Note: Descriptions are shown in the official language in which they were submitted.
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USE OF VIRUS-LIKE PARTICLES AS ADJWANTS
Technical Field
The present invention relates generally to
agents which enhance the immune response to a selected
l0 antigen. In particular, the invention pertains to the
use of virus-like particles (VLPs) as adjuvants and
coadjuvants, formulations including the VLPs, and
methods for making and using the compositions of the
invention.
Backctround
Vaccine compositions often include
immunological adjuvants to enhance cell-mediated and
humoral immune responses. For example, depot
adjuvants are frequently used which adsorb and/or
precipitate administered antigens and which can retain
the antigen at the injection site. Typical depot
adjuvants include aluminum compounds and water-in-oil
emulsions. However, depot adjuvants, although
increasing antigenicity, often provoke severe
persistent local reactions, such as granulomas,
abscesses and scarring, when injected subcutaneously
or intramuscularly. Other adjuvants, such as
lipopolysacharrides and muramyl dipeptides, can elicit
pyrogenic responses upon injection and/or Reiter~s
symptoms (influenza-like symptoms, generalized joint
discomfort and sometimes anterior uveitis, arthritis
and urethritis).
Particulate carriers with adsorbed or
entrapped antigens have been used in an attempt to
circumvent these problems. Such carriers present
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multiple copies of a selected antigen to the immune
system and promote trapping and retention of antigens
in local lymph nodes. The particles can be
phagocytosed by macrophages and can enhance antigen
presentation through cytokine release. Examples of
particulate carriers include those derived from
polymethyl methacrylate polymers, as well as
microparticles derived from poly(lactides) and
poly(lactide-co-glycolides), known as PLG. While
offering significant advantages over other more toxic
systems, antigen-containing PLG microparticles suffer
from some drawbacks. For example, the production of
microparticles is difficult and involves the use of
harsh chemicals that can denature the antigen and
destroy the immunogenicity thereof. Furthermore,
antigen instability can occur due to the high shear
forces used to prepare small microparticles and due to
interfacial effects within the emulsions used.
Liposomes have also been employed in an
effort to overcome these problems. Liposomes are
microscopic vesicles formed from lipid constituents
such as phospholipids which are used to entrap
pharmaceutical agents. Although the use of liposomes
as a drug delivery system alleviates some of the
problems described above, liposomes exhibit poor
stability during storage and use, and large scale
production and manufacturing of liposomes is
problematic.
Viral particles can be used as a matrix for
the proper presentation of an antigen entrapped or
associated therewith to the immune system of the host.
For example, U.S. Patent No. 4,722,840 describes
hybrid particles comprised of a particle-forming
fragment of a structural protein from a virus, such as
a particle-forming fragment of hepatitis B virus (HBV)
surface antigen (HBsAg), fused to a heterologous -
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polypeptide. Tindle et al., Virology (1994) 200:547-
557, describes the production and use of chimeric HBV
core antigen particles containing epitopes of human
papillomavirus (HPV) type 16 E7 transforming protein.
Adams et al., Nature (1987) 329:68-70,
describes the recombinant production of hybrid
HIVgp120:Ty VLPs in yeast and Brown et al., Virology
(1994) 198:477-488, the production of chimeric
proteins consisting of the VP2 protein of human
parvovirus B19 and epitopes from human herpes simplex
virus type 1, as well as mouse hepatitis virus A59.
Wagner et al., Virology (1994) 200:162-175, Brand et
al., J. Virol. Meth. (1995) 51:153-168 and Wagner et
al., Virology (1996) 220:128-140, describe the
~15 assembly of chimeric HIV-1 p55gag particles. U.S.
Patent No. 5,503,833 describes the use of rotavirus
VP6 spheres for encapsulating and delivering
therapeutic agents.
Despite the above antigen-presentation
systems, there is a continued need for effective and
safe adjuvants for use in a variety of pharmaceutical
compositions and vaccines.
Summary of the Invention
The inventors herein have found,
surprisingly, that combining selected antigens with
VLPs, wherein the antigens are distinct from the VLP,
provides for enhanced immune responses to the antigen
in question.
In one embodiment, then, the subject
invention is directed to a vaccine composition
comprising a VLP adjuvant, a selected antigen and a
pharmaceutically acceptable excipient. The selected
antigen is distinct from the VLP. In particularly
preferred embodiments, the VLP is derived from one or
more particle-forming polypeptides of a hepatitis _
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surface antigen or from a particle-forming polypeptide
of papillomavirus L1 and/or L2 protein and the
selected antigen is a MenC/Hib oligosaccharide
conjugate or an HPV E7.
In another embodiment, the subject invention
is directed to a method for producing an enhanced
immune response in a vertebrate subject comprising
administering to the vertebrate subject a VLP adjuvant
and a selected antigen. The antigen is distinct from
the VLP and the VLP is administered in an amount
effective for eliciting an enhanced immune response to
the antigen in the vertebrate subject. The VLP can be
administered to the subject prior or subsequent to, or
concurrent with, the antigen.
In yet another embodiment, the invention is
directed to a method for preparing an adjuvant
formulation comprising providing a VLP and combining
the VLP with a pharmaceutically acceptable excipient.
These and other embodiments of the present
invention will readily occur to those of ordinary
skill in the art in view of the disclosure herein.
Brief Description of the Figures
Figure 1 depicts antibody responses of
infant baboons to vaccines including hepatitis B virus
VLPs (HBV). The results are presented as the
geometric mean of antibody concentrations in IU/ml.
Figure 2 shows antibody responses of infant
baboons to vaccines including HBV VLPs. The results
are presented as the geometric mean of antibody
concentrations in IU/ml.
Figure 3 shows anticapsular antibody
responses of infant baboons to vaccines including HBV
VLPs. The results are presented as the geometric mean
of antibody concentrations in IU/ml.
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Figure 4 depicts anticapsular antibody
responses of infant baboons to vaccines including HBv
VLPs. The results are presented as the geometric mean
of antibody concentrations in IU/ml.
Figures 5A and 5B show serum antibody
responses of infant baboons to vaccines including HBV
VLPs. Figure 5A shows the IgG anticapsular antibody
responses determined by ELISA. Figure 5B depicts
complement-mediated bactericidal antibody responses.
Figure 6 is a graph showing the effect of a
combined HPV6b L1 VLP and E7 vaccine on rabbit humoral
immune responses.
Detailed Description of the Invention
The practice of the present invention will
employ, unless otherwise indicated, conventional
methods of virology, chemistry, biochemistry,
recombinant technology, immunology and pharmacology,
within the skill of the art. Such techniques are
2o explained fully in the literature. See, e.g.,
Virology, 3rd Edition, vol. I & II (B.N. Fields and
D.M. Knipe, eds., 1996); Remington's Pharmaceutical
Sciences, 18th Edition (Easton, Pennsylvania: Mack
Publishing Company, 1990); Methods In Enzymology (S.
Colowick and N. Kaplan, eds., Academic Press, Inc.);
Handbook of Experimental Immunology, Vols. I-IV (D. M.
Weir and C.C. Blackwell, eds., 1986, Blackwell
Scientific Publications); Sambrook et al., Molecular
Cloning: A Laboratory Manual (2nd Edition, 1989); and
DNA Cloning: A Practical Approach, vol. I & II {D.
Glover, ed.).
As used in this specification and the
appended claims, the singular forms "a," "an" and
"the" include plural references unless the content
clearly dictates otherwise.
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A. Definitions
In describing the present invention, the
following terms will be employed, and are intended to
be defined as indicated below.
As used herein, the term "virus-like
particle" or "VLP" refers to a nonreplicating, empty
viral shell, derived from any of several viruses
discussed further below. VLPs are generally composed
of one or more viral proteins, such as, but not
limited to those proteins referred to as capsid, coat,
shell, surface and/or envelope proteins, or particle-
forming polypeptides derived from these proteins.
VLPs can form spontaneously upon recombinant
expression of the protein in an appropriate expression
system. Methods for producing particular VLPs are
known in the art and discussed more fully below. The
presence of VLPs following recombinant expression of
viral proteins can be detected using conventional
techniques known in the art, such as by electron
microscopy, X-ray crystallography, and the like. See,
e.g., -Baker et al., Biophys. J. (1991) 60:1445-1456;
Hagensee -et al., J. Virol. (1994) 68:4503-4505. For
example, cryoelectron microscopy can be performed on
vitrified aqueous samples of the VLP preparation in
question, and images recorded under appropriate
exposure conditions.
By "particle-forming polypeptide" derived
from a particular viral protein is meant a full-length
or near full-length viral protein, as well as a
fragment thereof, or a viral protein with internal
deletions, which has the ability to form VLPs under
conditions that favor VLP formation. Accordingly, the
polypeptide may comprise the full-length sequence,
fragments, truncated and partial sequences, as well as
analogs and precursor forms of the reference molecule.
The term therefore intends deletions, additions and-
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substitutions to the sequence, so long as the
polypeptide retains the ability to form a VLP. Thus,
the term includes natural variations of the specified
polypeptide since variations in coat proteins often
occur between viral isolates. The term also includes
deletions, additions and substitutions that do not
naturally occur in the reference protein, so long as
the protein retains the ability to form a VLP.
Preferred substitutions are those which are
conservative in nature, i.e., those substitutions that
take place within a family of amino acids that are
related in their side chains. Specifically, amino
acids are generally divided into four families: (1)
acidic -- aspartate and glutamate; (2) basic --
lysine, arginine, histidine; (3) non-polar -- alanine,
valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan; and (4) uncharged polar --
glycine, asparagine, glutamine, cystine, serine
threonine, tyrosine. Phenylalanine, tryptophan, and
tyrosine are sometimes classified as aromatic amino
acids. For example, it is reasonably predictable that
an isolated replacement of leucine with isoleucine or
valine, an aspartate with a glutamate, a threonine
with a serine, or a similar conservative replacement
of an amino acid with a structurally related amino
acid, will not have a major effect on the biological
activity. Proteins having substantially the same
amino acid sequence as the reference molecule, but
possessing minor amino acid substitutions that do not
substantially affect the immunogenicity of the
protein, are therefore within the definition of the
reference polypeptide.
A VLP is "distinct from" from a selected
antigen when the antigen is not entrapped within the
VLP and/or the antigen and VLP are not expressed
together as a fusion protein. However, a VLP is
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considered "distinct from" a selected antigen even if
there is a loose physical association between the
antigen and VLP.
An "antigen" refers to a molecule containing
one or more epitopes (either linear, conformational or
both) that will stimulate a host's immune system to
make a humoral and/or cellular antigen-specific
response. The term is used interchangeably with the
term "immunogen." Normally, a B-cell epitope will
include at least about S amino acids but can be as
small as 3-4 amino acids. A T-cell epitope, such as a
CTL epitope, will include at least about 7-9 amino
acids, and a helper T-cell epitope at least about 12-
amino acids. The term "antigen" denotes both
15 subunit antigens, i.e., antigens which are separate
and discrete from a whole organism with which the
antigen is associated in nature, as well as killed,
attenuated or inactivated bacteria, viruses, fungi,
parasites or other microbes. Antibodies such as anti-
20 idiotype antibodies, or fragments thereof, and
synthetic peptide mimotopes, which can mimic an
antigen or antigenic determinant, are also captured
under the definition of antigen as used herein.
Similarly, an oligonucleotide or polynucleotide which
expresses an antigen or antigenic determinant in vivo,
such as in gene therapy and DNA immunization
applications, is also included in the definition of
antigen herein.
For purposes of the present invention,
antigens can be derived from any of several known
viruses, bacteria, parasites and fungi, as described
more fully below. The term also intends any of the
various tumor antigens. Furthermore, for purposes of
the present invention, an "antigen" refers to a
protein which includes modifications, such as
deletions, additions and substitutions (generally _
_g_
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conservative in nature), to the native sequence, so
long as the protein maintains the ability to elicit an
immunological response, as defined herein. These
modifications may be deliberate, as through site-
s directed mutagenesis, or may be accidental, such as
through mutations of hosts which produce the antigens.
An "immunological response" to an antigen or
composition is the development in a subject of a
humoral and/or a cellular immune response to the
antigen present in the composition of interest. For
purposes of the present invention, a "humoral immune
response" refers to an immune response mediated by
antibody molecules, while a "cellular immune response"
is one mediated by T-lymphocytes and/or other white
blood cells. One important aspect of cellular
immunity involves an antigen-specific response by
cytolytic T-cells ("CTL"s). CTLs have specificity for
peptide antigens that are presented in association
with proteins encoded by the major histocompatibility
complex (MHC) and expressed on the surfaces of cells.
CTLs help induce and promote the intracellular
destruction of intracellular microbes, or the lysis of
cells infected with such microbes. Another aspect of
cellular immunity involves an antigen-specific
response by helper T-cells. Helper T-cells act to
help stimulate the function, and focus the activity
of, nonspecific effector cells against cells
displaying peptide antigens in association with MHC
molecules on their surface. A "cellular immune
response" also refers to the production of cytokines,
chemokines and other such molecules produced by
activated T-cells and/or other white blood cells,
including those derived from CD4+ and CD8+ T-cells.
A composition or vaccine that elicits a
cellular immune response may serve to sensitize a
vertebrate subject by the presentation of antigen in
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association with MHC molecules at the cell surface.
The cell-mediated immune response is directed at, or
near, cells presenting antigen at their surface. In
addition, antigen-specific T-lymphocytes can be
generated to allow for the future protection of an
immunized host.
The ability of a particular antigen to
stimulate a cell-mediated immunological response may
be determined by a number of assays, such as by
lymphoproliferation (lymphocyte activation) assays,
CTL cytotoxic cell assays, or by assaying for T-
lymphocytes specific for the antigen in a sensitized
subject. Such assays are well known in the art. See,
e.g., Erickson et al., J. Immunol. (1993) 151:4189-
4199; Doe et al., Eur. J. Immunol. (1994} 24:2369-
2376.
Thus, an immunological response as used
herein may be one which stimulates the production of
CTLs, and/or the production or activation of helper T-
cells. The antigen of interest may also elicit an
antibody-mediated immune response. Hence, an
immunological response may include one or more of the
following effects: the production of antibodies by B-
cells; and/or the activation of suppressor T-cells
and/or ~yb T-cells directed specifically to an antigen
or antigens present in the composition or vaccine of
interest. These responses may serve to neutralize
infectivity, and/or mediate antibody-complement, or
antibody dependent cell cytotoxicity (ADCC) to provide
protection to an immunized host. Such responses can
be determined using standard immunoassays and
neutralization assays, well known in the art.
A vaccine composition which contains a
selected antigen along with a VLP adjuvant of the
present invention, or a vaccine composition which is
coadministered with the subject VLP adjuvant, displays
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"enhanced immunogenicity" when it possesses a greater
capacity to elicit an immune response than the immune
response elicited by an equivalent amount of the
antigen administered without the VLP adjuvant or
coadjuvant. Thus, a vaccine composition may display
"enhanced immunogenicity" because the antigen is more
strongly immunogenic or because a lower dose or fewer
doses of antigen are necessary to achieve an immune
response in the subject to which the antigen is
administered. Such enhanced immunogenicity can be
determined by administering the VLP composition and
antigen controls to animals and comparing antibody
titers and/or cellular-mediated immunity against the
two using standard assays such as radioimmunoassay and
ELISAs, well known in the art.
For purposes of the present invention, an
"effective amount" of a VLP adjuvant will be that
amount which enhances an immunological response to a
coadministered antigen.
By "vertebrate subject" is meant any member
of the subphylum chordata, including, without
limitation, humans and other primates, including non-
human primates such as chimpanzees and other apes and
monkey species; farm animals such as cattle, sheep,
pigs, goats and horses; domestic mammals such as dogs
and cats; laboratory animals including rodents such as
mice, rats and guinea pigs; birds, including domestic,
wild and game birds such as chickens, turkeys and
other gallinaceous birds, ducks, geese, and the like.
The term does not denote a particular age. Thus, both
adult and newborn individuals are intended to be
covered. The system described above is intended for
use in any of the above vertebrate species, since the
immune systems of all of these vertebrates operate
similarly.
By "pharmaceutically acceptable" or
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"pharmacologically acceptable" is meant a material
which is not biologically or otherwise. undesirable,
i.e., the material may be administered to an
individual along with the VLP adjuvant formulation
without causing any undesirable biological effects or
interacting in a deleterious manner with any of the
components of the composition in which it is
contained.
By "physiological pH" or a "pH in the
physiological range" is meant a pH in the range of
approximately 7.2 to 8.0 inclusive, more typically in
the range of approximately 7.2 to 7.6 inclusive.
"Recombinant" as used herein to describe a
nucleic acid molecule means a polynucleotide of
genomic, cDNA, semisynthetic, or synthetic origin
which, by virtue of its origin or manipulation: (1) is
not associated with all or a portion of the
polynucleotide with which it is associated in nature;
and/or (2) is linked to a polynucleotide other than
that to which it is linked in nature. The term "re-
combinant" as used with respect to a protein or
polypeptide means a polypeptide produced by expression
of a recombinant polynucleotide. "Recombinant host
cells " "host cells " "cells " "cell lines " "cell
cultures," and other such terms denoting procaryotic
microorganisms or eucaryotic cell lines cultured as
unicellular entities, are used interchangeably, and
refer to cells which can be, or have been, used as
recipients for recombinant vectors or other transfer
DNA, and include the progeny of the original cell
which has been transfected. It is understood that the
progeny of a single parental cell may not necessarily
be completely identical in morphology or in genomic or
total DNA complement to the original parent, due to
accidental or deliberate mutation. Progeny of the
parental cell which are sufficiently similar to the-
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parent to be characterized by the relevant property,
such as the presence of a nucleotide sequence encoding
a desired peptide, are included in the progeny
intended by this definition, and are covered by the
above terms.
As used herein, "treatment" refers to any of
(i) the prevention of infection or reinfection, as in
a traditional vaccine, (ii) the reduction or
elimination of symptoms, and (iii) the substantial or
complete elimination of the pathogen in question.
Treatment may be effected prophylactically (prior to
infection) or therapeutically (following infection).
B. General Methods
Central to the present invention is the
discovery that VLPs can serve as adjuvants or
coadjuvants to enhance humoral and/or cell-mediated
immune responses in a vertebrate subject when the VLPs
are administered with a selected antigen.
Surprisingly, the antigen need not be entrapped within
the VLP in order for enhanced immunogenicity to
result. Thus, the present invention does not require
the use of linking agents, harsh chemical treatments,
and the like, since the antigen of interest is not
encapsulated or chemically conjugated to the VLP.
Additionally, antigen size is not limited since the
system does not depend on encapsulation of the
antigen. Accordingly, the present system is useful
with a wide variety of antigens and provides a
powerful tool to prevent and/or treat a large number
of infections.
VLPs for use as adjuvants can be formed from
any viral protein, particle-forming polypeptide
derived from the viral protein, or combination of
viral proteins or fragments thereof, that have the
capability of forming particles under appropriate -
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conditions. The requirements for the particle-forming
viral protein are that if the particle is formed in
the cytoplasm of the host cell, the protein must be
sufficiently stable in the host cell in which it is
expressed such that formation of virus-like structures
will result, and that the polypeptide will
automatically assemble into a virus-like structure in
the cell of the recombinant expression system used.
If the protein is secreted into culture media,
conditions can be adjusted such that VLPs will form.
Furthermore, the particle-forming protein should not
be cytotoxic in the expression host and should not be
able to replicate in the host in which the VLP will be
used.
For example, it is known that some proteins
can spontaneously form VLPs when pH is brought to an
appropriate level. Similarly, some proteins require
sufficient amounts of protein to be present in order
for VLPs to form.
Methods and suitable conditions for forming
particles from a wide variety of viral proteins are
known in the art. For example, proteins derived from
the hepatitis B virus (HBV) surface, such as the
surface antigen, sAg, as well as the presurface
sequences, pre-S1 and pre-S2 (formerly called pre-S),
and any combination of these sequences, can
spontaneously form particles upon expression in a
suitable host cell, such as upon expression in
mammalian, insect, yeast or Xenopus cells. Thus, HBV
VLPs for use in the present invention can include
particle-forming polypeptides of sAg, pre-S1 or pre-
S2, as well as particle-forming polypeptides from any
combination of the above, such as sAg/pre-S1, sAg/pre-
S2, sAg/pre-S1/pre-S2, and pre-S1/pre-S2. See, e.g.,
"HBV Vaccines - from the laboratory to license: a case
study" in Mackett, M. and Williamson, J.D., Human -
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Vaccines and Vaccination, pp. 159-176, for a
discussion of HBV structure; and U.S. Patent Nos.
4,722,840, 5,098,704, 5,324,513, Beames et al., J.
Virol. (1995) 69:6833-6838, Birnbaum et al., J. Virol.
{1990} 64:3319-3330, Zhou et al., J. Virol. (1991)
65:5457-5464, for descriptions of the recombinant
production of various HBV particles.
Additionally, particle-forming polypeptides
derived from papillomavirus structural proteins L1 and
L2, either alone or in combination, will find use
herein as VLP adjuvants and coadjuvants. L1 and L2
proteins for use with the present invention can be
derived from any of the various human and other animal
papillomaviruses and subgroups thereof, including but
not limited to, HPV-1, HPV-2, HPV-5, HPV-6, HPV-8,
HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-45.
The sequences for L1 and L2 are known for various HPV
types. See, e.g., Human Papillomaviruses: A
Compilation and Analysis of Nucleic Acid and Amino
Acid Sequences {Myers, G. et al., eds.) Los Alamos
National Laboratory, Los Alamos, New Mexico, 1994-
1996, for the sequences of various HPV types.
Methods for forming VLPs from L1 and L2,
either alone or in combination, are known and reported
in, e.g., U.S. Patent No. 5,437,951 {production of Ll
VLPs in insect cells); WO 95/20659 (production of L2
VLPs in eucaryotic cells using vaccinia virus
vectors); WO 93/02184 (production of L1/L2 VLPs in
eucaryotic cells using vaccinia virus vectors);
Kirnbauer et al. J. Virol. (1993) 67:6929-6936
(production of L1 and L1/L2 VLPs in insect cells);
Heino et al., Virology (1995) 214:349-359 {production
of L1 and L1/L2 VLPs in mammalian cells); Sasagawa et
al., Virology (1995) 206:126-135 (production of L1 and
L1/L2 VLPs in yeast cells); Zhou et al., Virology
(1991) 185:251-257 (production of L1/L2 VLPs in -
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epithelial cells infected with vaccinia virus
vectors).
Similarly, some rotavirus proteins are known
to form VLPs upon expression. For example, rotavirus
VP6 VLPs can be produced as described in U.S. Patent
No. 5,503,833. Generally, VP6 spherical particles
spontaneously form when the pH of a lysate containing
recombinantly produced VP6 is brought to about pH 4.0
or greater. Bovine rotavirus VP2 spontaneously forms
VLPs upon expression in baculovirus systems. See,
e.g., Labbe et al., J. Virol. (1991) 65:2946-2952.
The yeast retrotransposon, Ty, encodes a set
of proteins that assembles into VLPs. See, e.g.,
Mellor et al., Nature (1985) 318:583-586; Garfinkel et
al., Cell (1985) 42:507-517; Adams et al., Cell (1987)
49:111-119. Ty VLPs can be formed from the primary
translation product of the Ty element, pl, as well as
the combination of pl with p3 (pre-Ty VLP), and
particle-forming proteolytic products of p1 and p3,
upon expression in yeast. See, e.g., Adams et al.,
Nature (1987) 329:68-70.
VLPs for use in the present invention can
also be derived from viral proteins from human
parvovirus B19, such as the viral proteins VP1 and/or
VP2 expressed in insect cells (see, Brown et al.,
Virology (1994) 198:477-488) and insect cell-expressed
VP1, VP2 and/or VP3 proteins from polyomavirus (Delos
et al., Virology (1993) 194:393-398; Forstova et al.,
Human Gene Therapy (1995) 6:297-306). Retrovirus gag
proteins, such as those derived from Rous sarcoma
virus (RSV) and Moloney murine leukemia virus (MLV)
will also find use herein, as will the HIV-1 group-
specific core antigen, p55gag, and deletion mutants
thereof, which spontaneously form VLPs when expressed
in eucaryotic cells. See, e.g., Wagner et al., Arch.
Virol. (1992) 127:117-137; Wagner et al., Virology -
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_..T_~..~-~ _- _ _ .....
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{1994) 200:162-175; Brand et al., J. Virol. Meth.
(1995) 51:153-168 and Wagner et al., Virology (1996)
220:128-140.
As explained above, vLPs can spontaneously
form when the particle-forming polypeptide of interest
is recombinantly expressed in an appropriate host
cell. Thus, the VLPs for use in the present invention
are conveniently prepared using recombinant
techniques, well known in the art. In this regard,
genes encoding the particle-forming polypeptide in
question can be isolated from DNA libraries or
directly from cells and tissues containing the same,
using known techniques. See, e.g., Sambrook et al.,
supra, for a description of techniques used to obtain
and isolate DNA. The genes encoding the particle-
forming polypeptides can also be produced
synthetically, based on the known sequences. The
nucleotide sequence can be designed with the
appropriate codons for the particular amino acid
sequence desired. In general, one will select
preferred codons for the intended host in which the
sequence will be expressed. The complete sequence is
generally assembled from overlapping oligonucleotides
prepared by standard methods and assembled into a
complete coding sequence. See, e.g., Edge, Nature
(1981) 292:756; Nambair et al., Science (1984)
223:1299; Jay et al., J. Biol. Chem. (1984) 259:6311.
Once coding sequences for the desired
particle-forming polypeptides have been isolated or
synthesized, they can be cloned into any suitable
vector or replicon for expression. Numerous cloning
vectors are known to those of skill in the art, and
the selection of an appropriate cloning vector is a
matter of choice. See, generally, Sambrook et al,
supra. The vector is then used to transform an
appropriate host cell. Suitable expression systems-
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include, but are not limited to, bacterial, mammalian,
baculovirus/insect, vaccinia, Semliki Forest virus
(SFV), mammalian, yeast and Xenopus expression
systems, well known in the art. Particularly
preferred expression systems are mammalian, vaccinia,
insect and yeast systems.
For example, a number of mammalian cell
lines are known in the art and include immortalized
cell lines available from the American Type Culture
Collection (ATCC), such as, but not limited to,
Chinese hamster ovary (CHO) cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS),
human hepatocellular carcinoma cells (e. g., Hep G2),
as well as others. Similarly, bacterial hosts such as
E. coli, Bacillus subtilis, and Streptococcus spp.,
will find use with the present expression constructs.
Yeast hosts useful in the present invention include
inter alia, Saccharomyces cerevisiae, Candida
albicans, Candida maltosa, Hansenula polymorpha,
Kluyveromyces fragilis, Kluyveromyces lactis, Pichia
guillerimondii, Pichia pastoris, Schizosaccharomyces
pombe and Yarrowia lipolytica. Insect cells for use
with baculovirus expression vectors include, inter
alia, Aedes aegypti, Autographa californica, Bombyx
mori, Drosophila melanogaster, Spodoptera frugiperda,
and Trichoplusia ni. See, e.g., Summers and Smith,
Texas Agricultural Experiment Station Bulletin No.
1555 (1987).
Viral vectors can be used for the production
of particles in eucaryotic cells, such as those
derived from the pox family of viruses, including
vaccinia virus and avian poxvirus. Additionally, a
vaccinia based infection/transfection system, as
described in Tomei -et al., J. Virol. (1993) 67:4017-
4026 and Selby et al., J. Gen. Virol. (1993)
74:1103-1113, will also find use with the present -
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invention. In this system, cells are first
transfected in vitro with a vaccinia virus recombinant
that encodes the bacteriophage T7 RNA polymerise.
This polymerise displays exquisite specificity in that
it only transcribes templates bearing T7 promoters.
Following infection, cells are transfected with the
DNA of interest, driven by a T7 promoter. The
polymerise expressed in the cytoplasm from the
vaccinia virus recombinant transcribes the transfected
DNA into RNA which is then translated into protein by
the host translational machinery. The method provides
for high level, transient, cytoplasmic production of
large quantities of RNA and its translation
product ( s ) .
Depending on the expression system and host
selected, the VLPS are produced by growing host cells
transformed by an expression vector under conditions
whereby the particle-forming polypeptide is expressed
and VLPs can be formed. The selection of the
appropriate growth conditions is within the skill of
the art. If the VLPs are formed intracellularly, the
cells are then disrupted, using chemical, physical or
mechanical means, which lyse the cells yet keep the
VLPs substantially intact. Such methods are known to
those of skill in the art and are described in, e.g.,
Protein Purification Applications: A Practical
Approach, (E.L.V. Harris and S. Angal, Eds., 1990)
The particles are then isolated using
methods that preserve the integrity thereof, such as
by gradient centrifugation, e.g., cesium chloride
(CsCl) and sucrose gradients, and the like (see, e.g.,
Kirnbauer et al. J. Virol. (1993) 67:6929-6936), as
well as standard purification techniques including,
e.g., ion exchange and gel filtration chromatography.
Once obtained, the VLP adjuvants of the
present invention can be incorporated into vaccine
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compositions containing the desired antigen, or can be
administered separately, either simultaneously with,
just prior to, or subsequent to, an antigen-containing
composition. The vaccine compositions can be used
both for treatment and/or prevention of infection.
Furthermore, the adjuvant formulations of the
invention may be used to enhance the activity of
antigens produced in vivo, i.e., in conjunction with
DNA immunization.
The VLP adjuvants can be used in
compositions for immunizing a vertebrate subject
against one or more selected pathogens or against
subunit antigens derived therefrom, or for priming an
immune response to one or several antigens. Antigens
that can be administered with the VLP adjuvants
include proteins, polypeptides, antigenic protein
fragments, oligosaccharides, polysaccharides, and the
like. Similarly, an oligonucleotide or
polynucleotide, encoding a desired antigen, can be
administered with the VLP adjuvants for in vivo
expression.
Antigens can be derived from a wide variety
of viruses, bacteria, fungi, plants, protozoans and
other parasites. For example, the present invention
will find use for stimulating an immune response
against a wide variety of proteins from the
herpesvirus family, including proteins derived from
herpes simplex virus (HSV) types 1 and 2, such as HSV-
1 and HSV-2 gB, gD, gH, VP16 and VP22; antigens
derived from varicella zoster virus (VZV), Epstein-
Barr virus (EBV) and cytomegalovirus (CMV) including
CMV gB and gH; and antigens derived from other human
herpesviruses such as HHV6 and HHV7. (See, e.g. Chee
et al., Cytomegaloviruses (J. K. McDougall, ed.,
Springer-Verlag 1990) pp. 125-169, for a review of the
protein coding content of cytomegalovirus; McGeoch Et
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al., J. Gen. Virol. (1988) 69:1531-1574, for a
discussion of the various HSV-1 encoded proteins; U.S.
Patent No. 5,171,568 for a discussion of HSV-1 and
HSV-2 gB and gD proteins and the genes encoding
therefor; Baer et al., Nature (1984) 310:207-211, for
the identification of protein coding sequences in an
EBV genome; and Davison and Scott, J. Gen. Virol.
(1986) 67:1759-1816, for a review of VZV.)
Additionally, immune responses to antigens
from the hepatitis family of viruses, including
hepatitis A virus (HAV), hepatitis B virus (HBV),
hepatitis C virus (HCV), the delta hepatitis virus
(HDV), hepatitis E virus (HEV), and hepatitis G virus,
can also be conveniently enhanced using the VLP
adjuvants. By way of example, the HCV genome encodes
several viral proteins, including E1 (also known as E)
and E2 (also known as E2/NSI), which will find use
with the present invention (see, Houghton et al.
Hepatology (1991) 14:381-388, for a discussion of HCV
proteins, including E1 and E2). The b-antigen from
HDV can also be used with the present VLP adjuvant
system (see, e.g., U.S. Patent No. 5,389,528, for a
description of the b-antigen).
Similarly, influenza virus is another
example of a virus far which the present invention
will be particularly useful. Specifically, the
envelope glycoproteins HA and NA of influenza A are of
particular interest for generating an immune response.
Numerous HA subtypes of influenza A have been
identified (Kawaoka et al., Virology (1990) 179:759-
767; Webster et al. "Antigenic variation among type A
influenza viruses," p. 127-168. In: P. Palese and D.W.
Kingsbury (ed.), Genetics of influenza viruses.
Springer-Verlag, New York). Thus, the immune response
to any of these antigens may be enhanced when they are
administered with the subject VLP adjuvants. -
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Other antigens of particular interest to be
used in combination with the VLP adjuvants include
antigens and polypeptides derived therefrom from human
papillomavirus (HPV), such as one or more of the
various early proteins including E6 and E7, tick-borne
encephalitis viruses, HIV-1 (also known as HTLV-III,
LAV, ARV, hTLR, etc.), including but not limited to
antigens from the isolates HIVIIIb~ HIVSFa, HIV~~, HIV~,I,
HIV,",,) such as gp120, gp4l, gp160, gag and pol (see,
e.g., Myers et al. Los Alamos Database, Los Alamos
National Laboratory, Los Alamos, New Mexico (1992);
Myers et al., Human Retroviruses and Aids, 1990, Los
Alamos, New Mexico: Los Alamos National Laboratory;
and Modrow et al., J. Virol. (1987) 61:570-578, for a
comparison of the envelope gene sequences of a variety
of HIV isolates) .
Proteins derived from other viruses will
also find use in the claimed methods, such as without
limitation, proteins from members of the families
Picornaviridae (e. g., polioviruses, etc.);
Caliciviridae; Togaviridae {e. g., rubella virus,
dengue virus, etc.); Flaviviridae; Coronaviridae;
Reoviridae; Birnaviridae; Rhabodoviridae (e. g., rabies
virus, etc.); Filoviridae; Paramyxoviridae (e. g.,
mumps virus, measles virus, respiratory syncytial
virus, etc.); Orthomyxoviridae (e. g., influenza virus
types A, B and C, etc.}; Bunyaviridae; Arenaviridae;
Retroviradae, e.g., HTLV-I; HTLV-II; HIV-1; HIV-2;
simian immundeficiency virus (SIV) among others. See,
e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988);
Fundamental Virology, 2nd Edition (B.N. Fields and
D.M. Knipe, eds. 1991), for a description of these and
other viruses.
Particularly preferred bacterial antigens
are derived from organisms that cause diphtheria,
tetanus, pertussis, meningitis, and other pathogenic-
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states, including, without limitation, antigens
derived from Corynebacterium diphtheriae, Clostridium
tetani, Bordetella pertusis, Neisseria meningitidis,
including serotypes Meningococcus A, B, C, Y and WI35
(MenA, B, C, Y and WI35), Haemophilus influenza type B
(Hib), and Helicobacter pylori. Examples of parasitic
antigens include those derived from organisms causing
malaria and Lyme disease.
Combinations of antigens derived from the
organisms above can be conveniently used to elicit
immunity to multiple pathogens in a single vaccine.
For example, a particularly preferred combination is a
combination of bacterial surface oligosaccharides
derived from MenC and Hib, conjugated to a nontoxic
mutant carrier derived from a bacterial toxin, such as
a nontoxic mutant of diphtheria toxin known as CRM19,.
This conjugate is useful for preventing bacterial
meningitis and is described in International
Publication No. WO 96/14086, published May 17, 1996.
Furthermore, the methods described herein
provide means for treating a variety of malignant
cancers. For example, the system of the present
invention can be used to enhance both humoral and
cell-mediated immune responses to particular proteins
specific to a cancer in question, such as an activated
oncogene, a fetal antigen, or an activation marker.
Such tumor antigens include any of the various MAGEs
(melanoma associated antigen E), including MAGE 1, 2,
3, 4, etc. (Boon, T. Scientific American (March
1993):82-89); any of the various tyrosinases; MART 1
(melanoma antigen recognized by T cells), mutant ras;
mutant p53; p97 melanoma antigen; CEA
(carcinoembryonic antigen), among others.
It is readily apparent that the subject
invention can be used to mount an immune response to a
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wide variety of antigens and hence to treat or prevent
a large number of diseases.
As explained above, the VLP formulations may
or may not contain one or more antigens of interest.
For example, antigens can be administered separately
from the VLP compositions at the same or at different
sites. In any event, one or more selected antigens
will be administered in a "therapeutically effective
amount" such that an immune response can be generated
in the individual to which it is administered. The
exact amount necessary will vary depending on the
subject being treated; the age and general condition
of the subject to be treated; the capacity of the
subject's immune system to synthesize antibodies
and/or mount a cell-mediated immune response; the
degree of protection desired; the severity of the
condition being treated; the particular antigen
selected and its mode of administration, among other
factors. An appropriate effective amount can be
readily determined by one of skill in the art. Thus,
a "therapeutically effective amount" will fall in a
relatively broad range that can be determined through
routine trials. In general, a "therapeutically
effective" amount of antigen will be an amount on the
order of about 0.1 ~.g to about 1000 ~,g, more
preferably about 1 ug to about 100
Similarly, the VLP adjuvant will be present
in an amount such that the antigen displays "enhanced
immunogenicity," as defined above, as compared to
administration of the antigen alone, without the VLP
adjuvant. Amounts which are effective for eliciting
an enhanced immune response can be readily determined
by one of skill in the art.
The compositions may additionally contain
one or more "pharmaceutically acceptable excipients or
vehicles" such as water, saline, glycerol, ethanol, -
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etc. Additionally, auxiliary substances, such as
wetting or emulsifying agents, biological buffers, and
the like, may be present in such vehicles. A
biological buffer can be virtually any solution which
is pharmacologically acceptable and which provides the
adjuvant formulation with the desired pH, i.e., a pH
in the physiological range. Examples of buffer
solutions include saline, phosphate buffered saline,
Tris buffered saline, Hank's buffered saline, growth
media such as Eagle's Minimum Essential Medium
("MEM"), and the like.
The antigen is optionally associated with a
carrier which is a molecule that does not itself
induce the production of antibodies harmful to the
individual receiving the composition. Suitable
carriers are typically large, slowly metabolized
macromolecules such as proteins, polysaccharides,
polylactic acids, polyglycollic acids, polymeric amino
acids, amino acid copolymers, lipid aggregates (such
as oil droplets or liposomes), and inactive virus
particles. Such carriers are well known to those of
ordinary skill in the art. Additionally, these
carriers may function as additional immunostimulating
agents. Furthermore, the antigen may be conjugated to
a bacterial toxoid, such as toxoid from diphtheria,
tetanus, cholera, etc.
Coadjuvants in addition to the VLPs of the
present invention, may also be used to enhance the
effectiveness of the pharmaceutical compositions.
Such coadjuvants include, but are not limited to: (1)
aluminum salts (alum), such as aluminum hydroxide,
aluminum phosphate, aluminum sulfate, etc.; (2) oil-
in-water emulsion formulations (with or without other
specific immunostimulating agents such as muramyl
peptides (see below) or bacterial cell wall
components), such as for example (a) MF59 -
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(International Publication No. WO 90/14837),
containing 5% Squalene, 0.5% Tween 80, and 0.5% Span
85 (optionally containing various amounts of MTP-PE
(see below), although not required) formulated into
submicron particles using a microfluidizer such as
Model 110Y microfluidizer (Microfluidics, Newton, MA),
(b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%
pluronic-blocked polymer L121, and thr-MDP {see below)
either microfluidized into a submicron emulsion or
vortexed to generate a larger particle size emulsion,
and (c) RibiT"" adjuvant system (RAS) , (Ribi Immunochem,
Hamilton, MT) containing 2% Squalene, 0.2% Tween 80,
and one or more bacterial cell wall components from
the group consisting of monophosphorylipid A (MPL),
trehalose dimycolate (TDM), and cell wall skeleton
(CWS) , preferably MPL + CWS (Detox~") ; (3) saponin
adjuvants, such as StimulonT"" (Cambridge Bioscience,
Worcester, MA) may be used or particle generated
therefrom such as ISCOMs (immunostimulating
complexes); (4) Complete Freunds Adjuvant (CFA) and
Incomplete Freunds Adjuvant (IFA); (5) cytokines, such
as interleukins (IL-1, IL-2, etc.), macrophage colony
stimulating factor (M-CSF), tumor necrosis factor
(TNF), etc.; (6) mucosal adjuvants such as those
derived from cholera toxin (CT), pertussis toxin {PT),
E. coli heat labile toxin (LT), and mutants thereof
(see, e.g., International Publication Nos. WO
95/17211, WO 93/13202, and WO 97/02348); and (7) other
substances that act as immunostimulating agents to
enhance the effectiveness of the composition. Alum
and MF59 are preferred.
Muramyl peptides include, but are not
limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine
(thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme
(nor-MDP), N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-
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alanine-2-{1'-2'-dipalmitoyl-sn-glycero-3-
huydroxyphosphoryloxy)-ethylamine (MTP.-PE), etc.
Once formulated, the compositions of the
invention can be administered parenterally, e.g., by
injection. The compositions can be injected either
subcutaneously, intraperitoneally, intravenously or
intramuscularly. Other modes of administration
include oral and pulmonary administration,
suppositories, mucosal and transdermal applications.
Dosage treatment may be a single dose schedule or a
multiple dose schedule. A multiple dose schedule is
one in which a primary course of vaccination may be
with 1-10 separate doses, followed by other doses
given at subsequent time intervals, chosen to maintain
and/or reinforce the immune response, for example at
1-4 months for a second dose, and if needed, a
subsequent doses) after several months. The dosage
regimen will also, at least in part, be determined by
the need of the subject and be dependent on the
judgment of the practitioner. Furthermore, if
prevention of disease is desired, the vaccines are
generally administered prior to primary infection with
the pathogen of interest. If treatment is desired,
e.g., the reduction of symptoms or recurrences, the
vaccines are generally administered subsequent to
primary infection.
C. Experimental
Below are examples of specific embodiments
for carrying out the present invention. The examples
are offered for illustrative purposes only, and are
not intended to limit the scope of the present
invention in any way.
Efforts have been made to ensure accuracy
with respect to numbers used (e. g., amounts,
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temperatures, etc.), but some experimental error and
deviation should, of course, be allowed for.
Example 1
In order to evaluate the immunogenicity in
infant baboons of vaccines containing Hepatitis B
virus (HBV) VLPs and Haemophilus influenzae type b
(Hib) and/or Hib/Meningococcal C (MenC) conjugates,
the following study was done.
Materials and Methods
A. Vaccines
The Hib/MenC conjugate vaccine and Hib
conjugate vaccines used in the study included
oligosaccharides independently conjugated to the
protein carrier CRM19~, and were produced as described
in International Publication No. WO 96/14086,
published May 17, 1996.
The VLPs used were recombinant Hepatitis B
virus (HBV) Pre-S2/S antigens. To produce the VLPs, a
Pre-S2/sAg gene was incorporated into an expression
vector and used to transfect DG44 Chinese hamster
ovary (CHO) cells which had also been transfected with
the dhfr gene. Growth of the cells and expression of
the HBV particles was performed essentially as
described in Michel et al., Bio/Technology (June 1985)
3:561-566 and Patzer et al., Bio/Technology (July
1986) 4:630-636.
The control vaccine formulation included a
yeast-derived HBV S antigen adsorbed to alum.
On the day of vaccination, the
glycoconjugate vaccines and the HBV VLP formulations
were reconstituted together to provide a dose of
approximately 5 ~.g of each of the saccharides, a total
of 20 ~g of CRMl9., protein, and 2 . 5 ~.g of HBV VLPs .
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B. Vaccine Groups
Infant baboons, 1.5 to 4 months of age at
study initiation, were assigned to groups of 5 animals
each, as shown in Table 1.
Table
1
Vaccine
Groups
Group' HBV Hib MenC Adjuvant
1 Control Vaccine --- --- Alum
2.5 ~tg
2 HBV VLPs --- --- MF59
3 HBV VLPs 5 ~.g - - - MF5 9
4 HHV VLPs 5 ~Cg 5 ~.g MF59
5 ___ 5 ~,g 5 ~.~g MF59
'N=6 animals per group (1.5 to 4 mos. of age at study
initiation)
Blood samples were collected prior to each
immunization and 2 weeks after the third immunization.
Results
Figures 1 and 2 show the serum antibody
responses of the infant baboons to HBV VLPs as
determined by a commercial EIA (Abbott). The results
are presented as the geometric mean of the antibody
concentrations in IU/ml, ~95% C1, on a log scale, for
the four groups that were given vaccines containing
HBV VLPs.
As can be seen, HBV VLPs alone, given with
MF59, were highly immunogenic, achieving geometric
mean titers of greater than 100 IU/ml after a single
injection, greater than 1000 IU/ml after a second
injection, and greater than 100,000 IU/ml after a
third injection.
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In contrast, the control vaccine given with
alum was much less immunogenic, with no response to 1
injection, and 10-fold lower antibody responses to the
second and third injections, compared to the
experimental vaccines. Although not shown, there were
no detectable hepatitis antibody responses in the
fifth group which was given the MenC/Hib conjugate
vaccines without VLPs. These results parallel the
differences in immunogenicity between these two
vaccines observed in clinical trials in healthy
adults.
The presence of Hib, or Hib/MenC conjugate,
decreased the hepatitis antibody responses, compared
to those of the group receiving the Hepatitis B/MF59
alone.
This was true particularly after the first or second
injections. However, after the third injection of the
MF59-adjuvanted combination vaccines, the respective
antibody responses to HBV were still 10-fold higher
than those of the animals vaccinated with the control
vaccine (geometric means 49,000 and 38,000 IU/ml
versus 5000 IU/ml for the control HBV) (p s 0.01).
Serum anticapsular antibody responses of the
infant baboons to the Hib conjugate vaccine, were
determined using a radioantigen binding assay.
Figures 3 and 4 show the geometric mean antibody
concentrations in ~.g/ml on a log scale for the three
groups given vaccines containing Hib/MenC given with
MF59, HBV/Hib and MF59, and HBV/Hib/MenC with MF59.
All three groups showed robust antibody
responses to Hib. Interestingly, the presence of HBV
VLPs appeared to augment the Hib antibody responses.
For example, after the third injection, the geometric
mean antibody concentration was 35 ~g/ml in the group
receiving Hib/MenC and HBV with MF59, versus 10.8
~,g/ml for the group receiving Hib/MenC/MF59 without -
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HBV. The Hib response to HBV/Hib without MenC was
even higher (100 ~g/ml).
Serum antibody responses of the infant
baboons to the MenC conjugate vaccine were also
measured. Results are shown in Figures 5A and 5B.
Figure 5A shows the IgG anticapsular antibody
responses determined by ELISA. Figure 5B depicts
complement-mediated bactericidal antibody responses.
The antibody responses to the MF59-adjuvanted MenC
combination vaccine containing HBV VLPs were
equivalent or higher than those of animals given the
Hib/MenC vaccine alone with MF59. This was true for
both IgG binding antibody determined by ELISA, and
antibody functional activity determined with the
bactericidal assay.
Table 2 summarizes the geometric antibody
responses after the third injection for all groups.
As can be seen, the animals given the control vaccine
adsorbed with alum, or the experimental HBV VLP
preparation with MF59, responded only to HBV.
Further, the magnitude of the response was >20-fold
higher to the experimental MF59 adjuvanted vaccine.
The HBV antibody responses to the two
combination vaccines containing Hib, or Hib and MenC
were approximately 2-fold lower than those of animals
given HBV/MF59 alone. Nevertheless, they were still
8- to 10-fold higher than those observed with the
control vaccine. As already noted, the Hib antibody
responses to the two combination vaccines containing
HBV VLPs were higher than those of animals given
Hib/MenC/MF59 without HBV VLPs (group 5).
With respect to MenC conjugate, there also
was evidence that HBV VLPs may augment the IgG MenC
antibody responses.
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Table 2
Serum Antibody
Responses
Geo. Mean
Antibody
(Post-3)
HBV VLPs Hib MenC
Group/Dose, (mIU/ml) (~g/ml) IgG, U/ml 1/Cidal
fig
Control 5,400 0.05 0.1 ND
vaccine
MF59 with:
HBV VLPs 120,000 0.07 0.1 ND
VLP/Hib 49,600 128.0" 0.1 ND
VLP/Hib/MenC 37,600 35.2' 53.5t 1,550
Hib/MenC 4 10.8' 26.51 1,079
~p=0_07 tPs0.03
Example 2
An experiment was also done in rabbits in
order to evaluate the immunogenicity of vaccines
containing human papillomavirus 6b (HPV 6b) L1 VLPs
and HPV E7 as follows.
Materials and Methods
A' Vaccines
The recombinant proteins used in the
vaccines of this study were cloned from HPV 6b as
described. See, Schwarz, EMBO J. (1983) 2:2341-2348.
The virus-like particle (VLP) vaccine consisted of
baculovirus-derived Late 1 (L1) protein that had self-
assembled into particles, that were purified through
ion exchange and gel filtration chromatography. The
VLPs were approximately 50nm in size, and closely
resembled HPV virions (Greer, J. Cain. Micro. (1995)
33:2058-2063). The second vaccine consisted of E.
coli-derived Early 7 (E7) protein.
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On the day of vaccination, the vaccines were
prepared such that a dose of VLP vaccine would contain
17 ~.g of HPV 6 L1 VLPs, while the E7 vaccine dose
would contain 50 ~,g, and the L1/E7 combined vaccine
dose would contain 17 ~Cg of VLP and 50 ~g of E7. The
total volume of all the vaccines was 0.5 ml of which
0.25 mI was MF59 coadjuvant.
B. Vaccine Groups
Young adult, female New Zealand white
rabbits were assigned to one of three possible groups
(20 animals per group) as shown in Table 3. The
animals received vaccines containing either VLP, E7,
or a combination of L1 and E7.
Table
3
Vaccine
Groups
Group Number L1 E7 Co- Total Site of
of Antigen Antigen adjuvant Vaccine Injec-
Animals Volume tion
2 L1 20 17 ~.g --- MF59 0.5 ml Hind leg
0
L1/E7 20 17 ~g 50 ug MF59 0.5 ml Hind leg
E7 20 --- 50 ~.g MF59 0.5 ml Hind leg
C. Immunization and Bleedincr Schedules
The rabbits were given intramuscular (hind
leg} vaccinations during week 0, 3, and 6. Blood
samples were collected during weeks 0, 4, 5, 7 and 8.
D. Determination of Antigen-Specific Antibody
Response
Antibody responses were measured using
antigen-specific ELISAs. Titers were determined for
each serum at the dilution that resulted in an optical
density of 0.5.
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CA 02288129 1999-10-26
WO 98/50071 PCT/US98/08146
Results
The rabbit serum titer results for E7 and L1
antigens are summarized in Figure 6. The results
presented on a log scale, are the arithmetic mean of
the antibody titers for the groups. The antibody
titers generated from the E7 alone with MF59 vaccine
were significantly lower than the VLPs after the
second and third vaccinations (313 versus 106,000;
p<0.0001; and 1,400 versus 65,000; p<0.0001).
However, anti-E7 mean titers generated by the E7/VLP
combination appeared augmented in the animals injected
with combined E7/L1 vaccine (and MF59) as compared to
E7 alone with MF59 (825 versus 323; 1,567 versus
1,363). In addition, the median titer of E7/L1 was
significantly higher at weeks 5 and 8 at the 5% level
(p-values 0.0018 and 0.0125, respectively).
Furthermore, the number of non-responders was
statistically significantly lower for E7/L1 than for
E7 (p-value 0.023 Fisher's exact test).
The data indicates that the VLPs alone with
MF59 were highly immunogenic, resulting in titers of
482 after the first injection and 106,000 and 65,000
after the second and third injections. However, anti-
VLP mean titers for the combined VLP/E7 vaccine group
after the second immunization were reduced (106,000
versus 89,000) and reduced significantly after the 3
immunization (65,000 versus 35,000; p=0.001).
Thus, novel VLP adjuvant compositions and
methods for using and making the same are disclosed.
Although preferred embodiments of the subject
invention have been described in some detail, it is
understood that obvious variations can be made without
departing from the spirit and the scope of the
invention as defined by the appended claims.
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