CA2135201A1 - Universal coronavirus vaccine - Google Patents

Abstract

A universal vaccine is disclosed which elicits a protective immune response in different host species and against different coronaviruses. A polypeptide which elicits protective antibodies against a homologous sequence found in the C terminal portion of coronavirus S proteins is disclosed. Vaccines comprising either the polypeptide or nucleic acids which encode the polypeptide are also disclosed. Methods of protecting a host against coronavirus infection are disclosed.

Description

WOg3/2~21 PCT/US~3/~365 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~

- ?. - ' 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~

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~

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 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 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.

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WO 93/23421 P(~/US93/04365 2i35201 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

~- 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

~- 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
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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 ~ ' ....... ......................................................... .......... ...............

r--WO 93/23421 2 1 3 $ 2 0 1 PCr/US93/0436~

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 Arg Leu Asp Glu Leu Ser Ala Asp ~la Gln Val Asp Arg Leu Ile Thr Gly Arg Leu Thr Ala Leu ~sn Ala Phe Val Ser Gln Thr Leu Thr Arg : Gln Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Lys Asp Lys Val Asn Glu Cys Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly 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 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 Leu Thr Pro Arg Thr Met Tyr Gln :~ 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 ' 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 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 Gly Arg Leu Thr Ala Leu Asn Ala Phe Val Ser Gln Thr Leu Thr Arg , 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.

Glu Cy8 Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly Thr Hi~ Leu Phe Ser Leu Ala Asn Ala Ala Pro A~3n Gly Met Ile Phe Phe Hi8 Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp Ser Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val Lya Asp Val Gln Leu Thr Leu Phe Arg Asn Leu A3p Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln 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 ~ ~: 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 3 0 Arg Leu Asp Glu Leu Ser Ala Asp Ala Gln Val Asp Arg Leu Ile Thr 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 Glu Cys Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly Thr His Leu Phe Ser Leu Ala Asn Ala Ala Pro Asn Gly Met Ile Phe 4 0 Phe His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp Ser Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val W O 93/2342l 213~ PCT/~593/04365 Lys Asp Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln 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 Ser Gln Gly Leu Ala Thr Val Ala Ly~ Ala Leu Ala Lys Val Gln A~p 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 : 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 Lys Val Asn Glu Cys Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly Thr His Leu Phe Ser Leu Ala Asn Ala Ala Pro Asn Gly Met Ile Phe Phe His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp Ser Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val Lys Asp Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln `

(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 Ser Gln Gly Leu Ala Thr Val Ala Lys Ala Leu Ala Lys Val Gln Asp 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 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 Gln Ala Glu Val Arg Ala Ser Arg Gln Leu Ala Lys Asp Lys Val Asn Glu Cy9 Val Arg Ser Gln Ser Gln Arq Phe Gly Phe Cy9 Gly Asn Gly Thr Hi5 Leu Phe Ser Leu Ala ~sn Ala Ala Pro Asn Gly Met Ile Phe Phe His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp Ser Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val Lys Asp Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr 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 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 WO 93/23~21 2 1 3 5 2`~ 1 : PCI/US93/0436~
` . .

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 Glu Cy8 Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly 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 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 ~:

. (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 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 Leu Ser Arg Leu Asp Ala Leu Glu Ala Gln Ala Gln Ile Asp Arg Leu Ile Asn Gly Arg Leu Thr Ala Leu Asn Val Tyr Val Ser Gln Gln Leu 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 Gly Asn His Ile Ile Ser Leu Val Gln Asn Ala Pro Tyr Gly Leu Tyr Phe Ile His Phe Ser Tyr Val Pro Thr Lys Tyr Val Thr Ala Lys Tyr ~:

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-Gly Tyr ~he Val Asn Val Asn Asn Thr Trp Met Phe Thr Gly Ser Gly 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 ¦ Ser Gln Ala Leu Gln Thr Val Ala Thr Ala Leu Asn Lys Ile Gln Asp Val Val Asn Gln Gln Gly Asn Ser Leu Asn His Leu Thr Ser Gln Leu Arg Gln Asn Phe Gln Ala Ile Ser Ser Ser Ile ~ln Ala Ile Tyr Asp Arg Leu Asp Thr Ile Gln Ala Asp Gln Gln Val AQP Arg Leu Ile Thr Gly Arg Leu Ala Ala Leu Asn Val Phe Val Ser His Thr Leu Thr Lys Tyr Thr Glu Val Arg Ala Ser Arg Gln Leu Ala Gln Gln Lys Val Asn Glu Cys Val Ly~ Ser Gln Ser Lys Arg Tyr Gly Phe Cys Gly Asn Gly - 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 , Ser Gly Leu Cys Val Asp Gly Thr Asn Gly Tyr Val Leu Arg Gln Pro Asn Leu Ala Leu Tyr Lys Glu Gly A~n Tyr Tyr Arg Ile Thr Ser Arg Ile Met Phe Glu J

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~

(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 Gln Gln Phe Aqp Ala Ile Gln Ala Asn Ala Gln Val Asp Arg Leu Ile 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 Arg His Val Leu Thr Ile Pro Gln Asn ~la Pro Asn Gly Ile Val Phe Ile His Phe Ser Tyr Thr Pro Aqp Ser Phe Val Asn Val Thr Ala Ile Val Gly Phe Cys Val Lys Pro Ala Asn Ala Ser Gln Ala Ile Val Pro Ala A~n Gly Arg Gly Ile Phe Ile Gln Val Asn Gly Ser Tyr Tyr Ile Thr Ala Arg Asp Met Tyr Met (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 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 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 A~n Gly Asn His Ile Leu Ser Leu Val Gln Asn Ala Pro Tyr Gly Leu Tyr Phe Ile Hi~ Phe Ser Tyr Val Pro Il.e Ser Phe Thr Thr Ala Asn Val Ser Pro Gly Leu Cye Ile Ser Gly Asp Arg Gly Leu Ala Pro Lyq~

Ala Gly Tyr Phe Val Gln Asp A~p Gly Glu Trp Lys Phe Thr Gly Ser 15Ser Tyr Tyr Tyr (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 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 : 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 Asn Gly Asn His Ile Leu Ser Leu Val Gln Asn Ala Pro Tyr Gly Leu ~: 40 Cys Phe Ile Hi~ Phe Ser Tyr Val Pro Thr Ser Phe LYS Thr Ala Asn Val Ser Pro Gly Leu Cys Ile Ser Gly Asp Arg Gly Leu Ala Pro Lys WO 93~23'~21 2 1 3 5 2 0 1 PCr/US93/W365 Ala Gly Tyr Phe Val Gln Aqp A~n Gly Glu Trp Lys Phe Thr Gly Ser Asn Tyr Tyr Tyr (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 Ser 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 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 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 Lys Val Asn Glu 10~ 105 110 Cys Val Arg Ser Gln Ser Gln Arg Phe Gly Phe Cys Gly Asn Gly Thr ~`

His Leu Phe Ser Leu Ala Asn Ala Ala Pro Asn Gly Met Ile Phe Phe His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala Trp Pro Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Val Lys AQP Val Gln Leu Thr Leu Phe Arg Asn Leu Asp Asp Lys Phe Tyr Leu Thr Pro Arg Thr Met Tyr Gln ?

(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:

(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:

~ ,.

Claims (6)

Claims

1. A polypeptide comprising a universal conserved domain of a coronavirus or an immunogenic fragment or derivative thereof; said polypeptide having less than a complete amino acid sequence of said S protein.

2. A vaccine comprising a pharmaceutically acceptable carrier or diluent and a polypeptide comprising a universal conserved domain of a coronavirus or an immunogenic fragment or derivative thereof; said polypeptide having less than a complete amino acid sequence of said S protein.

3. A nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising a universal conserved domain of a coronavirus or an immunogenic fragment or derivative thereof; said polypeptide having less than a complete amino acid sequence of said S protein.

4. A recombinant vaccine comprising a nucleic acid molecule, said nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising a universal conserved domain of a coronavirus or an immunogenic fragment or derivative thereof; said polypeptide having less than a complete amino acid sequence of said S protein.

5. A method of protecting an animal against coronavirus comprising administering a polypeptide comprising a universal conserved domain of a coronavirus or an immunogenic fragment or derivative thereof; said polypeptide having less than a complete amino acid sequence of said S protein.

6. A method of protecting an animal against coronavirus comprising administering a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising a universal conserved domain of a coronavirus or an immunogenic fragment or derivative thereof; said polypeptide ?O 93/23421 PCT/US93/04365 having less than a complete amino acid sequence of said S
protein.