolate a linear fragment with the cut
downstream of the pcDNA polyadenylation signal (the other PvuII
site is the CMV intron). The HincII fragment from the modified
pIRES1hyg vector was ligated into the PvuII cut vector. The
polyadenylation signal from the pcDNA derived transcription unit
was deleted by digesting with EcoRI (pcDNA nucleotide 955) and XhoI
(pIRES1hyg nucleotide 3472) and replaced with a synthetic
polyadenylation sequence. The synthetic polyadenylation signal was
described in Levitt et al., Genes and Development 3:1019-1025
(1989)).
[0186] Two oligos were annealed to produce a fragment that
contained a polylinker and polyadenylation signal with EcoRI and
XhoI cohesive ends. The oligos were:
2 AATTCGGATATCCAAGCTTGATGAATAAAAGATCAGAGCTCTAGTGATCTGTGTGTTG (SEQ
ID NO:84) GTTTTTTTGTGTGC and
TCGAGCACACAAAAAACCAACACACAGATCACTAGAGCTCTGATCTTTTTATTCATC (SEQ ID
NO:85) AAGCTTGGATATCCG.
[0187] The resulting vector is named pEP2 and contains two separate
transcription units. Both transcription units use the same CMV
promoter but each contains different intron, polylinker, and
polyadenylation sequences.
[0188] The pEP2 vector contains two transcription units. The first
transcription unit contains the CMV promoter initially from pcDNA
(nucleotides 210-862 in FIG. 19), CMV intron A sequence
(nucleotides 900-1728 in FIG. 19), polylinker cloning site
(nucleotides 1740-1760 in FIG. 19) and synthetic polyadenylation
signal (nucleotides 1764-1769 in FIG. 19). The second transcription
unit, which was initially derived from pIRES1hyg, contains the CMV
promoter (nucleotides 3165-2493 in FIG. 19), intron sequence
(nucleotides 2464-2173 in FIG. 19), polylinker clone site
(nucleotides 2126-2095 in FIG. 19) and bovine growth hormone
polyadenylation signal (nucleotides 1979-1974 in FIG. 19). The
kanamycin resistance gene is encoded in nucleotides 4965-4061 (FIG.
19).
[0189] The DNA constructs described above were digested with NheI
and KpnI and cloned into the XbaI and KpnI sites of pEP2 (the
second transcription unit).
[0190] Additional vectors were also constructed. To test for the
effect of co-expression of MHC class I epitopes with MHC class II
epitopes, an insert was generated, designated AOS, that contains
nine MHC class I epitopes. The AOS insert was initially constructed
in the vector pMIN.0 (FIG. 20; SEQ ID NO:36). Briefly, the AOS
insert contains nine MHC class I epitopes, six restricted by HLA-A2
and three restricted by HLA-A11, and the universal MHC class II
epitope PADRE. The vector pMIN.0 contains epitopes from HBV, HIV
and a mouse ovalbumin epitope. The MHC class I epitopes appear in
pMIN.0 in the following order: consensus mouse Ig Kappa signal
sequence (pMIN.0 amino acid residues 1-20, nucleotides 16-81)
MQVQIQSLFLLLLWVPGSRG (SEQ ID NO:86) encoded by nucleotides ATG CAG
GTG CAG ATC CAG AGC CTG TTT CTG CTC CTC CTG TGG GTG CCC GGG TCC AGA
GGA (SEQ ID NO:87); HBV pol 149-159 (A11 restricted) (pMIN.0 amino
acid residues 21-31, nucleotides 82-114) HTLWKAGILYK (SEQ ID NO:88)
encoded by nucleotides CAC ACC CTG TGG AAG GCC GGA ATC CTG TAT AAG
(SEQ ID NO:89); PADRE-universal MHC class II epitope (pMIN.0 amino
acid residues 32-45, nucleotides 115-153) AKFVAAWTLKAAA (SEQ ID
NO:52) encoded by nucleotides GCC AAG TTC GTG GCT GCC TGG ACC CTG
AAG GCT GCC GCT (SEQ ID NO:90); HBV core 18-27 (A2 restricted)
(pMIN.0 amino acid residues 46-55, nucleotides 154-183) FLPSDFFPSV
(SEQ ID NO:91) encoded by nucleotides TTC CTG CCT AGC GAT TTC TTT
CCT AGC GTG (SEQ ID NO:92); HIV env 120-128 (A2 restricted) (pMIN.0
amino acid residues 56-64, nucleotides 184-210) KLTPLCVTL (SEQ ID
NO:93) encoded by nucleotides AAG CTG ACC CCA CTG TGC GTG ACC CTG
(SEQ ID NO:94); HBV pol 551-559 (A2 restricted) (pMIN.0 amino acid
residues 65-73, nucleotides 211-237) YMDDVVLGA (SEQ ID NO:95)
encoded by nucleotides TAT ATG GAT GAC GTG GTG CTG GGA GCC (SEQ ID
NO:96); mouse ovalbumin 257-264 (K.sup.b restricted) (pMIN.0 amino
acid residues 74-81, nucleotides 238-261) SIINFEKL (SEQ ID NO:97)
encoded by nucleotides AGC ATC ATC AAC TTC GAG AAG CTG (SEQ ID
NO:98); HBV pol 455-463 (A2 restricted) (pMIN.0 amino acid residues
82-90, nucleotides 262-288) GLSRYVARL (SEQ ID NO:99) encoded by
nucleotides GGA CTG TCC AGA TAC GTG GCT AGG CTG (SEQ ID NO:100);
HIV pol 476-84 (A2 restricted) (pMIN.0 amino acid residues 91-99,
nucleotides 289-315) ILKEPVHGV (SEQ ID NO:101) encoded by
nucleotides ATC CTG AAG GAG CCT GTG CAC GGC GTG (SEQ ID NO:102);
HBV core 141-151 (A11 restricted) (pMIN.0 amino acid residues
100-110, nucleotides 316-348) STLPETTVVRR (SEQ ID NO: 103) encoded
by nucleotides TCC ACC CTG CCA GAG ACC ACC GTG GTG AGG AGA (SEQ ID
NO:104); HIV env 49-58 (Al1 restricted) (pMIN.0 amino acid residues
111-120, nucleotides 349-378) TVYYGVPVWK (SEQ ID NO: 105) encoded
by nucleotides ACC GTG TAC TAT GGA GTG CCT GTG TGG AAG (SEQ ID
NO:106); and HBV env 335-343 (A2 restricted) (pMIN.0 amino acid
residues 121-129, nucleotides 378-405) WLSLLVPFV (SEQ ID NO: 107)
encoded by nucleotides TGG CTG AGC CTG CTG GTG CCC TTT GTG (SEQ ID
NO: 108).
[0191] The pMIN.0 vector contains a KpnI restriction site (pMIN.0
nucleotides 406-411) and a NheI restriction site (pMIN.0
nucleotides 1-6). The pMIN.0 vector contains a consensus Kozak
sequence (nucleotides 7-18) (GCCGCCACCATG; SEQ ID NO: 109) and
murine Kappa Ig-light chain signal sequence followed by a string of
10 MHC class I epitopes and one universal MHC class II epitope. The
pMIN.0 sequence encodes an open reading frame fused to the Myc and
His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
The pMIN.0 vector was constructed with eight oligonucleotides: Min1
oligo GAGGAGCAGAAACAGGCTCTGGATCTGCACCTGCATTC- CCATGGTGGCGGCGCTAGC
AAGCTTCTTGCGC (SEQ ID NO:110); Min2 oligo
CCTGTTTCTGCTCCTCCTGTGGGTGCCCGGGTCCAGAGGACACACCCTGTGGAAGGC
CGGAATCCTGTATA (SEQ ID NO:111); Min3 oligo
TCGCTAGGCAGGAAAGCGGCAGCCTTCAGGGTCCAGGCAGCCACG- AACTTGGCCTT
ATACAGGATTCCGG (SEQ ID NO:112); Min4 oligo
CTTTCCTGCCTAGCGATTTCTTTCCTAGCGTGAAGCTGACCCCACTGTGCGTGACCCT
GTATATGGATGAC (SEQ ID NO:113); Min5 oligo
CGTACCTGGACAGTCCCAGCTTCTCGAAGTTGATGATGCTGGCTC- CCAGCACCACGT
CATCCATATACAG (SEQ ID NO: 114); Min6 oligo
GGACTGTCCAGATACGTGGCTAGGCTGATCCTGAAGGAGCCTGTGCACGGCGTGTCC
ACCCTGCCAGAGAC (SEQ ID NO: 115); Min7 oligo
GCTCAGCCACTTCCACACAGGCACTCCATAGTACACGGTCCTCC- TCACCACGGTGGT
CTCTGGCAGGGTG (SEQ ID NO:116); Min8 oligo
GTGGAAGTGGCTGAGCCTGCTGGTGCCCTTTGTGGGTACCTGATCTAGAGC (SEQ ID
NO:117).
[0192] Additional primers were flanking primer 5', GCG CAA GAA GCT
TGC TAG CG (SEQ ID NO: 118) and flanking primer 3', GCT CTA GAT CAG
GTA CCC CAC (SEQ ID NO:119).
[0193] The original pMIN.0 minigene construction was carried out
using eight overlapping oligos averaging approximately 70
nucleotides in length, which were synthesized and HPLC purified by
Operon Technologies Inc. Each oligo overlapped its neighbor by 15
nucleotides, and the final multi-epitope minigene was assembled by
extending the overlapping oligos in three sets of reactions using
PCR (Ho et al., Gene 77:51-59 (1989).
[0194] For the first PCR reaction, 5 .mu.g of each of two oligos
were annealed and extended: 1+2, 3+4, 5+6, and 7+8 were combined in
100 .mu.l reactions containing 0.25 mM each dNTP and 2.5 units of
Pfu polymerase in Pfu polymerase buffer containing 10 mM KCl, 10 mM
(NH.sub.4).sub.2SO.sub.- 4, 20 mM Tris-chloride, pH 8.75, 2 mM
MgSO.sub.4, 0.1% TRITON X-100 and 100 mg/ml BSA. A Perkin/Elmer
9600 PCR machine was used and the annealing temperature used was
5.degree. C. below the lowest calculated T.sub.m of each primer
pair. The full length dimer products were gel-purified, and two
reactions containing the product of 1-2 and 3-4, and the product of
5-6 and 7-8 were mixed, annealed and extended for 10 cycles. Half
of the two reactions were then mixed, and 5 cycles of annealing and
extension carried out before flanking primers were added to amplify
the full length product for 25 additional cycles. The full length
product was gel purified and cloned into pCR-blunt (Invitrogen) and
individual clones were screened by sequencing. The Min insert was
isolated as an NheI-KpnI fragment and cloned into the same sites of
pcDNA3.1 (-)/Myc-His A (Invitrogen) for expression. The Min protein
contains the Myc and His antibody epitope tags at its
carboxyl-terminal end.
[0195] For all the PCR reactions described, a total of 30 cycles
were performed using Pfu polymerase and the following conditions:
95.degree. C. for 15 seconds, annealing temperature for 30 seconds,
72.degree. C. for one minute. The annealing temperature used was
5.degree. C. below the lowest calculated T.sub.m of each primer
pair.
[0196] Three changes to pMIN.0 were made to produce pMIN.1 (FIG.
21; SEQ ID NO:37, also referred to as pMIN-AOS). The mouse ova
epitope was removed, the position 9 alanine anchor residue (#547)
of HBV pol 551-560 was converted to a valine which increased the in
vitro binding affinity 40-fold, and a translational stop codon was
introduced at the end of the multi-epitope coding sequence. The
changes were made by amplifying two overlapping fragments and
combining them to yield the full length product.
[0197] The first reaction used the 5' pcDNA vector primer T7 and
the primer Min-ovaR (nucleotides 247-218)
TGGACAGTCCCACTCCCAGCACCACGTCAT (SEQ ID NO: 120). The 3' half was
amplified with the primers: Min-ovaF (nucleotides 228-257)
GCTGGGAGTGGGACTGTCCAGGTACGTGGC (SEQ ID NO:121) and Min-StopR
(nucleotides 390-361) GGTACCTCACACAAAGGGCACCAGCAGGC (SEQ ID
NO:122)
[0198] The two fragments were gel purified, mixed, denatured,
annealed, and filled in with five cycles of PCR. The full length
fragment was amplified with the flanking primers T7 and Min-Stop
for 25 more cycles. The product was gel purified, digested with
NheI and KpnI and cloned into pcDNA3.1 for sequencing and
expression. The insert from pMin. I was isolated as an NheI-KpnI
fragment and cloned into pEP2 to make pEP2-AOS.
Example II
Assay for T Helper Cell Activation
[0199] This example shows methods for assaying T helper cell
activity. One method for assaying T helper cell activity uses
spleen cells of an immunized organism. Briefly, a spleen cell
pellet is suspended with 2-3 ml of red blood cell lysis buffer
containing 8.3 g/liter ammonium chloride in 0.001 M Tris-HCl, pH
7.5. The cells are incubated in lysis buffer for 3-5 min at room
temperature with occasional vortexing. An excess volume of 50 ml of
R10 medium is added to the cells, and the cells are pelleted. The
cells are resuspended and pelleted one or two more times in R2
medium or R10 medium.
[0200] The cell pellet is suspended in R10 medium and counted. If
the cell suspension is aggregated, the aggregates are removed by
filtration or by allowing the aggregates to settle by gravity. The
cell concentration is brought to 10.sup.7/ml, and 100 .mu.l of
spleen cells are added to 96 well flat bottom plates.
[0201] Dilutions of the appropriate peptide, such as pan DR epitope
(SEQ ID NO:145), are prepared in R10 medium at 100, 10, 1, 0.1 and
0.01 .mu.g/ml, and 100 .mu.l of peptide are added to duplicate or
triplicate wells of spleen cells. The final peptide concentration
is 50, 5, 0.5, 0.05 and 0.005 .mu.g/ml. Control wells receive 100
.mu.l R10 medium.
[0202] The plates are incubated for 3 days at 37.degree. C. After 3
days, 20 .mu.l of 50 .mu.Ci/ml .sup.3H-thymidine is added per well.
Cells are incubated for 18-24 hours and then harvested onto glass
fiber filters. The incorporation of .sup.3H-thymidine into DNA of
proliferating cells is measured in a beta counter.
[0203] A second assay for T helper cell activity uses peripheral
blood mononuclear cells (PBMC) that are stimulated in vitro as
described in Alexander et al., supra and Sette (WO 95/07,707), as
adapted from Manca et al, J. Immunol. 146:1964-1971 (1991), which
is incorporated herein by reference. Briefly, PBMC are collected
from healthy donors and purified over Ficoll-Plaque (Pharmacia
Biotech; Piscataway, N.J.). PBMC are plated in a 24 well tissue
culture plate at 4.times.10 cells/ml. Peptides are added at a final
concentration of 10 .mu.g/ml. Cultures are incubated at 37.degree.
C. in 5% CO.sub.2.
[0204] On day 4, recombinant interleukin-2 (IL-2) is added at a
final concentration of 10 ng/ml. Cultures are fed every 3 days by
aspirating 1 ml of medium and replacing with fresh medium
containing IL-2. Two additional stimulations of the T cells with
antigen are performed on approximately days 14 and 28. The T cells
(3.times.10.sup.5/well) are stimulated with peptide (10 .mu.g/ml)
using autologous PBMC cells (2.times.10.sup.6 irradiated
cells/well) (irradiated with 7500 rads) as antigen-presenting cells
in a total of three wells of a 24 well tissue culture plate. In
addition, on day 14 and 28, T cell proliferative responses are
determined under the following conditions: 2.times.10.sup.4 T
cells/well; 1.times.10.sup.5 irradiated PBMC/well as
antigen-presenting cells; peptide concentration varying between
0.01 and 10 .mu.g/ml final concentration. The proliferation of the
T cells is measured 3 days later by the addition of 3H-thymidine (1
.mu.Ci/well) 18 hr prior to harvesting the cells. Cells are
harvested onto glass filters and .sup.3H-thymidine incorporation is
measured in a beta plate counter. These results demonstrate methods
for assaying T helper cell activity by measuring .sup.3H-thymidine
incorporation.
Example III
Assay for Cytotoxic T Lymphocyte Response
[0205] This example shows a method for assaying cytotoxic T
lymphocyte (CTL) activity. A CTL response is measured essentially
as described previously (Vitiello et al., Eur. J. Immunol.
27:671-678 (1997), which is incorporated herein by reference).
Briefly, after approximately 10-35 days following DNA immunization,
splenocytes from an animal are isolated and co-cultured at
37.degree. C. with syngeneic, irradiated (3000 rad) peptide-coated
LPS blasts (1.times.10.sup.6 to 1.5.times.10.sup.6 cells/ml) in 10
ml R10 in T25 flasks. LPS blasts are obtained by activating
splenocytes (1.times.10.sup.6 to 1.5.times.10.sup.6 cells/ml) with
25 .mu.g/ml lipopolysaccharides (LPS) (Sigma cat. no. L-2387; St.
Louis, Mo.) and 7 .mu.g/ml dextran sulfate (Pharmacia Biotech) in
30 ml R10 medium in T75 flasks for 3 days at 37.degree. C. The
lymphoblasts are then resuspended at a concentration of
2.5.times.10.sup.7 to 3.0.times.10.sup.7/ml, irradiated (3000 rad),
and coated with the appropriate peptides (100 .mu.g/ml) for 1 h at
37.degree. C. Cells are washed once, resuspended in R10 medium at
the desired concentration and added to the responder cell
preparation. Cultures are assayed for cytolytic activity on day 7
in a .sup.51Cr-release assay.
[0206] For the .sup.51Cr-release assay, target cells are labeled
for 90 ml at 37.degree. C. with 150 .mu.l sodium .sup.51chromate
(.sup.51Cr) (New England Nuclear; Wilmington Del.), washed three
times and resuspended at the appropriate concentration in R10
medium. For the assay, 10.sup.4 target cells are incubated in the
presence of different concentrations of effector cells in a final
volume of 200 .mu.l in U-bottom 96 well plates in the presence or
absence of 10 .mu.g/ml peptide. Supernatants are removed after 6 h
at 37.degree. C., and the percent specific lysis is determined by
the formula: percent specific lysis=100.times.(experimental
release-spontaneous release)/(maximum release-spontaneous release).
To facilitate comparison of responses from different experiments,
the percent release data is transformed to lytic units 30 per 106
cells (LU30/10.sup.6), with 1 LU30 defined as the number of
effector cells required to induce 30% lysis of 10.sup.4 target
cells in a 6 h assay. LU values represent the LU30/10.sup.6
obtained in the presence of peptide minus LU30/10.sup.6 in the
absence of peptide. These results demonstrate methods for assaying
CTL activity by measuring .sup.51Cr release from cells.
Example IV
T Cell Proliferation in Mice Immunized with Expression Vectors
Encoding MHC Class II Epitopes and MHC Class II Targeting
Sequences
[0207] This example demonstrates that expression vectors encoding
MHC class II epitopes and MHC class II targeting sequences are
effective at activating T cells.
[0208] The constructs used in the T cell proliferation assay are
described in Example I and were cloned into the vector pEP2, a CMV
driven expression vector. The peptides used for T cell in vitro
stimulation are: Ova 323-339, ISQAVHAAHAEINEAGR (SEQ ID NO:48);
HBVcorel28, TPPAYRPPNAPILF (SEQ ID NO:124); HBVenyl82,
FFLLTRILTIPQSLD (SEQ ID NO:50); and PADRE, AKFVAAWTLKAAA (SEQ ID
NO:52).
[0209] T cell proliferation was assayed essentially as described in
Example II. Briefly, 12 to 16 week old B6D2 F1 mice (2 mice per
construct) were injected with 100 .mu.g of the indicated expression
vector (50 .mu.g per leg) in the anterior tibialis muscle. After
eleven days, spleens were collected from the mice and separated
into a single cell suspension by Dounce homogenization. The
splenocytes were counted and one million splenocytes were plated
per well in a 96-well plate. Each sample was done in triplicate.
Ten .mu.g/ml of the corresponding peptide encoded by the respective
expression vectors was added to each well. One well contained
splenocytes without peptide added for a negative control. Cells
were cultured at 37.degree. C., 5% CO.sub.2 for three days.
[0210] After three days, one .mu.Ci of .sup.3H-thymidine was added
to each well. After 18 hours at 37.degree. C., the cells were
harvested onto glass filters and .sup.3H incorporation was measured
on an LKB .beta. plate counter. The results of the T cell
proliferation assay are shown in Table 9. Antigenspecific T cell
proliferation is presented as the stimulation index (SI); this is
defined as the ratio of the average .sup.3H-thymidine incorporation
in the presence of antigen divided by the .sup.3H-thymidine
incorporation in the absence of antigen.
[0211] The immunogen "PADRE+IFA" is a positive control where the
PADRE peptide in incomplete Freund's adjuvant was injected into the
mice and compared to the response seen by injecting the MHC class
II epitope constructs containing a PADRE sequence. As shown in
Table 9, most of the expression vectors tested were effective at
activating T cell proliferation in response to the addition of
PADRE peptide. The activity of several of the expression vectors
was comparable to that seen with immunization with the PADRE
peptide in incomplete Freund's adjuvant. The expression vectors
containing both MHC class I and MHC class II epitopes, pEP2-AOS and
pcDNA-AOS, were also effective at activating T cell proliferation
in response to the addition of PADRE peptide.
[0212] These results show that expression vectors encoding MHC
class II epitopes fused to a MHC class II targeting sequence is
effective at activating T cell proliferation and are useful for
stimulating an immune response.
Example V
In Vivo Assay Using Transgenic Mice
[0213] A. Materials and methods
[0214] Peptides were synthesized according to standard F-moc solid
phase synthesis methods which have been previously described
(Ruppert et al., Cell 74:929 (1993); Sette et al., Mol. Immunol.
31:813 (1994)). Peptide purity was determined by analytical
reverse-phase HPLC and purity was routinely >95%. Synthesis and
purification of the Theradigm-HBV lipopeptide vaccine is described
in (Vitiello et al., J. Clin. Invest. 95:341 (1995)).
[0215] Mice
[0216] HLA-A2.1 transgenic mice used in this study were the F1
generation derived by crossing transgenic mice expressing a
chimeric gene consisting of the .alpha.1, .alpha.2 domains of
HLA-A2.1 and .alpha.3 domain of H-2 K.sup.b with SJL/J mice
(Jackson Laboratory, Bar Harbor, Me.). This strain will be referred
to hereafter as HLA-A2.1/K.sup.b-H-2.sup.bxs. The parental
HLA-A2.1/K.sup.b transgenic strain was generated on a C57BL/6
background using the transgene and methods described in (Vitiello
et al., J. Exp. Med. 173:1007 (1991)). HLA-A 1/K.sup.b transgenic
mice used in the current study were identical to those described in
(Alexander et al., J. Immunol. 159:4753 (1997)).
[0217] Cell Lines, MHC Purification, and Peptide Binding Assay
[0218] Target cells for peptide-specific cytotoxicity assays were
Jurkat cells transfected with the HLA-A2.1/K.sup.b chimeric gene
(Vitiello et al., J. Exp. Med. 173:1007 (1991)) and 0.221 tumor
cells transfected with HLA-A11/K.sup.b (Alexander et al., J.
Immunol. 159:4753 (1997)).
[0219] To measure presentation of endogenously processed epitopes,
Jurkat-A2.1/K.sup.b cells were transfected with the pMin.1 or
pMin.2-GFP minigenes then tested in a cytotoxicity assay against
epitope-specific CTL lines. For transfection, Jurkat-A2.1/K.sup.b
cells were resuspended at 10.sup.7 cells/ml and 30 .mu.g of DNA was
added to 600 .mu.l of cell suspension. After electroporating cells
in a 0.4 cm cuvette at 0.25 kV, 960 .mu.Fd, cells were incubated on
ice for 10 min then cultured for 2 d in RPMI culture medium. Cells
were then cultured in medium containing 200 U/ml hygromycin B
(Calbiochem, San Diego Calif.) to select for stable transfectants.
FACS was used to enrich the fraction of green fluorescent protein
(GFP)-expressing cells from 15% to 60% (data not shown).
[0220] Methods for measuring the quantitative binding of peptides
to purified HLA-A2.1 and -A11 molecules is described in Ruppert et
al., Cell 74:929 (1993); Sette et al., Mol. Immunol. 31:813 (1994);
Alexander et al., J. Immunol. 159:4753 (1997).
[0221] All tumor cell lines and splenic CTLs from primed mice were
grown in culture medium (CM) that consisted of RPMI 1640 medium
with Hepes (Life Technologies, Grand Island, N.Y.) supplemented
with 10% FBS, 4 mM L-glutamine, 5.times.10.sup.-5 M 2-ME, 0.5 mM
sodium pyruvate, 100 .mu.g/ml streptomycin, and 100 U/ml
penicillin.
[0222] Construction of Minigene Multi-Epitope DNA Plasmids
[0223] pMIN.0 and pMIN.1 (i.e., pMIN-AOS) were constructed as
described above and in U.S. S No. 60/085,751.
[0224] pMin.1-No PADRE and pMin.1-Anchor. pMin.1 was amplified
using two overlapping fragments which was then combined to yield
the full length product. The first reaction used the 5' pcDNA
vector primer T7 and either primer ATCGCTAGGCAGGAACTTATACAGGATTCC
(SEQ ID NO:126) for pMin.1-No PADRE or TGGACAGTCCGGCTCCCAGCACCACGT
(SEQ ID NO:127) for pMin.1-Anchor. The 3' half was amplified with
the primers TTCCTGCCTAGCGATTTC (SEQ ID NO:128) (No PADRE) or
GCTGGGAGCCGGACTGTCCAGGTACGT (SEQ ID NO:129) (Anchor) and Min-StopR.
The two fragments generated from amplifying the 5' and 3' ends were
gel purified, mixed, denatured, annealed, and filled in with five
cycles of PCR. The full length fragment was further amplified with
the flanking primers T7 and Min-StopR for 25 more cycles.
[0225] Min.1-No Sig. The Ig signal sequence was deleted from pMin.1
by PCR amplification with primer
GCTAGCGCCGCCACCATGCACACCCTGTGGAAGGC CGGAATC (SEQ ID NO: 130) and
pcDNA rev (Invitrogen) primers. The product was cloned into
pCR-blunt and sequenced.
[0226] pMin.1-Switch. Three overlapping fragments were amplified
from pMin.1, combined, and extended. The 5' fragment was amplified
with the vector primer T7 and primer
GGGCACCAGCAGGCTCAGCCACACTCCCAGCACCACGTC (SEQ ID NO: 131). The
second overlapping fragment was amplified with primers
AGCCTGCTGGTGCCCTTTGTGATCCTGAAGGAGCCTGTGC (SEQ ID NO: 132) and
AGCCACGTACCTGGACAGTCCCTTCCACACAGGCACTCCAT (SEQ ID NO: 133). Primer
TGTCCAGGTACGTGGCTAGGCTGTGAGGTACC (SEQ ID NO: 134) and the vector
primer pcDNA rev (Invitrogen) were used to amplify the third (3')
fragment. Fragments 1, 2, and 3 were amplified and gel purified.
Fragments 2 and 3 were mixed, annealed, amplified, and gel
purified. Fragment 1 was combined with the product of 2 and 3, and
extended, gel purified and cloned into pcDNA3.1 for expression.
[0227] pMin.2-GFP. The signal sequence was deleted from pMin.0 by
PCR amplification with Min.0-No Sig-5'
GCTAGCGCCGCCACCATGCACACCCTGTGGAAGGCCGG- AATC (SEQ ID NO:135) plus
pcDNA rev (Invitrogen) primers. The product was cloned into
pCR-blunt and sequenced. The insert containing the open reading
frame of the signal sequence-multi-deleted epitope construct was
cut out with NheI plus HindIII and ligated into the same sites of
pEGFPN1 (Clontech). This construct fuses the coding region of the
signal-deleted pMin.0 construct to the N-terminus of green
fluorescent protein (GFP).
[0228] Immunization of Mice
[0229] For DNA immunization, mice were pretreated by injecting 50
.mu.l of 10 .mu.M (Sigma Chem. Co., #C9759) bilaterally into the
tibialis anterior muscle. Four or five days later 100 .mu.g of DNA
diluted in PBS were injected in the same muscle.
[0230] Theradigm-HBV lipopeptide (10 mg/ml in DMSO) that was stored
at -20.degree. C., was thawed for 10 min at 45.degree. C. before
being diluted 1:10 (v/v) with room temperature PBS. Immediately
upon addition of PBS, the lipopeptide suspension was vortexed
vigorously and 100 .mu.l was injected s.c. at the tail base (100
.mu.g/mouse).
[0231] Immunogenicity of individual CTL epitopes was tested by
mixing each CTL epitope (50 .mu.g/mouse) with the HBV core 128-140
peptide (TPPAYRPPNAPIL (SEQ ID NO:49), 140 .mu.g/mouse) which
served to induce I-A.sup.b-restricted Th cells. The peptide
cocktail was then emuslifed in incomplete Freund's adjuvant (Sigma
Chem. Co.) and 100 .mu.l of peptide emulsion was injected s.c. at
the tail base.
[0232] In Vitro CTL Cultures and Cytotoxicity Assays
[0233] Eleven to 14 days after immunization, animals were
sacrificed and a single cell suspension of splenocytes prepared.
Splenocytes from cDNA-primed animals were stimulated in vitro with
each of the peptide epitopes represented in the minigene.
Splenocytes (2.5-3.0.times.10.sup.7- /flask) were cultured in
upright 25 cm.sup.2 flasks in the presence of 10 .mu.g/ml peptide
and 10.sup.7 irradiated spleen cells that had been activated for 3
days with LPS (25 .mu.g/ml) and dextran sulfate (7 .mu.g/ml).
Triplicate cultures were stimulated with each epitope. Five days
later, cultures were fed with fresh CM. After 10 d of in vitro
culture, 2-4.times.10.sup.6 CTLs from each flask were restimulated
with 10.sup.7 LPS/dextran sulfate-activated splenocytes treated
with 100 .mu.g/ml peptide for 60-75 min at 37.degree. C., then
irradiated 3500 rads. CTLs were restimulated in 6-well plates in 8
ml of cytokine-free CM. Eighteen hr later, cultures received
cytokines contained in con A-activated splenocyte supernatant
(10-15% final concentration, v/v) and were fed or expanded on the
third day with CM containing 10-15% cytokine supernate. Five days
after restimulation, CTL activity of each culture was measured by
incubating varying numbers of CTLs with 1 .sup.51Cr-labelled target
cells in the presence or absence of peptide. To decrease
nonspecific cytotoxicity from NK cells, YAC-1 cells (ATCC) were
also added at a YAC-1:.sup.51Cr-labeled target cell ratio of 20:1.
CTL activity against the HBV Pol 551 epitope was measured by
stimulating DNA-primed splenocytes in vitro with the native
A-containing peptide and testing for cytotoxic activity against the
same peptide.
[0234] To more readily compare responses, the standard E:T ratio vs
% cytotoxicity data curves were converted into LU per 106 effector
cells with one LU defined as the lytic activity required to achieve
30% lysis of target cells at a 100:1 E:T ratio. Specific CTL
activity (ALU) was calculated by subtracting the LU value obtained
in the absence of peptide from the LU value obtained with peptide.
A given culture was scored positive for CTL induction if all of the
following criteria were met: 1) .DELTA.LU>2; 2)
LU(+peptide).div.LU(-peptide)>3; and 3) a >10% difference in
% cytotoxicity tested with and without peptide at the two highest
E:T ratios (starting E:T ratios were routinely between
25-50:1).
[0235] CTL lines were generated from pMin. 1-primed splenocytes
through repeated weekly stimulations of CTLs with peptide-treated
LPS/DxS-activated splenocytes using the 6-well culture conditions
described above with the exception that CTLs were expanded in
cytokine-containing CM as necessary during the seven day
stimulation period.
[0236] Cytokine Assay
[0237] To measure IFN-.gamma. production in response to
minigene-transfected target cells, 4.times.10.sup.4 CTLs were
cultured with an equivalent number of minigene-transfected
Jurkat-A2.1/K.sup.b cells in 96-well flat bottom plates. After
overnight incubation at 37.degree. C., culture supernatant from
each well was collected and assayed for IFN-.gamma. concentration
using a sandwich ELISA. Immulon II microtiter wells (Dynatech,
Boston, Mass.) were coated overnight at 4.degree. C. with 0.2 .mu.g
of anti-mouse IFN-.gamma. capture Ab, R4-6A2 (Pharmingen). After
washing wells with PBS/0.1% Tween-20 and blocking with 1% BSA,
Ab-coated wells were incubated with culture supernate samples for 2
hr at room temperature. A secondary anti-IFN-.gamma. Ab, XMG1.2
(Pharmingen), was added to wells and allowed to incubate for 2 hr
at room temperature. Wells were then developed by incubations with
Avidin-DH and finally with biotinylated horseradish peroxidase H
(Vectastain ABC kit, Vector Labs, Burlingame, Calif.) and TMB
peroxidase substrate (Kirkegaard and Perry Labs, Gaithersberg,
Md.). The amount of cytokine present in each sample was calculated
using a rIFN-.gamma. standard (Pharmingen).
[0238] B. Results
[0239] Selection of Epitopes and Minigene Construct Design
[0240] In the first series of experiments, the issue was whether a
balanced multispecific CTL response could be induced by simple
minigene cDNA constructs that encode several dominant HLA class
I-restricted epitopes. Accordingly, nine CTL epitopes were chosen
on the basis of their relevance in CTL immunity during HBV and HIV
infection in humans, their sequence conservancy among viral
subtypes, and their class I MHC binding affinity (Table 10). Of
these nine epitopes, six are restricted by HLA-A2.1 and three
showed HLA-AL 1-restriction. One epitope, HBV Pol 551, was studied
in two alternative forms: either the wild type sequence or an
analog (HBV Pol 551-V) engineered for higher binding affinity.
[0241] As referenced in Table 10, several independent laboratories
have reported that these epitopes are part of the dominant CTL
response during HBV or HIV infection. All of the epitopes
considered showed greater than 75% conservancy in primary amino
acid sequence among the different HBV subtypes and HIV clades. The
MHC binding affinity of the peptides was also considered in
selection of the epitopes. These experiment addressed the
feasibility of immunizing with epitopes possessing a wide range of
affinities and, as shown in Table 10, the six HBV and three HIV
HLA-restricted epitopes covered a spectrum of MHC binding
affinities spanning over two orders of magnitude, with IC.sub.50%
concentrations ranging from 3 nM to 200 nM.
[0242] The immunogenicity of the six A2.1- and three A11-restricted
CTL epitopes in transgenic mice was verified by co-immunization
with a helper T cell peptide in an IFA formulation. All of the
epitopes induced significant CTL responses in the 5 to 73 ALU range
(Table 10). As mentioned above, to improve the MHC binding and
immunogenicity of HBV Pol 551, the C-terminal A residue of this
epitope was substituted with V resulting in a dramatic 40-fold
increase in binding affinity to HLA-A2.1 (Table 10). While the
parental sequence was weakly or nonimmunogenic in HLA transgenic
mice, the HBV Pol 551-V analog induced significant levels of CTL
activity when administered in IFA (Table 10). On the basis of these
results, the V analog of the HBV Pol 551 epitope was selected for
the initial minigene construct. In all of the experiments reported
herein, CTL responses were measured with target cells coated with
the native HBV Pol 551 epitope, irrespective of whether the V
analog or native epitope was utilized for immunization.
[0243] Finally, since previous studies indicated that induction of
T cell help significantly improved the magnitude and duration of
CTL responses (Vitiello et al., J. Clin. Invest. 95:341 (1995);
Livingston et al., J. Immunol. 159:1383 (1997)), the universal Th
cell epitope PADRE was also incorporated into the minigene. PADRE
has been shown previously to have high MHC binding affinity to a
wide range of mouse and human MHC class II haplotypes (Alexander et
al., Immunity 1:751 (1994)). In particular, it has been previously
shown that PADRE is highly immunogenic in H-2b mice that are used
in the current study (Alexander et al., Immunity 1:751 (1994)).
[0244] pMin.1, the prototype cDNA minigene construct encoding nine
CTL epitopes and PADRE, was synthesized and subcloned into the
pcDNA3.1 vector. The position of each of the nine epitopes in the
minigene was optimized to avoid junctional mouse H-2b and HLA-A2.1
class I MHC epitopes. The mouse Ig K signal sequence was also
included at the 5' end of the construct to facilitate processing of
the CTL epitopes in the endoplasmic reticulum (ER) as reported by
others (Anderson et al., J. Exp. Med. 174:489 (1991)). To avoid
further conformational structure in the translated polypeptide gene
product that may affect processing of the CTL epitopes, an ATG stop
codon was introduced at the 3' end of the minigene construct
upstream of the coding region for c-myc and poly-his epitopes in
the pcDNA3.1 vector.
[0245] Immunogenicity of pMin.1 in Transgenic Mice
[0246] To assess the capacity of the pMin. 1 minigene construct to
induce CTLs in vivo, HLA-A2.1/K -H-2bxs transgenic mice were
immunized intramuscularly with 100 .mu.g of naked cDNA. As a means
of comparing the level of CTLs induced by cDNA immunization, a
control group of animals was also immunized with Theradigm-HBV, a
palmitolyated lipopeptide consisting of the HBV Core 18 CTL epitope
linked to the tetanus toxin 830-843 Th cell epitope.
[0247] Splenocytes from immunized animals were stimulated twice
with each of the peptide epitopes encoded in the minigene, then
assayed for peptide-specific cytotoxic activity in a .sup.51Cr
release assay. A representative panel of CTL responses of
pMin.1-primed splenocytes, shown in FIG. 22, clearly indicates that
significant levels of CTL induction were generated by minigene
immunization. The majority of the cultures stimulated with the
different epitopes exceeded 50% specific lysis of target cells at
an E:T ratio of 1:1. The results of four independent experiments,
compiled in Table 11, indicate that the pMin.1 construct is indeed
highly immunogenic in HLA-A2.1/K.sup.b-H-2.sup.bxs transgenic mice,
inducing a broad CTL response directed against each of its six
A2.1-restricted epitopes.
[0248] To more conveniently compare levels of CTL induction among
the different epitopes, the % cytotoxicity values for each
splenocyte culture was converted to ALU and the mean ALU of CTL
activity in positive cultures for each epitope was determined (see
Example V, materials and methods, for positive criteria). The data,
expressed in this manner in Table 11, confirms the breadth of CTL
induction elicited by pMin.1 immunization since extremely high CTL
responses, ranging between 50 to 700 .DELTA.LU, were observed
against the six A2.1-restricted epitopes. More significantly, the
responses of several hundred .DELTA.LU observed for five of the six
epitopes approached or exceeded that of the Theradigm-HBV
lipopeptide, a vaccine formulation known for its high CTL-inducing
potency (Vitiello et al., J. Clin. Invest. 95:341 (1995);
Livingston et al., J. Immunol. 159:1383 (1997)). The HBV Env 335
epitope was the only epitope showing a lower mean .DELTA.LU
response compared to lipopeptide (Table 11, 44 vs 349
.DELTA.LU).
[0249] Processing of Minigene Epitopes by Transfected Cells
[0250] The decreased CTL response observed against HBV Env 335 was
somewhat unexpected since this epitope had good A2.1 binding
affinity (IC50%, 5 nM) and was also immunogenic when administered
in IFA. The lower response may be due, at least in part, to the
inefficient processing of this epitope from the minigene
polypeptide by antigen presenting cells following in vivo cDNA
immunization. To address this possibility, Jurkat-A2.1/K.sup.b
tumor cells were transfected with pMin.1 cDNA and the presentation
of the HBV Env 335 epitope by transfected cells was compared to
more immunogenic A2.1-restricted epitopes using specific CTL lines.
Epitope presentation was also studied using tumor cells transfected
with a control cDNA construct, pMin.2-GFP, that encoded a similar
multi-epitope minigene fused with GFP which allows detection of
minigene expression in transfected cells by FACS.
[0251] Epitope presentation of the transfected Jurkat cells was
analyzed using specific CTL lines, with cytotoxicity or IFN-.gamma.
production serving as a read-out. It was found that the levels of
CTL response correlated directly with the in vivo immunogenicity of
the epitopes. Highly immunogenic epitopes in vivo, such as HBV Core
18, HIV Pol 476, and HBV Pol 455, were efficiently presented to CTL
lines by pMin.1- or pMin.2-GFP-transfected cells as measured by
IFN-.gamma. production (FIG. 23A, >100 pg/ml for each epitope)
or cytotoxic activity (FIG. 23C, >30% specific lysis). In
contrast to these high levels of in vitro activity, the stimulation
of the HBV Env 335-specific CTL line against both populations of
transfected cells resulted in less than 12 pg/ml IFN-.gamma. and 3%
specific lysis. Although the HBV Env 335-specific CTL line did not
recognize the naturally processed epitope efficiently, this line
did show an equivalent response to peptide-loaded target cells, as
compared to CTL lines specific for the other epitopes (FIGS. 23B,
D). Collectively, these results suggest that a processing and/or
presentation defect associated with the HBV Env 335 epitope that
may contribute to its diminished immunogencity in vivo.
[0252] Effect of the Helper T cell Epitope PADRE on Minigene
Immunogenicity
[0253] Having obtained a broad and balanced CTL response in
transgenic mice immunized with a minigene cDNA encoding multiple
HLA-A2.1-restricted epitopes, next possible variables were examined
that could influence the immunogenicity of the prototype construct.
This type of analysis could lead to rational and rapid optimization
of future constructs. More specifically, a cDNA construct based on
the pMin.1 prototype was synthesized in which the PADRE epitope was
deleted to examine the contribution of T cell help in minigene
immunogenicity (FIG. 24A).
[0254] The results of the immunogenicity analysis indicated that
deletion of the PADRE Th cell epitope resulted in significant
decreases in the frequency of specific CTL precursors against four
of the minigene epitopes (HBV Core 18, HIV Env 120, HBV Pol 455,
and HBV Env 335) as indicated by the 17 to 50% CTL-positive
cultures observed against these epitopes compared to the 90-100%
frequency in animals immunized with the prototype pMin.1 construct
(FIG. 25). Moreover, for two of the epitopes, HBV Core 18 and HIV
Env 120, the magnitude of response in positive cultures induced by
pMin.1-No PADRE was 20- to 30-fold less than that of the pMin.1
construct (FIG. 25A).
[0255] Effect of Modulation of MHC Binding Affinity on Epitope
Immunogenicity
[0256] Next a construct was synthesized in which the V anchor
residue in HBV Pol 551 was replaced with alanine, the native
residue, to address the effect of decreasing MHC binding on epitope
immunogenicity (FIG. 24B).
[0257] Unlike deletion of the Th cell epitope, decreasing the MHC
binding capacity of the HBV Pol 551 epitope by 40-fold through
modification of the anchor residue did not appear to affect epitope
immunogenicity (FIG. 25B). The CTL response against the HBV Pol 551
epitope, as well as to the other epitopes, measured either by LU or
frequency of CTL-positive cultures, was very similar between the
constructs containing the native A or improved V residue at the MHC
binding anchor site. This finding reinforces the notion that
minimal epitope minigenes can efficiently deliver epitopes of
vastly different MHC binding affinities. Furthermore, this finding
is particularly relevant to enhancing epitope immunogenicity via
different delivery methods, especially in light of the fact that
the wild type HBV Pol 551 epitope was essentially nonimmunogenic
when delivered in a less potent IFA emulsion.
[0258] Effect of the Signal Sequence on Minigene Construct
Immunogenicity
[0259] The signal sequence was deleted from the pMin.1 construct,
thereby preventing processing of the minigene polypeptide in the ER
(FIG. 24C). When the immunogenicity of the pMin.1-No Sig construct
was examined, an overall decrease in response was found against
four CTL epitopes. Two of these epitopes, HIV Env 120 and HBV Env
335, showed a decrease in frequency of CTL-positive cultures
compared to pMin.1 while the remaining epitopes, HBV Pol 455 and
HIV Pol 476, showed a 16-fold (from 424 to 27 .DELTA.LU) and 3-fold
decrease (709 to 236 .DELTA.LU) in magnitude of the mean CTL
response, respectively (FIG. 25C). These findings suggest that
allowing ER-processing of some of the epitopes encoded in the
pMin.1 prototype construct may improve immunogenicity, as compared
with constructs that allow only cytoplasmic processing of the same
panel of epitopes.
[0260] Effect of Epitope Rearrangement and Creation of New
Junctional Epitopes
[0261] In the final construct tested, the immunogenicity of the HBV
Env 335 epitope was analyzed to determine whether it may be
influenced by its position at the 3' terminus of the minigene
construct (FIG. 24D). Thus, the position of the Env epitope in the
cDNA construct was switched with a more immunogenic epitope, HBV
Pol 455, located in the center of the minigene. It should be noted
that this modification also created two potentially new epitopes.
As shown in FIG. 25D, the transposition of the two epitopes
appeared to affect the immunogenicity of not only the transposed
epitopes but also more globally of other epitopes. Switching
epitopes resulted in obliteration of CTL induction against HBV Env
335 (no positive cultures detected out of six). The CTL response
induced by the terminal HBV Pol 455 epitope was also decreased but
only slightly (424 vs 78 mean .DELTA.LU). In addition to the
switched epitopes, CTL induction against other epitopes in the
pMin.1-Switch construct was also markedly reduced compared to the
prototype construct. For example, a CTL response was not observed
against the HIV Env 120 epitope and it was significantly diminished
against the HBV Core 18 (4 of 6 positive cultures, decrease in mean
.DELTA.LU from 306 to 52) and HBV Pol 476 (decrease in mean
.DELTA.LU from 709 to 20) epitopes (FIG. 25D).
[0262] As previously mentioned, it should be noted that switching
the two epitopes had created new junctional epitopes. Indeed, in
the pMin.1-Switch construct, two new potential CTL epitopes were
created from sequences of HBV Env 335-HIV Pol 476 (LLVPFVIL (SEQ ID
NO:123), H-2K -restricted) and HBV Env 335-HBV Pol 551 (VLGVWLSLLV
(SEQ ID NO: 136), HLA-A2.1-restricted) epitopes. Although these
junctional epitopes have not been examined to determine whether or
not they are indeed immunogenic, this may account for the low
immunogenicity of the HBV Env 335 and HIV Pol 476 epitopes. These
findings suggest that avoiding junctional epitopes may be important
in designing multi-epitope minigenes as is the ability to confirm
their immunogenicity in vivo in a biological assay system such as
HLA transgenic mice.
[0263] Induction of CTLs against A11 epitopes encoded in pMin.1
[0264] To further examine the flexibility of the minigene vaccine
approach for inducing a broad CTL response against not only
multiple epitopes but also against epitopes restricted by different
HLA alleles, HLA-A11/K.sup.b transgenic mice were immunized to
determine whether the three A11 epitopes in the pMin.1 construct
were immunogenic for CTLs, as was the case for the A2.1-restricted
epitopes in the same construct. As summarized in Table 12,
significant CTL induction was observed in a majority of cultures
against all three of the HLA-A11-restricted epitopes and the level
of CTL immunity induced for the three epitopes, in the range of 40
to 260 .DELTA.LU, exceeded that of peptides delivered in IFA (Table
10). Thus, nine CTL epitopes of varying HLA restrictions
incorporated into a prototype minigene construct all demonstrated
significant CTL induction in vivo, confirming that minigene DNA
plasmids can serve as means of delivering multiple epitopes, of
varying HLA restrictions and MHC binding affinities, to the immune
system in an immunogenic fashion and that appropriate transgenic
mouse strains can be used to measure DNA construct immunogenicity
in vivo.
[0265] CTLs were also induced against three A11 epitopes in
A11/K.sup.b transgenic mice. These responses suggest that minigene
delivery of multiple CTL epitopes that confers broad population
coverage may be possible in humans and that transgenic animals of
appropriate haplotypes may be a useful tools in optimizing the in
vivo immunogenicity of minigene DNA. In addition, animals such as
monkeys having conserved HLA molecules with cross reactivity to CTL
and HTL epitopes recognized by human MHC molecules can be used to
determine human immunogenicity of HTL and CTL epitopes (Bertoni et
al., J. Immunol. 161 :4447-4455 (1998)).
[0266] This study represents the first description of the use of
HLA transgenic mice to quantitate the in vivo immunogenicity of DNA
vaccines, by examining response to epitopes restricted by human HLA
antigens. In vivo studies are required to address the variables
crucial for vaccine development, that are not easily evaluated by
in vitro assays, such as route of administration, vaccine
formulation, tissue biodistribution, and involvement of primary and
secondary lymphoid organs. Because of its simplicity and
flexibility, HLA transgenic mice represent an attractive
alternative, at least for initial vaccine development studies,
compared to more cumbersome and expensive studies in higher animal
species, such as nonhuman primates. The in vitro presentation
studies described above further supports the use of HLA transgenic
mice for screening DNA constructs containing human epitopes
inasmuch as a direct correlation between in vivo immunogenicity and
in vitro presentation was observed. Finally, strong CTL responses
were observed against all six A 2.1 restricted viral epitopes and
in three A11 restricted epitopes encoded in the prototype pMin.1
construct. For five of the A2.1 restricted epitopes, the magnitude
of CTL response approximated that observed with the lipopeptide,
Theradigm-HBV, that previously was shown to induce strong CTL
responses in humans (Vitiello et al., J. Clin. Invest. 95:341
(1995); Livingston et al., J. Immunol. 159:1383 (1997)). 1
3TABLE 1 HBV derived HTL epitopes SEQ ID Peptide Sequence Source
NO: 1298.06 KQAFTFSPTYKAFLC HBV POL 661 137 F107.03 LQSLTNLLSSNLSWL
HBV POL 412 138 1280.06 AGFFLLTRILTIPQS HBV ENV 180 139 1280.09
GTSFVYVPSALNPAD HBV POL 774 140 CF-08 VSFGVWIRTPPAYRPPNAPI HBV NUC
120 141 27.0280 GVWIRTPPAYRPPNA HBV NUC 123 142 1186.25
SFGVWIRTPPAYRPP HBV NUC 121 143 27.0281 RHYLHTLWKAGILYK HBV POL 145
144 F107.04 PFLLAQFTSAICSVV HBV POL 523 145 1186.15 LVPFVQWFVGLSPTV
HBV ENV 339 146 1280.15 LHLYSHPIILGFRKI HBV POL 501 147 1298.04
KQCFRKLPVNRPIDW HBV POL 615 148 1298.07 AANWILRGTSFVYVP HBV POL 764
149 857.02 PHHTALRQAILCWGELMTLA HBV CORE 50 150 35.0100
LCQVFADATPTGWGL HBV POL 683 151 35.0096 ESRLVVDFSQFSRGN HBV POL 387
152 35.0093 VGPLTVNEKRRLKLI HBV POL 96 153 1186.18 NLSWLSLDVSAAFYH
HBV P0L 422 154
[0267]
4TABLE 2 HBV derived CTL epitopes Supertype Peptide Sequence Source
SEQ ID NO: A2 924.07 FLPSDFFPSV HBV core 18-27 91 1013.0102
WLSLLVPFV HBVadr-ENV (S Ag 335-343) 107 777.03 FLLTRILTI HBV ENV
ayw 183 155 927.15 ALMPLYACI HBV ayw pol 642 156 1168.02 GLSRYVARL
HBV POL 455 99 927.11 FLLSLGIHL HBV pol 562 157 A3 1147.16
HTLWKAGILYK HBV POL 149 88 1083.01 STLPETTVVRR HBV core 141 103
1090.11 SAICSVVRR HBV pol 531 158 1090.10 QAFTFSPTYK HBV pol 665
159 1069.16 NVSIPWTHK HBV pol 47 160 1069.20 LVVDFSQFSR HBV pol 388
161 1142.05 KVGNFTGLY HBV adr POL 629 162 1069.15 TLWKAGILYK HBV
pol 150 163 B7 1145.04 IPIPISSWAF HBV ENV 313 164 988.05 LPSDFFPSV
HBV core 19-27 165 1147.04 TPARVTGGVF HBV POL 354 166 A2 1069.06
LLVPFVQWFV HBV env 338-347 167 1147.13 FLLAQFTSAI HBV POL 513 168
1147.14 VLLDYQGMLPV HBV ENV 259 169 1132.01 LVPFVQWFV HBV ENV 339
170 1069.05 LLAQFTSAI HBV pol 504-512 171 927.42 NLSWLSLDV HBV pol
411 172 927.41 LLSSNLSWL HBV pol 992 173 927.46 KLHLYSHPI HBV pol
489 174 1069.071 FLLAQFRSA HBV pol 503 175 1142.07 GLLGWSPQA HBV
ENV 62 176 927.47 HLYSHPIIL HBV ayw pol 1076 177 1069.13 PLLPIFFCL
HBV env 377-385 178 1013.1402 VLQAGFFLL HBV adr-ENV 177 179 1090.14
YMDDVVLGA HBV pol 538-546 95 A3 26.0539 RLVVDFSQFSR HBV pol 376 180
26.0535 GVWIRTPPAYR HBV X niuc fus 299 181 A3 26.0153 SSAGPCALR HBV
X 64 182 1.0993 KVFVLGGCR HBV adr "X" 1548 183 26.0149 CALRFTSAR
HBV X 69 184 26.0023 VSFGVWIR HBV x nuc fus 296 185 26.0545
TLPETTVVRRR HBV x nuc fus 318 186 20.0131 SVVRRAFPH HBV POL 524 187
1.0219 FVLGGCRHK HBV adr "X" 1550 188 26.0008 FTFSPTYK HBV pol 656
189 20.0130 AFTESPTYK HBV POL 655 190 B7 1147.05 FPHCLAFSYM HBV POL
530 191 1147.08 YPALMPLYA HBV POL 640 192 1147.06 LPVCAFSSA HBV X
58 193 1147.02 HPAAMPHLL HBV POL 429 194 26.0570 YPALMPLYACI HBV
pol 640 195 19.0014 YPALMPLY HBV POL 640 196 1145.08 FPHCLAFSY HBV
POL 541 197 Other 1090.02 AYRPPNAPI HBV NUC 131 198 1.0519
DLLDTASALY HBV adr CORE 419 199 13.0129 EYLVSFGVWI HBV NUC 117 200
20.0254 FAAPFTQCGY HBV POL 631 201 2.0060 GYPALMPLY HBV ALL 1224
202 1069.04 HTLWKAGILY HBV pol 149 203 1069.08 ILLLCLIFLL HBV env
249-258 204 1.0166 KVGNFTGLY HBV adr POL 629 162 1069.23 KYTSFPWLL
HBV POL 745 205 1069.01 LLDTASALY HBV core 59 26 2.0239 LSLDVSAAFY
HBV ALL 1000 207 2.0181 LYSHPIILGF HBV POL 492 208 1039.01
MMWYWGPSLY HBV 360 209 2.0126 MSTTDLEAY HBV adr 1521 210 1069.03
PLDKGIKPYY HBV pol 124 211 1090.09 PTTGRTSLY HBV pol 808 212
20.0138 PWTHKVGNF HBV POL 51 213 20.0135 RWMCLRRFI HBV ENV 236 214
20.0269 RWMCLRRFII HBV ENV 236 215 20.0139 SFCGSPYSW HBV POL 167
216 Other 1069.02 SLDVSAAFY HBV pol 427 217 20.0136 SWLSLLVPF HBV
ENV 334 218 20.0271 SWPKFAVPNL HBV POL 392 219 20.0137 SWWTSLNFL
HBV ENV 197 220 2.0173 SYQHFRKLLL HBV POL 4 221 13.0073 WFHISCLTF
HBV NUC 102 222 1.0774 WLWGMDIDPY HBV adw CORE 416 223 1039.06
WMMWYWGPSLY HBV env 359 224 924.14 FLPSDFFPSI HBv 18-27 I.sub.10
var. 225 1090.77 YMDDVVLGV HBV pol 538-546 sub 462 941.01
FLPSDYFPSV HBc 18-27 analog 226 1083.02 STLPETYVVRR HBV core
141-151 analog 227 1145.05 FPIPSSWAF HBV ENV 313 analog 228 1145.11
FPHCLAFSL HBV POL 541 analog 229 1145.24 FPHCLAFAL HBV POL 541
analog 230 1145.06 IPITSSWAF HBV ENV 313 analog 231 1145.23
IPIPMSWAF HBV ENV 313 analog 232 1145.07 IPILSSWAF HBV ENV 313
analog 233 1145.09 FPVCLAFSY HBV POL 541 analog 234 1145.10
FPHCLAFAY HBV POL 541 analog 235
[0268]
5TABLE 3 HCV derived HTL epitopes Peptide Sequence Source SEQ ID
NO: AAYAAQGYKVLVLNPSVAATLGFGAY HCV NS3 1242-1267 236 P98.03
AAYAAQGYKVLVLNPSVAAT HCV NS3 1242 237 P98.04 GYKVLVLNPSVAATLGFGAY
HCV NS3 1248 238 P98.05 GYKVLVLNPSVAAT HCV NS3 1248 239 1283.21
GYKVLVLNPSVAATL HCV NS3 1253 240 1283.20 AQGYKVLVLNPSVAA HCV NS3
1251 241 GEGAVQWMNRLIAFASRGNHVS HCV NS4 1914-1935 242 F134.08
GEGAVQWMNRLIAFASRGNHV HCV NS4 1914 243 1283.44 MNRLIAFASRGNHVS HCV
NS4 1921 244 1283.16 SKGWRLLAPITAYAQ HCV NS3 1025 245 1283.55
GSSYGFQYSPGQRVE HCV NS5 2641 246 F134.05 NFISGIQYLAGLSTLPGNPA HCV
NS4 1772 247 1283.61 ASCLRKLGVPPLRVW HCV NS5 2939 248 1283.25
GRHLIFCHSKKKCDE HCV NS3 1393 249 35.0107 TVDFSLDPTFTIETT HCV 1466
250 35.0106 VVVVATDALMTGYTG HCV 1437 251
[0269]
6TABLE 4 HCV derived CTL epitopes SEQ ID Supertype Peptide Sequence
Source NO: A2 1090.18 FLLLADARV HCV NS1/E2 728 252 1073.05
LLFNILGGWV HCV NS4 1812 253 1013.02 YLVAYQATV HCV NS3 1590 254
1013.1002 DLMGYIPLV HCV Core 132 255 1090.22 RLIVFPDLGV HCV NS5
2611 256 24.0075 VLVGGVLAA HCV NS4 1666 257 24.0073 WMNRLIAFA HCV
NS4 1920 258 1174.08 HMWNFISGI HCV NS4 1769 259 1073.06 ILAGYGAGV
HCV NS4 1851 260 24.0071 LLFLLLADA HCV NS1/E2 726 261 1073.07
YLLPRRGPRL HCV Core 35 262 1.0119 YLVTRHADV HCV NS3 1136 263 A3
1.0952 KTSERSQPR HCV Core 51 264 1073.10 GVAGALVAFK HCV NS4 1863
265 1.0123 LIFCHSKKK HCV NS3 1391 266 1.0955 QLFTFSPRR HCV E1 290
267 1073.11 RLGVRATRK HCV Core 43 268 1073.13 RMYVGGVEHR HCV NS1/E2
635 269 24.0090 VAGALVAFK HCV NS4 1864 270 F104.01 VGIYLLPNR HCV
NS5 3036 271 B7 1145.12 LPGCSFSIF HCV Core 168 272 29.0035 IPFYGKAI
HCV 1378 273 Other 1069.62 CTCGSSDLY HCV NS3 1128 274 24.0092
FWAKHMWNF HCV NS4 1765 275 13.0019 LSAFSLHSY HCV NS5 2922 276 A3
24.0086 LGFGAYMSK HCV NS3 1267 277 1174.21 RVCEKMALY HCV NS5 2621
278 1174.16 WMNSTGFTK HCV NS1/E2 557 279 1073.04 TLHGPTPLLY HCV NS3
1622 280 B7 16.0012 FPYLVAYQA HCV NS3 1588 281 15.0047 YPCTVNFTI
HCV NS1/E2 623 282 Other 24.0093 EVDGVRLHRY HCV NS5 2129 283 3.0417
LTCGFADLMGY HCV 126 284 1073.01 NIVDVQYLY HCV E1 700 285 1.0509
GLSAFSLHSY HCV NS5 2921 286 1073.17 MYVGDLCGSVF HCV E1 275 287
1073.18 MYVGGVEHRL HCV NS1/E2 633 288 13.075 QYLAGLSTL HCV NS4 1778
289 1145.13 FPGCSFSIF HCV Core 168 290 1145.25 LPGCMFSIF HCV Core
168 291 1292.24 LPGCSFSII HCV Core 169 292 1145.14 LPVCSFSIF HCV
Core 168 293 1145.15 LPGCSFSYF HCV Core 168 294
[0270]
7TABLE 5 HIV derived HTL epitopes Peptide Sequence Source SEQ ID
NO: GEIYKRWIILGLNKIVRMYSPTSILD HIV1 GAG 294-319 295
KRWIILGLNKIVRMYSPTSILD HIV gag 298-319 296 27.0313 KRWIILGLNKIVRMY
HIV1 GAG 298 297 27.0311 GEIYKRWIILGLNKI HIV1 GAG 294 298 27.0354
WEFVNTPPLVKLWYQ HIV1 POL 596 299 27.0377 QKQITKIQNFRVYYR HIV1 POL
956 300 EKVYLAWVPAHKGIGG HIV1 POL 711-726 301 1280.03
KVYLAWVPAHKGIGG HIV POL 712 302 27.0361 EKVYLAWVPAHKGIG HIV1 POL
711 303 PIVQNIQGQMVHQAISPRTLNA HIV1 gag 165-186 304 27.0304
QGQMVHQAISPRTLN HIV1 GAG 171 305 27.0297 QHLLQLTVWGIKQLQ HIV1 ENV
729 306 27.0344 SPAIFQSSMTKILEP HIV1 POL 335 307 F091.15
IKQFINMWQEVGKAMY HIV1 ENV 566 308 27.0341 FRKYTAFTIPSINNE HIV1 POL
303 309 27.0364 HSNWRAMASDFNLPP HIV1 POL 758 310 27.0373
KTAVQMAVFIHNFKR HIV1 POL 915 311 DRVHPVHAGPIAPGQMREPRGS HIV GAG 245
312 AFSPEVIPMFSALSEGATPQDLNTML HIV gag 195-216 313
AFSPEVIPMFSALSEGATPQDL HIV gag 195-216 314 200.06 SALSEGATPQDLNTML
HIV gag 205 315 27.0307 SPEVIPMFSALSEGA HIV gag 197 316
LQEQIGWMTNNPPIPVGEIYKR HIV gag 275 317 27.0310 QFQIGWMTNNPPIPV HIV
gag 276 318 35.0135 YRKILRQRKIDRLID HIV VPU 31 319 35.0131
WAGIKQEFGIPYNPQ HIV POL 874 320 35.0127 EVNIVTDSQYALGII HIV POL 674
321 35.0125 AETFYVDGAANRETK HIV POL 619 322 35.0133 GAVVIQDNSDIKVVP
HIV POL 989 323
[0271]
8TABLE 6 HIV derived CTL epitopes Supertype Peptide Sequence Source
SEQ ID NO: A2 25.0148 MASDFNLPPV HIV1 POL 70 324 1069.32 VLAEAMSQV
HIV gag 397 325 1211.04 KLTPLCVTL HIV ENV 134 326 25.0062
KILVGKLNWA HIV1 POL 87 463 25.0039 LTFGWCFKL HIV1 NEF 62 327
941.031 ILKEPVHGV HIV1 pol 476-484 101 25.0035 MTNNPPIPV HIV1 GAG
34 328 25.0057 RILQQLLFI HIV1 VPR 72 329 A3 1.0944 AVFIHNFKR HIV
POL 1434 330 1.1056 KIQNFRVYYR HIV POL 1474 331 1069.49 QMAVFLHNFK
HIV pol 1432 332 966.0102 AIFQSSMTK HIV pol 337 333 1150.14
MAVFIHNFK HIV pol 909 334 940.03 QVPLRPMTYK HIV nef 73-82 335
25.0175 TTLFCASDAK HIV1 ENV 81 336 1069.43 TVYYGVPVWK HIV env 49
105 25.0209 VTIKIGGQLK HIV1 POL 65 337 B7 1146.01 FPVRPQVPL HIV nef
84-92 338 29.0060 IPIHYCAPA HIV env 293 339 15.0073 FPISPIETV HIV
POL 171 340 29.0056 CPKVSFEPI HIV env 285 341 29.0107 IPYNPQSQGVV
HIV pol 883 342 A2 25.0151 CTLNFPISPI HIV1 POL 96 343 25.0143
LTPGWCFKLV HIV1 NEFP 62 344 25.0043 YTAFTIPSI HIV1 POL 83 345
25.0055 AIIRILQQL HIV1 VPR 76 346 25.0049 ALVEICTEM HIV1 POL 52 347
25.0032 LLQLTVWGI HIV1 ENV 61 348 25.0050 LVGPTPVNI HIV1 POL 100
349 25.0047 KAACWWAGI HIV1 POL 65 350 25.0162 KMIGGIGGFI HIV1 POL
96 351 25.0052 RAMASDFNL HIV1 POL 78 352 1211.09 SLLNATDIAV HIV ENV
814 353 A2 25.0041 TLNFPISPI HIV1 POL 96 354 A3 1.0046 IVIWGKTPK
HIV POL 1075 355 25.0064 MVHQAISPR HIV1 GAG 45 356 1.0062 YLAWVPAHK
HIV POL 1227 357 1.0942 MTKILEPFR HIV POL 859 358 25.0184
QMVHQAISPR HIV1 GAG 45 359 1069.48 AVFIHNFKRK HIV pol 1434 360
1069.44 KLAGRWPVK HIV pol 1358 361 1069.42 KVYLAWVPAHK HIV pol 1225
362 1.0024 NTPVFAIKK HIV pol 752 363 25.0062 RIVELLGRR HIV1 ENV 53
364 25.0095 TIKIGGQLK HIV1 POL 65 365 25.0078 TLFCASDAK HIV1 ENV 82
366 25.0104 VMIVWQVDR HIV1 VIF 83 367 1069.47 VTVYYGVPVWK HIV env
48 368 B7 15.0268 YPLASLRSLF HIV GAG 507 369 1292.13 HPVHAGPIA HIV
GAG 248 370 19.0044 VPLQLPPL HIV con. REV 71 371 Other 1.0431
EVNIVTDSQY HIV POL 1187 372 1.0014 FRDYVDRFY HIV GAG 298 373
25.0113 IWGCSGKLI HIV1 ENV 69 374 25.0127 IYETYGDTW HIV1 VPR 92 375
1069.60 IYQEPFKNL HIV pol 1036 376 2.0129 IYQYMDDLY HIV pol 359 377
25.0128 PYNEWTLEL HIV1 VPR 56 378 25.0123 PYNTPVFAI HIV1 POL 74 379
1069.57 RYLKDQQLL HIV env 2778 380 1069.58 RYLRDQQLL HIV env 2778
381 1069.59 TYQIYQEPF HIV pol 1033 382 1069.27 VIYQYMDDLY HIV pol
358 383 1069.26 VTVLDVGDAY HIV pol 265 384. 25.0115 VWKEATTTL HIV1
ENV 47 385 25.0218 VWYEATITLF HIV1 ENV 47 386 25.0219 YMQATWIPEW
HIV1 POL 96 387 A2 1211.4 SLLNATAIAV HIV MN gp 160 814(a) 388 A3
F105.21 AIFQRSMTR HIV pol 337(a) 389 F105.17 AIFQSSMTR HIV pol
337(a) 390 F105.02 GIFQSSMTY HIV pol 337(a) 391 F105.03 AAFQSSMTK
HIV pol 337(a) 392 F105.04 AIAQSSMTK HIV pol 337(a) 393 F105.05
AIFASSMTK HIV pol 337(a) 394 F105.06 AIFQASMTK HIV pol 337(a) 395
F105.07 AIFQSAMTK HIV pol 337(a) 396 F105.08 AIFQSSATK HIV pol
337(a) 397 F105.09 AIFQSSMAK HIV pol 337(a) 398 F105.11 FIFQSSMTK
HIV pol 337(a) 399 F105.12 SIFQSSMTK HIV pol 337(a) 400 F105.16
AIFQCSMTK HIV pol 337(a) 401 B7 1145.03 FPVRPQFPL HIV nef 84-92
analog 402 1181.03 FPVRPQVPI HIV nef 84-92(a) 403 1292.14 HPVHAGPII
HIV GAG 248 404 1292.09 FPLSPIETI HIV POL 179 405 1145.02 FPVFQVPL
HIV nef 84-92 analog 406 1145.22 TPVRMQVPL HIV nef 84-92 analog 407
1181.04 FPVRPQVPM HIV nef 84-92(a) 408 1181.01 FPVRPQVPA HIV nef
84-92(a) 409 1181.02 FPVRPQVPV HIV nef 84-92(a) 410 1181.05
FPVRPQVPF HIV nef 84-92(a) 411 1181.06 FPVRPQVPW HIV nef 84-92(a)
412
[0272]
9TABLE 7 P. falciparum derived HTL epitopes Peptide Sequence Source
SEQ ID NO: F125.04 RHNWVNHAVPLAMKLI Pf SSP2 61 473 1188.34
HNWVNHAVPLAMKLI Pf SSP2 62 414 1188.16 KSKYKLATSVLAGLL Pf EXP1 71
415 LVNLLIFHINGKIIKNSE Pf LSA1 13 416 F125.02 LVNLLIFHINGKIIKNS Pf
LSA1 13 417 27.0402 LLIFHINGKIIKNSE Pf LSA1 16 418 1188.32
GLAYKFVVPGAATPY Pf SSP2 512 419 27.0392 SSVFNVVNSSIGLIM Pf CSP 410
420 27.0417 VKNVIGPFMKAVCVE Pf SSP2 223 421 27.0388 MRKLAILSVSSFLFV
Pf CSP 2 422 27.0387 MNYYGKQENWYSLKK Pf CSP 53 423 1188.38
KYKIAGGIAGGLALL Pf SSP2 494 424 1188.13 AGLLGNVSTVLLGGV Pf EXP1 82
425 27.0408 QTNFKSLLRNLGVSE Pf LSA1 94 426 35.0171 PDSIQDSLKESRKLN
Pf SSP2 165 427 35.0172 KCNLYADSAWENVKN Pf SSP2 211 428
[0273]
10TABLE 8 P. falciparum derived CTL epitopes SEQ ID Supertype
Peptide Sequence Source NO: A2 1167.21 FLIFFDLFLV Pf SSP2 14 429
1167.08 GLIMVLSFL Pf CSP 425 430 1167.12 VLAGLLGNV Pf EXP1 80 431
1167.13 KILSVFFLA Pf EXP1 2 432 1167.10 GLLGNVSTV Pf EXP1 83 433
1167.18 ILSVSSFLFV Pf CSP 7 434 1167.19 VLLGGVGLVL Pf EXP 191 435
A3 1167.36 LACAGLAYK Pf SSP2 511 436 1167.32 QTNFKSLLR Pf LSAI 94
437 1167.43 VTCGNGIQVR Pf CSP 375 438 1167.24 ALFFIIFNK Pf EXP1 10
439 1167.28 GVSENIFLK Pf LSA1 105 440 1167.47 HVLSHNSYEK Pf LSA1 59
441 1167.51 LLACAGLAYK Pf SSP2 510 442 1167.46 FILVNLLIFH Pf LSA1
11 443 B7 1101.03 MPLETQLAI Pf SHEBA 77 444 1167.61 TPYAGEPAPF Pf
SSP2 539 445 A2 1167.14 FLIFFDLFL Pf SSP2 14 446 1167.16 FMKAVCVEV
Pf SSP2 230 447 1167.15 LIFFDLFLV Pf SSP2 15 448 1167.17 LLMDCSGSI
Pf SSP2 51 449 1167.09 VLLGGVGLV Pf EXP1 91 450 B7 19.0051 LPYGRTNL
Pf SSP2 126 451 Other 16.0245 FQDEENIGIY Pf LSA1 1794 452 16.0040
FVEALFQEY Pf CSP 15 453 1167.54 FYFILVNLL Pf LSA1 9 454 1167.53
KYKLATSVL Pf EXP1 73 455 1167.56 KYLVIVFLI Pf SSP2 8 456 15.0184
LPSENERGY Pf LSA1 1663 457 16.0130 PSDGKCNLY Pf SSP2 207 458
16.0077 PSENERGYY Pf LSA1 1664 459 1167.57 PYAGEPAPF Pf SSP2 528
460 1167.55 YYIPHQSSL Pf LSA1 1671 461
[0274]
11TABLE 9 Activation of T Cell Proliferation by Expression Vectors
Encoding MHC Class II Epitopes Fused to MHC Class II Targeting
Sequences Stimulating Peptide.sup.1 Immunogen PADRE OVA 323 CORE
128 peptide + CFA.sup.2 3.0 (1.1) 2.7 (1.2) 3.2 (1.4)
pEP2.(PAOS).(-) -- -- -- pEP2.(AOS).(-) 5.6 (1.8) -- --
pEP2.(PAOS).(sigTh) 5.0 (2.9) -- 2.6 (1.5)
pEP2.(PAOS).(Ig.alpha.Th) 5.6 (2.1) -- 3.0 (1.6)
pEP2.(PAOS).(LampTh) 3.8 (1.7) -- 3 pEP2.(PAOS).(IiTh) 5.2 (2.0)
3.2 (1.5) 3.7 (1.5) pEP2.(PAOS).(H2M) 3.3 (1.3) -- 2.8
.sup.1Geometric mean of cultures with SI .gtoreq. 2.
.sup.2Proliferative response measured in the lymph node.
[0275]
12TABLE 10 CTL Epitopes in CDNA Minigene Immunogenicity In Vivo
(IFA) CTL MHC Response Binding No. CTL- (Geo.Mean MHC Affinity
Positive .times./.div.SD).sup.b Epitope Sequence Restrict. IC30 %
(nM) Cultures .DELTA.LU SEQ ID NO: HBV Core 18 FLPSDFFPSV A2.1 3
6/6 73.0 (1.1) 91 HBV Env 335 WLSLLVPFV A2.1 5 4/6 5.3 (1.6) 107
HBV Pol 455 GLSRYVARL A2.1 76 ND.sup.c ND 99 HIV Env 120 KLTPLCVTL
A2.1 102 2/5 6.4 (1.3) 93 HIV Pol 476 ILKEPVHGV A2.1 192 2/5 15.2
(2.9) 101 HBV Pol 55 1-A YMDDVVLGA A2.1 200 0/6 -- 95 HBV Pol 55
1-V YMDDVVLGV A2.1 5 6/6 8.2 (2.3) 462 HIV Env 49 TVYYGVPVWK A11 4
28/33 13.4 (3.1) 105 HBV Core 141 STLPETRVVRR A11 4 6/6 12.1 (2.6)
103 HBV Pol 149 HTLWKAGILYK A11 14 6/6 13.1 (1.2) 88 .sup.aPeptide
tested in HLA-A2.1/K.sup.bH-2.sup.bxs transgenic mice by
co-immunizing with a T helper cell peptide in IFA. .sup.bGeometric
mean CTL response of positive cultures. .sup.cND, not done.
[0276]
13TABLE 11 Summary of Immunogenicity of pMin.1 DNA construct in HLA
A2.1/K.sup.b transgenic mice CTL Response.sup.a No. Positive Geo.
Mean Response Positive Epitope Cultures/Total.sup.b Cultures
[.times./.div.SD] .DELTA.LU HBV Core 18 9/9 455.5 [2.2] HIV Env 120
12/12 211.9 [3.7] HBV Pol 551-V 9/9 126.1 [2.8] HBV Pol 455 12/12
738.6 [1.3] HIV Pol 476 11/11 716.7 [1.5] HBV Env 335 12/12 43.7
[1.8] HBV Core 18 10/10 349.3 [1.8] (Theradigm).sup.c .sup.aMice
were immunized with pMin.1 DNA or Theradigm-HBV lipopeptide and CTL
activity in splenocyte cultures was determined after in vitro
stimulation with individual peptide epitopes. Results from four
independent experiments are shown. .sup.bSee Example V, Materials
and Methods for definition of a CTL-positive culture.
.sup.cResponse of mice immunized with Theradigm-HBV lipopeptide
containing the HBV Core 18 epitope.
[0277]
14TABLE 12 Summary of immunogenicity in HLA A11/K.sup.b transgenic
mice CTL Response.sup.a No. Positive Geo. Mean Response Positive
Epitope Cultures/Total.sup.b Cultures [.times./.div.SD] .DELTA.LU
HBV Core 141 5/9 128.1 [1.6] HBV Pol 149 6/9 267.1 [2.2] HIV Env 43
9/9 40.1 [2.9] .sup.aMice were immunized with pMin.1 DNA and CTL
activity in splenocyte cultures was determined after in vitro
stimulation with individual A11-restricted epitopes. The geometric
mean CTL response from three independent experiments are shown.
.sup.bDefinition of a CTL-positive culture is described in Example
V, Materials and Methods.
* * * * *
References