U.S. patent application number 09/781124 was filed with the patent office on 2002-01-24 for prophylactic and therapeutic monoclonal antibodies.
Invention is credited to Hooper, Jay W., Schmaljohn, Alan L., Schmaljohn, Connie S..
Application Number | 20020009447 09/781124 |
Document ID | / |
Family ID | 22666929 |
Filed Date | 2002-01-24 |
United States Patent
Application |
20020009447 |
Kind Code |
A1 |
Hooper, Jay W. ; et
al. |
January 24, 2002 |
Prophylactic and therapeutic monoclonal antibodies
Abstract
In this application are described vaccinia monoclonal
antibodies. Also provided are mixtures of antibodies of the present
invention, as well as methods of using individual antibodies or
mixtures thereof for the detection, prevention, and/or
therapeutical treatment of vaccinia virus infections in vitro and
in vivo.
Inventors: |
Hooper, Jay W.; (New Market,
MD) ; Schmaljohn, Alan L.; (Frederick, MD) ;
Schmaljohn, Connie S.; (Frederick, MD) |
Correspondence
Address: |
U.S. Army Medical Research and Materiel Command
ATTN: MCMR-JA(Ms. Elizabeth Arwine-PATENT ATTY)
504 Scott Street
Fort Detrick
MD
21702-5012
US
|
Family ID: |
22666929 |
Appl. No.: |
09/781124 |
Filed: |
February 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60182066 |
Feb 11, 2000 |
|
|
|
Current U.S.
Class: |
424/147.1 ;
530/388.3 |
Current CPC
Class: |
C07K 16/081 20130101;
A61P 31/20 20180101; A61K 2039/505 20130101 |
Class at
Publication: |
424/147.1 ;
530/388.3 |
International
Class: |
A61K 039/42; C07K
016/00; C12P 021/08 |
Claims
What is claimed is:
1. A composition comprising one or more monoclonal antibody
directed against a vaccinia virus antigen.
2. The composition of claim 1 wherein said vaccinia virus antigen
is L1R.
3. The composition of claim 1 wherein said vaccinia virus antigen
is A33R.
4. The composition of claim 2 wherein said composition further
comprises one or more monoclonal antibody directed against vaccinia
A33R.
5. The composition of claim 4 wherein said composition further
comprises one or more monoclonal antibody directed against an
antigen chosen from the group consisting essentially of: vaccinia
H3L, D8L, B5R, A27L and A17L.
6. The composition of claim 4 wherein said composition inhibits
vaccinia virus infection in a subject in vivo.
7. The composition of claim 6 wherein said subject is avian or
mammalian.
8. The composition of claim 4 wherein said composition ameliorates
symptoms of vaccinia virus infection when said composition is
administered to a subject after infection with vaccinia virus.
9. The composition of claim 8 wherein said subject is avian or
mammalian.
10. The composition of claim 2 wherein said monoclonal antibody
immunoprecipitates L1R in vitro.
11. The composition of claim 3 wherein said monoclonal antibody
immunoprecipitates A33R in vitro.
12. A therapeutic composition for ameliorating symptoms of vaccinia
virus infection comprising the composition of claim 4, and a
pharmaceutically acceptable excipient.
13. A passive vaccine against vaccinia virus infection comprising
the composition of claim 4.
14. An anti-vaccinia composition, comprising one or more monoclonal
antibodies, wherein at least two of said monoclonal antibodies are
directed against L1R and A33R, in an amount effective for
inhibiting vaccinia virus infection, and a pharmaceutically
acceptable carrier.
15. A method of treating vaccinia virus infection comprising
administering to a patient in need of said treatment an effective
amount of a composition according to claim 4.
16. The composition according to claim 1 wherein said vaccinia
virus antigen is chosen from the vaccinia strain Connaught, IHD-J,
Brighton, WR, Lister, Copenhagen, Ankara, Dairen I, L-IPV, LC16M8,
LC16MO, LIVP, Tian Tan, WR 65-16, Wyeth.
17. A poxvirus monoclonal antibody composition comprising
monoclonal antibodies against a homolog of a vaccinia antigen
chosen from the group consisting of L1R and A33R, said poxvirus
chosen from the group consisting of: orthopoxvirus, parapoxvirus,
avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus,
molluscimpoxvirus, and yatapoxvirus.
Description
[0001] This application claims benefit from an earlier filed
Provisional application Ser. No. 60/182,066 filed on Feb. 11,
2000.
INTRODUCTION
[0002] Viruses in the family Poxviridae, including vaccinia virus
(VACV) and variola virus, are characterized by a large linear
double-stranded DNA genome (130-300 kb) packaged in a relatively
large virion (.about.350.times.270 nm), and a cytoplasmic site of
replication (reviewed by Moss, 1996, In "Fields Virology", D. M.
Knipe et al. Eds., vol. 3, pp 2637-2671. Lippincott-Raven,
Philadelphia). Assembly of VACV virions begins with condensation of
dense granular material into membrane-wrapped particles called
intracellular mature virions (IMV). Recent findings indiate the IMV
are wrapped by a single membrane (Hollingshead et al., 1999, J.
Virol. 73, 1503-1517) rather than a double membrane as previously
reported. IMV are then enveloped in two additional membranes
derived from the trans Golgi to form multiple membrane-warpped
particles called intracellular enveloped virions (IEV) (Schmelz et
al., 1994, J. Virol. 68, 130-147). IEV are moved, possibly by actin
polymerization (Cudmore et al., 1995, Nature 378, 636-638), to the
cell periphery, where the outermost membrane fuses with the cell
plasma membrane, exposing a cell-associated eneveloped virion (CEV)
(Blasco and Moss, 1991, J. Virol. 65, 5910-5920). CEV are released
from the cell as extracellular enveloped virions (EEV), which play
a role in long-range spread of the virus (Payne, 1980, J. Gen.
Virol. 50, 89-100). IMV released from disrupted cells and EEV are
both infectious forms of VACV.
[0003] The primary therapeutic tool for the control and eradication
of infection with VACV include a live virus vaccine to prevent
disease, and a vaccinia immune globulin (VIG) to treat dissminated
infections. The existing VIG product is derived from human donors
who have been vaccinated with the smallpox vaccine, vaccinia virus.
As with all human products, the existing VIG must be tested
exhaustively for blood borne human pathogens such as human
immunodeficiency virus and hepatitus B. Therefore, the existing VIG
suffers from several drawbacks including the necessity for using
human volunteers, i.e. the use of a live virus as an immunogen
which could cause infectious lesions that scar in healthy
individuals and severe disseminated life-threatening infection in
immunocompromised individuals. And, despite continuous screening of
the donor population to assure consistency which is very expensive,
product lots can vary significantly between batches and geographic
regions.
[0004] Therefore, there is a need to provide an immune globulin
composition which is safe and precisely defined, and which does not
rely on human donors. However, it is not known which components of
the vaccinia VIG are important for protection nor how many of the
.about.200 genes contained in the vaccinia genome encode proteins
that would elicit a protective response upon passive transfer of
monoclonal antibodies directed to such proteins.
SUMMARY OF THE INVENTION
[0005] This application satisfies the need mentioned above. This
application describes a vaccinia immunoglobulin composition which
can serve as a replacement for the presently used VIG. The vaccinia
immunoglobulin composition of the present invention is composed of
one or more monoclonal antibody against vaccinia antigens defined
to be important for protection. To identify potential targets for
poxvirus therapeutics, we generated and characterized a panel of
400 VACV-specific monoclonal antibodies (MAbs) in mice. The
monoclonal antibodies were first tested for their ability to
neutralize virus and then were tested for their ability to protect
mice from challenge. Two challenge models were used, one that
involves dissemination of the virus (in suckling mice) and a
challenge that involves a massive challenge dose (by
intraperitoneal injection). To our surprise, the ability of the
MAbs to inhibit plaque formation by vaccinia virus, a standard
assay of virus neutralization, did not always predict their
protective efficacy. Moreover, the monoclonal antibodies differed
in their ability to provide protection depending on the challenge
model.
[0006] We found that the majority of moderately neutralizing
monoclonal antibodies were directed against a 34 KDa protein later
determined to be D8L which on its own did not provide protection in
mice. Another monoclonal antibody which was neutralizing did not
protect against challenge when given alone to mice and was directed
against the protein A27L. Neutralizing MAbs binding to the 29-KDa
protein (e.g. MAb-10F5, and MAB-7D11), protected mice against
intraperitoneal challenge and were found to react with the IMV
product of the L1R gene first described in Wolffe, E. J. et al.,
1995, Virology 211, 53-63). Nonneutralizing MAbs binding to 23 to
28-kDA protein (e.g. MAb-1G10) protected against challenge with
VACV (strain WR) in suckling mice. The target of MAb-1G10 was the
EEV product of the A33R gene (Roper et al., 1996, J. Virol. 70,
3753-3762).
[0007] The LIR and A33R gene product will be called L1R and A33R,
respectively. L1R is an essential myristoylated protein associated
with the IMV membrane and is thought to play a role in IMV
attachment or penetration (Franke et al., 1990, J. Virol. 64,
5988-5996; Ravanello et al., 1993, J. Gen. Virol. 75, 1479-1483;
Ichihashi et al., 1994, Virology 202, 834-843; Ravanello and Hruby,
1994, J. Gen. Virol. 75, 1479-1483; Wolffe et al., 1995, supra).
A33R is a nominally nonessential glycosylated/palmitated protein
that forms dimers and is incorporated into the outer membrane of
EEV (Payne, 1992, Virology 187, 251-260; Roper et al., 1996,
supra). A33R is thought to be involved in facilitating direct
cell-to-cell spread via actin-containing microvilli (Roper et al.,
1998, J. Virol. 72, 4192-4204). Homologs of L1R and A33R are
present in other Orthopoxviruses, e.g. between VACV and variola,
L1R identity is 99.6% and A33R is 94.1% (Massung et al., 1994,
Virology 201, 215-240).
[0008] Therefore, it is an object of the present invention to
provide a composition of one or more monoclonal antibody directed
against at least one, preferably two or more, vaccinia virus
antigens. Antigens preferably include L1R and A33R.
[0009] It is another object of the present invention to provide
monoclonal antibodies which protect against vaccinia virus
infection and bind to epitopes on L1R and A33R gene products. The
monoclonal antibodies described below recognize epitopes on the
VACV strain Connaught L1R sequence (Genebank #Af226617) and the
Connaught strain A33R gene sequence (Genebank #Af226618). L1R and
A33R homologs from other poxviruses can be used as immunogens to
produce monoclonal antibodies which would most likely be protective
since the homologs in other poxviruses have high identity with the
VACV proteins. Other poxviruses include other Orthopoxviruses such
as variola virus, monkeypox virus, cowpox virus, Parapoxviruses
such as orf virus, paravaccinia virus, and unclassified poxviruses
such as Tanapoxvirus, Yabapoxvirus and Molluscum contagiosum.
[0010] It is yet another object of the present invention to provide
a composition comprising humanized monoclonal antibodies of the
present invention for example anti-L1R antibody, or anti-A33R
antibody or a mixture thereof, as a vaccinia immunoglobulin
replacement. The vaccinia immunoglobulin replacement may further
contain other antibodies specific for vaccinia antigens shown to be
effective for eliciting neutralizing/protective antibodies, for
example H3L, D8L, B5R, A27L, and A17L. In addition, MAbs against
L1R and A33R homologs from other poxviruses can be used alone or in
combination with the vaccinia MAbs to provide a therapeutic and
prophylactic composition.
[0011] It is another object of the invention to provide for
antibodies that are functionally equivalent to the antibodies
listed above. These functionally equivalent antibodies
substantially share at least one major functional property with an
antibody listed above and herein described comprising: binding
specificity to L1R and A33R, immunoreactivity in vitro, protection
against vaccinia challenge when administered prophylactically or
therapeutically, competition for same binding site on L1R and A33R.
The antibodies can be of any class such as IgG, IgM, or IgA or any
subclass such as IgG1, IgG2a, and other subclasses known in the
art. Further, the antibodies can be produced by any method, such as
phage display, or produced in any organism or cell line, including
bacteria, insect, mammal or other type of cell or cell line which
produces antibodies with desired characteristics, such as humanized
antibodies. The antibodies can also be formed by combining an Fab
portion and a Fc region from different species.
[0012] It is another object of the present invention to provide for
mixtures of antibodies according to the present invention, as well
as to methods of using individual antibodies, or mixtures thereof
for the prevention and/or therapeutic treatment of vaccinia virus
infections in vitro and in vivo, and/or for improved detection of
vaccinia infections.
[0013] It is yet another object of the present invention to treat
or prevent vaccinia virus infection by administering a
therapeutically or prophylactically effective amount of one
antibody of the present invention or a mixture of antibodies of the
present invention to a subject in need of such treatment.
[0014] It is another object of the present invention to provide
passive vaccines for treating or preventing vaccinia virus
infections comprising a therapeutically or prophylactically
effective amount of the antibodies of the present invention which
protect against vaccinia virus, in combination with a
pharmaceutically acceptable carrier or excipient.
[0015] It is yet another object of the present invention to provide
a method for diagnosis of vaccinia virus infection by assaying for
the presence of vaccinia in a sample using the antibodies of the
present invention.
[0016] It is still another object of the present invention to
provide novel immunoprobes and test kits for detection of vaccinia
virus infection comprising antibodies according to the present
invention. For immunoprobes, the antibodies are directly or
indirectly attached to a suitable reporter molecule, e.g., and
enzyme or a radionuclide. The test kit includes a container holding
one or more antibodies according to the present invention and
instructions for using the antibodies for the purpose of binding to
vaccinia virus to form an immunological complex and detecting the
formation of the immunological complex such that presence or
absence of the immunological complex correlates with presence or
absence of vaccinia virus.
[0017] It is another object of the present invention to provide
anti-idiotypic antibodies raised against one of the present
monoclonal antibodies for use as a vaccine to elicit an active
anti-vaccinia response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] These and other features, aspects, and advantages of the
present invention will become better understood with reference to
the following description and appended claims, and accompanying
drawings where:
[0019] FIG. 1. Passive transfer of L1R-specific MAb protects
against lethal intraperitoneal challenge with VACV (strain WR).
Mice were injected with the indicated antibody and then, after 24
hrs, were challenged with VACV (strain WR). A group of 5 previously
immunized mice (tail-scarified) served as positive controls.
MAb-7D1is a L1R-specific mouse MAb. MAb-1G10 is a A33R-specific
mouse MAb. MAb-8E10 is a negative control mouse MAb.
DETAILED DESCRIPTION
[0020] In the description that follows, a number of terms used in
recombinant DNA, virology and immunology are extensively utilized.
In order to provide a clearer and consistent understanding of the
specification and claims, including the scope to be given such
terms, the following definitions are provided.
[0021] The term "antibody" is art-recognized terminology and is
intended to include molecules or active fragments of molecules that
bind to known antigens. Examples of active fragments of molecules
that bind to known antigens include Fab and F(ab').sub.2 fragments.
These active fragments can be derived from an antibody of the
present invention by a number of techniques. For example, purified
monoclonal antibodies can be cleaved with an enzyme, such as
pepsin, and subjected to HPLC gel filtration. The appropriate
fraction containing Fab fragments can then be collected and
concentrated by membrane filtration and the like. For further
description of general techniques for the isolation of active
fragments of antibodies, see for example, Khaw, B. A. et al. J.
Nucl. Med. 23:1011-1019 (1982). The term "antibody" also includes
bispecific and chimeric antibodies.
[0022] The language "monoclonal antibody" is art-recognized
terminology. The monoclonal antibodies of the present invention can
be prepared using classical cloning and cell fusion techniques. The
immunogen (antigen) of interest, is typically administered (e.g.
intraperitoneal injection) to wild type or inbred mice (e.g.
BALB/c) or transgenic mice which produce desired antibodies, rats,
rabbits or other animal species which can produce native or human
antibodies. The immunogen can be administered alone, or mixed with
adjuvant, or expressed from a vector (VEE replicon vector,
vaccinia), or as DNA, or as a fusion protein to induce an immune
response. Fusion proteins comprise the peptide against which an
immune response is desired coupled to carrier proteins, such as
.beta.-galactosidase, glutathione S-transferase, keyhole limpet
hemocyanin (KLH), and bovine serum albumin, to name a few. In these
cases, the peptides serve as haptens with the carrier proteins.
After the animal is boosted, for example, two or more times, the
spleen is removed and splenocytes are extracted and fused with
myeloma cells using the well-known processes of Kohler and Milstein
(Nature 256:495-497 (1975)) and Harlow and Lane (Antibodies: A
Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988)).
The resulting hybrid cells are then cloned in the conventional
manner, e.g. using limiting dilution, and the resulting clones,
which produce the desired monoclonal antibodies, cultured.
[0023] Monoclonal antibodies raised against vaccinia antigens L1R,
A33R, H3L, D8L, B5R, A27L, and A17L are part of the present
invention. These monoclonal antibodies were generated from the
vaccinia Connaught vaccine strain. Other strains of vaccinia are
expected to contain sequences at least 90% identical and which will
likely produce antigens capable of eliciting
protective/neutralizing antibodies. Such strains include IHD-J,
Brighton, WR, Lister, Copenhagen, Ankara, Dairen I, L-IPV, LC16M8,
LC16MO, LIVP, Tian Tan, WR 65-16, Wyeth. Other homologs having at
least 90% identity exist in other poxviruses in the genera
orthopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus,
leporipoxvirus, suipoxvirus, molluscipoxvirus and Yatapoxvirus
which members include variola major and minor virus, monkeypox
virus, camelpox virus, raccoonpox virus, ectromelia virus, sealpox
virus, contagious ecthyma virus, canarypox virus, juncopox virus,
pigeonpox virus, turkeypox virus, penguinpox virus, sheepox virus,
goatpox, swinepox virus, buffalopox virus, cowpox virus, rabbit
fibroma virus, myxoma virus, and molluscum contagiosum (genus
Molluscipoxvirus) which is 59% identical and 77% similar to
vaccinia (Altschul, S. F. et al. 1997, Nucl. Acids Res. 25,
3389-3402, fowlpox (genus Avipoxvirus), Yata-tumor like virus
(Yatapoxvirus), among others (Fenner, Frank, Poxviruses, In
"Virology" B. N. Fields et al., eds. Raves Press, Ltd. New York,
1990, pp. 2113-2133). Monoclonal antibodies against homologs from
these poxviruses would likely be protective against challenge with
the source of immunogen virus.
[0024] The term "epitope" is art-recognized. It is generally
understood by those of skill in the art to refer to the region of
an antigen, such as L1R for example, that interacts with an
antibody. An epitope of a peptide or protein antigen can be formed
by contiguous or noncontinguous amino acid sequences of the
antigen. L1R and A33R, like many proteins, contains many epitopes.
The epitopes or peptides recognized by the antibodies of the
present invention and conservative substitutions of these peptides
which are still recognized by the antibody are an embodiment of the
present invention. These peptides offer a convenient method for
eluting the vaccinia antigen bound to the respective antibody on
immunoaffinity columns. For example, when an antibody which
recognizes the epitope for L1R is used in an immunoaffinity column
to purify L1R, the peptide recognized by the antibody can be added
to the immunoaffinity column to elute the L1R. Further truncation
of these epitopes may be possible since antigenic epitopes have
been reported to be represented by as few as five amino acid
residues.
[0025] The antibodies described in the Examples below are
characterized in that the antibody binds to the appropriate
immunogen as measured by assays such as ELIA, immunoprecipitation,
or immunofluorescence. Also, the L1R-specific MAbs must neutralize
vaccinia virus as measured by plaque redution neutralization test
(PRNT). Any monoclonal antibody retaining these characteristics is
related to the present invention.
[0026] By further mapping of the binding site of the monoclonal
antibodies described in this application other peptides useful as a
vaccine or a therapeutic can be predicted. Therefore, in another
aspect, this invention relates to a method for identifying
protective antigenic epitopes the method comprising (i) reacting a
monoclonal antibody described in this application to different
overlapping fragments encompassing the complete antigen, (ii)
identifying a fragment to which the protective antibody binds,
(iii) narrowing the region containing sites further by reacting the
monoclonal with smaller overlapping fragments encompassing the
region identified in (ii), and (iv) choosing peptides to which the
antibody binds as possible antigenic epitopes. The peptides can
then be assayed for their ability to protect an animal from
disease, or to reduce the severity of disease.
[0027] The epitopes or peptides on the vaccinia antigen to which
the monoclonal antibodies bind can constitute all or part of an
eventual active vaccine candidate. An active vaccine or therapeutic
candidate might comprise these peptide sequences and others. These
might be delivered as synthetic peptides, or as fusion proteins,
alone or co-administered with cytokines and/or adjuvants or
carriers safe for human use, e.g. aluminum hydroxide, to increase
immunogenicity. In addition, sequences such as ubiquitin can be
added to increase antigen processing for more effective immune
responses.
[0028] The present invention also pertains to hybridomas producing
antibodies which bind to an epitope of vaccinia antigens. The term
"hybridoma" is art recognized and is understood by those of
ordinary skill in the art to refer to a cell produced by the fusion
of an antibody-producing cell and an immortal cell, e.g. a multiple
myeloma cell. This hybrid cell is capable of producing a continuous
supply of antibody. See the definition of "monoclonal antibody"
above and the Examples below for a more detailed description of the
method of fusion.
[0029] The present invention still further pertains to a method for
detecting vaccinia in a sample suspected of containing vaccinia.
The method includes contacting the sample with an antibody which
binds an epitope of a vaccinia antigen, allowing the antibody to
bind to the vaccinia antigen to form an immunological complex,
detecting the formation of the immunological complex and
correlating the presence or absence of the immunological complex
with the presence or absence of vaccinia antigen in the sample. The
sample can be biological, environmental or a food sample.
[0030] The language "detecting the formation of the immunological
complex" is intended to include discovery of the presence or
absence of vaccinia antigen in a sample. The presence or absence of
vaccinia antigen can be detected using an immunoassay. A number of
immunoassays used to detect and/or quantitate antigens are well
known to those of ordinary skill in the art. See Harlow and Lane,
Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New
York 1988 555-612). Such immunoassays include antibody capture
assays, antigen capture assays, and two-antibody sandwich assays.
These assays are commonly used by those of ordinary skill in the
art. In an antibody capture assay, the antigen is attached to solid
support, and labeled antibody is allowed to bind. After washing,
the assay is quantitated by measuring the amount of antibody
retained on the solid support. A variation of this assay is a
competitive ELISA wherein the antigen is bound to the solid support
and two solutions containing antibodies which bind the antigen, for
example, serum from a vaccinia virus vaccinee and a monoclonal
antibody of the present invention, are allowed to compete for
binding of the antigen. The amount of monoclonal bound is then
measured, and a determination is made as to whether the serum
contains anti vaccinia antigen antibodies. This competitive ELISA
can be used to indicate immunity to known protective epitopes in a
vaccinee following vaccination.
[0031] In an antigen capture assay, the antibody is attached to a
solid support, and labeled antigen is allowed to bind. The unbound
proteins are removed by washing, and the assay is quantitated by
measuring the amount of antigen that is bound. In a two-antibody
sandwich assay, one antibody is bound to a solid support, and the
antigen is allowed to bind to this first antibody. The assay is
quantitated by measuring the amount of a labeled second antibody
that can bind to the antigen.
[0032] These immunoassays typically rely on labeled antigens,
antibodies, or secondary reagents for detection. These proteins can
be labeled with radioactive compounds, enzymes, biotin, or
fluorochromes. Of these, radioactive labeling can be used for
almost all types of assays and with most variations.
Enzyme-conjugated labels are particularly useful when radioactivity
must be avoided or when quick results are needed. Biotin-coupled
reagents usually are detected with labeled streptavidin.
Streptavidin binds tightly and quickly to biotin and can be labeled
with radioisotopes or enzymes. Fluorochromes, although requiring
expensive equipment for their use, provide a very sensitive method
of detection. Antibodies useful in these assays include monoclonal
antibodies, polyclonal antibodies, and affinity purified polyclonal
antibodies. Those of ordinary skill in the art will know of other
suitable labels which may be employed in accordance with the
present invention. The binding of these labels to antibodies or
fragments thereof can be accomplished using standard techniques
commonly known to those of ordinary skill in the art. Typical
techniques are described by Kennedy, J. H., et al.,1976 (Clin.
Chim. Acta 70:1-31), and Schurs, A. H. W. M., et al. 1977 (Clin.
Chim Acta 81:1-40). Coupling techniques mentioned in the latter are
the glutaraldehyde method, the periodate method, the dimaleimide
method, and others, all of which are incorporated by reference
herein.
[0033] The language "biological sample" is intended to include
biological material, e.g. cells, tissues, or biological fluid. By
"environmental sample" is meant a sample such as soil and water.
Food samples include canned goods, meats, and others.
[0034] Yet another aspect of the present invention is a kit for
detecting vaccinia virus in a biological sample. The kit includes a
container holding one or more antibodies which binds an epitope of
a vaccinia antigen and instructions for using the antibody for the
purpose of binding to vaccinia antigen to form an immunological
complex and detecting the formation of the immunological complex
such that the presence or absence of the immunological complex
correlates with presence or absence of vaccinia virus in the
sample. Examples of containers include multiwell plates which allow
simultaneous detection of vaccinia virus in multiple samples.
[0035] As described in greater detail in the examples, the present
inventors have isolated monoclonal antibodies which bind to at
least two different vaccinia virus antigens, L1R and A33R, and
display in vitro and/or in vivo vaccinia virus protective
properties. Significantly, the reactivity of the MAbs is applicable
against a broad variety of different wild type and laboratory
vaccinia strains of different types.
[0036] Given these results, monoclonal antibodies according to the
present invention are suitable both as therapeutic and prophylactic
agents for treating or preventing vaccinia infection in susceptible
vaccinia-infected subjects. Subjects include rodents such as mice
or guinea pigs, birds or avian, and mammals, including humans.
[0037] In general, this will comprise administering a
therapeutically or prophylactically effective amount of one or more
monoclonal antibodies of the present invention to a susceptible
subject or one exhibiting vaccinia infection. Any active form of
the antibody can be administered, including Fab and F(ab').sub.2
fragments. Antibodies of the present invention can be produced in
any system, including insect cells, baculovirus expression systems,
chickens, rabbits, goats, cows, or plants such as tomato, potato,
banana or strawberry. Methods for the production of antibodies in
these systems are known to a person with ordinary skill in the art.
Preferably, the antibodies used are compatible with the recipient
species such that the immune response to the MAbs does not result
in clearance of the MAbs before virus can be controlled, and the
induced immune response to the MAbs in the subject does not induce
"serum sickness" in the subject. Preferably, the MAbs administered
exhibit some secondary functions such as binding to Fc receptors of
the subject.
[0038] Treatment of individuals having vaccinia infection may
comprise the administration of a therapeutically effective amount
of vaccinia antibodies of the present invention. The antibodies can
be provided in a kit as described below. The antibodies can be used
or administered as a mixture, for example in equal amounts, or
individually, provided in sequence, or administered all at once. In
providing a patient with antibodies, or fragments thereof, capable
of binding to vaccinia antigen, or an antibody capable of
protecting against vaccinia virus in a recipient patient, the
dosage of administered agent will vary depending upon such factors
as the patient's age, weight, height, sex, general medical
condition, previous medical history, etc.
[0039] In general, it is desirable to provide the recipient with a
dosage of antibody which is in the range of from about 1 pg/kg-100
pg/kg, 100 pg/kg-500 pg/kg, 500 pg/kg-1 ng/kg, 1 ng/kg-100 ng/kg,
100 ng/kg-500 ng/kg, 500 ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100
ug/kg-500 ug/kg, 500 ug/kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100
mg/kg, 100 mg/kg-500 mg/kg, 500 mg/kg-1 g/kg, 1 g/kg-5 g/kg, 5
g/kg-10 g/kg (body weight of recipient), although a lower or higher
dosage may be administered.
[0040] In a similar approach, another therapeutic use of the
monoclonal antibodies of the present invention is the active
immunization of a patient using an anti-idiotypic antibody raised
against one of the present monoclonal antibodies. Immunization with
an anti-idiotype which mimics the structure of the epitope could
elicit an active anti-L1R or anti-A33R responses (Linthicum, D. S.
and Farid, N. R., Anti-Idiotypes, Receptors, and Molecular Mimicry
(1988), pp 1-5 and 285-300).
[0041] Likewise, active immunization can be induced by
administering one or more antigenic and/or immunogenic epitopes as
a component of a subunit vaccine. Vaccination could be performed
orally or parenterally in amounts sufficient to enable the
recipient to generate protective antibodies against this
biologically functional region, prophylactically or
therapeutically. The host can be actively immunized with the
antigenic/immunogenic peptide in pure form, a fragment of the
peptide, or a modified form of the peptide. One or more amino
acids, not corresponding to the original protein sequence can be
added to the amino or carboxyl terminus of the original peptide, or
truncated form of peptide. Such extra amino acids are useful for
coupling the peptide to another peptide, to a large carrier
protein, or to a support. Amino acids that are useful for these
purposes include: tyrosine, lysine, glutamic acid, aspartic acid,
cyteine and derivatives thereof. Alternative protein modification
techniques may be used e.g., NH.sub.2-acetylation or COOH-terminal
amidation, to provide additional means for coupling or fusing the
peptide to another protein or peptide molecule or to a support.
[0042] The antibodies capable of protecting against vaccinia virus
are intended to be provided to recipient subjects in an amount
sufficient to effect a reduction in the vaccinia virus infection
symptoms. An amount is said to be sufficient to "effect" the
reduction of infection symptoms if the dosage, route of
administration, etc. of the agent are sufficient to influence such
a response. Responses to antibody administration can be measured by
analysis of subject's vital signs
[0043] A composition is said to be "pharmacologically acceptable"
if its administration can be tolerated by a recipient patient. Such
an agent is said to be administered in a "therapeutically effective
amount" if the amount administered is physiologically significant.
An agent is physiologically significant if its presence results in
a detectable change in the physiology of a recipient patient.
[0044] The compounds of the present invention can be formulated
according to known methods to prepare pharmaceutically useful
compositions, whereby these materials, or their functional
derivatives, are combined in admixture with a phamaceutically
acceptable carrier vehicle. Suitable vehicles and their
formulation, inclusive of other human proteins, e.g., human serum
albumin, are described, for example, in Remington's Pharmaceutical
Sciences (16 th ed., Osol, A. ed., Mack Easton Pa. (1980)). In
order to form a pharmaceutically acceptable composition suitable
for effective administration, such compositions will contain an
effective amount of the above-described compounds together with a
suitable amount of carrier vehicle.
[0045] Additional pharmaceutical methods may be employed to control
the duration of action. Control release preparations may be
achieved through the use of polymers to complex or absorb the
compounds. The controlled delivery may be exercised by selecting
appropriate macromolecules (for example polyesters, polyamino
acids, polyvinyl, pyrrolidone, ethylenevinylacetate,
methylcellulose, carboxymethylcellulose, or protamine sulfate) and
the concentration of macromolecules as well as the method of
incorporation in order to control release. Another possible method
to control the duration of action by controlled release
preparations is to incorporate the compounds of the present
invention into particles of a polymeric material such as
polyesters, polyamino acids, hydrogels, poly(lactic acid) or
ethylene vinylacetate copolymers. Alternatively, instead of
incorporating these agents into polymeric particles, it is possible
to entrap these materials in microcapsules prepared, for example,
interfacial polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly(methylmethacylate)-microcapsules,
respectively, or in colloidal drug delivery systems, for example,
liposomes, albumin microspheres, microemulsions, nanoparticles, and
nanocapsules or in macroemulsions. Such techniques are disclosed in
Remington's Pharmaceutical Sciences (1980).
[0046] Administration of the antibodies disclosed herein may be
carried out by any suitable means, including parenteral injection
(such as intraperitoneal, subcutaneous, or intramuscular
injection), in ovo injection of birds, orally, or by topical
application of the antibodies (typically carried in a
pharmaceutical formulation) to an airway surface. Topical
application of the antibodies to an airway surface can be carried
out by intranasal administration (e.g., by use of dropper, swab, or
inhaler which deposits a pharmaceutical formulation intranasally).
Topical application of the antibodies to an airway surface can also
be carried out by inhalation administration, such as by creating
respirable particles of a pharmaceutical formulation (including
both solid particles and liquid particles) containing the
antibodies as an aerosol suspension, and then causing the subject
to inhale the respirable particles. Methods and apparatus for
administering respirable particles of pharmaceutical formulations
are well known, and any conventional technique can be employed.
Oral administration may be in the form of an ingestable liquid or
solid formulation.
[0047] The treatment may be given in a single dose schedule, or
preferably a multiple dose schedule in which a primary course of
treatment may be with 1-10 separate doses, followed by other doses
given at subsequent time intervals required to maintain and or
reinforce the response, for example, at 1-4 months for a second
dose, and if needed, a subsequent dose(s) after several months.
Examples of suitable treatment schedules include: (i) 0, 1 month
and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv)
0 and 6 months, or other schedules sufficient to elicit the desired
responses expected to reduce disease symptoms, or reduce severity
of disease.
[0048] The present invention also provides kits which are useful
for carrying out the present invention. The present kits comprise a
first container means containing the above-described antibodies.
The kit also comprises other container means containing solutions
necessary or convenient for carrying out the invention. The
container means can be made of glass, plastic or foil and can be a
vial, bottle, pouch, tube, bag, etc. The kit may also contain
written information, such as procedures for carrying out the
present invention or analytical information, such as the amount of
reagent contained in the first container means. The container means
may be in another container means, e.g. a box or a bag, along with
the written information.
[0049] The contents of all cited references (including literature
references, issued patents, published patent applications, and
co-pending patent applications) cited throughout this application
are hereby expressly incorporated by reference.
[0050] Other features of the invention will become apparent in the
course of the following descriptions of exemplary embodiments which
are given for illustration of the invention and are not intended to
be limiting thereof.
EXAMPLE 1
[0051] Testing L1R- and A33R-specific mouse monoclonal antibodies
(MAbs) in vaccinia virus (strain WR) intraperitoneal lethal
challenge model.
[0052] A passive transfer experiment was performed to determine if
mouse MAbs to either L1R or A33R could protect against a lethal
intraperitoneal challenge with VACV (strain WR). Groups of 5 BALB/c
mice (14-16 weeks old) were anesthetized, bled, and then injected
(subcutaneous, behind the base of the neck, using a 1 cc 25G 5/8
tuberculin syringe) with 200 ul of either L1R-specific MAb
(MAb-7D11 ascites fluid) or A33R-specific MAb (MAb-1G10 ascities
fluid) or a combination of MAb-7D11 plus MAb-1G10, or a negative
control MAb (MAb-8E10, ascites fluid). As positive controls, 5 mice
were tail-scarified with 10.sup.6 pfu of VACV, strain WR,
approximately 3 weeks earlier. Twenty-four hours after the antibody
injection, the mice were challenged with 12.5 LD.sub.50
(5.times.10.sup.8 pfu in 200 ul)of VACV (strain WR).
[0053] The results are shown in FIG. 1. All of the mice injected
with MAb-7D11, either alone or in combination with MAb-1G10,
survived challenge. Only one of the five mice injected with
MAb-1G10 survived. All of the mice vaccinated with the negative
control MAb-8E10 died. Four of the five positive control mice
lived.
[0054] Thus, these data indicate that the L1R-specific MAb can
confer protection against a lethal challenge with VACV via the
intraperitoneal route. The A33R-specific MAb failed to protect
against this challenge.
EXAMPLE 2
[0055] Neonatal ICR mice were injected with the indicated antibody,
as ascitic fluid or protein purified antibody, and then challenged
with vaccinia virus (strain IHD-J) by the subcutaneous route. The
results indicate that A33R-specific MAb-1G10 protectes against
vaccinia virus (strain IHD-J) injected by the subcutaneous route
whereas the L1R-specific MAb-1F5 does not. Mouse hyperimmune
ascitic fluid (HMAF) also protected.
[0056] Thus, these data indicate that the L1R-specific MAb can
confer protection against a lethal challenge with VACV via the
intraperitoneal route. The A33R-specific MAb failed to protect
against this challenge. This is contrast to this MAbs capacity to
protect against a disseminated VACV (strain IHD-J) infection in
suckling mice (Table 1).
1TABLE 1 Survivors/total challenged VACV challenge Antibody
transferred Strain (route) PFU (log 10) diluent HMAF Mab-10F5
MAb-1G10 10F5 + 1G10 IHD-J (s.c.) 3.9 0/13 25 ug Ab 25 ug Ab 25 ug
Ab 25 ug Ab 9/12 0/12 10/12 9/11 25 ul AF 50 ug AB 50 ug Ab 50 ug
Ab IHD-J (s.c.) 3.9 0/10 8/10 0/10 9/11 (ea) combined results 0/23
17/22 0/22 19/23 9/11 % survival 0% 77% 0% 83% 82% IHD-J = Vaccinia
virus strain IHD-J s.c. = subcutaneous PFU = plaque forming units
HMAF = Vaccinia (stain Connaught) hyperimmune ascites fluid
MAb-10F5 = L1R-specific MAb MAb-1G10 = A33R-specific MAb Ab =
antibody AF = ascitic fluid
* * * * *