U.S. patent application number 08/699716 was filed with the patent office on 2007-02-22 for recombinant f1-v plague vaccine.
Invention is credited to GEORGE W. JR. ANDERSON, ARTHUR M. FRIEDLANDER, DAVID G. HEATH, SUSAN L. WELKOS.
Application Number | 20070043215 08/699716 |
Document ID | / |
Family ID | 37768115 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070043215 |
Kind Code |
A1 |
HEATH; DAVID G. ; et
al. |
February 22, 2007 |
RECOMBINANT F1-V PLAGUE VACCINE
Abstract
A recombinant vaccine is described which is composed of one
recombinant protein comprising F1 and V antigen of Yersinia pestis.
This vaccine protects experimental mice against pneumonic as well
as bubonic plague produced by either F1.sup.+ or F1.sup.- strains
of Y. pestis and may provide the basis for an improved human plague
vaccine.
Inventors: |
HEATH; DAVID G.; (LANSTUHL,
DE) ; FRIEDLANDER; ARTHUR M.; (GAITHERSBURG, MD)
; ANDERSON; GEORGE W. JR.; (FREDERICK, MD) ;
WELKOS; SUSAN L.; (FREDERICK, MD) |
Correspondence
Address: |
MCMR SGRD-JA;US Army MRMC
504 Scott Street
Fort Detrick
MD
21702-5012
US
|
Family ID: |
37768115 |
Appl. No.: |
08/699716 |
Filed: |
August 27, 1996 |
Current U.S.
Class: |
536/23.5 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 31/04 20180101; C07K 14/24 20130101; C07K 2319/00
20130101 |
Class at
Publication: |
536/023.5 |
International
Class: |
C07H 21/04 20060101
C07H021/04 |
Claims
1-30. (canceled)
31. An isolated and purified DNA fragment according to claim 1
further comprising a V antigen from other Yersinia species chosen
from the group consisting of Yersinia pseudotuberculosis and
Yersinia enterocolitica.
Description
[0001] Yersinia pestis is the causative organism of plague in a
wide range of animals including man. Bubonic plague in man is most
commonly acquired from the bite of an infected flea and is
characterized by the swelling of local lymph nodes which form
buboes. One of the complications of bubonic plague is secondary
pneumonia, and in these cases plague can be transmitted between
humans by airborne droplets. Pneumonic plague, particularly, is
extremely virulent and results in high mortality rates.
[0002] Plague is endemic in regions of North and South America,
Africa, China and Asia and, as evidenced by the recent outbreak of
pneumonic plague in India [Centers for Disease Control. (1994).
Update: Human Plague-India MMWR 43:722-723], epidemics of enormous
consequences remain a potential for this organism. Thus, there is a
clear need for a vaccine which would protect individuals living and
traveling in endemic areas.
[0003] The current human, licensed, vaccines available for
prevention of plague are whole cell vaccines. A number of
formulations exist. The plague USP vaccine, comprising formaldehyde
killed Y. pestis bacilli, which is administered to the body via
intramuscular injection, produces local and systemic side-effects,
ranging from mild headaches to severe malaise and fever.
Additionally, the vaccine does not provide complete immunity, since
vaccinated individuals can contract pneumonic plague, indicating
inadequate immunity at mucosal surfaces.
[0004] The live attenuated vaccine EV76 [Meyer et al. (1974) J.
Infect. Dis. 129 suppl., 13-18] was tested extensively and used in
the former Soviet Union from 1939, although its efficacy in evoking
an immune response in man is questionable [Meyer et al. (1974) J.
Infect. Dis. 129 suppl., 85-120]. The virulence of EV76 differs in
several animal species, and non-human primates are particularly
susceptible to a chronic infection with this strain. In the Western
World, the vaccine is considered to be unsuitable for mass
vaccination due to the severity of the side-effects and the
possibility of the strain reverting to full virulence.
[0005] Efforts to develop a more effective vaccine have focused on
a cellular subunits from Y. pestis as immunogens. Two of the
candidate subunits are the F1 and V antigens. The capsule
surrounding Y. pestis cells is composed of a protein component
known as Fraction 1 (F1) [Baker et al. (1952) J. Immunol. 68:
131-145] which is only fully expressed at 37.degree. C. and encoded
on the 100 kb pFra plasmid [Protsenko et al. (1983) Genetika 19:
1081-1090]. This complex confers resistance to phagocytosis.
Detection of antibodies to F1 is the basis of standard serological
tests for the surveillance and diagnosis of plague as infected
animals and humans produce a strong humoral response to the antigen
[Shepherd et al. (1986) J. Clin. Microbiol. 24: 1075-1078; Williams
et al. (1982) Bull. World Health Organ 64: 745-752].
[0006] V antigen, postulated to act as a virulence factor, is a 37
kDa secreted protein which acts as a cytoplasmic regulator of Yops
(Yersinia outer membrane protein) expression. The V antigen is
encoded on a homologous 75 kb low-calcium response (LCR) plasmid
present in Y. pestis, Y. pseudotuberculosis and Y. enterocolitica.
This plasmid mediates the growth restriction of the organism
observed in vitro at 37.degree. C. in the presence of less than 2.5
mM Ca2.sup.+. Under such conditions the cells fail to synthesize
bulk vegetative proteins although a series of stress proteins and
virulence factors are expressed. The V antigen provides both active
and passive immunity against experimental infection with F1.sup.+
strains [Lawton et al. (1963) J. Immunol. 91: 179-184; Leary et al.
(1995) Infect. Immun. 63: 2854-2858; Nakajima et al. (1995) Infect.
Immun. 63: 3021-3029].
[0007] Vaccines which have focused on the capsule protein, F1,
expressed either as the purified protein [Baker et al. (1952) J.
Immunol. 68: 131-145; Simpson et al. (1990) Am. J. Trop. Med. Hyg.
43: 389-396] or in aroA mutant of Salmonella typhimurium [Oyston et
al. (1995) Infect. Immun. 63:563-568] protected mice against
virulent Y. pestis. However, Y. pestis strains lacking or deficient
in F1 were isolated from immunized animals after challenge with
F1.sup.+ organisms from wild rodents [Welkos et al. (1995) Contrib.
Microbiol. Immunol. 1 3:299-305] and from a fatal human case of
plague [Winter et al. (1960) Bull. WHO 23: 408-409]. Most
importantly, F1.sup.+ strains are virulent in mice [Worsham et al.
(1995) Contrib. Microbiol. Immunol. 13: 325-327] and nonhuman
primates [Friedlander et al. (1995) Clin. Infect. Dis. 21: (Suppl
2), S178-181] and we found in the present study that the human
plague vaccine does not protect against infection with F1.sup.-
organisms. This implies that, as originally suggested by Burrows
[Burrows, T. W. (1957) Nature 179: 1246-1247], an improved plague
vaccine protective against both F1.sup.- and F1.sup.+ strains of Y.
pestis must not rely solely on F1.
[0008] Therefore, there is a need to develop an improved vaccine
protective against both F1.sup.- and F1.sup.+ strains of Y. pestis
suitable for human administration.
SUMMARY
[0009] The present invention is directed to a vaccine that
satisfies this need. The vaccine of the present invention is
protective against both F1.sup.- and F1.sup.+ strains of Y. pestis.
The vaccine of the present invention is composed of a fusion
between a portion of the F1 protein and another protective
immunogen, the V antigen. This invention is novel because it is a
single constructed protein, F1-V, composed of two unique proteins,
the entire F1 capsule antigen and V antigen. It induces an
immunological response against both F1 protein and V antigen.
[0010] The invention was designed to be used in a vaccine affording
protection against plague, and to solve the problem of protecting
humans against both bubonic and pneumonic plague caused by
infection by the subcutaneous (insect bite) and aerosol routes,
respectively, with F1.sup.+ or F1.sup.- plague organisms, or with
strains which may vary in their V antigen.
[0011] The advantages of using this protein over the present whole
cell vaccine are as follows:
[0012] The current licensed vaccine does not protect mice against
subcutaneous challenge with F1.sup.- strains of Y. pestis, which
have been shown to cause fatal disease in both humans and
experimental animals infected by a peripheral, non-respiratory
route. The new F1-V vaccine does protect mice against bubonic
plague caused by subcutaneous challenge (insect bite) with F1.sup.-
organisms.
[0013] The current licensed vaccine does not protect mice against
pneumonic plague induced by aerosol challenge with F1.sup.- strains
of Y. pestis. The new F1-V vaccine does protect mice against
pneumonic plague caused by aerosol challenge with F1.sup.-
strains.
[0014] The current licensed vaccine does not protect mice against
pneumonic plague when challenged by the respiratory route with
F1.sup.+ strains of Y. pestis. The new F1-V vaccine does protect
mice against pneumonic plague caused by aerosol challenge with
F1.sup.+ strains.
[0015] The new F1-V vaccine is expected to protect humans against
pneumonic plague produced by strains of Y. pestis, either naturally
occurring or genetically engineered, which may be altered in their
content or composition of V antigen, but which still contain F1.
This is because the F1-V vaccine also contains F1. The current
licensed vaccine does not protect against pneumonic plague induced
by either F1.sup.- or F1.sup.+ organisms when given by the aerosol
route.
[0016] The new F1-V vaccine is composed of two antigens, both of
which have been shown to be protective. The combination of both
antigens should provide better protection against F1.sup.+ strains
than either F1 or V when used alone as vaccines. This is possible
because the immunity induced by F1 and by V occur by different
mechanisms which may be additive or synergistic.
[0017] Approximately 8% of humans immunized with the current
licensed human plague vaccine fail to develop an immune response to
F1 [Marshall et al. (1974) J. Inf. Dis. 1 29:S26-S29]. These
non-responders may well be at risk for development of plague. The
inclusion of two different protective antigens in the same vaccine
will help to eliminate the problem of non-responders to either of
the individual antigens and so increase the overall efficacy of
vaccination in a human population.
[0018] The new F1-V vaccine is composed of highly purified
recombinant proteins which are very well defined. This contrasts
with the present human licensed vaccine composed of whole bacteria
The nature of the protective immunogen(s) in the present licensed
vaccine is completely unknown. The present licensed vaccine is
known to contain and induce antibodies to F1 but it does not induce
antibodies to V antigen in mice, suggesting that V antigen is
absent.
[0019] The F1-V protein was constructed so that a single protein
could be purified as a vaccine component rather than having to
produce and purify F1 and V antigen separately. The purification of
a single protein as opposed to two separate proteins could result
in considerable savings when manufacturing a vaccine.
[0020] Therefore, it is an object of the present invention to
provide a Y. pestis DNA fragment encoding 1563 bp of a fusion
protein comprising the F1 protein fused at its carboxyl terminus to
the amino terminus of the entire V antigen useful in the production
of a diagnostic agent and a vaccine.
[0021] It is another object of the present invention to provide an
amino acid sequence for Y. pestis F1-V protein encoding 521 amino
acids.
[0022] It is another object of the invention to provide a
recombinant vector comprising a vector and the above described DNA
fragment for use as a DNA vaccine.
[0023] It is a further object of the present invention to provide a
host cell transformed with any of the above-described recombinant
DNA constructs for use as a live bacterial vaccine when the host
cell is a bacteria such as Salmonella, BCG, or a live viral vaccine
when the host cell is a virus such as adenovirus, or Venezuelan
Equine Encephalitis virus. These transformed cells, bacteria and
viruses can also be used as a source for the Y. pestis F1-V
protein.
[0024] It is another object of the present invention to provide a
method for producing Y. pestis F1-V fusion protein which comprises
culturing a host cell under conditions such that a recombinant
vector comprising a vector and the Y. pestis F1-V protein DNA
fragment is expressed and F1-V protein is thereby produced, and
isolating F1-V protein for use as a vaccine or a diagnostic
agent.
[0025] It is still another object of the invention to provide a
purified Y. pestis F1-V protein useful as a vaccine and a
diagnostic agent.
[0026] It is a further object of the present invention to provide
an antibody to the above-described F1-V protein for use as a
therapeutic agent and a diagnostic agent.
[0027] It is yet another object of the invention to provide a Y.
pestis vaccine comprising a F1-V protein effective for eliciting an
antigenic and immunogenic response resulting in the protection of a
mammal against Y. pestis infection by subcutaneous and aerosol
route.
[0028] It is yet another object of the present invention to provide
a method for the diagnosis of Y. pestis infection comprising the
steps of:
[0029] (i) contacting a sample from an individual suspected of
having the infection with antibodies which recognize F1-V protein;
and
[0030] (ii) detecting the presence or absence of a complex formed
between Y. pestis F1 and/or V antigen and antibodies specific
therefor.
[0031] It is a further object of the present invention to provide a
diagnostic kit comprising a F1-V protein antibody and ancillary
reagents suitable for use in detecting the presence Y. pestis in
mammalian sputum, serum, or tissues.
[0032] It is yet another object of the present invention to provide
a therapeutic method for the treatment or amelioration of symptoms
of infection by Y. pestis and other species of Yersinia such as Y.
enterocolitica and Y. pseudotuberculosis, said method comprising
providing to an individual in need of such treatment an effective
amount of sera from individuals immunized with F1-V protein in a
pharmaceutically acceptable excipient.
[0033] It is further another object of the present invention to
provide a therapeutic method for the treatment or amelioration of
symptoms of Yersinia infection, said method comprising providing to
an individual in need of such treatment an effective amount of
antibodies against F1-V protein of Yersinia pestis and all or a
portion of V antigen of Yersinia enterocolitica and Yersinia
pseudotuberculosis in a pharmaceutically acceptable excipient.
[0034] It is still another object of the present invention to
provide antigenic epitopes of F1-V protein, which are useful in
peptide vaccine design.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] 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:
[0036] FIG. 1 shows plasmid pF1V showing an in-frame insertion of
the F1 and V open reading frames at the NdeI and BamHI sites of
pEr19b separated by an inframe EcoRI site. F1-V was transcribed by
using the T7 promoter (arrow) within pET19b after induction of T7
polymerase by isopropyl-.beta.-D-thiogalactopyranoside (IPTG). The
F1-V amino acid sequence begins with a His-Tag and enterolinase
cleavage site derived from pFT19b before the methionine start codon
of F1. The F1 portion consists of 170 amino acids followed by two
amino acids, asparagine and glutamine (encoded by the BcoRI site)
and the entire sequence of the V antigen. F1-V, therefore, has 521
amino acids with a predicted molecular mass of 57,926 daltons.
[0037] FIG. 2 shows the gel electrophoresis of F1-V fusion protein.
The F1-V fusion protein expressed from pEr19b was isolated with 6M
urea as recommended (Novagen). Residual endotoxin was removed by
passing F1-V over an endotoxin removing gel column (Pierce,
Rockford, Ill.). After the column, F1-V contained 202 endotoxin
units per mg of protein by Limulus amoebocyte lysate assay (Sigma,
St. Louis, Mo.) (1 endotoxin unit=0.1 ng of E. coli 055:B5 LPS
standard). Recombinant F1 and V were cloned separately and purified
as described in Andrews et al. (1996) Inf. Immun. 64:2180-2187.
F1-V, V and F1 proteins (2 .mu.g each) were subjected to SDS-PAGE
on 10% tricine gels (Novex, San Diego, Calif.) and visualized by
Coomassie brilliant blue staining.
DESCRIPTION
[0038] In one embodiment, the present invention relates to a DNA or
cDNA segment which encodes Y. pestis recombinant F1-V protein
consisting of the F1 protein fused at its carboxyl terminus to the
amino terminus of the entire V antigen. The sequence of the 1563
nucleotide DNA segment is specified in SEQ ID NO: 1.
[0039] DNA or polynucleotide sequences to which the invention also
relates include fragments of F1 or V containing protective epitopes
[Motin et al. (1994) Infect Immun. 62:4192-4201].
[0040] The derived polynucleotide is not necessarily physically
derived from the nucleotide sequence shown in SEQ ID NO:1, but may
be generated in any manner, including for example, chemical
synthesis or DNA replication or reverse transcription or
transcription, which are based on the information provided by the
sequence of bases in the region(s) from which the polynucleotide is
derived. In addition, combinations of regions corresponding to that
of the designed sequence may be modified in ways known in the art
to be consistent with an intended use. The sequences of the present
invention can be used in diagnostic assays such as hybridization
assays and polymerase chain reaction assays for the detection of F1
or V sequences of Y. pestis.
[0041] In another embodiment, the present invention relates to a
recombinant DNA molecule that includes a vector and a DNA sequence
as described above. The vector can take the form of a plasmid such
as pET19b, pMBa/pPBac, pSSV1 or any broad host range expression
vector such as viral vectors such as adenovirus or Venezuelan
Equine Encephalitis virus and others known in the art.
[0042] In a further embodiment, the present invention relates to
host cells stably transformed or transfected with the above
described recombinant DNA constructs. The host cell can be
prokaryotic such as Bacillus or E. coli, or eukaryotic such as
Saccharomyces or Pichia, or mammalian cells or insect cells. The
vector containing the F1-V protein sequence is expressed in the
bacteria and the expressed product used for diagnostic procedures
or as a vaccine. Please see e g., Maniatis, Fitsch and Sambrook,
Molecular Cloning: A Laboratory Manual (1982) or DNA Cloning,
Volumes I and II (D. N. Glover ed. 1985) for general cloning
methods. The DNA sequence can be present in the vector operably
linked to a highly purified IgG molecule, an adjuvant, a carrier,
or an agent for aid in purification of F1-V protein. The
transformed or transfected host cells can be used as a source of
DNA sequences described above. When the recombinant molecule takes
the form of an expression system, the transformed or transfected
cells can be used as a source of the protein described below.
[0043] In another embodiment, the present invention relates to a
DNA sequence incorporated into a vector which can be used as a DNA
vaccine in animals, including humans, or which can be used in a
live bacterial or viral vaccine, e.g. Salmonella, BCG, adenovirus,
or Venezuelan Equine Encephalitis virus.
[0044] In another embodiment, the present invention relates to a Y.
pestis F1-V fusion protein having an amino acid sequence
corresponding to SEQ ID NO: 2 and encompassing 521 amino acids or
any allelic variation thereof.
[0045] A polypeptide or amino acid sequence derived from the amino
acid sequence in SEQ ID NO:2, refers to a polypeptide having an
amino acid sequence identical to that of a polypeptide encoded in
the sequence, or a portion thereof wherein the portion consists of
at least 2-5 amino acids, and more preferably at least 810 amino
acids, and even more preferably at least 11-15 amino acids, or
which is immunologically identifiable with a polypeptide ended in
the sequence. The present invention relates to a F1-V protein where
the F1 is fused by its carboxy-terminus to the amino-terminus of V
antigen constructed with a polyhistidine and enterokinase site and
where the F1 signal sequence is present. Also embodied in this
invention is a F1-V protein wherein the F1 is fused to the amino
terminus of V, with or without any of the polyhistidine,
enterokinase sites and the F1 signal sequence. In addition, a
linker of additional amino acids can be fused in frame between the
F1 and V antigen (or V antigen and F1) sequences or fragments
thereof for convenience as long as the changes do not affect the
immunological activity of the fusion protein.
[0046] A recombinant or derived polypeptide is not necessarily
translated from a designated nucleic acid sequence, or the sequence
in SEQ ID NO:1; it may be generated in any manner, including for
example, chemical synthesis, or expression of a recombinant
expression system. In addition the polypeptide can be fused to
other proteins or polypeptides for the pub of transport or for
secretion from the cell or for increasing the protective efficacy
in a vaccine. Some examples include the Yersinia outer proteins
(Yops) of Yersinia species or fragments thereof, or the
amino-terminal protective antigen binding domain of anthrax toxin
lethal and edema factors to name a few.
[0047] In a further embodiment, the present invention relates to a
method of producing F1-V protein which includes culturing the
above-described host cells, under conditions such that the DNA
fragment is expressed and F1-V is produced F1-V can then be
isolated using methodology well known in the art or by the
production method described below. F1-V protein can be used as a
vaccine for immunity against infection with the Y. pestis or as a
diagnostic tool for detection of Y. pestis infection. The
transformed host cells can be used to analyze the effectiveness of
drugs and agents which inhibit Y. pestis, such as host proteins or
chemically derived agents or other proteins which may interact with
the bacteria to down-regulate or alter the expression of F1 protein
or V antigen.
[0048] In another embodiment, the present invention relates to
antibodies specific for the above-described F1-V protein. For
instance, an antibody can be raised against the complete F1-V or
against a portion thereof. Persons with ordinary skill in the art
using standard methodology can raise monoclonal and polyclonal
antibodies to F1-V of the present invention, or a unique portion
thereof. Material and methods for producing antibodies are well
known in the art (see for example Goding, in, Monoclonal
Antibodies: Principles and Practice. Chapter 4, 1986). In addition,
the protein or polypeptide can be fused to or combined with other
proteins or polypeptides or adjuvants which increase its
antigenicity, thereby producing higher titers of neutralizing
antibody when used as a vaccine. Examples of such proteins or
polypeptides include cholera toxin B subunit and any adjuvants or
carriers safe for human use, such as aluminum hydroxide.
[0049] In a further embodiment, the present invention relates to a
method of detecting the presence of Y. pestis infection or
antibodies against Y. pestis in a sample. Using standard
methodology well known in the art, a diagnostic assay can be
constructed by coating on a surface (i.e. a solid support) for
example, a microtitration plate or a membrane (e.g. nitrocellulose
membrane), all or a unique portion of the F1-V protein described
above, and contacting it with the serum of a person suspected of
having plague. The presence of a resulting complex formed between
F1-V protein and antibodies specific for either F1 or V in the
serum can be detected by any of the known methods common in the
art, such as fluorescent antibody spectroscopy or colorimetry. This
method of detection can be used, for example, for the diagnosis of
bubonic and pneumonic plague.
[0050] Similarly, antibodies to F1-V protein can be used in a rapid
diagnostic assay to detect the presence of F1 and/or V antigen in
the serum of patients infected with Yersinia pestis. Such a test
may also be of value in the rapid diagnosis of infection in humans
or animals with other Yersinia species by detection of V antigen in
serum or tissue samples.
[0051] The ability of an individual to fight Y. pestis infection is
dependent on the individual's ability to produce antibodies against
Y. pestis. Diagnostic assays, similar to those described above,
designed to measure the production of protective antibodies against
F1-V can be used to measure an individual's response to receiving a
plague vaccine.
[0052] In another embodiment, the present invention relates to a
diagnostic kit which contains F1-V protein from Y. pestis and
ancillary reagents that are suitable for use in detecting the
presence of antibodies to Y. pestis, Y. enterocolitica and Y.
pseudotuberculosis in serum or a tissue sample. Tissue samples
contemplated can be rodents and human, or other mammals. Ancillary
reagents would include standard anti-rodent or anti-human
antibodies.
[0053] In another embodiment, the present invention relates to a
vaccine for protection against Y. pestis infections by aerosol or
subcutaneous route (insect bite). The vaccine comprises F1-V
protein, or an immunogenic portion thereof, from a specific strain
or species of Yersinia pestis. It could also contain V antigen from
Y. enterocolitica and Y. pseudotuberculosis. The vaccine can be
prepared by inducing expression of a recombinant expression vector
comprising F1-V protein sequence and purifying the resulting
protein. The purified F1-V protein is prepared for administration
to mammals by methods known in the art, which can include filtering
to sterilize the solution, diluting the solution, adding an
adjuvant and stabilizing the solution. The vaccine can be
lyophilized to produce a vaccine against Y. pestis in a dried form
for ease in transportation and storage. Further, the vaccine may be
prepared in the form of a mixed vaccine which contains the F1-V
protein described above and at least one other antigen as long as
the added antigen does not interfere with the effectiveness of the
vaccine and the side effects and adverse reactions are not
increased additively or synergistically.
[0054] The vaccine may be stored in a sealed vial, ampule or the
like. The present vaccine can generally be administered in the form
of a liquid or suspension. In the case where the vaccine is in a
dried form, the vaccine is dissolved or suspended in sterilized
distilled water before administration. Generally, the vaccine may
be administered orally, subcutaneously, intradermally or
intramuscularly but preferably in nasally in a dose effective for
the production of neutralizing antibody and protection from
infection or disease.
[0055] In another embodiment, the present invention relates to a
method of reducing Y. pestis infection symptoms in a patient with
bubonic or pneumonic plague by administering to said patient an
effective amount of F1-V protein antibodies including those made in
humans, either polyclonal or combinations of monoclonals to F1 and
V antigen, as described above. When providing a patient with F1-V
antibodies, the dosage administered will vary depending upon such
factors as the patient's age, weight, height, sex, general medical
condition, previous medical history, etc. In general, it is
desirable to provide the recipient with a dosage of the above
compounds which is in the range of from about 1 pg/kg to 500 mg/kg
(body weight of patient), although a lower or higher dosage may be
administered.
[0056] Described below are examples of the present invention which
are provided only for illustrative purposes, and not to limit the
scope of the present invention. In light of the present disclosure,
numerous embodiments within the scope of the claims will be
apparent to those of ordinary skill in the art
[0057] The following Materials and Methods were used in the
Examples below.
[0058] DNA methods All restriction enzymes used were purchased from
Life Technologies (Gaithersburg, Md.). Plasmid DNA samples were
purified using a Qiagen plasmid purification kit (Qiagen, Inc.,
Chatsworth, Calif.) All oligonucleotide primers were synthesized on
an Applied Biosystems model 391 DNA synthesizer (Foster City,
Calif.) and the polymerase chain reaction (PCR) was performed using
a DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, Conn.). DNA
sequencing was performed by the dideoxy-chain termination method
[Sanger et al. (1977) Proc. Natl. Acad. Sci. 74: 5463-54671 using
[.alpha.-.sup.35S]dATP (Amersham, Arlington Heights, Ill.). Genetic
manipulations were performed by standard procedures [Sambrook et
al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]
[0059] Analytical methods. Protein content was determined by the
bicinchoninic acid-Lowry method with BSA as a standard. (Pierce).
Purified F1-V was analyzed by sodium dodecyl sulfate
(SDS)-polyacrylainide gel electrophoresis (PAGE) on 10% tricine
gels (Novex, San Diego, Calif.) and visualized by Coomassie blue
R-250 (Bio-Rad Laboratories, Hercules, Calif.) staining.
EXAMPLE 1
[0060] Construction, Purification, and Characterization of
Recombinant F1-V Fusion Protein
[0061] To first isolate the F1 structural gene minus its stop
codon, plasmid pYPR1 (kindly provided by T. Schwan, Rocky Mountain
Laboratories, Hamilton, Mont.) containing the F1 operon [Simpson et
al. (1990) Am. J. Trop. Med. Hyg. 43:389-396] was used as template
DNA in a PCR using as the forward oligonucleotide primer,
GGCGCGGCATATGAAAAAAATCAGTTCC (SEQ ID NO:3), containing an internal
Nde I restriction site (underlined), and the reverse primer,
CTCGAATTCTTGGTTAGATACGGT (SEQ ID NO:4), containing an internal Eco
R1 site (underlined). The V antigen gene was then isolated by PCR
using plasmid DNA from a pigmentation negative derivative (Pgm) of
Y. pestis CO92, the forward oligonucleotide primer,
CGCGAATTCATGATTAGAGCCTACGAA (SEQ ID NO: 5), containing an internal
Eco RI site (underlined), and the reverse primer,
CGCGGATCCTCATTTACCAGACGTGTCA (SEQ ID NO.6) containing an internal
Bam HI site (underlined). The purified F1 PCR product was then
digested with Nde I and Eco RI while the V antigen purified PCR
product was digested with Eco RI and Bam HI. Both restricted
fragments were then ligated to the Nde I and Bam H1 digested
expression vector pET19b (Novagen, Madison, Wis.) and used to
transform Escherichia coli strain BLR (Novagen) to create plasmid
pF1V. The final protein contains an amino-terminal 10 histidines
and enterokinase site from pEr19b followed by the F1-V protein. The
F1 portion consists of 170 amino acids followed by two amino acids,
glutamic acid and phenylalanine (the Eco RI site) and the entire
sequence of the V antigen. F1-V, therefore, has 521 amino acids
with a predicted molecular mass of 57,926 daltons. The nucleotide
sequence of the F1-V portion of pFIV was verified by sequencing.
Two nucleotide differences were found between the V sequence
present in F1-V and that reported previously for the V antigen
[Price et al. (1989) J. Bacteriol. 171: 5646-5653]. A G was
replaced by an A at base 247 of the open reading frame, resulting
in a change from an alanine to a threonine. At base 324, a G was
replaced with a C with no change in the amino acid at that
site.
[0062] The F1-V fusion protein expressed from pEr19b was isolated
with 6 M urea as recommended (Novagen). Residual endotoxin was
removed by passing F1-V over an endotoxin removing gel column
(Pierce, Rockford, Ill.). After the column, F1-V contained 202
endotoxin units per mg of protein by Limulus amoebocyte lysate
assay (Sigma, St. 2 Louis, Mo.) (1 endotoxin unit=0.1 ng of E. coli
055:B5 LPS standard).
[0063] The recombinant F1-V fusion protein constructed in pET19b
consists of the F1 protein fused at its carboxyl terminus to the
amino terminus of the entire V antigen F1-V had a relative
molecular weight of 58,000 on SDS-PAGE (FIG. 2) which agreed with
its predicted size of 57,926. The F1-V protein reacted on Western
blot with both rabbit polyclonal antibody to F1 as well as mouse
monoclonal antibodies directed against V antigen (data not shown).
Recombinant F1 and V antigen are also shown in FIG. 2.
[0064] After establishing that F1-V had a relative molecular weight
consistent with its DNA coding sequence and contained both F1 and V
specific epitopes, we tested its ability to protect mice against
plague.
EXAMPLE 2
[0065] Animal Immunization and challenge with Y. pestis. Groups of
female 8-10 week old Swiss Webster (Hsd:ND4) mice (Harlan Sprague
Dawley, Indianapolis, Ind.) were immunized subcutaneously on days 0
and 28 with 0.2 ml of the F1-V, F1, or V antigen preparation
adsorbed to the aluminum hydroxide adjuvant, Alhydrogel (1.3%,
Superfos Biosector, Vedbaek, Denmark, 0.19 mg aluminum per dose),
the human whole-cell plague vaccine U. S. Pharmacopeia (USP) (Greer
Irbories, Lenoir, N.C.) or Alhydrogel alone as a control. Serum
obtained on day 58 after initial immunization was assayed for
anti-F1 and anti-V IgG antibody by standard EUSA on individual
animals and group geometric mean titers determined. Titers were
determined as the reciprocal of the maximum dilution giving an
absorbance greater than 0.1 units after subtraction of nonspecific
binding in normal serum.
[0066] The immunized animals were then challenged on day 78 by
either the subcutaneous or aerosol route with wild-type F1.sup.+Y.
pestis, CO92 (kindly provided by T. Quan, Center for Disease
Control, Ft Collins, Colo.) or C12, an F1.sup.- isogenic derivative
of CO92 with a deletion in the F1 structural gene [Worsham et al.
(1995) Contrib. Microbiol. Imunnol. 13: 325-327]. The inocula for
s.c. and aerosol challenge were prepared and the animals challenged
by s.c. and aerosol routes as previously described [Welkos et al.
(1995) Contrib. Microbiol. Immunol. 13: 299-305]. The s.c.
LD.sub.50 is 9.1 and 1.9 CFU for F1.sup.- C12 (37) and F1.sup.+
C092 (32) strains, respectively. The aerosol LD.sub.50 is
1.1.times.10.sup.5 and 2.times.10.sup.4 CFU (32) for F1.sup.- C12
and F1.sup.+ C092 strains, respectively.
[0067] All animal experiments were conduct in accordance with the
Guide for the Care and Use of Laboratory Animals (Committee on Care
and Use of Laboratory Animals of the Institute of Laboratory Animal
Resources, National Research Council. 1985. Guidelines for the care
and use of laboratory animals revised. National Institutes of
Health publication no 86-23. National Institutes of Health,
Bethesda, Md.) and animals were provided food and fresh water ad
libitum during the experiment
[0068] Efficacy of F1-V Against Bubonic Plague.
[0069] In two separate experiments (Table 1), mice immunized with
13.6 .mu.g of F1-V were protected (90-100% survival) against a
subcutaneous challenge with a moderate (57 LD.sub.50) or high
(1.1.times.10.sup.6LD.sub.50) dose of the F1.sup.- Y. pestis stain,
C12, while all control animals died Another group of animals
immunized with 27.2 .mu.g of F1-V all survived (100%) the high-dose
challenge. As expected, F1 alone, when adsorbed to Alhydrogel, did
not protect animals against challenge with the F1.sup.- C12 strain.
Animals given 10 .mu.g of V were afforded the same degree of
protection (90% survival) as with F1-V, against the high-dose
challenge. Thus, the protective efficacy of the F1-V fusion protein
against infection with an F1.sup.- Y. pestis strain was equivalent
at this challenge dose, to that provided by V alone. In marked
contrast, in a separate experiment, the current human, whole-cell
plague vaccine USP, failed to protect against a low dose challenge;
none of nine challenged animals survived. TABLE-US-00001 TABLE 1
Efficacy of F1-V vaccination against a lethal subcutaneous Y.
pestis infection of mice. Treatment Group Strain Challenge
dose.sup.a Survivors/total Alhydrogel alone F1.sup.- C12 57 0/10
13.6 .mu.g F1-V '' 57 10/10 10 .mu.g F1 '' 60 0/10 Plague USP.sup.b
'' 124 0/9 Alhydrogel alone '' 1.1 .times. 10.sup.6 0/10 13.6 .mu.g
F1-V '' 1.1 .times. 10.sup.6 9/10 27.2 .mu.g F1-V '' 1.1 .times.
10.sup.6 10/10 10 .mu.g V '' 1.1 .times. 10.sup.6 9/10 .sup.aNumber
of LD.sub.50 .sup.bThe licensed, human, whole-cell plague
vaccine.
[0070] Efficacy of F1-V Against Pneumonic Plague.
[0071] We next determined the efficacy of F1-V against pneumonic
plague induced by an aerool challenge (Table 2). Mice immunized
with 13.6 .mu.g of F1-V were completely protected (100% survival)
against a moderate (91 LD.sub.50) or high (590 LD.sub.50) aerosol
dose of the F1.sup.- Y. pestis strain, C12 Animals given 27.2 .mu.g
of F1-V were also completely protected against the high-dose
challenge. Similarly, V protected animals exposed to the high-dose
aerosol challenge, with 80% of animals surviving. However, as with
the s.c. challenge, the plague vaccine USP failed to project a s
fatal pneumonic plague; none of eight challenged animals survived.
Thus, the whole-cell plague vaccine USP failed to protect mice
against challenge with the F1.sup.- strain by either the s.c. or
aerosol route, while it does protect against s.c. challenge
[Andrews et al. (1996) Infect. Immun. 64:2180-2187; Simpson et al.
(1990) Am. J. Trop. Med. Hyg. 43:389-396] and partially protects
with prolongation of time to death against aerosol challenge with
F1 strains [Andrews et al., ibid.; Pitt et al. (1994) Abstr. E-45.
In: Abstracts of the 94th General Meeting of the American Society
for Microbiology 1994. American Society for Microbiology,
Washington, D.C.; Smith and Packman (1966) Brit. J. Exp. Path 47:
25-34]. This differential protection against F1.sup.+ and F1.sup.-
strains and the absence of an immune response to V antigen in the
plague vaccine USP group (Table 2) in agreement with other studies
[Chen et al. (1961) J. Immunol. 87:64-71;Williamson et al. (1995)
FEMS Immunol. Med. Macrobiol. 12:-223-230], strongly suggest that
the major protective immunogen in the plague vaccine USP is F1 and
that V antigen and other possible immunogens are absent.
[0072] The F1-V also protected (100% survival) against a high-ose
aerosol challenge with the F1.sup.+ Y. pestis, CO92 strain isolated
from a fatal human pneumonic case. While these studies were in
progress, a report described the increased effectiveness of
co-immunization with F1 and V antigen in protecting against
subcutaneous challenge with an F1.sup.+ strain [Williamson et al.,
ibid.]. No studies were performed with F1 strains or against an
aerosol challenge. TABLE-US-00002 TABLE 2 Efficacy of F1-V
vaccination against a lethal aerosol Y. pestis infection of mice
Geometric mean Treatment Challenge Survivors/ antibody titer.sup.b
Group Strain dose.sup.a Total F1 V Alhydrogel F1.sup.- C12 91 0/9
.sup. NT.sup.c NT alone 13.6 .mu.g F1-V '' 91 10/10 NT NT
Alhydrogel '' 590 0/14 <640 <640 alone 13.6 .mu.g F1-V '' 590
10/10 66,540 432,376 27.2 .mu.g F1-V '' 590 10/10 108,094 432,376
10 .mu.g V '' 590 8/10 NT 655,360 Plague USP.sup.d '' 590 0/8
55,738 <640 Alhydrogel F1.sup.+ CO92 761 1/10 NT NT alone 13.6
.mu.g F1-V '' 761 10/10 NT NT .sup.aNumber of LD.sub.50 .sup.bSerum
obtained on day 58 after the initial vaccine dose was assayed for
anti-F1 and anti-V IgG antibody by ELISA on individual animals and
group geometric mean titers determined as described in Materials
and Methods. .sup.cNot tested. .sup.dThe licensed, human,
whole-cell plague vaccine.
[0073] Our results clearly demonstrate that nice immunized with a
fusion protein, consisting of the F1 capsular antigen fused at its
carboxyl terminus to the amino terminus of the V antigen of Y.
pestis, were provided with excellent protection against both
parental and aerosol challenge with an F1.sup.- Y. pestis strain.
The F1-V fusion protein also protected mice against an aerosol
challenge with an F1.sup.+ Y. pestis strain. Other workers, as
noted previously, showed that V antigen protects animals against
challenge with virulent F1.sup.+ strains, results which we have
confirmed and extended to aerosol challenge. This raises the issue
of whether the F1 portion of F1-V is immunogenic and contributes to
the protection against challenge with the F1.sup.+ Y. pestis strain
CONE. Several lines of evidence suggest that the F1 portion is
immunogenic and protective. The development of high levels of
antibody to F1 after immunization with F1 -V (Table 2) and the
numerous reported studies showing a strong correlation between the
level of antibody to F1 and protection against infection [Williams
and Cavanaugh (1979) Bull. WHO 57-309-313) suggest the F1 portion
of F1-V helps protect against challenge with F1.sup.+ Y. pestis.
Further support for a protective role for the F1 portion of F1-V
was provided by our observations with a smaller F1-V fusion protein
we constructed that consisted of the entire F1 gene fused at its
carboxyl terminus to amino acids 168 to 275 of V antigen. This
fusion protein was unable to effectively immunize mice against a
subcutaneous challenge with the F1.sup.- Y. pestis C12 strain (3
survivors out of 10 mice challenged with 55 LD.sub.50, data not
shown), showing that the V segment of the fusion protein was not
protective. However, this same fusion protein was able to protect
mice against the F1.sup.+ Y. pestis CO92 strain (10 survivors out
of 10 mice challenged subcutaneously with63 LD.sub.50, data not
shown). These results indicate the F1 portion of this fusion
protein was immunogenic, enabling mice to survive challenge against
infection with the F1.sup.+ Y. pestis CO92, while the V segment
failed to elicit a significant protective immune response against
challenge with the V antigen expressing but F1.sup.- Y. pestis C12
strain. The value of a combined immune response to both F1 and V
for combating infection with F1.sup.+ Y. pestis strains is
supported by the studies of Burrows and Bacon [Burrows, T. W.
(1963) Ergeb. Mikrobiol. Immunitatsforch. Exp. Ther. 37:59-113;
Burrows and Bacon (1958) Brit. J. Exp. Pathol. 39: 278-291] who
found that serum from rabbits immunized with attenuated strains of
Y. pestis expressing both F1 and V antigen provided better passive
protection in mice against wild type F1.sup.+ Y. pestis than serum
from rabbits immunized with attenuated Y. pestis strains expressing
only V antigen. It is also supported by the recent study showing
increased protection by co-immunization of F1 with V antigen
[Williamson et al., ibid.]. Beter protection by antibody directed
against both F1 and V might occur by counteracting both the
anti-phagocytic activity associated with F1 [Burrows, T. W., ibid.]
and the virulence-enhancing activity associated with secreted V
antigen [Nakajima et al. (1995) Infect. Immun. 63: 3021-3029].
[0074] An additional advantage of a vaccine containing both F1 and
V is that F1 should protect against variant strains of Y. pestis
which might be altered in the amount or composition of V antigen,
in a manner analogous to that by which V protects against F1.sup.-
strains. Indeed, isolates deficient in V, determined
immunologically, have been cultured from immunized animals infected
with V-containing, wild-type Y. pestis [Williams et al. (1974)
Trans. Roy. Soc. Trop. Med. Hyg. 68:1711. Furthermore, variability
in the structural gene for V has been described for Y.
pseudotuberculosis [Motin et al. (1994) Infect. Immun.
62:4192-4201], although to date, this has not been reported for Y.
pestis.
[0075] Another potential advantage of an F1-V multicomponent
vaccine would be to protect individuals who may be non-responders
to one component of a vaccine. Indeed, some recipients of the
current plague vaccine USP fail to develop an antibody response to
F1 [Marshall et al. (1974) J. Infect. Dis. 129:S26-S29]. A similar
approach to the development of recombinant multicomponent vaccines
may be of value against other infectious diseases.
[0076] In summary, we constructed an F1-V fusion protein to provide
optimal protective immunity against pneumonic as well as bubonic
plague due to either wild-type F1.sup.+ Y. pestis or fully virulent
F1.sup.- Y. pestis strains which may occur naturally or may develop
after infection of vaccinated individuals. The new vaccine was
shown to be effective against both F1.sup.- as well as F1.sup.+ Y.
pestis strains. Most importantly, it prevented both fatal pneumonic
as well as bubonic infection. This vaccine candidate may lead to
the development of an improved human plague vaccine.
Sequence CWU 0
0
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 6 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 1566 bp (B) TYPE: Nucleic acid (C)
STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) SEQUENCE
DESCRIPTION: SEQ ID NO:1: ATGGGCCATC ATCATCATCA TCATCATCAT
CATCACAGCA 40 GCGGCCATAT CGACGACGAC GACAAGCATA TGAAAAAAAT 80
CAGTTCCGTT ATCGCCATTG CATTATTTGG AACTATTGCA 120 ACTGCTAATG
CGGCAGATTT AACTGCAAGC ACCACTGCAA 160 CGGCAACTCT TGTTGAACCA
GCCCGCATCA CTCTTACATA 200 TAAGGAAGGC GCTCCAATTA CAATTATGGA
CAATGGAAAC 240 ATCGATACAG AATTACTTGT TGGTACGCTT ACTCTTGGCG 280
GCTATAAAAC AGGAACCACT AGCACATCTG TTAACTTTAC 320 AGATGCCGCG
GGTGATCCCA TGTACTTAAC ATTTACTTCT 360 CAGGATGGAA ATAACCACCA
ATTCACTACA AAAGTGATTG 400 GCAAGGATTC TAGAGATTTT GATATCTCTC
CTAAGGTAAA 440 CGGTGAGAAC CTTGTGGGGG ATGACGTCGT CTTGGCTACG 480
GGCAGCCAGG ATTTCTTTGT TCGCTCAATT GGTTCCAAAG 520 GCGGTAAACT
TGCAGCAGGT AAATACACTG ATGCTGTAAC 560 CGTAACCGTA TCTAACCAAG
AATTCATGAT TAGAGCCTAC 600 GAACAAAACC CACAACATTT TATTGAGGAT
CTAGAAAAAG 640 TTAGGGTGGA ACAACTTACT GGTCATGGTT CTTCAGTTTT 680
AGAAGAATTG GTTCAGTTAG TCAAAGATAA AAATATAGAT 720 ATTTCCATTA
AATATGATCC CAGAAAAGAT TCGGAGGTTT 760 TTGCCAATAG AGTAATTACT
GATGATATCG AATTGCTCAA 800 GAAAATCCTA GCTTATTTTC TACCCGAGGA
TACCATTCTT 840 AAAGGCGGTC ATTATGACAA CCAACTGCAA AATGGCATCA 880
AGCGAGTAAA AGAGTTCCTT GAATCATCGC CGAATACACA 920 ATGGGAATTG
CGGGCGTTCA TGGCAGTAAT GCATTTCTCT 960 TTAACCGCCG ATCGTATCGA
TGATGATATT TTGAAAGTGA 1000 TTGTTGATTC AATGAATCAT CATGGTGATG
CCCGTAGCAA 1040 GTTGCGTGAA GAATTAGCTG AGCTTACCGC CGAATTAAAG 1080
ATTTATTCAG TTATTCAAGC CGAAATTAAT AAGCATCTGT 1120 CTAGTAGTGG
CACCATAAAT ATCCATGATA AATCCATTAA 1160 TCTCATGGAT AAAAATTTAT
ATGGTTATAC AGATGAAGAG 1200 ATTTTTAAAG CCAGCGCAGA GTACAAAATT
CTCGAGAAAA 1240 TGCCTCAAAC CACCATTCAG GTGGATGGGA GCGAGAAAAA 1280
AATAGTCTCG ATAAAGGACT TTCTTGGAAG TGAGAATAAA 1320 AGAACCGGGG
CGTTGGGTAA TCTGAAAAAC TCATACTCTT 1360 ATAATAAAGA TAATAATGAA
TTATCTCACT TTGCCACCAC 1400 CTGCTCGGAT AAGTCCAGGC CGCTCAACGA
CTTGGTTAGC 1440 CAAAAAACAA CTCAGCTGTC TGATATTACA TCACGTTTTA 1480
ATTCAGCTAT TGAAGCACTG AACCGTTTCA TTCAGAAATA 1520 TGATTCAGTG
ATGCAACGTC TGCTAGATGA CACGTCTGGT 1560 AAATGA 1566 (2) INFORMATION
FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 521 (B)
TYPE:Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii)
SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Gly His His His His His His
His His His His Ser Ser Gly 1 5 10 15 His Ile Asp Asp Asp Asp Lys
His Met Lys Lys Ile Ser Ser Val 20 25 30 Ile Ala Ile Ala Leu Phe
Gly Thr Ile Ala Thr Ala Asn Ala Ala 35 40 45 Asp Leu Thr Ala Ser
Thr Thr Ala Thr Ala Thr Leu Val Glu Pro 50 55 60 Ala Arg Ile Thr
Leu Thr Tyr Lys Glu Gly Ala Pro Ile Thr Ile 65 70 75 Met Asp Asn
Gly Asn Ile Asp Thr Glu Leu Leu Val Gly Thr Leu 80 85 90 Thr Leu
Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn 95 100 105 Phe
Thr Asp Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser 110 115 120
Gln Asp Gly Asn Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys 125 130
135 Asp Ser Arg Asp Phe Asp Ile Ser Pro Lys Val Asn Gly Glu Asn 140
145 150 Leu Val Gly Asp Asp Val Val Leu Ala Thr Gly Ser Gln Asp Phe
155 160 165 Phe Val Arg Ser Ile Gly Ser Lys Gly Gly Lys Leu Ala Ala
Gly 170 175 180 Lys Tyr Thr Asp Ala Val Thr Val Thr Val Ser Asn Gln
Glu Phe 185 190 195 Met Ile Arg Ala Tyr Glu Gln Asn Pro Gln His Phe
Ile Glu Asp 200 205 210 Leu Glu Lys Val Arg Val Glu Gln Leu Thr Gly
His Gly Ser Ser 215 220 225 Val Leu Glu Glu Leu Val Gln Leu Val Lys
Asp Lys Asn Ile Asp 230 235 240 Ile Ser Ile Lys Tyr Asp Pro Arg Lys
Asp Ser Glu Val Phe Ala 245 250 255 Asn Arg Val Ile Thr Asp Asp Ile
Glu Leu Leu Lys Lys Ile Leu 260 265 270 Ala Tyr Phe Leu Pro Glu Asp
Thr Ile Leu Lys Gly Gly His Tyr 275 280 285 Asp Asn Gln Leu Gln Asn
Gly Ile Lys Arg Val Lys Glu Phe Leu 290 295 300 Glu Ser Ser Pro Asn
Thr Gln Trp Glu Leu Arg Ala Phe Met Ala 305 310 315 Val Met His Phe
Ser Leu Thr Ala Asp Arg Ile Asp Asp Asp Ile 320 325 330 Leu Lys Val
Ile Val Asp Ser Met Asn His His Gly Asp Ala Arg 335 340 345 Ser Lys
Leu Arg Glu Glu Leu Ala Glu Leu Thr Ala Glu Leu Lys 350 355 360 Ile
Tyr Ser Val Ile Gln Ala Glu Ile Asn Lys His Leu Ser Ser 365 370 375
Ser Gly Thr Ile Asn Ile His Asp Lys Ser Ile Asn Leu Met Asp 380 385
390 Lys Asn Leu Tyr Gly Tyr Thr Asp Glu Glu Ile Phe Lys Ala Ser 395
400 405 Ala Glu Tyr Lys Ile Leu Glu Lys Met Pro Gln Thr Thr Ile Gln
410 415 420 Val Asp Gly Ser Glu Lys Lys Ile Val Ser Ile Lys Asp Phe
Leu 425 430 435 Gly Ser Glu Asn Lys Arg Thr Gly Ala Leu Gly Asn Leu
Lys Asn 440 445 450 Ser Tyr Ser Tyr Asn Lys Asp Asn Asn Glu Leu Ser
His Phe Ala 455 460 465 Thr Thr Cys Ser Asp Lys Ser Arg Pro Leu Asn
Asp Leu Val Ser 470 475 480 Gln Lys Thr Thr Gln Leu Ser Asp Ile Thr
Ser Arg Phe Asn Ser 485 490 495 Ala Ile Glu Ala Leu Asn Arg Phe Ile
Gln Lys Tyr Asp Ser Val 500 505 510 Met Gln Arg Leu Leu Asp Asp Thr
Ser Gly Lys 515 520 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 28 bp (B) TYPE: Nucleic acid (C)
STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) SEQUENCE
DESCRIPTION: SEQ ID NO:3: GGCGCGGCAT ATGAAAAAAA TCAGTTCC 28 (2)
INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 24 bp (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D)
TOPOLOGY: Linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO:4: CTCGAATTCT
TGGTTAGATA CGGT 24 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 27 bp (B) TYPE: Nucleic acid (C)
STRANDEDNESS: Single
(D) TOPOLOGY: Linear (ii) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CGCGAATTCA TGATTAGAGC CTACGAA 27 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 bp (B) TYPE: Nucleic
acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) SEQUENCE
DESCRIPTION: SEQ ID NO:6: CGCGGATCCT CATTTACCAG ACGTGTCA 28
* * * * *