JP2017141234A - Synthetic peptide-based marker vaccine and diagnostic system for effective control of porcine reproductive and respiratory syndrome (prrs) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide-based marker vaccine against Porcine Reproductive and Respiratory Syndrome (PRRS); and a PRRS-preventing method and immunodiagnostic method.SOLUTION: A PRRS vaccine formulation comprises a mixture of peptides derived from PRRSV GP2, GP3, GP4, or GP5 proteins. Each of the peptides individually contains a B-cell PRRSV neutralizing/receptor binding epitope, which is individually linked to an artificial T helper epitope for enhancement of the respective peptide's immunogenicity; and which can be supplemented with a mixture of peptides representing the T helper epitopes derived from the PRRSV GP4, GP5, M and nucleocapsid proteins to provide cell mediated immunity.SELECTED DRAWING: None

Description

  The present disclosure relates to a peptide-based marker vaccine for porcine genital respiratory syndrome (PRRS) and a set of immunodiagnostic tests for monitoring and control of porcine genital respiratory syndrome virus (PRRSV).

  Porcine reproductive and respiratory syndrome virus (PRRSV) was discovered in the late 1980s as a cause of severe reproductive failure in mature sows and young sows and is one of the most important pathogens in the pig industry. Infection of mature and young sows can cause late miscarriage, premature labor, and delivery of sick piglets, while infected boars show reduced sperm quality and viral shedding in semen.

  PRRSV has also been found to be involved in porcine respiratory disease syndrome in juvenile pigs, causing respiratory disorders with secondary viral and bacterial infections. The virus exhibits limited in vivo cell orientation and alveolar macrophages are the main target cells.

  PRRSV is an enveloped single-stranded RNA virus that belongs to the family Artidoviridae Nidovirus (1), is approximately 15 kb in length, and consists of nine open reading frames (ORFs). Virions consist of a nucleocapsid core composed of nucleocapsid proteins (open reading frame 7, encoded by ORF7) linked to viral RNA. The nucleocapsid is surrounded by a lipid envelope in which six structural proteins: glycoproteins GP2 (ORF2a), GP3 (ORF3), GP4 (ORF4) and GP5 (ORF5), and non-glycosylated proteins M (ORF6) and E (ORF2b) is embedded. GP5 and M are considered to be the most abundant proteins in the envelope, while other envelope proteins are present in lower amounts. ORF1a and ORF1b located at the 5 'end of the genome encode nonstructural proteins.

  Like many other RNA viruses, PRRSV exhibits high genetic variability and is reflected in pathogenic mutations, interactions with the immune system, and antigenic properties of viral proteins. Although high variability exists between genotypes, virus strains are usually classified into European (EU) and North American (NA) genotypes based on ORF5 and / or ORF7 sequences.

  PRRSV has acquired numerous properties that can circumvent the host's protective immunity. These properties are the delayed production of virus-specific antibodies 1-2 weeks after infection; such antibodies reduce in vitro virus replication in primary porcine alveolar macrophages (PAM). The virus-neutralizing antibodies that are really needed are too late to affect acute viremia because they appear at low levels approximately 3-4 weeks after infection (1, 2).

  Despite this weak virus-neutralizing antibody response, a sufficient amount of such virus-neutralizing antibodies at the start of infection protects against viral replication in the lungs, viremia, and transplacental transmission of the virus. It has been shown that PRRSV-specific neutralizing antibodies can contribute in part to protective immunity (2, 3).

  PRRS viremia was found in the blood of infected pigs along with neutralizing antibodies, indicating that humoral immune response alone did not confer robust protection. Cellular immunity (CMI) has been shown to play an important role in the elimination of PRRSV (4). Progression of the CMI response in infected pigs was found to be delayed as determined by lymphocyte blastogenesis and adaptive cytokine production (eg, interferon γ; IFN-γ), and peripheral blood mononuclear cells (PBMC) In vitro recall responses were detectable approximately 4-8 weeks after infection, which correlated with the development of neutralizing antibodies (5-7). IFN-γ plays an important role in the cellular immune response against various cytopathic virus infections in animals. In pigs infected with PRRSV, IFN-γ mRNA was detected in lymph nodes, lungs and peripheral blood mononuclear cells (7).

  Exploring antigenic regions across the PRRSV structural proteome for B cell epitopes that induce virus neutralizing antibodies and T cell epitopes that induce IFN-γ has been the most challenging in veterinary virus immunology over the past 20 years This is one of the topics. As a result accumulated by the global PRRSV research group, representative papers showing such epitope mapping results are provided herein for reference (8-10).

  Despite the availability of modified live vaccines (MLV) and inactivated PRRSV virus lysate vaccines, the control of PRRSV-related diseases remains problematic. One major problem is the effectiveness in that the PRRSV vaccine is effective against homogeneous attacks but not effective against heterogeneous attacks. Also, safety issues regarding MLV have been reported in this field. The modified live vaccine is not suitable for use in boar pigs because it is not suitable for use in pregnant mature sows and young sows and may cause vaccination of vaccine virus in semen by vaccination. The modified live virus vaccine can persist in the vaccinated animal. Infection of unvaccinated animals and subsequent vaccine-virus-induced disease has been reported. There is an urgent need to develop a marker vaccine that can distinguish between infected and vaccinated pigs, thereby facilitating tracking and control of PRRSV infection.

  In summary, design of immunogenic PRRSV peptides with different functional B cell epitopes and T cell epitopes capable of inducing protective antibodies and cellular immune responses, and those that allow cross protection of PRRSV strains in pigs There is still an urgent need to design vaccine formulations with these designer peptides. Along with the availability of these rationally designed and molecularly characterized immunogenic peptides, there is also a need to identify antigenic peptides that can be recognized by antibodies from infected pigs, and these designer peptides There is also a need to develop a set of diagnostic tests (and thus a diagnostic system) to serologically distinguish between infected and vaccinated animals and allow effective control of PRRSV infection. Finally, there is a need to develop a means for low-cost production and quality control of such peptide-based marker vaccines, as well as a versatile diagnostic system to effectively monitor and control PRRS disease .

References
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BRIEF DESCRIPTION OF THE INVENTION The present disclosure relates to a peptide-based marker vaccine for porcine genital respiratory syndrome (PRRS) and a set of immunodiagnostic tests for the prevention, monitoring and control of porcine genital respiratory syndrome virus (PRRSV).

  Despite the availability of scientific information about the antigenic domain across the structural PRRSV proteome, research organizations have been able to develop no successful peptide-based vaccines against infectious agents at all for decades, There is an opinion that for most pathogens synthetic peptide vaccines are unlikely to be produced in the future. The inventor has learned a great deal from first generation biological vaccines, where a rational design approach involving selective PRRSV B- and T-cell epitopes provides a synthetic peptide-based PRRSV for the desired virus strain. It should be possible to validate and develop companion diagnostic tests for vaccines and disease monitoring and control. It is important to recognize that B cell neutralizing epitopes are mostly conformational and must be considered when designing their B cell epitope-related peptide immunogens. Identification and design of relevant B antigenic epitopes requires an understanding of the relationship between the structure of the target molecule and its function. This discipline in understanding the structure and function of the target molecule that serves the immunogen design is referred to by the inventor as “functional antigenic”. In addition, immune responses are complex and multifaceted. Vaccines against rapidly evolving RNA viruses that exist as pseudo-species populations stimulate as many different protective immune mechanisms as possible to minimize the risk of the emergence of viral variants that evade the host immune system Should always be the goal.

  Successful synthetic peptide-based vaccines will have components that stimulate the appropriate components of the immune system, including adaptive and innate immunity. Another major obstacle in the development of epitope-based peptide vaccines is due, in part, to the non-immunogenic nature of short peptides that present these epitopes. It is also necessary to include a broad repertoire of B and T epitopes to allow for universal cross-protection vaccines arising from pathogen genetic variation and host genetic variability. With vast efforts and experimental validation of synthetic peptide-based vaccines and diagnostic tests against various diseases, the inventor has developed serological studies in vivo and in vitro supplemented with T cell epitopes suitable for enhancing the desired immunogenicity. Emphasis is placed on rational design of peptide immunogens mimicking antigenic sites on natural target molecules and eliciting antibodies for functional studies. This has led to optimized antigens, immunogens, and vaccine formulations in their respective target diseases for clinical and commercial applications (11-20).

  Vaccine formulations according to various aspects of the present invention comprise a mixture of peptides derived from PRRSV GP2, GP3, GP4, or GP5 protein; each peptide individually has a B cell PRRSV neutralization / receptor binding epitope, Epitopes individually bind to artificial T helper epitopes to enhance the immunogenicity of each peptide; a mixture of peptides presenting T helper epitopes derived from PRRSV GP4, GP5, M and nucleocapsid proteins in a mixture of peptides Can be replenished to confer cellular immunity. Such viral peptide compositions can be prepared in a delivery system that is acceptable as a vaccine formulation and can provide cross protection from infection to pigs that do not have PRRSV antibodies when loaded with PRRSV.

  A diagnostic system according to various aspects of the present invention includes a set of diagnostic tests, one test providing two overlapping antigenic peptides encoding PRRSV ORF7 in an ELISA immunoassay format for optimal antibody recognition from PRRSV-infected animals. However, other tests provide GP2, GP3, GP4 and GP5-derived vaccine target peptides in an ELISA immunoassay format for optimal antibody recognition from PRRSV peptide vaccine immunized animals. These diagnostic tests are combined to form a diagnostic system for the discrimination between infected and vaccinated animals (DIVA) and thus effective monitoring and control of the disease.

  It also relates to a method for producing such a vaccine that protects pigs from PRRSV infection, and a method for producing a set of immunodiagnostic tests for DIVA and thus enabling effective control of the disease.

  The peptide or peptide composition of the present invention is a marker vaccine for cross-protecting piglets that do not have a PRRSV antibody from infection when PRRSV is loaded by cross-reactivity with a natural virus, and an infected animal and a vaccinated animal. Are designed and optimized by a vast serological verification process in the target species for the development of a set of peptide-based ELISAs to distinguish between

  The present disclosure relates to PRRSV vaccines and, in particular, to PRRSV vaccines having peptides and peptide compositions derived from the B epitope of the PRRSV GP2, GP3, GP4 or GP5 protein, each peptide optionally binding to a T helper epitope Often, the immunogenicity of the peptide may be enhanced to induce humoral immunity, including high-titer antibodies cross-reactive with PRRSV, into the immunized host.

  The peptide or peptide composition is further supplemented with additional peptides presenting TRS epitopes of PRRSV GP4, GP5, M and nucleocapsid proteins in the vaccine formulation to initiate a cellular immune response.

  Various aspects of the invention are provided. One set of embodiments relates to peptides and peptide compositions that are effective as antigens for detecting antibodies to nucleocapsid (NC) proteins from infected animals. The second set of aspects relates to peptides and peptide compositions effective for the detection of antibodies against marker vaccines targeting the GP2, GP3, GP4 and GP5 epitopes from vaccinated animals. These diagnostic tests constitute the DIVA diagnostic system and thus the effective control of the disease.

  There are another set of aspects relating to the peptide (s), homologues and analogs thereof derived from both the B cell epitope and the T cell epitope of the PRRSV protein. Such PRRSV peptides may optionally, but preferentially, bind to artificial combinatorial T helper epitopes to enhance their individual immunogenicity.

  Also, in another set of embodiments, to protect pigs against PRRSV infection, elicit both antibody and cell-mediated immune responses that are cross-reactive with natural PRRSV protein antigens together. The invention relates to vaccine formulations having peptides and peptide compositions derived from peptide immunogens derived from PRRSV B cell epitopes and T cell epitopes.

Each of such designed peptides can be chemically synthesized and quality controlled on a milligram to kilogram scale for industrial use.
Delivery vehicles and other components normally included in vaccine formulations are also provided by the various aspects of the invention.

FIG. A shows a mechanism for detecting antibodies against PRRSV GP5 protein in the cytoplasm of co-transfected HTK cell line cells by immunofluorescence according to aspects of the present invention. FIG. 1B is an immunofluorescence assay (IFA) performed by the mechanism described in FIG. 1A. Cotransfected HTK cell line cells with antibodies to PRRSV GP5 protein bound to GP5 in the cytoplasm are detected. Identification of the antigenic peptide (SEQ ID NO: 1) used for UBI PRRSV NC ELISA to detect the localization of the antigenic site on the nucleocapsid (NC) protein of PRRSV MD001 strain (Accession No. AF121131) and the antibody against the NC protein in infected animals. Identification of the antigenic peptide (SEQ ID NO: 2) used for UBI PRRSV NC ELISA to detect the localization of the antigenic site on the nucleocapsid (NC) protein of PRRSV MD001 strain (Accession No. AF121131) and the antibody against the NC protein in infected animals. Alignment of homologous nucleocapsid protein sequences from PRRSV strains MD001 (SEQ ID NO: 3), JXA1 (SEQ ID NO: 4), NA (SEQ ID NO: 5) and EU (SEQ ID NO: 6). Localization of selected B cell epitopes and T cell epitopes in the PRRSV ORF2-ORF7 protein of PRRSV JXA1 strain (Accession No. AY2G2352) suitable for marker vaccine design. Serum reactivity of porcine sera from different sources tested in the UBI PRRSV NC _NA ELISA. Detected by UBI's diagnostic system (ie, a test to detect PRRSV infected pigs and a test to detect animals vaccinated with a designer peptide immunogen derived from GP2, GP3, and GP4 proteins) and As identified, a UBI PRRSV marker vaccine formulation that elicits specific high titer antibodies against target peptide immunogens in PRRSV infected pigs. Detected by UBI's diagnostic system (ie, a test to detect PRRSV infected pigs, and a test to detect animals vaccinated with a designer peptide immunogen derived from GP3, GP4, and GP5 proteins) As identified, a UBI PRRSV marker vaccine formulation that elicits specific high titer antibodies against target peptide immunogens in PRRSV infected pigs. FIG. 7A is a photograph showing a histopathological lesion after loading with PRRS virus. In the control group of animals that developed interstitial pneumonia, the alveolar wall thickened by lymphocytes in the lungs. FIG. 7B is a photograph showing a histopathological lesion after loading with PRRS virus. Animals immunized with UBI PRRS GP5 peptide vaccine formulation. The lungs maintain a normal alveolar wall.

Detailed Description of the Invention Peptide antigens can detect immune responses, and certain peptide antigens can also stimulate immune responses. Many peptide antigens can be used for sensitive and specific detection of immune responses, but in most cases they do not act as immunogens by themselves. Peptide immunogens are a special class of peptide antigens that can be used to stimulate immune responses and detect them. According to one aspect of the invention, the peptide antigen in the PRRSV vaccine is peptide immunity having both a B cell (B) epitope and a T helper cell (Th) epitope that act together to stimulate the development of a protective immune response. There is also another set of peptide antigens that are original and capable of detecting an immune response to PRRSV infection.

  One method of B cell epitope identification utilizes a set of overlapping peptides that are nested peptides of varying lengths, typically 20-60 residues or longer. To do. These longer peptides are synthesized by a cumbersome series of independent solid phase peptide synthesis. The resulting nested and overlapping peptide set is then used for antibody binding studies to identify the peptide that best presents the immunodominant determinants including non-contiguous conformational B cell epitopes. Can do. One aspect of the invention provides a nucleocapsid peptide encoded by two overlapping PRRSV ORF7s, one having a 70 amino acid sequence (SEQ ID NO: 1, also shown in FIG. 2A) and the other a 73 amino acid sequence. (SEQ ID NO: 2, also shown in FIG. 2B) and each independently has a cluster of B cell epitopes for optimal antibody recognition. These antigenic peptides were identified empirically and optimized using serum samples from an PRRSV infected piglet and an ELISA immunoassay format. By using any immunoassay format (eg, ELISA) that is compatible with an antibody capture phase that includes a peptide antigen, an antibody that binds to a specific fragment of the PRRSV nucleocapsid protein in a blood, serum, or plasma sample from a PRRSV infected pig It can be detected and quantified.

  In certain embodiments, an optimized PRRSV antigenic peptide (SEQ ID NO: 1) of about 70 amino acids, corresponding to amino acid residues 2-71 of the full-length PRRSV nucleocapsid protein, and amino acid residues 51-123 of the full-length PRRSV nucleocapsid protein Another optimized PRRSV antigenic peptide (SEQ ID NO: 2) of about 73 amino acids corresponding to was identified. These two peptides were mixed in equal proportions in the mixture to constitute an optimal antibody capture phase for detection of antibodies against PRRSV by ELISA in infected pigs. Both these two highly antigenic peptides were found to have a cluster of immunodominant B cell epitopes and had the most prominent and consistent antigenicity against the PRRSV positive serum panel. The manufacture and use of diagnostic test kits comprising PRRSV nucleocapsid peptides (eg, SEQ ID NOs: 1 and 2) are within the scope of various exemplary embodiments of the invention.

  Certain embodiments of the PRRSV antigenic peptides of the present invention are further defined as being immunologically functional homologues of SEQ ID NOs: 1 and 2, which correspond to corresponding sequences from mutants and variants of PRRSV and It has a three-dimensional structure element. Homologous PRRSV antigenic peptides have amino acid residues that are substantially correlated with positions 2-71 and 51-123 of the nucleocapsid protein of the mutant PRRSV North American strain that originated. Such homologues are readily demonstrated by sequence alignment programs such as ClustalW (created by Julie D. Thompson, Toby Gibson of European Molecular Biology Laboratory, Germany and Desmond Higgins of European Bioinformatics Institute, Cambridge, UK. Algorithmic). FIG. 3 shows PRRSV MD001 Taiwan / 99Y / AF121131 (SEQ ID NO: 3), JXA1 Being / 06Y / EF112445 (SEQ ID NO: 4), NA / NJ-a / 04Y / AY37282 (SEQ ID NO: 5), EU / Lena / 08Y / 4 shows the alignment by ClustalW of four antigen sequences obtained from various strains of EU909691 (SEQ ID NO: 6). The PRRSV strain that is the origin of the nucleocapsid homologs aligned in FIG. 3 contains the virus of the European strain. Table 1 also shows the homologues of antigen peptide SEQ ID NO: 1 and SEQ ID NO: 2 (North American strain / MD001 / TW / AAC98536) using the original PRRSV strain EU (European strain 08V204 / Belgium / EU / GU737266). (SEQ ID NO: 7 and SEQ ID NO: 8) are exemplified. In certain embodiments, a homologue to SEQ ID NO: 1 (SEQ ID NO: 7) has an amino acid sequence from about amino acid 2 to about amino acid 72 of the PRRSV NC protein from the EU strain. In another embodiment, the homologue (SEQ ID NO: 8) has an amino acid sequence from about amino acid position 52 to about amino acid position 128 of the PRRSV NC protein from the EU strain. These two homologous peptides can also be mixed in equal proportions in the mixture to constitute an optimal antibody capture phase for detection of antibodies against PRRSV (preferably European strains) by ELISA in infected pigs.

  The homologues of the present invention are further defined as having at least 50% identity with SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments, the mutant homologue (SEQ ID NO: 7) has about 50% identity with SEQ ID NO: 1. In another embodiment, the mutant homologue (SEQ ID NO: 8) has about 64% identity with SEQ ID NO: 2.

  PRRSV GP2, GP3, GP4 and GP5 corresponding to neutralizing and / or receptor binding functional sites in addition to antigenic peptides identified from PRRSV NC protein useful for detecting antibodies from serum samples of infected animals There is an antigenic region present on the protein. Various peptide immunogens are elicited against native PRRSV proteins to assess their immunogenicity by target peptide-based ELISA and, more importantly, by immunofluorescence assay (IFA) according to embodiments of the present invention. Designed around these epitope regions for antibody cross-reactivity. There are also well-documented immunodominant T cell epitopes present on PRRSV GP4, GP5, M and NC proteins. Peptides derived from these T helper sites cause cytokine production, including IFN-γ, which causes a lymphocyte proliferative response in pigs and plays an important role in the cellular immune response to various cytopathic virus infections in animals.

  The embodiment as shown in FIG. 4 is selected according to the present invention in proteins (GP2, GP3, GP4, GP5, M, and N) encoded by PRRSV ORF2 to ORF7 based on the sequence of PRRSV JXA1 (Accession No. AY2G2352) The distribution and location of B cell epitopes and T cell epitopes will be described.

  Another aspect of the present invention is the sequence of four optimized and selected PRRSV B cell epitope cluster peptides: GP5.3 (V21-E65) (SEQ ID NO: 9), GP2B (V111-L136) (SEQ ID NO: 10), GP3B (C57-C75) (SEQ ID NO: 11), and GP4B (C52-C69) (SEQ ID NO: 12). These B cell epitope clusters are located around sites that have neutralizing and receptor binding properties.

  Other aspects of the invention provide immunologically functional analogs of these four PRRSV B cell epitope cluster peptides. Table 3 shows the origin strains MD001, JXA1, NA and EU, using homologous GP5 (SEQ ID NO: 13-15), GP2 (SEQ ID NO: 17-19), GP3 (SEQ ID NO: 21-23), and The alignment of the B epitope cluster peptide sequence derived from GP4 (SEQ ID NO: 25-27) is shown. The consensus sequence for each of the B cell epitope cluster peptides (SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28) is also shown, and the amino acids assigned to variable positions are most frequently applied to those positions. It is an amino acid.

  Immunologically functional analogs of B cell epitope cluster peptides are SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and homologs that retain substantially the same immunological properties as the original antigenic peptide. Including variants. For example, a variant that is a functional analog or homologue of SEQ ID NO: 9 is a conservative substitution at one amino acid position; a change in total charge; a covalent bond to another moiety; or a small addition, insertion, deletion or conservation And / or any combination thereof. Thus, an antibody that binds to the PRRSV GP5.3 B epitope (V21-E65) antigen peptide (eg, SEQ ID NO: 9) is also substantially an immunologically functional analog of that PRRSV GP5.3 B epitope antigen peptide. Will bind with similar potency. In some embodiments, the functional analog has at least 40% identity to SEQ ID NO: 9 or homolog. In another embodiment, the functional analog has at least 56% identity to SEQ ID NO: 11 or homolog. In yet another embodiment, the functional analog has at least 72% identity to SEQ ID NO: 12 or homolog. In yet another embodiment, the functional analog has at least 80% identity to SEQ ID NO: 10 or homolog. In yet another embodiment, the functional analog has at least 94% identity to SEQ ID NO: 12 or homolog.

  In certain embodiments, as shown in Table 3, an immunologically functional analog of PRRSV GP5.3 B epitope cluster peptide (V21-E65) is modified by conservative substitutions and by insertions or deletions. Including a version of the PRRSV GP5.3 B epitope cluster peptide. In this embodiment, an immunologically functional analog can be modified by conservative substitutions from SEQ ID NO: 9 or from a homologue of SEQ ID NO: 9.

  A conservative substitution is one in which one amino acid residue is replaced with another amino acid residue having similar chemical properties. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine Positively charged (basic) amino acids include arginine, lysine and histidine; negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

  In another embodiment, as shown in Table 3 and Table 4, the immunologically functional analog is modified by amino acid addition at the N-terminus, C-terminus, and / or by insertion into the middle of the peptide. Can do. In various aspects of the invention, the addition is an addition to the N-terminus or C-terminus of the peptide. The addition can be an addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues (SEQ ID NOs: 9 and 30). Such additions may constitute amino acid sequences that are not present in the PRRSV protein and do not alter the immunogenicity of the PRRSV B epitope cluster peptide. As an addition not present in the PRRSV B epitope cluster peptide, a small charged sequence (eg, lysine-lysine-lysine), an amino acid capable of forming a branched chain structure (eg, εN-lysine), or an amino acid capable of forming a cyclized structure (For example, cysteine), but is not limited thereto. In one embodiment of the present invention, the addition of an amino acid sequence that is not present in PRRSV is an addition of 5 amino acids or less. Amino acid additions can be either classical or non-classical amino acids, or mixtures thereof.

  In another specific embodiment, the immunologically functional analog can be modified by amino acid deletions to the N-terminus, C-terminus, and / or intermediate portion of the peptide. In various embodiments, the deletion is a deletion relative to the N-terminus or C-terminus of the peptide. The deletion can be a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues. In certain embodiments as shown in Table 4, the amino acid sequence deletion is a deletion of 9 amino acids or less (SEQ ID NOs: 33 and 34).

  In another embodiment, as shown in Table 7, an immunologically functional analog of a PRRSV B epitope cluster peptide includes a PRRSV B epitope cluster antigen peptide that has been modified by alteration of charge. Such charge alterations may be the result of amino acid substitutions, additions or deletions, or covalent attachment of charged molecules. Changing the charge may result in the peptide becoming more basic, more acidic, or more neutral compared to the unmodified peptide. In certain embodiments, the peptide is made more basic by adding 1-5 lysine residues to the N-terminus or C-terminus. In a more specific embodiment, the peptide is made more basic by adding three lysine residues to the N-terminus.

  As a non-limiting example, an immunologically functional analog of a peptide of the invention can have from 1 to about 5 additional amino acids (classical and non-classical) appended to the terminal amino acid. For example, the sequence Lys-Lys-Lys can be added to the amino terminus of this PRRSV B cell epitope cluster peptide to alter the charge.

  The peptides can be readily synthesized using standard techniques such as the Merrifield solid phase synthesis method and the myriad available improvements to the process. The peptide can also be produced using recombinant DNA technology. Thus, nucleic acid molecules encoding PRRSV B cell epitope cluster antigen peptides and immunologically functional analogs of PRRSV B cell epitope cluster antigen peptides, and their complements, are various exemplary embodiments of the present invention. It is included by this aspect. Also encompassed by the various exemplary aspects of the invention are vectors, particularly expression vectors, comprising nucleic acid molecules encoding PRRSV B cell epitope cluster antigen peptides and immunologically functional analogs. Host cells containing these vectors are also encompassed by various exemplary embodiments of the invention.

  Various exemplary aspects of the invention also include methods of producing immunologically functional analogs of PRRSV antigenic peptides and PRRSV B cell antigenic peptides. For example, the method can include treating a host cell comprising an expression vector comprising a PRRSV antigen peptide and / or a nucleic acid molecule encoding an immunologically functional analog of the PRRSV antigen peptide with the immunology of the PRRSV peptide and / or PRRSV peptide. Incubating under conditions such that a functionally functional analog is expressed.

  One aspect of the invention provides a peptide composition produced by solid phase synthesis. This embodiment can use a controlled and well-defined immunogen derived from the lysate or secretion of infected cells. The quality of the antigen produced by this aspect of the chemical process is controlled and defined, so that the antigenicity, immunogenicity and yield reproducibility can be ensured as a result. Also, since no biohazardous material is used in the production of peptide antigens, the risk is reduced and the need for costly biological containment is eliminated. The site-specific immunogen presents a high molar concentration of the selected epitope, thus ensuring both the safety and immune efficacy of the vaccine with the PRRSV antigen peptide composition.

  In certain embodiments, the peptides of the invention are synthesized. By using the defined PRRSV antigen synthetic peptides, false positive results are minimized when used as antigens for antibody detection and diagnosis in piglets. When used as an immunogenic component of a vaccine, by using a defined synthetic peptide having a known B cell epitope and Th epitope as an immunogen, derived from a host cell infected with PRRSV or infected with a recombinant virus As well as unwanted non-PRRSV specific immune reactions caused by the presence of antigenic material that may be co-purified with the PRRS virus and / or recombinant protein from the recombinant protein expression system. For example, serum from pigs may have antibodies against host cells or against recombinant Escherichia coli, yeast or baculovirus, which are later used for diagnostic tests based on biologically derived antigens Such immune responses produced by vaccines that are cross-reactive with antigenic material and contain these foreign immunogens as components are not protective. In contrast, pigs vaccinated with the PRRSV peptide vaccines of the present invention can be isolated from proteins derived from host cells or expression vectors, such as recombinant Escherichia coli, yeast or baculo that are co-purified with biologically derived PRRSV antigens. It will produce a focused immune response that is free of adverse antibodies and other immune responses to proteins from the virus.

  This embodiment of the synthetic peptide also minimizes interference from impurities that occur during production. In the case of long synthesis, peptide analogs are also generated due to events during the elongation cycle, including amino acid insertions, deletions, substitutions, and premature termination, despite strict control of coupling efficiency, so the target A plurality of peptide analogs are produced together with the peptide synthesis. Nonetheless, such peptide analogs are both antigenic and immunogenic when used in immunological applications, either as solid phase antigens for immunodiagnostics or as immunogens for vaccination. It is still suitable in peptide preparations as a contributor.

  In 25 years of experience in the immunological use of synthetic peptides, the inventors have determined that the range of structural variability that allows retention of the intended immunological activity is specific drug activity with small molecule drugs, or Found to be much more flexible than the range of structural variability that allows retention of the desired activity and undesirable toxicity seen in macromolecules produced simultaneously with biologically derived drugs. It was. This is either a peptide analog that was either intentionally designed or inevitably produced by a mistake in the synthesis process as a mixture of deleted sequence by-products with similar chromatographic and immunological properties to the intended peptide This is why it is often as effective as a purified preparation of the desired peptide. As long as a discriminating QC procedure is developed that monitors both the manufacturing process and the product evaluation process to ensure the reproducibility and effectiveness of the final product using these peptides, a designed analog And unintended analog mixtures are effective.

  In other aspects of the invention, endogenous PRRSV Th peptides and their homologues (SEQ ID NOs: 47-79) can be included in the vaccine composition. The presence of the Th peptide can improve the immunogenicity of the PRRSV peptide vaccine. Immunogenic peptides derived from the PRRSV B epitope (including the homologues and analogs described above) can be mixed with the endogenous PRRSV Th epitope.

  In other aspects of the invention, the endogenous PRRSV Th peptide can be presented as a combinatorial sequence, where the combination of amino acid residues is determined based on the sequence of homologues to the PRRSV Th peptide. Represented in position. Aggregates of combinatorial peptides can be synthesized in one synthetic process by adding a mixture of designed protected amino acids instead of one specific amino acid at a specific position during the synthetic process. Such combinatorial PRRSV Th peptide aggregates allow a broad T helper epitope coverage for animals of diverse genetic backgrounds. Representative combinatorial sequences of PRRSV Th peptides are shown in SEQ ID NOs: 80-90 in Table 7, as derived from SEQ ID NOs: 47-79 in Table 6 for each of the PRRSV Th epitopes.

  In some embodiments, described as SEQ ID NOs: 47, 51, 55, 59, 61, 63, 67, 70, 74, 76 based on the JXA1 sequence (also shown in Table 6) and bind to the PRRSV B peptide immunogen. Complementing the immunogenicity of the PRRSV B epitope peptide immunogen with a Th peptide having a cluster of immunodominant PRRSV Th epitopes derived from ORF4, ORF5, ORF6 and ORF7, as described in Example 5 The immunogenicity of the PRRSV vaccine formulation can be enhanced. By including SEQ ID NOs: 47, 51, 55, 59, 61, 63, 67, 70, 74, 76 as free peptides without covalently binding to the B epitope peptide immunogen, the immunogenicity of these vaccine formulations is increased. Can be improved. In another embodiment as described in Example 10, SEQ ID NOs: 47, 51, 55, 59, 61, 63, 67, 70, 74, 76 for group 2 without covalently binding to the B epitope peptide immunogen. And for groups 1, 3, and 4, the immunogenicity of these vaccine formulations can be improved by including SEQ ID NOs: 80-90 as free peptides.

  In another aspect, the PRRSV peptide (including homologs and analogs described above) can be covalently linked to a peptide comprising a sequence known to contain a Th epitope, with or without a spacer. . This aspect can provide enhanced immunogenicity over equivalent immunogens that are not covalently linked to a Th epitope. In certain embodiments, the peptide comprising a Th epitope is covalently linked to the N-terminus (SEQ ID NO: 38) and / or C-terminus (SEQ ID NO: 39) of the PRRSV peptide. In another specific embodiment, the spacer has the sequence Lys-Lys-Lys-εNLys (SEQ ID NO: 36), or a single amino acid εNLys, and is also shown in Table 5 (SEQ ID NOs: 43 and 42, respectively). In certain embodiments, the peptide comprising a Th epitope is covalently linked to the amino terminus of the PRRSV peptide. In certain embodiments, a peptide (SEQ ID NO: 40) as shown in Table 5 comprising a Th epitope is an artificial combinatorial Th peptide sequence linked to the amino terminus via a Lys-Lys-Lys-εNLys spacer (SEQ ID NO: 36). No. 35 (shown in Table 5), shown as SEQ ID NO: 40.

Various aspects of the invention relate to vaccine compositions for protecting pigs against PRRSV. In an exemplary embodiment, the vaccine has an immunogenic peptide antigen or peptide immunogenic composition and an acceptable delivery vehicle or adjuvant. In various embodiments, the PRRSV vaccine composition has a peptide antigen or peptide antigen composition and a veterinarily acceptable delivery vehicle or adjuvant, the peptide antigen comprising:
a) PRRSV B cell epitope cluster peptide antigens GP5.3 (V21-E65) (SEQ ID NO: 9), GP2B (V111-L136) (SEQ ID NO: 10), GP3B (C57-C75) (SEQ ID NO: 11) and GP4B (C52) -Any one of (C69) (SEQ ID NO: 12);
b) a homolog of (a);
c) an antigenically and immunologically functional analog of (a) or (b);
d) from (a), (b), or (c) having at least one conservative amino acid substitution, amino acid addition, and / or amino acid deletion; and e) from any combination of (a)-(d) Having an amino acid sequence selected from the group consisting of

  In the PRRSV vaccine embodiment, the charge of the peptide antigen is altered by adding or deleting 1 to 5 amino acids. In another embodiment of PRRSV cutin, the antigenically and immunologically functional homolog or analog is derived from GP2, GP3, GP4 and GP5, PRRSV B cell epitope cluster peptide antigen GP5.3 (V21-E65) (SEQ ID NO: 9), an amino acid derived from any one of GP2B (V111-L136) (SEQ ID NO: 10), GP3B (C57-C75) (SEQ ID NO: 11) and GP4B (C52-C69) (SEQ ID NO: 12) It has at least 50% identity with the antigen of the sequence. In certain embodiments, the peptide antigen has an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12.

  In another embodiment of the PRRSV vaccine, the peptide antigen further has a T helper epitope covalently linked to the N-terminus or C-terminus of the peptide antigen. In certain embodiments, the T helper epitope is covalently linked to the amino terminus of the peptide antigen. In another specific embodiment, the T helper epitope is covalently linked to the peptide antigen via a spacer having at least one amino acid. In certain embodiments, the T helper epitope is SEQ ID NO: 35. In yet another specific embodiment, the spacer is Lys-Lys-Lys-εNLys (SEQ ID NO: 36). In yet another specific embodiment, the spacer is εNLys. In certain embodiments, the peptide antigen is SEQ ID NO: 42, 43, 44, 45 or 46.

  In various exemplary embodiments, any amount of immunogenic peptide antigen can be used to elicit an immune response in an animal. In certain embodiments, the amount of peptide antigen is from about 0.1 μg to about 100 mg. In another specific embodiment, the amount of peptide antigen is from about 1 μg to about 10 mg. In yet another embodiment, the amount of peptide antigen is from about 10 μg to about 1 mg.

  In various embodiments of the PRRSV vaccine composition, the composition comprises an equimolar of 11 PRRSVT helper epitope peptides of SEQ ID NOs: 47, 51, 52, 55, 59, 61, 63, 67, 70, 74, and 76. It further has a mixture. In certain embodiments, the amount of an equimolar mixture of SEQ ID NOs: 47, 51, 52, 55, 59, 61, 63, 67, 70, 74, and 76 is about 0.1 μg to about 1 mg. In a more particular embodiment, the amount of an equimolar mixture of SEQ ID NOs: 47, 51, 52, 55, 59, 61, 63, 67, 70, 74, and 76 is from about 1 μg to about 100 μg.

  In various exemplary embodiments, any type or amount of delivery vehicle or adjuvant can be used. In certain embodiments, the delivery vehicle and adjuvant are Montanide ™ ISA 50V (an oily vaccine adjuvant composition consisting of vegetable oil and mannide oleate to produce a water-in-oil emulsion). , Tween® 80 (also known as polysorbate 80 or polyoxyethylene (20) sorbitan monooleate), CpG oligonucleotides, and / or any combination thereof.

  In certain embodiments, the PRRSV vaccine composition has a peptide antigen of SEQ ID NO: 33 and a veterinary acceptable delivery vehicle or adjuvant, wherein the amount of peptide antigen is from about 10 μg to about 1 mg.

  Another aspect of the invention relates to a method of protecting a PRRSV maternally derived antibody (MDA) positive or non-positive piglet against PRRSV infection, which method includes any of the exemplary aspects described above. Administering a vaccine to be administered.

  Using a mixture of two PRRSV NC peptides of SEQ ID NO: 1 and SEQ ID NO: 2 prepared according to the present disclosure, this peptide can be used in an antigenically effective amount in the capture phase of an immunoassay, eg, in a solid phase immunosorbent in an ELISA test kit. By using it, a PRRSV antibody can also be detected. According to certain aspects of the invention, any compatible immunoassay format can be used with the peptide of interest. Such formats are well known to those skilled in the art and are described in many standard immunology manuals and texts. See, for example, Harlow et al., 1988 (24). These include, among other well-known immunoassay formats, enzyme-linked immunosorbent assay (ELISA), enzyme immunodot assay, aggregation assay, antibody-peptide-antibody sandwich assay, peptide-antibody-peptide sandwich assay. In certain embodiments, the immunoassay is an ELISA using a solid phase coated with a peptide composition comprising two PRRSV NC antigen peptides (SEQ ID NO: 1 and SEQ ID NO: 2).

  According to one aspect of the present invention, the peptide allows sera from mature sows and young sows, mature boars and castrated pigs, and piglets to be tested for PRRSV infection by screening ELISA before vaccination. Can be evaluated for maternally derived anti-PRRSV antibody levels and to measure the level of immune response in vaccinated piglets against vaccines using PRRSV antigenic peptides Can do.

In certain embodiments, an ELISA immunoassay can be used to test porcine blood, serum or plasma samples for the presence of anti-PRRSV antibodies, the immunoassay comprising:
i. Attaching a mixture of two PRRSV NC peptides (SEQ ID NO: 1 and SEQ ID NO: 2) to a solid support;
ii. Exposing the peptide attached to the solid support to a porcine blood, serum or plasma sample containing the antibody under conditions that promote binding of the antibody to the peptide; and iii. Detecting the presence of an antibody bound to the peptide attached to the solid support;
Have

In another specific embodiment, an ELISA immunoassay can be used to test porcine blood, serum or plasma samples for the presence of anti-PRRSV antibodies, the assay comprising:
i. Attaching a mixture of two PRRSV NC peptides (SEQ ID NO: 7 and SEQ ID NO: 8) to a solid support as a homologue of SEQ ID NO: 1 and SEQ ID NO: 2 derived from a European strain sequence;
ii. Exposing the peptide attached to the solid support to a porcine blood, serum or plasma sample containing the antibody under conditions that promote binding of the antibody to the peptide; and iii. Detecting the presence of an antibody bound to the peptide attached to the solid support;
Have

  In an exemplary use of the subject ELISA kit, after dilution of the porcine serum sample to be tested with the sample diluent, one or more of the above PRRSV peptides and any antibodies, if present for a period of time, are peptide-sensitized. Contact under conditions that bind to the solid phase. After removal of unbound material (eg, by washing with phosphate buffered saline), the secondary complex is converted to labeled antibody or labeled protein A, labeled protein G, or labeled protein A against porcine specific IgG. Contact with / G. These antibodies or proteins A, G or A / G bind to this secondary complex to form a tertiary complex, and these secondary antibodies or protein A or G or A / G are labeled with a reporter molecule. Therefore, the tertiary complex is detected when provided to the detection means. Reporter molecules can be enzymes, radioisotopes, fluorophores, bioluminescent molecules, chemiluminescent molecules, biotin, avidin, streptavidin, and the like. In the case of an ELISA, the reporter molecule is preferably an enzyme.

Specific embodiments of the present invention include, but are not limited to:
(1) A porcine genital respiratory syndrome (PRRS) vaccine composition comprising a peptide antigen and a veterinary acceptable delivery vehicle or adjuvant, wherein the peptide antigen is a) SEQ ID NO: 9, SEQ ID NO: 10, Having an amino acid sequence selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 12, and any combination thereof; b) a homolog of (a); and c) any combination of (a) or (b); PRRS vaccine composition.
(2) The PRRS vaccine according to (1), wherein the peptide antigen has the amino acid sequence of SEQ ID NO: 9.
(3) The PRRS vaccine according to (1), wherein the peptide antigen has the amino acid sequence of SEQ ID NO: 10.
(4) The PRRS vaccine according to (1), wherein the peptide antigen has the amino acid sequence of SEQ ID NO: 11.
(5) The PRRS vaccine according to (1), wherein the peptide antigen has the amino acid sequence of SEQ ID NO: 12.
(6) The PRRS vaccine according to (1), wherein the peptide antigen is altered by adding or deleting 1 to 5 amino acids.
(7) The PRRS vaccine according to (1), further having a T helper epitope covalently bound to the amino terminus or carboxyl terminus of the peptide antigen.
(8) The PRRS vaccine according to (7), wherein the T helper epitope is SEQ ID NO: 35.
(9) The PRRS vaccine according to (7), wherein the T helper epitope is covalently bound to the peptide antigen via a spacer having an ε-lysine residue.
(10) The PRRS vaccine according to (9), wherein the spacer is SEQ ID NO: 36.
(11) The PRRS vaccine according to (1), further comprising a T helper epitope that is not bound to an antigen peptide, wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 47 to 90.
(12) The PRRS vaccine according to (1), wherein the total amount of peptide antigens is about 10 μg to about 1 mg.
(13) The PRRS vaccine according to (1), wherein the delivery vehicle and adjuvant are selected from the group consisting of Montanide ISA 50V, polyoxyethylene (20) sorbitan monooleate, and CpG oligonucleotides.
(14) A method for protecting a piglet against PRRS infection, comprising a step of administering the vaccine according to (1).
(15) a) a peptide antigen and a veterinary acceptable delivery vehicle or adjuvant, b) a peptide antigen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 31, and combinations thereof, And c) a PRRSV vaccine composition comprising a PRRSV Th peptide selected from the group consisting of SEQ ID NOs: 80-90 and combinations thereof.
(16) a) a peptide antigen and a veterinary acceptable delivery vehicle or adjuvant, b) a peptide antigen having an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 44, 45, 46, and combinations thereof; And c) a PRRSV vaccine composition comprising a PRRSV Th peptide selected from the group consisting of SEQ ID NOs: 80-90 and combinations thereof.
(17) a) attaching the mixture of SEQ ID NO: 1 and SEQ ID NO: 2 to a solid support, b) under the conditions that promote the antibody binding to the peptide attached to the solid support, A method of diagnosing PRRS infection, comprising: exposing a porcine blood, serum or plasma sample comprising an antibody; and c) detecting the presence of an antibody bound to the peptide attached to the solid support.
(18) a) attaching the mixture of SEQ ID NO: 7 and SEQ ID NO: 8 to a solid support, b) under conditions that promote the antibody binding to the peptide attached to the solid support, For testing the presence of an anti-PRRSV antibody comprising: exposing to a porcine blood, serum or plasma sample comprising the antibody; and c) detecting the presence of an antibody bound to the peptide attached to the support. ELISA immunoassay.

  The following examples serve to illustrate the invention and should not be used to limit the scope of the invention.

For evaluation of immune serum titers for cross-reactivity between target PRRSV B cell epitope cluster peptide antigens and native PRRSV protein antigens by immunofluorescence assay (IFA) using PRRSV co-transfected HTK cells Serological assay

  Antibodies against the PRRSV subunit proteins, GP3, GP4 and GP5, are co-transfected with plasmids encoding PRRSV orf3, orf4 and orf5, respectively, downstream of the recombinant vaccinia virus rVVT7 (T7 polymerase recombinant vaccinia virus) and the T7 promoter. It can be detected by immunofluorescence using HTK cells. This method results in transient expression of these PRRSV proteins in mammalian cells with high fidelity along with their original viral replication. Since PRRSV GP3, GP4 and GP5 consist of transmembrane domains and side chain modifications, the best PRRSV protein structure can only be generated de novo. Each PRRSV subunit protein, GP2, GP3, GP4 or GP5, was de novo synthesized and co-transfected HTK cells were subjected to intracellular staining with immune sera for evaluation in an immunoassay for the presence of specific antibodies against PRRSV. Used as target cells to do. This immunofluorescence assay system allows the best conditions for the detection of antibodies against the native PRRSV antigen with high specificity and sensitivity.

  According to the mechanism illustrated in FIG. 1A, HTK cell line cells were infected with T7 polymerase recombinant vaccinia virus (rVVT7) (21) and PRRSV-orf3, orf4 and orf5 by lipofectamine ™ (Invitrogen). Co-transfected with plasmid (22). Specifically, the construction of the pRRSV plasmid for expression of the native PRRSV protein mediated by the T7 polymerase promoter was performed as follows: The full-length pRRSV gene (from Taiwan PRRSV MD001 strain (Accession No. AF035409)) was obtained. Amplified by polymerase chain reaction (PCR) and cloned into pCR2.1Topo® plasmid vector (Invitrogen). The expression ability of the pRRSV plasmid was confirmed by sequencing showing the complete nucleotide sequence for orf3, orf4 and orf5 from PRRSV strain MD001.

  HTK cells are grown in 96-well plates to 80% confluuency, infected with rVVT7 (22) and then individually with pRRSV orf3, orf4 and orf5 plasmids by Lipofectamine ™ (Invitrogen). Co-transfected. Twenty-four hours after cotransfection, cells were fixed with 80% acetone and the plates were stored at −80 ° C. for detection of antibodies to PRRSV protein by immunofluorescence assay (IFA).

  The diagram in FIG. 1A shows an HTK cell line cell infected with T7 polymerase recombinant vaccinia virus (T7 / vac) (21) and co-transfected with the pCR-orf5 plasmid by Lipofectamine ™ (Invitrogen). . Recombinant GP5 protein is transported to the cytoplasm after translation (22). The anti-GP5 antibody binds to the cytoplasm of the transfected HTK host cell according to one embodiment of the invention and is detected by immunofluorescence of labeled secondary antibody. By this method, antibodies to authentic PRRSV GP5 protein are detected with high specificity by transient eukaryotic expression of full-length PRRSV GP5 protein in HTK cells via the prokaryotic T7 promoter. Similar transfections with pCR-full-length PRRSV genomes were also performed for PRRSV GP2, GP3, GP4, M and N proteins by transient eukaryotic expression in HTK cells of full-length PRRSV genome via the prokaryotic T7 promoter. It can be used for detection with high specificity.

Determination of PRRSV antibody titer by immunofluorescence assay (IFA) First , a serum sample was diluted 10-fold with PBS, and then 2-fold serial dilution was performed. For each test run, expression of PRRSV nucleocapsid protein by the pRRSV orf-7 plasmid was verified, including both positive control sera from PRRSV infected SPF pigs and negative control sera from uninfected SPF pigs. Serum samples (shown in FIG. 1B) that produced a fluorescent signal localized in the cytoplasm at dilutions higher than 1:10 were scored as having an IFA titer of>10; from these titers, infection with PRRSV Animals were shown. For sera from animal vaccines that contain “target peptide” specific antibodies as a result of vaccination, the cross-reactivity of those antibodies against specific target proteins or the complete PRRSV genome is compared to the corresponding target native PRRSV proteins (eg, GP2 It can be evaluated by immunofluorescence assay (IFA) using 3, 4, or 5). All tests of serum samples taken from naturally infected pigs or from pigs vaccinated with the PRRSV peptide based vaccine were performed under the code.

PRRSV B cell epitope cluster peptide antigen-based ELISA for evaluation of immunogenicity of designer peptides presenting neutralization / receptor binding sites of PRRSV
The wells of a 96 well plate were individually coated for 1 hour at 37 ° C. with 100 μL of individual target peptide at 2 μg / mL unless otherwise specified in 10 mM NaHCO ₃ buffer (pH 9.5).

  Peptide-coated wells were incubated with 250 μL of 3 wt% gelatin in PBS for 1 hour at 37 ° C. to block non-specific protein binding sites and then PBS containing 0.05% by volume TWEEN® 20 Washed 3 times with and dried. Porcine serum and negative control serum positive for IRS for PRRSV antibody at 1:20 with PBS containing 20 volume% normal goat serum, 1 weight% gelatin and 0.05 volume% TWEEN® 20 unless otherwise noted. Diluted. 100 μL of this diluted specimen was added to each well and reacted at 37 ° C. for 60 minutes.

The wells were then washed 6 times with 0.05 volume% TWEEN® 20 in PBS to remove unbound antibody. Horseradish peroxidase-conjugated goat anti-pig IgG was used as a labeled tracer to bind to the antibody / peptide antigen complex formed in the positive wells. 100 μL of peroxidase-labeled goat anti-pig IgG at the optimal titer previously titrated in PBS solution of 1 volume% normal goat serum containing 0.05 volume% TWEEN® 20 was added to each well. Incubated for an additional 30 minutes. The wells were washed 6 times with 0.05 volume% TWEEN® 20 in PBS to remove unbound antibody, and 0.04 wt% 3 ′, 3 ′, 5 ′, in sodium citrate buffer. The mixture was further reacted for 15 minutes with 100 μL of the substrate mixture containing 5′-tetramethylbenzidine (TMB) and 0.12% by volume hydrogen peroxide. This substrate mixture was used to detect peroxidase label by forming a colored product. The reaction was stopped by adding 100 μL of 1.0 MH ₂ SO ₄ and the absorbance at 450 nm (A ₄₅₀ ) was measured.

Serum dilution was performed according to the purpose of detecting PRRSV antibodies in animal serum. (A) Use a 1:20 dilution to identify potential natural infections, record A ₄₅₀ measurements, and use intrinsic intrinsic negative controls for cut-off calculations; or ( b) To determine the antibody titer of pigs inoculated with peptide-based PRRSV vaccine formulations, 10-fold serial dilutions of sera from 1:10 to 1: 10,000 were tested and the titer of the tested sera (Log ₁₀ represented by) as calculated by linear regression analysis of the a _450.

Identification of optimal PRRSV antigenic peptides for diagnostic use to distinguish between infected and vaccinated animals. From PRRSV isolates JXA1, LV, EU, NA already published sequences and PRRSV genomic sequences from Taiwan strain MD001. Used to estimate the protein sequence from the open reading frame and use the data obtained from the protein sequences encoded by ORFs 2, 3, 4, 5, 6 and 7 to detect antibodies in sera from animals infected with PRRSV An antigen peptide presenting a functional neutralization / receptor binding site for use in a marker vaccine formulation to detect specific antibodies elicited in animals administered a marker vaccine formulation Designed.

We use bioinformatics information and classical immunization experiments to determine the number of peptides that are screened to identify optimal B and T cell epitope clustering peptides for diagnostic and vaccine applications. A limiting strategy was used.
The development of an urgently required tool for distinguishing between infected and vaccinated animals (referred to as the “DIVA” system) complements the marker vaccine design process.

  Most antibodies produced during PRRSV infection in pigs are nucleocapsid protein (NC) proteins encoded by PRRSV ORF7 (the major antigenic determinant for which is relatively well conserved among strains from the same continent) Because of its specificity, this NC protein was targeted as a suitable candidate for detection of virus specific antibodies and diagnosis of infection and disease. Advantages of using synthetic peptide-based antigens for antibody capture are due to the absence of cellular components unrelated to PRRSV that cause false positives, and due to the inherent nature of epitope cluster peptides It is well known that there is an improvement in sensitivity.

  Positive sera from animals with known PRRSV infection were used to screen for overlapping PRRSV ORF7-encoded NC peptides for potent and consistent antigenicity that may aid in antibody detection. Negative sera were collected from normal pigs and SPF pigs known to be free of PRRSV infection. Data on epitope mapping of NC protein of North American MD001 strain is summarized in FIG. An indirect ELISA was used in which the designer peptide was used for plate coating at 2 mL / mL at 0.1 mL per well. Two series (ae) of peptides were synthesized in which the sequence from the MD001 NC protein was gradually lengthened. A 4171 series peptide (shown in FIG. 2A) was synthesized at the C-terminus beginning at residue 71, and a 4172 series peptide (shown in FIG. 2B) was synthesized at the C-terminus beginning at residue 123. These peptides were individually used for plate coating and tested for sensitivity and specificity for each peptide with 3 pooled PRRSV positive sera with a panel of 12 validated PRRSV negative sera.

  As shown in FIG. 2A, among peptides from this 4171 series tested with 11mer antigenic epitopes having the sequence “PNNNNGKQQKKK” (SEQ ID NO: 91) located at the N-terminus of peptide 4171e, peptide 4171e (SEQ ID NO: 1) Was found to be the most antigenic.

  As shown in FIG. 2B, peptide 4172e (SEQ ID NO: 2) is the most antigenic among peptides derived from this 4172 series tested with an 18mer antigen epitope having the sequence “EKPHFPPLATTEDVRRHHFT” (SEQ ID NO: 92) located at the N-terminus. It turns out that the nature is high.

  The 18-mer antigen epitope “EKPHFPPLATTEDVRRHHFT” (SEQ ID NO: 92) was not antigenic when presented within peptides 4171a-d. However, this 18-mer peptide epitope presents itself within the full-length 4171e molecule (SEQ ID NO: 1) and forms a large conformational epitope when combined with the 11-mer antigen epitope “PNNNNGKQQKKK” on SEQ ID NO: 1. To do. Furthermore, the unexpected immunodominant antigenicity of the two peptides of 70mer and 73mer (SEQ ID NOs: 1 and 2, respectively) presents a large exposed surface on the NC protein that presents a sequence of long continuous and non-contiguous epitopes Consistent with the long-chain peptide.

  Antigen peptides 4171e (SEQ ID NO: 1) and 4172e (SEQ ID NO: 2) were used for plate coating at a mixing ratio of 2: 1 at 0.1 μg / well at 3 μg / mL, which captured antibodies against the PRRSV North American strain / Part of UBI PRRSV NC ELISA for detection.

  Homologous NC protein sequences from various PRRSV strains (MD001, JXA1, NA and EU) were aligned as shown in FIG. Table 1 based on the amino acid sequence of the PRRSV nucleocapsid protein of the originating European strain, despite the significant substitution, insertion and deletion of amino acid residues in the originating EU sequence compared to the parent MD001 sequence Substituted peptide homologues as shown in (SEQ ID NO: 3 and SEQ ID NO: 4) are similarly used as antigens in UBI PRRSV NC EU ELISA for capture / detection of antibodies against PRRSV European strains.

  The sensitivity and specificity of the UBI PRRSV NC ELISA was further verified using a panel of 30 sera previously characterized for anti-PRRSV NC protein reactivity by IFA testing as a positive serum panel. As shown in Table 2, the UBI PRRSV NC ELISA has roughly the same level of sensitivity for the NC protein as the IFA test when the limit of detection by IFA was about 1:16 dilution. Using the UBI PRRSV NC ELISA test as an auxiliary serology tool, as described in other examples, assessing field samples, distinguishing infected and vaccinated animals, and using PRRSV marker peptide-based vaccines evaluated.

PRRSV GP2 with serological verification peptide design and sequence of synthetic about 10 to about 70 amino acids in length to identify the antigenic peptide used in PRRSV marker vaccine, GP3, GP4, GP5, M and NC protein derived from PRRSV B A broad repertoire of PRRSV antigenic peptides was designed that present both cellular and T cell epitope cluster sites. For some peptides, amino acid substitutions at the appropriate sites allowed the formation of cyclic peptides that constrained local structure maintenance to maximize cross-reactivity with the corresponding native protein. Other antigenic peptides were conjugated with artificial combinatorial Th peptides (eg, UBITh3, SEQ ID NO: 15) to increase their respective immunogenicity. All peptides are subjected to a laborious and time-constrained “serologic validation” process, including repetitive cycles of peptide synthesis, vaccine formation, animal immunization, and serological testing, and each diagnosis and Candidate peptides were obtained for further testing in target animals that received our initial application for vaccines.

  All peptides used for immunogenicity tests were synthesized using Applied BioSystems Peptide Synthesizer Models 430A, 431 and 433 using Fmoc chemistry. Each peptide was generated by independent synthesis on a solid support using an N-terminal Fmoc protection and a trifunctional amino acid side chain protecting group. The completed peptide was cleaved from the solid support and the side chain protecting group was removed with 90% trifluoroacetic acid. Synthetic peptide preparations were characterized by matrix-assisted laser desorption time-of-flight (MALTDTOF) mass spectrometry for correct composition and by reverse phase HPLC for content including synthesis profile and concentration. For long syntheses, peptide analogs were also generated due to events during the elongation cycle (including amino acid insertions, deletions, substitutions, and premature termination), despite the tight control of coupling efficiency. A plurality of peptide analogs were produced together with the peptide. Nevertheless, such peptide analogs contribute to antigenicity and immunogenicity when used in immunological applications, either as antibody capture antigens for immunodiagnostics or as immunogens for vaccination. It was still suitable in peptide preparations. As long as a discriminating QC procedure is developed to monitor both the manufacturing process and the product evaluation process to ensure the reproducibility and effectiveness of the final product using these peptides, it is deliberately designed Such peptide analogs produced as a by-product mixture during the synthesis process are typically as effective as purified preparations of the desired peptide.

  FIG. 4 shows selection on proteins encoded by PRRSV2-ORF7 (GP2, GP3, GP4, GP5, M and NC proteins) through our initial investigation and validation for later use in vaccine formulations. 2 shows the distribution / localization of B cell epitopes and T cell epitopes. The design goal of B cell epitope cluster peptide immunogens is to create antigenic peptides that can induce neutralizing antibodies or mimic selected functional sites associated with the PRRSV receptor that bind to target cells. . If the specific GP5 B cell epitope cluster peptide is based on a single PRRSV strain sequence or is derived from several PRRSV strain sequences, the native PRRSV GP5 as a combinatorial sequence for a wide range of viral coverage To test their relative immunogenicity and cross-reactivity to protein antigens, the four amino acid frames GP5.1, 5.2, 5., 5. around the receptor binding / neutralization site as shown in Table 4. Designed over 3 and 5.4 (SEQ ID NOs: 9-14).

  GP2 B epitope (V111-L136), GP3 B epitope (C57-C75) and GP4 B epitope (C52-C69) are named based on the PRRSV XXA1 sequence and designed around the neutralization site derived from the structural proteins GP2-GP4. Further B cell cluster antigen peptides are shown in Table 3. GP2 B (V111-L136) is presented as a linear peptide (SEQ ID NO: 6), while GP3 B epitope (C57-C75) and GP4 B epitope (C52-C69) are cyclic peptides (SEQ ID NO: 7 and 8). By being presented, local constraints for maintaining the three-dimensional structure are possible. These antigenic peptides also have a C-terminal (SEQ ID NO: 5) for GP5.1 and GP5.2 frames as shown in Table 5 to enhance immunogenicity due to the short length of these peptide antigens. 29 and 30) or at the N-terminus (SEQ ID NO: 39-46) individually bound to the artificial combinatorial Th peptide (SEQ ID NO: 15). These B cell epitope cluster peptide immunogens were incorporated into vaccine formulations, guinea pigs or pigs were immunized according to well-designed protocols, and immune sera were collected in a timely manner for extensive serological evaluation. These immune sera were tested for immunogenicity by testing with each target peptide antigen-based ELISA for antibody titers against each target peptide antigen.

  Detection of cross-reactivity against the PRRSV native protein antigen by immunofluorescence assay (IFA) was performed as described in Example 1. This is to evaluate that the designed peptide immunogen is suitable for use in the final vaccine formulation. Regarding these peptide antigens recognized as peptide immunogens, sequences derived from PRRSV strains (eg, MD001, JXA1, NA and EU strains) of origin as shown in Table 3, or consensus (cons) sequences, or Peptide immunogens derived from peptide homologs having combinatorial sequences as shown in Table 4 as SEQ ID NOs: 32, 33 and 34 can elicit antibodies in immunized hosts for broad cross-reactivity with different strains of PRRSV As further designed.

  The design of a T cell epitope cluster peptide immunogen is much simpler. Ability to induce IFN-γ responses in cultures of peripheral blood mononuclear cells (PBMC) obtained from pigs immunized with PRRSV and then virus-loaded to broaden the range of cellular immune responses in immunized hosts Well-characterized immunodominant T cell epitopes based on the above were selected for incorporation into vaccine formulations. The alignment of T cell epitope cluster peptides based on the PRRSV JXA1 strain, derived from GP4, GP5, M and NC proteins, and the alignment of homologous PRRSV T helper epitope sequences of various PRRSV strains (MD001, NA and EU) is shown in SEQ ID NO: 47 As shown in Table 6 as ~ 79. In order to improve the solubility of these slightly hydrophobic peptide antigens, three lysine residues (KKK) were added to the N-terminus of individual T helper peptides against them to act as peptide immunogens. . All identified Th peptide immunogens (SEQ ID NOs: 47-79) are cellular as supplements (T cell epitope 1) to further enhance the immunogenicity of PRRSV B cell cluster peptide immunogens and themselves As a PRRSV T cell peptide immunogen for immunization, a pool of these T cell cluster peptides was created by mixing in equal proportions in the vaccine formulation.

  In order to further extend the T cell epitope coverage across the diverse genetic backgrounds of animals, specific epitope combinatorial peptides (based on four homologous T epitope sequences) are prepared for each T cell epitope. Similarly, as shown in Table 7, three lysine residues (KKK) were added to the N-terminus of each combinatorial T epitope cluster peptide immunogen (SEQ ID NOs: 80-90). These pools of T-epitope cluster combinatorial peptide immunogens (Pool 2) can be used as supplements in vaccine formulations to further enhance the immunogenicity of PRRSV B cell cluster peptide immunogens and as such are cellular immunity Used as a PRRSV T cell peptide immunogen.

Guinea pig or pig immunization ectodomains using vaccine formulations containing PRRSV B epitope cluster peptide antigens derived from GP5, GP2, GP3 and GP4 proteins to assess immunogenicity and cross-reactivity with native PRRSV proteins are: , A domain of membrane proteins that extends into the extracellular space (the space outside the cell). The ectodomain is usually the part of the viral protein that initiates contact with the target cell surface and causes attachment to and entry into the cell during infection. Virus invasion-related domains are important for the induction of neutralizing antibodies because neutralizing antibodies block the interaction of the virus with its cellular receptors. Therefore, when developing the next generation of effective PRRSV vaccines, it is important to evaluate the functionality of PRRSV viral entry related domains. Sialoadhesin (CD163), a macrophage-specific lectin, is a critical viral receptor on macrophages. Using a soluble form of sialoadhesin, it was found that the disulfide bond heterodimer of the PRRSV GP5 and M complex is a ligand for sialoadhesin. This ligand-receptor interaction is dependent on the lectin activity of sialoadhesin and sialic acid on the GP5 glycoprotein. Therefore, to investigate the effect of M on the ectodomain of the PRRSV GP5 protein and the immunogenicity of GP5 and to induce immunity that blocks the interaction between the viral M / GP5 and the host cell receptor sialoadhesin, An optimal GP5 peptide immunogen for use in the PRRSV marker vaccine formulation was identified.

  More than 45 peptide antigens were designed around the receptor binding / neutralization site of the PRRSV GP5 protein ectodomain and grouped into four amino acid sequence frames for immunogenicity evaluation.

  The first frame, based on the sequence of PRRSV strain MD001, begins with the C-terminal residue “E” (glutamic acid) of the GP5 ectodomain and includes 5 additional amino acid residues in the signal sequence of the GP5 protein, By extending to the N-terminus of the ectodomain, a 40-mer peptide antigen as shown in Table 4 was prepared as a GP5.1 MD001 (A26-E65) peptide antigen (SEQ ID NO: 29). In this design, if necessary, the central Cys residue of the GP5 peptide antigen can form a disulfide bond with the ectodomain of the M peptide.

  The second domain is a 45 mer peptide GP5.2 MD001 obtained by extension at the N-terminus with another 5 amino acid residues into the signal sequence of the GP5.1 MD001 (A26-E65) peptide antigen. V21-E65) (SEQ ID NO: 30), and the intramolecular disulfide bond between C24 and C48 made it possible to maintain the local conformation of the ectodomain of GP5. This design allowed the induction of specific antibodies against most of the ectodomain of GP5 in the absence of M protein.

  The third frame, apart from the exclusion of the 11-mer GP5 HV2 region of the ectodomain, was modeled on the second frame peptide antigen GP5.2 MD001 (V21-E65) and the 34-mer peptide antigen GP5.3 (V21 -D54) (SEQ ID NO: 9) was obtained. This design allows B cell recognition of the highly conserved central region of the GP5 ectodomain.

The fourth frame (GP5.4) targeted the loop structure located in the middle of the ectodomain of GP5 protein and became a 25mer cyclic peptide GP5.4 (C24-C48) based on the PRRSV strain MD001 sequence. In order to adapt to mutations between strains, a combinatorial peptide modeled on peptide antigens of the 3rd and 4th amino acid frames according to the PRRSV strain sequences of North American type JXA1 / MD001 and JXA1 / NJ-a, GP5.3 JXA1 / MD001 (V21-D54) (SEQ ID NO: 32), GP5.3 JXA1 / NJ-a (V21-D54) (SEQ ID NO: 33) and GP5.4 NJ-a / JJA1 / MD001 (C24-C48) (SEQ ID NO: 34) Designed as. To assess the effect of M protein ectodomain on GP5 ectodomain peptide immunogenicity, a 26mer ectodomain peptide having the following sequence based on MD001 sequence: “MGSSLDDR C HDSTAPQKVLLAFSITY” (SEQ ID NO: 93) was evaluated. Designed for (M1-Y26).

  The structure of PRRSV consists of a nucleocapsid protein bound to viral RNA. This nucleoprotein is surrounded by a lipid envelope in which the following six structural proteins are embedded: GP2, GP3, GP4, GP5 and the non-glycosylated proteins M and E (ORF2b). GP5 and M are the most abundant proteins in the envelope, with less abundance of other envelope proteins. GP5 has continued to be a major target for PRRSV protective immunity, while PRRSV variability and GP2 and GP3 have also been implicated in some clinical and virological defense, while derived from GP2, GP3 and GP4 In view of the latest knowledge that the demonstrated polyclonal antibodies that bind to the antigenic peptides of the present invention also show neutralizing activity against PRRSV, selected antigenic peptides derived from GP2, GP3 and GP4 are also included for incorporation into the PRRSV vaccine formulation Designed, screened and identified. Specifically, antigen peptide GP2B epitope (V111-L136) (SEQ ID NO: 10), GP3 B epitope (C57-C75) (SEQ ID NO: 11), GP4 B epitope (C52-C69) (sequence) as shown in Table 3 No. 12) was designed based on the PRRSV JXA1 strain sequence for immunogenicity and cross-reactivity evaluation. B epitope sequences derived from homologous GP5, GP2, GP4 and GP4 of various PRRSV strains are also listed in Table 3 as an example for reference of peptide antigen design.

  These B epitope cluster peptide antigens derived from GP5, GP2, GP3, and GP4 are also C-terminal (SEQ ID NO: 37 and 38) or N-terminal (SEQ ID NO: 37) for GP5.1 and GP5.2 frames, as shown in Table 5. No. 39-46) individually bound to the artificial combinatorial Th peptide (SEQ ID NO: 35), overcoming the short nature of these lengths to enhance the immunogenicity of these peptide antigens.

Vaccine Formulation In an exemplified use of the peptide-based PRRSV vaccine of the present invention, an artificial combinatorial Th sequence UBITh® via a Lys-Lys-Lys-εNLys (SEQ ID NO: 36) or εNLys spacer at the amino terminus or carboxyl terminus. Receptor binding / neutralizing sites or T helper epitope cluster sites as listed in Tables 3-7 as SEQ ID NOs: 9-90, which bind to or do not bind to exogenous Th epitopes such as 3 (SEQ ID NO: 35) A vaccine formulation comprising as an immunogenic peptide having was formulated into a water-in-oil emulsion using the commercial oil based vaccine delivery vehicle, Montanide ISA 50V2, based on the supplier's recommended procedure. Montanide® ISA 50V2 (Seppic, Paris, France), an oily adjuvant composition of mannide oleate, and mineral oil normally used in swine vaccines in equal volumes of aqueous phase peptide PBS solution Emulsified. Peptide immunogens were formulated into each emulsion at about 25-75 μg / mL according to specific protocols (eg, peptide ratio and total peptide concentration in the emulsion). These emulsion-based vaccine formulations were injected intramuscularly in guinea pigs at 0.25 mL to 0.5 mL per site or 1 mL per site unless otherwise stated.

Immunization of guinea pigs and piglets with designer peptide vaccine formulations For immunogenicity studies conducted in mature sensitized male and female Duncan-Hartley guinea pigs (body weight 300-350 g) at 3 animals per group Using. Animals were immunized with a specific vaccine formulation administered intramuscularly (IM) at 0 wpi and 3 wpi as two doses. These vaccine formulations must be left at room temperature for about 30 minutes and stirred for about 10-15 seconds prior to immunization. Blood was collected for serum samples at 0, 3 and 5 weeks (wpi) after the initial immunization. For the immunogenicity evaluation of peptide immunogens and vaccine formulations, as described in Examples 1 and 2, cotranslation was performed by target peptide-based ELISA for direct binding titers and by PRRSV for cross-reactive titers. These samples were tested with a transfected cell-based IFA.

  Approximately 4-week-old piglets from specific pathogen-free (SPF) farms were earmarked for immunogenicity studies and grouped according to the study protocol (3-5 piglets / group). These groups were immunized with intramuscular vaccine formulations at 0 and 4 weeks. In one study, a combination of diagnostic ELISA tests was used to distinguish infected pigs from pigs vaccinated with the marker vaccine formulation of the present invention using normal farm-derived piglets previously infected with PRRSV. Used to assess the distinct immunogenicity of designer PRRSV peptide antigens in the presence of anti-PRRSV antibodies and to measure the response of these pigs to PRRSV marker vaccine formulations.

  Blood samples were collected at the first immunization, at the first boost at 3 to 4 weeks, and 2 weeks after the boost at 5 to 6 weeks, and serum samples were prepared and detailed in Examples 1 and 2. As such, it was subjected to multiple serological tests for evaluation of immunogenicity and cross-reactivity.

Optimization and ranking of peptide immunogens designed from PRRSV GP5 protein ectodomain sequence Nine out of a number of prepared GP5 ectodomain peptide immunogens were formulated into emulsion formulations and after immunization schedules for initial and booster immunizations Their relative immunogenicity was illustrated and ranked. As can be seen from Table 8, the peptide immunogen 4020Kc (SEQ ID NO: 38) based on the PRRSV MD001 sequence is based on both the target peptide ELISA (> 1 log10) and the IFA titer 4020 Kb (SEQ ID NO: 37). More immunogenic than Thus, the amino acid sequence frame 2 (GP5.2) design is superior to frame 1 (GP5.1). Peptide immunogen 4020 Kc was used in most of the PRRSV loading studies with PRRSV MD001 as detailed in Example 6. As shown in Table 8, the peptide immunogen 4048 Kb (SEQ ID NO: 39) designed in frame 3 (GP5.3) having a sequence derived from PRRSV MD001 was found to have approximately the same immunogenicity as peptide 4020 Kc. However, its IFA titer was higher than that of peptide immunogen 4020 Kc. Another peptide immunogen 4050 Kb (Sequence) designed based on the third frame (GP5.3) is similar immunogenicity as demonstrated by both ELISA and IFA when compared to peptide immunogen 4048 Kb. No. 40). Frame 3 was therefore considered a more advantageous design frame for GP5 B cell epitope cluster antigen presentation.

  In order to broaden the scope of the virus strains, we based on the amino acid sequence frame 3, we have several methods such as peptide immunogen 4052 Kb (SEQ ID NO: 41) and peptide 4124 Kb (SEQ ID NO: 42). Proceeding to the design of the relevant combinatorial peptide antigen, it was tested for the breadth of antibody reactivity when compared to the single sequence GP5 peptides 4048a (SEQ ID NO: 9) and 4050a (SEQ ID NO: 31). A dual reactivity with the GP5.3 frame sequence from MD001 and JXA1 was found for immune sera obtained by immunization with peptide 4052 Kb (SEQ ID NO: 41), indicating that both MD001 and JXA1 The breadth of coverage was demonstrated. GP5.3 peptide antigens 4124a (SEQ ID NO: 30) and 4124 Kb (SEQ ID NO: 42) were designed to further expand the combinatorial nature of peptide immunogens and compared for their respective immunogenicity. As shown in Table 8, a significant enhancement of immunogenicity as shown by both ELISA and IFA was found with peptide 4124 Kb when compared to peptide antigen 4124a and artificial combinatorial when bound to peptide antigen 4124a The immunogenicity enhancing effect of Th peptide (SEQ ID NO: 35) was found. Design of peptide antigen 4094a (SEQ ID NO: 35) and its higher immunogenic peptide antigen 4094 Kb (SEQ ID NO: 43) by reducing amino acids at both the N-terminus and C-terminus to maintain the loop structure of GP5 Got. Since both peptide antigens showed reduced immunogenicity after such sequence trimming, the artificially constructed loop structure (C24-C48) left a critical part of the GP5 ectodomain. Thus, the enhanced immunogenicity can be optimally incorporated into the structure of peptide antigens 4124a and 4124 Kb by extending from both the N-terminus and C-terminus.

  In all evaluations, an artificial combinatorial Th peptide (SEQ ID NO: 35) attached to the C-terminus as seen with peptide antigens 4020b and 4020c (resulting in 4020 Kb and 4020 Kc), or N such as 4124a Terminal attachment (resulting in 4124 Kb) all promoted each peptide antigen to become a better peptide immunogen. Peptide 4094 and 4124 series peptide antigens were prepared in emulsion vaccine formulations to follow PRRSV GP5-based loading studies with the PRRSV JXA1 strain as described in Example 7. Peptide immunogen 4124 Kb demonstrated successful full protection (20/20 = 100%) of naive piglets from infection with highly pathogenic PRRSV JXA1 strain despite single administration of emulsion (Group 5) On the other hand, the peptide immunogen 4094 series provided suboptimal protection (4/5/5 = 80%) after two doses of immunization.

Effect of PRRSV ectodomain M peptide on the immunogenicity of GP5 B epitope cluster peptide antigens GP5 and M are the most abundant proteins within the PRRSV envelope, and these two proteins are disulfide bonded in their ectodomain It forms a heterodimer and acts as a ligand for the sialoadhesin receptor (CD163) on the cell surface of macrophages. Full length M ectodomain peptide antigen (26 mer length) was prepared and mixed with the full length GP5 ectodomain peptide immunogen 4020Kb or 4020Kc at a ratio of 1: 1 and 1:10 to give GP5 ectodomain peptide antigen 4020Kb and The effect of this ectodomain M peptide on both 4020 Kc immunogenicity was evaluated.

  As shown in Table 9, the 26mer ectodomain M peptide was found to be surprisingly immunogenic when mixed in an equal ratio with the GP5 peptide immunogens 4020Kb and 4020Kc, and the GP5 peptide immunogen 4020Kb and The relative immunogenicity of M to 4020 Kc was ranked approximately 1000-fold and 100-fold, respectively, as measured by peptide-based ELISA (group 1 and group 2 comparison; group 3, group 4 and group 5) comparison). The IFA titer against GP5 peptide immunogen 4020Kc was also significantly suppressed in a dose-dependent manner (comparison of groups 3, 4 and 5). Since the ligand-receptor interaction is dependent on the lectin activity of sialoadhesin on macrophages and sialic acid on the GP5 glycoprotein, GP5 immunity allows the induction of antibodies to bind to this major protein in the PRRSV envelope. It is important to preserve the originality. Thus, the present invention in B cell epitope cluster peptide antigen-based components despite the fact that M peptide is suggested to be covalently linked to GP5 on the envelope and is part of the GP5 / M complex as a ligand. Excluded in their follow-up PRRSV vaccine design.

Effect of various doses of PRRSV Th pool on IFA titer of PRRSV GP5 B cell epitope cluster peptide antigen GP5 peptide antigen 4094a (C24-C48) presenting the central loop structure of GP5 ectodomain with suboptimal immunogenicity We used this study in piglets to investigate the effect of various doses of PRRSV Th peptide on the immunogenicity of B cell epitope cluster peptide antigens. An equal ratio mixture of PRRSV Th epitope cluster peptides was further supplemented with 4094a peptide antigen (SEQ ID NO: 31) at 5%, 10%, 20% -50%. A vaccine formulation comprising both a B cell epitope cluster antigen peptide and a T cell epitope cluster peptide antigen is administered to a piglet by standard immunization protocols including initial and boost immunization protocols at a suboptimal dose of 30 μg / mL The effect of such PRRSV Th peptides on the immunogenicity of antigen 4094a was monitored (Group 1 to Group 4). The immunogenicity measured by ELISA did not change much for these monitored groups, but the cross-reactive dose of anti-peptide antibody elicited in these piglets against native PRRSV GP5 protein by IFA titer Dependent improvements have been demonstrated. For comparison, a GP5 B cell epitope cluster peptide immunogen 4094 Kb (SEQ ID NO: 43) containing a combinatorial Th epitope covalently linked to 4094a (SEQ ID NO: 31) (Group 5) is compared to peptide antigen 4094a alone. It has also been shown to have improved immunogenicity. These short PRRSV Th peptides (SEQ ID NOs: 47-79) are known to initiate cellular immunity against PRRSV by a recall response when the immunized host is exposed to PRRSV after stimulation, but B cells It did not covalently bind to the epitope cluster peptide antigen. The improved quality of antibody reactivity exhibited by improved IFA titers suggested the benefits of such a T cell immune response. In order to initiate extensive cellular immunity including cytokine production against PRRSV upon exposure to PRRSV, these carefully selected PRRSV Th peptides were PRRSV B cells at 20% (weight / weight) in all follow-up loading studies. It was included in the vaccine formulation along with the epitope cluster peptide immunogen.

Identification and design of GP2, GP3 and GP4 B cell cluster peptides showing high immunogenicity around antigenic sites having neutralizing activity Polyclonal antibodies that bind to antigen peptides derived from GP2, GP3 and GP4 proteins also have neutralizing activity against PRRSV In view of the latest findings that these PRRSV envelope trace proteins are also involved in some clinical and virological protection against PRRSV, specific antigenic peptide GP2B epitopes (V111) as shown in Table 3 are shown. -L136) (SEQ ID NO: 6), GP3 B epitope (C57-C75) (SEQ ID NO: 11), GP4 B epitope (C52-C69) (SEQ ID NO: 12) for immunogenicity and cross-reactivity evaluation, Designed based on the PRRSV JXA1 strain sequence, Identified as original. These B epitope cluster peptide antigens are individually conjugated at their N-terminus to an artificial combinatorial Th peptide (SEQ ID NO: 35), and GP2, 3, and 4 B cell epitope cluster antigen peptides (for evaluation of immunogenicity in guinea pigs) ( SEQ ID NOs: 44 to 46).

  Vaccine preparations individually comprising GP2, GP3 and GP4 B cell cluster peptides, specifically designed around antigenic sites with neutralizing activity, which are bound or not bound to artificial combinatorial Th sequences, Tests in guinea pigs for evaluation of each immunogenicity. Of the many peptide immunogens designed, three peptides (SEQ ID NOs: 44, 45, and 46) conjugated to an artificial combinatorial Th peptide at their N-terminus were isolated at 30 μg dose as shown in Table 11. Even a single dose showed surprisingly high immunogenicity. Therefore, these highly immunogenic peptides are outstanding candidates for incorporation into the final formulation for the PRRSV vaccine.

Immunogenicity assessment for complex GP2, GP3, GP4 and GP5 epitope cluster peptide immunogens in guinea pigs After initial design improvements for each of the BRS epitope cluster peptide antigens derived from PRRSV GP2, 3, 4 and 5 proteins These peptide combinations mixed in equal proportions in standard emulsion-based vaccine formulations were mixed with other peptide antigens at a total peptide concentration of 30 μg / mL and for follow-up boosters at 15 μg / mL at 3 wpi. ) In order to test the immunogenicity of individual peptide antigens and to assess and avoid any unintentional suppression of the immunogenicity of one or more antigens in the mixture. Sex was also evaluated. This evaluation was performed to avoid the negative effects of M peptide on the GP5 / M peptide antigen mixture.

  As shown in Table 12, GP4 B epitope cluster peptide antigen (SEQ ID NO: 46) was combined with GP3 B epitope cluster peptide antigen (SEQ ID NO: 45) as a GP3 / 4 combination formulation, and GP3 as GP3 / 4/5 combination formulation. (SEQ ID NO: 45) and GP5 (SEQ ID NO: 42), or when mixed with GP2 (SEQ ID NO: 44) and GP3 (SEQ ID NO: 45) as a GP2 / 3/4 combination product, as determined by target peptide-based ELISA The immunogenicity of the GP4 peptide antigen remained strong when mixed with the GP3 target peptide, but was significantly weakened when in the three peptide antigen mixture. The immunogenicity of the GP2 and GP3 epitope cluster peptide antigens maintained the same immunogenicity as the single peptide antigen compared to the GP4 peptide antigen. Thus, GP2 and GP3 epitope cluster peptide antigens can be more reliably used in any PRRSV peptide antigen mixture to broaden the range of immune response coverage. Regardless of the fact that GP2, GP3 and GP4 are minor components on the viral envelope, a reasonable IFA titer showing cross-reactivity with the corresponding native PRRSV protein antigen is a peptide in the absence of the GP5 antigen peptide. Obtained by antigen mixture (eg, groups 1, 2 and 4).

A peptide-based PRRSV vaccine derived from GP5 supplemented with PRRSV Th epitope cluster peptide antigen is effective for PRRSV GP5 B cell epitope cluster peptide antigen in protecting pigs from PRRSV infection that protected piglets from loading with PRRSV MD001 strain In order to prove this, three consecutive immunization and challenge studies were conducted by the Taiwan Institute of Animal Technology (ATIT). All formulations were encoded in order to objectively evaluate vaccine formulations produced by the PRRSV GP5 B epitope cluster peptide antigen. SPF pigs were regularly monitored to ensure that there were no pathogens including swine fever, pseudorabies, atrophic rhinitis, Mycoplasma hyopneumoniae, foot-and-mouth disease, swine dysentery, scabies, and actinobacillus pluronia. . In all groups of the PRRS 1001S, 1002S and 1003S studies, GP5 B epitope cluster peptide immunogens 4020Kc (SEQ ID NO: 38) and 4052 Kb (SEQ ID NO: 41) were used and there were slight variations in some formulation parameters. These formulations were considered equivalent in groups 1 and 2 as 4020 Kc antigen peptides and in groups 3 and 4 as 4052 Kb antigen peptide-derived vaccine formulations. Pooled equal ratios of PRRSV Th epitope peptide mixtures (SEQ ID NO: 47, 51, 52, 55, 59, 61, 63, 67, 70, 74, 76 based on JXA1 sequence) at 10% by weight in each vaccine formulation The immunogenicity of the PRRSV GP5 peptide antigen was further enhanced by recruiting and conferring cellular immunity.

  These studies were conducted in two locations to avoid contaminating the transgenic SPF farm. That is, only the immunization portion was performed on transgenic SPF farms that provided 4 week old SPF pigs for participation in these immunization / load studies. This farm is operated by ATIT and is located in an isolated area of Hsian Shan, Hsin Chu in northern Taiwan. After the completion of the four week primary and boost immunization protocol, these SPF pigs were moved to another well-managed farm in southern Taiwan for the load study. Since US strains of PRRSV are prevalent in Taiwan, two US strains MD-001 and AMERVAC-PRRS were employed for these stress studies. Serum samples were taken after viral challenge for IFA and viremia testing. Finally, these animals were euthanized and subjected to gross and pathological examinations. SPF piglets were grouped according to study design, with 5 per group. Blood samples were collected as shown in Table 13. All collected serum samples were tested by PRRSV-ORF5 IFA test (by rVV-PRRS ORF5 expression method).

Clinical observation after injection All piglets before and after each vaccination were treated with local (including reactive and allergic reactions) and systemic (dyspnea, appetite, diarrhea, cough, CNS / Clinical observations on reactions (including SS and allergic reactions) were made. During the study, swelling associated with local reactiveness was occasionally found but lasted for only 3-4 days, and no further differences were observed and returned to normal. No systemic adverse reactions were observed for all animals studied.

Clinical observation after viral challenge All immunized pigs showed no clinical signs specific to PRRS during the monitoring period following the viral challenge.

IFA detection after immunization and challenge and specific antibodies against lung lesions PRRSV were generated after immunization, as detected by IFA at 4 and 6 wpi (weeks after immunization) as shown in Table 14 Reached a high titer. As shown in Table 14, all animals in the control group were also PRRSV GP5 even after the PRRSV challenge study, largely due to the poor immunogenicity of the PRRSV GP5 protein when represented by the stressed virus stock. It did not elicit antibodies against the protein. The vaccinated group maintained a high IFA titer at 2 wpc (weeks after challenge) after virus challenge. Eighteen out of 20 piglets immunized with the PRRSV GP5 B epitope cluster peptide antigen vaccine formulation (4020 KC for groups 1 and 2 and 4052 Kb for groups 3 and 4) were completely detected without detection of one lesion. (Protection rate 90%). Minor lesions were detected in two of the group 3 and 4 piglets. In contrast, as shown in FIGS. 7A and 7B, 80% (4/5) pigs had severe lesions of interstitial pneumonia. Based on extensive challenge tests conducted in the PRRSV laboratory at ATIT, higher IFA titers of PRRSV GP5 protein correlated well with full protection against PRRSV load.

When the PR-PCR method was used for viremia detection PRRSV viral load detection, no detectable viral load was observed in all vaccinated pigs. This is consistent with the defense effectiveness results in the lungs. These two virus strains did not cause serious symptoms after infection, but there was no viremia and no pathological results in 18/20 vaccinated pigs Demonstrated the proof of concept of protective efficacy conferred by the PRRSV GP5-based vaccine formulation.

A peptide-based PRRSV vaccine derived from GP5 supplemented with PRRSV Th epitope cluster peptide antigen is highly virulent PRRSV (NVDC-JJA1 strain) after a single immunization schedule and after both primary and boost immunization schedules. Protected piglets from loads

China PRRSV JXA1 load model background:
In 2006, in China, there was an unprecedented large-scale epidemic of so-called “high fever” disease with PRRS symptoms, which was not known at first, which spread to more than 10 provinces (autonomous cities or autonomous regions) The disease occurred in more than 2,000,000 pigs, and about 400,000 fatal cases occurred. Unlike typical PRRS, many mature sows were also infected with "high fever" disease. This outbreak of atypical PRRS was initially identified as a swine fever-like disease that exhibited neurological symptoms (eg, tremors), high fever (40-42 ° C.), erythematous whitening rash, and the like. An autopsy performed with immunological analysis clearly showed that multiple organs were infected with highly pathogenic PRRSV, and severe pathological changes were observed. A porcine infection model has been established in China in collaboration with Kegong Tian et al. (2007 PLosOne doi: 10.1371 / journal.pone.0000526) in “Emergence of Fatal PRRSV Variants: Unparalleled Outbreaks of Atypical PRRS in China and Molecular Dissection of the Unique Hallmark” Because of the load of 12 SPF pigs (4 piglets per group), three representative PRRSV isolates (JXA1, HEB1, and HUB2) with different origins were used to isolate PRRSV Reproduced high pathogenicity.

  In each group, two piglets were injected intravenously and both died within 6-8 days, suggesting the high pathogenicity of the PRRSV strain tested. Similarly, when the other two piglets in each group are inoculated intranasally, they will show prominent signs of “high fever” disease (eg, high fever, blood spots, punctate bleeding, tremors, and so on) within 3-6 days. Both showed lamping) and both died 10 days after injection. The virus isolate was then successfully recovered from the infected pig and confirmed by PCR detection and EM. An autopsy was performed to assess immunological effects and pathological lesions. Since nearly the same pathological changes (in lungs, heart, brain, kidney, liver, etc.) were observed in pigs slaughtered during the “high fever” epidemic, the 2006 pandemic of “high fever” disease in China It was confirmed that it was caused by highly pathogenic PRRAV infection in the swine population. One of the PRRSV isolates, JXA1, is currently used as a standard virus in standardized load studies to verify the protective efficacy of the PRRSV vaccine in piglets according to the People's Republic of China (PRC) guidelines.

Definition of valid PRRAV load and vaccine efficacy according to PRC Government Vaccine Product Guidelines Four week old piglets were vaccinated once or twice with peptide-based PRRSV vaccine preparations at two week intervals, whereas one group Was kept as an unvaccinated control. All of those piglets were challenged intramuscularly behind the ear with 3 mL of PRRSV NVDC-JJA1 high antigenic virus (including 10E5 TCID50). Body temperature was observed daily for 21 days. This challenge study is considered valid if 5 out of 5 animals become ill with fever when loaded and at least 2 out of 5 animals die. In order for the vaccine to be considered protective, it is necessary that at least 4 out of 5 immunized pigs remain healthy.

A burden study conducted by UBI in collaboration with the Animal Health Vaccine company in Nanjing, PRC 2011 summary report:
A total of 35 pigs were screened for seronegative according to the guidelines published by the Department of Agriculture of PRC as described above, 30 of which were selected for this challenge study. Detailed animal selection, randomization, grouping, immunization and challenge studies, temperature observation and recording, and deaths are described below.

  We selected healthy 28-day-old piglets from a farm in Wuxi, Jiangsu Province, China. The study was conducted at an animal health vaccine company based in the Nanjing Vaccine Efficacy / Virus Load Testing Center. PRRS antibody test kit (LSI, France), lot number 2-VERPRA-001, Exp. 2012-01 and PRRS antigen test kit: PRRSV-free animals were selected using PRRS RT-PCR in vitro diagnostic kit (NSP2 1594-1680 mutant strain). The PRRS pathogenic strain (NVCD-JXA1) was made into a 10-fold dilution of the stock and used for stress testing and was administered to animals at 3 ml per animal.

  A total of 35 healthy piglets from the farm were selected for screening and 30 were enrolled in the study. All selected piglets gave negative results for serum PRRS antigens and antibodies. All enrolled piglets were randomly assigned to 6 groups, groups 1-6, 5 per group. Intramuscular injections were made at a site located just behind the ears next to the porcine neck muscles. The immunization doses and groups are listed in Table 15 (done under code to be objective).

  Group 1 has a 20% by weight pool of an equal ratio of PRRSV endogenous T helper epitope peptides (SEQ ID NOs: 47, 51, 52, 55, 59, 61, 63, 67, 70, 74, 76 based on the JXA1 sequence). Immunized with a supplemented vaccine based on the PRRSV GP5 peptide (GP5 B epitope cluster peptide antigen 4094 series, SEQ ID NO: 43).

  For groups 2-5, PRRSV GP5 peptide (GP5 B epitope cluster peptide antigen 4124, SEQ ID NO: 42) was used as the immunogen. The final peptide concentration in the vaccine formulation was 30 μg / mL. This formulation also contains a pool of PRRSV endogenous T helper epitope peptides (SEQ ID NOs: 47, 51, 52, 55, 59, 61, 63, 67, 70, 74, 76 based on the JXA1 sequence) mixed in an equal ratio. , 10% by weight for groups 2, 3 and 4 (ie 27.5 μg B: 2.5 μg T pool), 20% by weight (ie 25 μg B: 5 μg T pool) and 50% respectively. % (Ie 20 μg B: 10 μg T pool) supplemented to the PRRSV B epitope immunogen (SEQ ID NO: 42). Group 5 animals received the same vaccine formulation as Group 4, except that they received only a single dose.

  Group 6 was a negative control group that did not receive immunization with any of the peptides.

  The antibody screening results for all 30 piglets initially enrolled in this study are shown in Table 16. The results of all samples from pigs tested for PRRSV antigen by RT-PCR prior to immunization showed that 30 of 30 were negative, whereas the positive control of the test kit was an 185 base pair amplification band This confirmed the effectiveness of the test system. All animal numbers after randomization are shown in Table 17. The results of the OD value of the PRRSV antibody are shown in Table 18 for the 0th day and the 28th day after the first immunization. The body temperature and the number of deaths after PRRSV JXA1 loading were observed at the temperature (° C.) shown in Table 19.

  In summary, as shown in Table 19, all piglets in the control group (Group 6) became ill during the first few days after the viral challenge, and 2 died as shown in Table 20 ( 2 out of 5 = 40% mortality). The results of the control group that did not receive vaccination under this challenge study met the validity criteria and guidelines for the challenge study established by the Agriculture Department of PRC. In contrast to the control group, group 1 animals were protected with 80% survival (4/5) and only 20% mortality. Note that all animals in groups 2-5 (20 of 20) were protected, thus providing 100% protection.

  When all 25 animals that received a GP5 B epitope cluster peptide antigen-based vaccine formulation as one major study were combined (ie, groups 1-5), 24 of 25 animals died only 4% Survived a load by the highly pathogenic PRRSV virus strain JXA1 at a rate. This result is surprising when compared to a control group that resulted in 100% mortality after 40% of pigs died in a few days under load and all five piglets became ill. is there.

The multi-component PRRSV peptide vaccine PRRSV for inducing broadly protective antibodies against PRRSV is a major glycoprotein, GP5, and three other minor glycoproteins, namely GP2a, GP3, and GP4, which are virion envelopes. All of these are necessary for the production of infectious virions. A strong interaction was found to exist between GP4 and GP5 proteins, but weak interactions between other trace amounts of envelope glycoproteins (GP2 and GP3) and GP5 were also detected, The result is the formation of a multiprotein complex. Overall, the GP4 protein is important for mediating interactions between glycoproteins, along with GP2a in addition to GP5, which is responsible for mediating interactions with CD163 for viral entry into susceptible host cells. The conclusion was reached that it plays a role as a virus attachment protein.

  Due to the high variability of PRRSV, the development of a broadly effective PRRSV vaccine requires the protection of multiple virus strains. As the basis for the PRRSV marker vaccine component, the endogenous PRRSV Th peptide (for pool 1, SEQ ID NOs: 47, 51, 52, 55, 59, 61, 63, 67, 70, 74, 76 and for pool 2) 4 GP5-based PRRSV vaccine formulations (GP5 B epitope peptide antigen 4020Kc of SEQ ID NO: 38, peptide antigen 4052Kb of SEQ ID NO: 41), which have been supplemented with a pool of SEQ ID NO: 80 to 90) and have already demonstrated protective efficacy Antigen peptide presenting a functional neutralization / receptor binding site demonstrated in this peptide mixture to elicit polyclonal antibodies with multiprotein receptor complexes using 4094 Kb of No. 43 and 4124 Kb of SEQ ID No. 42) By incorporating the virus It would be highly desirable to be able to develop a powerful and powerful multi-component PRRSV vaccine.

  Peptide antigens were designed around these selected regions using GP2, GP3, and GP4 antigen peptide-binding antibodies that exhibit significant neutralizing properties against PRRSV (8), and the most from each protein as shown in Example 5. A strong peptide immunogen was screened. These peptide immunogens were incorporated into various vaccine formulations as multi-component PRRSV peptide vaccines aimed at a broad range of virus strains. Streamlined reproducible vaccine formulations customized for local use (eg North America vs. Europe) with sophisticated analytical tools LC / MS / MS tools and well-managed GMP peptide manufacturing processes Can be developed.

Application of UBI DIVA system and epitope-based marker vaccine to identify PRRSV infected animals, vaccinated PRRSV absent animals, or vaccinated PRRSV infected animals As shown in Example 1, a mixture for plate coating By using a protein-HRP conjugate as a tracer to detect PRRSV-infected animals after combining two antigenic peptides (4171e, SEQ ID NO: 1 and 4172e, SEQ ID NO: 2) derived from PRRSV nucleocapsid protein as An NC ELISA test kit was established. This test is used in conjunction with a customized target peptide-based ELISA to monitor the immunogenicity of an effective PRRSV marker peptide vaccine to form a PRRSV DIVA system for identification of infected and vaccinated animals be able to.

  As shown in FIG. 5, sera from normal piglets (n = 109) known to be PRRSV negative prior to entering the PRRSV vaccine immunogenicity study were tested in the UBI PRRSV NC ELISA test. Showed high specificity (109-1 / 109 = 99%). Subsequent to this group, it has been shown to contain antibodies with high titers against GP5 as shown by the target peptide-based ELISA with cross-reactivity to native PRRSV GP5 protein (Log 10 titer> = 3). Sera from pigs (n = 45) that received the PRRSV GP5 marker vaccine formulation were scored negative by the PRRSV NC ELISA test, further demonstrating the specificity of this test. The ELISA measurements for the three serogroups shown in the middle are of pure IFA positive sera from infected animals. These samples showed ELIA OD450nm measurements increasing in parallel with IFA titers. The rightmost serum was a sample (n = 100) taken from a US farm known to have PRRSV infection. A seropositive rate of> 56% was found in the UBI PRRSV NC ELISA, indicating a high prevalence of PRRSV infection in this farm.

  A combination of UBI PRRSV NC ELISA and UBI PRRSV B epitope marker vaccine target peptide-based ELISA was used to monitor the immunogenicity of PRRSV marker vaccine formulations in a Taiwanese farm known to have PRRSV infection. Insightful information was observed.

  First, as shown in FIGS. 6A and 6B, all pigs enrolled in the PRRSV vaccine immunogenicity study are positive, showing a s / c ratio much higher than the cut-off value in the PRRSV NS ELISA I understood that. Such an infection is shown in a diagram highlighted with a gray background and all animals tested were infected. Second, despite the presence of high levels of PRRSV NC-reactive antibodies (mostly maternally derived (MDA)) in these piglets, all peptide-based PRRSV vaccine formulations have their respective target peptides Significant immunogenicity was detected as detected by the base ELISA. As shown in FIGS. 6A and 6B, all animals were monitored for their peptide-specific antibody titers against component peptide antigens contained in vaccine formulations received throughout weeks 0, 4 and 6 after initial immunization. . In vaccine formulations (GP2 + GP3, GP3, GP3 + GP4, GP4, or GP5 peptide antigens) each containing these peptide antigens at a total of 30 μg / mL, each dose for the initial and 0 and 4 week boost immunization schedules Independent high immunogenicity was found to be associated with almost all PRRSV GP2 and GP3 neutralization site-derived peptide vaccine formulations, and with the “GP5 peptide alone” vaccine formulation, Site-derived peptide antigens were slightly impaired in immunogenicity when combined with other PRRSV (GP3) peptide antigens.

  Incorporates a PRRSV B epitope cluster peptide immunogen designed around the neutralization and receptor binding sites from the GP2, GP3, GP4 and GP5 proteins to elicit neutralizing antibodies and endogenous to promote cellular immunity Combinatorial peptide-based PRRSV vaccines supplemented with a mixture of sex PRRSV T helper peptides are planned for challenge studies with PRRSV MD001 and JXA1 strains (both North American strains).

UBI PRRSV peptide-based marker vaccine for eradication of PRRSV infection on SPF farms and adoption of its diagnostic DIVA system The PRRSV B epitope cluster peptide antigen used in the vaccine formulation does not contain the antigen peptide from the PRRSV nucleocapsid protein Because infected pigs usually express early antibodies against this major structural protein, the diagnostic system for distinguishing infected and vaccinated animals, and thus the diagnostic DIVA system, includes PRRSV NC ELISA and PRRSV B epitope marker vaccines. Incorporating a target peptide-based ELISA is logical.

  UBI's PRRSV peptide-based marker vaccine can induce neutralization on PRRSV protein and high titer IFA positive cross-reactive antibodies to receptor binding sites. Such marker vaccine formulations can prevent PRRSV infection more effectively than MLV or virus lysate based PRRSV vaccines. This is because the immunogenicity of these proteins is very weak when presented in current biological vaccine formats.

  The combined use of UBI's diagnostic DIVA system and its PRRSV marker vaccine will be implemented at an SPF farm in Taiwan based on the following strategy to overcome the long-term problems caused by PRRSV infection. This SPF farm continues strict monitoring records for all pathogens and has only PRRSV infection. This farm cannot eliminate its current PRRSV infection with conventional biological vaccines without incurring significant biosecurity risks. UBI's PRRSV peptide-based marker vaccine does not present any such biosecurity risk due to the chemical nature of the vaccine.

Strategies for PRRSV eradication on PRRSV infected SPF farms This farm will continue a vaccination program with UBI's PRRSV peptide-based marker vaccine for at least two years. All pigs on the farm are vaccinated using the immunization schedule for 0, 4 and 8 weeks and monitored for at least 6 months. Serum antibody titers against NC and marker vaccine target peptides are monitored by assays including PRRSV NC ELISA, IFA and quantitative PCR (qPCR) along with PRRSV viremia levels. Six months after the start of the vaccination program, piglets born from vaccinated mothers are not vaccinated and monitored for infection rates in these piglets by UBI PRRSV NC for 1-3 months. If all piglets show negative reactivity with the UBI PRRSV NC ELISA, the farm can be considered under control for PRRSV infection. The farm then dismisses all pigs previously infected with PRRSV. Until such withdrawal, all pigs on the farm are continuously vaccinated with the PRRSV peptide-based marker vaccine.

  This farm will continue vaccination of all pigs with a PRRSV peptide-based marker vaccine to enhance pig immunity against PRRSV.

  If newly introduced piglets and mature sows are found to be absent from PRRSV based on seronegative reactivity in the PRRSV NC ELISA, the farm can be declared absent from PRRSV, Therefore, the success of the PRRSV eradication program can be declared.

Indicators used for monitoring UBI's PRRSV NC ELISA and PRRSV marker vaccine target peptide-based ELISA, and thus DIVA, can be used to monitor sera from all animals on this farm for reactivity consistency. The percent positive and average OD values derived from the PRRSV NC ELISA are recorded during vaccination and monitoring.

  Pigs younger than one month that may have a transit antibody against the PRRSV NC protein (thus showing positive in the PRRSV NC ELISA) decrease with time. If there is no PRRSV circulating in the environment, ie no new infection, these piglets will also be absent from PRRSV. The farm rushes out the PRRSV-infected mature sow. Treatment with the PRRSV marker peptide vaccine further reduces the residual risk for the release of PRRSV from the previously infected sows to the environment, thus allowing the entire farm to reach the PRRSV absence state Become. Table 21 sets the sample size requirement for such tests with 95% confidence for the probability of detecting one PRRSV positive pig.

Pilot PRRSV exclusion studies using DIVA and multi-component PRRSV peptide immunogen-based vaccine formulations Monitored by serological means by DIVA, pilot PRRSV exclusion studies are a multi-component PRRSV peptide immunogen in normal PRRSV-infected farms. The potential for PRRSV NC antibody negation by animals receiving such multi-component PRRSV peptide immunogen-based vaccine formulations was evaluated by using a water-in-oil (ISA50) emulsion-based vaccine formulation containing .

  More specifically, a total of 15 piglets aged 4 weeks that were positive by PRRSV NC ELISA were classified into 5 experimental groups for this study. These piglets were immunized based on the immunization and bleeding schedule for 0, 4 and 8 weeks and monitored for 13 weeks from the day of the first immunization. PRRSV B peptide immunogens derived from GP5 (SEQ ID NO: 42), GP2 (SEQ ID NO: 44), GP3 (SEQ ID NO: 45) and GP4 (SEQ ID NO: 46) can be combined in various combinations (eg, GP5 for groups 1 and 2, GP2, GP3, and GP4; GP5, GP3, and GP4 for group 3; GP3 and GP4 for group 4), and at an equal ratio, PRRSV B peptide immunogens were mixed at a total dose of 25 μg / mL. In these formulations, the PRRSV Th peptide for Group 2 (also in the same ratio, SEQ ID NOs: 47, 51, 52, 55, 59, 61, 63, 67, 70, 74, and 76) For 3 and 4, PRRSV Th combinatorial peptide (SEQ ID NOs: 80-90) was further supplemented at 20 wt% (ie, 5 μg / mL per dose). Serum antibody reactivity against NCs is monitored by PRRSV NC ELISA to identify PRRSV infection, while serum antibody titers against PRRSV marker peptide vaccine B epitope components are matched for immunogenicity provided by various vaccine formulations PRRSV were monitored by GP2, GP3, GP4 and GP5 peptide-based ELISA. Group 5 animals did not receive the PRRSV peptide-based vaccine formulation and served as a negative control group.

It was detected by the respective PRRSV peptide-based ELISA that all pigs in groups 1-4 expressed antibodies against the respective marker vaccine target PRRSV B GP2, GP3, GP4 and GP5 peptides, and the specific antibody titer was Higher than 3 Log ₁₀ after the second immunization, 4 weeks after the first immunization, and remained high for the 13 weeks monitored. However, as shown in Table 22, antibody reactivity to the PRRSV NC protein increased to maximum reactivity approximately 6-8 weeks after the first immunization and then reached near baseline by 10 weeks after the first immunization. Decreased and remained at baseline 13 weeks after the first immunization during the monitoring period.

  In contrast, all sera from Group 5 animals that did not receive vaccine formulations containing PRRSV peptide-based immunogens remained highly reactive to PRRSV NC protein. As antibodies against PRRSV NC protein are known to be present in infected pigs, the surprising finding of such negatives against PRRSV NC in pigs vaccinated with multicomponent PRRSV peptide immunogen-based vaccine formulations Further validated the effectiveness of these multi-component PRRSV peptide-based vaccine formulations. Thus, DIVA applications and these multi-component PRRSV peptide immunogen-based vaccine formulations have broad application and meet the urgent need for monitoring, prevention, elimination and eradication of PRRSV infection.

Claims (18)

A porcine genital respiratory syndrome (PRRS) vaccine composition having a peptide antigen and a veterinary acceptable delivery vehicle or adjuvant, wherein the peptide antigen comprises:
a) SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and any combination thereof;
a PRRS vaccine composition having an amino acid sequence selected from the group consisting of b) a homolog of (a); and c) any combination of (a) or (b).   The PRRS vaccine of claim 1, wherein the peptide antigen has the amino acid sequence of SEQ ID NO: 9.   The PRRS vaccine according to claim 1, wherein the peptide antigen has the amino acid sequence of SEQ ID NO: 10.   The PRRS vaccine according to claim 1, wherein the peptide antigen has the amino acid sequence of SEQ ID NO: 11.   The PRRS vaccine according to claim 1, wherein the peptide antigen has the amino acid sequence of SEQ ID NO: 12.   The PRRS vaccine according to claim 1, wherein the peptide antigen is altered by adding or deleting 1 to 5 amino acids.   The PRRS vaccine of claim 1, further comprising a T helper epitope covalently linked to the amino terminus or carboxyl terminus of the peptide antigen.   The PRRS vaccine of claim 7, wherein the T helper epitope is SEQ ID NO: 35.   The PRRS vaccine according to claim 7, wherein the T helper epitope is covalently bound to a peptide antigen via a spacer having an ε-lysine residue.   The PRRS vaccine of claim 9, wherein the spacer is SEQ ID NO: 36.   The PRRS vaccine according to claim 1, further comprising a T helper epitope not bound to the antigenic peptide, wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 47 to 90.   2. The PRRS vaccine according to any one of the preceding claims, wherein the total amount of the peptide antigen is about 10 [mu] g to about 1 mg.   The PRRS vaccine of claim 1, wherein the delivery vehicle and adjuvant are selected from the group consisting of Montanide ISA 50V, polyoxyethylene (20) sorbitan monooleate, and CpG oligonucleotides.   A method for protecting piglets against PRRS infection, comprising administering the vaccine of claim 1. a) a peptide antigen and a veterinary acceptable delivery vehicle or adjuvant,
b) a peptide antigen having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 31, and combinations thereof; and c) SEQ ID NOs: 80 to 90 and combinations thereof. A PRRS vaccine composition comprising a PRRSV Th peptide selected from the group. a) a peptide antigen and a veterinary acceptable delivery vehicle or adjuvant,
b) a peptide antigen having an amino acid sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and combinations thereof; and c) consisting of SEQ ID NOs: 80-90 and combinations thereof. A PRRS vaccine composition comprising a PRRSV Th peptide selected from the group. a) attaching a mixture of SEQ ID NO: 1 and SEQ ID NO: 2 to a solid support;
b) exposing the peptide attached to the solid support to a porcine blood, serum or plasma sample containing the antibody under conditions that promote antibody binding to the peptide; and c) the solid support. Detecting the presence of an antibody bound to the peptide attached to the body. An ELISA immunoassay for testing the presence of anti-PRRSV antibodies,
a) attaching a mixture of SEQ ID NO: 7 and SEQ ID NO: 8 to a solid support;
b) exposing the peptide attached to the solid support to a porcine blood, serum or plasma sample having the antibody under conditions that promote antibody binding to the peptide; and c) the support. Detecting the presence of an antibody bound to the peptide attached to the ELISA immunoassay.

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