Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOCONJUGATES COMPRISING POXVIRUS-DERIVED
PEPTIDES AND ANTIBODIES AGAINST ANTIGEN-PRESENTING
CELLS FOR SUBUNIT-BASED POXVIRUS VACCINES
Related Applications
[001] This application claims priority to U.S. Patent Applications Serial Nos.
12/754,140,
filed April 5, 2010; 12/754,740, filed April 6, 2010, and U.S. Provisional
Patent Applications
Nos. 61/258,369, filed November 5, 2009; 61/258,729, filed November 6, 2009;
and
61/378,059, filed August 30, 2010. The text of each priority application is
incorporated
herein by reference in its entirety.
BACKGROUND
Field of the Invention
[002] The present invention relates to the design, generation and use of
subunit-based
vaccines for the treatment and/or prevention of poxvirus infections, including
but not limited
to smallpox. In preferred embodiments, the vaccines comprise an
immunoconjugate of a
subunit antigenic peptide derived from one or more viral proteins. In more
preferred
embodiments the viral proteins are immunomodulating factors, such as the viral
IL-18
binding protein (vIL 18BP), although alternative viral proteins may be used,
such as viral
envelope proteins. In other alternative embodiments, subunit-based vaccines
may comprise
combinations of antigenic peptides from more than one viral protein, such as
an
immunomodulating factor and an envelope protein. The viral antigenic peptide
is attached to
an antibody or antigen-binding fragment thereof that targets the subunit to
antigen-producing
cells (APC5). In most preferred embodiments, the subunit-based vaccine
incorporates an
antibody or antibody fragment against the HLA-DR antigen, such as the L243
antibody;
although the skilled artisan will realize that other APC targeting antibodies
are known and
may be used. Use of the immunoconjugate provides substantially increased
immunogenicity
and improved immune system response against viral antigens, while avoiding the
possibility
of infection of immunocompromised individuals exposed to live virus-based
vaccines.
Preferably, the subunit-based vaccine is effective to induce immunity against
and to prevent
infection by smallpox and/or other poxviruses in vivo.
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Related Art
[003] The Orthopoxviruses, a group of complex viruses with cross-reacting
antigens,
includes vaccinia virus (VV), monkeypox virus, and the virus that causes
smallpox (variola,
VAR). Smallpox is no longer a naturally occurring infection, having been
eradicated by a
massive immunization program up to 1978, when routine vaccination of the
world's
population ceased (Minor, 2002, British Med J 62:213-224). At that time, the
remaining
stocks of virus were deposited in the U.S. and the former Soviet Union. The
recent threat of
bioterrorism, a recent outbreak of monkeypox (CDC MMWR 2003), and the deadly
nature of
smallpox disease, especially in a world where it is estimated that less than
half the population
is vaccinated, has stimulated renewed interest in development of vaccine
protection against
VAR, or other dangerous members of this family of viruses.
[004] Smallpox vaccine, which employs active VV, currently represents the most
effective
means to immunize against smallpox (Rosenthal et al, 2001, Emerg. Infect. Dis.
7: 920-926).
This vaccine produces a transient viremia which is resolved in most
individuals, and which
leaves long-lasting immunity. However, this vaccine also raises safety issues
because of
serious adverse reactions, which include systemic viremia and death (He et al,
2007, JID 196:
1026-1032; Rosenthal et al, 2001). Therefore, development of alternative
vaccination
strategies is required if circumstances necessitate immunization of the
population.
[005] Several approaches to alternative vaccines have been tried, or are in
development.
Attenuated forms of poxvirus, such as the Akhara Modified Vaccinia (MVA), with
deleted or
mutated genes (Grandpre et al., Vaccine 27:1549-56, 2009), may confer partial
immunity to
highly virulent strains of poxvirus. Immunization with inactivated virus has
been
investigated, but it does not confer the same degree of protection as live
virus; 103-104 more
units of inactivated virus are required to protect mice from challenge,
regardless of the
inactivation method used (Turner et al. 1970, J Hyg. Camb 68:197-210). This
fact implies
that the protection derived from immunization with active virus includes
factors that are not
produced by, or are not present in, inactive virus. Poxviruses produce a
spectrum of secreted
host immune response modifying factors which neutralize host cytokines and
innate defense
mechanisms. By weakening the host's first line of defense, the virus may be
able to establish
infection (e.g. in the mucosa) and begin the first phase of infectious
replication in host cells.
[006] A need exists for vaccines against poxviruses, such as smallpox, that
are more
effective than inactivated virus but which avoid the safety issues seen with
live virus
vaccines.
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SUMMARY OF THE INVENTION
[007] The present invention discloses improved compositions and methods of use
of subunit
vaccines against poxviruses, such as smallpox. In preferred embodiments, the
vaccine
comprises one or more subunit antigenic peptides conjugated to an antibody or
antibody
fragment that binds to antigen presenting cells (APCs), such as dendritic
cells (DCs), to form
an immunoconjugate. Administration of the vaccine to subjects induces immunity
against the
poxvirus and is effective to treat or prevent poxvirus infection. Optionally,
the vaccine may
incorporate one or more adjuvants, such as aluminum hydroxide, CpG DNA,
calcium
phosphate or bacterial-based adjuvant (e.g., L. delbroeckii/bulgaricus)..
[008] One host immune modulating factor produced by both VV and VAR is the
viral
interleukin-18 binding protein (vIL18BP, vaccinia virus C12L gene). Like other
viral host
defense modulating factors, this gene is expressed in the early phase of
infection, and it
cripples host immunity by neutralizing a key pro-inflammatory cytokine, IL-18,
which
stimulates NK, CD8, and Thl CD4 cells to produce interferon-gamma (IFN), which
in turn
activates antigen presenting cells (APCs), and other cells and which directs
immune
responses toward the Thl type (Born et al, 2000, J. Immunol. 164: 3246-3254;
Scott, 1991, J
Immunol 147:3149-3155; Pien et al, 2002,. Immunol. 169:5827-5837; Xiang and
Moss,
1999, Proc. Natl. Acad. Sci. USA 96:11537-11542). In preferred embodiments,
the subunit
antigenic peptide is selected to mimic an epitope of vIL18BP. Other exemplary
host immune
modulating factors and their locus-tag identifiers are provided in Table 1.
[009] In other preferred embodiments the subunit antigenic peptide is derived
from a viral
immunomodulating protein. The skilled artisan will realize that various viral
immunomodulating proteins are known and may be of use. Non-limiting examples
include
the interferon-gamma (IFN-gamma) receptor homolog (B8R gene), complement
control
protein homolog (B5R gene) and serine protease inhibitors (B 13R, B 14R, B22R
genes). A
wide variety of poxvirus immunomodulatory proteins have been reported,
although their
effect on viral immunogenicity has not been well characterized. (See, e.g.,
Jackson et al. J
Virol 79:6554-59, 2005; B12R gene (ser/thr protein kinase); B15R gene (IL-1
and IL-6
receptor); B 16R gene (IL-1 receptor), B 18R gene (IFN-a receptor), B 19R gene
(IL-1 and IL-
6 receptor, IFN inhibitor.)
Table 1. Poxvirus Immunomodulating Proteins
VACWR001 TNF-alpha receptor-like
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VACWROI I apoptosis
VACWR012 zinc(Zn)-finger-like
VACWR013 (VAC WR C12L) IL-18 binding protein
VACWR025 blocks C3b/C4b complement activation
VACWR028 intracellular signal transduction inhibitor
VACWR033 serine protease inhibitor
VACWR034 interferon resistance
VACWR059 ds RNA binder; interferon binder
VACWR172 Toll-like receptor modulator
VACWR190 IFN-gamma receptor-like
VACWR208 Zn-finger-like
VACV WR 215 TNF-alpha R
VACWR217 TNF-alpha receptor-like
VACV COP B19R (VACWR200) IFN-type I binder
VACV COP A39R semaphoring-like
VACV COP A40R type II membrane protein
VACV COP A41 L secreted glycoprotein
VACV A4L immunodominant antigen
VACV A27L (VACWR150) surface binding heparin sulfate
VACV D8L (VACWR113) surface binding chondroitin sulfate
VACV B5R (VACWR187) essential for membrane wrapping of IMV in trans-Golgi
[0010] In alternative embodiments, the subunit antigenic peptide may be
derived from a viral
envelope protein or other viral proteins. Non-limiting examples include the
protein products
of the D8L, A27L, L1R and A33R genes. The skilled artisan will realize that
the DNA and
amino acid sequences of the various poxviral genes and proteins are well known
in the art
and are publicly available (see, e.g., GenBank Accession No. AY243312 for the
complete
genomic sequence of Vaccinia virus WR, along with the encoded protein
sequences).
[0011] The antibody component of the immunoconjugate directs the complex to
APCs,
where the antigenic peptide component is processed to invoke an immune
response against
poxviruses and/or infected cells expressing the target antigen. Various APC
targeting
antibodies are known in the art, such as antibodies that bind to an antigen
selected from the
group consisting of HLA-DR, CD74, CD209 (DC-SIGN), CD34, CD74, CD205, TLR 2
(toll-
like receptor 2), TLR 4, TLR 7, TLR 9, BDCA-2, BDCA-3 and BDCA-4. In more
preferred
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embodiments, the antibody binds to an antigen selected from HLA-DR and CD74.
In most
preferred embodiments, the antibody binds to HLA-DR.
[0012] In certain preferred embodiments, the poxvirus vaccine comprises a
humanized,
human or chimeric anti-HLA-DR antibody, such as the L243 antibody. The L243
antibody
has been described (e.g., U.S. Patent No. 7,612,180, the Examples section of
which is
incorporated herein by reference) and is characterized by having heavy chain
complementarity determining region (CDR) sequences CDR1 (NYGMN, SEQ ID NO:1),
CDR2 (WINTYTREPTYADDFKG, SEQ ID NO:2), and CDR3 (DITAVVPTGFDY, SEQ
ID NO:3) and light chain CDR sequences CDR1 (RASENIYSNLA, SEQ ID NO:4), CDR2
(AASNLAD, SEQ ID NO:5), and CDR3 (OHFWTTPWA, SEQ ID NO:6). However, other
anti-HLA-DR antibodies known in the art may be used (see, e.g., U.S. Patent
Nos. 6,416,958,
6,894,149; 7,262,278, the Examples section of each of which is incorporated
herein by
reference).
[0013] In other preferred embodiments, the poxvirus vaccine comprises a
humanized, human
or chimeric anti-CD74 antibody, such as the LL1 antibody. The LLI antibody has
been
described (e.g., U.S. Patent No. 7,312,318, the Examples section of which is
incorporated
herein by reference) and is characterized by having light chain CDR sequences
CDR1
(RSSQSLVHRNGNTYLH; SEQ ID NO:7), CDR2 (TVSNRFS; SEQ ID NO:8), and CDR3
(SQSSHVPPT; SEQ ID NO:9) and heavy chain CDR sequences CDRI (NYGVN; SEQ ID
NO:10), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO: 11), and CDR3
(SRGKNEAWFAY; SEQ ID NO: 12). Alternatively, other anti-CD74 antibodies or
antibodies against other APC- or DC-associated antigens may be utilized (see,
e.g., LifeSpan
Biosciences Inc., Seattle, WA; BioLegend, San Diego, CA; Abcam, Cambridge,
MA).
[0014] In various embodiments, the antibody or antigen-binding fragment
thereof may be
chimeric, humanized or human. The use of chimeric antibodies is preferred to
the parent
murine antibodies because they possess human antibody constant region
sequences and
therefore do not elicit as strong a human anti-mouse antibody (HAMA) response
as murine
antibodies. The use of humanized antibodies is even more preferred, in order
to further
reduce the possibility of inducing a HAMA reaction. As discussed below,
techniques for
humanization of murine antibodies by replacing murine framework and constant
region
sequences with corresponding human antibody framework and constant region
sequences are
well known in the art and have been applied to numerous murine anti-cancer
antibodies.
Antibody humanization may also involve the substitution of one or more human
framework
amino acid residues with the corresponding residues from the parent murine
framework
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region sequences. As also discussed below, techniques for production of human
antibodies
are also well known and such antibodies may be incorporated into the subject
poxvirus
vaccine constructs.
[0015] Still other embodiments relate to DNA sequences encoding fusion
proteins, such as
antibody-subunit antigenic peptide fusion proteins, vectors and host cells
containing the DNA
sequences, and methods of making fusion proteins for the production of
poxvirus vaccines.
In certain embodiments, where DNL (dock-and-lock) technology is used to make
the subunit
vaccine, the fusion proteins may comprise DDD (dimerization and docking
domain) moieties
or AD (anchoring domain) moieties. In alternative embodiments, the
immunoconjugate may
be formed by chemical cross-linking of, for example, an antibody or antibody
fragment and
an antigenic peptide.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0016] As used herein, the terms "a", "an" and "the" may refer to either the
singular or
plural, unless the context otherwise makes clear that only the singular is
meant.
[0017] As used herein, the term "about" means plus or minus ten percent (10%)
of a value.
For example, "about 100" would refer to any number between 90 and 110.
[0018] An antibody refers to a full-length (i.e., naturally occurring or
formed by normal
immunoglobulin gene fragment recombinatorial processes) immunoglobulin
molecule (e.g.,
an IgG antibody) or an immunologically active, antigen-binding portion of an
immunoglobulin molecule, like an antibody fragment.
[0019] An antibody fragment is a portion of an antibody such as F(ab')2,
F(ab)2, Fab', Fab,
Fv, scFv and the like. Regardless of structure, an antibody fragment binds
with the same
antigen that is recognized by the intact antibody. Therefore the term is used
synonymously
with "antigen-binding antibody fragment." The term "antibody fragment" also
includes
isolated fragments consisting of the variable regions, such as the "Fv"
fragments consisting of
the variable regions of the heavy and light chains and recombinant single
chain polypeptide
molecules in which light and heavy variable regions are connected by a peptide
linker ("scFv
proteins"). As used herein, the term "antibody fragment" does not include
portions of
antibodies without antigen binding activity, such as Fc fragments or single
amino acid
residues. Other antibody fragments, for example single domain antibody
fragments, are
known in the art and may be used in the claimed constructs. (See, e.g.,
Muyldermans et al.,
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TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; Maass
et al., J
Immunol Methods 324:13-25, 2007).
[00201 The term antibody fusion protein may refer to a recombinantly produced
antigen-
binding molecule in which one or more of the same or different single-chain
antibody or
antibody fragment segments with the same or different specificities are
linked. Valency of
the fusion protein indicates how many binding arms or sites the fusion protein
has to a single
antigen or epitope; i.e., monovalent, bivalent, trivalent or multivalent. The
multivalency of
the antibody fusion protein means that it can take advantage of multiple
interactions in
binding to an antigen, thus increasing the avidity of binding to the antigen.
Specificity
indicates how many antigens or epitopes an antibody fusion protein is able to
bind; i.e.,
monospecific, bispecific, trispecific, multispecific. Using these definitions,
a natural
antibody, e.g., an IgG, is bivalent because it has two binding arms but is
monospecific
because it binds to one epitope. Monospecific, multivalent fusion proteins
have more than
one binding site for an epitope but only bind with one epitope. The fusion
protein may
comprise a single antibody component, a multivalent or multispecific
combination of
different antibody components or multiple copies of the same antibody
component. The
fusion protein may additionally comprise an antibody or an antibody fragment
and a subunit
peptide antigen. However, the term is not limiting and a variety of protein or
peptide
effectors may be incorporated into a fusion protein. In another non-limiting
example, a
fusion protein may comprise an AD or DDD sequence for producing a DNL
construct as
discussed below.
[00211 A chimeric antibody is a recombinant protein that contains the variable
domains
including the complementarity determining regions (CDRs) of an antibody
derived from one
species, preferably a rodent antibody, while the constant domains of the
antibody molecule
are derived from those of a human antibody. For veterinary applications, the
constant
domains of the chimeric antibody may be derived from that of other species,
such as a cat or
dog.
[00221 A humanized antibody is a recombinant protein in which the CDRs from an
antibody
from one species; e.g., a rodent antibody, are transferred from the heavy and
light variable
chains of the rodent antibody into human heavy and light variable domains
(e.g., framework
region sequences). The constant domains of the antibody molecule are derived
from those of
a human antibody. In certain embodiments, a limited number of framework region
amino
acid residues from the parent (rodent) antibody may be substituted into the
human antibody
framework region sequences.
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[00231 A human antibody is, e.g., an antibody obtained from transgenic mice
that have been
"engineered" to produce specific human antibodies in response to antigenic
challenge. In this
technique, elements of the human heavy and light chain loci are introduced
into strains of
mice derived from embryonic stem cell lines that contain targeted disruptions
of the
endogenous murine heavy chain and light chain loci. The transgenic mice can
synthesize
human antibodies specific for particular antigens, and the mice can be used to
produce human
antibody-secreting hybridomas. Methods for obtaining human antibodies from
transgenic
mice are described by Green et al., Nature Genet. 7:13 (1994), Lonberg et al.,
Nature 368:856
(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully human antibody
also can be
constructed by genetic or chromosomal transfection methods, as well as phage
display
technology, all of which are known in the art. See for example, McCafferty et
al., Nature
348:552-553 (1990) for the production of human antibodies and fragments
thereof in vitro,
from immunoglobulin variable domain gene repertoires from unimmunized donors.
In this
technique, antibody variable domain genes are cloned in-frame into either a
major or minor
coat protein gene of a filamentous bacteriophage, and displayed as functional
antibody
fragments on the surface of the phage particle. Because the filamentous
particle contains a
single-stranded DNA copy of the phage genome, selections based on the
functional properties
of the antibody also result in selection of the gene encoding the antibody
exhibiting those
properties. In this way, the phage mimics some of the properties of the B
cell. Phage display
can be performed in a variety of formats, for review, see e.g. Johnson and
Chiswell, Current
Opinion in Structural Biology 3:5564-571 (1993). Human antibodies may also be
generated
by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, the
Examples
sections of which are incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[00241 FIG. 1. Binding and uptake of peptides derived from vIL18BP sequence
(SEQ ID
NO:23). (A) vIL18BP110 (SEQ ID NO:16) bound to T2 cells. Indicated peptides
(TT830,
SEQ ID NO:19; vA4L229, SEQ ID NO:18; vIL18BP008, SEQ ID NO:13; vIL18BP105,
SEQ ID NO:15; vIL18BP110, SEQ ID NO:16; vIL18BP117, SEQ ID NO:17) were
incubated
with T2 cells for 24 h. Relative abundance of HLA-A02 on T2 cells is shown.
Each bar, left
to right, represents increasing concentrations of peptide from 0 to 40 g/mL
in 10- g/mL
increments. (B) vIL18BP105 (SEQ ID NO: 15) demonstrated the highest uptake by
donor
PBMCs. Duplicate samples were evaluated after incubation with the indicated
biotinylated
peptides for 24 h. NJO1, NJ04, NJ07 and NJ08. Results were analyzed by flow
cytometry
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after addition of an avidin-FITC conjugate. Fluorescence value for each
peptide equals
fluorescence value of peptide-treated cells minus the fluorescence value of
untreated cells in
the same experiment. Peptide concentration was 20 g/mL.
[0025] FIG. 2. PBMCs from vaccinated donors proliferate when incubated with
viral
peptides. CFSE-loaded PBMCs from vaccinated (A) and unvaccinated (B) human
donors
were incubated with 10 g/mL of the designated peptide (vA27L003, SEQ ID
NO:20;
vD8L118, SEQ ID NO:22; vIL18BP105, SEQ ID NO:15 or control) for 5 days. Cells
were
harvested and analyzed by flow cytometry (means + SD). Bars shown in order:
open bars,
medium control; solid black bars, 2.5 mg/mL peptide (or SEA); horizontal-hatch
light-grey
bars, 5.0 mg/mL peptide; vertical-hatch dark-grey bars, 10.0 mg/mL peptide.
(C) Results
from separate experiments where cells from the designated samples were
incubated with
vD8L1 18 (SEQ ID NO:22) to determine intracellular cytokine and activation
marker
expression. The results are shown in the embedded table (C) (D8L, vD8L 118
peptide, SEQ
ID NO:22). * Group average P < 0.05 vs. medium controls (t-test).
[0026] FIG. 3. The responding CD8+ IFN-y+ cells have the phenotype of TEM or
CD45RA-
terminally differentiated cells. CD8+ PBMCs from vaccinated donors were
assessed for
CD45RA and CCR7 expression. The numbers represent percentage of total cells. P
< 0.019
vD8L1 18 vs. medium controls (ANOVA) for the TEM population (lower left
quadrant).
[0027] FIG. 4. CD107a expression by CD8+ cells. CD8+ cell population was
assessed for
IFN-y, IL-2 vs. degranulation potential marker CD 107a. Numbers and bar values
represent
percentage of gated cells for (A) CD8+IFN-7+ cells, (B) CD8+IFN-y- cells, and
(C) CD8+IL-
2+ cells. * group average P < 0.04 vs. medium control (t-test).
[0028] FIG. 5. Antibody to peptides is present in serum from vaccinated
donors. Serum
from unvaccinated or vaccinated donors was diluted 1:200 and incubated with
peptide
immobilized on 96-well plates in a modified ELISA for (A) peptide vA27L003
(SEQ ID
NO:20), (B) peptide vD8L1 10 (SEQ ID NO:21), and (C) peptide vIL18BP102 (SEQ
ID
NO:14). Dots represent the A450 for each donor. * P < 0.03 vs. unvaccinated
(ANOVA).
Unvaccinated donors: 213, 704, 220; vaccinated donors: 05, 12A, 12B, 19, 26,
720, 308,
416, and 920. Peptides vD8L110 and vIL18BP105 were 25-mers which included the
full
sequences of vD8L118 and vIL18BP105.
[0029] FIG. 6. HLA-DR04 tg splenocyte proliferation to v1L 18BP 105. HLA-DR04
tg mice
were immunized with vIL18BP105-L243 conjugate (conj) or free vIL18BP105
(Free), naive
HLA-DR04 tg mice (HLA-DR04 tg naive) and wild type C57BL/6J (WT naive) (n=3
mice/group). Assays were performed in triplicate with CFSE- labeled
splenocytes incubated
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with varied concentrations of peptides. Results are typical of 3 separate
experiments (n=3,
means + SD).
[0030] FIG. 7. Peptide-specific serum antibody production in HLA-DR04 tg mice
immunized with CIL18BP105 (Conjugate) and IL18BP105 (Free) 7 and 14 days
following
the first boost. Naive HLA-DR04 tg mouse serum was used as control (Naive).
Experiments
were performed in triplicate with pooled sera (n=3, means + SD).
[0031] FIG. 8. Binding of serum antibodies from immunized mice to intact
vIL18BP
protein. Serum from mice immunized with L243 antibody alone, vIL18BP105
peptide (SEQ
ID NO:15), the viral IL18BP105 peptide conjugated to L243 antibody
(CIL18BP105),
medium alone, or naive mice was tested for antibodies recognizing intact
vIL18BP protein.
[0032] FIG. 9. Liposome based immunoconjugate for subunit vaccine. (A)
Liposome-
displayed peptide-L243 antibody conjugate. (B) Liposome-displayed bare peptide
without
antibody.
Poxvirus Vaccines
Subunit Antigenic Peptides
[0033] Poxviruses produce a spectrum of secreted host immune-response
modifying factors
which neutralize host cytokines and innate defense mechanisms. Weakening the
host's first
line of defense allows the virus to establish infection (e.g., in the mucosa)
and then begin the
first phase of infectious replication. One factor produced by VV and other
poxviruses is the
viral interleukin-18 binding protein (vIL18BP, vaccinia virus C12L gene),
expressed in the
early phase of infection (Born et al. J Immunol 164:3246-54, 2000). It works
by neutralizing
a key pro-inflammatory cytokine, IL-18, which stimulates NK, CD8, and Thl CD4
cells to
produce interferon-y (IFN-y), which directs acquired immunity toward the Th 1
type
(Livingston et al., J Immunol 168:5499-5506, 2002; Pien et al., J Immunol
160:5827-37,
2002; Scott, J Immunol 147:3149-55, 1991; Turner et al., J Hyg Camb 68:197-
210, 1970;
Xiang and Moss, PNAS USA 96:11537-542, 1999).
[0034] The studies described in the Examples below were addressed to the
question of
whether or not host response against vIL18BP is involved in resistance to
poxvirus infection.
If so, an alternative vaccine strategy should include this factor and/or
similar antigens. It has
recently been reported that another orthopoxvirus host defense-modulating
factor, type-I IFN-
binding protein, was essential for virulence (Xu et al., J Exp Med 205:981-92,
2008) and may
be a candidate for inclusion in a subunit poxvirus vaccine. As discussed
below, other known
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viral proteins may also be candidates for inclusion as subunit antigenic
peptides for a subunit-
based poxvirus vaccine.
[0035] As described in the following Examples, the subunit peptide-based
vaccine approach
to human immunity was tested by investigating whether vIL18BP antigen peptides
were able
to elicit recall responses by peripheral blood mononuclear cells (PBMCs), and
serum of
vaccinated human subjects. The importance of cell-mediated immunity in
resistance to
poxvirus remains under investigation, but it is fairly well established that
antibody response is
required for immunity (Chaudhri et al., J Virol 80:6339-44, 2006; Combadiere
et al., J Exp
Med 199, 1585-89, 2004; Kim et al., Clin Vaccine Immunol 13:1172-74, 2006).
[0036] In addition to the vIL 18BP-derived peptides, peptides derived from
other VV genes,
D8L and A27L, were also tested. The D8L protein is important for viral
attachment and entry
into cells, and has been shown to elicit strong protective immunity in mouse
models of
poxvirus infection (Kan-Mitchell et al., J Immunol 172:5249-61, 2010; Berhanu
et al., J Virol
82:3517-29, 2008). The A27L protein is also important for viral attachment and
assembly,
and antibodies against it provide protective immunity (Berhanu et al., J Virol
82:3517-29,
2008; Chun get al., J Virol 72:1577-85, 1998; Scott, J Immunol 147:3149-55,
1991).
[0037] The overall goal of this invention was to select and develop T-cell
(HLA-binding) and
B- cell antigen peptides for inclusion in a multi-epitopic vaccine format.
Peptides have the
advantages of being relatively easy to synthesize, modify, and combine into
multi-antigen
complexes. To enhance their immunogenicity, the peptides were attached to
antibodies
targeting APCs, such as antibodies against the HLA-DR antigen.
APC- Targeting Antibodies
[0038] As the professional antigen-presenting cells, dendritic cells (DCs)
play a pivotal role
in orchestrating innate and adaptive immunity, and have been harnessed to
create effective
vaccines (Vulink et al., Adv Cancer Res. 2008, 99:363-407; O'Neill et al., Mol
Biotechnol.
2007, 36:131-41). In vivo targeting of antigens to APCs and DCs represents a
promising
approach for vaccination, as it can bypass the laborious and expensive ex vivo
antigen loading
and culturing, and facilitate large-scale application of immunotherapy (Tacken
et al., Nat Rev
Immunol. 2007, 7:790-802). More significantly, in vivo APC and/or DC targeting
vaccination is more efficient in eliciting anti-tumor immune response, and
more effective in
controlling tumor growth in animal models (Kretz-Rommel et al., J Immunother
2007,
30:715-726).
[0039] In addition to DCs, B cells are another type of potent antigen-
presenting cells capable
of priming Thl/Th2 cells (Morris et al, J Immunol. 1994, 152:3777-3785;
Constant, J
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Immunol. 1999, 162:5695-5703) and activating CD8 T cells via cross-
presentation (Heit et
al., J Immunol. 2004, 172:1501-1507; Yan et al., Int Immunol. 2005, 17:869-
773). It was
recently reported that in vivo targeting of antigens to B cells breaks immune
tolerance of
MUC 1 (Ding et al., Blood 2008, 112:2817-25).
[0040] In various embodiments of the present invention, antibodies against
antigens
expressed by APCs in general and DCs in particular may be incorporated into
immunoconjugate vaccines to target subunit antigenic peptides to immune system
cells. Two
exemplary APC antigens are HLA-DR and CD74. HLA-DR is a major
histocompatibility
complex class II cell surface receptor which functions in antigen presentation
to elicit T-cell
immune responses. HLA-DR is found on a wide variety of antigen presenting
cells, such as
macrophages, B-cells and dendritic cells. As discussed above, antibodies
against HLA-DR,
including the L243 antibody, are known in the art. Such antibodies may be
conjugated to
subunit antigenic peptides for delivery to APCs.
[00411 Another APC expressed antigen is CD74, which is a type II integral
membrane
protein essential for proper MHC II folding and targeting of MHC II-CD74
complex to the
endosomes (Stein et al., Clin Cancer Res. 2007, 13:5556s-5563s; Matza et al.,
Trends
Immunol. 2003, 24(5):264-8). CD74 expression is not restricted to DCs, but is
found in
almost all antigen-presenting cells (Freudenthal et al., Proc Natl Acad Sci U
S A. 1990,
87:7698-702; Clark et al., J Immunol. 1992, 148(11):3327-35). The wide
expression of
CD74 in APCs may offer some advantages over sole expression in myeloid DCs, as
targeting
of antigens to other APCs like B cells has been reported to break immune
tolerance (Ding et
al., Blood 2008, 112:2817-25), and targeting to plasmacytoid DCs cross-
presents antigens to
naive CD8 T cells. More importantly, CD74 is also expressed in follicular DCs
(Clark et al.,
J Immunol. 1992, 148(11):3327-35), a DC subset critical for antigen
presentation to B cells
(Tew et al., Immunol Rev. 1997, 156:39-52). This expression profile makes CD74
an
excellent candidate for in vivo targeting vaccination. A variety of anti-CD74
antibodies are
known in the art, such as the LL1 antibody (Leung et al., Mol Immunol. 1995,
32:1416-1427;
Losman et al., Cancer 1997, 80:2660-2666; Stein et al., Blood 2004, 104:3705-
11).
Antibodies and Antibody Fragments
[0042] In various embodiments, antibodies or antigen-binding fragments of
antibodies may
be incorporated into the poxvirus vaccine. Antigen-binding antibody fragments
are well
known in the art, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and the like,
and any such
known fragment may be used. As used herein, an antigen-binding antibody
fragment refers
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to any fragment of an antibody that binds with the same antigen that is
recognized by the
intact or parent antibody. Techniques for preparing conjugates of virtually
any antibody or
fragment of interest are known (e.g., U.S. Patent No. 7,527,787).
[0043] Techniques for preparing monoclonal antibodies against virtually any
target antigen,
such as HLA-DR or CD74, are well known in the art. See, for example, Kohler
and Milstein,
Nature 256:495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN
IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly,
monoclonal antibodies can be obtained by injecting mice with a composition
comprising an
antigen, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes
with
myeloma cells to produce hybridomas, cloning the hybridomas, selecting
positive clones
which produce antibodies to the antigen, culturing the clones that produce
antibodies to the
antigen, and isolating the antibodies from the hybridoma cultures.
[0044] MAbs can be isolated and purified from hybridoma cultures by a variety
of well-
established techniques. Such isolation techniques include affinity
chromatography with
Protein-A SEPHAROSE , size-exclusion chromatography, and ion-exchange
chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3.
Also, see Baines et al., "Purification of Immunoglobulin G (IgG)," in METHODS
IN
MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).
[0045] After the initial raising of antibodies to the immunogen, the
antibodies can be
sequenced and subsequently prepared by recombinant techniques. Humanization
and
chimerization of murine antibodies and antibody fragments are well known to
those skilled in
the art. The use of antibody components derived from humanized, chimeric or
human
antibodies obviates potential problems associated with the immunogenicity of
murine constant
regions.
Chimeric Antibodies
[0046] A chimeric antibody is a recombinant protein in which the variable
regions of a
human antibody have been replaced by the variable regions of, for example, a
mouse
antibody, including the complementarity-determining regions (CDRs) of the
mouse antibody.
Chimeric antibodies exhibit decreased immunogenicity and increased stability
when
administered to a subject. General techniques for cloning murine
immunoglobulin variable
domains are disclosed, for example, in Orlandi et al., Proc. Nat'l Acad. Sci.
USA 86:3833
(1989). Techniques for constructing chimeric antibodies are well known to
those of skill in
the art. As an example, Leung et al., Hybridoma 13:469 (1994), produced an LL2
chimera
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by combining DNA sequences encoding the V. and VH domains of murine LL2, an
anti-
CD22 monoclonal antibody, with respective human x and IgG1 constant region
domains.
Humanized Antibodies
[00471 Techniques for producing humanized MAbs are well known in the art (see,
e.g., Jones
et al., Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988),
Verhoeyen et al.,
Science 239:1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285
(1992), Sandhu,
Crit. Rev. Biotech. 12:437 (1992), and Singer et al., J. Immun. 150:2844
(1993)). A chimeric
or murine monoclonal antibody may be humanized by transferring the mouse CDRs
from the
heavy and light variable chains of the mouse immunoglobulin into the
corresponding variable
domains of a human antibody. The mouse framework regions (FR) in the chimeric
monoclonal antibody are also replaced with human FR sequences. As simply
transferring
mouse CDRs into human FRs often results in a reduction or even loss of
antibody affinity,
additional modification might be required in order to restore the original
affinity of the murine
antibody. This can be accomplished by the replacement of one or more human
residues in the
FR regions with their murine counterparts to obtain an antibody that possesses
good binding
affinity to its epitope. See, for example, Tempest et al., Biotechnology 9:266
(1991) and
Verhoeyen et al., Science 239:1534 (1988). Generally, those human FR amino
acid residues
that differ from their murine counterparts and are located close to or
touching one or more
CDR amino acid residues would be candidates for substitution. Humanized forms
of the
L243 and LL1 antibodies are known (see, e.g., U.S. Patent Nos. 7,612,180 and
7,312,318).
Human Antibodies
[00481 Methods for producing fully human antibodies using either combinatorial
approaches
or transgenic animals transformed with human immunoglobulin loci are known in
the art
(e.g., Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller,
2005, Comb.
Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr. Opin.
Phamacol.
3:544-50). A fully human antibody also can be constructed by genetic or
chromosomal
transfection methods, as well as phage display technology, all of which are
known in the art.
See for example, McCafferty et al., Nature 348:552-553 (1990). Such fully
human
antibodies are expected to exhibit even fewer side effects than chimeric or
humanized
antibodies and to function in vivo as essentially endogenous human antibodies.
In certain
embodiments, the claimed methods and procedures may utilize human antibodies
produced
by such techniques.
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[0049] In one alternative, the phage display technique may be used to generate
human
antibodies (e,g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40).
Human antibodies
may be generated from normal humans or from humans that exhibit a particular
disease state,
such as cancer (Dantas-Barbosa et al., 2005). The advantage to constructing
human
antibodies from a diseased individual is that the circulating antibody
repertoire may be biased
towards antibodies against disease-associated antigens.
[0050] In one non-limiting example of this methodology, Dantas-Barbosa et al.
(2005)
constructed a phage display library of human Fab antibody fragments from
osteosarcoma
patients. Generally, total RNA was obtained from circulating blood lymphocytes
(1d).
Recombinant Fab were cloned from the t, y and K chain antibody repertoires and
inserted
into a phage display library (Id.). RNAs were converted to cDNAs and used to
make Fab
cDNA libraries using specific primers against the heavy and light chain
immunoglobulin
sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97). Library construction
was
performed according to Andris-Widhopf et al. (2000, In: Phage Display
Laboratory Manual,
Barbas et al. (eds), 1St edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY
pp. 9.1 to 9.22). The final Fab fragments were digested with restriction
endonucleases and
inserted into the bacteriophage genome to make the phage display library. Such
libraries may
be screened by standard phage display methods, as known in the art (see, e.g.,
Pasqualini and
Ruoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J. Nucl.
Med. 43:159-
162).
[0051] Phage display can be performed in a variety of formats, for their
review, see e.g.
Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).
Human
antibodies may also be generated by in vitro activated B-cells. See U.S.
Patent Nos.
5,567,610 and 5,229,275, incorporated herein by reference in their entirety.
The skilled
artisan will realize that these techniques are exemplary and any known method
for making
and screening human antibodies or antibody fragments may be utilized.
[0052] In another alternative, transgenic animals that have been genetically
engineered to
produce human antibodies may be used to generate antibodies against
essentially any
immunogenic target, using standard immunization protocols. Methods for
obtaining human
antibodies from transgenic mice are disclosed by Green et al., Nature Genet.
7:13 (1994),
Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579
(1994). A non-
limiting example of such a system is the XenoMouse (e.g., Green et al., 1999,
J. Immunol.
Methods 231:11-23) from Abgenix (Fremont, CA). In the XenoMouse and similar
animals,
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the mouse antibody genes have been inactivated and replaced by functional
human antibody
genes, while the remainder of the mouse immune system remains intact.
[0053] The XenoMouse was transformed with germline-configured YACs (yeast
artificial
chromosomes) that contained portions of the human IgH and Igkappa loci,
including the
majority of the variable region sequences, along accessory genes and
regulatory sequences.
The human variable region repertoire may be used to generate antibody
producing B-cells,
which may be processed into hybridomas by known techniques. A XenoMouse
immunized
with a target antigen will produce human antibodies by the normal immune
response, which
may be harvested and/or produced by standard techniques discussed above. A
variety of
strains of XenoMouse are available, each of which is capable of producing a
different class
of antibody. Transgenically produced human antibodies have been shown to have
therapeutic
potential, while retaining the pharmacokinetic properties of normal human
antibodies (Green
et al., 1999). The skilled artisan will realize that the claimed compositions
and methods are
not limited to use of the XenoMouse system but may utilize any transgenic
animal that has
been genetically engineered to produce human antibodies.
Antibody Fragments
[0054] Antibody fragments which recognize specific epitopes can be generated
by known
techniques. Antibody fragments are antigen binding portions of an antibody,
such as F(ab')2,
Fab', F(ab)2, Fab, Fv, sFv and the like. F(ab')2 fragments can be produced by
pepsin digestion
of the antibody molecule and Fab' fragments can be generated by reducing
disulfide bridges
of the F(ab')2 fragments. Alternatively, Fab' expression libraries can be
constructed (Huse et
al., 1989, Science, 246:1274-1281) to allow rapid and easy identification of
monoclonal Fab'
fragments with the desired specificity. F(ab)2 fragments may be generated by
papain
digestion of an antibody and Fab fragments obtained by disulfide reduction.
[0055] A single chain Fv molecule (scFv) comprises a VL domain and a VH
domain. The
VL and VH domains associate to form a target binding site. These two domains
are further
covalently linked by a peptide linker (L). Methods for making scFv molecules
and designing
suitable peptide linkers are described in US Patent No. 4,704,692, US Patent
No. 4,946,778,
R. Raag and M. Whitlow, "Single Chain Fvs." FASEB Vol 9:73-80 (1995) and R.E.
Bird and
B.W. Walker, "Single Chain Antibody Variable Regions," TIBTECH, Vol 9:132-137
(1991).
[0056] Techniques for producing single domain antibodies are also known in the
art, as
disclosed for example in Cossins et al. (2006, Prot Express Purif 51:253-259).
Single domain
antibodies (VHH) may be obtained, for example, from camels, alpacas or llamas
by standard
immunization techniques. (See, e.g., Muyldermans et al., TIBS 26:230-235,
2001; Yau et al.,
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17
J Immunol Methods 281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25,
2007).
The VHH may have potent antigen-binding capacity and can interact with novel
epitopes that
are inaccessible to conventional VH-VL pairs. (Muyldermans et al., 2001).
Alpaca serum
IgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs) (Maass
et al.,
2007). Alpacas may be immunized with known antigens, such as TNF-a, and VHHs
can be
isolated that bind to and neutralize the target antigen (Maass et al., 2007).
PCR primers that
amplify virtually all alpaca VHH coding sequences have been identified and may
be used to
construct alpaca VHH phage display libraries, which can be used for antibody
fragment
isolation by standard biopanning techniques well known in the art (Maass et
al., 2007).
[0057] An antibody fragment can be prepared by proteolytic hydrolysis of the
full length
antibody or by expression in E. coli or another host of the DNA coding for the
fragment. An
antibody fragment can be obtained by pepsin or papain digestion of full length
antibodies by
conventional methods. These methods are described, for example, by Goldenberg,
U.S.
Patent Nos. 4,036,945 and 4,331,647 and references contained therein. Also,
see Nisonoff et
al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959),
Edelman et
al., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and
Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
Known Antibodies
[0058] Although antibodies against HLA-DR or CD74 are preferred, the poxvirus
vaccine can
alternatively be made by using an antibody that binds to or is reactive with
another antigen on
the surface of the target cell. Preferred additional MAbs may comprise a
humanized, chimeric
or human MAb reactive with CD209 (DC-SIGN), CD34, CD205, TLR 2 (toll-like
receptor 2),
TLR 4, TLR 7, TLR 9, BDCA-2, BDCA-3 or BDCA-4.
[0059] Such antibodies may be obtained from public sources like the American
Type Culture
Collection or from commercial antibody vendors. For example, antibodies
against
CD209(DC-SIGN), CD34, BDCA-2, TLR2, TLR 4, TLR 7 and TLR 9 may be purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against CD205
and
BDCA-3 may be purchased from Miltenyi Biotec Inc. (Auburn, CA). Numerous other
commercial sources of antibodies are known to the skilled artisan.
[0060] These are exemplary only and a wide variety of other antibodies and
their hybridomas
are known in the art. The skilled artisan will realize that antibody sequences
or antibody-
secreting hybridomas against almost any APC-associated antigen may be obtained
by a
simple search of the ATCC, NCBI and/or USPTO databases for antibodies against
a selected
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target antigen of interest. The antigen binding domains of the cloned
antibodies may be
amplified, excised, ligated into an expression vector, transfected into an
adapted host cell and
used for protein production, using standard techniques well known in the art.
Immunoconjugates
[0061] In various embodiments, the poxvirus vaccine may be administered as an
immunoconjugate. Many methods for making covalent or non-covalent conjugates
with
antibodies or fusion proteins are known in the art and any such known method
may be
utilized.
[0062] For example, an antigenic peptide can be attached at the hinge region
of a reduced
antibody component via disulfide bond formation. Alternatively, such agents
can be attached
using a heterobifunctional cross-linker, such as N-succinyl 3-(2-
pyridyldithio)propionate
(SPDP). Yu et al., Int. J. Cancer 56:244 (1994). General techniques for such
conjugation are
well-known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al.,
"Modification of Antibodies by Chemical Methods," in MONOCLONAL ANTIBODIES:
PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss,
Inc.
1995); Price, "Production and Characterization of Synthetic Peptide-Derived
Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge University Press
1995).
Dock and Lock (DNL) method
[00631 In alternative embodiments, subunit-based vaccines comprising
immunoconjugates
may be made by other techniques. One technique for conjugating virtually any
protein or
peptide to any other protein or peptide is known as the dock-and-lock (DNL)
technique. The
DNL method exploits specific protein/protein interactions that occur between
the regulatory
(R) subunits of cAMP-dependent protein kinase (PKA) and the anchoring domain
(AD) of A-
kinase anchoring proteins (AKAPs) (Baillie et al., FEBS Letters. 2005;
579:3264. Wong and
Scott, Nat. Rev. Mol. Cell Biol. 2004; 5:959).
[0064] PKA, which plays a central role in one of the best studied signal
transduction
pathways triggered by the binding of the second messenger cAMP to the R
subunits, was first
isolated from rabbit skeletal muscle in 1968 (Walsh et al., J. Biol. Chem.
1968;243:3763).
The structure of the holoenzyme consists of two catalytic subunits held in an
inactive form by
the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are
found with two
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types of R subunits (RI and RII), and each type has a and (3 isoforms (Scott,
Pharmacol.
Ther. 1991;50:123). The R subunits have been isolated only as stable dimers
and the
dimerization domain has been shown to consist of the first 44 amino-terminal
residues
(Newlon et al., Nat. Struct. Biol. 1999;6:222). Binding of cAMP to the R
subunits leads to
the release of active catalytic subunits for a broad spectrum of
serine/threonine kinase
activities, which are oriented toward selected substrates through the
compartmentalization of
PKA via its docking with AKAPs (Scott et al., J. Biol. Chem. 1990;265;21561).
[0065] Since the first AKAP, microtubule-associated protein-2, was
characterized in 1984
(Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs
that
localize to various sub-cellular sites, including plasma membrane, actin
cytoskeleton,
nucleus, mitochondria, and endoplasmic reticulum, have been identified with
diverse
structures in species ranging from yeast to humans (Wong and Scott, Nat. Rev.
Mol. Cell
Biol. 2004;5:959). The AD of AKAPs for PKA is an amphipathic helix of 14-18
residues
(Carr et al., J. Biol. Chem. 1991;266:14188). The amino acid sequences of the
AD are quite
varied among individual AKAPs, with the binding affinities reported for RII
dimers ranging
from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA. 2003;100:4445).
Interestingly,
AKAPs will only bind to dimeric R subunits. For human RIIa, the AD binds to a
hydrophobic surface formed by the 23 amino-terminal residues (Colledge and
Scott, Trends
Cell Biol. 1999; 6:216). Thus, the dimerization domain and AKAP binding domain
of human
RIla are both located within the same N-terminal 44 amino acid sequence
(Newlon et al.,
Nat. Struct. Biol. 1999;6:222; Newlon et al., EMBO J. 2001;20:1651), which is
termed the
DDD herein.
DDD of Human RIIa and AD of AKAPs as Linker Modules
[0066] We have developed a platform technology to utilize the DDD of human
RIIa and the
AD of a AKAPs as an excellent pair of linker modules for docking any two
entities, referred
to hereafter as A and B, into a noncovalent complex, which could be further
locked into a
stably tethered structure through the introduction of cysteine residues into
both the DDD and
AD at strategic positions to facilitate the formation of disulfide bonds. The
general
methodology of the "dock-and-lock" approach is as follows. Entity A is
constructed by
linking a DDD sequence to a precursor of A, resulting in a first component
hereafter referred
to as a. Because the DDD sequence would effect the spontaneous formation of a
dimer, A
would thus be composed of a2. Entity B is constructed by linking an AD
sequence to a
precursor of B, resulting in a second component hereafter referred to as b.
The dimeric motif
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of DDD contained in a2 will create a docking site for binding to the AD
sequence contained
in b, thus facilitating a ready association of a2 and b to form a binary,
trimeric complex
composed of alb. This binding event is made irreversible with a subsequent
reaction to
covalently secure the two entities via disulfide bridges, which occurs very
efficiently based
on the principle of effective local concentration because the initial binding
interactions should
bring the reactive thiol groups placed onto both the DDD and AD into proximity
(Chmura et
al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically.
[0067] In certain alternative embodiments, the poxvirus vaccine
immunoconjugates are based
on a variation of the alb structure, in which each heavy chain of an anti-HLA-
DR or anti-
CD74 antibody or F(ab')2 or F(ab)2 antibody fragment is attached at its C-
terminal end to one
copy of an AD moiety. Since there are two heavy chains per antibody or
fragment, there are
two AD moieties per antibody or fragment. A subunit antigenic peptide is
attached to a
complementary DDD moiety. After dimerization of DDD moieties, each DDD dimer
binds
to one of the AD moieties attached to the IgG antibody or F(ab')2 or F(ab)2
fragment, resulting
in a stoichiometry of four antigenic peptides per IgG or F(ab')2 or F(ab)2
unit. However, the
skilled artisan will realize that alternative complexes may be utilized, such
as attachment of
the antigenic peptide to the AD sequence and attachment of the anti-HLA-DR or
anti-CD74
MAb or fragment to the DDD moiety, resulting in a different stoichiometry of
effector
moieties. For example, by attaching a DDD sequence to the C-terminal end of
each heavy
chain of an IgG antibody or F(ab')2 fragment, and attaching an AD sequence to
the antigenic
peptide, a DNL complex may be constructed that comprises one antigenic peptide
and one
antibody or fragment.
[0068] By attaching the DDD and AD away from the functional groups of the two
precursors, such site-specific ligations are expected to preserve the original
activities of the
two precursors. This approach is modular in nature and potentially can be
applied to link,
site-specifically and covalently, a wide range of substances.
[0069] In preferred embodiments, the DDD or AD moiety is covalently attached
to an
antibody or antigenic peptide to form a fusion protein or peptide. A variety
of methods are
known for making fusion proteins, including nucleic acid synthesis,
hybridization and/or
amplification to produce a synthetic double-stranded nucleic acid encoding a
fusion protein
of interest. Such double-stranded nucleic acids may be inserted into
expression vectors for
fusion protein production by standard molecular biology techniques (see, e.g.
Sambrook et al.,
Molecular Cloning, A laboratory manual, 2nd Ed, Cold Spring Harbor Press, Cold
Spring
Harbor, NY, 1989). In such preferred embodiments, the AD and/or DDD moiety may
be
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attached to either the N-terminal or C-terminal end of a protein or peptide.
However, the skilled
artisan will realize that the site of attachment of an AD or DDD moiety may
vary. For example,
although an AD or DDD moiety may be attached to either the N- or C-terminal
end of an
antibody or antibody fragment while retaining antigen-binding activity,
attachment to the C-
terminal end positions the AD or DDD moiety farther from the antigen-binding
site and appears
to result in a stronger binding interaction (e.g., Chang et al., Clin Cancer
Res 2007, 13:5586s-
91 s). Site-specific attachment of a variety of effector moieties may be also
performed using
techniques known in the art, such as the use of bivalent cross-linking
reagents and/or other
chemical conjugation techniques.
Methods of Therapeutic Treatment
Formulations
[0070] The poxvirus vaccine can be formulated according to known methods to
prepare
pharmaceutically useful compositions, whereby the poxvirus vaccine is combined
in a mixture
with a pharmaceutically suitable excipient. Sterile phosphate-buffered saline
is one example
of a pharmaceutically suitable excipient. Other suitable excipients are well-
known to those in
the art. See, for example, Ansel et at., PHARMACEUTICAL DOSAGE FORMS AND
DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing
Company 1990), and revised editions thereof.
[0071] The poxvirus vaccine is preferably administered either subcutaneously
or nasally.
More preferably, the poxvirus vaccine is administered as a single or multiple
boluses via
subcutaneous injection. Formulations for administration can be presented in
unit dosage
form, e.g., in ampoules or in multi-dose containers, with an added
preservative. The
compositions can take such forms as suspensions, solutions or emulsions in
oily or aqueous
vehicles, and can contain formulatory agents such as suspending, stabilizing
and/or
dispersing agents. Alternatively, the active ingredient can be in powder form
for constitution
with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0072] Additional pharmaceutical methods may be employed to control the
duration of action
of the poxvirus vaccine. Control release preparations can be prepared through
the use of
polymers to complex or adsorb the poxvirus vaccine. For example, biocompatible
polymers
include matrices of poly(ethylene-co-vinyl acetate) and matrices of a
polyanhydride
copolymer of a stearic acid dimer and sebacic acid. Sherwood et at.,
Bio/Technology
10:1446 (1992). The rate of release from such a matrix depends upon the
molecular weight
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22
of the poxvirus vaccine, the amount of poxvirus vaccine within the matrix, and
the size of
dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et
al., supra.
Other solid dosage forms are described in Ansel et al., PHARMACEUTICAL DOSAGE
FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and
Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack
Publishing Company 1990), and revised editions thereof.
[0073] Generally, the dosage of an administered poxvirus vaccine for humans
will vary
depending upon such factors as the patient's age, weight, height, sex, general
medical
condition and previous medical history. It may be desirable to provide the
recipient with a
dosage of poxvirus vaccine that is in the range of from about 1 mg/kg to 25
mg/kg as a single
administration, although a lower or higher dosage also may be administered as
circumstances
dictate. A dosage of 1-20 mg/kg for a 70 kg patient, for example, is 70-1,400
mg, or 41-824
mg/m2 for a 1.7-m patient. The dosage may be repeated as needed for induction
of an
immune response.
[0074] In alternative embodiments, therapeutic peptides may be administered by
an
inhalational route (e.g., Sievers et al., 2001, Pure Appl. Chem. 73:1299-
1303). Supercritical
carbon dioxide aerosolization has been used to generate nano or micro-scale
particles out of a
variety of pharmaceutical agents, including proteins and peptides (Id.)
Microbubbles formed
by mixing supercritical carbon dioxide with aqueous protein or peptide
solutions may be
dried at lower temperatures (25 to 65 C.) than alternative methods of
pharmaceutical powder
formation, retaining the structure and activity of the therapeutic peptide
(Id.) In some cases,
stabilizing compounds such as trehalose, sucrose, other sugars, buffers or
surfactants may be
added to the solution to further preserve functional activity. The particles
generated are
sufficiently small to be administered by inhalation. In still other
alternatives, nasal
administration of an aqueous solution may be utilized.
Kits
[0075] Various embodiments may concern kits containing components suitable for
treating or
diagnosing diseased tissue in a patient. Exemplary kits may contain at least
one or more
poxvirus vaccine immunoconjugates as described herein. If the composition
containing
components for administration is not formulated for delivery via nasal
administration or
inhalation, a device capable of delivering the kit components through
subcutaneous injection
may be included. One type of device is a syringe that is used to inject the
composition into
the body of a subject. In certain embodiments, a therapeutic agent may be
provided in the
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form of a prefilled syringe or autoinjection pen containing a sterile, liquid
formulation or
lyophilized preparation.
[0076] The kit components may be packaged together or separated into two or
more
containers. In some embodiments, the containers may be vials that contain
sterile,
lyophilized formulations of a composition that are suitable for
reconstitution. A kit may also
contain one or more buffers suitable for reconstitution and/or dilution of
other reagents. Other
containers that may be used include, but are not limited to, a pouch, tray,
box, tube, or the
like. Kit components may be packaged and maintained sterilely within the
containers.
Another component that can be included is instructions to a person using a kit
for its use.
Expression Vectors
[0077] Still other embodiments may concern DNA sequences comprising a nucleic
acid
encoding a poxvirus vaccine immunoconjugate, or its constituent proteins.
Fusion proteins may
comprise an anti-HLA-DR antibody attached to a subunit antigenic peptide.
Alternatively the
encoded fusion proteins may comprise a DDD or AD moiety attached to an
antibody or
antigenic peptide.
[0078] Various embodiments relate to expression vectors comprising the coding
DNA
sequences. The vectors may contain sequences encoding the light and heavy
chain constant
regions and the hinge region of a human immunoglobulin to which may be
attached chimeric,
humanized or human variable region sequences. The vectors may additionally
contain
promoters that express the encoded protein(s) in a selected host cell,
enhancers and signal or
leader sequences. Vectors that are particularly useful are pdHL2 or GS. More
preferably, the
light and heavy chain constant regions and hinge region may be from a human EU
myeloma
immunoglobulin, where optionally at least one of the amino acid in the
allotype positions is
changed to that found in a different IgGi allotype, and wherein optionally
amino acid 253 of the
heavy chain of EU based on the EU number system may be replaced with alanine.
See Edelman
et al., Proc. Natl. Acad. Sci USA 63:78-85 (1969). In other embodiments, an
IgGI sequence
may be converted to an IgG4 sequence.
[0079] The skilled artisan will realize that methods of genetically
engineering expression
constructs and insertion into host cells to express engineered proteins are
well known in the
art and a matter of routine experimentation. Host cells and methods of
expression of cloned
antibodies or fragments have been described, for example, in U.S. Patent Nos.
7,531,327;
7,537,930 and 7,608,425, the Examples section of each incorporated herein by
reference.
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EXAMPLES
[0080] The following examples are provided to illustrate, but not to limit,
the claims of the
present invention.
Example 1. Immune Response to Poxvirus Subunit Antigenic Peptides
Materials and Methods
[0081] Peptide design. 9-mer or 15-mers peptide sequences bearing multiple
potential
binding sites for both HLA class I and/or HLA class II molecules were derived
from poxvirus
open reading frames by visual screening for HLA anchor residues at the correct
spacing, or
by use of web-based methods (e.g., BIMAS or SYFPEITHI [Parker et al., J
Immunol
152:163-75, 1994; Rammensee et al., Immunogenetics 50:213-19, 1999]), with
selection
based on high potential for specific HLA-binding (Table 2). The nucleotide and
amino acid
sequences of vIL18BP (CJ2L), A4L (Boulanger et al., J Virol 72:170-79, 1998),
A27L
(Chung et al., J Virol 72:1577-85, 1998), or D8L (Hsaio et al., J Virol
73:8750-61) VV
antigens were retrieved from NIH GenBank, Accession number: AY243312. These
peptides
are designated by their gene source and a number (e.g., vIL18BP105 or
vD8L118).
Table 2. Amino acid number and HLA restriction of poxvirus vIL18BP-derived
peptides, A4L229, and TT830.
Peptide Peptide length (amino acids) Potential HLA binding@
TT830* 15-mer HLA-A02, (A03, DR04, DR15)
vA4L229 9-mer HLA-A02, (A03, A 11)
vIL18BP008 9-mer HLA-A02, (A03, A11)
vIL 18BP 110 9-mer HLA-A02, (A03, A 11)
vIL18BP105 15-mer HLA-A02, A03, (Al 1), A35,
vIL18BP102 25-mer DRO1, DR04, DR07, DR 11, DR15
vA27L003 15-mer HLA-A01, A02, A03, (A11),
DROI, DR03, DR04, DR07, DR15
vD8L1 18 15-mer HLA-A01, A02, A03, A11, DRO1,
vD8L110 25-mer (DR03), DR04, DR07, DR11,
(DR15)
*TT830, Tetanus toxoid T-cell helper peptide; A4L229, epitope from vaccinia
A4L ORF;
vIL18BP, poxvirus IL-18 binding protein-derived peptides (vaccinia C12L).
@Binding
potential without parentheses >_ 15 (www.SYNPEITHI.de); within parentheses,
binding
probability of 10-14. 15-mers may contain more than one potential HLA class I
binding
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epitope in addition to HLA class II epitopes. Only a limited number of HLA
types are
shown.
[0082] Peptide amino acid sequences are shown below. Peptides were screened
for
similarities with the human genome, using the NIH Blast server
(http://www.ncbi.nlm.nih.gov/blasto. Peptides with homology to the human
proteome were
discarded. All newly designed peptides were commercially synthesized at 95%
purity
(Sigma-Genosys, Woodlands, TX USA, New England Peptide, Gardner, MA).
vIL18BP118
CVLTTLNGV (SEQ ID NO:13)
vIL18BP102
KFAHYRFTCVLTTLNGVSKKNIVVLK (SEQ ID NO:14)
vIL18BP105
HYRFTCVLTTLNGVS (SEQ ID NO:15)
vIL18BP110
CVLTTLNGV (SEQ ID NO:16)
vIL18BP117
GVSKKNIWL (SEQ ID NO:17)
vA4L229 (variola virus)
ALKDLMSSV (SEQ ID NO:18)
TT830 (Clostridium tetani)
QYIKANAKFIGITEL (SEQ ID NO:19)
vA27L003-027
GTLFPGDDDLAIPAT (SEQ ID NO:20)
vA27L003-012
GTLFPGDDDLAIPATEFFSTKAAKK (SEQ ID NO:28)
vA27L004-012
TLFPGDDDL (SEQ ID NO:29)
vD8L110-134
HDDGLIIISIFLQVLDHKNVYFQKI (SEQ ID NO:21)
vD8L118-132
SIFLQVLDHKNVYFQ (SEQ ID NO:22)
vD8L116-124
IISIFLQVL (SEQ ID NO:33)
vB5R001-025
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MKTISVVTLLCVLPAVVYSTCTVPT (SEQ ID NO:30)
vBSRO04-018
ISVVTLLCVLPAVVY (SEQ ID NO:31)
vBSRO08-016
TLLCVLPAV (SEQ ID NO:32)
[0083] Donor samples Buffy coats were obtained from the Blood Center of New
Jersey
(NJBB) (West Orange, NJ USA). Other PBMC samples were obtained from local
donors
after approval for use of human blood by the New England Institutional Review
Board
(Wellesley, MA USA), or from Cellular Technology Limited (CTL) (Shaker Hts, OH
USA).
Table 3 summarizes the donor HLA types, age, and vaccine status. Due to
limited numbers
of cells in each sample, not all samples were included in every assay.
Table 3. Summary of blood donor vaccine status, age, and HLA type.
Vaccine status Average age SD % (HLA allele)
(range)
Vaccinated 43 11 14% (A01), 45% (A02), 9% (A03), 0% (All), 11%
(N = 22) (18-66) (DR01), 16% (DR04), 9% (DR07), 2% (DR11), 9%
(DR15)
Unvaccinated 30 12 7% (A01), 50% (A02), 0% (A03), 4% (All), 4%
(N = 14) (17-49) (DR01), 0% (DR04),14% (DR07), 14%(DRl1), 7%
(DR15)
[0084] DNA from donor PBMCs was amplified according to HLA-Typing kit
(Biotest,
Dreieich, Germany) specifications. HLA type was provided for the CTL, Inc.,
samples.
Vaccinated donors were persons who either stated that they had previously
received the live
smallpox vaccine, or vaccination status was presumed based on age, while
unvaccinated
donors were persons who stated they had not received a smallpox vaccination or
were born
after vaccination ceased in the U.S. Due to limited numbers of cells in most
samples, not all
samples were tested in all assays. When the HLA type of the donors was not
determined by
the supplier, PBMCs were typed for HLA by SSP-PCR using the Biotest kit
(Biotest,
Dreieich, Germany).
[0085] Peptide screening Transporter associated with antigen-processing
protein-1 and -2
(TAP1 and 2)-deficient human B/T hybridoma cell line, T2 cells (ATCC,
Manassas, VA
USA), which expresses surface HLA-A02 exclusively, and which increases its
expression
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when stabilized by peptide in the antigen presentation groove (Nijman et al.,
Eur J Immunol
23:1215-19, 1993), was incubated with beta-2-microglobulin and peptides at the
indicated
concentrations. Due to TAP deficiency, peptides are not processed, and so must
be of a length
that allows binding to HLA-A02 (9-mer). Analysis of HLA was performed using
FITC-
labeled W6/32 (BD Pharmingen, San Diego, CA USA) and a FACSCALIBURTM flow
cytometer (Becton Dickinson, San Jose, CA USA). Binding of the peptide
epitopes to human
PBMCs obtained from donors was detected by incubation of PBMCs at 1x106/mL
with
biotinylated peptides, followed by addition of avidin-FITC conjugate to fixed
cells, and flow
cytometry.
[0086] T-cell proliferation and phenotype analysis For evaluation, peptides
were screened in
vitro against PBMCs from smallpox-vaccinated and naive donors, using a
carboxyfluorescein
diacetate succinimidyl ester (Invitrogen, Carlsbad, CA USA) based cell
proliferation assay
(Younes et al., J Exp Med 198:1909-22, 2003). For comparison purposes,
peptides derived
from the immunodominant poxvirus protein, A4L (Boulanger et al., J Virol
72:170-79, 1998),
another from Tetanus Toxoid (TT830) (Demotz et al., J Immunol 142:394-402,
1989), or the
HIV gag protein (HIVgag) (Kan-Mitchell et al., J Immunol 172:5249-61, 2010),
were
included. Briefly, 10-50 x 106 PBMCs were labeled with CFSE (1.5 M). 2x105
cells (200
L) were incubated with indicated concentrations of peptides, Staphylococcus
aureus
enterotoxin (SEA) (10 ng/mL), or phytohemagglutinin (2.5 g/mL) (PHA, both
from Sigma-
Aldrich). Cells were stained with antibodies against CD8 or CD3, and for
viability (7-AAD)
after 5 days. 20,000 events, gated on live CD3+ lymphocytes, were collected by
flow
cytometry, and analyzed using Flow-Jo software (Mountain View, CA USA).
Proliferation
was evaluated based on the reduction of CFSE fluorescence. The fluorescence
index (FI) of
proliferating cells was calculated by dividing the number of cells losing CFSE
dye in the
presence of the stimulating peptide (test) by the number of cells
proliferating in the absence
of the peptide (control).
[0087] For phenotype analysis, PBMCs in GOLGIPLUGTM (Brefeldin A, 1 g/mL)
were
incubated with 10 g/mL of the indicated peptides, medium control (with PBS
added in same
volume as peptide stock), or SEA or PHA, for 14 h. Cells were then surface- or
intracellularly-stained (after permeabilization) with the indicated
fluorescently-labeled
antibodies (IFN-y or IL-2). Cells were also stained for CD8, CD45RA to
determine prior
encounter with antigen, CCR7 (lymph node homing marker) (38), or CD107a
(cytolytic
capacity marker) (1). The percentages of CD8+ or CD8- effector-memory (TEM) or
terminally differentiated T cells (both CD45RA-CCR7-), central-memory T cells
(TcM)
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(CD45RA-CCR7+), and cytokine-driven differentiated T cells (TEMRA)
(CD45RA+CCR7-)
(12) in peptide-stimulated and control assays were determined. CD8-negative T
cells were
considered to contain the CD4+ population.
[0088] Antibody analysis A modified ELISA-based method (Makabi-Panzu et al.,
Vaccine
16:1504-10, 1998) was used to assess serum antibody. Briefly, ELISA plate
wells were
coated with 10 g/mL of target peptide. After blocking and washing, test sera
were added in
2-fold serial dilutions in PBS. Binding of antibody was detected with
peroxidase-conjugated
anti-human antibody. Plates were developed with o-phenylenediamine
dihydrochioride
peroxidase substrate (Sigma-Aldrich, St. Louis, MO USA) and the optical
density of wells
was measured at 490 nm with an ELISA reader.
[0089] Data analysis The significance of differences observed under the
experimental
conditions was determined by Student's t-test with Fisher's corrections for
multiple
comparisons using Statview+SE software (Abacus Concepts, Berkeley, CA USA), or
Analysis of Variance (ANOVA) (Excel, Microsoft Corp., Redmond, WA USA) as
indicated.
P < 0.05 was considered significant.
Results
[0090] Poxvirus peptide design and screening Poxvirus vIL18BP (SEQ ID NO:23)
was
parsed into 9-, 15-, or 25-mer peptides based on a high score for HLA-binding
potential
according to the ranking system of SYFPEITHI or BIMAS, with emphasis on HLA-
A02- and
HLA-DR04-binding. The vIL 18BP-derived peptides were tested for binding to the
TAP-
deficient T2 hybridoma, which increases expression of HLA-A02 when stabilized
by a
peptide in the antigen-presenting groove. The 9-mer peptides, vIL18BP110 (SEQ
ID
NO:16), vIL18BP117(SEQ ID NO:17), and A4L (SEQ ID NO:18) all contain sequences
with
potential HLA-A0201 binding capability (without processing). Of these
peptides, vA4L
(SEQ ID NO:18), and vIL18BP110 (SEQ ID NO:16) bound HLA-A02 on T2 cells in a
concentration-dependent manner (FIG. IA). The vIL18BP117 (SEQ ID NO:17)
peptide,
despite moderate to high probability of binding HLA-A02, did not. Nor did the
15-mer
peptides incorporating the sequence of vIL18BP110 (SEQ ID NO:16), which T2
cells cannot
process (vIL18BP008, SEQ ID NO:13 and 105, SEQ ID NO:15).
vIL18BP sequence
MRILFLIAFMYGCVHSYVNAVETKCPNLDIVTSSGEFYCSGCVEHMSKFSYMYWLA
KDMKSDEYTKFIEHLGDGIKEDETIRTTDGGITTLRKVLHVTDTNKFAHYRFTCVLTT
LNGVSKKNIWLK (SEQ ID NO:23)
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[0091] The vIL18BP peptides were also predicted to bind several other HLA
haplotypes
(Table 2), most of which were represented in the PBMC donor population,
summarized in
Table 3. When peptides were tested in binding to donor cells, vIL18BP008 (SEQ
ID NO:13,
15-mer) and vIL18BP110 (SEQ ID NO:16, 9-mer) demonstrated strong binding to
PBMCs
from Donors NJ04 (A01/03, DR04) and NJO1 (All, DR15), and relatively weak
binding to
NJ07 (A01, DR16) and NJ08 (A01/02,DR16) PBMCs (FIG. 1B). Taking into account
donor
HLA-types, the predicted HLA target of the peptides, and the T2 results, it
can be concluded
that vIL 18BP 110 (SEQ ID NO:16), a 9-mer, does not bind HLA-A01, but binds
HLA-A02, -
A03, and -A11, all of which were represented by T2 cells, or the donor panel.
[0092] The 15-mer, vIL18BP105 (SEQ ID NO:15), was predicted to bind HLA class
II DR04
and DR 15 (NJO1 and NJ04), and all of class I HLA types represented by the
donors except
HLA-A01. In addition to HLA-A01, donor NJ08 is HLA-A02-positive, thus HLA-A02
may
account for the measured binding. Evidence for binding of vIL18BP105 (SEQ ID
NO: 15) to
HLA-DR 16 is suggested by the strong signal from Donor NJ07, which expresses
HLA-DR 16
and non-binding HLA-A01. While the consensus motif for HLA-DR16 has not been
well-
characterized (Onion et al., J Gen Virol 88:2417-25, 2007), there is at least
one report that
suggests the binding motifs of HLA-DR15 and -DR16 share similarities (Zeng et
al., J Virol
70:3108-17, 1996).
[0093] Immunoreactivity of peptides as antigen mimics for T cells was assessed
by 5-day
CFSE-based proliferation assays, where CFSE-loaded PBMCs from vaccinated or
unvaccinated donors were incubated with vIL18BP105 (SEQ ID NO: 15) and
peptides from
two other poxvirus genes, vD8L1 18 (SEQ ID NO:22) and vA27L003 (SEQ ID NO:20).
The
results for all the vIL18BP105 (SEQ ID NO:15), assays are summarized in Table
4. Results
for concurrent assays for vIL18BP105 (SEQ ID NO:15), vD8L118 (SEQ ID NO:22),
and
vA27L003 (SEQ ID NO:20) are shown in FIG. 2A (vaccinated donors) and FIG. 2B
(unvaccinated donors).
[0094] Overall, vIL18BP105 (SEQ ID NO:15), induced significant proliferation
of PBMCs
from vaccinated donors (Table 4) at a concentration of 10 g/mL. Vaccinated
donor cells
also proliferated when incubated with vD8L118 (SEQ ID NO:22) (6 of 7) and
vA27L003
(SEQ ID NO:20) (4 of 7, FIG. 2A). These results indicate that vIL18BP105 (SEQ
ID
NO:15), , vD8L118 (SEQ ID NO:22), and vA27L003 (SEQ ID NO:20) include epitopes
that
are recognized by lymphocytes from smallpox-vaccinated donors. Cells from
unvaccinated
donors were overall unresponsive to the poxvirus peptides (FIG. 2B). When
samples from
vaccinated donors (12A, 416, 417) and unvaccinated donors (213, 704, 706) were
assayed for
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markers of activation and intracellular cytokine production in separate
experiments (14-h
assays), an IFN-'y response was noted in both CD4+ and CD8+ cells. CD8+ cells
also
expressed the cytolytic capacity marker, CD107a (P < 0.05 vs. medium controls,
FIG. 2C).
Table 4. Summary table of PBMC proliferative responses to vIL18BP105
Donor vaccine status Fluorescence index FI
vIL18BP105 PHA or SEA
Yes(N=11) 5.07 3.37 25.27 14.15
No (N = 10) 1.00 0.46 49.68 24.39
Concentrations of 10 g/mL of vIL18BP105 peptide, 2.5 g/mL of PHA (P), or 10
ng/mL
SEA (S) were used. Fluorescence Index (FI) is estimated by dividing the number
of cells
proliferating in the presence of peptide (or P or S) by the number of cells
proliferating in the
absence of peptide (or P or S) using the CFSE-based cell proliferation assay
described in
Materials and Methods. N = number of individual donors. All samples were
assayed in
triplicate ( mean + SD). P < 0.001, vaccinated vs. unvaccinated for vIL18BP105
(ANOVA).
[0095] Phenotype of proliferating To determine the CD4 or CD8 phenotype of the
proliferating cells, CFSE-loaded PBMCs from vaccinated and unvaccinated
controls
incubated with either vA27L003 (SEQ ID NO:20) or vIL18BP105 (SEQ ID NO:15), (5
days) were probed for CD4 or CD8 expression. Both CD4+ (4/5) and CD8+ (2/5)
cells
proliferated in samples from vaccinated donors, with little to no
proliferation of either subset
of cells in the unvaccinated donor samples (0 of 3, Table 5).
[0096] Further determinations of responding cells' phenotype were performed in
14-hour
intracellular cytokine staining assays. Increased IFN-,y production in the
CD8+ T cell
population was found in samples incubated with vD8L1 18 (SEQ ID NO:22) (2/5)
or
vIL18BP105 (SEQ ID NO:15) (2/5) (Table 6, P < 0.05). Despite stimulating
proliferation in
the 5-day CFSE-based assay, vA27L003 (SEQ ID NO:20) did not stimulate IFN-y or
IL-2
increases (not shown). IFN-,y production did not significantly increase in the
CD4+ T cells,
but isolated samples responded. However, IL-2 production increased
significantly in the
CD4+ population (vD8L118, SEQ ID NO:22) and in CD8+ cells (vIL18BP105, SEQ ID
NO:15) (Table 6; P < 0.04).
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Table 5. CD4+ or CD8+ phenotype of proliferating T cells incubated with
vA27L003 or
vIL18BP105 peptides (5-day assay).
CD4 CD8
Cont PHA vA27L vIL18BP Cont PHA vA27L vIL18BP
UNVACCINATED
NJO1 7.73 37.44 Nd 1.10 1.31 20.38 nd 0.94
E 0.61 11.46 1.11 nd 0.15 5.91 0.15 nd
G 0.92 2.40 0.63 nd 0.21 0.46 0.00 nd
VACCINATED
NJ04 1.66 12.57 Nd 6.02 1.01 6.91 nd 3.64
D 3.52 20.84 3.52 nd 1.65 29.22 1.22 nd
F 1.08 17.67 8.30 nd 1.12 19.80 7.85 nd
H 4.46 49.20 Nd 25.73 2.75 11.00 nd 15.14
NJ08 3.10 26.47 Nd 6.25 3.76 36.29 nd 7.26
Stored human PBMCs were thawed, incubated with CFSE for 24 h, followed by
incubation
with either PHA-P (2.5 g/mL) or 20 g/mL peptide as indicated, after which
they were
stained for CD4 or CD8 expression. Analysis was by flow cytometry. Column
headings:
Cont: medium control; vA27L: vA27L003; vIL18BP: vIL18BP105. Values in bold-
face are
> 1.5-fold vs. control.
[0097] CD8+/IFN-y-producing T cells from the same vaccinated donors were
further
analyzed for markers related to memory phenotype by staining for CD45RA, a
marker of
naive and a subset of effector CD8 cells (TEMRA), and CCR7, a lymph node
homing marker.
This analysis differentiates between TcM (CCR7+CD45RA-), precursors
(CCR7+CD45RA+),
TEMRA (CCR7-CD45RA+), and TEM and terminally differentiated (CCR7-CD45RA-)
cell
populations. The cell types that developed were CCR7-CD45RA- (TEM or
terminally-
differentiated effector) (FIG. 3). The vD8L118 (SEQ ID NO:22) antigen peptide
was most
active in generating these cell types (P < 0.019 vs. medium controls). In
addition, 2 donors in
each assay also responded similarly to vIL18BP105 (SEQ ID NO:15), and vA27L003
(SEQ
ID NO:20).
Table 6. IL-2 or IFN-y production by CD4 and CD8 T cells incubated with
poxvirus
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peptides for 14 h.
Medium PHA vD8L118 vIL18BP105
control
IFN- CD4 CD8 CD4 CD8 CD4 CD8 CD4 CD8
Donor:
NJ291 0.06 0.01 1.25 0.48 0.16 0.08 0.04 0.02
NJ663 0.49 0.06 4.68 0.31 0.40 0.10 0.60 0.10
NJ652 1.17 0.2 8.05 0.62 0.97 0.37 1.34 0.25
12B 0.3 0.01 5.66 2.28 0.45 0.11 0.48 0.02
920 0.37 0.02 7.05 0.49 0.13 0.02 0.41 0.02
IL-2 *
NJ291 0.02 0.01 0.26 0.02 0.21 0.01 0.02 0.04
NJ663 0.22 0.01 1.97 0.11 2.93 0.11 0.17 0.05
NJ652 0.14 0.03 0.60 0.15 2.44 0.27 0.20 0.08
12B 0.03 0 0.30 0.03 0.08 0.01 0.06 0.01
920 0.06 0.01 0.71 0.06 1.20 0.04 0.08 0.02
Values shown are percent positive cells for each cytokine for each donor after
14 h of
incubation with the designated peptide. Cells were stained for CD8, and the
CD8-negative
lymphocytes were considered CD4+. CD8+ cells consisted of 10-30% of total
lymphocytes.
All donors were vaccinated against smallpox. Concentration of peptides: 10
g/mL; PHA,
2.5 g/mL. Values in bold-face are > 1.5-fold above medium control. Response
to HIV
peptide and vA27L003 was not significantly different than medium-only
controls. *P < 0.05
for CD8/IFN-7/vD8L118 and vIL18BP105 vs. medium control: P < 0.04 for CD4/IL-
2/vD81118 vs. medium control; P < 0.013 for CD4/IL-2/vIL18BP105 vs. medium
control (t-
test).
[0098] The capacity of the CD8+ effector cell population to degranulate, i.e.,
their ability to
perform effector function, was assayed by determination of the expression of
CD 107a
(Berhanu et al., J Virol 82:3517-29, 2008) (FIG. 4). In both the CD8+IFN-'y+
and the
CD8+IFN-y- populations, CD107a expression increased 2-7-fold in 3 of 5 PBMC
samples
incubated with vD8L1 18 (SEQ ID NO:22) (P < 0.04). Increased CD107a was also
measured
in the CD8+IL-2+ population when incubated with vIL18BP105 (SEQ ID NO:15), (P
<
0.01) and vD8L 118 (SEQ ID NO:22), although the latter did not achieve
significance.
[0099] CD8+IFN--y+ cells from unvaccinated donors were unresponsive to the
peptides in
similar 14-hour intracellular cytokine staining assays (not shown).
[00100] Serum antibody titers Antibody against poxvirus is required for
protection upon
secondary exposure, and the presence of anti-vaccinia antibody is maintained
in 90% of
vaccinees for decades after vaccination (Hammarlund et al., Nat Med 9:1131-37,
2003).
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33
Therefore, serum antibody from previously vaccinated patients would be
directed toward
immunologically relevant B-cell epitopes. To determine if the antigen
peptides' sequence
included recognizable B-cell epitopes, 1:200 diluted sera from vaccinated and
unvaccinated
donors were tested with the peptides vA27L003 (SEQ ID NO:20) (15-mer),
vIL18BP102,
and vD8L110 (25-mers). The results (FIG. 5) show that serum antibody to the
vD8L110 and
vA27L003 (SEQ ID NO:20) peptides was higher overall, and significantly above
that from
unvaccinated individuals (P < 0.05). Although 3 donors produced antibody that
recognized
vIL18BP102, the overall results did not achieve significance. The results
suggest that the
experimental peptides contained one or several sequences that are B-cell
epitopes. The
presence of anti-peptide antibodies did not differ according to age of donor
or time since
vaccination (not shown).
Discussion
[00101] Inclusion of antigenic peptides in an alternative poxvirus vaccine
requires that they
be relevant targets of human immunity. The results described above determined
whether or
not specific peptides derived from poxvirus antigens were able to elicit
memory responses in
PBMCs from vaccinated donors. The epitopes were derived from three poxvirus
antigens,
including an antigen (vIL 18BP) that is uncharacterized in host immunity, as
well as the
known poxviral envelope antigens, A27L and D8L, which are characterized for
host
protection, but for which specific epitopes are not characterized (Chung et
al., J Virol
72:1577-85, 1998; Hsaio et al., J Virol 73:8750-61, 1999). The types of
responses elicited by
each peptide varied.
[00102] Poxvirus IL18BP modulates host innate immunity by neutralizing NK cell
IL-18
which, in turn, prevents IFN-y production. Recognition of one set of peptides
from vIL18BP,
vIL18BP105 (SEQ ID NO:15), and its derivatives, by CD4+ and CD8+ cells, and
serum
antibody from vaccinated donors, confirms the hypothesis that this, and most
likely other,
transiently-expressed viral host-response modulators are targets of host
immunity.
Neutralization of vIL18BP may aid in protection from initial infection, and
therefore,
establishment of infection, as was demonstrated recently for poxvirus type I
IFN-binding
protein (Xu et al., J Exp Med 205:981-92, 2008).
[00103] The design of an antigen epitope-based vaccine strategy requires that
the viral
components interact with HLA for T-cell development. The peptides in this
study were
predicted to bind several defined HLA haplotypes. But, despite predictions of
HLA-binding,
only some epitopes bound, as was demonstrated by the peptides from the C 12L
sequence
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34
(vIL 18BP).
[00104] Peptides from C 12L (vIL18BP), as well as the antigens A27L and D8L,
elicited
proliferation by CD4+ and CD8+ cells from vaccinated donors, indicating that
APCs take up
the peptides and process them for presentation in the context of both HLA
class I for CD8+,
and class II for CD4+ lymphocytes.
[00105] Immunity to poxvirus is dependent on both cellular and humoral
immunity (Dunne
et al., Blood 100:933-40, 2002; Ferrier-Rembert et al., Viral Immunol 20:214-
20, 2007;
Meseda et al., Clin Vaccine Immunol 16:1261-71, 2009; Xu et al., J Immunol
172:6265-71,
2004), both of which require T-cell help for class switching and affinity
maturation. The
proximity of T and B epitopes within a polypeptide may impact antibody
production due to
differences in their presentation. However, natural B-cell epitopes are quite
often proximal to
HLA-binding regions (Simitsek et al., J Exp Med 181:1957-63, 1995). A number
of factors
may influence the formation of antibody, including HLA type (Quaratino et al.,
J Immunol
174:557-63, 2005).
[00106] In the studies described above, we demonstrated measurable antibody in
a sub-set
of 1 /3 of the vaccinated donors. In addition, the peptides encompassed linear
sequences, and
therefore the antibody recognized linear epitopes, which can include as few as
3 amino acids
(Tahtinen et al., Virology 187:156-64, 1992). In the case of vIL18BP105 (SEQ
ID NO:15),
further evidence of the presence of a relevant B-cell epitope is provided by
mouse studies,
where serum antibody against vIL18BP105 (SEQ ID NO: 15) recognized full length
recombinant vIL 18BP (C 12L) protein (not shown). Overall, these results show
that the
antigen peptides used in this study present T- and B-cell targets of human
response.
[00107] Cytokine and marker production revealed that vIL18BP105 (SEQ ID NO:15)
and
vD8L118 (SEQ ID NO:22) elicited IL-2 production, which preserves the
proliferation
capacity of T cells, even in the absence of CD4 help (Zimmerli et al., Proc
Natl Acad Sci
USA 102:7239-44, 2005). This supports the proliferation data and further
demonstrates the
utility of vILl 8BP peptides for subunit-based vaccines. IFN--y production by
peptide-
stimulated CD8+ TEM cells has multiple effects, including induction of anti-
viral effector
function. Thus, when vD8L118 (SEQ ID NO:22) stimulated CD8+ T cells with
effector and
proliferative potential, the results were similar to that reported for cells
incubated with virus
(Laouar et al., Plos One 3:e4089, 2008). IFN-y production also characterizes
generation of a
Thl response, which is necessary for development of cytotoxicity against
vaccinia (Meseda
et al., Clin Vaccine Immunol 16:1261-71, 2009; Xu et al., J Immunol 172:6265-
71, 2004).
Additionally, production of IL-2 and IFN-'y by CD4+ cells implicates helper
Thi-oriented T-
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cell participation. The A27L peptide did not stimulate increased IFN-y or IL-2
in CD8+ or
CD4+ cells, even though T cells proliferated when incubated with this peptide.
The lack of
activity in the A27L samples may be due to different kinetics of response, or
stimulation of
alternate populations of cells which produce different cytokines or
interleukins, such as IL-4,
for which we did not assay.
[00108] Our results with respect to CD 107a, a marker of cytolytic capacity,
are similar to a
recent study, where a subset of CD4+ cells from revaccinated donors expressed
CD 107a upon
stimulation by vaccinia virus (Puissant-Lubrano et al., J Clin Invest 120:1636-
44, 2010). In
our study, CD8+ cells from vaccinated donors demonstrated enhanced expression
of this
marker upon stimulation by the peptides.
[00109] In contrast to the finding of Combadiere et al. (J Exp Med 199:1585-
89, 2004), we
did not observe loss of ability to produce cytokines in response to vaccinia
antigens when
smallpox vaccination took place more than 45 years previously.
[00110] In summary, we have presented evidence that subunit antigenic peptides
from 3
poxvirus antigens are capable of stimulating recall responses from vaccinated
donors,
including T-cell proliferation, expression of cytokines, and serum antibody
recognition of B-
cell epitopes. One antigenic epitope was from a heretofore uncharacterized
host defense
modulator produced by vaccinia, the IL 18BP. The results presented here show
that
development of an alternative vaccine against poxvirus using select peptide
epitopes could
produce immunity without the hazards of vaccination with active virus. An
advantage of this
virus-free approach over immunization with attenuated forms of poxvirus, the
virulence
genes of which are often deleted or mutated, is that the immunologically-
relevant portions of
any poxvirus gene, as well as altered genes, can be included.
Example 2. Conjugation of APC-Targeting Antibody to Subunit Antigenic Peptides
for
Poxvirus Vaccines
Summary
[00111] The vIL18BP105 (SEQ ID NO:15) was conjugated to the anti-HLA-DR
antibody,
L243, for better presentation to the immune system, and used to immunize HLA-
DR04-
expressing transgenic (tg) mice. Conjugated vIL18BP105 (CIL18BP105) was more
readily
taken up by human and HLA-DR transgenic mouse cells than free vIL18BP105 (SEQ
ID
NO:15). Splenocytes from HLA-DR04 transgenic mice immunized with CIL18BP105
proliferated in vitro when stimulated with vIL18BP105 (SEQ ID NO:15).
Proliferation of
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CIL18BP105-inoculated mouse splenocytes involved CD3+CD4+CD45RA- cells.
Proliferation was accompanied by interferon-y production (quantitative
sandwich ELISA).
CIL18BP105-innoculated mice also showed early and rapidly rising titers of
peptide-specific
antibodies, 4 times that of vIL18BP105-injected controls at day 7 after the
first boost. At a
later time, both CIL18BP105 and vIL18BP105 (SEQ ID NO:15) induced IgG2a and
IgG1,
suggesting the initiation of both Thl and Th2 immunity. Serum antibody from
CIL18BP105-
immunized mice recognized whole recombinant C 12L protein. These results
demonstrate
that conjugation of antigenic peptides to anti-HLA-DR antibody boosts
immunogenicity and
enhances peptide delivery to antigen-presenting cells expressing HLA-DR.
Methods
[00112] HLA-DR antibody-conjugates Peptides that were found to stimulate
proliferation
of immune donor PBMCs were conjugated with L243 antibody using the
heterobifunctional
cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate
(Sulfo-
SMCC), "SMCC", containing H-hydroxysuccinimide (NHS) ester and maleimide
groups,
following the manufacturer's protocol (Pierce, Rockford, IL, USA). SMCC
interacts with
primary amine of the antibody through its NHS ester groups to form amide
bonds, and the
maleimide groups form thioester bonds with the free sulfhydryl group of a C-
terminal
cysteine on the peptide. Briefly, 1 ml of a 1 mg/ml solution of antibody in
PBS was reacted
with 20 L of a 1 mg/ml solution of freshly prepared SMCC in PBS for 2 hours
at 40 C.
Following incubation; excess SMCC was removed through a PBS pre-equilibrated
desalting
column. The activated antibody was collected and then incubated with the
peptide (which
bore a C-terminal cysteine) for another 2 hours (or overnight) at 40 C. The
conjugate was
purified by size exclusion using a P60 fine cross-linked bead column (BioRad,
Hercules, CA,
USA) to remove free peptide. Peptide conjugation efficiency was assessed by
SDS-PAGE
using a 5%-20% gradient gel. Before being injected into mice, conjugate
preparations were
filter-sterilized through a 0.22- m PVDF filter (Millipore, Bedford, MA), and
emulsified in
incomplete Freund's adjuvant (IFA).
[00113] Immunization of mice Six-to-eight week old female C57BL/6J (B6)
transgenic (tg)
mice expressing HLA-DR04 (HLA-DR tg) were obtained from Taconic (Germantown,
NY,
USA). Mice were maintained in a pathogen-free area of our facility. For
immunizations,
groups of 3 mice were primed, and then boosted twice at two-week intervals by
the
subcutaneous route, with 25 gg of vIL18BP105 (SEQ ID NO:15) peptide emulsified
in IFA
in either free, or antibody-conjugated, form. Mice injected with IFA-
emulsified PBS served
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as naive controls. Blood was collected at one-week intervals from priming to
sacrifice, which
was 7 days after the final boost. Spleen samples were collected at sacrifice.
Serum for
antibody detection and isotyping by ELISA was prepared from blood after
overnight
coagulation at 40 C. Splenocytes used in CFSE-based T-cell proliferation
assays and TCR
repertoire analysis, were isolated by mechanical disruption of spleens through
stainless steel
mesh.
[00114] Antibody production analysis and isotyping determination A modified
ELISA-
based method from a previous report was used (Makabi-Panzu et al, 1998) to
assess antibody
production and isotype. Briefly, ELISA plate wells were coated with 10 gg/ml
of peptide in
PBS and incubated overnight at 4 C. They were then blocked with skim milk/PBS
for 30
minutes at 37 C and washed with PBS containing 0.05% Tween 20 (PBST). Test
sera were
either added in 2-fold serial dilution for antibody titer, or as a 1:200
dilution for isotype
determination. Plates were incubated with sera for 2 h at room temperature.
Excess serum
was removed by washing three times with PBST, and peroxidase-conjugated goat
anti-mouse
(or peroxidase-conjugated sheep anti-mouse IgA, IgGI, IgG2a, IgG2b, IgG3 and
IgM in case
of isotyping) at 1:1000 dilution was added for 45 min at room temperature.
Following this
incubation, wells were washed, peroxidase substrate was added, and after
development, the
OD of wells was measured at 490nm with an ELISA reader.
[00115] T-cell proliferation assay and TCRVI3 repertoire analysis T-cell
proliferation for
either donor PBMCs or murine splenocytes was assessed using a 5-day CFSE-based
cell
proliferation assay as reported previously (Younes et al, 2003). Briefly, 10-
50 x 106 PBMC
or splenocytes were labeled with CFSE at a final concentration of 1.5 M.
Cells were washed
twice in PBS and re-suspended in complete RPMI medium at 106 cells/ml. 2X105
cells were
incubated with indicated concentrations of peptides or PHA (2.5 gg/ml) for
positive control
wells. Cells were stained with CD4-APC, CD8-PE with 7-AAD or CD3-perCp after 5
days of
in vitro incubation at 37 C in a 5% CO2 atmosphere. A minimum of 20,000
events gated on
live CD3+ lymphocytes were collected on a FACScalibur flow cytometer, and
analyzed using
Flow Jo software. T-cell proliferation was evaluated based on the reduction of
CFSE
fluorescence of growing cells. An integrated cell proliferation Flow Jo
program was used for
analysis. The fluorescence index of proliferating cells was calculated by
dividing the number
of cells losing the CFSE dye in the presence of the stimulating peptide (test)
by the number of
cells proliferating in the absence of the peptide (control).
[00116] For the TCRVB repertoire analysis, washed splenocytes from immunized
or naive
mice were washed again with complete RPMI- 1640 medium and with staining
buffer, then
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pre-stained for T-cell surface markers as described above, for 20 min at 40 C,
before being
incubated again for 15 min at 40 C with the blocking 2.4G2 anti-FcRIII/I mAb.
The cells
were then stained with an appropriate fluorescently labeled anti-TCRVf
antibody without
removal of the FcR-blocking mAb. Following this last incubation, the cells
were washed with
stain buffer and analyzed by flow cytometry.
[00117] Data analysis The significance of differences observed under the
experimental
conditions was determined using one way analysis of variance followed as
appropriate by a t-
test with Fisher's corrections for multiple comparisons using Statview+SE
software (Abacus
Concepts, Berkeley, CA). P < 0.05 was considered significant.
Results
[00118] In vitro T- cell proliferation in response to vIL18BP105 (SEQ ID
NO:15) peptide
To test whether conjugation of a sub-unit antigen to an APC-targeting mAb
would generate
an enhanced immune response, the peptide vIL18BP105 (SEQ ID NO:15) was
conjugated
chemically to the mAb L243 (CIL18BP105) and used to immunize mice. The results
were
compared to mice given PBS/IFA (naive) and free vIL18BP105 (SEQ ID NO:15) in
IFA.
Using a 5-day CFSE-based in vitro cell proliferation assay, splenocytes from
CIL18BP105-
immunized mice proliferated in a concentration-dependent manner. Cells from
naive or free-
peptide immunized mice were relatively unresponsive (FIG. 6). The TCRV[3
repertoire of
CD4-positive splenocytes from HLA-DR04 tg mice following immunization with
either form
of vIL18BP105 (SEQ ID NO:15) skewed toward TCRV(3 8.3 (not shown).
[00119] Antibody production in response to vIL18BP105 (SEQ ID NO:15) Peptide
Humoral immunity to poxvirus is essential for protection against infection.
Therefore, the
antibody response against CIL18BP105 versus vIL18BP105 (SEQ ID NO:15) was
investigated in the immunized HLA-DR04 tg mice. The results are shown in FIG.
7. At day 7
after the first boost (day 21 after priming), CIL18BP105-injected mice
displayed higher
peptide-specific antibody production than mice injected with vIL18BP105 (SEQ
ID NO:15).
But, at 14 days after the first boost, the amounts of antibody were similar.
Both IgGI and
IgG2a isotypes were induced by CIL18BP105 and vIL18BP105 (SEQ ID NO:15), but
CIL18BP105 caused more production of IgGI antibodies than its free counterpart
(not
shown). The production of IgGI and IgG2a suggests a mature antibody response
with T-cell
help. Both ThI and Th2 helper cell participation is also suggested by this
antibody response.
Serum antibody from CIL18BP105-immunized mice reacted strongly with whole
vIL18BP
protein (C12L) (FIG. 8), indicating that subunit antigenic peptide conjugated
to anti-APC
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antibody is capable of inducing a systemic immune response against intact
virions.
Immunization with CIL 18BP 105 was more effective than immunization with vIL
18BP 105
(SEQ ID NO: 15) at promoting interferon-y production from splenocytes
stimulated in vitro
with vIL18BP105 (SEQ ID NO:15) peptide (Table 7).
[00120] These results show that the poxvirus sub-unit peptide, vIL 18BP 105
(SEQ ID
NO: 15), induced both cellular and humoral immune responses in HLA-DR04 tg
mice when
conjugated with the anti-HLA-DR antibody, L243. T-cell proliferative
responses, which are
indicative of cell-mediated immunity, were especially enhanced by the antigen-
L243
conjugate. Antibody production rose more quickly in the CIL18BP105-immunized
mice.
The peptide-antibody conjugate induced higher titers of antibody earlier than
the free peptide.
These results show that T-cell response against a relatively small peptide
antigen can be
elicited successfully by conjugation to L243.
Table 7. Interferon-y Production From Immunized Mice
Treatment CIL18BP105- L18BP105- give-
s lenocto s lenoc to s lenoc to
Medium 00.00 + 00.00 00.00 + 00.00 00.00 + 00.00
IL1813P105, 10 ug/ml 101.03 + 11.61 * 25.78 + 11.27 38.32 + 11.27
Con A, 5 ug/ml 373.15 + 4.23 84.68 + 10.00 149.00 + 12.46
SEA, 100 ng/mi 363.00 + 21.44 148.24 + 00.00 66.46 + 17.55
Conclusions
[00121] A vaccine against poxvirus requires Thl and Th2 immune responses, cell-
mediated
and humoral immunity, and a suitable pool of memory CD4 T cells (Belyakoc et
al, Proc.
Natl. Acad. Sci. USA 100: 9458-9463, 2003). The results presented show that
sub-unit
antigens conjugated to APC-targeting antibody can enhance and to induce Thl,
Th2, and
humoral immune responses.
Example 3. Nasal Administration of Subunit Vaccine
[00122] Mice (HLA-DR04 Tg) are anesthetized and vaccine is administered (15-25
g
peptide total) intranasally (i.n.) (10 l/nostril). Vaccine is either free
peptide, or peptide-
L243 conjugate. For these experiments, the adjuvant is the calcium phosphate
adjuvant
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described by He et al. (Clin Diagnos Lab Immunol 9:1021-1024, 2002) (10
g/dose of
antigen). Controls consist of unimmunized (naive) mice, mice immunized with
the whole
viral protein (i.n.), systemically immunized mice (peptide, sub-cutaneous
(s.c.)), and mice
immunized with carrier/adjuvant only (i.n.). Equal amounts of peptide are
administered in
each case. Mice are boosted twice using the same route as prime, at weeks 2
(d14) and 4
(d28) after priming. Combinations of route of immunization may be employed
(e.g., s.c.
prime, followed by i.n. immunization on day 14).
[00123] Five mice from each treatment group are sacrificed at day 35 after
prime
immunization (25 of the 75 mice). Serum is harvested before priming
immunization (d0), at
day 7, 28, and 56. In addition nasal lavage (NL) fluids or bronchoalveolar
lavage (BAL) and
splenocytes are harvested at sacrifice.
[00124] Antigen-specific antibody in the respiratory tract fluids (gathered by
NL or BAL
upon sacrifice), and in the serum are titred by serial dilution and
application to ELISA, with
immobilized whole recombinant antigen (or vaccinia proteins), peptide, non-
relevant peptide
control and serially diluted serum from all treatment groups, including naive
mice. Isotype of
specific antibodies is determined.
[00125] Neutralizing antibodies are present in mice immunized by either nasal
or
subcutaneous administration. The antibodies react with both antigenic peptide
and whole
viral protein. Nasal administration is more efficient to promote a mucosal
immune response,
while subcutaneous administration is more efficient to promote a systemic
immune response
against poxvirus.
Example 4. Alternative Methods of Preparing Immunoconjugates by the Dock-and-
Lock (DNL) Technique
DDD and AD Fusion Proteins
[00126] The DNL technique can be used to make dimers, trimers, tetramers,
hexamers, etc.
comprising virtually any antibodies or fragments thereof or other protein or
peptide moieties.
For certain preferred embodiments, IgG antibodies, F(ab')2 antibody fragments
and subunit
antigenic peptides, may be produced as fusion proteins containing either a
dimerization and
docking domain (DDD) or anchoring domain (AD) sequence. Although in preferred
embodiments the DDD and AD moieties are produced as fusion proteins, the
skilled artisan
will realize that other methods of conjugation, such as chemical cross-
linking, may be
utilized within the scope of the claimed methods and compositions.
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[00127] DNL constructs may be formed by combining, for example, an Fab-DDD
fusion
protein of an anti-HLA-DR or anti-CD74 antibody with a vIL18BP105-AD fusion
protein.
Alternatively, constructs may be made that combine IgG-AD fusion proteins with
vIL18BP105-DDD fusion proteins. The technique is not limiting and any protein
or peptide
of use may be produced as an AD or DDD fusion protein for incorporation into a
DNL
construct. Where chemical cross-linking is utilized, the AD and DDD conjugates
are not
limited to proteins or peptides and may comprise any molecule that may be
cross-linked to an
AD or DDD sequence using any cross-linking technique known in the art.
[00128] Independent transgenic cell lines may be developed for each DDD or AD
fusion
protein. Once produced, the modules can be purified if desired or maintained
in the cell
culture supernatant fluid. Following production, any DDD-fusion protein module
can be
combined with any AD-fusion protein module to generate a DNL construct. For
different
types of constructs, different AD or DDD sequences may be utilized. Exemplary
DDD and
AD sequences are provided below.
DDD1: SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:24)
DDD2: CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:25)
AD I: QIEYLAKQIVDNAIQQA (SEQ ID NO:26)
AD2: CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:27)
Expression Vectors
[00129] The plasmid vector pdHL2 has been used to produce a number of
antibodies and
antibody-based constructs. See Gillies et al., J Immunol Methods (1989),
125:191-202;
Losman et al., Cancer (Phila) (1997), 80:2660-6. The di-cistronic mammalian
expression
vector directs the synthesis of the heavy and light chains of IgG. The vector
sequences are
mostly identical for many different IgG-pdHL2 constructs, with the only
differences existing
in the variable domain (VH and VL) sequences. Using molecular biology tools
known to
those skilled in the art, these IgG expression vectors can be converted into
Fab-DDD or Fab-
AD expression vectors. To generate Fab-DDD expression vectors, the coding
sequences for
the hinge, CH2 and CH3 domains of the heavy chain are replaced with a sequence
encoding
the first 4 residues of the hinge, a 14 residue Gly-Ser linker and the first
44 residues of human
RIIa (referred to as DDD 1). To generate Fab-AD expression vectors, the
sequences for the
hinge, CH2 and CH3 domains of IgG are replaced with a sequence encoding the
first 4
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residues of the hinge, a 15 residue Gly-Ser linker and a 17 residue synthetic
AD called
AKAP-IS (referred to as AD 1), which was generated using bioinformatics and
peptide array
technology and shown to bind RIIa dimers with a very high affinity (0.4 nM).
See Alto, et
al. Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.
[00130] Two shuttle vectors were designed to facilitate the conversion of IgG-
pdHL2
vectors to either Fab-DDD 1 or Fab-AD 1 expression vectors. Using this
technique, we have
produced AD and/or DDD fusion proteins and encoding plasmids for Fab
expression of a
wide variety of known antibodies, such as hLL1, hLL2, hPAM4, hRl, hRS7, hMN-
14, hMN-
15, hA19, hA20 and many others.
[001311 Trimeric DNL construct are obtained by reacting a DDD fusion protein
comprising,
e.g., an IgG antibody or F(ab) antibody fragment with an AD fusion protein
comprising, e.g.,
a subunit antigenic peptide, at a molar ratio of between 1.4:1 and 2:1. The
total protein
concentration is 1.5 mg/ml in PBS containing 1 mM EDTA. Subsequent steps may
involve
TCEP reduction, HIC chromatography, DMSO oxidation, and affinity
chromatography to
obtain the purified DNL construct. Addition of 5 mM TCEP rapidly results in
the formation
of alb complex. Binding assays show that the antibody moiety and antigenic
peptide
moieties retain their functional properties of respectively antigen-binding
and antigenicity.
[00132] Using this technique, virtually any antibody or antibody fragment may
be attached
to any subunit antigenic peptide by preparing appropriate fusion proteins of
each, comprising
complementary DDD and AD moieties.
[00133] The following peptides are made as AD2 modules incorporating a linking
sequence
attaching a subunit vaccine peptide. The AD2-peptide fusion is combined with
DDD2-linked
IgG or Fab moieties to provide a subunit based vaccine incorporating an APC-
targeting
antibody or antibody fragment.
ND8L
SIFLQVLDHKNVYFQGGGSCGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:34)
CD8L
CGQIEYLAKQIVDNAIQQAGCGGGSSIFLQVLDHKNVYFQ (SEQ ID NO:35)
NILI8BP
HYRFTCVLTTLNGVSGGGSCGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:36)
CIL 18BP
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CGQIEYLAKQIVDNAIQQAGCGGGSHYRFTCVLTTLNGVS (SEQ ID NO:37)
CSCRD8L
CGQIEYLAKQIVDNAIQQAGCGGGSYHQFVIDQLKLSVNF (SEQ ID NO:38)
CSCRIL18BP105
CGQIEYLAKQIVDNAIQQAGCGGGSGNCTFVTYLRHLSTV (SEQ ID NO:39)
Example 5. Liposome Formulation for Nasal Administration of Subunit Based
Vaccine
[00134] A liposome formulation of antigenic peptide conjugated to L243
antibody was
prepared by standard techniques. The intranasal peptides were designed with
linkers at both
the C-terminal and N-terminal ends. The C-terminal linker was used for
conjugation of the
L243 antibody. The N-terminal linker was used to facilitate attachment to the
liposome, via
palmitoylation. The peptide conjugates were as indicated below. The CD8L118
peptide was
not a lipoprotein and was encapsulated into liposomes.
LIR183
GVQFYMIVIGVIILAALF (SEQ ID NO:40)
Conjugated L1 R183
KKKKGVQFYMIVIGVIILAALFPSEC (SEQ ID NO:41)
Conjugated A27L3
KSGTLFPGDDDLAIPATEFFSTKAAKKPSEC (SEQ ID NO:42)
Conjugated ILI8BP105
KSHYRFTCVLTTLNGVSPESC (SEQ ID NO:43)
CD8L118
HDDGLIIISIFLQVLDHKNVYFQKIGGGSC (SEQ ID NO:44)
[00135] FIG. 9(A) shows the results of nasal administration of a liposome
formulated
subunit vaccine. Peptides were prepared and conjugated to antibody as
described in
Examples 1 and 2 above. The results presented in FIG. 9 show T cell
proliferation in
response to incubation with the designated peptide in vitro after nasal
immunization of mice.
The strongest effect on T cell proliferation (FIG. 9A) was observed with the
L1R183
antigenic peptide (SEQ ID NO:39) derived from the LIR antigen, an
immunodominant
intracellular mature virion (IMV) protein that offers post-exposure
prophylaxis.
Immunization of mice with liposome-displayed bare peptide alone (FIG. 9B)
produced little
effect on T cell proliferation, regardless of the tested peptide. Immunization
with free peptide
in the absence of liposome or with empty liposomes also had little to no
effect on T cell
proliferation (data not shown). The results demonstrate that nasal
administration of a subunit
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based poxvirus vaccine, using conjugation to an APC targeting antibody, is
effective to
induce an immune response.
[00136] All of the COMPOSITIONS and METHODS disclosed and claimed herein can
be
made and used without undue experimentation in light of the present
disclosure. While the
compositions and methods have been described in terms of preferred
embodiments, it is
apparent to those of skill in the art that variations maybe applied to the
COMPOSITIONS and
METHODS and in the steps or in the sequence of steps of the METHODS described
herein
without departing from the concept, spirit and scope of the invention. More
specifically,
certain agents that are both chemically and physiologically related may be
substituted for the
agents described herein while the same or similar results would be achieved.
All such similar
substitutes and modifications apparent to those skilled in the art are deemed
to be within the
spirit, scope and concept of the invention as defined by the appended claims.
