JPH07508176A - universal coronavirus vaccine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] universal coronavirus vaccine Simultaneous application interaction The present invention is based on United States patent application Ser. No. 07/613,066 (November 1990). United States Patent Application No. 07/6989, a partially pending application (filed on the 14th) No. 27 (filed May 13, 1991), a pending U.S. Partially pending U.S. Patent Application No. 07/882,171 (filed May 8, 1992) This is an application.

field of invention The present invention provides a method for activating different species against infections caused by different host-specific coronaviruses. Concerning universal vaccines useful for protecting against diseases.

Background of the invention Coronavirus is an enveloped virus with a single-stranded positive-sense genome It is a type of host-specific RNA virus. For example, coronaviruses are specific to cats. Feline infectious peritonitis virus (FIPV) and feline small intestinal coronavirus (FECV), canine coronavirus (CCV), which is specific to dogs, and canine coronavirus (CCV), which is specific to dogs. Transmissible gastroenteritis virus (TGEV): a bovine coronavirus specific to Virus (BCV), human coronavirus specific to humans, specific to Fuzumi Fuzumi hepatitis virus (MHV), and infectious bronchitis specific to Including, but not limited to, viruses (IBV). these Host-specific coronaviruses cannot cross-infect between different animal species. corona virus Symptoms of viral infection of the host by this virus range from mild heat haze to a severe and debilitating illness. and may even lead to death.

The coronavirus has a spike or S protein'M (also called pepromere protein). have common structural features including The S protein protrudes from the surface of the virus particle. It is a glycoprotein that is released. S protein binds pirion to host cell receptor mediates membrane fusion. Additionally, it is a target for virus-neutralizing antibodies.

The S protein consists of an N-terminal/gnal sequence, a C-terminal transmembrane segment, and a polygonal sequence. Contains a tensile N-linked glyconylation site. Different coronavirus S proteins A comparison shows that there is little homology at the N-terminus, that is, no similarity, and high amino acids at the C-terminus. Sequence conservation can be seen. Histological attributes and symptoms vary considerably between types of this virus. The pathogenesis of coronaviruses is determined by the N-terminal encoded sequence. The more conserved C-terminus is an important structural feature common to all coronaviruses. It is thought to be a hood. The carboquino terminus of S protein is thought to be involved in fusion. It will be done.

The structure of S protein has already been studied. Cavanagh, (19 83) Nyanar of Noeneral Virorono (J, Gen, Vir ol, ) 64: 2577-2583 (Parts of this specification are incorporated herein by reference) ), the C-terminal half of the protein forms the stalk, and the N-terminal half forms the stalk. We propose a model of the coronavirus spike forming a globular protein. De deGroot et al. (1987), Glossy of Molecule - Biol/- (J, Mo1. Biol,) 197 (hereinafter referred to by reference) (regarded as part of the book) has a coiled structure that is similar to the globular part of the S protein and the virus. The model assumes that bonds between films are formed. This model has 7 repeats sequence, i.e. periodic (a-b-c-d-e-f-g) in which the amino acids are hydrophobic. Based on existence. Britton (1991) Nature (\a ture) 353: 394 (Considered to be part of this specification by citation) is the carboxyl terminus of the S-glycoprotein of coronavirus whose spike sequence is known. TGEV FS772/ which reports the presence of leunon 7 nopa motif in 70 (amino acids 1342-1377), FIPVWSU1146 (amino acids 1345-1380), vIHV to 59 (amino acids 1217-1252), Human coronavirus 229E (amino acids 1067-1102), BCVMeb us (amino u 1266-1294) and infectious bronchitis virus Bea udette (amino acids 1059-1079). Roynon zipper motif is , terminating at 10 residues upstream of the conserved KWP motif before the transmembrane region. Ru.

Lack of effort to develop vaccines against various host-specific coronaviruses It's been coming. Attenuated live virus vaccines, inactivated vaccines, subunit vaccines and attempts have been made to develop vaccines based on recombinant nucleic acids.

In the case of bulrushes, the developed vaccines do not cross-protect pond host animals. coronawi Vaccines currently available to protect against R. ruses are directed against certain coronaviruses. Specific to defense. Such vaccines require the use of other species that can infect other species. There is no cross-protection that protects the host against coronaviruses. Furthermore, this Such vaccines cross-protect other animals against coronaviruses that are susceptible to infection. It's good There is a need for vaccines that can protect against coronavirus infections.

In particular, vaccines are useful in protecting hosts against different coronaviruses. needed and useful to protect different hosts against different coronaviruses A vaccine that is 2.

Summary of the invention The present invention has been found to be highly conserved among coronaviruses and to stimulate protective immune responses. Amino acid sequence derived from the C-bundle end of the coronavirus S protein that could have arisen in the past. The present invention relates to a polypeptide consisting of: This arrangement is called a universally conserved region. Polymer of the present invention The peptide has less than the complete amino acid sequence of the S protein.

The present invention provides polypeptides containing universally conserved regions and fewer than the complete amino acid sequence of S protein. Relating to a vaccine consisting of peptide.

The present invention contains universally conserved region polypeptides and is based on the complete amino acid sequence of S protein. Relating to isolated nucleic acid molecules having nucleic acid sequences encoding small polypeptides .

The present invention is based on W1! ! Contains conserved regions and contains fewer points than the complete amino acid sequence of S protein. It relates to a vaccine consisting of a nucleic acid molecule coated with a repeptide.

The present invention provides for administering an effective amount of a polypeptide to elicit a protective immune response. This article relates to a method for protecting animals from infectious diseases caused by coronaviruses. Applicable person The polypeptide administered in this method consists of universally conserved regions and contains the entire S protein. less than the amino acid sequence.

The present invention provides an amount of nucleic acid encoding a polypeptide effective to elicit a protective immune response. Protecting animals from infections caused by coronaviruses by administering acid molecules Regarding the method. the polypeptide encoded by the nucleic acid molecule administered in the method; It consists of universally conserved regions and has less than the complete amino acid sequence of S protein.

Detailed description of the invention According to the present invention, highly conserved regions of the spike protein can be used as vaccine components or When provided as a product, it is used to protect animals against coronavirus infections. It was identified that it is useful as a universal immunogen for humans. The vaccine of the present invention Any animal susceptible to infections caused by viruses belonging to the family Naviridae It is also used to inoculate. Therefore, the present invention can be manufactured in a single manufacturing process. To provide a vaccine that can be administered to different species of animals. With the vaccine of the invention The resulting cross-protection protects animals against different viruses of the Coronaviridae family. There is no need to manufacture different vaccines to control

As used herein, the term "polypeptide" refers to peptides, polypeptides, molecules containing peptides or protein molecules, peptides, polypeptides or protein molecules. or refers to a molecule containing amino acid residues linked by non-peptide bonds.

As used herein, “universally conserved region” (“UCD”) refers to TGEV , in the C-terminal part of the S protein from CCV and feline coronavirus strains. refers to the same 124 amino acid segment seen. In addition, “UCD” The term refers to the corresponding coronaviruses of other coronaviruses that have different but homologous amino acid sequences. means an amino acid segment. Such a corresponding sequence is found in the S protein. Due to their location, i.e. downstream of the globular N-terminal region, the transmembrane upstream of the region and a high level of amino acid sequence similarity with the 124 amino acid sequence described above. Identified by gender. Furthermore, the term "UCD-" also refers to the comparison of corresponding sequences. and by measuring the statistical average of amino acid residues at a given position in the sequence means the consensus sequence obtained. In this way, you can compare several different sequences. The most common residue at a given position is selected for that position in the consensus sequence. Ru.

The conservation of the UCD sequence suggests that it plays a major role in viral structure and/or replication. It suggests that you will fulfill your share. Regions of complete homology are found in other coronavirus S genes When compared, the size has decreased. For example, bovine and human coronaviruses sequence in this conserved region than sequences from Fuzumi and avian coronaviruses. It is similar to the feline, canine, and porcine coronavirus S protein genes.

Table 1 shows the ratio of corresponding amino acid sequences from the C-terminal region of various coronaviruses. Show a comparison. SEQ ID NO: 1 is the amino acid sequence derived from FI PV Wsue2 strain. (Toxic, type ■: Genbank accession number X06170). Sequence number 2 is the amino acid sequence derived from the FIPVDf2e2 strain (virulence, ■ type). Sequence number/ 3 is the amino acid sequence derived from FIPVTse2 strain (temperature-sensitive mutant of Df2). ). SEQ ID NO: 4 is the amino acid sequence derived from FECV Fecve2 strain (non-toxic 1683 strains). SEQ ID NO: 5 is the amino acid sequence derived from TGEV Tgeνe2 strain. Yes (Purdue strain: Genbank number DOO118), sequence Number 6 is the amino acid sequence derived from FT PV Tgeve2f2 strain (Mil ler strain; G6bank accession number M56002).

SEQ ID NO: 7 is the amino acid sequence derived from BCV Bcve2 strain (Genbank The receiving number is M30613). Sequence number 8 is HCV Hcve,! Stock-derived a It is a amino acid sequence (G enbank acceptance number X16816), SEQ ID NO. ・9 is the amino acid sequence derived from the IBV Ibbspi strain (Genbank The receiving number is X16816). Sequence number 10 is MHV Mhve 2 a 5 This is the amino acid sequence derived from 9 strains (Genbank accession number X51939).

SEQ ID NO: 11 is the amino acid sequence derived from FIPVMhvS strain (Genbank The receiving number is X04797). SEQ ID NO: 12 provides the optimal UCD amino acid sequence This is the consensus sequence for

124 completely conserved among TGEV, CCV and feline coronaviruses The amino acid residue sequences are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. , sequence (indicated from residue 37 to residue 160 in i11 number 5. In the consensus sequence A certain SEQ ID NO: 12 also has this 124 amino acid sequence from residue 37 to residue 160. This 124 amino acid sequence is currently unknown. A preferred UCD arrangement of the invention. Complete 199 amino acid consensus sequence is a preferred UCD-containing peptide.

No matter which coronavirus-derived amino acid information is used, those skilled in the art can Containers corresponding to the 124 amino acid sequences found in CV and feline coronaviruses It is possible to identify the area in which the data exists. As illustrated in Table 1, coronavirus C. Comparing the amino acid sequences from the terminal parts, TGEV, CCV and cat coronavirus Sequences corresponding to virus-derived UCDs can be identified. The method is simple, another U This can be done to obtain CD and flanking sequences.

Corresponding products derived from coronaviruses other than CCV, TGEV, and feline coronaviruses The conserved region is determined by its position on the S protein and when compared to the 124 amino acids mentioned above. Identified by high levels of sequence homology. An example of such comparison and identification is Shown in Table 1. Table 1 lists various S proteins upstream of the transmembrane region. Sequences from the C-terminal region of white matter have been compared and homologous sequences have been identified.

PLOTSTMILARITY software Group (Genetics Computer Group), Ricecon using widely available computer programs such as , positioning UCD during coronavirus.

In addition, such software can be used to simplify the generation of consensus sequences. can be done. This software was originally developed by Wilbur and Lipmano (V Ilbur and Lipman), and later Smith and Water. Man C3lIj th and Wa+erzan),! , ;Rabini Needleman and original modified by Needleman and Wunsch. It depends on the logic. Using these well-known guy lines, one skilled in the art can to arrive at the statistical average or most common residue occupying a given position.

PLOTSIill, ^RIT~ software automates this function. be. A consensus sequence is thus obtained. Like array number 12 In addition to the resulting consensus sequence, another concept derived from the comparison of corresponding sequences sequence is disclosed in a co-owned, pending application (title of invention: ``Coronavirus''). "Compositions and Methods for Vaccination against Viruses", filed on the same day as this application; One inventor (Miller, Timono-Noei; Nyons, Elaine Bui, Reed, Albert Bee: and Talefer. Miller.

Timothy J: Jones, Elaine V: Reed , ^1bert P, : Klepfer, 5haron@R) ; Applicant Identification number H85009-1: incorporated herein by reference).

Therefore, the present invention provides polypeptides containing UCD or fragments or derivatives thereof. Regarding petit de. That is, the present invention relates to TGEV, CCV, and feline coronavirus. or other coronaviruses corresponding to the 124 amino acid sequence Constructs derived from comparisons of different amino acid sequences or corresponding regions from viruses. consisting of a census sequence, or an immunogenic fragment or derivative thereof Concerning polypeptides.

The polypeptides of the present invention may also be used with other flanking sequences derived from coronaviruses. sequence or the flanking sequences of corresponding regions from different coronaviruses. The present invention relates to a polypeptide consisting of flanking sequences expressed as sequence sequences.

As used herein, the term "immunogenic fragment" refers to Eliciting a protective immune response against coronaviruses in susceptible animals refers to a polypeptide containing an incomplete UCD that has the ability to Immunogenic fragments The component consists of a sequence having 9 or more amino acids derived from the UCD. may contain different amino acid sequences.

As used herein, the term "immunogenic derivative" refers to derivatives that undergo conservative amino acid substitutions. In animals that have a large UCD or part thereof and are susceptible to coronavirus infection. refers to molecules that have the ability to elicit a protective immune response against coronaviruses. . Those skilled in the art will easily understand the UCD sequence with conservative amino acid substitutions. It is possible to design derivatives with For example, day hots regarding amino acid substitutions Hof’s attention 11 (Da>hof’s rule) (Daikhonov, M.D. Dayhof, M., D.) (1978) Found (Nat, Biomed, Res, Found, ), Wannon According to Tono D, C, Vol, 5.5upp, 3), amino acids in the peptide sequence Noic acid residues can be replaced with comparable amino acid residues. Such substitutions are well known based on the charge and structural properties of each amino acid.

Using standard methods and readily available starting materials, one skilled in the art can and whether the derivative is an immunogenic fragment or an immunogenic derivative, respectively. can be determined. Briefly, polypeptides can be produced by standard methods and provide protective immunity. It can be determined by testing whether it can elicit an infectious response. from vaccinated animals to detect the presence of antibodies in the culture medium that can inhibit infection of cells. Can be done. Additionally, challenge studies were conducted to show that animals inoculated with the polypeptide to determine whether they are protected from subsequent infection with wild-type virus. Ru. Those skilled in the art will routinely generate and screen fragments and derivatives. , it is possible to investigate the effectiveness of such vaccine components in eliciting a protective immune response. Wear. Similarly, large molecules can be screened in the same way to elicit protective immune responses. can detect the ability to

UCD is located near the transmembrane region of S protein. This region of the S protein is Glycoprotein secondary structure, associated with receptor binding and virus-induced cell fusion Therefore, UCD plays a role in the function of S protein and the formation of infectious viruses. play an important role. Eliciting an immune response to this region is caused by the S-glycoprotein prevents it from folding into its proper configuration. Circulating antibodies against this region If present, it will bind to either the virus expressing the glycoprotein on its surface or the infected cells. It will fit. Virus complexed with antibodies cannot bind to receptors on susceptible cells and and/or the pathway necessary to obtain entry that involves a conformational change in the S protein. Unable to start. Recognition of this region on the surface of infected cells targets their elimination . Antibodies that bind to conserved regions on the surface of the S protein expressed by infected cells are likely may prevent cell fusion and interfere with virus assembly. Apart from the mechanism, the The immune response to the UCD of the Ronavirus S protein control the spread of virus and limit virus infection.

The polypeptides of the invention consist of less than the complete S protein sequence. In particular, The polypeptide does not include the entire N-terminus of the S protein, preferably the N-terminal globule of the protein. Contains little or no amino acid sequences derived from conditions. Furthermore, the port The repeptide preferably does not include the entire transmembrane region of the S protein.

In some preferred embodiments, the polypeptide comprises a transmembrane region. From about 2 amino acids upstream of the region to only 400 upstream (from the C-terminus to the N-terminus) Consists of an amino acid sequence. In some preferred embodiments, the polypeptide is , from about 5 amino acids upstream of the transmembrane region (from the C-terminus to the N-terminus) It consists of a sequence of only 300 amino acids.

In some preferred embodiments, UCD, or a derivative thereof and/or is a polypeptide consisting of its fragments, and even in coronaviruses. Consists of flanking sequences of the visible UCD. For example, some preferred specifics In examples, the polypeptides described by deGroot et al. For example, 10 repeats in FIPVWSU1146 strain. flanked by and optionally including the sequences 67 to 1380. It consists of part of the Moyoi S protein. In some preferred embodiments, polypep Tido is a leumenzi as reported by Britton. at the carbokino side by the par motif, or from a part of the S protein containing this. Become. In some preferred embodiments, the polypeptide is a transmembrane From about 300 residues upstream of the transmembrane region to about 5 amino acid residues upstream of the transmembrane region. It consists of part of the S protein up to the base.

In some preferred embodiments, the polypeptide has about 124 amino acids. Consists of length [,'CD. In some preferred embodiments, the polypeptide consists of an immunogenic fragment of LJCD approximately 100 amino acids in length.

In some preferred embodiments, the polypeptide is about 50 amino acids long. It consists of an immunogenic fragment of the UCD. Some preferred embodiments The polypeptide is an immunogenic fragment of tJcD approximately 25 amino acids in length. Consists of In some preferred embodiments, the polypeptide has about 15 consists of an immunogenic fragment of the UCD of amino acids in length. some preferences In certain embodiments, the polypeptide has a UCD of approximately 10 amino acids in length. Consists of primary fragments.

(In some preferred embodiments, UCD is the amino acid residue of SEQ ID NO: 12. It consists of 37 to 160. Additionally, a preferred embodiment includes SEQ ID NO:12.

Another preferred embodiment of the present invention is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, Contains SEQ ID NO:4 or SEQ ID NO:5. Other preferred specific examples include 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 including.

In addition to the UCD and, optionally, another flanking segment derived from the S protein. In addition to segments, other peptide segments can also be included in the polypeptides of the invention. Wear. Such separate peptide segments may be used to protect against coronavirus and/or other may consist of other immunogenic targets from pathogens and/or stability. whether supplied to improve UCD epitopes, provide UCD epitopes or facilitate production/purification. good. The resulting polypeptide is considered to be a chimeric or fusion polypeptide. Ru.

The vaccine of the present invention is used to vaccinate animals against infections caused by coronaviruses. or at least to prevent the symptoms associated with such infections. Can be used. Such vaccines protect against multiple coronaviruses and confer interspecific cross-protection. Vaccines can be protein-based or nucleic acid-based produced in either In both cases, vaccinated animals exposed to an immunogenic polypeptide consisting of D. Animals against coronavirus A protective immune response sufficient to protect is elicited.

The vaccine of the present invention is (a) a composition comprising a polypeptide comprising a universally conserved region; or (b) a universally conserved region. consisting of a nucleic acid molecule containing a nucleotide sequence encoding a polypeptide containing a region Composition Either. In both types of vaccines, the polypeptide is the complete S protein. rather than eliciting a protective immune response in the animal.

In protein-based or subunit vaccines, polypeptides with UCD protein production using standard techniques including recombinant DNA techniques or Produced by tide synthesis. In a preferred embodiment, the subunit work of the invention The polypeptides used in Cutino are produced by recombinant DNA methods. .

The nucleic acid sequence of the coronavirus S gene is widely known. Those skilled in the art will understand standard techniques and a polypeptide comprising a UCD, using widely available starting materials. DNA encoding can be obtained. S protein from several coronavirus strains The quality-related nucleotide and amino acid sequences are published in the shared published PCT application P CT/US9110852.5 (United States Patent Application No. 6 Claims priority to No. 13066 and No. 698927, and cites each of them. (herein incorporated by reference). Nucleotide and amino acid sequences of S protein Column also published European Patent Application No. 0524672A1, No. 0411 682 No. A2, No. 0264979A1: No. 0138242A1, and It is disclosed in application number EP91303737. These European patent applications are , each of which is incorporated herein by reference. In addition, some coronau Nucleotide and amino acid sequences and consensus of S protein from viruses The nucleotide and amino acid sequences of the sequences are disclosed in a co-pending patent application. has been done. (Name of the invention Composition and method for vaccination against coronavirus, book Filed on the same day as the application 1 Same inventor as the invention (Miller, Timono G.I.: John Z.

Elaine Bui, Reed, Albert Peay: and Talefer. Tsuyaron A Rule01i11er, Timothy J,: Jones, Elaine V, ; Reed, ＾1bert P, ; and Kle Taru ■■ Chichi B 5haron R): Identification number by applicant H85009-1: With reference to the source (considered a part of this specification).

There are various nucleic acid molecules encoding part or all of the S protein derived from coronavirus. produced by technology. In such molecules, the nucleotide encoding the UCD The code sequence is identified. For example, using polymerase chain reaction (PCR), We designed primers that flank both sides of the region to generate multiple copies of the UCD. Used for production. In addition, using restriction enzymes, tJcD can be converted to encode S protein. I'Ii away from the DNA. Furthermore, nucleic acid molecules encoding UCD are also known in the art. It can be synthesized using techniques known to those skilled in the art.

One skilled in the art can use known techniques to synthesize such NA molecules in known expression systems. It can be inserted into the commercially available expression vector used. For example, the commercially available plasmid pSE4 20 (Invitrogen, San Diego, California) n) Production of DNA encoding a polypeptide containing UCD in E. coli It can be used for. Commercially available plasmid l)’l’Es2 (California , San Diego, Invitrogen), for example, by fermentation. It can be used for production in the mother N. cerevisiae strain. complete baculo will Commercially available MaxBac expression system? 1' (San Diego, California, IN) Vitrogen), for example, can be used for production in insect cells. commercially available Plasmid pcDNA + (Invitroge, San Diego, California) for example, in mammalian cells such as Chinese hamster ovary cells. It can be used for production. Those skilled in the art will be familiar with these commercially available expression vectors and using conventional methods and readily available starting materials. It can produce repeptides.

(For example, Sambrook et al., Molecular Cloning ・A Laboratory Manual (ilolecular Cloning a) Laboratory Manual), 2nd edition.

Cold Spring Harbor Press or Press) (1989), incorporated herein by reference. vinegar). In this way, desired proteins can be prepared in both prokaryotic and eukaryotic systems, and The result is a series of processed forms of the protein.

Details of the construction of expression systems suitable for the desired host are known to those skilled in the art. Simply, egg For recombinant production of white matter, the DNA encoding the polypeptide is suitably , ligated into the expression vector of choice. DNA in the selected host It can be linked to all regulatory elements necessary for expression of the DNA. person skilled in the art can prepare expression vectors for recombinant production of polypeptides using known techniques. Wear.

This method is applied using an expression vector containing DNA encoding a polypeptide consisting of UCD. A compatible host is transformed and then cultured to obtain conditions under which expression of the foreign DNA occurs. Keep it under control. The protein of the present invention thus produced is lysed using a cell lysing method. or, as appropriate, from a culture medium known to those skilled in the art. A person skilled in the art can use known techniques to Isolating the UCD-containing polypeptide produced using such four-pronged filament Can be done.

In addition to producing these proteins by recombinant techniques, automated peptide synthesizers can also be used to produce these proteins. can be used to produce a polypeptide containing a UCD. Such technology is within the skill of the art. It is known to those skilled in the art that derivatives with substitution operations can be used in the production of DNA-encoded proteins. This is useful when it is not available.

The subunit vaccines of the present invention contain polypeptides that contain a UCD but are not a complete S protein. and a pharmaceutically acceptable carrier or diluent. Vaccine if desired may include additional immunogenic proteins, additional vaccine components, e.g. It may consist of a vaccine and/or an adjuvant.

In nucleic acid molecule-based, i.e., recombinant vaccines, polypeptides containing UCD The encoding nucleotide sequence is inserted into a vector and administered to an animal. vector delivers the genetic material to the animal, where it is transcribed and translated into immunogenic molecules. Produces repeptides. Vectors used as vaccines are known and non-pathogenic. Includes viruses and eukaryotes. suitable for delivering genetic material Vectors are either readily available or prepared from standard starting materials. Produced using industrial technology. Useful for delivering genetic material as vaccines Two examples of vectors that can be used are recombinant box vectors or non-pathogenic Salmonella strains. It is. The nucleotide sequence encoding the immunogenic polypeptide contains the necessary components for expression. It can be linked to regulatory elements and inserted into a vector. Also, vector vector at the site where it is located under the control of essential regulatory elements already present in the incorporated into the computer. DNA that is not enveloped by a membrane can also be used for vaccine delivery/ Can be used as a stem.

Recombinant vaccines can be used in combination with other vaccines. Furthermore , genetic material encoding a polypeptide consisting of UCD may be encodes other peptide sequences capable of eliciting immunogenic responses against other pathogens. Additional l; may consist of a coding sequence.

Both subunit and recombinant vaccines contain buffers, stabilizers, preservatives, Process normally with solubilizers and compositions used to promote sustained release. be treated. Generally, additives for isostrength are sodium chloride, dextrose , mannitol, sorbitol and lactose. The stabilizer is Includes gelatin and aldimine. aluminum or magnesium hydroxide You may use an anouvant such as. Vaccines may be kept in solution and are Alternatively, in some cases, particularly in the case of recombinant vaccines, it may be lyophilized.

Lyophilized vaccines can be conveniently stored and combined with a sterile solution prior to administration.

The amount of polypeptide administered depends on the size of the polypeptide, the species, age, weight, and size of the animal. and general physical characteristics, and the composition of the vaccine. Each change Determination of optimal doses in terms of numbers is carried out by conventional methods. Generally, the present invention subunit vaccines are 0.05 to 5000 per milliliter of sterile solution. micrograms, preferably 10 to 1000 micrograms of polypeptide. have In general, the recombinant vaccines of the present invention will be administered per milliliter of sterile solution. Contains approximately 105 to 108 dyeing units. Contains about 0.5 to 2 milliliters of polypeptide. Administer liquid solution.

subunit vaccines and genetic material-based vaccines by appropriate routes, e.g. For example, it is administered orally, intranasally, intramuscularly, intraperitoneally or subcutaneously. intranasal or Subcutaneous administration may be preferred. After the first vaccination, animals should not be revaccinated. and boost with antigen.

Example Example 1 Cloning of coronavirus conserved region in pMGl Bacterial expression vector pMG-1 is a partial sequence of the NSI gene derived from influenza virus. , the foreign protein's gene so that it is fused with its first 81 encoded amino acids. Express. This vector is known from European Patent Application No. 366238 (1999). published on May 2, 2013) (which is hereby incorporated by reference with reference to it). I reckon).

Primers are DF2FIP for expression in this vector as follows: to amplify the S gene region encoding amino acids 1115 to 1238 of the V strain. design. The upstream primer contains Nco+ and Ndel restriction sites and is 340 The amplification was started at 6 base pairs (amino acid 1115), and the amplification was started at SEQ ID NO: 13. The amplified fragment (412 bp), which is SEQ ID NO: °14, was terminated into pT, Manufacturer's Note in Blue Vector! Clone accordingly. 1) TV Gibble - Amino acids 1115-123 are ligated and dephosphorylated. host cell AR120 and AR58 were transformed with ligation mix to detect the presence of clone-bearing inserts. Confirm by restriction enzyme digestion.

Example 2 - Cloning of coronavirus conserved region in pscll Wakunoni A. By genetically manipulating the recombinant, WT DF2 FIPV 1115-1238 Contain amino acid conserved regions. Conserved region in vaccinia expression vector p sc11, isolated from the pT1 blue clone described in Example 12. cloned with a blunt-ended 412 base pair N co I/S tu I fragment. Loan. Transform the ligation mix into HBIOI host cells. Full length BamHI and BamHI of m1ni-prepDNA, respectively. 9. Orient the digest with respect to the vector.

Sequence list (1) General information; (i) Applicant Miller, Timothy Jay J.) Jones, Elaine V. Reed, Albert P. , Sharon (Klepfer, 5 haron) (ii) Name of invention: Universal Coro Number of navirus vaccine (iii) sequences, 14 (iv) Contact information: (A) Name SmithKline Beecham Corporation (SmithKl) ine Beecham Corporation) (B) street name, suede Land Road (Svedeland Road) No. 709 (city name: Kin CKing of Prussian (D) State name: Pencil Benia state (E) Country name: United States of America (F) Postal code: 19406-2799 (computer reader COMPUTER READABLE FORM (A) Media format: Floating PC (B) Computer: Compatible IBM PC (operating system: PC-DO8/MS-DO8(D) Software Patent-in Release ( PatentIn　Re1ease)　$1.0゜Version #1.25 (vl) Current application data: (^) Application number: US (B) Application mill (C) Classification: (vii) Previous application data.

(A) Application number: US 07/882.171 (B) Application: May 8, 1992 Day (vii) Previous application data: ′ (A) Application number: US 07/698.927CB) Application: May 1, 1991 3 days (vii) Previous application data; (^) Application number/US 07/613,066 (B) Application mill + November 1990 14th (viii) Agent information; (A) Name: Schreck, Patrick ia^, ) (B) Registration number: 33.777 (C) Processing number: SBC/PAS/WWOOI (2) Sequence number; Regarding 1 information (i) Characteristics of array: (^) Sequence length: 200 amino acids (B) Sequence type diamino acid (D) Topology, linear (ii) Molecule type protein (xi) Sequence description: Sequence number, 1: Asn　Ile　Thr　Gln^la　Phe　Gly　Lys　Val^s n Asp^la Ile His Gin Thrl 5 10 15 Ser　Gin　Gly　Leu　Ala　Dinghr　Val　Ala　Lys Ala　Leu　Ala　Lys　Val　Gin　AspVal　Val　A sn Thr Gln Gly Gln^la Leu Ser His Le u Thr %’al Gln LeuGin Asn Asn Phe Gi n^Ia rIe Ser Ser Ser He Ser Asp 11e Tyr As.

＾rg Leu Asp Glu Leu Ser Ala Asp Ala Gin Val Asp Arg Leu Ile Thr65’70 7 5 80 Gly Arg Leu Thr^la Leu Asn Ala Phe V al Ser Gln Thr Leu Thr ArgGin^la Glu Vat Arg^la Ser Arg Gin Leu^la Lys A sp　Lys　Val　AsnGlu　Cys　Val＾rg　Ser　Gln Ser Gin Arg Phe Gly Phe Cys Gly Asn GlyThr His Leu Phe Ser Leu^la Asn A la^la Pro Asn Gly Met Ile PhePhe His Thr Val Leu Leu Pro Thr Ala Tyr Glu Thr Val Thr Ala TrpSer Gly Ile Cys Ala Ser Asp Gly Asp Arg Thr Phe Gly Leu Val Va1Lys Asp Val Gin Leu Thr L eu Phe Arg Asn Leu Asp^sp Lys Phe Ty rLeu Thr Pro Arg Thr Met Tyr G1n (2) arrangement Information about column number 2 (i) Characteristics of array: (^) Sequence length: 200 amino acids (B) Sequence type/amino acid (D) Topology/linear (ii) Type of molecule: protein (xi) Sequence description: Sequence number = 2 ^sn　Ile　Thr　Gin　Ala　Phe　Gly　Lys　Val^ sn Asp Ala Ile His Gin Thrl 5 10 15 Ser　Gin　Gly　Leu^la　Thr　Val^la　Lys^la Leu Ala Lys Val Gin AspVal Val Asn Thr Gin Gly Gin Ala Leu Ser His Leu Thr　Val　Gln　LeuGln　Asn　Asn　Phe　Gln＾l a Ile Ser Ser Ser Ile Ser Asp Ile Ty r　AsnArg　Leu　Asp　Glu　Leu　Ser^la　Asp　 Ala Gln Val Asp Arg Leu Ile ThrGly A rg Leu Thr^la Leu Asn^la Phe Val Ser Gin Thr Leu Thr ArgGin Ala Glu Val Arg^la Ser Arg Gln Leu Ala Lys Asp L ys Val^5nGlu Cys Val^rg Ser Gin Ser Gin Arg Phe Gly Phe Cys Gly Asn GlyT hr　tlis　Leu　Phe　Ser　Leu^la　Asn^la　Al a Pro Asn Gly 1let Ile PhePhe His Th r Val Leu Leu Pro Thr Ala Tyr Glu Th r　Val　Thr^la　TrpScr　Gly　Ile　Cys^la　S er Asp Gly Asp Arg Thr Phe Gly Leu V al　Va1Lys　Asp　Val　Gin　Leu　Thr　Leu　Ph e Arg Asn Leu Asp Asp Lys Phe TyrLeu Thr Pro Arg Thr Met Tyr G1n (2) Sequence number・ Information about 3. (i) Characteristics of array: (^) Sequence length: 200 amino acids (B) Sequence type Amino acid (D) Topology: linear (11) Molecule type/protein (xl) Sequence description/Sequence number; 3: Asn　Ile　Thr　Gin^la　Phe　Gly　Lys　Val^s n Asp^la Ile His Gln Thrl 5 10 15 Ser　Gin　Gly　Leu^la　Thr　Val^la　Lys　Al a Leu Ala Lys Val Gin AspVal Val^sn Thr　Gin　Gly　Gln^la　Leu　Ser　His　Leu　T hr Val Gin LeuGln Asn Asn Phe Gin^la Ile Ser Ser Ser Ile Ser Asp Ile Tyr AsnArg Leu Asp Glu Leu Ser^la Asp A la Gln Vat^sp Arg Leu Ile ThrLeu Thr About Pro Arg Thr Met Tyr G1n (2) Sequence number 5 Information/ (i) Characteristics of array・ (A) Sequence length: 200 amino acids (B) Sequence type diamino acid (D) Topology/linear (ii) Type of molecule: protein (X]) Sequence description Sequence number 5 People sn Tle Thr Gln Ala Phe Gly Lys Val Asn Asp Ala Ile His Gin Thrl 5 10 15 Ser　Gln　Gly　Leu^la　Thr　Val^la　Lys　Al a Leu Ala Lys Val Gin AspVal Val^sn Thr　Gln　Gly　Gin^la　Leu　Ser　His　Leu　T hr Mal Gin LeuGin Asn Asn Phe Gln Al a Ile Ser Ser Ser Ile Ser Asp Ile Ty r　Asn＾rg　Leu　Asp　Glu　Leu　Ser　Ala　Asp Ala Gin Val Asp Arg Leu Ile ThrGly Arg Leu Thr^la Leu Asn Ala Phe Val S er Gin Thr Leu Thr ArgGln Ala Glu Va l Arg Ala Ser Arg Gin Leu Ala Lys As p Lys Val Asn1.00 105 110 Glu Cys Val Arg Ser Gln Ser Gin Arg Phe Gly Phe Cys Gly Asn GlyThr His L eu Phe Ser Leu^la Asn Ala^la Pro^sn Gly Met Ile PhePhe His Thr Val Leu L eu Pro Thr Ala Tyr Glu Thr Val Thr A la TrpSer Gly Ile Cys Ala Ser Asp G1 ．． y Asp Arg Thr Phe Gly Leu Val VaP Lys Asp Val Gin Leu Thr Leu I’he Arg Asn Leu Asp Asp Lys Phe Ty■ Leu Thr Pro human rg Thr Met Tyr G1n (2) sequence number Information about issue 6.

(i) Sequence characteristics.

(^) Sequence length, 200 amino acids (B) Sequence type, amino acid (D) Topolono: linear (11) Molecule type, protein (X]) Sequence description, Sequence number 6゜ 8sn Ile Thr Gin Ala Phe Gly Lys Val Person sn Asp Ala Ile His Gln Thrl 5 10 15 Ser Gin Gly Leu Ala Tbr Val Ala Lys Ala Leu Ala Lys Val Gln AspSer Pro G ly　Leu　Cys　Ile　Ala　Gly　Asp　Arg　Gly　r le^la Pro Lys 5erGly Tyr Phe Vat^sn Val^sn Asn Thr Trp Met Phe Thr Gly S er　GlyTyr　Tyr　Tyr (2) Information about sequence number 8 (i) Characteristics of array: (^) Sequence length: 196 amino acids (B) Sequence type, amino acid (D) Topology/linear (]1) Molecule type/protein (xl) Sequence description Sequence number, 8. Asn Ile Val^sp^la Phe Thr Gly Vat^sn Asp^la Ile Thr Gin Thrl 5 10 15 Ser Gin^la Leu Gln Thr Val A], a Thr^ la Leu Asn Lys Ile Gin AspVal l’al^s n Gin Gin Gly Asn Ser Leu Asn His Le u Thr Ser Gin LeuArg Gln Asn Phe Gln ^la Ile Ser Ser Ser Ile Gln Ala Ile Tyr　Asp＾rg　Leu　Asp　Thr　Ile　Gin　Ala　A sp　Gin　Gin　Val^sp　Arg　Leu　Ile　ThrGly Arg Leu Ala Ala Leu Asn Val Phe Vat Ser　His　Thr　Leu　Thr　LysTyr　Thr　Glu　 Val^rg Ala Ser Arg Gln Leu Ala Gin G in Lys Val AsnGlu Cys Val Lys Ser Gl n Ser　Lys　Arg　Tyr　Gly　Phe　Cys　Gly　As n　GlyThr　His　Ile　Phe　Ser　Ile　Val^sn^ la Ala Pro Glu Gly Leu Val PheLeu Hi s Thr Val Leu Leu Pro Thr Gin Tyr Ly s Asp Val Glu Ala TrpSer Gly Leu Cys Val^sp Gly Thr Asn Gly Tyr Val Leu Arg　Gin　Pr.

Person sn Leu Ala Leu Tyr Lys Glu Gly Asn Tyr　Tyr　Arg　Ile　Thr　Ser　Arg+80　185　1 90 11e Met Phe Glu (2) Information about SEQ ID NO: 9.

(i) Features of array (^) Sequence length: 183 amino acids (B) Sequence type, amino acid (D) Topology: Ii chain (11) Molecule type protein (xl) Sequence description: Sequence number: 9゜ His Met Gin Glu Gly Phe Arg Ser Thr Ser　Leu　Ala　Leu　Gin　Gin　l1eGin　Asp　V al　Vat　Ser　Lys　Gin　Ser^la　Ile　Leu　Th r Glu Thr Met AlaSer Leu Asn Lys Asn Phe GlyAla Ile Ser Ser Val Ile Gin Glu　l1eGin　Gin　Phe　Asp^la　Ile　Gin　Al a　Asn^1aG1nva1^sp　Arg　Leu　l1eThr　Gly Arg Leu Ser Ser Leu Ser Val Leu^la Ser^la Lys Gin^1aGlu Ile Arg Val Ser Gin Gin Arg Glu Leu^la Thr Gin Lys Ile AsnGlu Cys Val Lys Ser Gin Ser I le Arg Tyr Ser Phe Cys Gly Asn Glylo o +05 1l1 0Ar　His　l’al　Leu　Thr　Ile　Pro　Gin　Asn ^la Pro Asn Gly Ile Val PheIce His P he Ser Thr Thr Pro Asp Ser Phe Val A sn Val Th’r Ala l1■ Val　Gly　Phe　Cys　l’al　Lys　Pro　Ala　Asn Ala Ser Gin Ala Ile Val PrB Ala Asn Gly Arg Gly Ile Phe Ile Gin Val　Asn　Gly　Ser　Tyr　Tyr　l1eThr　Ala^r Information about g Asp Met Tyr Met (2) SEQ ID NO: 10.

(i) Characteristics of array: (^) Sequence length: 180 amino acids (B) Sequence type amino acid (D) Topology: linear (11) Molecule type/protein (xi) Sequence description Sequence number 10. Ala　Ile　Gln　Asp　Gly　Phe　Asp^la　Thr　A sn Ser^la Leu Gly Lys l1eGin Ser Val Val　Asn^la　Asn　Ala　Glu^la　Leu　Asn　A sn Leu Leu AsnGin Leu Ser Asn Arg Ph e　GlyAla　lie　Ser^la　Ser　Leu　Gin　Glu　 l1eLeu Thr Arg Leu Glu^la Val Glu^la Lys^la Gin Ile Asp Arg Leu11e Asn G ly　Arg　Leu　Thr　Ala　Leu　Asn　Ala　Tyr　I le Ser Lys Gin LeuSer Asp Ser Thr Le u Ile Lys Val Ser Ala Ala Gin Ala Il e Glu Lys1’al Asn Glu Cys val Lys Se r Gln Thr Thr Arg Ile Asn Phe Cys Gl ■ Asn Gly Asn His Ile Leu Ser Leu Val Gin Asn Ala Pro Tyr Gly LeuTyr Phe I le Royis Phe Ser Tyr Val Pro Ile Ser P he Thr Thr Ala AsnVal Ser Pro Gly Le u Cys Ile Ser Gly Asp Arg Gly Leu Al a Pro LysAla Gly Dingyr Phe Val Gln Asp Asp Gly Glu Trp Lys Phe Thr Gly 5er Ser Tyr Tyr Tyr (2) Information about sequence number 11 (i) Characteristics of array・ (^) Sequence length: 180 amino acids (B) Sequence type diamino acid (D) Topology linear (ii) Molecule type protein (xi) Sequence description, SEQ ID NO: 11 ^1a Tle Gin Glu Gly Phe Asp^la Thr A sn Ser Ala Leu Gly Lys l1eGln Ser Va l Val Asn Ala Asn Ala Glu Ala Leu As n Asn Leu Leu Asn Gln Leu Ser Asn Arg Phe Gly Ala Tle Ser Ala Ser Leu Gln Glu l1eLeu Thr Arg Leu Asp^la Val G lu^la Lys^la Gin Ile Asp Arg Leu11e Asn　Gly　Arg　Leu　Thr^la　Leu　Asn^la　Ty r　Ile　Ser　Lys　Gin　LeuSer　Asp　Ser　Thr Leu Ile Lys Phe Ser^1a^la Gin^la Il e Glu LysVal Asn Glu Cys Val Lys Ser Gin Thr Thr Arg Ile Asn Phe Cys Gly loo 105 1.10 ^sn　Gly　Asn　His　Leu　Leu　Ser　Leu　l’al Gin Asn^la Pro Tyr Gly LeuCys Phe I le His Phe Ser Tyr Val Pro Thr Ser P he Lys Thr^la AsnVal Ser Pro Gly Leu Cys Ile Ser Gly Asp Arg Gly Leu^la Pro Lys＾1a Gly Tyr Phe l’al Gin Asp Asn Gly Glu Trp Lys Phe Thr Gly Se■ +65 170 175 Asn Tyr Tyr Tyr (2) Information about sequence number 12 (1) Characteristics of array (A) Sequence length, 199 amino acids (B) Sequence type/amino acid (D) Topology linear (ii) Molecule type/protein (xi) Sequence description/Sequence number, 12 Asn Ile Thr Gln Al a Phe Gly Lys Val Asn Asp Ala Ile Hi s Gin Thrl 5 10 15 Ser　Gly　Leu^la　Thr　Val^la　Lys　Ala　Le u^la Lys Val Gln Asp Va1Val Asn Thr Gin Gly Gln Ala Leu Ser His Leu Thr Val　Gln　Leu　GlyAsn　Asn　Phe　Gln＾1a　Il e Ser　Ser　Ser　Ile　Ser　Asp　Ile　Tyr　As n　ArgLeu　Asp　Glu　Leu　Ser^la　Asp　Ala　 Gin Val^sp Arg Leu Ile Thr Gly^rg Le u Thr^la Leu Asn^la Phe val Ser Gin Thr　Leu　Thr　Arg　G1n^1a　Glu　l’al　Arg^ la Ser Arg Gin Leu^la Lys Asp Lys Va l Asn Gluloo 105 110 Cys Val Arg Ser Gin Ser Gln Arg Phe Gly　Phe　Cys　Gly　Asn　Gly　ThrHis　Leu　P he Ser Leu^la Asn^1a^la Pro Asn Gly Met　Ile　Phe　PheHis　Thr　Val　Leu　Leu　P ro Thr Ala Tyr Glu Thr ~’al Dhr Ala Trp PrB Gly Ile Cys^la Ser Asp Gly Asp Arg T hr　Phe　Gly　Leu　Val　Val　LysAsp　Val　Gl n Leu Thr Leu Phe Arg Asn Leu Asp As p Lys Phe Tyr LeuThr Pro＾rg Thr Met Information about Tyr　Gln(2) SEQ ID NO: 13: (i) Sequence characteristics: (A) Sequence length = 53 base pairs (B) Sequence type/nucleic acid (C) Number of chains - heavy chain (D) Topology/linear (11) Molecule type: cDNA (xl) Sequence description/Sequence number, 13 GTTGTCAACA　CACCATGGAT　CATATGCAAG　GGC ^^GCTTT^^GTCACCTT ACA 5(2) Regarding sequence number 14 Description of (1) Characteristics of array (A) Sequence length: 42 base pairs (B) Sequence type/nucleic acid (C) Number of chains - heavy chain (D) Topology linear (11) Molecule type/c DNA (xl) Sequence description Sequence number 14 AAATACCTGA GGCCTCCAAG CTGTTACAGT TTC Continuation of AT^^GCT GT 42 front page (51) Int, C1,6 identification symbol Internal office reference number Cl2P 21102 C9282-4B (72) Inventor Kloepfer, Sharon USA Penn 113 Lindbergh Avenue, Blue Mall, Sylvania 19008 I (72) Inventor Reed, Albert Paul Pennsylvania, USA 117 Baker Circle, Exton, 19341 (72) Inventor: Joe Winwood, Pennsylvania 19096, United States 1217 Ashido Bar Road

Claims (6)

[Claims] 1. Universally conserved regions of coronaviruses or immunogenic fragments or derivatives thereof A polypeptide consisting of the body with fewer sequences than the complete amino acid sequence of S protein. A polypeptide characterized by having a sequence. 2. Pharmaceutically acceptable carriers or diluents and coronavirus universal storage areas or or an immunogenic fragment or derivative thereof. in which the polypeptide contains less than the complete amino acid sequence of the S protein. A vaccine characterized by having: 3. Universally conserved regions of coronaviruses or immunogenic fragments or derivatives thereof A nucleic acid molecule consisting of a nucleotide sequence that encodes a polypeptide consisting of Therefore, the polypeptide may have a sequence less than the complete amino acid sequence of the S protein. A nucleic acid molecule characterized by. 4. The nucleic acid molecule is a universally conserved region of coronaviruses or immunogenic fragments thereof or a set of nucleotide sequences encoding a polypeptide consisting of a derivative thereof. A recombinant vaccine in which the polypeptide contains less than the complete amino acid sequence of the S protein. A recombinant vaccine characterized in that it has a sequence that does not contain any of the following. 5. Universally conserved regions of coronaviruses or immunogenic fragments or derivatives thereof Animal protection against coronavirus consisting of administering polypeptides from the body A method of protection, wherein the polypeptide has less than the complete amino acid sequence of S protein. A defense method characterized by having an array. 6. Universally conserved regions of coronaviruses or immunogenic fragments or derivatives thereof Administering a nucleic acid molecule consisting of a nucleotide sequence encoding a polypeptide consisting of A method of protecting an animal against a coronavirus, the method comprising: The amino acid sequence is characterized by having less sequence than the complete amino acid sequence of S protein. Defense method.