Note: Descriptions are shown in the official language in which they were submitted.
WOg3/2~21 PCT/US~3/~365
2135201
Uni~er~al Coronavirus Vaccine
cros9 reference to related application~
This application is a continuation-in-part
application of U.S. application serial number Q7/882,171,
filed May 8, 1992, panding, which is a continuation-in-part
of U.S. application serial number 07/698,927, filed May 13,
1991, which is a continuation-in-part of U.S. application
serial number 07/613,066, filed November 14, 1990, each of
which is incorporated herein by reference.
`~ 10 Field o~ the invention
The present invention relates to a universal vaccine
useful to protect different species of animals against
infection by different host-specific coronaviruses.
~;Background of the invention
~- 15Coronaviruses are a family of host-specific
enveloped RNA viruses with a single-stranded positive sense
,,
genome. Examples of coronaviruses include, but are not
limited to: feline infectious peritonitis (FIPV) and feline
~enteric coronavirus ~FECV) which are specific to felines;
-~ 20 canine coronavirus (CCV) which is specific to canines;
~-~transmissible gastroenteritis coronavirus (TGEV) which is
specific to swine; bovine coronavirus (BCV) which is specific
to bovine species; human coronavirus which is specific to
humans; mouse hepatitis virus (MHV) which is specific to
murine species; and infectious bronchitis virus (IBV) which
is~ specific to avian species. These host-specific
coronaviruses cannot cross infect different species of
animals. Viral infection of the host by a coronavirus can
cause symptoms ranging from mild enteritis to severe
debilating disease to, in some cases, death.
Coronaviruses share common structural features
including a spike or S protein (also referred to as a peplomer
protein). The S protein is a glycoprotein which protrudes
.
WO93~2~21 ; PCT/US93/0436~
2135201
- ?. - '
from the surface of the virus particle. The S protein
mediates the binding of virions to the host cell receptor and
is involved in membrane fusion. In addition, it is the target
of virus neutralizing antibodies.
S proteins contain an N-terminal signal sequence,
a C-terminal transmembrane segment and potential N-linked
glycosylation sites. Comparison of different coronavirus S
proteins show little homology, i.e. similarity, at the N
terminus and highly conserved amino acid sequences at the C
terminus. Because the tissue tropism and disease
symptomatology is quite varied among this virus family, it is
speculated that the pathogenesis of coronaviruses is
- determined by the sequences encoded at the N-terminus while
the more conserved C-terminus encodes critical structural
features common to all coronaviruses. The carboxy terminus
of the S protein is believed to be involved in fusion~
The structure of the S protein has been studied.
~ Cavanagh (1983) J. Gen. Virol. 64:2577-2583, which is
¦~ ~ incorporated herein by reference, proposed a model for the
coronavirus spike in which the C-terminal half of the protein
forms its stalk and the N-terminal half, its bulbous protein.
deGroot et al., (1987) J . Mol . Biol . 197 :, which is
incorporated herein by reference, have postulated a model in
which a coiled-coil structure forms the connection between the
globular part of the S protein and the viral membrane. This
- model is based on the occurrence of heptad repeats, i.e., a
periodicity (a-b-c-d-e-f-g) in which the amino acids are
hydrophobic. Britton (1991)` Nature 353:394, which is
incorporated herein by reference, reported the presence of a
leucine zipper motif at the carboxyl end of the S glycoprotein
of coronaviruses for which the spike sequence is available:
TGEV FS772/70 (amino acids 1342-1377), FIPV WSU 1146 (amino
acids 1345-1380), MHV A59 (amino acids 1217-1252), human
coronavirus 229E (amino acids 1067-1102), BCV Mebus (amino
acids 1266-1294), and infectious bronchitis virus Beaudette
(amino acids 1059-1079). The leucine zipper motif terminates
: .
:
K' :`
WO93/2~21 2 1 3 5 2 0 1 PCT/US93/~36~
ten residues upstream of the consPrYed KWP motif preceding the
transmembrane domain.
Efforts have been made to develop vaccines against
various host-specific coronaviruses. Attempts have been made
with varying success to develop attenuated live virus
vaccines, inactivated vaccines, subunit vaccines and
recombinant nucleic acid based vaccines. In each case, the
vaccine developed did not cross-protect other host animals.
Vaccines currently available for protection against
coronavirus are specific for protection against a given member
of the coronavirus family. Such vaccines do not provide cross
protection to protect a host against other members of the
coronavirus family which are able to infect the species.
Furthermore, such vaccines do not cross protect other animals
against coronaviruses for which they are susceptible to
infection.
There is a need for a vaccine which can protect
against coronavirus infection. In particular, there is a need
for a vaccine which can be useful to protect a host species
against different coronaviruses and there is a need for a
vaccine which can be useful to protect different host species
against different coronaviruses.
~ummary of the invention
The present invention relates to a polypeptide
comprising an amino acid sequence from the C terminal portion
of a coronavirus S protein which has been found to be highly
conserved among coronaviruses and which is capable of
eliciting a protective immune response. This sequence is
re~erred to as a universal conserved domain. The polypeptides
of the present invention have less than a complete amino acid
sequence of an S protein.
The present invention relates to a vaccine
comprising a polypeptide which includes an universal conserved
domain and which has less than a complete amino acid sequence
of an S protein.
r ~ ~
WO93/2~1 2 1 3 5 2 0 1 ` PCT/US93/0436
_ 4 _
The present invention relates to an isolated nucleic
acid molecule having a nucleic acid sequence which encodes a
polypeptide that includes a universal conserved domain
polypeptide and that has less than a complete amino acid
sequence of an S protein.
The present invention relates to a vaccine
comprising a nucleic acid molecule that encodes a polypeptide
which includes an universal conserved domain and which has
less than a complete amino acid sequence of an S protein.
The present invention relates to a method of
protecting an animal from infection by a coronavirus
comprising administering an amount of a polypeptide effective
_ to elicit a protective immune response. The polypeptide
administered in the method comprises a universal conserved
domain and has less than a complete amino acid sequence of an
S protein.
The present invention relates to a method of
protecting an animal from infection by a coronavirus
comprising administering an amount of a nucleic acid molecule
which encodes a polypeptide effective to elicit a protective
; immune response. The polypeptide encoded by the nucleic acid
molecule administered in the method comprises a universal
conserved domain and has less than a complete amino acid
se~uence of an S protein.
-~ 25 Detailed description of the invention
According to the present invention, a highly
conserved region of the spike protein has been identified
which, when presented as a vaccine component or product, is
useful as a universal immunogen to protect an animal against
coronavirus infection. The vaccine of the present invention
may be used to vaccinate any animal susceptible to infection
by virus that is a member of the coronavirus family.
Accordingly, the present invention provides vaccines which can
be produced in a single manufacturing process and administered
to different species of animals. The cross-protection
afforded by vaccines of the present invention eliminates the
:
W093/2~21 2 1 3 5 2 0 1 - PCT/US93/~365
need to produce different vaccines to protect animals against
different members of the coronavirus family.
As used herein, the term "polypeptide" is meant to
refer to a peptide, polypeptide or protein molecule; a
molecule which includes a peptide, polypeptide or protein
molecule; or a molecule that contains amino acid residues
which are linked by non-peptide bonds.
As used herein, the term "universal conserved
domain" ~"UCD") is meant to refer to the identical 124 amino
acid segment found in the C terminal portion of S proteins
from TGEV, CCV and strains of feline coronaviruses. In
addition, the term ''UCD'I is meant to refer to the
corresponding amino acid segments of other coronavirus which
have different but homologous amino acid sequences. Such
corresponding sequences may be identified by their location
in the S protein, i.e. downstream of the bulbous N-terminal
region and upstream of the transmembrane region and the high
level of amino acid sequence similarity to the 12~ amino acid
sequence described abo~e. Furthermore, the term "UCD" is
additionally meant to refer to consensus sequences are
generated by comparing corresponding sequences and determining
the statistically average amino acid residue at a given
position in the sequence. Thus, when several different
sequences are compared, the most common residue at a given
position is assigned to that position in a consensus sequence.
The conservation of UCD sequences suggests that they
play a major role in virus structure and/or replication. The
region of perfect homology decreases in size as other
coronavirus S genes are included in the comparison. For
example, bovine and human coronavirus are more closely aligned
to the feline, canine and porcine coronavirus S genes in this
conserved region than are sequences from the murine and avian
coronaviruses.
Table 1 contains a comparison of corresponding amino
acid sequences from the C terminal portion of various
coronaviruses. SEQ ID NO:1 is an amino acid sequence from
FIPV strain Wsue2 (Virulent, Type II; Genbank accession number
WO93/~21 213 5 2 0 ~ PCT/US93/~36~
X06170). SEQ ID NO:2 is an amino acid sequence from F~PV
strain Df2e2 (Virulent, Type II). SEQ ID N0:3 is an amino
acid sequence from FIPV strain Tse2 (Temperature sensitive
mutant of Df2). SEQ ID NO:4 is an amino acid sequence from
FECV strain Fecve2 (Avirulent strain 1683). SEQ ID NO:5 is
an amino acid sequence from TGEV strain Tgeve2 (Purdue strain;
Genbank accession number D00118). SEQ ID NO:6 is an amino
acid sequence from FIPV strain Tgeve2f2 (Miller strain;
Genbank accession number M56002). SEQ ID N0:7 is an amino
acid sequence from BCV strain Bcve2 (Genbank accession number
M30613). SEQ ID N0:8 is an amino acid sequence from HCV
strain Hcve2 (Genbank accession number X16816). SEQ ID NO:9
_ is an amino acid sequence from IBV strain Ibbspi (Genbank
accession number X16816). SEQ ID N0:10 is an amino acid
sequence from MHV strain Mhve2a59 (Genbank accession number
X51939 SEQ ID NO:ll is an amino acid sequence from FIPV strain
Mhvs (Genbank accession number X04797). SEQ ID NO:12 is a
consensus sequence which has been designed to provide an
optimum UCD amino acid sequence.
The 124 residue amino acid sequence which is
completely conserved in TGEV, CCV and feline coronaviruses is
shown in SEQ ID N0:1l SEQ ID N0:2, SEQ ID N0:3, SEQ ID NO:4
and SEQ ID NO:5 from residue 37 to residue 160. The consensus
sequence, SEQ ID N0:12, also contains this 124 amino acid
sequence in its entirety from residue 37 to residue 160. This
124 amino acid sequence is currently a preferred UCD sequence
of the present invention. The entire 199 amino acid consensus
sequence is a preferred UCD-containing peptide.
Using amino acid sequence information from any
coronavirus, one having ordinary skill in the art can identify
the conserved region corresponding to the 124 amino acid
sequence found in TGEV, CCV and feline coronaviruses. As i;
exemplified in Table 1, the amino acid sequences from the C
terminal portion of coronaviruses can be compared to identify
the sequence which corresponds to the UCD from TGEV, CCV and
feline coronaviruses. The procedure is straightforward and
W093/2~21 ~- 2 1 3 S 2 0 1 j PCT/US93/~36~
can be performed to provide additional UCD sequences and
flanking sequences.
Corresponding conserved regions from coronaviruses
other than CC~, TGEV and feline coronaviruses may be
identified ~y their location on the S protein and the high
level of sequence homology the possess when compared to the
124 amino acid sequence referred to above. An example of such
comparison and ide~tification is shown in Table 1 in which
sequences from the C terminal regions of various S proteins
upstream from the transmembrane region are compared and
homologous sequences identified. Widely available computer
programs such as PLOTSIMILARITY software (Genetics Computer
Group, Madison WI) may be employed to locate a UCD in a
coronavirus.
In addition, such software may be employed to
expedite the generation of consensus sequences. This software
relies on the principles originally set out by Wilbur and
Lipman and later refined by Smith and Waterman and by
Needleman and Wunsch. Using these well known guidelines,
having ordinary skill in the art may compare sequences and
arrive at the statistically average or most common residue
occupying a given position. The PLOTSIMILARITY software
automates this function. Consensus sequences are thus
generated. In addition to the consensus sequence provided as
SEQ ID NO:12, a different consensus sequence derived from a
comparison of corresponding sequences is disclosed in the co-
owned, co-pending patent application: which is filed on the
same day as the present application; which is entitled
"Compositions and Methods for Vaccinating Coronaviruses";
, 30 which names the same inventors as the present application
(Miller, Timothy ~; Jones, Elaine V.; Reed, Albert P.; and
Klepfer, Sharon R); which has been designated docket number
H85009-1 by Applicants; and which is incorporated herein by
reference.
Accordingly, the present invention relates to
polypeptides which comprise a UCD or a fragment or a
derivative thereof. That is, the present invention relates
~ :
WO93/2~21 2~ ~ 5 2 01 . PCT/US93/~365
to polypeptides which comprise: the lZ4 amino acid sequence
form TGEV, CCV and feline coronaviruses; or the different
amino acid sequences from other coronaviruses which correspond
to the 124 amino acid sequence; or a consensus sequence
generated from comparison of correspondiny regions; or
immunogenic fragments or immunogenic derivatives thereof.
Polypeptides according to the present may further
comprise additional flanking sequences from coronavirus or
flanking sequences designed as a consensus sequence of the
flanking sequences of corresponding regions from different
coronaviruses.
As used herein, the term "immunogenic fragment" is
meant to refer to polypeptides which include an incomplete UCD
which is capable of eliciting a protective immune response
against coronavirus in an animal susceptible to coronavirus
infection. Immunogenic fragments may comprise a sequence
having nine or more amino acids from a UCD, and may include
additional amino acid sequences.
As used herein, the term "immunogenic derivatives"
is meant to refer to molecules which have a UCD or portions
thereof with conservative amino acid substitutions and which
are capable of eliciting a protective immune response against
a coronavirus in an animal susceptible to coronavirus
infecti~n. Those having ordinary skill in the art can readily
design derivatives having UCD sequences with conservative
substitutions for amino acids. For example, following what
~ are referred to as Dayhof's rules for amino acid substitution
-~ (Dayhof, M.D. (1978) Nat. Biomed. Res. Found., Washington,
D.C. Vol. 5, supp. 3), amino acid residues in a peptide
sequence may be substituted with comparable amino acid
residues. Such substitutions are well known and are based the
upon charge and structural characteristics of each amino acid.
Using standard procedures and readily available
starting materials, one having ordinary skill in the art can
determine whether a fragment and derivative is an immunogenic
fragment or an immunogenic derivative, respectively. Briefly,
polypeptides can be produced by standard methodologies and
W093~2~21 2 1 3 5 2 0 1 ` PCT/US93/~365
_ g
tested to determine whether they are capable of eliciting a
protective immune response. Sera from vaccinated animals can
be analyzed to detect the pre6ence of antibodies capable of
inhibiting infection of cells in culture. Furthermore,
challenge studies can be performed to determine if animals
vaccinated with a polypeptide are protected from subsequent
infection by wild type virus. One having ordinary skill in
the art can routinely produce and screen fragments and
derivatives to determine the effectiveness of such vaccine
components to elicit protective immune responses. Similarly,
larger molecules may also be screened by the same means to
detect their ability to elicit a protective immune response.
The UCD lies near the transmembrane region of the
S protein. Because this region of the S protein is purported
to be involved in the secondary structure of the glycoprotein,
in receptor binding and in virus-induced cell fusion, the UCD
plays an important role in the function of the S protein and
in the formation of infectious virus. Inducing an immune
.,-: ::
-~ response against this region will interfere with the folding
of the S glycoprotein into its proper conformation. The
presence of circulating antibodies to this region could bind
to either virus or infected cells expressing the glycoprotein
on the surface. Virus complexed with antibody may be unable
~to bind to receptors on susceptible cells and/or initiate the
;; 25 pathway required to gain entry which involves a conformational
change of the S protein. Recognition of this region on the
surface of infected cells would target them for clearance.
Antibody binding to the conserved region of the S protein
surface expressed by infected cells would, most likely,
i 30 prevent cell fusion and interfere with virus assembly.
- Regardless of mechanism, an immune response to the UCD of a
coronavirus S protein will inhibit virus spread from cell to
cell and limit virus infection.
~- Polypeptides according to the present invention
comprise less than a complete S protein sequence. In
particular, the polypeptides do not comprise a complete N-
¦ terminal portion of an S protein and preferably comprise few
1~
1~ .
WO93/2~21 PCT/US93/~36~
5201 --
-- 10 --
or no amino acid sequences from the N terminal bulbous portion
of the protein. Furthermore, the polypeptides preferably do
not comprise a complete transmembrane domain of an S protein.
In some preferred embodiments, polypeptides comprise no more
than a 400 amino acid se~uence upstream (from the C terminus
to the N terminus) from about 2 amino acids upstream from the ~ J
transmembrane domain. In some preferred embodiments,
polypeptides comprise no more than a 300 amino acid sequence
upstream (from the C terminus to the N terminus) from about
5 amino acids upstream from the transmembrane domain.
In some preferred embodiments, polypeptides which
comprise a UC~, or derivatives and/or fragments thereof
further comprise flanking sequences of the UCD found in
coronavirus. For example, in some preferred embodiments, the
polypeptide comprises portions of the S protein flanked by and
optionally including the heptad repeats reported by deGroot
et al., such as, for example, in FIPV strain WSU 1146 from
residues 1067 to 1380. In some preferred embodiments, the
polypeptide comprises portions of the S protein flanked on the
carboxy side by and may also include a leucine zipper motif
as reported by Britton. In some preferred embodiments, the
polypeptide comprises portions of the S protein from about 300
residues upstream of the transmembrane region to about 5 amino
acid residues upstream from the transmembrane domain.
In some preferred embodiments, the polypeptide
comprises a UCD about 124 amino acids in length. In some
preferred embodiments, the polypeptide comprises an
- immunogenic fragment of a UCD about 100 amino acids in length.
In some preferred embodiments, the polypeptide comprises an
immunogenic fragment of a UCD about 50 amino acids in length.
In some preferred embodiments, the polypeptide comprises an
immunogenic fragment of a UCD about 25 amino acids in length.
In some preferred embodiments, the polypeptide comprises an
immunogenic fragment of a UCD about 15 amino acids in length.
In some preferred embodiments, the polypeptide comprises an
immunogenic fragment of a UCD about 10 amino acids in length.
: ~
- ~
WO93/2~21 2 1 ~ ~ 2 o 1 PCT/US9~/~36~
In some preferred embodiments, a UCD comprises amino
acid residues 37-160 of SEQ ID NO:12. Additional preferred
embodiments comprise SEQ ID NO:12. Other preferred
embodiments of the invention comprise SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. Other
preferred embodiments comprise SEQ ID NO:6, SEQ ID NO:7, SEQ
ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.
In addition to a UCD and, optionally, additional
flanking segments from an S protein, other peptide segments
may also be included in the polypeptide of the present
invention. Such additional peptide segments may comprise
other immunogenic targets from coronavirus and/or other
pathogens, and/or they may be provided for improved stability,
UCD epitope presentation or production/purification
facilitation. The resulting polypeptide is considered a
chimeric or fusian polypeptides.
Vaccines according to the present invention can be
; employed to vaccinate animals against infection by
coronaviruses or at least to prevent the clinical symptoms
associated with such infections. Such vaccines will provide
protection against multiple coronaviruses and cross species
protection. Vaccines may be produced which are either
protein-based or nucleic acid-based. In both cases, the
vaccinated animal is exposed to an immunogenic polypeptide
which comprises a UCD. A protective immune response is
elicited which is sufficient to protect the animal against
~ coronavirus.
-~ Vaccines according to the present invention can be
either: 1
~ 30 a) compositions which comprise a polypeptide that
; includes a universal conserved domain; or
b) compositions which comprise a nucleic acid
molecu~e that includes a nucleotide sequence which encodes
a polypeptide that includes a universal conserved domain. In
3S both types of vaccines, the polypeptide is not a complete S
protein and it elicits a protective immune response in
animals.
~.
!
WO93/2~21 2 1 3 5 2 0 1 PCT/US93/~36~
- 12 -
In protein based, i.e. subunit vaccines,
polypeptides having a UCD may by produced using standard
techniques including recombinant DNA techniques for protein
production or by peptide synthesis. In preferred em~odiments,
5 polypeptides used in subunit vaccines according to the present
invention are produced by recombinant DNA methodology.
The nuoleic acid sequences of coronavirus S genes
are widely known. One having ordinary skill in the art may
routinely obtain ~NA that encodes a polypeptide including a
10 UCD using standard techniques and widely available starting
materials. The nucleotide and amino acid sequences for S
proteins from several types and strains of coronaviruses can
_be found in the co-owned published PCT application
PCT/US91/08525 which claims priority to U.S. Patent
Application Serial Numbers 613,066 and 698,927; each of these
- applications are incorporated herein by reference. Nucleotide
~ and amino acid sequences of S proteins can also be found in
I published European Patent Applications publication numbers:
0,524,672 Al; 0,411,684 A2; 0,264,979 Al; 0,138,242 A1; and
application number EP 91 30 3737. Each of these European
-~ patent applications are incorporated herein by reference. In
-~ addition, nucleotide and amino acid sequences of S proteins
- from several coronaviruses as well as nucleotide and amino
acid sequences of a consensus sequence is disclosed in the co-
25 owned, co-pending patent application: which is filed on the
~ same day as the present application; which is entitled
;~ "Compositions and Methods for Vaccinating Coronaviruses";
which names the same inventors as the present application
(Miller, Timothy J.; Jones, Elaine V.; Reed, Albert P.; and
' 30 Klepfer, Sharon R); which has been designated docket number
H85009-1 by Applicants; and which is incorporated herein by
reference.
Nucleic acid molecules encoding some or all of an
S protein from a coronavirus may be generated by a variety of 5
35 techniques. For such molecules, a nucleotide sequence that
encodes a UCD may be identified. Using, for example,
Polymerase Chain Reaction (PCR) methodology, primers flanking
W093~2~2l 2 1 3 5 2 0 1 ~ PCT/US93/~365
- 13 -
both sides the region of interest may be designed and used to
produce multiple copies of the UCD routinely. Alternatively,
using restriction enzymes, a UCD may be isolated from DNA
encoding an S protein. Moreover, nucleic acid molecules that
S encode a UCD may also be synthesized using techniques well
known to those having ordinary skill in the art.
One having ordinary skill in the art can, using well
known techniques, insert such DNA molecules into a
commercially available expression vector for use in well known
expression systems. For example, the commercially available
plasmid pSE420 ~Invitrogen, San Diego, CA) may be used for
production of a DNA encoding a polypeptide including a UCD in
E. coli. The commercially available plasmid pYES2
(Invitrogen, San Diego, CA) may, for example, be used for
production in S. cerevisiae strains of yeast. The
commercially available MaxBac~ (Invitrogen, San Diego, CA)
complete baculovirus expression system may, for example, be
~ used for production in insect cells. The commercially
- available plasmid pcDNA I tInvitrogen, San Diego, CA) may, for
example, be used for production in mammalian cells such as
Chinese Hamster Ovary cells. One having ordinary skill in the
art can use these commercial expression vectors and systems
~ or others to produce a polypeptide including a UCD using
¦; routine techniques and readily available starting materials.
(See e.g., Sambrook et al., Molecular Cloning a Laboratory
- ~ Manual, Second Ed. Cold Spring Harbor Press (lg89) which is j
-~ incorporated herein by reference.) Thus, the desired proteins
can be prepared in both prokaryotic and eukaryotic systems,
resulting in a spectrum of processed forms of the protein.
The particulars for the construction of expression
~ systems suitable for desired hosts are known to those in the
-~ art. Briefly, for recombinant production of the protein, the
DNA encoding the polypeptide is suitably ligated into the
expression vector of choice. The DNA is operably linked to
all regulatory elements which are necessary for expression of
the DNA in the selected host. One having ordinary skill in
WO93/2~21 21~3 5 2 0 1 PCT/US93/0436~ i
- l4 -
the art can, using well known techniques, prepare expres~ion
vectors for recombinant production of the polypeptide.
The expression vector including the DNA that encodes
the polypeptide comprising a UCD is used to transform the
compatible host which is then cultured and maintained under
conditions wherein expression of the foreign DNA takes place.
~ The protein of the present invention thus produced is
¦- recovered from the culture, either by lysing the cells or from
the culture medium as appropriate and known to those in the
art. One having ordinary skill in the art can, using well
known techniques, isolate the polypeptide that includes a UCD
; produced using such expression systems.
In addition to producing these proteins by
recombinant techniques, automated peptide synthesizers may
lS also be employed to produce polypeptides that include a UCD.
Such techniques are well known to those having ordinary skill
-~ - in the art and are useful if derivatives which have
substitutions not provided for in DNA-encoded protein
production.
Subunit vaccines according to the invention comprise
a polypeptide the includes a UCD but which is not a complete
S protein and a pharmaceutically acceptable carrier or
diluent. Optionally, the vaccine may comprise additional
immunogenic proteins, additional vaccine components such as
non-subunit vaccines, and/or an adjuvant.
In nucleic acid molecule based, i.e. recombinant
vaccines, a nucleotide sequences which encode polypeptides
that include a UCD is inserted into a vector and administered
to the animal. The vector delivers genetic material to the
animal where it is transcribed and translated to produce the
immunogenic polypeptide. Vectors for use as vaccines are well
known and include non-pathogenic viruses and prokaryotic
organisms. Suitable vectors for delivering genetic material
are readily available or may be produced from readily
available starting materials using standard techniques. Two
examples of vectors useful for delivering genetic material as
a vaccine are the recombinant pox vectors or non-pathogenic
:
. `.
WO 93/23421 ~ ~ . PCr/US93/0436~
213~01
Salmonella strains. The nucleotide sequence that encodes the
immunogenic polypeptide is operably linked to regulatory
elements required for expression and inserted within the
vsctor. Alternatively, it is incorporated into the vector at
a site where it is placed under the contro} of the necessary
regulatory elements already present in the vector. Naked DNA
may also be used as a vaccine delivery system.
Recombinant vaccines may be used in combination with
~ other vaccines. Further, the genetic material which encodes
-~ 10 the polypeptide that comprises the UCD may further comprise
; additional~ coding sequences which encode other peptide
-- ~ sequences capable of eliciting an immunogenic response against
coronavirus or another pathogen.
Both subunit and recombinant vaccines may be
formulated following accepted convention using buffers,
stabilizers, preservative, solubilizers and compositions used
to facilitate sustained release. Generally, additives for
isotonicity can include sodium chloride, dextrose, mannitol,
sorbitol and lactose. Stabilizers include gelatin and
20~ albumin. Adjuvants such as aluminum or magnesium hydroxide
, ~
may be employed. Vaccines may be maintained in solution or,
in some cases, particularly recombinant vaccines, lyophilized.
Lyophi}ized vaccine may be stored conveniently and combined
with sterile solution before administration.
~ The amount of polypeptide administered depends upon
such factors as the size of the polypeptide, the species, age,
weight, and general physical characteristics of the animal,
and ~by the~ composition of the vaccine. Determination of
optimum dosage for each parameter may be made by routine
j 30 methods. Generallv, subunit vaccines according to tha present
~ invention contain between 0.05-5000 micrograms of polypeptide
r: ~ per milliliter of sterile solution, preferably 10-1000
micrograms. Gençrally, recombinant vaccines according to the
present invention contain between 105-lOa infectious units per
milliliter of sterile solution. About .5-2 milliliter of
~ polypeptide-containing solution is administered.
.,
.
..
W093/~2l 2 1 3 S ~ 1 PCTtUS~3/~365
- 16 -
Subunit vaccines and genetic material based vaccines
may be administered by an appropriate route such as, for
example, by oral, intranasal, intramuscular, intraperitoneal
or subcutaneous administration. In some embodiments,
intranasal or subcutaneous administration is preferred.
Subsequent to initial vaccination, animals may be boosted by
revaccination.
Examples
Example 1 Cloning of Coronavirus Conserved Region in pMG1
The bacterial expression vector, pMG-1, allows a
gene expressing a foreign protein to be fused to a partial
sequence of the NS1 gene from influenza virus, the first 81
encoding amino acids thereof. This vector is described in
European Patent Application No. 366,238, published May 2, ~-
1990, which is incorporated herein by reference.
Primers were designed to amplify a S gene region
encoding amino acids 1115-1238 of the DF2 FIPV strain for
expression in this vector as follows. The upstream primer
~ contains NcoI and NdeI restriction sites and initiates
; ~20 amplification at base pair 3406 (amino acid 1115), and is SEQ
ID N0:13:
- 5'- ,
I GTTGTCAACACACCATGGATCATATGCAAGGGCAAGCTTTAAGTCACCTTACA.
NcoI NdeI
~ i
The downstream primer contains a StuI site and terminates
amplification at base pair 3777 (amino acid 1238), and is SEQ
ID N0: 14:
5'-AAATACCTGAGGCCTCCAAGCTGTTACAGTTTCATAAGCTGT.
StuI
The amplified fragment (412 bp) was cloned into the pT7 Blue
vector according to the manufacturer's instructions. A
plasmid containing amino acids 1115-1238 in pT7 Blue was
digested with NcoI/StuI, the 412 base pair insert isolated,
and ligated overnight at 15C to plasmid vector pMGl digested
with NcoI/StuI and dephosphorylated. Host cells AR120 and
AR58 were transformed with the ligation mix and the presence
;- ~
W093/2~21 2 13 5 2 0 1 PCT/US93/~365
- ~7
of insert bearing clones was confirmed by diagnostic
restriction enzyme digestions.
Example 2 - Cloning of Coronavirus Conserved Region in pSC11
Vaccinia recombinants were engineered to contain the
1115-1238 amino acid conserved region of WT DF2 FIPV. The
conserved region was cloned into the vaccinia expression
vector p5C11 by blunt-ending the 412 base pairs NcoI/StuI
fragment isolated from the pT7 Blue clone described in Example
- 10 12, end-filling by incubation with Klenow polymerase, and
inserting it into the SmaI site downstream of the 7.5K
vaccinia promoter. The ligation mix was transformed into
HB101 host cells. Full-length clones were identified and
oriented with respect to vector by BamHI and ScaI digests of
mini-prep DNAs, respectively.
:~
WO 93/23421 P(~/US93/04365
2i35201
-- 18 --
Table 1
W~ue2 NITQAFGKVN DAIHQTSQGL ATVAKALAXV QD WNTQGQA LSHLTVQLQN
Df2e2 NITQAFGKVN DAIHQTSQGL ATVAKALAKV QDWNTQGQA LSHLTVQLQN
5 Tse2 NITQAFGKVN DAIHQTSQGL ATVAKALAKV QDWNTQGQA LSHLTVQLQN
Fecve2 NITQAFGKVN DAIHQTSQGL ATVAKALAKV QD WNTQGQA LSHLTVQLQN
Tgeve2 NITQAFGKVN DAIHQTSQGL ATVAKALAKV QDWNTQGQA LSHLTVQLQN
Tgeve2f2 NITQAFGKVN DAIHQTSQGL ATVAKALAXV QDWNTQGQA LSHLTVQLQN
Bcve2 AIQEGFDATN S.............. ..... ALVKI QAWNANAEA LNNLLQQLSN
Hcve2 NIVDAFTGVN DAITQTSQAL QTVATALNKI QDWNQQGNS LNHLTSQLRQ
Ibbspi HMQE.......... ........ GF RSTSLALQQI QDWSKQSAI LTETMASLNK
Mhve2aS9 AIQDGFDATN S........... ..... ALGKI QSVVNANAEA LNNLLNQLSN
Mhvs AIQEGFDATN S............... ..... ALGKI QSWNANAEA LNNLLNQLSN
CONSENSUS NITQAFGKVN DAIHQTS.GL ATVAKALAKV QDWNTQGQA LSHLTVQLGN
51 100
~- Wsue2 NFQAISSSIS DIYNRLDELS ADAQVDRLIT GRLTALNAFV SQTLTRQAEV
Df2e2 NFQAISSSIS DIYNRLDELS ADAQVDRLIT GRLTALNAFV SQTLTRQAEV
Tse2 NFQAISSSIS DIYNRLDELS ADAQVDRLIT GRLTALNAFV SQTLTRQAEV
Fecve2 NFQAISSSIS DIYNRLDELS ADAQVDRLIT GRLTALNAFV SQTLTRQAEV
Tqeve2 NFQAISSSIS DIYNRLDELS ADAQVDRLIT GRLTALNAFV SQTLTRQAEV
Tgeve2f2 NFQAISSSIS DIYNRLDELS ADAQVDRLIT GRLTALNAFV SQTLTRQAEV
~ Bcve2 RFGAISSSLQ EILSRLDALE AQAQIDRLIN GRLTALNVYV SQQLSDSTLV
Hcve2 NFQAISSSIQ AIYDRLDTIQ ADQQVDRLIT GRLAALNVFV SHTLTKYTEV
ibbspi NFGAISSVIQ EIUQQFDAIQ ANAQVDRLIT GRLSSLSVLA SAXQAEUIRV
Mhve2aS9 RFGAISASLQ EILTRLEAVE AKAQIDRLIN GRLTALNAYI SKQLSDSTLI
Mhvs RFGAISASLQ EILTRLDAVE AKAQIDRLIN GRLTALNAYI SKQLSDSTLI
-~ ~ CONSENSUS NFQAISSSIS DIYNRLDELS ADAQVDRLIT GRLTALNAFV SQTLTRQAEV
101 1 5 0
~- Wsue2 RASRQLAKDK VNECVRSQSQ RFGFCGNGTH LFSLANAAPN GMIFFHTVLL
30 Df2e2 RASRQLAKDK VNECVRSQSQ RFGFCGNGTH LFSLANAAPN GMIFFHTVLL
-~ Tse2 RASRQLAKDK VNECVRSQSQ RFGFCGNGTH LFSLANAAPN GMIFF~TVLL
- ~ Fecve2 RASRQLAKDK VNECVRSQSQ RFGFCGNGTH LFSLANAAPN GMIFFHTVLL
Tgeve2 RASRQLAK~DK VNECVRSQSQ RFGFCGNGTH LFSLANAAPN GMIFFHTVLL
Tgève2f2 RASRQLAXDK VNECVRSQSQ RFGFCGNGTH LFSLANAAPN GMIFFHTVLL
- 35 ~Bcve2 KFSAA,QAMEX VNECVKSQSS RINFCGNGNH IISLVQNAPY GLYFIHFSYV
- ~ Hcve2 RASRQL~AQQK VNECVKSQSK RYGFCGNGTH IFSIVNAAPE GLVFLHTVLL
~S~ Ibbspi SQQRELATQK INECVKSQSI RYSFCGNGRH VLTIPQNAPN GIVFIHFSYT
Mhve2a59 XVSAAQAIEK VNECVKSQTT RINFCGNGNH ILSLVQNAPY GLYFIHFSYV
Mhvs KFSAAQAIEX VNECVKSQTT RINFCGNGNH ILSLVQNAPY GLCFIHFSYV
-~` 40 ~ CONSENSUS RASRQLAKDK VNECVRSQSQ RFGFCGNGTH LFSLANAAPN GMIFFHTVLL
~ 151 200
'~ Wsue2 PTAYETVTAW SGICASDGDR TFGLW KDVQ LTLFRNLDDK FYLTPRTMYQ
Df2e2 PTAYETVTAW SGICASDGDR TFGLW ~DVQ LTLFRNLDDK FYLTPRTMYQ
Tse2 PTAYETVTAW SGICASDGDR TFGLW KDVQ LTLFRNLDDK FYLTPRTMYQ
45 Fecve2 PTAYETVTAW SGICASDGDR TFGLW KDVQ LTLFRNLDDK FYLTPRTMYQ
Tgeve2 PTAYETVTAW SGICASDGDR TFGLVVKDVQ LTLFRNLDDK FYLTPRTMYQ
Tgeve2f2 PTAYETVTAW SGICASDGDR TFGLVVKDVQ LTLFRNLDDK FYLTPRTMYQ
Bcve2 PTKYVTAKYS PGLCIA.GDR GIA..... PK SGYFVNVNNT WMFTGSGYYYHcve2 ' PTQYKDVEAW SGLC...VDG TNGYVLRQPN LALYKE.GNY YRITSRIMFE
Ibbspi PDSFVNVTAI VGFCVKPANA SQUAIVPANG RGIFIQVNGS YYITARDMYM
Mhve2aS9 PISFTTANVS PGLCIS.GDR GLA.. PK AGYFVQDDGE WKFTGSSYYYMhvs PTSFKTANVS PGLCIS.GDR GLA...... PK AGYFVQDNGE WKFTGSNYYYCONSENSUS PTAYETVTAW PGICASDGDR TFGLVVKDVQ LTLFRNLDDK FYLTPRTMYQ
?
.~ t
,:
': ~
'~
''
.':
WO !?3~23421 2 1 ~ ~ 2 1~ 1 Pcr/US93/o436~;
_ 19 _
SEQUENCE LISTING
~1) GENERAL INFORMATION:
(i) APPLICANT: Miller, Ti~othy J.
Jones, Elaine V.
Reed, Albert P.
Xlepfer, Sharon R.
~ TITLE OF INVENTION: Univer~al Coronaviru~ Vaccine
(iii) NUMBER OF SEQUENCES: 14
~iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKline Beecham Corporation
(8) STREET: 709 Swedeland Road
~C) CITY: Xing of Prussia
(D) STATE: PA
(E) COUNTRY: USA
~F) ZIP: 19406-2799
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUM8ER:
. (B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/882,171
(8) FILING DATE: 08-MAY-1992
(~ii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/698,927
(B) FILING DATE: 13-MAY-l991
(~ii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/613,066
(B) FILING DATE: 14-NOV-1990
(viii) ATTORNEY/AGENT INFORMATION:
. (A) NAME: Schreck, Patricia A.
(B) ~EGISTRATION NUMBER: 33,777
(C) REFERENCE/DOCXET NUMBER: SBC/PAS/WW001
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acidq
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Aqn Ile Thr Gln Ala Phe Gly Lys Val Asn Asp Ala ~le His Gln Thr
1 5 10 15
~ '
....... ......................................................... .......... ...............
r--
WO 93/23421 2 1 3 $ 2 0 1 PCr/US93/0436~
-- 20 --
Ser Gln Gly Leu Ala Thr Val Ala Lys Ala Leu Ala Lys Val Gln Asp.
Val Val Asn Thr Gln Gly Gln Ala Leu Ser His Leu Thr Val Gln Leu
Gln A~n Asn Phe Gln Ala Ile Ser Sex Ser Ile Ser Asp Ile Tyr Asn
50 55 60
Arg Leu Asp Glu Leu Ser Ala Asp ~la Gln Val Asp Arg Leu Ile Thr
65 70 75 80
Gly Arg Leu Thr Ala Leu ~sn Ala Phe Val Ser Gln Thr Leu Thr Arg
85 90 95
: Gln Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Lys Asp Lys Val Asn
100 105 110
Glu Cys Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly
115 120 125
Thr His Leu Phe Ser Leu Ala Asn Ala Ala Pro Asn Gly Met Ile Phe
- 130 1~5 140
Phe His Th~ Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp
145 150 155 160
Ser Gly Ile Cy~ Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val
2~ 165 170 175
Lys Asp Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr
180 185 190
Leu Thr Pro Arg Thr Met Tyr Gln
195 200
:~ 25 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQU~NCE DESCRIPTION: SEQ ID NO:2:
A~n Ile Thr Gln Ala Phe Gly Lys Val Asn Asp Ala Ile His Gln Thr
1 5 10 15
' Ser Gln Gly Leu Ala Thr Val Ala Lys Ala Leu Ala Lys Val Gln Asp
20 25 30
Val Val Asn Thr Gln Gly Gln Ala Leu Ser His Leu Thr Val Gln Leu
Gln Asn Asn Phe Gln Ala Ile Ser Ser Ser Ile Ser Asp Ile Tyr Asn
Arg Leu Asp Glu Leu Ser Ala Asp Ala Gln Val Asp Arg Leu Ile Thr
65 70 75 80
Gly Arg Leu Thr Ala Leu Asn Ala Phe Val Ser Gln Thr Leu Thr Arg
85 90 95
,
WO 93/23421 2 1 3 5 2 0 1 PCI/US93/0"36:~
-- 21 -- .
Gln Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Lys Asp Lys Val Asn.
100 105 110
Glu Cy8 Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly
115 120 125
Thr Hi~ Leu Phe Ser Leu Ala Asn Ala Ala Pro A~3n Gly Met Ile Phe
130 135 140
Phe Hi8 Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp
145 150 155 160
Ser Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val
165 170 175
Lya Asp Val Gln Leu Thr Leu Phe Arg Asn Leu A3p Asp Lys Phe Tyr
180 185 190
Leu Thr Pro Arg Thr Met Tyr Gln
195 200
15 ~2) INFORMATION FOR SEQ ID NO:3:
( i ) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 200 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
2 0 ( ii ) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Asn Ile Thr Gln Ala Phe Gly Lys Val Asn Asp Ala Ile His Gln Thr
: Ser Gln Gly Leu Ala Thr Val Ala Lys Ala Leu Ala Lys Val Gln Asp
20 25 30
~ ~: Val Val Asn Thr Gln Gly Gln Ala Leu Ser His Leu Thr Val Gln Leu
- 35 40 45
: ~ Gln Asn Asn Phe Gln Ala Ile Ser Ser Ser Ile Ser Asp Ile Tyr Asn
50 55 60
3 0 Arg Leu Asp Glu Leu Ser Ala Asp Ala Gln Val Asp Arg Leu Ile Thr
65 70 75 80
Gly Arg Leu Thr Ala Leu Asn Ala Phe Val Ser Gln Thr Leu Thr Arg
85 90 . 95
~, Gln Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Lys Asp Lys Val Asn
3 5 100 105 110
Glu Cys Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly
115 120 12S
Thr His Leu Phe Ser Leu Ala Asn Ala Ala Pro Asn Gly Met Ile Phe
130 135 140
4 0 Phe His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp
145 150 155 160
Ser Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val
165 170 175
W O 93/2342l 213~ PCT/~593/04365
Lys Asp Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr
180 185 190
Leu Thr Pro Arg Thr Met Tyr Gln
195 200
t2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCS DESCRIPTION: SEQ ID NO:4:
A8n Ile Thr Gln Ala Phe Gly Lys Val Asn Asp Ala Ile His Gln Thr
1 5 10 15
Ser Gln Gly Leu Ala Thr Val Ala Ly~ Ala Leu Ala Lys Val Gln A~p
20 25 30
Val Val Asn Thr Gln Gly Gln Ala Leu Ser His Leu Thr Val Gln Leu
Gln Asn Asn Phe Gln Ala Ile Ser Ser Ser Ile Ser Asp Ile Tyr Asn
Arg Leu Asp Glu Leu Ser Ala A5p Ala Gln Val Asp Arg Leu Ile Thr
65 70 75 80
: Gly Arg Leu Thr Ala Leu Asn Ala Phe Val Ser Gln Thr Leu Thr Arg
- 85 90 95
Gln Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Lys Asp Lys Val Asn
100 105 110
Glu Cys Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly
115 120 125
Thr His Leu Phe Ser Leu Ala Asn Ala Ala Pro Asn Gly Met Ile Phe
130 135 140
Phe His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp
145 150 155 160
Ser Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val
165 170 175
Lys Asp Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr
180 185 190
Leu Thr Pro Arg Thr Met Tyr Gln `
195 200
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
WO 93/23421 2 1 3 5 2 0 1 - ^` - PCTtUS~3/04365
; 23 --
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
A~n Ile Thr Gln Ala Phe Gly Ly~ Val Asn Asp Ala Ile ~i~ Gln Thr
1 5 10 15
Ser Gln Gly Leu Ala Thr Val Ala Lys Ala Leu Ala Lys Val Gln Asp
20 25 30
Val Val A~n Thr Gln Gly Gln Ala Leu Ser Hi~ Leu Thr Val Gln Leu
Gln Asn Asn Phe Gln Ala Ile Ser Ser ~er Ile Ser A~p Ile Tyr Asn
S0 5S 60
Arg Leu ABP Glu Leu Ser Ala Asp Ala Gln Val Asp Arg Leu Ile Thr
~5 70 75 80
Gly Arg Leu Thr Ala Leu Asn Ala Phe Val Ser Gln Thr Leu Thr Arg
85 90 95
Gln Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Lys Asp Lys Val Asn
100 105 110
Glu Cy9 Val Arg Ser Gln Ser Gln Arq Phe Gly Phe Cy9 Gly Asn Gly
115 120 125
Thr Hi5 Leu Phe Ser Leu Ala ~sn Ala Ala Pro Asn Gly Met Ile Phe
130 135 140
Phe His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp
145 150 155 160
Ser Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val
165 170 175
Lys Asp Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr
180 185 190
Leu Thr Pro Arg Thr Met Tyr Gln
: 195 200
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Asn Ile Thr Gln Ala Phe Gly Lys Val Asn Asp Ala Ile His Gln Thr
Ser Gln Gly Leu Ala Thr Val Ala Lys Ala Leu Ala Lys Val Gln Asp
20 25 30
Val Val Asn Thr Gln Gly Gln Ala Leu Ser His Leu Thr Val Gln Leu
35 40 45
Gln Asn Asn Phe Gln Ala Ile Ser Ser Ser Ile Ser Asp Ile Tyr Asn
WO 93/23~21 2 1 3 5 2`~ 1 : PCI/US93/0436~
` . .
- 24 -
Arg Leu Asp Glu Leu Ser Ala Asp Ala Gln Val Asp Arg Leu Ile Thr
7~ - 75 80
Gly Arg Leu Thr Ala Leu Asn Ala Phe Val Ser Gln Thr Leu Thr Arg
Gln Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Lys Asp Ly~ Val A3n
100 105 110
Glu Cy8 Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly
115 120 125
Thr His Leu Phe Ser Leu ~la Asn Ala Ala Pro Asn Gly Me~ Ile Phe
1~ 130 135 140
Phe His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp
145 150 ~55 160
Ser Gly Ile Cy~ Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val
165 170 175
Ly~ Asp Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr
~ 180 185 190
Leu Thr Pro Arg Thr Met Tyr Gln ~:
195 200
. (2) INFORMATION FOR SEQ ID NO:7:
: :
~:i 20 (i) SEQUENCE CHARACTERISTICS:
: (A) LENGT~: 179 amino acid~
-~ ~B) TYPE: amino acid
r~ (D) TOPOLOGY: linear
~ (ii) MOLECULE TYPE: protein "
.~
~: 25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
.~
. ~ Ala I}e Gln Glu Gly Phe Asp Ala Thr Asn Ser Ala Leu Val Lys Ile
1 5 10 15
Gln Ala Val Val Asn Ala Asn Ala Glu Ala Leu Asn Asn Leu Leu Gln
~ 20 25 30
: ~ 30 Gln Leu Ser Asn Arg Phe Gly Ala Ile Ser Ser Ser Leu Gln Glu Ile
35 40 45
Leu Ser Arg Leu Asp Ala Leu Glu Ala Gln Ala Gln Ile Asp Arg Leu
50 55 60
Ile Asn Gly Arg Leu Thr Ala Leu Asn Val Tyr Val Ser Gln Gln Leu
65 70 75 80
Ser Asp Ser Thr Leu Val Lys Phe Ser Ala Ala Gln Ala ~et Glu Lys
Val Asn Glu Cys Val Lys Ser Gln Ser Ser Arg Ile Asn Phe Gly Asn
100 105 110
Gly Asn His Ile Ile Ser Leu Val Gln Asn Ala Pro Tyr Gly Leu Tyr
115 120 125
Phe Ile His Phe Ser Tyr Val Pro Thr Lys Tyr Val Thr Ala Lys Tyr
130 135 140
~:
WO 93/23421 2 1 3`5 2 0 1 PCI`/US93/04365
-- 25 -- .
Ser Pro Gly Leu Cy9 Ile Ala Gly Asp Arg Gly Ile Ala Pro Lys Ser-
14S 150 155 160
Gly Tyr ~he Val Asn Val Asn Asn Thr Trp Met Phe Thr Gly Ser Gly
165 170 175
Tyr Tyr Tyr
(2) INFORMATION FOR SEQ ID NO:8:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 196 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID No:8:
_ Asn Ile Val Asp Ala Phe Thr Gly Val Asn Asp Ala Ile Thr Gln Thr
1 15 1 5 10 15
¦ Ser Gln Ala Leu Gln Thr Val Ala Thr Ala Leu Asn Lys Ile Gln Asp
20 25 30
Val Val Asn Gln Gln Gly Asn Ser Leu Asn His Leu Thr Ser Gln Leu
35 40 45
Arg Gln Asn Phe Gln Ala Ile Ser Ser Ser Ile ~ln Ala Ile Tyr Asp
50 55 60
Arg Leu Asp Thr Ile Gln Ala Asp Gln Gln Val AQP Arg Leu Ile Thr
65 70 75 80
Gly Arg Leu Ala Ala Leu Asn Val Phe Val Ser His Thr Leu Thr Lys
85 90 95
Tyr Thr Glu Val Arg Ala Ser Arg Gln Leu Ala Gln Gln Lys Val Asn
100 105 110
Glu Cys Val Ly~ Ser Gln Ser Lys Arg Tyr Gly Phe Cys Gly Asn Gly
115 12Q 125
- 30 Thr His Ile Phe Ser Ile Val Asn Ala Ala Pro Glu Gly Leu Val Phe
130 1~5 140
Leu His Thr Val Leu Leu Pro Thr Gln Tyr Lys Asp Val Glu Ala Trp
145 150 155 160
, Ser Gly Leu Cys Val Asp Gly Thr Asn Gly Tyr Val Leu Arg Gln Pro
165 170 175
Asn Leu Ala Leu Tyr Lys Glu Gly A~n Tyr Tyr Arg Ile Thr Ser Arg
180 185 190
Ile Met Phe Glu J
195
40 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENCTH: 183 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
,
WO 93J23421 -2il`3 ~ 2 0 1 PCI`/US93~436~
- 26 -
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Hi8 Met Gln Glu Gly Phe Arg Ser Thr Ser Leu Ala Leu Gln Gln Ile
Gln Asp Val Val Ser Ly~ ~ln Ser Ala Ile Leu Thr Glu Thr Met Ala
2~ 25 30
Ser Leu A0n Lys Asn Phe Gly Ala Ile Ser Ser Val Ile Gln Glu Ile
35 40 45
Gln Gln Phe Aqp Ala Ile Gln Ala Asn Ala Gln Val Asp Arg Leu Ile
50 55 60
Thr Gly Arg Leu Ser Ser Leu Ser Val Leu Ala Ser Ala Lys Gln Ala
~0
Glu Ile Arg Val Ser Gln Gln Arg Glu Leu Ala Thr Gln Lys Ile Asn
Glu Cy8 Val Lys Ser Gln Ser Ile Arg Tyr Ser Phe Cys Gly Asn Gly
100 105 110
Arg His Val Leu Thr Ile Pro Gln Asn ~la Pro Asn Gly Ile Val Phe
115 120 125
Ile His Phe Ser Tyr Thr Pro Aqp Ser Phe Val Asn Val Thr Ala Ile
130 135 140
Val Gly Phe Cys Val Lys Pro Ala Asn Ala Ser Gln Ala Ile Val Pro
145 150 155 160
Ala A~n Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile
165 170 175
Thr Ala Arg Asp Met Tyr Met
180
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) ~OLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ala Ile Gln Asp Gly Phe Asp Ala Thr A3n Ser Ala Leu Gly Lys Ile
1 5 10 15
Gln Ser Val Val Asn Ala Asn Ala Glu Ala Leu Asn Asn Leu Leu Asn
20 25 30 r
Gln Leu Ser Asn Arg Phe Gly Ala Ile Ser Ala Ser Leu Gln Glu Ile
Leu Thr Arg Leu Glu Ala Val Glu Ala Lys Ala Gln Ile Asp Arg Leu
wo g3,2342, ~ 2 1 3 5 2 0 I PCT/US93/0436~; ~
-- 2~ --
Ile Asn Gly Arg Leu Thr Ala Leu Asn Ala Tyr Ile Ser Ly~ Gln Leu
6S 70 75 80
Ser Asp Ser Thr Leu Ile Lys Val Ser Ala Ala Gln Ala Ile Glu Lys
Val Asn Glu Cys Val Lys Ser Gln Thr Thr Arg I le Asn Phe Cy3 Gly
100 105 110
A~n Gly Asn His Ile Leu Ser Leu Val Gln Asn Ala Pro Tyr Gly Leu
115 120 125
Tyr Phe Ile Hi~ Phe Ser Tyr Val Pro Il.e Ser Phe Thr Thr Ala Asn
0 130 135 140
Val Ser Pro Gly Leu Cye Ile Ser Gly Asp Arg Gly Leu Ala Pro Lyq~
145 150 15S 160
Ala Gly Tyr Phe Val Gln Asp A~p Gly Glu Trp Lys Phe Thr Gly Ser
165 170 175
15Ser Tyr Tyr Tyr
180
(2) INFOR~ATION FOR SEQ ID NO:ll:
i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180 amino acids
2 0 ( B ) TYPE: amino acid
~: ~D) TOPOLOGY: linear .
~ii) MOLECULE TYPE: protein
'
( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
: ~ 25 Ala Ile Gln Glu Gly Phe Asp Ala Thr Asn Ser Ala Leu Gly Lys Ile
Gln Ser Val Val Asn Ala Asn Ala Glu Ala Leu Asn Asn Leu Leu Asn
:~ Gln Leu Ser Asn Arg Phe Gly Ala Ile Ser Ala Ser Leu Gln Glu Ile
35 40 45
Leu Thr Arg Leu Asp Ala Val Glu Ala Lys Ala Gln Ile Asp Arg Leu
50 55 60 '.
~;: Ile Asn Gly Arg Leu Thr Ala Leu Asn Ala Tyr Ile Ser LYS Gln Leu
65 70 75 80
: j Ser Asp Ser Thr Leu Ile Lys Phe Ser Ala Ala Gln Ala Ile Glu Lys
Val Asn Glu Cys Val LYB Ser Gln Thr Thr Arg I le Asn Phe Cys Gly
100 105 110
Asn Gly Asn His Ile Leu Ser Leu Val Gln Asn Ala Pro Tyr Gly Leu
115 120 125
~: 40 Cys Phe Ile Hi~ Phe Ser Tyr Val Pro Thr Ser Phe LYS Thr Ala Asn
130 135 140
Val Ser Pro Gly Leu Cys Ile Ser Gly Asp Arg Gly Leu Ala Pro Lys
145 150 155 160
WO 93~23'~21 2 1 3 5 2 0 1 PCr/US93/W365
-- 28 --
Ala Gly Tyr Phe Val Gln Aqp A~n Gly Glu Trp Lys Phe Thr Gly Ser
165 170 175
Asn Tyr Tyr Tyr
180
(2) INFORMATION FOR SEQ ID NO:12:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 199 amino acids
(8) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Asn Ile Thr Gln Ala Phe Gly Lys Val Asn Asp Ala Ile His Gln Thr
1 5 10 15
Ser Gly Leu Ala Thr Val Ala Lys Ala Leu Ala Lys Val Gln Asp Val
20 25 30
Val Asn Thr Gln Gly Gln Ala Leu Ser His Leu Thr Val Gln Leu Gly
Asn Asn Phe Gln Ala Ile Ser Ser Ser Ile Ser Asp Ile Tyr Aqn Arg
Leu Asp Glu Leu Ser Ala A~p Ala Gln Val Asp Arg Leu Ile Thr Gly
65 70 75 80
Arg Leu Thr Ala Leu Asn Ala Phe Val Ser Gln Thr Leu Thr Arg Gln
85 90 95
Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Lys Asp Lys Val Asn Glu
10~ 105 110
Cys Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly Thr ~`
115 120 125
His Leu Phe Ser Leu Ala Asn Ala Ala Pro Asn Gly Met Ile Phe Phe
130 135 140
His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp Pro
145 150 15S 160
Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val Lys
165 170 175
AQP Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr Leu
180 185 190
Thr Pro Arg Thr Met Tyr Gln ?
195
(2) INFORMATION FOR SEQ ID NO:13:
~i) SEQUENCE CHARACTERISTICS: s
(A) LENGTH: 53 baqe pairs
(B) TYPE: nucleic acid ?
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
WO 93/23421 2 1 3 5 2 0 i PCI`/US93/04365
-- 2g --
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GTTGTCAACA CACCATGGAT CATATGCAAG GGCAAGCTTT AAGTCACCTT ACA 53
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: qingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
0 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
AAATACCTGA GGCCTCCAAG CTGTTACAGT TTCATAAGCT GT 42
~ ,.
