EP4460327A1 - Triple gene-deleted pseudorabies virus vectored pcv2b and csfv - Google Patents

Triple gene-deleted pseudorabies virus vectored pcv2b and csfv

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Publication number
EP4460327A1
EP4460327A1 EP23737802.1A EP23737802A EP4460327A1 EP 4460327 A1 EP4460327 A1 EP 4460327A1 EP 23737802 A EP23737802 A EP 23737802A EP 4460327 A1 EP4460327 A1 EP 4460327A1
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EP
European Patent Office
Prior art keywords
prv
recombinant vector
csfv
virus
prvtmv
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EP23737802.1A
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German (de)
French (fr)
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EP4460327A4 (en
Inventor
Shafiqul Islam Chowdhury
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Louisiana State University
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Louisiana State University
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Publication of EP4460327A1 publication Critical patent/EP4460327A1/en
Publication of EP4460327A4 publication Critical patent/EP4460327A4/en
Pending legal-status Critical Current

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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    • A61K2039/543—Mucosal route intranasal
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16741—Use of virus, viral particle or viral elements as a vector
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    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24371—Demonstrated in vivo effect

Definitions

  • Tins invention was made with government support under 2019-67015-29867 awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.
  • the field of the invention relate to methods for creating recombinant vectors, mutant viruses, and vaccines for preventing or reducing symptoms associated with porcine viral diseases.
  • the invention relates to a Triple Gene Deleted Mutant Pseudorabies Virus (PRVtmv).
  • PC V2 Porcine circovirus type 2
  • PMWS post-weaning multisystemic wasting syndrome
  • PRRSV porcine reproductive and respiratory syndrome virus
  • CSF Classical swine fever
  • EU European Union
  • pseudorabies or Aujeszky's disease (AD), caused by PRV has a major impact in the pig production in many regions, especially in China and developing countries [10], Europe and North America have implemented extensive programs to control pseudorabies infections including compulsory vaccination campaigns of domesticated pigswith gene-deleted marker vaccines whichlead to the eradication of PRV. .
  • monitoring of the disease among the populations of free-roaming feral pig and wild boar has provided serological and virological evidence that PRV remains more widely distributed than previously assumed. .
  • PRV-modified live vaccines such as the Bartha-K61 strain
  • MMVs PRV-modified live vaccines
  • An embodiment of the invention relates to the construction of a triple gene-deleted PRV vector (PRVtmv), in which the TK, and envelope glycoproteins gG and gE genes were deleted.
  • PRVtmv triple gene-deleted PRV vector
  • the chimeric PCV2b capsid (Cap), CSFV E2, and a chimeric CSFV E ms -GM-CSF (E rils fused with granulocyte-macrophage colony-stimulating factor; GM-CSF) genes were inserted in the TK-, gE- and gG-deletion loci, respectively, resulting in recombinant PRVtmv vector expressing PCV2b Cap, CSFV E2 and E ms -GM-CSF (referred hereafter as PRVtmv+).
  • the PRVtmv+ in vitro characterization, its pathogenicity in pigs, its protective serum neutralizing antibody titers against PCV2b before and after the PCV2b challenge was ascertained and compared with a commercial inactivated PCV2 commercial vaccine (Fostera® Gold PCV; Zoetis Animal Health). Further, the CSFV -specific neutralizing antibody response in pigs vaccinated with PRVtmv+ was determined. Following the PCV2b challenge, viremia, virus load in the lymphoid tissues, leukocytes and lymphocyte counts, nasal- and fecal-virus shedding were compared between the control and PRVtmv+-vaccinated prototype and the commercial vaccine.
  • a commercial inactivated PCV2 commercial vaccine Festera® Gold PCV; Zoetis Animal Health
  • PRVtmv+ is highly attenuated and safe for vaccination in pigs.
  • PRVtmv+ yielded beter protection for vaccinated pigs than the commercial vaccine after the PCV2b challenge regarding viremia, viral loads in the lymphoid tissues, and leukocyte or lymphocyte counts.
  • the PRVtmv+ vaccinated pigs generated low to moderate levels of CSFV- specific neutralizing antibody titers.
  • PRV Pseudorabies virus
  • TG trigeminal ganglionic
  • PRVtmv+ was compared with its parent wild-type (wt) Becker strain following intranasal infection.
  • PRVtmv +- establishes latency and can reactivate in TG neurons just like wtPRV,
  • PRVtmv+ is avirulent because it no longer replicates in post mitotic terminally differentiated neurons, which makes it a safe and promising live viral subunit vaccine candidate to control infections affecting pigs.
  • the replication-deficient caractheristic of PRVtmv+ precludes the possibility of vaccine virus circulation in pigs and the risk of reversion to virulence.
  • a pseudorabies virus (PRV) recombinant vector comprising a deletion in the gene encoding glycoprotein gE, a deletion in the gene encoding glycoprotein gG and a deletion in the gene encoding tyrosine kinase (TK), wherein at least one heterologous antigen is inserted in at least one deletion locus.
  • PRV pseudorabies virus
  • the PRV recombinant vector of the preceding embodiment wherein the deletion in the gene encoding gG does not affect the transcription of the Us3 gene and the deletion in the gene encoding TK does not affect the transcription of the UL22 and UL24 genes.
  • PRV recombinant vector of any of the preceding embodiment, wherein TAATA boxes of the UL22 and UL24 genes are functional.
  • the PRV recombinant vector of the preceding embodiment wherein the at least three heterologous antigens are derived from proteins of any of the viruses selected from the group consisting of Porcine Circo virus type 2 (PCV2), Classical Swine Fever Virus (CSFV), and combinations thereof.
  • PCV2 Porcine Circo virus type 2
  • CSFV Classical Swine Fever Virus
  • the PRV recombinant vector of the preceding embodiment wherein at least one PCV2 antigen derived a PCV2 having a genotype selected from the group consisting of PCV2a, PCV2b, PCV2c, PCV2d, PCV2e, and combinations thereof.
  • VLP virus-like particle
  • the PRV recombinant vector of any preceding embodiment wherein at least one of the at least three heterologous antigens is expressed as a fusion protein with a fusion partner.
  • the PRV recombinant vector of any preceding embodiment wherein the coding sequence of the CSFV E2-derived antigen is inserted into the gE deletion locus, the coding sequence of the PCV2 Cap-derived antigen is inserted into the TK deletion locus, and the coding sequence of the CSFV Ems-GMSCF-derived antigen is inserted into the gG deletion locus.
  • the PRV recombinant vector of the preceding embodiment wherein the promotor is selected from the group consisting of a HCMV promotor, a human elongation factor 1 alpha promotor, a CMV IE promotor and a CAG synthetic promotor.
  • the PRV recombinant vector of any preceding embodiment further expressing a CSFV E2-derived antigen from a gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:2, a PCV2 Cap-derived antigen from a gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:3, and aCSFV Ems-GMSCF-derived antigen from a gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO: 4.
  • the PRV recombinant vector of any preceding embodiment further incuding the gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:2 is inserted in the gE deletion locus, the gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:3 is inserted into the TK deletion locus, and the gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:4 is inserted into the gG deletion locus.
  • the PRV recombinant vector of any preceding embodiment wherein the vector establishes latency and does not replicate in the TG neurons.
  • a composition includes a carrier and at least one PRV recombinant vector of any preceding embodiment.
  • composition of the preceding embodiment formulated for administration by an intranasal route.
  • the PRV recombinant vector of any preceding embodiment for use as a vaccine.
  • the PRV recombinant vector of any preceding embodiment for use in the prevention and/or the treatment of porcine infection, preferably diseases induced by at least one pathogen selected from the group consisting of PRV, PCV2, CSFV, SIV, mycoplasma sp, parvovirus, PRRSV and combinations thereof.
  • porcine infection is a viral porcine infection induced by at least one virus selected from the group consisting of PRV, PCV2, CSFV, SIV, and combinations thereof.
  • porcine infection is caused by PCV2 in combination with at least one pathogen selected from the group consisting of mycoplasma sp, parvovirus and porcine reproductive and respiratory syndrome virus (PRRSV), and combinations thereof.
  • pathogen selected from the group consisting of mycoplasma sp, parvovirus and porcine reproductive and respiratory syndrome virus (PRRSV), and combinations thereof.
  • porcine infection is the postweaning multisystemic wasting syndrome (PMWS).
  • PMWS postweaning multisystemic wasting syndrome
  • the swine is a domesticated pig, an experimental pig or a boar.
  • a live attenuated vaccine for protection against at least one porcine disease including at least one of the RPV recombinant vector of any of the preceding embodiment.
  • porcine infection is caused by at least one pathogen selected from the group consisting of PRV, PCV2, CSFV, SIV, mycoplasma sp, parvovirus, PRRSV, and combinations thereof.
  • the vaccine of the preceding embodiment further including a pharmaceutically acceptable vehicle or adjuvant.
  • a method of vaccinating a swine against a porcine infection including inoculating the swine with the vaccine of any preceding embodiment.
  • PCV2-infected swine is coinfected with at least one virus selected from the group consisting of SIV, CSFV, PRV, and combinations thereof.
  • the vaccination results in the reduction of the pathogenicity of at least one virus selected from the group consisting of SIV, CSFV, PRV, and combinations thereof.
  • Figure 1 shows a genomic configuration of pseudorabies virus (PRV) and the strategy of thymidine kinase (TK), glycoprotein G (gG) and gE gene deletion to generate a PRV triple mutant virus (tmv) recombinant vector.
  • PRV pseudorabies virus
  • TK thymidine kinase
  • G glycoprotein G
  • tmv PRV triple mutant virus
  • Figure 2 shows the cloning strategy of chimeric PCV2 cap, CSFV E rns -GM-CSF and CSFV E2 insertion in the TK deletion, gG deletion and gE deletion loci, respectively of PRVtmv genome to generate PRVtmv-CSFV E2-E ms -GM-CSF-PCV2b Cap (PRVtmv+).
  • Figure 3 discloses the organization of the nucleotide sequence of chimeric CSFV E2 gene expression cassette (pPRV gEA/CSFV E2-INS) ( SEQ ID NO: 2); restriction sites (bold, underlined); PRV gE flanking upstream and downstream (lower case); CAG promotor sequence; Kozak sequence (italic); PRV gD signal (underlined); CSFV E2 coding sequence (highlighted); V5 epitope coding sequence (lower case, italic); 6x His epitope coding sequence (bold); Stop codon TGA (bold, italic); bovine growth hormone terminator (italic, underlined).
  • pPRV gEA/CSFV E2-INS SEQ ID NO: 2
  • restriction sites bold, underlined
  • PRV gE flanking upstream and downstream lower case
  • CAG promotor sequence Kozak sequence (italic); PRV gD signal (underlined); CSFV E2 coding sequence (high
  • Figure 4 discloses the organization of the nucleotide sequence of chimeric CPV2b CAP gene expression cassette (pPRV TKA/PCV2 Cap-INS, SEQ ID NO: 3); restriction site (bold, underlined); PRV TK flanking upstream and downstream (lower case), start and stop codon of UL24 and start codon of TK are highlighted; three stop codons (underlined); hEF-lapromotor sequence; vii) Kozak sequence (italic); PRV gD signal (underlined); PCV2 Cap coding sequence (highlighted); V5 epitope coding sequence (lower case, italic); 6x His epitope coding sequence (bold); Stop codon TGA (bold, italic); bovine growth hormone terminator (italic, underlined); stop codon of TK and start codon of UL22 (gH) are highlighted.
  • Figure 5 discloses the organization of the nucleotide sequence of CSFV chimeric Ems- GMCSF gene expression cassette (pPRV gGA CSFV Ems-GM-CSF-INS, SEQ ID NO: 4); Restriction sites ( bold underlined); PRV gG flanking upstream and downstream (lower case); Us3/Us4 polyadenylation signal (lower case, bold and italic); Kozak sequence (italic); PRV gD signal (underlined); CSFV Ems coding sequence (highlighted); GM-CSF coding sequence (underlined, highlighted); Flag epitope coding sequence (lower case, italic); Stop codon TAA (bold, italic); SV40 terminator (italic, underlined).
  • CSFV gGA CSFV Ems-GM-CSF-INS SEQ ID NO: 4
  • Restriction sites bold underlined
  • PRV gG flanking upstream and downstream lower case
  • Figure 6 illustrates the vaccination, sample collection, challenge, and euthanasia scheme for animal experiments.
  • Figure 7 shows immunoblot-analysis of PRVtmv+ expressing chimeric CSFV E2, CSFV E rils -GM-CSF. and PCV2 cap proteins using an anti-CSFV E2 monoclonal antibody (mAbs) (left panel), an anti-CSFV E rns mAbs (middle panel), and a rabbit anti-PCV2 cap Ab (right panel), respectively.
  • Figure 8 shows transmission electron microscopy imaging of Mock-infected healthy swine kidney (SK) cell with normal cellular morphology (A and B), PCV2b-infected SK cells (C and D), and PRV wt-infected SK cells (E, F and G).
  • Panels B, D, F, and G are magnifications of A, C and E, respectively.
  • FIG 9 shows transmission electron microscopy imaging of PRVtmv+ vaccine virus- infected SK cells: PRVtmv+ vaccine virus particles are visible in the exocytic vesicles and accumulation of PCV2 virus-like particles (VLPs) can be seen within the vesicular structures in the cytoplasm.
  • VLPs PCV2 virus-like particles
  • Figure 10 shows the In vitro characterization of PRVtmv+:
  • A is a plaque size analysis of PRVtmv+ compared to that of PRV wt;
  • SD standard deviation
  • C is a One-step growth analysis of PRVtmv+ compared with PRV wt.
  • Figure 11 shows growth kinetics assay for PRV wild type and PRVtmv+ after infection.
  • Figure 12 provides clinical assessments of control-, fostera- and PRVtmv+-immunized pigs following immunization and challenge: (A) and (B) assess the rectal temperature and the body weight, respectively.
  • Figure 13 is a table providing data on rectal temperature for individual animals enrolled in the study.
  • Figure 14 is a table providing data on body weight for individual animals enrolled in the study.
  • Figure 15 shows dot plot graphs representing nasal shedding measured by qPCR (A) and virus isolation by plaque assay (B) of PRVtmv+-immunized pigs
  • Figure 16 shows dot plot graphs representing data of PRVtmv+in tonsil swab quantified by qPCR (A) and virus isolation by plaque assay (B) in immunized pigs.
  • Figure 17 is a table providing data on PRVtmv+ vaccine virus replication in individual immunized pigs (nasal and tonsil swabs) and data on PRV-specific qPCR and virus isolation in SK cells (in PRVtmv+-immunized pigs).
  • Figure 18 shows immunoblot analysis of PRVtmv+ vaccine virus after a passage in pigs expressing chimeric CSFV E2 and E ms proteins by using E2- (left panel) and E rns -specific (right panel) mAbs, respectively.
  • Figure 19 provides imaging of indirect immunofluorescence assay (IIFA) for serum samples collected from PRVtmv+ immunized pigs. (Magnifications 200X). Positive signals were indicated by bright apple-green fluorescent signals.
  • IIFA indirect immunofluorescence assay
  • Figure 21 is a table providing data of PRV -neutralizing antibody titer after PCV2b challenge in individuals immunized pigs.
  • Figure 22 is a table providing data of PCV2b-neutralizing antibody titer after PCV2b challenge in individuals immunized pigs.
  • Figure 23 is a table providing data of CSFV-neutralizing antibody titer after PCV2b challenge in individual immunized pigs.
  • Figure 24 are graphs showing the percent changes in leukocyte counts (A) and lymphocyte counts (B) following vaccination and challenge in all three groups.
  • Figure 25 is a table providing leucocyte counts and lymphocyte counts in individual pigs days-post vaccination and post challenge.
  • Figure 26 shows a dot plot graph of Fecal PCV2b shedding in control and vaccinated pigs following the PCV2b challenge quantified by qPCR.
  • Figure 27 is a table providing data of Fecal PCV2b shedding by PCV2b- capsid genespecific qPCR for individual pigs in all 3 groups.
  • Figure 28 shows a dot plot graph representing copy numbers of PCV2b genome in serum (cell-free) and PBMC (cell-associated viremia) in control and vaccinated pigs following challenge.
  • Figure 29 is a table providing quantification of PCV2b viremia in serum by PCV2b- capsid gene-specific qPCR for individual pigs in all 3 groups
  • Figure 30 is a table providing quantification of PBMC-associated PCV2b viremia by PCV2b- capsid gene-specific qPCR for individual pigs in all 3 groups
  • Figure 31 shows histopathology imaging following vaccination with Fostera or PRVtmv+ vaccines and subsequent challenge with PCV2, Control group (A and D), Fostera- vaccinated group (B and E), and PRVtmv+ -vaccinated group (C and F).
  • Figure 32 shows a dot plot graph representing the mean number of PCV2b viral genome copies in pig lymphoid tissues (tonsil, mesenteric LN, mediastinal LN, cervical LN, Peyer's patch, and spleen) from control-, fostera-, PRVtmv+- vaccinated pigs;
  • Figure 33 is a table providing the number of PCV2b viral genome copies (normalized viral genome copies per 10 6 cells) in different lymphoid tissues of individual pigs from control, fostera and PRVtmv+-vaccinated groups
  • Figure 35 shows a table listing the criteria used to determine the clinical score during the clinical assessment of the pigs.
  • Figure 36 is a table providing body temperature (°C) in pigs following PRV infection and latency-reactivation.
  • Figure 37 is a table providing clinical scores of the pigs after infection
  • Figure 38 shows in (A) a graph of the mean rectal temperature of each treatment wtPRV and PRVtmv+ group with SD and in (B) a graph of the mean clinical score of each group with SD
  • Figure 39 are photographs showing in (A) bilateral nasal discharge and tear staining of a wtPRV-infected pig on 3 dpi ; in (B) unilateral comeal ulcer and descemetocele on the right eye of a wt PRV -infected pig on 7 dpi.; in (C) healthy PRVtmv+ - vaccinated pigs on 3dpi without any clinical symptoms of viral infection
  • Figure 40 is a table providing data on nasal shedding following infection determined by pseudorabies virus-specific qPCR and virus isolation in swine kidney (SK) cells
  • Figure 41 is a table providing data on nasal shedding following latency reactivation determined by PRV-specific qPCR
  • Figure 42 is a graph showing nasal virus-shedding following Dex-induced latency reactivation in pseudorabies virus wtPRV-infected and PRVtmv+-vaccinated pigs
  • Figure 43 is a table providing the number of copies of targeted PRV genome in the TG neurons of infected/immunized pigs
  • Figure 44 shows that wtPRV and PRVtmv+ establish latency in the TG neurons and have the ability to reactivatate following Dex treatments
  • Figure 45 is a table providing the number of targeted PRV transcript copies in the TG neurons of infected/immunized pigs
  • Figure 46 shows that wtPRV expressed genes involved in viral replication but PRVtmv+ does not; quantification of ICPO, MCP and gG transcripts in TG samples
  • Figure 47 is a table providing PRV-specific serum neutralizing (SN) antibody titers in wtPRV-infected/PRVtmv+-vaccinated pigs following infection and Dex-induced latencyreactivation
  • Figure 48 shows that only wtPRV-infected pigs but not PRVtmv+ - inoculated pigs elicits a memory SN antibody response following Dex-induced latency -reactivation.
  • Figure 49 shows a table gathering data on PRV genomic DNA and transcript (cDNA) copies based on immediate early protein, ICPO- and late envelope glycoprotein, gC-specific qRT-PCR; the gene and transcript copy numbers were normalized to 10 6 neurons in TG
  • Figure 50 shows a graph representing the variation of body temperature of unvaccinated (control) group and PRVtmv+ immunized group following lethal CSFV challenge over time; data are presented in the table
  • Figure 51 shows that viremia is controlled in PRVtmv+ immunized pigs after CSFV challenge; data are presented in the table
  • Figure 52 shows a graph representing the neutralizing antibody titers in PRVtmv+ vaccinated and non-vaccinated control pigs following vaccination; data are presented in the table
  • Figure 53 shows a survival curve of vaccinated versus control pigs following lethal CSFV challenge data are presented in the table
  • Figure 54 shows a graph representing the percent reduction in white blood cells (WBC) count following lethal CSFV challenge in both vaccinated and control groups; data are presented in the table
  • Figure 55 shows a graph representing the percent reduction in lymphocyte count following lethal CSFV challenge in both vaccinated and control groups; data are presented in the table
  • Figure 56 shows a graph representing the percent reduction in platelet count following lethal CSFV challenge in both vaccinated and control groups; data are presented in the table
  • nucleotide and amino acid sequences are polynucleotides and polypeptides as part of the invention. It is to be understood that the specifically identified sequences adequately describe other sequences that contain less than 100% sequence identity but to the identified sequences that provide the same function.
  • a nucleotide sequence may have 90% sequence identity or 95% sequence identity with a polynucleotide specifically disclosed herein and still encode for an entirely equivalent or functionally equivalent polypeptide.
  • a polypeptide may contain less than 100% sequence identity to a polypeptide specifically identified herein and provide the same function.
  • a polypeptide may have 90% sequence identity or 95% sequence identity with a polypeptide specifically disclosed herein and still retain the same or sufficiently similar activity or functionality as the specifically identified polypeptide.
  • chimeric gene refers to a hybrid gene having a nucleotide sequence comprising at least two partial or complete sequences derived from, obtained from, or isolated from different genes that are not naturally adjoined.
  • a chimeric protein or chimeric antigen is the functional product of a chimeric gene.
  • Chimeric genes can further be modified by mutation, deletion, insertion or substitution of heterologous sequences, or by any means available using recombinant DNA technology.
  • a “recombinant vector” as used herein refers to a genetic material, for example a virus or a plasmid, used as a vehicle to artificially carry foreign genetic material into a host cell where it can be replicated and/or expressed. Such vehicle has been genetically engineered to produce new genetic combination.
  • mutant virus refers to a virus which has been genetically engineered by deletion, mutation or truncation of genetic sequences and/or by subsequent insertion or substitution of heterologous genetic sequences. When used as a vaccine, such mutant virus becomes less pathogenic, while still being able to elicit robust immune responses in a host. Given that the engineered triple mutant virus described herein is used as a recombinant vector to carry and express protective viral antigens, both terms “mutant virus” and “recombinant vector” can be used interchangeably throughout.
  • gene expression cassette and “expression cassette” which are used interchangeably throughout the specification, refer to a sequence comprising at least one gene of interest and regulatory elements such as, for example, promoter and enhancers controlling its expression, i.e., its transcription and the translation of the transcription product. Regulatory elements such as, for example, promoter and enhancers may be located upstream of the coding sequence of the gene of interest and may be operably linked thereto or may be separated therefrom by intervening sequence such as, for example, by the 5 '-untranslated region of the gene of interest.
  • the regulatory elements may also include a downstream 3' untranslated region comprising a polyadenylation site.
  • the expression cassette may additionally contain sequences critical for the expression of genes adjacent to the insertion locus into a recombinant vector, i.e., insertion site of the expression cassette and/or selectable markers and/or sequences critical to the cellular localization of the product of gene of interest.
  • the expression cassette is usually flanked by one or more sets of restriction sites to enable its insertion into a recombinant vector and/or its excision from a vector.
  • a recombinant vector according to the present invention may include more than one expression cassette, each expression cassette expressing at least one gene of interest.
  • a gene of interest according to the present invention may be a chimeric gene coding for a chimeric antigen and/or a heterologous antigen.
  • live-attenuated or “modified live” refer to a live organism such as a virus which has been weakened so that it is not virulent but can still induce protective immune responses in a host.
  • heterologous antigen refers to any antigenic protein, polypeptide or peptide capable of eliciting a immune response, which originates from a different viral strain, a virus of a different genotype, a bacteria, a mammal or any species different from that of the PRV.
  • cytopathic refers to a virus which causes the death of the infected cells
  • non-cytopathic refers to a virus which propagate without killing the infected cell
  • Vaccination campaigns with MLV containing CSFV C-strain and PRV Bartha-K61 strain have been efficient in preventing and controling classical swine fever and pseudorabies challenges for nearly two decades
  • CSFV has become endemic in some parts of the world including China and is responsible for intermittent CSF outbreaks.
  • the vaccine strain cannot be distinguished from the circulating endemic wild type strain.
  • the vaccine C-strain may persist in vaccinated animals which may contribute to the endemic CSFV problem, thus making the use of the MLV vaccine problematic.lt is possible that the C-strain mutates in vaccinated animals and revert to virulence,, leading to the emergence of viral strains that can evade their immune system.
  • PCV2 is also endemic in most, if not all, intense pig farms worldwide [61], Although PCV2 alone only causes a sub-clinical disease, its coinfection with other infectious agents like Mycoplasma sp, PRRSV, PPV, SIV, and PRV increases the pathogenicity and clinical symptoms of the viral infections [62], PCV2 infection induces immunosuppression which not only leads to increase susceptibility to other infectious agents but importantly reduces immune response to vaccines. In addition, the only vaccines against PCV2 commercially available are subunit protein or inactivated vaccines because PCV2 has one the highest mutation rates among DNA viruses. .. Unfortunately, such vaccines do not provide adequate protection .
  • Pestiviruses contain single-stranded RNA genomes and have higher mutation rates in vitro and in vivo. Therefore, vaccine production requires strict quality control protocols to maintain its genomic sequence integrity. Most importantly, production facilities for CSFV require highly contained environments due to biosecurity concerns. PCV2 is also difficult to grow in cell cultures with the highest virus titers in cell culture usually less than 10 4 PFUs/ml. The problems associated with large-scale vaccine production are overcome in the production of the PRVtmv+ live vaccine virus described herein.
  • the PCV2 single-stranded DNA genome comprises 1,767 or 1,768 nucleotides (nt) and is predicted to have 11 potential open reading frames (ORF1-11) [19], Despite being the smallest genome among other DNA viruses, the virus has the highest evolution rate. As PCV2 continues to evolve, currently, there are five different genotypes, PCV2ato PCV2e. During the mid-2000s, pig populations were mainly infected with the PCV2b genotype. Since 2012, however, the dominant PCV2b genotype has been replaced by the PCV2d genotype in most pig production countries.
  • CSFV is enveloped and has a positive-sense, single-stranded, 12.5 kb RNA genome [23],
  • the genome contains a single ORF that encodes a polyprotein composed of 3,898 amino acids that is cleaved proteolytically by viral and cellular proteases to yield up to 12 products (NH2-NproC-E ms -El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) [24].
  • C capsid
  • E rns a polyprotein composed of 3,898 amino acids that is cleaved proteolytically by viral and cellular proteases to yield up to 12 products
  • C capsid
  • E rns a polyprotein composed of 3,898 amino acids that is cleaved proteolytically by viral and cellular proteases to yield up to 12 products
  • C capsid
  • E rns envelope proteins
  • E rns is associated loosely with the envelope, the protein is also secreted [25]
  • E2 has been implicated, along with E ms and El in viral adsorption to host cells [26]
  • E2 is essential for CSFV replication, as virus mutants containing partial or complete deletions of the E2 gene are nonviable [27]
  • Both E2 and E rns elicit neutralizing antibodies and induce protective immunity independently [28]
  • countries free of CSFV, including the EU countries do not vaccinate their domesticated herds despite the fact that a commercially available CSFV- MLV confers adequate, rapid, and solid immune protection and limits the severity of the diseases in vaccinated animals [29]
  • Extensive vaccination with MLV is believed to be responsible for sporadic outbreaks and endemic forms of CSFV in China and Korea [9,30]
  • the PRV genome is approximately 145 kb and composed of numerous essential and non-essential genes for replication in vitro in epithelial cells
  • TK thymidine kinase
  • gE envelope glycoprotein E
  • gG gG
  • TK-, gE- and gG- deletion individually or simultaneously reduce the virulence in pigs [10,34,35]
  • the TK-deleted virus has defective replication in the non-dividing or post-mitotic cells, e.g., neurons [36] which lack the cellular TK gene.
  • the TK-deleted virus can enter the sensory nerve endings in the nasal mucosa and be transported retrogradely to the neuron cell bodies in the trigeminal ganglia (TG), and establish latency.
  • TG trigeminal ganglia
  • the latent virus upon reactivation in vivo following dexamethasone treatment doesn’t replicate [37],
  • PRV gE-deleted virus replicates efficiently both in cell culture in vitro and in nasal epithelium of pigs in vivo,' however, the gE-deleted virus is highly attenuated in pigs and does not produce clinical symptoms following intranasal (IN) infection [34,35], PRV gE possesses Fc receptor activity which prevents the complement-mediated antibody-dependent cellular cytotoxicity /lysis (ADCC) of virus -infected monocytes expressing gE antibody on the surface.
  • ADCC complement-mediated antibody-dependent cellular cytotoxicity /lysis
  • the gE-deleted virus lacks Fc receptor activity, virus-infected monocytes are lysed due to ADCC, reducing virulence [38], The gE is also not required for viral entry into the nerve endings of the trigeminal nerve (maxillary branch) in the nasal mucosa. Neither is it necessary for the retrograde axonal transport from nasal epithelium to cell bodies in the TG neurons, where the virus establishes life-long latency. However, upon reactivation from latency in the TG neurons, due to stress or induced by dexamethasone injection, its anterograde axonal transport from the neuron cell bodies to axon termini or second-order neurons in the CNS is severely inhibited.
  • PRV gE-deleted-vaccinated pigs can be differentiated from the infected pigs (DIVA). Therefore, PRV gE-deleted vaccine is used for eradication efforts in many countries [10],
  • latent gE-deleted live vaccine strains may also prevent superinfecting wild type (wt) strains from becoming latent.
  • PRV envelope glycoprotein gG is a viral chemokine binding protein [41]
  • the binding of PRV-gG to chemokines results in interference of chemokine- mediated lymphocyte, neutrophil, and monocyte migration to the site of virus infection, indicating that PRV gG may play a role in evading the host's immune response [41]
  • a triple mutant virus PRVtmv was constructed as lacking the entire gE gene and part of the TK and gG gene sequences. The strategy is disclosed in Figure 1.
  • the deletion of the gE ORF is a serological marker distinguishing the vaccinated from the wt virus-infected pigs (DIVA).
  • the TK and gG genes were inactivated.
  • chimeric CSFV E2 and E ms - GM-CSF genes were inserted in the gE- and gG- deletion loci, respectively.
  • a chimeric PCV2b Cap gene was inserted in the TK deletion locus to provide a live-attenuated genetically engineered trivalent “PRVtmv+” vaccine against PRV, PCV2b, and CSFV as disclosed in Figure 2.
  • PRVtmv+ was shown to be safe for pigs and capable of eliciting virus-neutralizing antibodies in the vaccinated pigs against all three viruses; PRV, PCV2b, and CSFV.
  • the PRVtmv+ induced approx. 2-fold higher PCV2b- specific neutralizing antibody titers after vaccination and after the PCV2b challenge.
  • Both PRVtmv+ and Fostera prevented fecal virus-shedding of the challenge virus.
  • only PRVtmv+ vaccination prevented pigs entirely from both cell-free and cell-associated viremia as compared to the Fostera vaccine.
  • one PRVtmv+ vaccinated pigs had minimal PCV2b viral load only in the mediastinal lymph.
  • several Fostera vaccinated pigs had low levels of viral load in the mediastinal and cervical lymph nodes.
  • one Fostera pig had high PCV2b genome copy numbers in the Peyer’s patches.
  • the chimeric PCV2b Cap protein lacks the nuclear localization domain, it can self-assembled as PCV2b VLPs in PRVtmv+ infected SK cells.
  • Thechimeric Cap protein according to embodiments described herein remains functionally and antigenically intact and as such induces a protective immune response against PCV2b in vaccinated pigs. Additionally, PRVtmv+ induced low to moderate CSFV neutralizing antibody titers.
  • the claimed PRVtmv+ virus retains its genomic stability for the PCV2b and CSFV chimeric genes after multiple cell culture passages and a single animal passage.Furthermore, the claimed PRVtmv+ does not reactivate from latency upon dexamethasone-induced latencyreactivation. Accordingly, the risk of vaccine virus transmission and circulation in the pig population is limited.
  • the PRVtmv+ virus is easier to produce in vitro because it replicates significantly better in cell cultures than the commercially available vaccine, with a titer ranging from approx. 5 x 10 7 - 1 x 10 8 PFUs/ml, relative to the PCV2b highest titers (5- 8 x 10 3 PFUs/ml).
  • the TK- or gE-deleted viruses replicate in vitro in epithelial cells like wtPRV and with similar virus yields.
  • Epithelial cells express TK which is used by tto compensates for the viral TK, and the cell-to-cell spread defect phenotype of gE gene deletion does not affect virus replication in cell culture.
  • the TK-deleted virus does not replicate because neurons do not express TK required for virus replication.
  • PRV gE is not required for virus entry into the axon termini of the maxillary branch of the trigeminal nerve in the nasal mucosa and retrograde axonal transport to neuron cell bodies in the TG. However, it is essential for anterograde axonal transport from neuron cell bodies in the TG to nerve endings in the nasal mucosa.
  • wtPRV and PRVtmv+ established latency and can reactivate in the TG neurons upon dexamethasone-induced latency-reactivation.
  • PRVtmv+ like wtPRV, enters the sensory nerve endings in the nasal epithelium and is transported retrogradely to the neuronal cell bodies in the TG.
  • TK-deficient PRVtmv+ is avirulent because it no longer replicates in terminally differentiated neurons [6,14], Consequently, no PRVtmv+ particles are produced in the neuron cell bodies.
  • PRVtmv DNA is detected in the TG neurons of latently infected pigs, there is no DNA replication-dependent, late viral genes-gC, and MCP transcriptions.
  • wt PRV DNA is detected in the nasal swabs of wtPRV - infected pigs after 4-5 days of Dex treatment and has seroconverted memory B cells following the Dex-induced latency-reactivation.
  • PRVtmv+ has the DIVA property
  • PRVtmv+ can be used as a safe subunit vaccine vector in countries where PRV has been eradicated from the domestic pig population.
  • Swine kidney (SK; #CRL-2842, ATCC®, Manassas, VA, USA), Madin Darby bovine kidney (MDBK; #CCL-22, ATCC®), 293T (#CRL-3216, ATCC®) and TK negative TK-tsl3 hamster (ATCC #1632) cells were propagated in Dulbecco's modified Eagle's medium (DMEM; #10-017-CV, Coming®, Coming, NY, USA) supplemented with 10% heat- inactivated fetal bovine serum (FBS; EqualFETAL, Atlas Biologicals, Fort Collins, CO, USA) and 1 x antibiotic-antimycotic solution (#30-004-CI, Coming®) (Growth medium).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS EqualFETAL, Atlas Biologicals, Fort Collins, CO, USA
  • FBS EqualFETAL, Atlas Biologicals, Fort Collins, CO, USA
  • 1 x antibiotic-antimycotic solution #30-004
  • Plasmids and viral DNA co-transfection in 293T cells were performed using Opti-MEM® (#31985-070; Gibco, Waltham, MA, USA) and lipofectamine 2000 (#11668030, Thermo Fisher Scientific®, Waltham, MA, USA).
  • PRV wt Becker strain is a vimlent field isolate from a dog at Iowa State University, with subsequent laboratory passage [43], Low passage vims stock was propagated and maintained at -80 °C.
  • PRV wt and the genetically engineered recombinant PRV viruses were propagated in SK cells and titrated in MDBK cells as described earlier [45], Aliquots of low passage viral stocks were maintained at -80°C.
  • the CSFV strain Brescia (genotype 1.1) was obtained from the CSF virus collection of the EU and OIE Reference Laboratory for CSF (Institute of Virology, University of Veterinary Medicine, Foundation, Hannover, Germany).
  • PRV-specific rabbit anti-gE antibody was kindly provided by Lynn W. Enquist, Princeton University, NJ, USA.
  • Mouse anti-PRV gC antibody was purchased from VMRD (#3G9F3, VMRD Inc., Pullman, WA, USA).
  • Monoclonal CSFV E2 (HC/TC 50/2/1 and E ms (HC/TC 169/2/3) specific antibodies were kindly provided by Paul Becher, University of Veterinary Medicine, Hannover, Germany.
  • Anti PCV2 Cap rabbit polyclonal antibody was generated commercially (Genscript, Piscataway, NJ, USA) against a PCV2 Cap-specific E.coli -expressed polypeptide having the sequence (SEQ ID NO: 5) (AMTYPRRRYRRNGIFDPYVNYSSRHTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKRN QLWLRLQTSRNVDHVGLGTAFENSKYDQDYNIRVTMYVQFREFNLKDPPLNP).
  • Anti-PCV2 capsid protein-specific mAh 36Fl was kindly provided by Dr. Zoltan, CEVA.
  • Mouse anti-V5 mAb (#R960-25, Thermo Fisher Scientific®, Waltham, MA, USA) and antiflag rabbit antibody (#F7425, Sigma-Aldrich®, St. Louis, MO, USA) were purchased.
  • CSFV NS3-specific mouse mAh antibody BVD/C16 from Institute for Virology, Hannover, Germany was used.
  • PRVtmv the gE-, TK-, and gG-deleted triple mutant virus (PRVtmv) vector was first constructed.
  • Figure 1 A PRV wild type (wt) Becker strain backbone (GenBank accession # JF797219 [43]) comprises - a unique long region (UL), a unique short region (Us), - a internal repeat region (IR) and - a terminal repeat region (TR).
  • a targeted deletion gEA was introduced in the gE locus of the PRV wt backbone (A) to generate PRV gEA mutant virus ( Figure 1 C)..
  • a plasmid construct pPRV TKA ( Figure ID) was used to incorporate a targeted TK deletion in TK locus ( Figure IB) of PRV gEA ( Figure 1C) in order to generate PRV gE/TK dual gene deleted mutant virus.
  • PRVtmv (gE/TK/gG-deleted) was constructed by incorporating the plasmid construct pPRV gGA ( Figure IE) into the gG locus of the PRV gE/TK dual gene deleted mutant virus backbone. ( Figure 1)
  • PRV gEA PRV gE-deleted virus
  • a PRV gEA was generated by homologous recombination of full-length PRV wt DNA and a gE deletion plasmid (pPRV gEA).
  • Figure 1C Briefly, the pPRV gEA plasmid was constructed by PCR amplification of a 1,167 bp EcoRI-Hindlll fragment (partial gl ORF and gE promoter sequence; GenBank accession #JX797219, nt 120858 to 122024) and 936 bp KpnI-BamHI fragment (gE-Us9 intergenic, Us9 ORF, and partial Us2 sequence; GenBank accession #JX797219, nt 123846..124781) using the wtPRV Becker DNA as a template and the corresponding PRV gE left-flanking (Fl-Rl) and right-flanking primer pairs (F2-R2), respectively (Tables 1A and Figures 1A, IB
  • Table 1A List of primers used to construct the gE-deleted recombinant viruses. Restriction enzyme cleavage sites are bold and underlined. Stop codons are highlighted in bold.
  • Table 2B List of primers used to construct TK-null/deleted recombinant viruses. Restriction enzyme cleavage sites are bold and underlined. Stop codons are highlighted in bold. 2.4.2. Construction of PRV gE/TK dual gene-deleted virus
  • TK upstream UL24 and TK downstream UL22 genes are located within the TK ORF coding sequences.
  • Figures 1A-B These two genes are essential for virus replication [46,47]
  • PRV nucleotide sequences (GenBank accession #JX797219) spanning nt 58548 - 59519 (primer pairs F3/R3) and nt 59571 - 60474 (primer pairs F4/R4) were amplified by PCR as EcoRI/Kpnl and Hindlll-Nsil fragments, respectively.
  • the PRV gG ORF is located downstream of the Us3 ORF (Protein kinase; PK) and upstream of Us6 (gD).
  • PK Protein kinase
  • gD Us6
  • Figures 1A, IB and IE To generate a PRV gGA plasmid (pPRV gGA; Figure IE), a 2041 bp long DNA fragment was synthesized and cloned into EcoRI (5')/HindIII (3') sites of pUC57 (BioMatik, Ontario, Canada).
  • This fragment consisted from 5' to 3' of: an EcoRI restriction site, a 1001 bp PRV gG upstream flanking sequence comprising 5 ’-3' direction, partial carboxy-terminal Us3 ORF, and Us3-Us4 intergenic sequences, (GenBank accession #JF797219; nt 116964..117964), a chimeric Us3 and Us4 polyadenylation (Poly A) signal (as it is in the genome (GenBank accession #JF797219, nt 119520..119531) but placed immediately upstream of the Us4 start codon (ATG), followed by the Kpnl restriction site (GGTACC), a 10 bp long non-genomic spacer sequence, the BamHI restriction site and the coding sequence for the PRV gG residues 72-, Us4 (gG)-Us6 (gD ) intergenic sequence, (GenBank accession #JF797219; nt 118177..119176),
  • Table 2 List of primers used to verify the PRV TK-deletion and PCV2 Cap chimeric geneinsertion by PCR and/or sequencing.
  • nt 117965 to 118176 coding for gG residues 1 to 71 were deleted and replaced with the chimeric polyA and KpnI/BamHI sites, followed by the gG ORF residues 72-499 coding sequence and a Hindlll restriction site. Consequently, in the context of the viral genome, the gG ORF sequence deletion would not affect the Us3 gene transcription. However, gG residues 72-499 aa will not be translated due to the insertion inactivation of the gG gene.
  • the KpnI-BamHI sites of SEQ ID NO: 1 can be utilized to insert the chimeric CSFV E ms -GM-CSF gene in a site-specific manner (see 2.5.3).
  • PRVtmv-CSFV E2-E ms -GM-CSF-PCV2b Cap PRVtmv+
  • the chimeric PCV2 cap, CSFV E ms -GM-CSF and CSFV E2 were inserted in the TK deletion, gG deletion and gE deletion loci, respectively of PRVtmv genome.
  • Figure 2A the genomic organization of PRV triple mutant virus (tmv) (GenBank accession # JF797219) shows the thymidine kinase (TK) (B), glycoprotein gG (C) and gE (C) deletions.
  • the PCV2 cap expression cassette was cloned into EcoRI-Nsil site of pPRV TKA to yield pPRV TKA/PCV2 Cap-INS.
  • Figure 2E The CSFV E rns -GM-CSF expression cassette was cloned into KpnI-BamHI site of pPRV gGA to yield pPRV gGA/CSFV E ms -GM-CSF-INS.
  • Figure 2F Figure 2F
  • the 2023 bp pPre CSFV E2 chimera sequence consisted from 5'- 3' direction as follows: a Kpnl site, the 1662 bp nucleotide sequence for the CAG promoter (GenBank accession #GU299216.1, position 3-1664, which include CAG enhancer 3-364), with a Nhel restriction site , a Ncol restriction site positioned after a 12 bp spacer, 6X His coding sequence and V5 epitope coding sequence (69 bp), followed by a stop codon, a bovine growth hormone (BGH) Poly A sequence (253 bp) and a BamHI restriction site.
  • BGH bovine growth hormone
  • a CSFV E2 chimeric ORF coding sequence was synthesized and cloned into pUC57 after codon optimization for pig (pCSFV E2 chimeric ORF; Genscript).
  • the 1194 bp CSFV E2 chimeric ORF coding sequence consisted from 5’-3' direction as follows: a Nhel site, a Kozak sequence, PRV glycoprotein D signal sequence (gD predicted aa residues 1-18; GenBank accession #YP068387), predicted 373 aa of CSFV E2 ORF coding sequence (GenBank accession #AAC62087 , aa 690..1062) and aNcoI site.
  • the 1194 bp Nhel/Ncol fragment containing the chimeric CSFV ORF was cloned into the corresponding Nhel/Ncol sites of pPre CSFV E2 chimera above.
  • the expression of the CSFV E2 ORF with the PRV gD signal sequence is controlled by the CAG promoter and fused in frame with the V5 epitope and poly His coding sequences at the carboxy end.
  • the nucleotide sequence of the PRV gEA CSFV E2 insertion plasmid (SEQ ID NO: 2) is organized as follow (5’ to 3’): i) EcoRI restriction site; ii) PRV gE flanking upstream; iii) Kpnl restriction site; iv) CAG promotor sequence; v) Nhel restriction site; vi) Kozak sequence; vii) PRV gD signal; viii) CSFV E2 coding sequence; ix) Ncol restriction site; x) V5 epitope coding sequence; xi) 6x His epitope coding sequence; xii) Stop codon (TGA); xiii) bovine growth hormone terminator; xiv) BamHI restriction site; xv) PRV gE flanking downstream; xvi) Hindlll restriction site. ( Figure 3)
  • the 1638 bp pre PCV2 chimeric sequence consists from 5'- 3' direction as follows: a Kpnl site, the nucleotide sequence for the human elongation factor la (hEF-la) promoter (GenBank accession #J04617), arestriction site for Nhel, aNotl restriction site after a 12 bp spacer, V5 epitope coding sequence, 6X His coding sequence, the simian virus 40 Poly A sequence (SV40), and a Hindlll restriction site.
  • hEF-la human elongation factor la
  • SV40 simian virus 40 Poly A sequence
  • the chimeric PCV2 Cap ORF coding sequences were synthesized (pPCV2 Cap chimeric ORF; Genscript) along with a Nhel site and Kozak sequence (at 5' end) and a Notl site (at 3' end).
  • the 654 bp chimeric PCV2 Cap chimeric ORF coding sequence consists of 16 predicted amino acids of the PRV glycoprotein D signal sequence lacking the putative cleavage site (GenBank accession #YP068387; 1-16 aa) and a 654 bp Nhel/Notl chimeric codon-optimized (for pig) sequence coding for 202 predicted amino acids of PCV2 Cap protein (aa 1-10 plus aa 42-233; GenBank accession # AAD45581) lacking its nuclear localization signal (residues 11- 41; GenBank accession # AAD45581).
  • the 654 bp Nhel/Notl fragment was cloned into the corresponding Nhel/Notl sites of pPre PCV2 Cap chimera synthesized above.
  • the chimeric PCV2 Cap gene is regulated by the strong hEF-la promoter and the PCV2 Cap ORF is fused in frame with the V5 epitope and poly His coding sequence at the carboxy end.
  • the nucleotide sequence of the PRV TKA PCV2 Cap insertion plasmid (pPRV TKA/PCV2 Cap-INS) (SEQ ID NO: 3) is organized as follow (5’ to 3’): i) EcoRI restriction site ii) PRV TK flanking upstream, start and stop codon of UL24 and start codon of TK are highlighted; iii) three stop codons; iv) Kpnl restriction site; v) hEF-lapromotor sequence; vi) Nhel restriction site; vii) Kozak sequence; viii) PRV gD signal; ix) PCV2 Cap coding sequence; x) Notl restriction site; xi) V5 epitope coding sequence; xii) 6x His epitope coding sequence; xiii) Stop codon (TGA); xiv) bovine growth hormone terminator; xv) Hindlll restriction site; xvi) PRV TK flanking downstream; stop codon
  • the 2002 bp KpnI/BamHI fragment consists of the following (5’ to 3’): a Kpnl restriction site, a sequence for the cytomegalovirus (CMV) promoter sequence (GenBank accession #U55763; nt 1..605), a Kozak sequence, the PRV gD signal (GenBank accession #JF797219; nt 119647..119700, GenBank accession #YP068387; aa 1..18), the nucleotide sequence for CSFV E rns (GenBank accession #AF091661; nt 1175..1855, GenBank accession #AAC62087; aa 268..494), followed by the nucleotide sequence of porcine GM-CSF (GenBank accession #AAM48280; aa 1, 18..144) fused inframe with the C-terminal E ms coding sequence but lacking the stop codon, the nucleotide sequence for a flag
  • the chimeric CSFV E ms -GM-CSF would be expressed as a partially secreted protein.
  • Figure 2F The nucleotide sequence of the pPRV gGA/CSFV E ms -GM-CSF- INS was verified and the expression of E ms -GM-CSF was verified by transfection of the plasmid DNA in the SK cells and immunoblotting with the CSFV E ms -specific mAbs.
  • the 4021 bp long nucleotide sequence of the PRV gGA CSFV Ems GM-CSF Flag insertion was assembled from plasmid pPRV gGA and pCSFV Ems GM-CSF chimeric gene cassette is organized as follow (5’ to 3’): i) EcoRI restriction site; ii) PRV gG flanking upstream; iii) Us3/Us4 polyadenylation signal; iv) Kpnl restriction site; v) CMV promotor sequence; vi) Kozak sequence; vii) PRV gD signal; viii) CSFV Ems coding sequence; ix) GM- CSF coding sequence; x) Flag epitope coding sequence; xi) Stop codon (TAA); xii) SV40 terminator; xiii) BamHI restriction site.
  • the PCV2b Cap chimeric gene was first incorporated in the PRVtmv viral genome by cotransfection and homologous recombination of PRVtmv genomic DNA and linearized pPRV TKA/PCV2 Cap-INS constructed above.
  • Figure 2B PCR identified putative recombinant viral plaques were plaque purified and verified further by sequencing and immunoblotting with PCV2 Cap-specific rabbit polyclonal antibody.
  • a selected PRVtmv expressing the PCV2b Cap was then chosen to incorporate sequentially the CSFV-E2 and E rils -GM-CSF chimeric genes by cotransfection of the PRVtmv-PCV2 Cap genomic DNA with the corresponding linearized insertion plasmids, pPRV gEA/CSFV E2-INS ( Figure 2D) and pPRV gGA/CSFV E rils -GM-CSF-INS. respectively.
  • the putative recombinants were verified by PCR followed by sequencing and immunoblotting using the corresponding CSFV E ms - or E2-specific mAbs, respectively.
  • Figure 7 One PRVtmv+ recombinant vims expressing all three subunit chimeric antigens was selected for further in vitro characterization.
  • PRV wt and the PRV recombinant vimses were titrated by plaque assay in MDBK cells as described previously [45], For PCV2, infected cells in 24-well titration plates were fixed with 3% paraformaldehyde (PFA; #30525-89-4; Acros Organics BVBA, Fair Lawn, NJ, USA) in phosphate-buffered saline (PBS; #P3813, Sigma-Aldrich®), and non-cytopathic viral plaques were visualized by indirect immunofluorescence assay (IF A) using anti-PCV2 capsid protein (Cap)-specific mAbs 36F1. Fluorescent antibody (FA) labeled, PCV2 plaques were counted under an inverted fluorescent microscope (Olympus 1X71, Shinjuku City, Tokyo, Japan).
  • the pigs in groups 2 and 3 were housed in pens, at least 100 feet apart, in the pole barn-large animal isolation facility at the School of Veterinary Medicine, Louisiana State University.
  • the pigs in the control group were housed in a separate swine bam, approx. 100 yards away from the pole bam. All sanitary precautions were taken to prevent cross-contamination between the groups. Footbaths were located at the entrance of the pole bam, and in front of each pen entrance. All bedding materials and excretions from pigs were sterilized before discarding.
  • Intranasal (IN) intranasal inoculation; Subcut - subcutaneous injection; PFU - plaque-forming units.
  • Each pig in the PRVtmv+ vaccine group was vaccinated intranasally (IN) with 4 * 10 7 PFUs per nostril (total 8 x 10 7 PFU) and subcutaneously (SC) with filtered (0.2 p pore size) 4 x 10 7 PFUs.
  • the pigs in the Fostera vaccine group were vaccinated intramuscularly (IM) with 2 ml of the vaccine.
  • the pigs in the control group were sham inoculated IN with 1.0 ml of cell culture media.
  • the pigs in the PRVtmv+ vaccine group received Noromycin® 300 LA (Norbrook, Lenexa, KS, USA) 20 mg/kg of body weight IM.
  • animals of all three groups were challenged with PCV2b IN with a total of 1.6 x 10 4 PFU (8 x 10 3 PFU/nostril) and SC with 6.75 x 10 3 PFUs.
  • SD standard deviation
  • SD standard deviation
  • EDTA Ethylenediaminetetraacetic acid
  • Figure 6 The scheme of sample collection (Ethylenediaminetetraacetic acid [EDTA] -blood, serum, nasal, fecal, and tonsil swabs) is shown in Figure 6.
  • the swabs were collected in 2 ml of DMEM, supplemented with 3 x antibiotic-antimycotic solution and 2% FBS. Collected swab samples were aliquoted and stored at -80°C until use. Blood samples collected for sera were processed, aliquoted, and stored at -80°C.
  • Peripheral blood mononuclear cells (PBMCs) were separated from EDTA-blood using Ficoll-PaqueTM Plus (GE Healthcare, Chicago, IL, USA) and cryopreserved in liquid nitrogen as described previously [45],
  • % change in leukocyte count ol pig 100 - - - — - : - — x 100
  • Pigs were euthanized with Euthasol® (Euthanasia Solution; pentobarbital sodium and phenytoin sodium) and xylazine at 21 dpc.
  • tissue samples were collected from tonsils, lungs, liver, spleen, kidney, Peyer's patches, and cervical, bronchial, mediastinal, and mesenteric lymph nodes (LN) for histopathological (10% formalin), virus isolation, and qPCR assays (dry ice).
  • Formalin-fixed tissues were paraffinized, sectioned, and processed either for histopathology (H&E staining) or immunohistochemistry.
  • Standard plaque reduction assay was performed to evaluate the PRV- and PCV2b- specific virus -neutralizing antibody titers in serum, using 100 PFUs, as described previously [45], The plaque reduction assay was performed as above with some modifications. Since the PCV2b is non-cytopathic, plaques were visualized by FA staining with a PCV2b Cap-specific mAbs and then counted under a fluorescent microscope as described earlier [45], The virusneutralizing antibody titers for each serum sample were estimated by calculating the highest dilutions of the serum that neutralized 50% of the average numbers of respective control virus plaques without serum.
  • CSFV-specific neutralization test for the PRVtmv+ vaccinated pigs sera was performed at EU and OIE Reference Laboratory for CSF (Institute of Virology, University of Veterinary Medicine, Foundation, Hannover, Germany). The test was performed according to the protocol of the Manual of Diagnostic Tests for Detection of CSF, which was composed by the EU and OIE Reference Laboratory for CSF and is available on the website of the EU and OIE Reference Laboratory for CSF (www.tiho-hannover.de/kliniken-institute/institute/institut- fuer-virologie/eu-and-oie-reference-laboratory, accessed on 22 October 2021) [51], The titration of the antisera started with a 1:2 dilution and was incubated with the CSFV strain Brescia (CSFV genotype 1.1).
  • CSFV antigen detection in the cells was performed by immune- peroxidase staining as described in the Manual of Diagnostic Tests for Detection of CSF [51] using NS3-specific monoclonal mouse antibody BVD/C16 (dilution 1:50) and the conjugate rabbit anti-mouse horseradish peroxidase (dilution 1:200). Serum neutralization dose (ND50) titers were calculated as described previously [52],
  • PRVtmv+ and PCV2b-specific genome copies were determined by TaqMan probe-based Real-time qPCR in ABI PRISMTM 7900HT Sequence Detection System (Applied Biosystems, Waltham, MA, USA), using major capsid protein (V5) ORF coding (PRV wt) and Cap-specific (PCV2b) primer pairs (Table 3). Each time, the PCR reaction setup was run with six standards of known quantity (10 1 to 10 6 copies per reaction). PRV or PCV2b genome copies in the samples were compared with the generated standard curves.
  • Viral genome copies were normalized to a standard curve generated with host-specific swine housekeeping gene, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GenBank accession #AF017079.1). The assay was performed in duplicates, and results were expressed as PRV or PCV2b genome copies per million cells with the given fact that each eukaryotic diploid cell of pig has two copies of the GAPDH gene.
  • GAPDH Glyceraldehyde 3-phosphate dehydrogenase
  • Table 3 List of primers, probes, and double standard gene blocks (ds-gblock as standard) used in quantitative PCR used for quantification of pseudorabies (PRV)Zporcine circovirus type 2b (PCV2b) genome in samples and subsequent normalization based on glyceraldehyde 3- phosphate dehydrogenase (GAPDH) housekeeping gene.
  • PRV pseudorabies
  • PCV2b pseudorabies
  • GPDH glyceraldehyde 3- phosphate dehydrogenase
  • Immunohistochemical analysis of paraffinized tissue sections was performed for comparing PCV2b antigen distribution in different groups of pigs using the anti-PCV2 Capspecific mAh 36F1. Following standard deparaffinization and rehydration procedures in xylene and alcohol, respectively, endogenous peroxidase activity was quenched by incubating slides in 3% hydrogen peroxide in methanol for 1 hr at RT. Antigen retrieval was performed by incubating tissue sections with proteinase K (20 pg/ml) in Tris-EDTA buffer (50mM Tris Base, ImM EDTA, 0.5% Triton X-100, pH 8.0) for 30 min at 37 °C.
  • Tris-EDTA buffer 50mM Tris Base, ImM EDTA, 0.5% Triton X-100, pH 8.0
  • SK cells were grown on a 13 mm electron microscopic coverslip (#174950, Thermanox plastic coverslip, Ted Pella Inc, Redding, CA, USA) and infected with PRVtmv or PRVtmv+ at a multiplicity of infection (MOI) 5 or PCV2b (0.1 MOI).
  • MOI multiplicity of infection
  • uninfected SK cells were grown on coverslip as healthy control cells.
  • PCV2b 72 h post-infection
  • cells were fixed with primary fixative (1.25% glutaraldehyde and 2% formaldehyde in 0.1M Cacodylate buffer) for 1 h.
  • lymphoid organs (cervical and mesenteric LN, spleen, tonsil, and Peyer’s patches) were investigated for lymphoid hyperplasia, lymphoid depletion, granulomatous inflammation, multinucleated giant cells and marginal zone cellularity (except Peyer’s patches). Tonsils were also investigated for crypt inflammation.
  • BALT Bronchus-associated lymphoid tissue
  • peribronchiolar lymphocytes peribronchiolar lymphocytes
  • bronchial inflammation alveolar macrophages
  • alveolitis and inflammation of the interlobular septa/pleura.
  • the livers were investigated for portal inflammation, hepatocellular damage, and parenchymal inflammation.
  • the kidneys were investigated for interstitial inflammation, tubular damage, glomerular damage, and pelvic inflammation.
  • Each pig in the PRV wt infection group 1 was intranasally (IN) infected with total 2 x 106 PFUs /nostril (total 4 x 106 PFUs/pig).
  • the pigs in the PRVtmv+ vaccine group were inoculated IN with 4 x 106 PFUs per nostril (total 8 x 106 PFUs/pig) and subcutaneously (S/C) with filtered (0.2 pm pore size) 4 x 106 PFUs.
  • S/C subcutaneously
  • filtered 0.2 pm pore size
  • pigs in both groups received the dexamethasone (Dex) by intravenously (I/V) (0.5 mg/kg) followed by two more S/C Dex injections (0.25 mg/kg) on 29 and 30 dpi.
  • Dex dexamethasone
  • Pigs were routinely monitored for obvious clinical illness, feed-, and water-intake. Body weight and temperature were recorded. Clinical assessment included coughing, sneezing, nasal discharge, depression, respiratory difficulties, and other systemic illnesses ( Figures 35 and 36).
  • PRVtmv+ genomic copies were determined by Taq-Man probe-based real-time qPCR in ABI PRISMTM 7900HT Sequence Detection System (Applied Biosystems, Waltham, MA, USA), using targeted genes - ICP0-, MCP- and gC- specific primers and probes (Suid herpesvirus 1 strain Becker, GenBank accession # JF797219.1; Table 3). Each time, the PCR reaction setup was run with six standards of known quantity (101 to 106 copies per reaction)
  • the PRV gene copies were calculated by normalizing the MCP-specific CT values against the standard curve generated based on the CT values obtained for the known housekeeping gene, GAPDH copies (two copies/cell) in the same cells. The mean copies of the PRV-MCP gene per one million cells are then plotted.
  • RNA samples collected and stored at - 80° C from previous vaccination experiment were used.
  • treated RNA samples without cDNA synthesis were also used as controls.
  • PRV genome copies in the TG were normalized to endogenous host-specific swine housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GenBank accession #AF017079.1, Table 3). The assays were performed in duplicate.
  • the qPCR, genome copy results are expressed as PRV genome copies per million cells.
  • the RT-qPCR, gene transcript copy results are expressed as transcript copies/ng of RNA.
  • the PRVtmv+ virus was assessed for its inability to grow in the TK- negative, TK-tsl3 hamster cells relative to its SK and MDBK cells growth.
  • the absence of the gE was verified by immunoblotting the infected cell lysates with a gE-specific antibody.
  • the PRVtmv+ virus was further analyzed for the expression of chimeric CSFV E2, CSFV E rils -GM-CSF (E rns +). and PCV2 Cap proteins by immunoblotting with the CSFV E2- and Ems- specific mAbs, and rabbit PCV2b-Cap-specific polyclonal antibodies.
  • Figure 7 depicts the immunoblot-analysis of PRVtmv+ expressing chimeric CSFV E2, CSFV E ms -GM- CSF, and PCV2 cap proteins using an anti-CSFV E2 monoclonal antibody (mAbs) (left panel), an anti-CSFV E rils mAbs (middle panel), and a rabbit anti-PCV2 cap Ab (right panel), respectively.
  • CSFV E2-specific mAbs detected an approx. 53-55 kDa (E2 monomer) and an approx.103-110 kDa (putative dimer) band in PRV TMV+ -infected SK cell lysates.
  • both bands corresponding to the monomer and dimer of the CSFV E2 chimeric protein were absent in the mock- and PRV Becker (wt)-infected SK cell lysates.
  • the monoclonal antibody against CSFV E ms detected several bands, at 41 kDa, 58 kDa, 80 kDa, and 120-160 kDa bands in the PRVtmv+ -infected cell lysates.
  • Native E ms expressed by CSFV form homodimers through a disulfide bond with approximately 100 kDa [28], Most likely, the 41 kDa and 58 kDa bands are the unprocessed and processed monomers of E rns . respectively.
  • the 80 kDa and 120-160 kDa bands are most likely the unprocessed and processed dimers of E rils . respectively.
  • the molecular weight of the chimeric PCV2 Cap V5 His protein was predicted at 28.1 kDa.
  • the rabbit anti-PCV2 Cap antibody detected approx. 56 kDa and 110 kDa bands in PRVtmv+ -infected cell lysates. Therefore, the 55 kDa and 110 kDa bands recognized by the Cap-specific antibody are most likely the dimeric and tetrameric forms of the chimeric PCV2b Cap, respectively.
  • bands corresponding to the chimeric E2, E rils . and Cap proteins were absent in the mock- and PRV Becker (wt)-infected cell lysates. ( Figure 7)
  • VLPs virus-like particles
  • SK cells were infected with PCV2b infected cells at an MOI of 0.1 and fixed at 72 hours post-infection (hpi). They showed accumulation of PCV2 viral particles within the vesicle-like structures in the cytoplasm, (red arrow, Figures 8C-D)
  • the PCV2b-VLPs were circular, and each measured about 20 nm in diameter with an inter-particle distance of 5 nm. ( Figures 8C- D)
  • PRV wt virus-infected cells showed typical enveloped herpesvirus particles (about 200 nm in diameter) within the vesicular structures of the cytoplasm. ( Figures 8E-G). The process of budding and release of several enveloped viruses were also noticed on the periphery of the cell near the plasma membrane, (blue arrow, Figures 8E-G) Released virus particles from the outer surface of the cells were accumulated in intercellular space, (green arrow, Figures 8E-G) The PRVtmv+ vaccine virus -infected SK cells at an MOI of 5 also showed enveloped PRVtmv+ virus particles (about 200 nm in diameter) in the cytoplasm at 18 hpi ( red arrow, Figures 9A, 9C and 9F).
  • PRVtmv+ vaccine virus replicates with a similar kinetics and virus yield in SK cells, in vitro, like the PRV wt but produces smaller plaques
  • PRVtmv+ vaccine virus is highly attenuated, safe, and retains its stability to express the PCV2b and CSFV chimeric genes in pigs
  • PRVtmv+ immunized pigs the vaccine virus replicated and shed in the upper respiratory tract as evidenced by viral plaque assay and PRV-specific qPCR.
  • Figures 15, 16 and 17 DNA was isolated from nasal swab following immunization with PRVtmv+ vaccine, and PRV-qPCR was performed.
  • PRV genome copy numbers were calculated according to the CT values of a standard curve. Shown are the mean copy numbers of PRV genome in 100 ng of DNA of two independent qPCR analyses of each animal from three vaccination groups on 0, 2, 4, 8 and 15 dpv.
  • Virus isolated from each animal's nasal swab following the immunization with PRVtmv+ vaccine was titrated in confluent SK cells by plaque assay.
  • the virus titers (in plaque-forming unit/ml of the nasal swab; PFU/ml) of each animal from the three groups were evaluated on 0, 2, 4, 8, and 15 dpv.
  • PRVtmv+ could be isolated in cell culture from two pigs’ nasal swab samples on 4 dpv. ( Figure 15B).
  • PRVtmv+ virus replication was also evaluated in the tonsils.
  • Virus isolated from each animal's tonsil swab following the immunization with PRVtmv+ vaccine was titrated in confluent SK cells by plaque assay.
  • the virus isolated from nasal swabs on 4 dpv was tested by immunoblotting with anti-CSFV E2 and E rils -specific mAbs.
  • the E2- and E ms -specific mAbs recognized the 53 kDa E2- and 56 kDa E ms -specific bands as seen on the left and right panels of Figure 18, respectively.
  • PRVtmv+ remains stable in pigs.
  • PRVtmv+ induces the production of PCV2b-specific antibodies in the vaccinated pigs, which can detect PCV2b-infected cells in culture
  • PRVtmv+ vaccine elicits a PRV-specific neutralizing antibody response in the vaccinated pigs.
  • PRVtmv+ vaccine A single dose of PRVtmv+ vaccine is sufficient to elicit a higher PCV2b-specific antibody response than the inactivated Fostera Gold PCV vaccine
  • Both PRVtmv+ and Fostera vaccine-induced detectable levels of PCV2b-specific neutralizing antibodies in the vaccinated pigs by 15 dpv Figure 20B; Figure 22.
  • the antibody level in the PRVtmv+ vaccine group was nearly two-fold higher than the Fostera groups' titer; mean SN titer of 15 for the PRVtmv+ versus 8 for the Fostera group.
  • the average SN antibody titers in the PRVtmv+ rose to 22 on the day of PCV2b challenge (32 dpv), while the corresponding average SN titer in the Fostera group was 16.
  • Figure 20 shows PRV-, PCV2b- and CSFV-specific serum neutralizing (SN) antibody titer developed in pigs after PRVtmv+ vaccination.
  • PRVtmv+ immunized pigs generate CSFV-specific neutralizing antibody titers
  • the NDso titers against the CSFV strain Brescia was determined for sera samples collected on 0, 15, 21 and 32 dpv.
  • the pigs in the PRVtmv+ group had average NDso titers of less than 2, but the average titers rose to 5, 8, and 11 on 15, 21 and 32 dpv, respectively ( Figure 20C; Figure 23). Therefore, the pigs seroconverted (a four-fold rise), NDso titers from less than 2 to 8 by 21 dpv and rose further to 11 on 32 dpv.
  • both vaccine groups had a similar delay in the seroconversion after challenge, but the average SN titers in the PRVtmv+ group were nearly two-fold higher than in the Fostera vaccine group (44 versus 28) (Figure 20B).
  • antibodies against PCV2b in the control group begin to appear in a few pigs (two pigs out of five) on 13 dpc and rose to 13 on 21 dpc ( Figure 20B).
  • PRVtmv+ protects pigs from leukopenia and lymphopenia after PCV2b challenge
  • PCV2b-infected piglets especially when developing PCV2b associated PMWS disease, have lymphopenia [55,56], Leukocyte and lymphocyte counts were determined in control unvaccinated, Fostera and PRVtmv+ vaccinated pigs before challenge (0 dpc) and after the PCV2 challenge (at 21 dpc).
  • Whole blood was collected from pigs on 32 dpv/Odpc, and 53 dpv/21 dpc, The data presented in Figure 24A-B and Figure 25 show that in the PRVtmv+ vaccinated pigs, both the leukocyte and lymphocyte counts increased by 21 dpc relative to 0 dpc (36% and 20%, respectively).
  • PRVtmv+ vaccinated pigs had a moderate increase in leukocyte and lymphocyte counts following the PCV2b challenge
  • pigs in the control unvaccinated and commercial, Fostera vaccine groups had a reduction in both counts (a moderate to low level of leukopenia and lymphopenia).
  • Fecal virus shedding is a transmission source of PCV2b virus in pigs [57], PCV2b shedding after vaccination and PCV2b challenge was assessed by qPCR in fecal swabs on 0, 13, 17 and 21 dpc. Low fecal PCV2b shedding levels were observed only in a few control group pigs on 17 and 21 dpc; PCV2 genome copy numbers ranged between 13-35 copies/200 ng of total DNA in two pigs on 17 dpc and one pig on 21 dpc (Figure 26; Data shown in Figure 27).
  • PCV2b infection causes both cell-free and cell-associated (monocytes and lymphocytes) viremia following replication in monocytes/macrophages and lymphoblasts in the lymphoid tissues [58], Protective effect of PRVtmv+ vaccination in pigs was evaluated from cell-free or cell-associated viremia after a PCV2b challenge.
  • DNA was isolated from serum and PBMC, and PCV2b qPCR was performed as described in 2.11. The mean copy numbers of PCV2b genome in serum (100 ng of DNA), and PBMC (normalized to 10 7 cells) was determined from two independent qPCR analysis of each animal from three vaccination group on 0, 13, 17 and 21 dpc.
  • the PRVtmv+ vaccinated group was again negative for PCV2b-specific DNA the entire time post-PCV2b challenge. Therefore, taking together the cell-free and cell-associated viremia data, the PRVtmv+ vaccine prevented viremia entirely.
  • the commercial vaccine "Fostera" did not; the viremia was reduced from the high level in the control animals to a low to moderate level.
  • pigs of the three treatment groups had no gross or histologic changes, with no evidence of lymphoid depletion and/or granulomatous inflammation within lymphoid tissues (Figure 31).
  • pig #2302 vaccinated with the PRVtmv+ rare multinucleated giant cells were observed in the mesenteric lymph node ( Figure 31 F and inset); however, there was no lymphoid depletion or histiocytic inflammation, PCV2 DNA, identifiable viral botryoid inclusion bodies, or viral antigen.
  • lymphoid depletion or histiocytic inflammation PCV2 DNA, identifiable viral botryoid inclusion bodies, or viral antigen.
  • PRVtmv+ vaccine protects pigs against PCV2b challenge better than the inactivated commercial vaccine Fostera
  • the pigs were clinically evaluated according scoring criteria as listed in Figure 35.
  • Figures 37 and 38B Rectal temperature of pigs were recorded following primary infection and Dex-induced latency-reactivation.
  • Figures 36 and 38A On 3 dpi, wtPRV-infected pigs started to show moderate fever of 40.6 °C and mild to moderate respiratory signs with a clinical score of 9.
  • Figures 36, 37 and 38A-B The clinical signs in wt-infected pigs were most severe 4 dpi with high fever ranging between 40.8°C and 41.8 °C.
  • the clinical score was 11.5 which was characterized by shivering, bilateral nasal discharge, coughing, sneezing, tear staining, and moderate to severe respiratory difficulties ( Figures 38A-B and 39A).
  • the pigs were off-fed for a day (4th dpi) and had reduced feed and water intake for two days (3rd and 5th dpi).
  • 7 dpi the clinical signs gradually subsided.
  • one wt-infected pig developed a unilateral comeal ulcer on the right eye, leading to descemetocele on 7 dpi ( Figures 39B), which persisted until the day of euthanasia on 33 dpi.
  • the PRVtmv — vaccinated pigs were clinically normal until euthanasia on 33 dpi, including 5 days post-reactivation ( Figures 38A- B and 39C).
  • Nasal swabs collected from pigs daily between 0 and 5-day post-dexamethasone injection (dp-Dex) were tested by qPCR targeting PRV -Maj or capsid protein (MCP) gene.
  • PRV genomic copy numbers were calculated according to the CT values of a standard curve. The mean copy numbers of PRV genome in 100 ng of total DNA from nasal swabs of two independent qPCR analyses of each animal from both groups on 0-, 1-, 2-, 3-, 4- and 5-dp-Dex injection.
  • TK-deficient PRVtmv+ is avirulent because it no longer replicates in terminally differentiated neurons.
  • PRV and other alpha herpesviruses replicate initially in TG neurons at a low level. Subsequently, progeny viruses travel anterogradely from neuron cell bodies down the axon to nerve endings in the nasal mucosa and replicate there, resulting in nasal virus shedding. Since the pigs were pre-exposed to PRV, there was a B cell response and generation of memory B cells. Consequently, upon virus reactivation in the TG and replication in the nasal epithelium, memory B cells are expected to undergo a recall immune response resulting in a rapid rise in SN antibody titers.
  • PRVtmv+ establishes latency in the TG neurons and reactivates when induced by dexamethasone (dex) injection but is unable to replicate in the TG neurons, and therefore no nasal virus shedding following the latency -reactivation.
  • the TK- or gE-deleted viruses replicate in vitro in epithelial cells like the PRV wt virus and with similar virus yields. This is because cellular TK compensates for the viral TK, and the cell-to-cell spread defect phenotype of gE gene deletion does not affect virus replication in cell culture. However, in the neurons in culture or in vivo in the animals, i.e., TG neurons, the TK-deleted virus does not replicate because neurons do not have the thymidine kinase required for virus replication.
  • PRV gE is not required for virus entry into the axon termini of the maxillary branch of the trigeminal nerve in the nasal mucosa and retrograde axonal transport to neuron cell bodies in the TG; however, it is essential for anterograde axonal transport from neuron cell bodies in the TG to nerve endings in the nasal mucosa.
  • PRVtmv+ (TK-, gE- and gG-deleted) replicated in the nasal mucosa, established latency in the TG neurons and reactivated following dex treatment.
  • PRVtmv+ did not replicate in the TG neurons. Consequently, PRVtmv+ did not shed in the nasal discharge following the dex treatment.
  • - induced reactivation, di early protein (ICPO) gene transcription (ICPO) gene transcription; however, the reactivated virus ( Figure 49).
  • This safety property of PRVtmv+ characterized by “no nasal virus shedding” following reactivation, would be improved further for use in PRV-free countries, including the US and EU.
  • PRVtmv+ vaccinated pigs survived a virulent CSFV Brescia challenge and were protected from severe clinical signs, thrombocytopenia, and lymphocytopenia.
  • the CSFV challenge experiment was performed at Plum Island Foreign Animal Disease Research.
  • SK6 cells free of bovine viral diarrhea virus (BVDV) which is genetically and serologically related to CSFV, were maintained in Dulbecco's Minimal Essential Media (DMEM) (Gibco, Grand Island, NY) containing 10% fetal calf serum (FCS) (Atlas Biologicals, Fort Collins, CO).
  • DMEM Dulbecco's Minimal Essential Media
  • FCS fetal calf serum
  • BICv Bacillus fetal calf serum
  • Titrations of CSFV were performed using SK6 cell cultures in 96-well plates (Costar, Cambridge, MA).
  • Prescence of viral infection was detected after 4 days in culture by immunoproxidase assay using the E2 specific CSFV monoclonal antibody WH303 and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Titers were calculated and expressed as TCID50/ml as described previously with a sensitivity of detection of >1.8 TCID50/ml.
  • Serum neutralization assay was per-formed with heat-inactivated serum samples (56 °C for 30 min) as described previously (Govicov et al., Virology 420 (2011) 135-145). Briefly, two-fold serial dilutions of serum were prepared in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and mixed with equal volumes of BICv containing 10 2 TCID50. Serum-virus mixtures were incubated for 1 h at 37 °C and then transferred to 96-well flat-bottom tissue culture plates (Coming) followed by additionof SK6 cells (1 x 104 per well). Plates were incubated at 37 °C and 5% CO2 for 4 days.
  • DMEM Dulbecco's modified Eagle's medium
  • PRVtmv+ vaccine group pigs (5 animals per group), of 30-401bs female Yorkshire cross-breed pigs, were vaccinated, intranasally (IN) with 4 x 107 PFUs per nostril (total 8 x 107 PFU) and subcutaneously (SC) with filtered (0.2 pm pore size) 4 x 107 PFUs..
  • An additional group (control group) of pigs were mock-vaccinated.
  • At 28 post-immunization pigs were intranasally (IN) inoculated with 105 TCID50 of CSFV Brescia strain.
  • PRVtmv+ vaccinated pig After challenge, PRVtmv+ vaccinated pig had slightly elevated viremia at 4 dpc which decreased at 7dpc. Notably, no viremia was detectable at 14 and 21 dpc, demonstrating a protective effect of the vaccination with PRVtmv+. In contrast, unvaccinated pigs had increased viremia at 4 dpc and 7 dpc to the extend that the pigs were euthanized at 7dpc due to the severity of the CSFV infection. (Figure 51)
  • both groups have reduced WBC, lymphocyts and platelets counts at 4 pdc. After 7 dpc, the lymphocyts and platelets counts stabilized in the vaccinated group while the lymphocytes and platelets counts in the control group remained very low with a decrease of 80 % and 65% of decrease, respectively. (See Figures 54, 55 and 56)
  • a low WBC count is often related to a decrease in a type of infection-fighting WBC called neutrophils.
  • lymphocytes are immune cells responsible for the adaptive immune response, the stabilization of their counts over time in the PRVtmv+ vaccinated groups shows that the disease is well controlled.
  • ADV Aujeszky's disease virus
  • Bovine herpesvirus 5 glycoprotein E is important for neuroinvasiveness and neuro virulence in the olfactory pathway of the rabbit. J Virol 2000, 74, 2094-2106, doi:10.1128/jvi.74.5.2094-2106.2000. Pannhorst, K.; Wei, H.; Yezid, H.; He, J.; Chowdhury, S.I. Bovine Herpesvirus 1 UL49.5 Interacts with gM and VP22 To Ensure Virus Cell-to-Cell Spread and Virion Incorporation: Novel Role for VP22 in gM-Independent UL49.5 Virion Incorporation. J Virol 2018, 92, doi:10.1128/JVI.00240-18.

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Abstract

The invention relates to a engineered pseudorabies virus (PRV) to express protective antigens derived from viruses associated with infection in swine. The PRV recombinant vector is a triple deletion mutant and includes a deletion in the gene encoding glycoprotein gE, a deletion in the gene encoding glycoprotein gG and a deletion in the gene encoding tyrosine kinase (TK), wherein at least one heterologous antigen is inserted in at least one deletion locus. The PRV recombinant vector can include at least three heterologous antigens inserted therein. Included are methods for creating recombinant vectors, mutant viruses, and vaccines for preventing or reducing symptoms associated with viral infection in swine, in particular classical swine fever (CSF) and Porcine circovirus type 2 (PCV2).

Description

TRIPLE GENE-DELETED PSEUDORABIES VIRUS VECTORED PCV2b AND
CSFV
This application claims priority to U.S. Provisional Application No. 63/297,102, filed January 6, 2022, the contents of which are incorporated herein by reference in their entirety.
Tins invention was made with government support under 2019-67015-29867 awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.
SEQUENCE LISTING
The instant application contains a Sequence Listing XML which has been submitted electronically in EFS and is hereby incorporated by reference in its entirety. Said XML copy, created on January 6, 2023 is named 144240570327. xml and is 52,472 bytes in size.
BACKGROUND
1. Field of the invention
The field of the invention relate to methods for creating recombinant vectors, mutant viruses, and vaccines for preventing or reducing symptoms associated with porcine viral diseases. In particular, the invention relates to a Triple Gene Deleted Mutant Pseudorabies Virus (PRVtmv).
2. Discussion of Related Art
Porcine circovirus type 2 (PC V2) is endemic worldwide, including in the United States, as evidenced by a remarkably high seroprevalence. The virus was first isolated from pigs with the post-weaning multisystemic wasting syndrome (PMWS) in the 1990s [1], Although PCV2b alone causes only a sub-clinical disease, PCV2 infection with Mycoplasma sp, parvovirus, and porcine reproductive and respiratory syndrome virus (PRRSV) may cause the clinical disease designated as PMWS [2], Also, the preexisting PCV2 infection decreases the efficacy of vaccines. Coinfection of PCV2 with swine influenza virus (SIV), classical swine fever virus (CSFV), and pseudorabies virus (PRV) also increases the pathogenicity of these infections [3], Consequently, PCV2-associated diseases pose a significant problem for the pig industry worldwide [4] and cause significant economic losses in many pig-producing countries, including the USA.
Classical swine fever (CSF) is a highly contagious disease affecting swine and remains endemic in many countries, including China, where half of the world's pig population is located. CSF is regarded as one of the most significant problems in pig-rearing countries, i.e., China and other Southeastern Asian countries, East European, and some European Union (EU) countries [5], Routine prophylactic vaccination campaigns have been relatively successful in preventing and controlling the spread of the disease in affected regions (e.g., China, Russia, and Southeast Asia). While several countries in Europe, the US, Canada, New Zealand, and Australia are currently declared CSF-free, the risk of outbreaks remains [6,7].. Several EU countries experienced a series of CSFV outbreaks in the 1980s and 1990s. In the Netherlands alone, the economic impact of the epizootic was significant with financial losses that exceeding. $ 2 billion [8], In China, the modified live attenuated CSFV vaccine is believed to cause and maintain CSFV endemic status [9],
Similarly, pseudorabies or Aujeszky's disease (AD), caused by PRV, has a major impact in the pig production in many regions, especially in China and developing countries [10], Europe and North America have implemented extensive programs to control pseudorabies infections including compulsory vaccination campaigns of domesticated pigswith gene-deleted marker vaccines whichlead to the eradication of PRV. . However, monitoring of the disease among the populations of free-roaming feral pig and wild boar has provided serological and virological evidence that PRV remains more widely distributed than previously assumed. . The risk of PRV transmission from infected wild pigs to domesticated herds poses a real threat to the pig industry and should be controlled [11], For quite some time, PRV-modified live vaccines (MLVs), such as the Bartha-K61 strain, have successfully controlled the disease in many countries, including China [12], However, in 2011, a highly pathogenic PRV strain emerged in Nothem China , causing endemic outbreaks in Bartha-K61 -vaccinated swine herds [13,14],
Since PCV2, CSFV and PRV are endemic, and co-infections are common in intensive pig farms in China [15], the severity and fatality of the disease and the emergence of a highly pathogenic PRV strain may be the result of complex epidemiological interactions between the PRV and co-infecting virus(es) [16], While CSFV and PRV have been eradicated in the US and many European countries, they are maintained in the wild pig populations in some, if not all, European countries [11], CSFV is also endemic in some South and Central American countries [17], While Mexico is free of CSF in the Northern provinces that border the United States and has control programs in other provinces, the roaming of pigs from endemic area led to an outbreak in the northern region in 2000. CSFV also reemerged in Cuba in 1993 and has since spread to Haiti (1996) and the Dominican Republic (1997) [18], There is an unmet need for a vaccine capable of protecting pigs against endemic and emerging viral diseases that are efficacious without having the side effects associated with commercially available vaccines.
INCORPORATION BY REFERENCE
All publications and patent applications identified herein are incorporated by reference in their entirety and to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
SUMMARY
The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art how to make and use the invention. In describing embodiments of the invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. The described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. Moreover, features described in connection with one embodiment of the invention may be used in conjunction with other embodiments, even if not explicitly stated above. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.
An embodiment of the invention relates to the construction of a triple gene-deleted PRV vector (PRVtmv), in which the TK, and envelope glycoproteins gG and gE genes were deleted. In addition, the chimeric PCV2b capsid (Cap), CSFV E2, and a chimeric CSFV Ems-GM-CSF (Erils fused with granulocyte-macrophage colony-stimulating factor; GM-CSF) genes were inserted in the TK-, gE- and gG-deletion loci, respectively, resulting in recombinant PRVtmv vector expressing PCV2b Cap, CSFV E2 and Ems-GM-CSF (referred hereafter as PRVtmv+). The PRVtmv+ in vitro characterization, its pathogenicity in pigs, its protective serum neutralizing antibody titers against PCV2b before and after the PCV2b challenge was ascertained and compared with a commercial inactivated PCV2 commercial vaccine (Fostera® Gold PCV; Zoetis Animal Health). Further, the CSFV -specific neutralizing antibody response in pigs vaccinated with PRVtmv+ was determined. Following the PCV2b challenge, viremia, virus load in the lymphoid tissues, leukocytes and lymphocyte counts, nasal- and fecal-virus shedding were compared between the control and PRVtmv+-vaccinated prototype and the commercial vaccine. PRVtmv+ is highly attenuated and safe for vaccination in pigs. PRVtmv+ yielded beter protection for vaccinated pigs than the commercial vaccine after the PCV2b challenge regarding viremia, viral loads in the lymphoid tissues, and leukocyte or lymphocyte counts. In addition, the PRVtmv+ vaccinated pigs generated low to moderate levels of CSFV- specific neutralizing antibody titers.
Pseudorabies virus (PRV) establishes lifelong latency in trigeminal ganglionic (TG) neurons. Upon stress, the latent viruses in the TG neurons reactivate and are transported anterograde from the neuron cell bodies to the nerve endings in the nasal mucosa, where they replicate and are discharged in nasal and oral secretions. The cycle of latency, reactivation and replication continues until the animal dies or is slaughtered. PRVtmv+ was compared with its parent wild-type (wt) Becker strain following intranasal infection. PRVtmv +- establishes latency and can reactivate in TG neurons just like wtPRV, However, due to its TK-deficiency, PRVtmv+ is avirulent because it no longer replicates in post mitotic terminally differentiated neurons, which makes it a safe and promising live viral subunit vaccine candidate to control infections affecting pigs. The replication-deficient caractheristic of PRVtmv+ precludes the possibility of vaccine virus circulation in pigs and the risk of reversion to virulence.
Further aspects are provided by the subject mater of the following embodiments.
A pseudorabies virus (PRV) recombinant vector comprising a deletion in the gene encoding glycoprotein gE, a deletion in the gene encoding glycoprotein gG and a deletion in the gene encoding tyrosine kinase (TK), wherein at least one heterologous antigen is inserted in at least one deletion locus.
The PRV recombinant vector of the preceding embodiment, wherein the deletion in the gene encoding gG does not affect the transcription of the Us3 gene and the deletion in the gene encoding TK does not affect the transcription of the UL22 and UL24 genes.
The PRV recombinant vector of the preceding embodiment, wherein the Us3 Poly A sequence of the Us3 gene is repositioned upstream of the gG deletion.
The PRV recombinant vector of any of the preceding embodiment, wherein the deletion in the gene encoding gG disrupts the chemokine binding ability of glycoprotein gG.
The PRV recombinant vector of the preceding embodiment, wherein the deletion in the gene encoding gG encompasses the sequence coding for amino-terminal amino acid residues 1 to 71.
The PRV recombinant vector of any of the preceding embodiment, wherein TAATA boxes of the UL22 and UL24 genes are functional.
The PRV recombinant vector of any of the preceding embodiment, wherein the deletion in the gene encoding TK disrupts the tyrosine kinase activity of TK. The PRV recombinant vector of the preceding embodiment, wherein the deletion in the gene encoding TK encompasses the sequence coding for amino acid residues 136 to 320.
The PRV recombinant vector of any of the preceding embodiment, wherein the deletion in the gene encoding gE disrupts the Fc binding activity of the complex gE-gl.
The PRV recombinant vector of the preceding embodiment, wherein the deletion in the gene encoding gE encompasses the complete ORF of gE.
The PRV recombinant vector of any of the preceding embodiment, wherein the deletion in the gene encoding Gg is replaced by a sequence having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:1.
The PRV recombinant vector of any preceding embodiment, , further including at least three heterologous antigens inserted therein.
The PRV recombinant vector of the preceding embodiment, wherein the at least three heterologous antigens are derived from proteins of any of the viruses selected from the group consisting of Porcine Circo virus type 2 (PCV2), Classical Swine Fever Virus (CSFV), and combinations thereof.
The PRV recombinant vector of the preceding embodiment, wherein at least one PCV2 antigen derived a PCV2 having a genotype selected from the group consisting of PCV2a, PCV2b, PCV2c, PCV2d, PCV2e, and combinations thereof.
The PRV recombinant vector of the preceding embodiment, wherein the PCV2 antigen is derived from the virus of the PCV2b genotype.
The PRV recombinant vector of any preceding embodiment, wherein the at least three heterologous antigens are derived from proteins selected from the group consisting of viral envelope glycoproteins, capsid proteins, and combinations thereof.
The PRV recombinant vector of the preceding embodiment, wherein the capsid protein lacks a nuclear localization signal.
The PRV recombinant vector of any preceding embodiment, wherein the capsid protein self-assembled as a virus-like particle (VLP).
The PRV recombinant vector of any preceding embodiment, wherein the at least three heterologous antigens are derived from proteins selected from the group selected from PCV2 Cap, CSFV Ems, CSFV El, CSFV E2, CSFV C, and combinations thereof.
The PRV recombinant vector of any preceding embodiment, wherein at least one of the at least three heterologous antigens is expressed as a fusion protein with a fusion partner. The PRV recombinant vector of the preceding embodiment, wherein the fusion partner is a cytokine that can potentiate a humoral and/or cellular immunity, preferably the cytokine being GM-CSF.
The PRV recombinant vector of any preceding embodiment wherein the fusion partner is selected from the group consisting of a gD signal sequence, GM-CSF, and combinations thereof.
The PRV recombinant vector of any preceding embodiment, wherein the coding sequence of the CSFV E2-derived antigen is inserted into the gE deletion locus, the coding sequence of the PCV2 Cap-derived antigen is inserted into the TK deletion locus, and the coding sequence of the CSFV Ems-GMSCF-derived antigen is inserted into the gG deletion locus.
The PRV recombinant vector of any preceding embodiment, wherein at least one of the at least three heterologous antigens is expressed from a heterologous promoter within a gene expression cassette.
The PRV recombinant vector of the preceding embodiment, wherein the heterologous promoter is selected from the group consisting of a viral promoter and a mammalian promoter.
The PRV recombinant vector of the preceding embodiment, wherein the promotor is selected from the group consisting of a HCMV promotor, a human elongation factor 1 alpha promotor, a CMV IE promotor and a CAG synthetic promotor.
The PRV recombinant vector of any preceding embodiment, further expressing a CSFV E2-derived antigen from a gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:2, a PCV2 Cap-derived antigen from a gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:3, and aCSFV Ems-GMSCF-derived antigen from a gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO: 4.
The PRV recombinant vector of any preceding embodiment, further incuding the gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:2 is inserted in the gE deletion locus, the gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:3 is inserted into the TK deletion locus, and the gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:4 is inserted into the gG deletion locus. The PRV recombinant vector of any preceding embodiment, wherein the vector establishes latency and does not replicate in the TG neurons.
A composition includes a carrier and at least one PRV recombinant vector of any preceding embodiment.
The composition of the preceding embodiment formulated for administration by an intranasal route.
The PRV recombinant vector of any preceding embodiment for use as a vaccine.
The PRV recombinant vector of any preceding embodiment for use in the prevention and/or the treatment of porcine infection, preferably diseases induced by at least one pathogen selected from the group consisting of PRV, PCV2, CSFV, SIV, mycoplasma sp, parvovirus, PRRSV and combinations thereof.
A method for treating a swine having or at risk of having an infection, in particular a porcine infection, by administering at least one PRV recombinant vector of any preceding embodiment.
The method of the preceding embodiment, wherein the porcine infection is a viral porcine infection induced by at least one virus selected from the group consisting of PRV, PCV2, CSFV, SIV, and combinations thereof.
The method of the preceding embodiment, wherein the porcine infection is caused by PCV2 in combination with at least one pathogen selected from the group consisting of mycoplasma sp, parvovirus and porcine reproductive and respiratory syndrome virus (PRRSV), and combinations thereof.
The method of the preceding embodiment, wherein the porcine infection is the postweaning multisystemic wasting syndrome (PMWS).
The method of the any preceding embodiment, whereinadministering at least one PRV recombinant vector prevents or reduces the incidence or severity of viral infection in a swine.
The method of the preceding embodiment, whereinadministering at least one PRV recombinant vector induces humoral and/or cellular immunity.
The method of the preceding embodiment, wherein administering at least one PRV recombinant vector induces humoral immunity against at least one virus selected from the group consisting of PRV, PCV2, CSFV, and combinations thereof.
The method of the any preceding embodiment, wherein administering at least one PRV recombinant vector induces cellular immunity against CSFV.
The method of the any preceding embodiment, wherein the swine is a domesticated pig, an experimental pig or a boar. A live attenuated vaccine for protection against at least one porcine disease including at least one of the RPV recombinant vector of any of the preceding embodiment.
The vaccine of the preceding embodiment, wherein the porcine infection is caused by at least one pathogen selected from the group consisting of PRV, PCV2, CSFV, SIV, mycoplasma sp, parvovirus, PRRSV, and combinations thereof.
The vaccine of the preceding embodiment, further including a pharmaceutically acceptable vehicle or adjuvant.
The vaccine of any preceding embodiment, wherein said vaccine prevents virus shedding.
A method of vaccinating a swine against a porcine infection, said method including inoculating the swine with the vaccine of any preceding embodiment.
The method of the preceding embodiemnt, wherein the PCV2-infected swine is coinfected with at least one pathogen selected from the group consisting of mycoplasma sp, parvovirus, PRRSV, and combinations thereof.
The method of any preceding embodiment, wherein the vaccinating results in prevention or reduction of the symptoms associated with post-weaning multisystemic wasting syndrome (PMWS).
The method of the preceding embodiment, wherein the PCV2-infected swine is coinfected with at least one virus selected from the group consisting of SIV, CSFV, PRV, and combinations thereof.
The method of the preceding embodiment, wherein the vaccination results in the reduction of the pathogenicity of at least one virus selected from the group consisting of SIV, CSFV, PRV, and combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a genomic configuration of pseudorabies virus (PRV) and the strategy of thymidine kinase (TK), glycoprotein G (gG) and gE gene deletion to generate a PRV triple mutant virus (tmv) recombinant vector. SEQ ID NO: 1 is disclosed. Arrows indicate the direction of the corresponding open reading frame (ORF); Part. - partial sequence; Inter. - intergenic sequence.
Figure 2 shows the cloning strategy of chimeric PCV2 cap, CSFV Erns-GM-CSF and CSFV E2 insertion in the TK deletion, gG deletion and gE deletion loci, respectively of PRVtmv genome to generate PRVtmv-CSFV E2-Ems-GM-CSF-PCV2b Cap (PRVtmv+). Figure 3 discloses the organization of the nucleotide sequence of chimeric CSFV E2 gene expression cassette (pPRV gEA/CSFV E2-INS) ( SEQ ID NO: 2); restriction sites (bold, underlined); PRV gE flanking upstream and downstream (lower case); CAG promotor sequence; Kozak sequence (italic); PRV gD signal (underlined); CSFV E2 coding sequence (highlighted); V5 epitope coding sequence (lower case, italic); 6x His epitope coding sequence (bold); Stop codon TGA (bold, italic); bovine growth hormone terminator (italic, underlined).
Figure 4 discloses the organization of the nucleotide sequence of chimeric CPV2b CAP gene expression cassette (pPRV TKA/PCV2 Cap-INS, SEQ ID NO: 3); restriction site (bold, underlined); PRV TK flanking upstream and downstream (lower case), start and stop codon of UL24 and start codon of TK are highlighted; three stop codons (underlined); hEF-lapromotor sequence; vii) Kozak sequence (italic); PRV gD signal (underlined); PCV2 Cap coding sequence (highlighted); V5 epitope coding sequence (lower case, italic); 6x His epitope coding sequence (bold); Stop codon TGA (bold, italic); bovine growth hormone terminator (italic, underlined); stop codon of TK and start codon of UL22 (gH) are highlighted.
Figure 5 discloses the organization of the nucleotide sequence of CSFV chimeric Ems- GMCSF gene expression cassette (pPRV gGA CSFV Ems-GM-CSF-INS, SEQ ID NO: 4); Restriction sites ( bold underlined); PRV gG flanking upstream and downstream (lower case); Us3/Us4 polyadenylation signal (lower case, bold and italic); Kozak sequence (italic); PRV gD signal (underlined); CSFV Ems coding sequence (highlighted); GM-CSF coding sequence (underlined, highlighted); Flag epitope coding sequence (lower case, italic); Stop codon TAA (bold, italic); SV40 terminator (italic, underlined).
Figure 6 illustrates the vaccination, sample collection, challenge, and euthanasia scheme for animal experiments.
Figure 7 shows immunoblot-analysis of PRVtmv+ expressing chimeric CSFV E2, CSFV Erils-GM-CSF. and PCV2 cap proteins using an anti-CSFV E2 monoclonal antibody (mAbs) (left panel), an anti-CSFV Erns mAbs (middle panel), and a rabbit anti-PCV2 cap Ab (right panel), respectively.
Figure 8 shows transmission electron microscopy imaging of Mock-infected healthy swine kidney (SK) cell with normal cellular morphology (A and B), PCV2b-infected SK cells (C and D), and PRV wt-infected SK cells (E, F and G). Panels B, D, F, and G are magnifications of A, C and E, respectively.
Figure 9 shows transmission electron microscopy imaging of PRVtmv+ vaccine virus- infected SK cells: PRVtmv+ vaccine virus particles are visible in the exocytic vesicles and accumulation of PCV2 virus-like particles (VLPs) can be seen within the vesicular structures in the cytoplasm.
Figure 10 shows the In vitro characterization of PRVtmv+: (A) is a plaque size analysis of PRVtmv+ compared to that of PRV wt; the Bar graph (B) shows normalized average plaque size (n=150) for each virus with standard deviation (SD); (C) is a One-step growth analysis of PRVtmv+ compared with PRV wt.
Figure 11 shows growth kinetics assay for PRV wild type and PRVtmv+ after infection.
Figure 12 provides clinical assessments of control-, fostera- and PRVtmv+-immunized pigs following immunization and challenge: (A) and (B) assess the rectal temperature and the body weight, respectively.
Figure 13 is a table providing data on rectal temperature for individual animals enrolled in the study.
Figure 14 is a table providing data on body weight for individual animals enrolled in the study.
Figure 15 shows dot plot graphs representing nasal shedding measured by qPCR (A) and virus isolation by plaque assay (B) of PRVtmv+-immunized pigs
Figure 16 shows dot plot graphs representing data of PRVtmv+in tonsil swab quantified by qPCR (A) and virus isolation by plaque assay (B) in immunized pigs.
Figure 17 is a table providing data on PRVtmv+ vaccine virus replication in individual immunized pigs (nasal and tonsil swabs) and data on PRV-specific qPCR and virus isolation in SK cells (in PRVtmv+-immunized pigs).
Figure 18 shows immunoblot analysis of PRVtmv+ vaccine virus after a passage in pigs expressing chimeric CSFV E2 and Ems proteins by using E2- (left panel) and Erns-specific (right panel) mAbs, respectively.
Figure 19 provides imaging of indirect immunofluorescence assay (IIFA) for serum samples collected from PRVtmv+ immunized pigs. (Magnifications 200X). Positive signals were indicated by bright apple-green fluorescent signals.
Figure 20A provides PRV-specific SN antibody titer from pigs after PRVtmv+ vaccination; (20B) PCV2b-specific SN antibody titer following PRVtmv+ immunization and PCV2b challenge; (20C) CSFV-specific neutralization dose (ND50) SN titers following PRVtmv+ immunization; dot plot graphs show each animal's mean values and individual titer with standard deviation (n=5). dpv - days-post vaccination; dpc - days-post challenge.
Figure 21 is a table providing data of PRV -neutralizing antibody titer after PCV2b challenge in individuals immunized pigs. Figure 22 is a table providing data of PCV2b-neutralizing antibody titer after PCV2b challenge in individuals immunized pigs.
Figure 23 is a table providing data of CSFV-neutralizing antibody titer after PCV2b challenge in individual immunized pigs.
Figure 24 are graphs showing the percent changes in leukocyte counts (A) and lymphocyte counts (B) following vaccination and challenge in all three groups.
Figure 25 is a table providing leucocyte counts and lymphocyte counts in individual pigs days-post vaccination and post challenge.
Figure 26 shows a dot plot graph of Fecal PCV2b shedding in control and vaccinated pigs following the PCV2b challenge quantified by qPCR.
Figure 27 is a table providing data of Fecal PCV2b shedding by PCV2b- capsid genespecific qPCR for individual pigs in all 3 groups.
Figure 28 shows a dot plot graph representing copy numbers of PCV2b genome in serum (cell-free) and PBMC (cell-associated viremia) in control and vaccinated pigs following challenge.
Figure 29 is a table providing quantification of PCV2b viremia in serum by PCV2b- capsid gene-specific qPCR for individual pigs in all 3 groups
Figure 30 is a table providing quantification of PBMC-associated PCV2b viremia by PCV2b- capsid gene-specific qPCR for individual pigs in all 3 groups
Figure 31 shows histopathology imaging following vaccination with Fostera or PRVtmv+ vaccines and subsequent challenge with PCV2, Control group (A and D), Fostera- vaccinated group (B and E), and PRVtmv+ -vaccinated group (C and F). H&E 100X total magnification, Bar = 100 micrometers.
Figure 32 shows a dot plot graph representing the mean number of PCV2b viral genome copies in pig lymphoid tissues (tonsil, mesenteric LN, mediastinal LN, cervical LN, Peyer's patch, and spleen) from control-, fostera-, PRVtmv+- vaccinated pigs; The dot plot graph represents the mean + individual values in each group (n=5).
Figure 33 is a table providing the number of PCV2b viral genome copies (normalized viral genome copies per 106 cells) in different lymphoid tissues of individual pigs from control, fostera and PRVtmv+-vaccinated groups
Figure 34 shows immunohistochemistry imaging of the presence or absence of PCV2b antigen in tonsil (A) and intestine (B) in a control-, Fostera- and PRVtmv+-immunized pig; right panels are 100X magnifications of left panels. Bar = 100 micrometers. Figure 35 shows a table listing the criteria used to determine the clinical score during the clinical assessment of the pigs.
Figure 36 is a table providing body temperature (°C) in pigs following PRV infection and latency-reactivation.
Figure 37 is a table providing clinical scores of the pigs after infection
Figure 38 shows in (A) a graph of the mean rectal temperature of each treatment wtPRV and PRVtmv+ group with SD and in (B) a graph of the mean clinical score of each group with SD
Figure 39 are photographs showing in (A) bilateral nasal discharge and tear staining of a wtPRV-infected pig on 3 dpi ; in (B) unilateral comeal ulcer and descemetocele on the right eye of a wt PRV -infected pig on 7 dpi.; in (C) healthy PRVtmv+ - vaccinated pigs on 3dpi without any clinical symptoms of viral infection
Figure 40 is a table providing data on nasal shedding following infection determined by pseudorabies virus-specific qPCR and virus isolation in swine kidney (SK) cells
Figure 41 is a table providing data on nasal shedding following latency reactivation determined by PRV-specific qPCR
Figure 42 is a graph showing nasal virus-shedding following Dex-induced latency reactivation in pseudorabies virus wtPRV-infected and PRVtmv+-vaccinated pigs
Figure 43 is a table providing the number of copies of targeted PRV genome in the TG neurons of infected/immunized pigs
Figure 44 shows that wtPRV and PRVtmv+ establish latency in the TG neurons and have the ability to reactivatate following Dex treatments
Figure 45 is a table providing the number of targeted PRV transcript copies in the TG neurons of infected/immunized pigs
Figure 46 shows that wtPRV expressed genes involved in viral replication but PRVtmv+ does not; quantification of ICPO, MCP and gG transcripts in TG samples
Figure 47 is a table providing PRV-specific serum neutralizing (SN) antibody titers in wtPRV-infected/PRVtmv+-vaccinated pigs following infection and Dex-induced latencyreactivation
Figure 48 shows that only wtPRV-infected pigs but not PRVtmv+ - inoculated pigs elicits a memory SN antibody response following Dex-induced latency -reactivation.
Figure 49 shows a table gathering data on PRV genomic DNA and transcript (cDNA) copies based on immediate early protein, ICPO- and late envelope glycoprotein, gC-specific qRT-PCR; the gene and transcript copy numbers were normalized to 106 neurons in TG Figure 50 shows a graph representing the variation of body temperature of unvaccinated (control) group and PRVtmv+ immunized group following lethal CSFV challenge over time; data are presented in the table
Figure 51 shows that viremia is controlled in PRVtmv+ immunized pigs after CSFV challenge; data are presented in the table
Figure 52 shows a graph representing the neutralizing antibody titers in PRVtmv+ vaccinated and non-vaccinated control pigs following vaccination; data are presented in the table
Figure 53 shows a survival curve of vaccinated versus control pigs following lethal CSFV challenge data are presented in the table
Figure 54 shows a graph representing the percent reduction in white blood cells (WBC) count following lethal CSFV challenge in both vaccinated and control groups; data are presented in the table
Figure 55 shows a graph representing the percent reduction in lymphocyte count following lethal CSFV challenge in both vaccinated and control groups; data are presented in the table
Figure 56 shows a graph representing the percent reduction in platelet count following lethal CSFV challenge in both vaccinated and control groups; data are presented in the table
DETAILED DESCRIPTION
Some embodiments of the current invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be use and other methods developed without departing from the broad concepts of the current invention. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated.
Definitions are included herein for the purpose of understanding the present subject matter and the appended claims. The abbreviations used herein have their conventional meanings within the chemical and biological arts.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.
The present description identifies certain nucleotide and amino acid sequences (polynucleotides and polypeptides) as part of the invention. It is to be understood that the specifically identified sequences adequately describe other sequences that contain less than 100% sequence identity but to the identified sequences that provide the same function. For example, a nucleotide sequence may have 90% sequence identity or 95% sequence identity with a polynucleotide specifically disclosed herein and still encode for an entirely equivalent or functionally equivalent polypeptide. Similarly, a polypeptide may contain less than 100% sequence identity to a polypeptide specifically identified herein and provide the same function. For example, a polypeptide may have 90% sequence identity or 95% sequence identity with a polypeptide specifically disclosed herein and still retain the same or sufficiently similar activity or functionality as the specifically identified polypeptide.
As used throughout, the term “chimeric gene” refers to a hybrid gene having a nucleotide sequence comprising at least two partial or complete sequences derived from, obtained from, or isolated from different genes that are not naturally adjoined. A chimeric protein or chimeric antigen is the functional product of a chimeric gene. Chimeric genes can further be modified by mutation, deletion, insertion or substitution of heterologous sequences, or by any means available using recombinant DNA technology.
In some embodiments, a “recombinant vector” as used herein refers to a genetic material, for example a virus or a plasmid, used as a vehicle to artificially carry foreign genetic material into a host cell where it can be replicated and/or expressed. Such vehicle has been genetically engineered to produce new genetic combination.
As used throughout, the term “mutant virus” refers to a virus which has been genetically engineered by deletion, mutation or truncation of genetic sequences and/or by subsequent insertion or substitution of heterologous genetic sequences. When used as a vaccine, such mutant virus becomes less pathogenic, while still being able to elicit robust immune responses in a host. Given that the engineered triple mutant virus described herein is used as a recombinant vector to carry and express protective viral antigens, both terms “mutant virus” and “recombinant vector” can be used interchangeably throughout.
The terms “gene expression cassette” and “expression cassette” which are used interchangeably throughout the specification, refer to a sequence comprising at least one gene of interest and regulatory elements such as, for example, promoter and enhancers controlling its expression, i.e., its transcription and the translation of the transcription product. Regulatory elements such as, for example, promoter and enhancers may be located upstream of the coding sequence of the gene of interest and may be operably linked thereto or may be separated therefrom by intervening sequence such as, for example, by the 5 '-untranslated region of the gene of interest. The regulatory elements may also include a downstream 3' untranslated region comprising a polyadenylation site. The expression cassette may additionally contain sequences critical for the expression of genes adjacent to the insertion locus into a recombinant vector, i.e., insertion site of the expression cassette and/or selectable markers and/or sequences critical to the cellular localization of the product of gene of interest. The expression cassette is usually flanked by one or more sets of restriction sites to enable its insertion into a recombinant vector and/or its excision from a vector. A recombinant vector according to the present invention may include more than one expression cassette, each expression cassette expressing at least one gene of interest. A gene of interest according to the present invention may be a chimeric gene coding for a chimeric antigen and/or a heterologous antigen.
As used throughout, the term “live-attenuated” or “modified live” refer to a live organism such as a virus which has been weakened so that it is not virulent but can still induce protective immune responses in a host.
As used throughout, the wording “heterologous antigen” refers to any antigenic protein, polypeptide or peptide capable of eliciting a immune response, which originates from a different viral strain, a virus of a different genotype, a bacteria, a mammal or any species different from that of the PRV.
As used throughout, the term “cytopathic” refers to a virus which causes the death of the infected cells, whereas the term “non-cytopathic” refers to a virus which propagate without killing the infected cell.
Vaccination campaigns with MLV containing CSFV C-strain and PRV Bartha-K61 strain have been efficient in preventing and controling classical swine fever and pseudorabies challenges for nearly two decades However, CSFV has become endemic in some parts of the world including China and is responsible for intermittent CSF outbreaks. Given that no serological marker specific to the CSFV C-strain has been identified so far, the vaccine strain cannot be distinguished from the circulating endemic wild type strain. The vaccine C-strain may persist in vaccinated animals which may contribute to the endemic CSFV problem, thus making the use of the MLV vaccine problematic.lt is possible that the C-strain mutates in vaccinated animals and revert to virulence,, leading to the emergence of viral strains that can evade their immune system.
In addition, a highly neurovirulent PRV strain was recently isolated in pigs vaccinated with PRV Bartha- K61, suggesting that the Bartha K-61 vaccine may not efficiently protect vaccinated pigs against such emerging neurovirulent PRV strain [13-16,59,60],
PCV2 is also endemic in most, if not all, intense pig farms worldwide [61], Although PCV2 alone only causes a sub-clinical disease, its coinfection with other infectious agents like Mycoplasma sp, PRRSV, PPV, SIV, and PRV increases the pathogenicity and clinical symptoms of the viral infections [62], PCV2 infection induces immunosuppression which not only leads to increase susceptibility to other infectious agents but importantly reduces immune response to vaccines. In addition, the only vaccines against PCV2 commercially available are subunit protein or inactivated vaccines because PCV2 has one the highest mutation rates among DNA viruses. .. Unfortunately, such vaccines do not provide adequate protection .
Coinfection rates of PRV with porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and classical swine fever virus (CSFV) are high in intensive pig farms. In China alone, the rate of PRV coinfected with PRRSV was 36.0% followed by 12.9% with PCV2 and 1.8% with CSFV, respectively [15], The emergence of highly neurovirulent PRV strains in Bartha K61 vaccinated pigs may have been due to complex epidemiological interactions between the circulating PRV and co-infecting endemic PCV2, PRRSV, and/or CSFV virus(es).
Even though CSFV and PRV have been effectively eradicated in North America and Europe, PRV is circulating, undetected, in the wild boar populations in Europe and the US. CSFV outbreaks in Germany in the 90’ s, and subsequently in the Netherlands, Italy, and Spain were link to indirect or direct contact with wild boar infected with CSF virus or swill feeding [5,63,64], The circulation of CSFV in the European wild boars remains a potential threat. Circulation of CSFV in wild boars, in China, East Europe, including Russia, South, and Central American countries, and the Caribbean, i.e. Cuba, Haiti, and the Dominican Republic, most likely also contributes to the endemic status of CSFV and occasional outbreaks of CSF [9,17,65], Because of these reasons and the lack of a serological marker, CSFV MLV is not allowed in the EU. Instead, strict surveillance of CSFV in wild boars and domesticated pigs is implemented.
Pestiviruses contain single-stranded RNA genomes and have higher mutation rates in vitro and in vivo. Therefore, vaccine production requires strict quality control protocols to maintain its genomic sequence integrity. Most importantly, production facilities for CSFV require highly contained environments due to biosecurity concerns. PCV2 is also difficult to grow in cell cultures with the highest virus titers in cell culture usually less than 104 PFUs/ml. The problems associated with large-scale vaccine production are overcome in the production of the PRVtmv+ live vaccine virus described herein.
The PCV2 single-stranded DNA genome comprises 1,767 or 1,768 nucleotides (nt) and is predicted to have 11 potential open reading frames (ORF1-11) [19], Despite being the smallest genome among other DNA viruses, the virus has the highest evolution rate. As PCV2 continues to evolve, currently, there are five different genotypes, PCV2ato PCV2e. During the mid-2000s, pig populations were mainly infected with the PCV2b genotype. Since 2012, however, the dominant PCV2b genotype has been replaced by the PCV2d genotype in most pig production countries. Nevertheless, both genotypes are antigenically related and produce cross-neutralizing antibodies [20], Previous studies have demonstrated that ORF2 encodes a 27.8 kDa capsid (Cap) protein. PCV2 Cap is highly immunogenic and produces neutralizing antibodies in pigs [21], Neutralizing pig sera also recognize the PCV2 Cap [22],
CSFV is enveloped and has a positive-sense, single-stranded, 12.5 kb RNA genome [23], The genome contains a single ORF that encodes a polyprotein composed of 3,898 amino acids that is cleaved proteolytically by viral and cellular proteases to yield up to 12 products (NH2-NproC-Ems-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) [24]. There are four virion-associated structural proteins, C (capsid) and Erns, El, and E2 (envelope proteins). Although Erns is associated loosely with the envelope, the protein is also secreted [25], E2 has been implicated, along with Ems and El in viral adsorption to host cells [26], E2 is essential for CSFV replication, as virus mutants containing partial or complete deletions of the E2 gene are nonviable [27], Both E2 and Erns elicit neutralizing antibodies and induce protective immunity independently [28], Countries free of CSFV, including the EU countries, do not vaccinate their domesticated herds despite the fact that a commercially available CSFV- MLV confers adequate, rapid, and solid immune protection and limits the severity of the diseases in vaccinated animals [29], Extensive vaccination with MLV is believed to be responsible for sporadic outbreaks and endemic forms of CSFV in China and Korea [9,30], The PRV genome is approximately 145 kb and composed of numerous essential and non-essential genes for replication in vitro in epithelial cells. Among the non-essential genes, thymidine kinase (TK), envelope glycoprotein E (gE), and gG can be deleted or replaced by heterogeneous genes without affecting the in vitro replication in cell culture. TK-, gE- and gG- deletion individually or simultaneously reduce the virulence in pigs [10,34,35], Although TK is non-essential for viral replication in vitro in epithelial cells, the TK-deleted virus has defective replication in the non-dividing or post-mitotic cells, e.g., neurons [36] which lack the cellular TK gene. Following intranasal infection, the TK-deleted virus can enter the sensory nerve endings in the nasal mucosa and be transported retrogradely to the neuron cell bodies in the trigeminal ganglia (TG), and establish latency. However, the latent virus upon reactivation in vivo following dexamethasone treatment doesn’t replicate [37],
PRV gE-deleted virus replicates efficiently both in cell culture in vitro and in nasal epithelium of pigs in vivo,' however, the gE-deleted virus is highly attenuated in pigs and does not produce clinical symptoms following intranasal (IN) infection [34,35], PRV gE possesses Fc receptor activity which prevents the complement-mediated antibody-dependent cellular cytotoxicity /lysis (ADCC) of virus -infected monocytes expressing gE antibody on the surface. Since the gE-deleted virus lacks Fc receptor activity, virus-infected monocytes are lysed due to ADCC, reducing virulence [38], The gE is also not required for viral entry into the nerve endings of the trigeminal nerve (maxillary branch) in the nasal mucosa. Neither is it necessary for the retrograde axonal transport from nasal epithelium to cell bodies in the TG neurons, where the virus establishes life-long latency. However, upon reactivation from latency in the TG neurons, due to stress or induced by dexamethasone injection, its anterograde axonal transport from the neuron cell bodies to axon termini or second-order neurons in the CNS is severely inhibited. Consequently, both nasal shedding and CNS invasion of gE-deleted virus following reactivation in the TG neurons do not usually occur [39] . PRV gE-deleted-vaccinated pigs can be differentiated from the infected pigs (DIVA). Therefore, PRV gE-deleted vaccine is used for eradication efforts in many countries [10],
Interestingly, there appears to be a correlation between pre-colonization of the TG neurons with PRV and failure of superinfecting strain to become latent in the TG neurons [40], Therefore, latent gE-deleted live vaccine strains may also prevent superinfecting wild type (wt) strains from becoming latent. PRV envelope glycoprotein gG is a viral chemokine binding protein [41], The binding of PRV-gG to chemokines results in interference of chemokine- mediated lymphocyte, neutrophil, and monocyte migration to the site of virus infection, indicating that PRV gG may play a role in evading the host's immune response [41], A triple mutant virus PRVtmv was constructed as lacking the entire gE gene and part of the TK and gG gene sequences. The strategy is disclosed in Figure 1. The deletion of the gE ORF is a serological marker distinguishing the vaccinated from the wt virus-infected pigs (DIVA). The TK and gG genes were inactivated. Subsequently, chimeric CSFV E2 and Ems- GM-CSF genes were inserted in the gE- and gG- deletion loci, respectively. Furthermore, a chimeric PCV2b Cap gene was inserted in the TK deletion locus to provide a live-attenuated genetically engineered trivalent “PRVtmv+” vaccine against PRV, PCV2b, and CSFV as disclosed in Figure 2. As demonstrated in the examples disclosed herein, the PRVtmv+ was shown to be safe for pigs and capable of eliciting virus-neutralizing antibodies in the vaccinated pigs against all three viruses; PRV, PCV2b, and CSFV. Notably, compared with the commercial Fostera Gold PCV vaccine, the PRVtmv+ induced approx. 2-fold higher PCV2b- specific neutralizing antibody titers after vaccination and after the PCV2b challenge. Both PRVtmv+ and Fostera prevented fecal virus-shedding of the challenge virus. However, only PRVtmv+ vaccination prevented pigs entirely from both cell-free and cell-associated viremia as compared to the Fostera vaccine. Furthermore, one PRVtmv+ vaccinated pigs had minimal PCV2b viral load only in the mediastinal lymph. In contrast, several Fostera vaccinated pigs had low levels of viral load in the mediastinal and cervical lymph nodes. Notably, one Fostera pig had high PCV2b genome copy numbers in the Peyer’s patches.
Like the control group pigs, most of the Fostera vaccinated pigs had leukopenia and lymphopenia. In contrast, the vaccinated pigs immunized with the PRVtmv+ vaccine according to the invention had lymphocytosis and leukocytosis, indicating a better lymphoproliferative immune-stimulation induced by the chimeric PCV2b Cap antigen. Taking together, the claimed PRVtmv+ vaccine provided better immune protection than the Fostera vaccine. Because the chimeric PCV2b Cap protein lacks the nuclear localization domain, it can self-assembled as PCV2b VLPs in PRVtmv+ infected SK cells. Thechimeric Cap protein according to embodiments described herein remains functionally and antigenically intact and as such induces a protective immune response against PCV2b in vaccinated pigs. Additionally, PRVtmv+ induced low to moderate CSFV neutralizing antibody titers.
The claimed PRVtmv+ virus retains its genomic stability for the PCV2b and CSFV chimeric genes after multiple cell culture passages and a single animal passage.Furthermore, the claimed PRVtmv+ does not reactivate from latency upon dexamethasone-induced latencyreactivation. Accordingly, the risk of vaccine virus transmission and circulation in the pig population is limited. Advantageously, the PRVtmv+ virus is easier to produce in vitro because it replicates significantly better in cell cultures than the commercially available vaccine, with a titer ranging from approx. 5 x 107 - 1 x 108 PFUs/ml, relative to the PCV2b highest titers (5- 8 x 103 PFUs/ml).
The TK- or gE-deleted viruses replicate in vitro in epithelial cells like wtPRV and with similar virus yields. Epithelial cells express TK which is used by tto compensates for the viral TK, and the cell-to-cell spread defect phenotype of gE gene deletion does not affect virus replication in cell culture. However, in the neurons in culture or in vivo in the animals, i.e., TG neurons, the TK-deleted virus does not replicate because neurons do not express TK required for virus replication. As indicated above, PRV gE is not required for virus entry into the axon termini of the maxillary branch of the trigeminal nerve in the nasal mucosa and retrograde axonal transport to neuron cell bodies in the TG. However, it is essential for anterograde axonal transport from neuron cell bodies in the TG to nerve endings in the nasal mucosa.
Following intranasal infection, wtPRV and PRVtmv+ established latency and can reactivate in the TG neurons upon dexamethasone-induced latency-reactivation. PRVtmv+, like wtPRV, enters the sensory nerve endings in the nasal epithelium and is transported retrogradely to the neuronal cell bodies in the TG. TK-deficient PRVtmv+ is avirulent because it no longer replicates in terminally differentiated neurons [6,14], Consequently, no PRVtmv+ particles are produced in the neuron cell bodies. While PRVtmv DNA is detected in the TG neurons of latently infected pigs, there is no DNA replication-dependent, late viral genes-gC, and MCP transcriptions. In contrast, wt PRV DNA is detected in the nasal swabs of wtPRV - infected pigs after 4-5 days of Dex treatment and has seroconverted memory B cells following the Dex-induced latency-reactivation. Given that PRVtmv+ has the DIVA property, PRVtmv+ can be used as a safe subunit vaccine vector in countries where PRV has been eradicated from the domestic pig population.
The invention is described herein by the following representative non-limiting example intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this example or specification should be considered as limiting the scope of the present invention. The specific embodiments of the invention described may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.
EXAMPLES
2. Materials and methods 2.1. Cells and medium
Swine kidney (SK; #CRL-2842, ATCC®, Manassas, VA, USA), Madin Darby bovine kidney (MDBK; #CCL-22, ATCC®), 293T (#CRL-3216, ATCC®) and TK negative TK-tsl3 hamster (ATCC #1632) cells were propagated in Dulbecco's modified Eagle's medium (DMEM; #10-017-CV, Coming®, Coming, NY, USA) supplemented with 10% heat- inactivated fetal bovine serum (FBS; EqualFETAL, Atlas Biologicals, Fort Collins, CO, USA) and 1 x antibiotic-antimycotic solution (#30-004-CI, Coming®) (Growth medium). Plasmids and viral DNA co-transfection in 293T cells were performed using Opti-MEM® (#31985-070; Gibco, Waltham, MA, USA) and lipofectamine 2000 (#11668030, Thermo Fisher Scientific®, Waltham, MA, USA).
2.2. Viruses
PRV wt Becker strain is a vimlent field isolate from a dog at Iowa State University, with subsequent laboratory passage [43], Low passage vims stock was propagated and maintained at -80 °C. Infectious porcine circovirus type 2b (PCV2b) plasmid DNA clone [44] was obtained from Dr. Rowland (University of Illinois). The vims was reconstituted by transfection into SK cell using polyethyleneimine (#23966, Polysciences, Warrington, PA, USA) as described previously [44], Reconstituted PCV2b virus stock was titrated in SK cells. PRV wt and the genetically engineered recombinant PRV viruses were propagated in SK cells and titrated in MDBK cells as described earlier [45], Aliquots of low passage viral stocks were maintained at -80°C. The CSFV strain Brescia (genotype 1.1) was obtained from the CSF virus collection of the EU and OIE Reference Laboratory for CSF (Institute of Virology, University of Veterinary Medicine, Foundation, Hannover, Germany).
2.3. Antibodies
PRV-specific rabbit anti-gE antibody was kindly provided by Lynn W. Enquist, Princeton University, NJ, USA. Mouse anti-PRV gC antibody was purchased from VMRD (#3G9F3, VMRD Inc., Pullman, WA, USA). Monoclonal CSFV E2 (HC/TC 50/2/1 and Ems (HC/TC 169/2/3) specific antibodies were kindly provided by Paul Becher, University of Veterinary Medicine, Hannover, Germany. Anti PCV2 Cap rabbit polyclonal antibody was generated commercially (Genscript, Piscataway, NJ, USA) against a PCV2 Cap-specific E.coli -expressed polypeptide having the sequence (SEQ ID NO: 5) (AMTYPRRRYRRNGIFDPYVNYSSRHTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKRN QLWLRLQTSRNVDHVGLGTAFENSKYDQDYNIRVTMYVQFREFNLKDPPLNP). Anti-PCV2 capsid protein-specific mAh 36Flwas kindly provided by Dr. Zoltan, CEVA. Mouse anti-V5 mAb (#R960-25, Thermo Fisher Scientific®, Waltham, MA, USA) and antiflag rabbit antibody (#F7425, Sigma-Aldrich®, St. Louis, MO, USA) were purchased. CSFV NS3-specific mouse mAh antibody BVD/C16 from Institute for Virology, Hannover, Germany was used. Secondary antibodies, donkey anti-mouse IgG Alexa fluor 488 (#A-21202, Thermo Fisher Scientific®), goat anti-mouse IgG HRP (#32430, Thermo Fisher Scientific®), goat antipig IgG HRP (#abl02135, Abeam, Waltham, MA, USA), rabbit anti-mouse HRP (Agilent Technologies, Waldbronn, Germany) and FITC-rabbit anti-porcine IgG-H+L (#61-9111, Invitrogen) were purchased. Goat anti-mouse horseradish peroxidase (HRP) conjugated antibody (#32430) and donkey anti-rabbit HRP conjugate (#31458) were purchased from Thermo Fisher scientific.
2.4. Construction of the gE-, TK-, and gG-deleted triple mutant virus PRVtmv
To generate the PRVtmv+ vaccine virus, the gE-, TK-, and gG-deleted triple mutant virus (PRVtmv) vector was first constructed. (Figure 1) As represented in Figure 1 A, PRV wild type (wt) Becker strain backbone (GenBank accession # JF797219 [43]) comprises - a unique long region (UL), a unique short region (Us), - a internal repeat region (IR) and - a terminal repeat region (TR). First, a targeted deletion gEA was introduced in the gE locus of the PRV wt backbone (A) to generate PRV gEA mutant virus (Figure 1 C).. Then, a plasmid construct pPRV TKA (Figure ID) was used to incorporate a targeted TK deletion in TK locus (Figure IB) of PRV gEA (Figure 1C) in order to generate PRV gE/TK dual gene deleted mutant virus. Finally, PRVtmv (gE/TK/gG-deleted) was constructed by incorporating the plasmid construct pPRV gGA (Figure IE) into the gG locus of the PRV gE/TK dual gene deleted mutant virus backbone. (Figure 1)
2.4.1. Construction of PRV gE-deleted virus (PRV gEA)
Initially, a PRV gEA was generated by homologous recombination of full-length PRV wt DNA and a gE deletion plasmid (pPRV gEA). (Figure 1C) Briefly, the pPRV gEA plasmid was constructed by PCR amplification of a 1,167 bp EcoRI-Hindlll fragment (partial gl ORF and gE promoter sequence; GenBank accession #JX797219, nt 120858 to 122024) and 936 bp KpnI-BamHI fragment (gE-Us9 intergenic, Us9 ORF, and partial Us2 sequence; GenBank accession #JX797219, nt 123846..124781) using the wtPRV Becker DNA as a template and the corresponding PRV gE left-flanking (Fl-Rl) and right-flanking primer pairs (F2-R2), respectively (Tables 1A and Figures 1A, IB and 1C). Subsequently, the 1,167 bp EcoRI/Hindlll and the 936 bp KpnI-BamHI fragments were cloned sequentially into the corresponding sites of the plasmid pGEM3Z. In the pPRVgEA clone, the entire gE ORF was deleted, and two restriction sites Hindlll-Kpnl sites were incorporated in the gE deletion locus for future directional CSFV E2 chimeric gene insertion (as described in 2.5.1 and Figure 2). A PRV gEA recombinant virus (Figure 1C) was plaque purified and selected to construct a PRV gE/TK dual gene-deleted virus.
Table 1A: List of primers used to construct the gE-deleted recombinant viruses. Restriction enzyme cleavage sites are bold and underlined. Stop codons are highlighted in bold.
Table 2B: List of primers used to construct TK-null/deleted recombinant viruses. Restriction enzyme cleavage sites are bold and underlined. Stop codons are highlighted in bold. 2.4.2. Construction of PRV gE/TK dual gene-deleted virus
The TAATA boxes for the TK upstream UL24 and TK downstream UL22 genes are located within the TK ORF coding sequences. (Figures 1A-B) These two genes are essential for virus replication [46,47], To construct a TK null/deletion plasmid (pTKA) without affecting the surrounding UL24 and UL22 promoter sequences, PRV nucleotide sequences (GenBank accession #JX797219) spanning nt 58548 - 59519 (primer pairs F3/R3) and nt 59571 - 60474 (primer pairs F4/R4) were amplified by PCR as EcoRI/Kpnl and Hindlll-Nsil fragments, respectively. (Table IB) The two fragments were cloned sequentially into the corresponding sites of plasmid pGEM7Z (Promega Corporation, Madison, MA, USA). In the resulting clone, plasmid pPRV TKA (Figure ID), 51 nt within the TK ORF (nt 59520..59570), immediately downstream of the TK amino acid (aa) residue 135 were deleted, and instead the KpnI-Hindlll sites were inserted. In addition, , three stop codons were incorporated immediately upstream of the Kpnl site. (Figure ID) Because of these deletion-insertion manipulations, the coding sequence of TK corresponding to the aa residues 136-320 were truncated, which resulted in a non-functional TK gene. The intended mutations within the TK coding region of the pPRV TKA DNA was verified by sequencing with a series of sequencing primers, F5-F8 and R5-R8. (Table 2) The two restriction sites Kpn and Hindlll which were incorporated in the TK ORF- deletion locus (Figure ID) allowed for the insertion of the chimeric PCV2b gene (as described in 2.5.2). One PRV gE/TK dual gene-deleted virus recombinant was selected to construct a PRV gE/TK/gG triple gene-deleted virus.
2.4.3. Construction of PRV gE/TK/gG triple gene-deleted (PRVtmv) vector
The PRV gG ORF is located downstream of the Us3 ORF (Protein kinase; PK) and upstream of Us6 (gD). (Figures 1A, IB and IE) To generate a PRV gGA plasmid (pPRV gGA; Figure IE), a 2041 bp long DNA fragment was synthesized and cloned into EcoRI (5')/HindIII (3') sites of pUC57 (BioMatik, Ontario, Canada). This fragment consisted from 5' to 3' of: an EcoRI restriction site, a 1001 bp PRV gG upstream flanking sequence comprising 5 ’-3' direction, partial carboxy-terminal Us3 ORF, and Us3-Us4 intergenic sequences, (GenBank accession #JF797219; nt 116964..117964), a chimeric Us3 and Us4 polyadenylation (Poly A) signal (as it is in the genome (GenBank accession #JF797219, nt 119520..119531) but placed immediately upstream of the Us4 start codon (ATG), followed by the Kpnl restriction site (GGTACC), a 10 bp long non-genomic spacer sequence, the BamHI restriction site and the coding sequence for the PRV gG residues 72-, Us4 (gG)-Us6 (gD ) intergenic sequence, (GenBank accession #JF797219; nt 118177..119176), and the restriction site for Hindlll. (Figures IB and IE) Table 2: List of primers used to verify the PRV TK-deletion and PCV2 Cap chimeric geneinsertion by PCR and/or sequencing. In the resulting plasmid pPRV gGA, nt 117965 to 118176 coding for gG residues 1 to 71 were deleted and replaced with the chimeric polyA and KpnI/BamHI sites, followed by the gG ORF residues 72-499 coding sequence and a Hindlll restriction site. Consequently, in the context of the viral genome, the gG ORF sequence deletion would not affect the Us3 gene transcription. However, gG residues 72-499 aa will not be translated due to the insertion inactivation of the gG gene. In addition, the KpnI-BamHI sites of SEQ ID NO: 1 can be utilized to insert the chimeric CSFV Ems-GM-CSF gene in a site-specific manner (see 2.5.3).
2.5. Construction of PRVtmv, + vector virus expressing the CSFV E2, E™S-GM-CSF and PCV2b Cap proteins
To generate PRVtmv-CSFV E2-Ems-GM-CSF-PCV2b Cap (PRVtmv+), the chimeric PCV2 cap, CSFV Ems-GM-CSF and CSFV E2 were inserted in the TK deletion, gG deletion and gE deletion loci, respectively of PRVtmv genome. (Figure 2). As disclosed in Figure 2A, the genomic organization of PRV triple mutant virus (tmv) (GenBank accession # JF797219) shows the thymidine kinase (TK) (B), glycoprotein gG (C) and gE (C) deletions. The PCV2 cap expression cassette was cloned into EcoRI-Nsil site of pPRV TKA to yield pPRV TKA/PCV2 Cap-INS. (Figure 2E) The CSFV Erns-GM-CSF expression cassette was cloned into KpnI-BamHI site of pPRV gGA to yield pPRV gGA/CSFV Ems-GM-CSF-INS. (Figure 2F) .
2.5.1. Construction of PRV gEA CSFV-E2 insertion plasmid (pPRVgEA/CSFV E2-INS)
To generate the PRV gEA CSFV E2 insertion plasmid (pPRV gEA/CSFV E2-INS, SEQ ID NO: 2) the chimeric CSFV E2-V5 expression cassette (pCSFV E2) was cloned into the KpnI-BamHI sites of pPRV gEA. (Figure 2D) First, an ORF-less 1635 bp Pre CSFV E2 chimeric sequence was synthesized and cloned into Kpnl (5')/BamHI (3') sites of pUC57 (Genscript) resulting in pPre CSFV E2 chimera (Genscript). The 2023 bp pPre CSFV E2 chimera sequence consisted from 5'- 3' direction as follows: a Kpnl site, the 1662 bp nucleotide sequence for the CAG promoter (GenBank accession #GU299216.1, position 3-1664, which include CAG enhancer 3-364), with a Nhel restriction site , a Ncol restriction site positioned after a 12 bp spacer, 6X His coding sequence and V5 epitope coding sequence (69 bp), followed by a stop codon, a bovine growth hormone (BGH) Poly A sequence (253 bp) and a BamHI restriction site. Second, a CSFV E2 chimeric ORF coding sequence was synthesized and cloned into pUC57 after codon optimization for pig (pCSFV E2 chimeric ORF; Genscript). The 1194 bp CSFV E2 chimeric ORF coding sequence consisted from 5’-3' direction as follows: a Nhel site, a Kozak sequence, PRV glycoprotein D signal sequence (gD predicted aa residues 1-18; GenBank accession #YP068387), predicted 373 aa of CSFV E2 ORF coding sequence (GenBank accession #AAC62087 , aa 690..1062) and aNcoI site. Next, the 1194 bp Nhel/Ncol fragment containing the chimeric CSFV ORF was cloned into the corresponding Nhel/Ncol sites of pPre CSFV E2 chimera above. In the resulting clone (pCSFV E2 chimeric gene cassette) the expression of the CSFV E2 ORF with the PRV gD signal sequence is controlled by the CAG promoter and fused in frame with the V5 epitope and poly His coding sequences at the carboxy end. Lastly, the KpnI/BamHI fragment of pCSFV E2 chimeric gene cassette containing the CSFV E2 ORF was cloned into the corresponding KpnI/BamHI sites of pPRV gEA resulting in pPRV gEA/CSFV E2-INS (SEQ ID NO:2). (Figures 2D, 2C and3)
The nucleotide sequence of the PRV gEA CSFV E2 insertion plasmid (SEQ ID NO: 2) is organized as follow (5’ to 3’): i) EcoRI restriction site; ii) PRV gE flanking upstream; iii) Kpnl restriction site; iv) CAG promotor sequence; v) Nhel restriction site; vi) Kozak sequence; vii) PRV gD signal; viii) CSFV E2 coding sequence; ix) Ncol restriction site; x) V5 epitope coding sequence; xi) 6x His epitope coding sequence; xii) Stop codon (TGA); xiii) bovine growth hormone terminator; xiv) BamHI restriction site; xv) PRV gE flanking downstream; xvi) Hindlll restriction site. (Figure 3)
2.5.2. Construction of PRV TK-deleted PCV2 Cap insertion plasmid (pPRV TKA/PCV2 Cap-INS)
To construct the pPRV TKA/PCV2 Cap insertion plasmid (pPRV TKA/PCV2 Cap- INS), an ORF-less 1638 bp pre-PCV2 Cap chimeric sequence (pPre PCV2 Cap) was synthesized (Genscript). The 1638 bp pre PCV2 chimeric sequence consists from 5'- 3' direction as follows: a Kpnl site, the nucleotide sequence for the human elongation factor la (hEF-la) promoter (GenBank accession #J04617), arestriction site for Nhel, aNotl restriction site after a 12 bp spacer, V5 epitope coding sequence, 6X His coding sequence, the simian virus 40 Poly A sequence (SV40), and a Hindlll restriction site. Second, after codon optimization for pig, the chimeric PCV2 Cap ORF coding sequences were synthesized (pPCV2 Cap chimeric ORF; Genscript) along with a Nhel site and Kozak sequence (at 5' end) and a Notl site (at 3' end). The 654 bp chimeric PCV2 Cap chimeric ORF coding sequence consists of 16 predicted amino acids of the PRV glycoprotein D signal sequence lacking the putative cleavage site (GenBank accession #YP068387; 1-16 aa) and a 654 bp Nhel/Notl chimeric codon-optimized (for pig) sequence coding for 202 predicted amino acids of PCV2 Cap protein (aa 1-10 plus aa 42-233; GenBank accession # AAD45581) lacking its nuclear localization signal (residues 11- 41; GenBank accession # AAD45581). Next, the 654 bp Nhel/Notl fragment was cloned into the corresponding Nhel/Notl sites of pPre PCV2 Cap chimera synthesized above. In the resulting clone (pPCV2 Cap chimeric gene cassette), the chimeric PCV2 Cap gene is regulated by the strong hEF-la promoter and the PCV2 Cap ORF is fused in frame with the V5 epitope and poly His coding sequence at the carboxy end. (Figure 2B) To assemble the pPRV TKA/PCV2 Cap-INS, the 2289 bp KpnI/Hindlll fragment containing the PCV2 Cap chimeric gene sequence was cloned into the corresponding KpnI/Hindlll sites of pPRV TKA . (Figures 2B, 2E and 4)
The nucleotide sequence of the PRV TKA PCV2 Cap insertion plasmid (pPRV TKA/PCV2 Cap-INS) (SEQ ID NO: 3) is organized as follow (5’ to 3’): i) EcoRI restriction site ii) PRV TK flanking upstream, start and stop codon of UL24 and start codon of TK are highlighted; iii) three stop codons; iv) Kpnl restriction site; v) hEF-lapromotor sequence; vi) Nhel restriction site; vii) Kozak sequence; viii) PRV gD signal; ix) PCV2 Cap coding sequence; x) Notl restriction site; xi) V5 epitope coding sequence; xii) 6x His epitope coding sequence; xiii) Stop codon (TGA); xiv) bovine growth hormone terminator; xv) Hindlll restriction site; xvi) PRV TK flanking downstream; stop codon of TK and start codon of UL22 (gH); xvii) Nsil restriction site. (Figure 4)
2.5.3. Construction of the gG deletion/CSFV Erns-GM-CSF insertion plasmid (pPRV gGA/CSFV Erns-GM-CSF-INS)
To construct the pPRV gGA/CSFV Erils-GM-CSF-INS. a 2002 bp codon-optimized (for pig) Kpnl (5’)/BamHI (3’) DNA fragment coding for the CSFV Erils-GM-CSF (porcine) chimeric ORF was synthesized and cloned into Kpnl (5')/BamHI (3') sites of pBluescript (BioMatik). The 2002 bp KpnI/BamHI fragment consists of the following (5’ to 3’): a Kpnl restriction site, a sequence for the cytomegalovirus (CMV) promoter sequence (GenBank accession #U55763; nt 1..605), a Kozak sequence, the PRV gD signal (GenBank accession #JF797219; nt 119647..119700, GenBank accession #YP068387; aa 1..18), the nucleotide sequence for CSFV Erns (GenBank accession #AF091661; nt 1175..1855, GenBank accession #AAC62087; aa 268..494), followed by the nucleotide sequence of porcine GM-CSF (GenBank accession #AAM48280; aa 1, 18..144) fused inframe with the C-terminal Ems coding sequence but lacking the stop codon, the nucleotide sequence for a flag tag (SEQ ID NO: 22) (GACTACAAAGACGATGACGACAAG), a stop codon (TAA), the SV40 Poly A site (GenBank #U55763; nt 1411..1640), and the restriction site for BamHI. The 2.002 kb KpnI/BamHI fragment of pCSFV Erils-GM-CSF chimeric gene cassette was cloned into the KpnI/BamHI sites of pPRV gGA resulting in pPRV gGA CSFV Ems-GM-CSF-INS. (Figures 2C, 2F and 5 Consequently, in the context of the viral genome, due to the inactivation of gG gene (deletion of the amino terminal amino-terminal 71 aa coding sequence as shown in Figure 2C and insertion of 24 nucleotides (SEQ ID NO: 1 of Figure 1) and the insertion of the chimeric CSFV Erils-GM-CSF. the chimeric CSFV Ems-GM-CSF would be expressed as a partially secreted protein. (Figure 2F) The nucleotide sequence of the pPRV gGA/CSFV Ems-GM-CSF- INS was verified and the expression of Ems-GM-CSF was verified by transfection of the plasmid DNA in the SK cells and immunoblotting with the CSFV Ems-specific mAbs.
The 4021 bp long nucleotide sequence of the PRV gGA CSFV Ems GM-CSF Flag insertion was assembled from plasmid pPRV gGA and pCSFV Ems GM-CSF chimeric gene cassette is organized as follow (5’ to 3’): i) EcoRI restriction site; ii) PRV gG flanking upstream; iii) Us3/Us4 polyadenylation signal; iv) Kpnl restriction site; v) CMV promotor sequence; vi) Kozak sequence; vii) PRV gD signal; viii) CSFV Ems coding sequence; ix) GM- CSF coding sequence; x) Flag epitope coding sequence; xi) Stop codon (TAA); xii) SV40 terminator; xiii) BamHI restriction site.
2.5.4. Construction of PRVtmv vector virus expressing CSFV E2 and Erns-GM-CSF, and PCV2b Cap chimeric genes (PRVtmv+)
To construct the PRVtmv+, the PCV2b Cap chimeric gene was first incorporated in the PRVtmv viral genome by cotransfection and homologous recombination of PRVtmv genomic DNA and linearized pPRV TKA/PCV2 Cap-INS constructed above. (Figure 2B) PCR identified putative recombinant viral plaques were plaque purified and verified further by sequencing and immunoblotting with PCV2 Cap-specific rabbit polyclonal antibody. A selected PRVtmv expressing the PCV2b Cap was then chosen to incorporate sequentially the CSFV-E2 and Erils-GM-CSF chimeric genes by cotransfection of the PRVtmv-PCV2 Cap genomic DNA with the corresponding linearized insertion plasmids, pPRV gEA/CSFV E2-INS (Figure 2D) and pPRV gGA/CSFV Erils-GM-CSF-INS. respectively. In each case, the putative recombinants were verified by PCR followed by sequencing and immunoblotting using the corresponding CSFV Ems- or E2-specific mAbs, respectively. (Figure 7) One PRVtmv+ recombinant vims expressing all three subunit chimeric antigens was selected for further in vitro characterization.
2.6. Virus titrations
PRV wt and the PRV recombinant vimses were titrated by plaque assay in MDBK cells as described previously [45], For PCV2, infected cells in 24-well titration plates were fixed with 3% paraformaldehyde (PFA; #30525-89-4; Acros Organics BVBA, Fair Lawn, NJ, USA) in phosphate-buffered saline (PBS; #P3813, Sigma-Aldrich®), and non-cytopathic viral plaques were visualized by indirect immunofluorescence assay (IF A) using anti-PCV2 capsid protein (Cap)-specific mAbs 36F1. Fluorescent antibody (FA) labeled, PCV2 plaques were counted under an inverted fluorescent microscope (Olympus 1X71, Shinjuku City, Tokyo, Japan).
2.7. Growth kinetics and plaque size assay
Growth kinetics of PRVtmv+ was evaluated and compared with that of the PRV wt by standard one-step growth kinetics assay as described previously [45,48], To determine the cell- to-cell spread property of PRVtmv+ compared with that of PRV wt, average plaque sizes of wt and PRVtmv+ viruses were determined by measuring approximately 50 randomly selected plaques of each virus group under a microscope with a graduated ocular objective, as previously described [49,50],
2.8. Experiments
2.8.1. Animals and experimental design
Animal handling, sample collection, immunization, challenge infection, and euthanasia protocols were approved by the LSU Institutional Animal care and Use Committee (Protocol #20-027). Fifteen four weeks-old healthy Yorkshire pigs were purchased from a PCV2 free supplier (Valley Brook Research, Madison, GA, USA). Before inclusion in the study, pigs were tested for bovine viral diarrhea-free status by the serological assay as described previously [45], After acclimatization for seven days, pigs were divided randomly into three groups of 5 pigs each. Group 1 (Control group; sham immunization), group 2 (immunized with Fostera® Gold PCV, Killed PCV type 1-type 2 chimera, and group 3 (PRVtmv+ vaccine group). The pigs in groups 2 and 3 were housed in pens, at least 100 feet apart, in the pole barn-large animal isolation facility at the School of Veterinary Medicine, Louisiana State University. The pigs in the control group were housed in a separate swine bam, approx. 100 yards away from the pole bam. All sanitary precautions were taken to prevent cross-contamination between the groups. Footbaths were located at the entrance of the pole bam, and in front of each pen entrance. All bedding materials and excretions from pigs were sterilized before discarding. Intranasal (IN) intranasal inoculation; Subcut - subcutaneous injection; PFU - plaque-forming units.
2.8.2. Vaccination and challenge
The vaccination and PCV2b challenge scheme are shown in Figure 6. Each pig in the PRVtmv+ vaccine group was vaccinated intranasally (IN) with 4 * 107 PFUs per nostril (total 8 x 107PFU) and subcutaneously (SC) with filtered (0.2 p pore size) 4 x 107 PFUs. According to the manufacturer's instructions, the pigs in the Fostera vaccine group were vaccinated intramuscularly (IM) with 2 ml of the vaccine. The pigs in the control group were sham inoculated IN with 1.0 ml of cell culture media. The pigs in the PRVtmv+ vaccine group received Noromycin® 300 LA (Norbrook, Lenexa, KS, USA) 20 mg/kg of body weight IM. At 32 days post-vaccination (dpv), animals of all three groups were challenged with PCV2b IN with a total of 1.6 x 104 PFU (8 x 103 PFU/nostril) and SC with 6.75 x 103 PFUs.
2.8.3. Clinical examination of pigs following vaccination and challenge
Pigs were routinely monitored for any visible clinical illness, feed, and water intake every day. Rectal temperature, body weight and clinical signs were recorded on 0, 2, 4, 5, 6, 8, 15, 21 dpv, and 32 dpv/ 0 days post-challenge (dpc). Following the PCV2b challenge, pigs were examined daily, and body temperatures and weights were recorded on 7, 13, 17, and 21 dpc. Clinical evaluation included rectal temperature, injection site reactions, depression, lethargy, sneezing, coughing, ocular-oro-nasal discharge, diarrhea, and systemic illness and lesions, if any. Rectal temperature was measured using a digital thermometer on indicated days. Mean temperature of each treatment group was reported with standard deviation (SD) (n=5). Body weight of pigs following immunization and challenge was measured using a digital weight balance on indicated days. Measurements were reported and shown the with standard deviation (SD) (n=5). dpv - days-post vaccination; dpc - days-post challenge,
2.8.4. Sample collection and processing from the vaccinated and control pigs following vaccination and PCV2b challenge
The scheme of sample collection (Ethylenediaminetetraacetic acid [EDTA] -blood, serum, nasal, fecal, and tonsil swabs) is shown in Figure 6. The swabs were collected in 2 ml of DMEM, supplemented with 3 x antibiotic-antimycotic solution and 2% FBS. Collected swab samples were aliquoted and stored at -80°C until use. Blood samples collected for sera were processed, aliquoted, and stored at -80°C. Peripheral blood mononuclear cells (PBMCs) were separated from EDTA-blood using Ficoll-Paque™ Plus (GE Healthcare, Chicago, IL, USA) and cryopreserved in liquid nitrogen as described previously [45],
2.8.5. Leukocyte and lymphocyte counting in whole blood
For counting the leukocytes and lymphocytes in whole blood-EDTA samples, an automatic hematological analyzer (Advia 120; Siemens Healthcare Diagnostics, Tarrytown, NY, USA) was used. On the day of- vaccination (0 dpv), challenge (0 dpc) and 21 dpc, total leukocyte and lymphocyte counts were determined and recorded. In addition, the percent decline in leukocyte and lymphocyte numbers in each pig was calculated as follows and described earlier [45],
% change in leukocyte count ol pig = 100 - - - — - : - — x 100
Leukocyte count of the animal on 0 dpc
2.8.6. Euthanasia, necropsy, tissue sample collection, and processing
Pigs were euthanized with Euthasol® (Euthanasia Solution; pentobarbital sodium and phenytoin sodium) and xylazine at 21 dpc. After a complete necropsy examination, tissue samples were collected from tonsils, lungs, liver, spleen, kidney, Peyer's patches, and cervical, bronchial, mediastinal, and mesenteric lymph nodes (LN) for histopathological (10% formalin), virus isolation, and qPCR assays (dry ice). Formalin-fixed tissues were paraffinized, sectioned, and processed either for histopathology (H&E staining) or immunohistochemistry.
2.9. Serum virus neutralization (SN) by plaque reduction assay for PRV and PCV2b
Standard plaque reduction assay was performed to evaluate the PRV- and PCV2b- specific virus -neutralizing antibody titers in serum, using 100 PFUs, as described previously [45], The plaque reduction assay was performed as above with some modifications. Since the PCV2b is non-cytopathic, plaques were visualized by FA staining with a PCV2b Cap-specific mAbs and then counted under a fluorescent microscope as described earlier [45], The virusneutralizing antibody titers for each serum sample were estimated by calculating the highest dilutions of the serum that neutralized 50% of the average numbers of respective control virus plaques without serum.
2.10. CSFV-specific SN assay.
CSFV-specific neutralization test for the PRVtmv+ vaccinated pigs sera was performed at EU and OIE Reference Laboratory for CSF (Institute of Virology, University of Veterinary Medicine, Foundation, Hannover, Germany). The test was performed according to the protocol of the Manual of Diagnostic Tests for Detection of CSF, which was composed by the EU and OIE Reference Laboratory for CSF and is available on the website of the EU and OIE Reference Laboratory for CSF (www.tiho-hannover.de/kliniken-institute/institute/institut- fuer-virologie/eu-and-oie-reference-laboratory, accessed on 22 October 2021) [51], The titration of the antisera started with a 1:2 dilution and was incubated with the CSFV strain Brescia (CSFV genotype 1.1). At 72 h post-infection, the cells were fixated by heat treatment for four hours at 80 °C. CSFV antigen detection in the cells was performed by immune- peroxidase staining as described in the Manual of Diagnostic Tests for Detection of CSF [51] using NS3-specific monoclonal mouse antibody BVD/C16 (dilution 1:50) and the conjugate rabbit anti-mouse horseradish peroxidase (dilution 1:200). Serum neutralization dose (ND50) titers were calculated as described previously [52],
2.11. DNA isolation and quantitative PCR (qPCR)
To quantify the PRVtmv+ and PCV2b genome copies following vaccination and challenge, respectively, from swabs, sera, PBMCs, and tissue samples, total DNA was isolated using the QIAamp® DNA mini kit (#51306, Qiagen, Hilden, North Rhine-Westphalia, Germany). In addition to the nasal and tonsil swabs (PRVtmv+ and PCV2b), fecal swabs were collected (PCV2b). For DNA isolation, 25 mg tissue were homogenized using 2.8mm ceramic beads (#15-340-154, Thermo Fisher Scientific®) in Precellys 24 homogenizer (#13112, Bertin Instruments, Rockville, MD, USA) and DNA was isolated as above. PRVtmv+ and PCV2b- specific genome copies were determined by TaqMan probe-based Real-time qPCR in ABI PRISM™ 7900HT Sequence Detection System (Applied Biosystems, Waltham, MA, USA), using major capsid protein (V5) ORF coding (PRV wt) and Cap-specific (PCV2b) primer pairs (Table 3). Each time, the PCR reaction setup was run with six standards of known quantity (101 to 106 copies per reaction). PRV or PCV2b genome copies in the samples were compared with the generated standard curves. Viral genome copies were normalized to a standard curve generated with host-specific swine housekeeping gene, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GenBank accession #AF017079.1). The assay was performed in duplicates, and results were expressed as PRV or PCV2b genome copies per million cells with the given fact that each eukaryotic diploid cell of pig has two copies of the GAPDH gene.
Table 3: List of primers, probes, and double standard gene blocks (ds-gblock as standard) used in quantitative PCR used for quantification of pseudorabies (PRV)Zporcine circovirus type 2b (PCV2b) genome in samples and subsequent normalization based on glyceraldehyde 3- phosphate dehydrogenase (GAPDH) housekeeping gene.
2.12. Immunohistochemistry
Immunohistochemical analysis of paraffinized tissue sections was performed for comparing PCV2b antigen distribution in different groups of pigs using the anti-PCV2 Capspecific mAh 36F1. Following standard deparaffinization and rehydration procedures in xylene and alcohol, respectively, endogenous peroxidase activity was quenched by incubating slides in 3% hydrogen peroxide in methanol for 1 hr at RT. Antigen retrieval was performed by incubating tissue sections with proteinase K (20 pg/ml) in Tris-EDTA buffer (50mM Tris Base, ImM EDTA, 0.5% Triton X-100, pH 8.0) for 30 min at 37 °C. After permeabilization with (0.2% Triton X-100 in TBS for 15 min and blocking with 4% nonfat dry milk (Blottinggrade blocker, #170-6404, Bio-Rad, Hercules, CA, USA) in Tris-buffered saline (TBS; 20 mM Tris and 150 mM sodium chloride; pH 7.4) for 1 h at 37 °C. The slides containing the tissue sections were washed 3-4 times (TBS with 0.05 % Tween-20). The slides were then incubated with anti-PCV2 mAh at 37 °C for 1 hr. Finally, slides were incubated with goat anti-mouse IgG peroxidase conjugate at 37 °C for 1 h, followed by Sigmafast™ 3,3'-Diaminobenzidine tablets (#D4293, Sigma-Aldrich®) in distilled water as per manufacturer's instruction and counter-stained with 0.5% methyl green for 5 min. Sections were dehydrated, cleared, and mounted with a glass coverslip. Dark brown positive signals detected the presence of PCV2b antigen in tissue sections under the microscope.
2.13. Transmission electron microscopy (TEM)
For TEM, SK cells were grown on a 13 mm electron microscopic coverslip (#174950, Thermanox plastic coverslip, Ted Pella Inc, Redding, CA, USA) and infected with PRVtmv or PRVtmv+ at a multiplicity of infection (MOI) 5 or PCV2b (0.1 MOI). Similarly, uninfected SK cells were grown on coverslip as healthy control cells. After 12 and 18 hrs post-infection (PRV wt and PRVtmv+, respectively), or 72 h post-infection (PCV2b), cells were fixed with primary fixative (1.25% glutaraldehyde and 2% formaldehyde in 0.1M Cacodylate buffer) for 1 h. Fixed cells were washed with 0.1M Cacodylate buffer for 10 min/three times each and post-fixed with 1% osmium tetroxide for 1 h. Following washing with 0. IM Cacodylate buffer with 5% sucrose (pH 7.4), the samples were stained with 0.5% uranyl acetate in 0.2M sodium acetate buffer (pH 3.5) overnight. Samples were dehydrated in an ascending series of ethanol, infiltrated with epoxy resin to propylene oxide, embedded beam capsules, and polymerized at 60 °C overnight. Semi- and ultrathin sections were cut, mounted on Nickel-grids, examined with an electron microscope (JEM- 1400 TEM; Louisiana State University, shared instrumentation facility), and captured digital images for analysis. N - Nucleus; C - Cytoplasm; RER - Rough endoplasmic reticulum; M - Mitochondria; L - Lysosomes; G - Golgi apparatus.
2.14. Histopathology
Formalin fixed tissues were processed, embedded in paraffin, and 4 pm sections were stained with H&E following standard procedures at the Louisiana Animal Disease Diagnostic Laboratory (LADDL). Slides were evaluated by a single veterinary pathologist for any lesion, who was blinded to the treatment groups. The lymphoid organs (cervical and mesenteric LN, spleen, tonsil, and Peyer’s patches) were investigated for lymphoid hyperplasia, lymphoid depletion, granulomatous inflammation, multinucleated giant cells and marginal zone cellularity (except Peyer’s patches). Tonsils were also investigated for crypt inflammation. Lungs were investigated for Bronchus-associated lymphoid tissue (BALT), peribronchiolar lymphocytes, bronchial inflammation, alveolar macrophages, alveolitis, and inflammation of the interlobular septa/pleura. The livers were investigated for portal inflammation, hepatocellular damage, and parenchymal inflammation. The kidneys were investigated for interstitial inflammation, tubular damage, glomerular damage, and pelvic inflammation.
2.15. Latency, Reactivation and viral replication
2.15.1. Primary virus infection, dexamethasone-induced latent virus reactivation and clinical evaluation
Each pig in the PRV wt infection group 1 was intranasally (IN) infected with total 2 x 106 PFUs /nostril (total 4 x 106 PFUs/pig). The pigs in the PRVtmv+ vaccine group were inoculated IN with 4 x 106 PFUs per nostril (total 8 x 106 PFUs/pig) and subcutaneously (S/C) with filtered (0.2 pm pore size) 4 x 106 PFUs. On the day of infection, each pig received Noromycin® 300 LA I/M injection (Norbrook, Lenexa, KS, USA; 20 mg/kg body weight) to prevent secondary bacterial infections. At 28 days post-infection (dpi), pigs in both groups received the dexamethasone (Dex) by intravenously (I/V) (0.5 mg/kg) followed by two more S/C Dex injections (0.25 mg/kg) on 29 and 30 dpi.
Pigs were routinely monitored for obvious clinical illness, feed-, and water-intake. Body weight and temperature were recorded. Clinical assessment included coughing, sneezing, nasal discharge, depression, respiratory difficulties, and other systemic illnesses (Figures 35 and 36).
2.15.2. DNA/RNA isolation, cDNA synthesis, and PRV-specific quantitative PCR (q-PCR) To determine the PRV-genome copies in the nasal swabs and in the TG, total DNA was isolated using the QIAamp® DNA mini kit (#51306, Qiagen, Hilden, North Rhine-Westphalia, Germany. Before DNA isolation from the TG, tissues were homogenized using 2.8mm ceramic beads (#15-340-154, Thermo Fisher Scientific®) in Pre-cellys 24 homogenizer (#13112, Bertin Instruments, Rockville, MD, USA). To determine targeted PRV genes - immediate early (infected cell protein 0 - ICP0) and late (major capsid protein - MCP , and gC) - transcription and quantification in the TG neurons, RNA was isolated. Briefly, after the homogenization of the TG, RNA was isolated using an RNeasy mini kit (#74104, Qiagen). DNA contamination in RNA sample was removed by RNase-Free DNase Set (#79254, Qiagen). Finally, cDNA synthesis was performed using Verso cDNA synthesis kit (AB-1453/A, Thermo Fisher Scientific®). PRVtmv+ genomic copies were determined by Taq-Man probe-based real-time qPCR in ABI PRISM™ 7900HT Sequence Detection System (Applied Biosystems, Waltham, MA, USA), using targeted genes - ICP0-, MCP- and gC- specific primers and probes (Suid herpesvirus 1 strain Becker, GenBank accession # JF797219.1; Table 3). Each time, the PCR reaction setup was run with six standards of known quantity (101 to 106 copies per reaction)
The PRV gene copies were calculated by normalizing the MCP-specific CT values against the standard curve generated based on the CT values obtained for the known housekeeping gene, GAPDH copies (two copies/cell) in the same cells. The mean copies of the PRV-MCP gene per one million cells are then plotted.
For the PRVtmv+ latency control, the TG samples collected and stored at - 80° C from previous vaccination experiment were used. To demonstrate that RNA samples treated with the DNAse are free from residual DNA, treated RNA samples without cDNA synthesis were also used as controls. PRV genome copies in the TG were normalized to endogenous host-specific swine housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; GenBank accession #AF017079.1, Table 3). The assays were performed in duplicate. The qPCR, genome copy results are expressed as PRV genome copies per million cells. The RT-qPCR, gene transcript copy results are expressed as transcript copies/ng of RNA.
3. Results
3.1.1. Characterization of PRVtmv+
The genomic sequence of one selected PRVtmv+ having gE-, TK-, and gG- deletion regions and genes including the CSFV E2, PCV2b Cap, and the chimeric CSFV Ems-GM-CSF were verified by sequencing. Nucleotide sequence results validated the results obtained during the single, dual, and triple deletion steps (Figures 1 and 2).
Additionally, the PRVtmv+ virus was assessed for its inability to grow in the TK- negative, TK-tsl3 hamster cells relative to its SK and MDBK cells growth. The absence of the gE was verified by immunoblotting the infected cell lysates with a gE-specific antibody.
3.1.2. Characterization of PRVtmv+ for the expression of the CSFV E2, PCV2b Cap, and chimeric CSFV Erns-GM-CSF
The PRVtmv+ virus was further analyzed for the expression of chimeric CSFV E2, CSFV Erils-GM-CSF (Erns+). and PCV2 Cap proteins by immunoblotting with the CSFV E2- and Ems- specific mAbs, and rabbit PCV2b-Cap-specific polyclonal antibodies. Figure 7 depicts the immunoblot-analysis of PRVtmv+ expressing chimeric CSFV E2, CSFV Ems-GM- CSF, and PCV2 cap proteins using an anti-CSFV E2 monoclonal antibody (mAbs) (left panel), an anti-CSFV Erils mAbs (middle panel), and a rabbit anti-PCV2 cap Ab (right panel), respectively. CSFV E2-specific mAbs detected an approx. 53-55 kDa (E2 monomer) and an approx.103-110 kDa (putative dimer) band in PRV TMV+ -infected SK cell lysates. As expected, both bands corresponding to the monomer and dimer of the CSFV E2 chimeric protein were absent in the mock- and PRV Becker (wt)-infected SK cell lysates. The monoclonal antibody against CSFV Ems detected several bands, at 41 kDa, 58 kDa, 80 kDa, and 120-160 kDa bands in the PRVtmv+ -infected cell lysates. Native Ems expressed by CSFV form homodimers through a disulfide bond with approximately 100 kDa [28], Most likely, the 41 kDa and 58 kDa bands are the unprocessed and processed monomers of Erns. respectively. The 80 kDa and 120-160 kDa bands are most likely the unprocessed and processed dimers of Erils. respectively. The molecular weight of the chimeric PCV2 Cap V5 His protein was predicted at 28.1 kDa. The rabbit anti-PCV2 Cap antibody detected approx. 56 kDa and 110 kDa bands in PRVtmv+ -infected cell lysates. Therefore, the 55 kDa and 110 kDa bands recognized by the Cap-specific antibody are most likely the dimeric and tetrameric forms of the chimeric PCV2b Cap, respectively. As expected, bands corresponding to the chimeric E2, Erils. and Cap proteins were absent in the mock- and PRV Becker (wt)-infected cell lysates. (Figure 7)
3.1.3. PRVtmv+ expressed chimeric PCV2b cap which self-assemble into virus-like particles in vitro
The morphology of virus-like particles (VLPs) was assessed in PCV2b, PRV wt, and PRVtmv+ infected SK cells by transmission electron microscopy. Specifically, the emphasis was to determine whether the chimeric PCV2 Cap expressed in the PRVtmv+-infected cells can self-assemble into well-formed PCV2b VLPs that resemble the PCV2b VLPs of the control PCV2b-infected SK cells. Mock-infected healthy SK cells showed normal cellular shape. (Figures 8A-B)
SK cells were infected with PCV2b infected cells at an MOI of 0.1 and fixed at 72 hours post-infection (hpi). They showed accumulation of PCV2 viral particles within the vesicle-like structures in the cytoplasm, (red arrow, Figures 8C-D) The PCV2b-VLPs were circular, and each measured about 20 nm in diameter with an inter-particle distance of 5 nm. (Figures 8C- D)
PRV wt virus-infected cells showed typical enveloped herpesvirus particles (about 200 nm in diameter) within the vesicular structures of the cytoplasm. (Figures 8E-G). The process of budding and release of several enveloped viruses were also noticed on the periphery of the cell near the plasma membrane, (blue arrow, Figures 8E-G) Released virus particles from the outer surface of the cells were accumulated in intercellular space, (green arrow, Figures 8E-G) The PRVtmv+ vaccine virus -infected SK cells at an MOI of 5 also showed enveloped PRVtmv+ virus particles (about 200 nm in diameter) in the cytoplasm at 18 hpi ( red arrow, Figures 9A, 9C and 9F). Secondary envelopment of intracytoplasmic PRVtmv+ capsids by budding into vesicles was visible (yellow arrows, Figures 9A-B). Most of the PRVtmv+ infected SK cells showed several accumulations of PCV2 virus-like particles (VLPs) and resembled the PCV2b virus particles as observed in the PCV2b-infected cells within the vesicular structures of the cytoplasm (swhite filled-red arrow, Figures 9A-F). The PCV2-VLPs were circular, and each measured about 20 nm in diameter. As shown by electron microscopic PCV2b capsid proteins expressed by the PRVtmv+ vectors self-assemble into PCV2b VLPs.
3.2. PRVtmv+ vaccine virus replicates with a similar kinetics and virus yield in SK cells, in vitro, like the PRV wt but produces smaller plaques
Two independent assays were performed to determine the plaque sizes and one-step growth kinetics of the PRVtmv+ virus relative to the PRV wt virus. As shown in Figure 10, the PRVtmv+ produced significantly smaller plaques (about 72% reduction in plaque size) compared to the PRV wt virus. (Figures 10A-B). The pictures of Figure 10A show areas containing representatives plaques of each virus. However, the one-step growth kinetics and virus yield of the PRVtmv+ virus was similar to that of the PRV wt which suggests that PRVtmv+ replicates with a similar kinetics than PRV wt. ( Figures 10C 11).
3.3. PRVtmv+ vaccine virus is highly attenuated, safe, and retains its stability to express the PCV2b and CSFV chimeric genes in pigs
All pigs were healthy and negative for PRV and PCV2b at the time of immunization. Two groups of five pigs were immunized either with Fostera Gold PCV (killed vaccine) or live PRVtmv+ (PCV2b/CSFV subunit vaccine). The pigs were assesed clinically following immunization and challenge; their rectal temperatures and weight were measured on indicated days, with their . Regardless of the vaccine used, none of the pigs showed clinical symptoms after vaccination and challenge. Body temperature in immunized pigs was within the physiological range (38.67-39.78 °C), except for three pigs in the Fostera group that had slightly elevated body temperature (40.0-40.2 °C) at 6 dpv. ( Figures 12A and 13) In the Fostera group, pigs did not gain weight until 6 dpv. (sFigures 12Band 14 ) However, by 6 dpv, the mean body weight of the control and PRVtmv+ vaccinated groups increased by 3% and 15%, respectively. 3.4. Nasal virus shedding following IN/Subcut vaccination with PRVtmv+ vaccine
In PRVtmv+ immunized pigs, the vaccine virus replicated and shed in the upper respiratory tract as evidenced by viral plaque assay and PRV-specific qPCR. (Figures 15, 16 and 17) DNA was isolated from nasal swab following immunization with PRVtmv+ vaccine, and PRV-qPCR was performed. PRV genome copy numbers were calculated according to the CT values of a standard curve. Shown are the mean copy numbers of PRV genome in 100 ng of DNA of two independent qPCR analyses of each animal from three vaccination groups on 0, 2, 4, 8 and 15 dpv. (Figure 15 A, dot plot graph represents mean + individual values in each group (n=5)) On 2 and 4 dpv, most pigs showed PRVtmv+ nasal shedding by qPCR. On day 8, only two pigs had virus nasal shedding. (Figure 15 A)
Virus isolated from each animal's nasal swab following the immunization with PRVtmv+ vaccine was titrated in confluent SK cells by plaque assay. The virus titers (in plaque-forming unit/ml of the nasal swab; PFU/ml) of each animal from the three groups were evaluated on 0, 2, 4, 8, and 15 dpv. Further, PRVtmv+ could be isolated in cell culture from two pigs’ nasal swab samples on 4 dpv. (Figure 15B).
After natural infection, PRV replicates in the nasal, pharyngeal, and tonsillar epithelium [53] and spreads to other tissues. Quantification of PRVtmv+ in tonsil swab of immunized pigs were determined by qPCR and virus isolation. DNA was isolated from tonsil swabs following immunization with the PRVtmv+ vaccine, and PRV-qPCR was performed. PRV genome copy numbers were calculated according to the CT values of a standard curve. The mean copy numbers of PRV genome in 100 ng of DNA from tonsil swab of two independent qPCR analyses of each animal from the three groups were determined on 0, 2, 4, 8, and 15 dpv. (Figure 16 A, the dot plot graph represents mean + individual values in each group (n=5)_
PRVtmv+ virus replication was also evaluated in the tonsils. Virus isolated from each animal's tonsil swab following the immunization with PRVtmv+ vaccine was titrated in confluent SK cells by plaque assay. The titers (in plaque-forming unit/ml of the nasal swab; PFU/ml) of each animal from all three groups were determined on 0, 2, 4, 8, and 15 dpv. (Figure 16B, the dot plot graph represents mean + individual values in each group (n=5))
The qPCR results show that on 2, 4, and 8 dpv, all the pigs in the PRVtmv+ vaccine group had the vaccine virus in their tonsils. However, the vaccine virus was isolated from four pigs on 2 dpv and from only one pig on 4 dpv (Figure 16A-B; Figure 17). As expected, both control and Fostera group pigs remained negative for PRVtmv+ by qPCR and virus isolation, indicating no cross-contamination. 3.5. The chimeric E2 and E, ns proteins are intact in the PRVtmv+ virus isolated from the vaccinated animals at 4 dpv
To determine the stability of the PRVtmv+ vaccine virus after a passage in pig, in vivo, the virus isolated from nasal swabs on 4 dpv was tested by immunoblotting with anti-CSFV E2 and Erils-specific mAbs. The E2- and Ems-specific mAbs recognized the 53 kDa E2- and 56 kDa Ems-specific bands as seen on the left and right panels of Figure 18, respectively. PRVtmv+ remains stable in pigs.
3.6. PRVtmv+ induces the production of PCV2b-specific antibodies in the vaccinated pigs, which can detect PCV2b-infected cells in culture
To test whether serum samples from the PRVtmv+ vaccinated pigs detected PCV2b- specific viral antigens in the PCV2b-infected SK cells, sera samples were collected from two vaccinated pigs collected at 0 dpv (before vaccination) and 32 dpv and tested by immunofluorescence assay (IF A). Swine kidney cells were transfected with PCV2b plasmid and fixed 72 hours post- transfection. While the serum of pig #2313 collected before the vaccination did not react with the PCV2b antigens in the PCV2b-infected cells, the serum of two pigs #2313 and 2304 which were collected on 32 dpv labeled the PCV2b non-cytopathic plaques in the PCV2b-infected SK cells , as visualized by fluorescent-tagged anti-pig antibodies. (Figure 19) PCV2b Cap-specific antibodies were detected in sera of PRVtmv+ vaccinated pigs
Taken together, the results in sections 3.5 and 3.6 validate the stability of the vaccine virus and the expression of all three chimeric proteins in vivo.
3.7. PRVtmv+ vaccine elicits a PRV-specific neutralizing antibody response in the vaccinated pigs.
At the time of immunization (0 dpv), none of the pigs among all three groups had any detectable levels of PRV- or PCV2b-specific SN antibody titers in the serum. However, by 8 dpv, levels of PRV-specific serum neutralizing antibodies (mean SN titer of 10) were detected ( Figures 20A and 21). The PRV-antibody levels rose four-fold to 40 on 15 dpv and maintained slightly lower SN titers until 53 dpv (24 at 42 dpv and dropped to 11 at 52 dpv). Notably, none of the pigs in the control and Fostera group developed PRV-specific antibodies in serum throughout the study.
3.8 A single dose of PRVtmv+ vaccine is sufficient to elicit a higher PCV2b-specific antibody response than the inactivated Fostera Gold PCV vaccine Both PRVtmv+ and Fostera vaccine-induced detectable levels of PCV2b-specific neutralizing antibodies in the vaccinated pigs by 15 dpv (Figure 20B; Figure 22). At this point, the antibody level in the PRVtmv+ vaccine group was nearly two-fold higher than the Fostera groups' titer; mean SN titer of 15 for the PRVtmv+ versus 8 for the Fostera group. The average SN antibody titers in the PRVtmv+ rose to 22 on the day of PCV2b challenge (32 dpv), while the corresponding average SN titer in the Fostera group was 16.
Figure 20 shows PRV-, PCV2b- and CSFV-specific serum neutralizing (SN) antibody titer developed in pigs after PRVtmv+ vaccination. (A) PRV-specific SN titers. The data represent the mean + standard deviation (n=5). (B) PCV2b-specific SN antibody titer following PRVtmv+ immunization and PCV2b challenge. PCV2b is non-cytopathic, and the viral plaques were visualized by IFA at 72h post-inoculation using the PCV2b specific mAbs 36F1 and fluorescent-tagged secondary antibody. (C) CSFV-specific neutralization dose (NDso) SN titers following PRVtmv+ immunization. NDso titers were calculated as described previously [54] . The dot plot graph shows each animal's mean values and individual titer with standard deviation (n=5).
3.9. PRVtmv+ immunized pigs generate CSFV-specific neutralizing antibody titers
To determine whether the PRVtmv+ vaccinated pigs also induced a CSFV-specific neutralizing antibody response, the NDso titers against the CSFV strain Brescia was determined for sera samples collected on 0, 15, 21 and 32 dpv. On the day of vaccination (0 dpv), the pigs in the PRVtmv+ group had average NDso titers of less than 2, but the average titers rose to 5, 8, and 11 on 15, 21 and 32 dpv, respectively (Figure 20C; Figure 23). Therefore, the pigs seroconverted (a four-fold rise), NDso titers from less than 2 to 8 by 21 dpv and rose further to 11 on 32 dpv. These results demonstrate that the chimeric Ems and E2 proteins expressed by the PRVtmv+ induced a low to modest CSFV strain Brescia-specific serum neutralizing antibody titers.
3.10. High SN antibody titers in the PRVtmv+ vaccinated group
Following the PCV2b challenge, the neutralizing antibody titers in both PRVtmv+ and Fostera groups continued to decline gradually through 13 dpc, from average SN titers of 23 (0 dpc) to 9 (PRVtmv+) and from average SN titers of 16 (0 dpc) to 6 (Fostera) (Figure 20B; Figure 22). However, by 21 dpc (day of euthanasia), pigs in both the groups SN titers increased more than a four-fold (seroconverted) 9 to 44 (PRVtmv+) and 6 to 28 (Fostera). Therefore, both vaccine groups had a similar delay in the seroconversion after challenge, but the average SN titers in the PRVtmv+ group were nearly two-fold higher than in the Fostera vaccine group (44 versus 28) (Figure 20B). Notably, antibodies against PCV2b in the control group begin to appear in a few pigs (two pigs out of five) on 13 dpc and rose to 13 on 21 dpc (Figure 20B).
Both the PRVtmv+ and the Fostera vaccinated pigs had similar PCV2b-specific seroconversion after the challenge, but the SN antibody titers in the PRVtmv+ vaccine group were two-fold higher than the commercial vaccine group.
3.11. PRVtmv+ protects pigs from leukopenia and lymphopenia after PCV2b challenge
PCV2b-infected piglets, especially when developing PCV2b associated PMWS disease, have lymphopenia [55,56], Leukocyte and lymphocyte counts were determined in control unvaccinated, Fostera and PRVtmv+ vaccinated pigs before challenge (0 dpc) and after the PCV2 challenge (at 21 dpc). Whole blood was collected from pigs on 32 dpv/Odpc, and 53 dpv/21 dpc, The data presented in Figure 24A-B and Figure 25 show that in the PRVtmv+ vaccinated pigs, both the leukocyte and lymphocyte counts increased by 21 dpc relative to 0 dpc (36% and 20%, respectively). At the same time, based on the criteria described in the Methods section for the leukopenia and lymphopenia, all five pigs in the control group developed a low to moderate level of leukopenia and lymphocytopenia averaging an approx. 29% decline (Figure 24A-B). Notably, four out of five pigs in the Fostera group also developed mild leukopenia and lymphocytopenia ranging between 4-36%, with an average decline of 20%. The remaining one pig in the Fostera group had a 36% increase in leukocyte count.
While PRVtmv+ vaccinated pigs had a moderate increase in leukocyte and lymphocyte counts following the PCV2b challenge, pigs in the control unvaccinated and commercial, Fostera vaccine groups had a reduction in both counts (a moderate to low level of leukopenia and lymphopenia).
3.12. Both PRVtmv+ and Fostera immunized pigs did not shed the PCV2b challenge virus in feces
Fecal virus shedding is a transmission source of PCV2b virus in pigs [57], PCV2b shedding after vaccination and PCV2b challenge was assessed by qPCR in fecal swabs on 0, 13, 17 and 21 dpc. Low fecal PCV2b shedding levels were observed only in a few control group pigs on 17 and 21 dpc; PCV2 genome copy numbers ranged between 13-35 copies/200 ng of total DNA in two pigs on 17 dpc and one pig on 21 dpc (Figure 26; Data shown in Figure 27). No PCV2b genomic DNA was detected in the fecal swabs of PRVtmv+ and Fostera- vaccinated groups after the PCV2b challenge through the day of euthanasia (21 dpc). Regardless of the experimental group, neither PCV2b infectious virus nor genomic DNA was detectable in the nasal and tonsil swabs after the challenge. 3.13. PRVtmv+ protects the vaccinated pigs from both cell-free and cell-associated viremia after PCV2b challenge
PCV2b infection causes both cell-free and cell-associated (monocytes and lymphocytes) viremia following replication in monocytes/macrophages and lymphoblasts in the lymphoid tissues [58], Protective effect of PRVtmv+ vaccination in pigs was evaluated from cell-free or cell-associated viremia after a PCV2b challenge. DNA was isolated from serum and PBMC, and PCV2b qPCR was performed as described in 2.11. The mean copy numbers of PCV2b genome in serum (100 ng of DNA), and PBMC (normalized to 107 cells) was determined from two independent qPCR analysis of each animal from three vaccination group on 0, 13, 17 and 21 dpc. After PCV2b challenge, PCV2b genomic DNA could not be detected by qPCR in the sera of PRVtmv+ -vaccinated pigs on 13, 17, and 21 dpc. (Figure 28A) In contrast, PCV2 genome copies were readily detected in the sera of three pigs (13 dpc) and two pigs (17 and 21 dpc) of the control group; mean copies 116 on 13 dpc, 73 on 17 dpc, and 18 on 21 dpc (Figure 28A Data shown in Figure 29). Regarding the Fostera-vaccinated group, PCV2b DNA was detected in sera of two pigs on both 13 dpc (44 and 38 copies, respectively) and 21 dpc (19 and 12 genomic copies). (Figure 28A) While PRVtmv+ vaccinated pigs were entirely protected from cell-free viremia, Fostera vaccinated pigs had significantly reduced cell-free viremia compared with the control pigs.
To determine whether pigs in the PRVtmv+ vaccine group were also protected entirely from cell-associated viremia relative to the commercial vaccine group, PBMCs collected on 0, 7, 13 and 21 dpc, from the vaccinated and unvaccinated control groups were analyzed by PCV2b-specific qPCR as above. The results depicted in Figure 28B (Data shown in Figure 30) show that while the control pigs had a high number of PCV2 genomic copies starting on 7 dpc until 21 dpc, when the pigs were euthanized, the pigs in the Fostera group had moderate numbers of PCV2b genomic copies only on the 13 dpc. Notably, the PRVtmv+ vaccinated group was again negative for PCV2b-specific DNA the entire time post-PCV2b challenge. Therefore, taking together the cell-free and cell-associated viremia data, the PRVtmv+ vaccine prevented viremia entirely. However, the commercial vaccine "Fostera" did not; the viremia was reduced from the high level in the control animals to a low to moderate level.
3.14. Histopathology following immunization with Fostera and PRVtmv+ vaccines and subsequent challenge with PCV2 in pigs
Overall, pigs of the three treatment groups had no gross or histologic changes, with no evidence of lymphoid depletion and/or granulomatous inflammation within lymphoid tissues (Figure 31). In pig #2302 vaccinated with the PRVtmv+), rare multinucleated giant cells were observed in the mesenteric lymph node (Figure 31 F and inset); however, there was no lymphoid depletion or histiocytic inflammation, PCV2 DNA, identifiable viral botryoid inclusion bodies, or viral antigen. Such result was not unexpected given that lesions are observed when PCV2 is coinfected with another infectious agents .
3.15. PRVtmv+ vaccine protects pigs against PCV2b challenge better than the inactivated commercial vaccine Fostera
To determine PCV2b viral genome copies tissues i.e., tonsil, mesenteric LN, mediastinal LN, cervical LN, Peyer's patch, and spleen from vaccinated and unvaccinated pigs were processed and quantified by qPCR. (See 2.11) The PCV2b genome copy numbers were calculated after normalization with the porcine house-keeping gene GAPDH. (Figures 32 and 33) Pigs in the control group had PCV2b genome in both tonsil and cervical LN with average copy numbers of 326 and 802 per one million cells, respectively. Three out of five control pigs had PCV2b in Peyer's patches and mesenteric LN (mean genome copy numbers of 5257 and 263, respectively). Finally, two of the five control pigs had PCV2b in mediastinal LN and spleen (mean genomic copy numbers 55 and 3000 copies, respectively. In contrast, no PCV2b viral genome copies were detected in any tissues of pigs immunized with PRVtmv+, except for pig #2302, which had very low levels of PCV2b restricted to mediastinal LN (22 genome copies). In the Fostera group, two out of five pigs had PCV2b genomes in mediastinal and cervical LN (mean genome copy numbers 48 and 14, respectively). Pig #2305 had 23 genomic copies in the tonsil and pig #2310 had 861 copies in the Peyer's patches. PRVtmv+ vaccine provides better immune protection than the Fostera vaccine as shown by the PCV2b loads in the lymphoid tissues (p<0.05).
3.16. No PCV2b Cap-specific antigens detected in lymphoid tissues of PRVtmv+ and Fostera vaccinated pigs
Immunohistochemistry was performed to assess the presence of PCV2b antigen in tonsils (Figure 34A) and intestine (Figure 34B) of vaccinated pigs. The presence of PCV2 antigens was confirmed by bright-golden brown positive signals in tissues. While PCV2b Cap antigen was detected in the tonsil and Peyer's patches of control unvaccinated pigs, none of the PRVtmv+ vaccinated pigs had detectable PCV2b Cap antigens in any tissues. In the Fostera vaccine group, only one pig (#2310) had detectable PCV2b Cap antigens in the Peyer's Patches. As noted above, pig #2310 also had 861 copies of PCV2b genome copies in the Peyer's Patch. (Figure 34A-B)
3.17. Comparison of the latency-reactivation and replication properties of PRVtmv+ with its parent wtPRV (Becker strain) following intranasal infection 3.17.1. Clinical evaluation of wtPRV-infected pigs and PRVtmv+ immunized pigs
The pigs were clinically evaluated according scoring criteria as listed in Figure 35. (Figures 37 and 38B) Rectal temperature of pigs were recorded following primary infection and Dex-induced latency-reactivation. (Figures 36 and 38A) On 3 dpi, wtPRV-infected pigs started to show moderate fever of 40.6 °C and mild to moderate respiratory signs with a clinical score of 9. (Figures 36, 37 and 38A-B) The clinical signs in wt-infected pigs were most severe 4 dpi with high fever ranging between 40.8°C and 41.8 °C. The clinical score was 11.5 which was characterized by shivering, bilateral nasal discharge, coughing, sneezing, tear staining, and moderate to severe respiratory difficulties (Figures 38A-B and 39A). The pigs were off-fed for a day (4th dpi) and had reduced feed and water intake for two days (3rd and 5th dpi). By 7 dpi, the clinical signs gradually subsided. However, one wt-infected pig developed a unilateral comeal ulcer on the right eye, leading to descemetocele on 7 dpi (Figures 39B), which persisted until the day of euthanasia on 33 dpi. In contrast, the PRVtmv — vaccinated pigs were clinically normal until euthanasia on 33 dpi, including 5 days post-reactivation (Figures 38A- B and 39C).
3.17.2. Nasal virus shedding following intranasal (IN) administration of wtPRV and PRVtmv+
Following primary intranasal infection of pigs, both wtPRV and PRVtmv+ replicated in the nasal mucosa and shed in nasal secretions. As shown in Figure 40, infectious viral plaques were recovered only from the nasal swabs of wtPRV-infected pigs on 3 dpi. Consistent with this result, on 3 dpi and 5 dpi, the corresponding genomic copy numbers in the nasal swabs of PRV wt-infected pigs were also significantly higher (30,000- folds) compared with the PRVtmv+ - infected pigs. (Figure 40) In additon, a low-level nasal virus shedding (approx. 2 x 102 - 5 x 102 genome copies) persisted until 21 dpi in the wtPRV-infected pigs but was cleared by 28 dpi.
3.17.3. Following Dex-induced reactivation, only wtPRV-infected pigs shed the virus
Nasal swabs collected from pigs daily between 0 and 5-day post-dexamethasone injection (dp-Dex) were tested by qPCR targeting PRV -Maj or capsid protein (MCP) gene. PRV genomic copy numbers were calculated according to the CT values of a standard curve. The mean copy numbers of PRV genome in 100 ng of total DNA from nasal swabs of two independent qPCR analyses of each animal from both groups on 0-, 1-, 2-, 3-, 4- and 5-dp-Dex injection.
On 28 dpi, nasal virus shedding was no longer detectable by qPCR, indicating that virus established latency in the TG neurons. Starting 28 dpi, Dex was administrated to all five pigs in order to induce the reactivation of latent PRVtmv+ and PRV wt viruses. Nasal swabs of pigs collected on 3 and 5 dp-Dex were tested by qPCR for virus shedding. On 4 dp-Dex one of the two pigs-infected with wtPRV shed the virus. On day 5 post-Dex, both the pigs shed the wtPRV virus. In contrast, none of the three pigs of the PRVtmv+ group shed detectable virus in nasal secretion following Dex treatments. (Figures 41 and 42)
3.17.4. Both wtPRV and PRVtmv+ establish latency in the TG neurons and reactivate following Dex injection: only wtPRV and not PRVtmv+ replicates in the TG neurons
To demonstrate that PRVtmv+ was transported retrogradely to the TG neurons following intranasal infection and established latency in the neuron cell bodies, viral genome copies in the TGs of wtPRV-infected and PRVtmv+ -vaccinated pigs were determined by qPCR targeting the PRV MCP gene. Specifically, the TGs collected from pigs at 53 days post- PRVtmv+ vaccination/21 days post PCV2b challenge from a recent PRVtmv+ vaccination- PCV2b challenge study, (see herein) The results revealed that a mean of 1643 PRV genome copies per million cells was detected in the PRVtmv+ latently-infected TGs. (Figures 43 and 44) There was no corresponding wt PRV TG samples. Nevertheless, wt PRV latency in the TG neurons was not questionable following primary intranasal infection. To determine whether the latent PRVtmv+ virus replicates in the TG neurons following latency -reactivation, PRVtmv+ genome copy numbers were quantified in the TGs of vaccinated pigs at 5 days post-Dex treatment and compared with the corresponding wtPRV genome copy numbers in wtPRV - infected pigs treated with Dex. At 5 days post-Dex treatment, 122,924 mean genome copies per million cells in the wtPRV-infected TGs were detected while the corresponding mean genome copy numbers in similarly treated PRVtmv+ - infected TGs were 1174/million cells, which is 100-fold less than the numbers obtained from the TGs of wt-infected pigs. (Figure 44) Remarkably, the comparison of genome copy numbers obtained from the TGs of PRVtmv+- vaccinated pigs either during latency or at 5 days post-Dex treatment (latency-reactivation) were very similar. Together, these results suggest that PRVtmv+ does not replicate in the TG after the Dex treatment, while wtPRV can replicate.
To confirm that latent PRVtmv+ reactivated in the TG neurons following Dex treatments, transcription of the viral immediate early gene, ICPO was assessed during latency (of PRVtmv+ - infected, negative control) and at 5-days post-Dex treatment (of PRVtmv+ and wtPRV-infected pigs). Regardless of wtPRV or PRV vaccine vector-infected pigs, there was ICPO transcription in TGs following the Dex treatment. Of note, the ICPO copy numbers in PRVtmv+-vaccinated pigs were slightly lower than that of wtPRV. (Figures 45 and 46A) Unlike cells of the nasal epithelium, the post-mitotic neuron does not have a complementary TK enzyme. Accordingly, PRVtmv+ which lacks a functional viral TK can not replicate in the TG neurons. To prove that PRVtmv+ do not replicate, transcription levels of the DNA replication-dependent late y genes, MCP-and gC- were assessed in the TG neurons, during latency (of PRVtmv+ - vaccinated) and at 5-days post-Dex treatment (of PRVtmv+ and wtPRV -infected pigs). The rationale was that detecting the MCP, and gC transcriptions or no transcription, following the Dex treatment would indirectly indicate that virus replicated or failed to replicate (abortive replication), respectively. MCP (Figure 46B) and gC (Figure 46C) gene transcriptions occurred only in the TGs of wtPRV-infected and not in PRVtmv+- vaccinated pigs. (Figures 45, 46 and 49) These results proved that both wtPRV and PRVtmv+ viruses can reactivate in the TG neurons upon Dex treatments. However, only the wtPRV virus is capable of replication in the TG.
TK-deficient PRVtmv+ is avirulent because it no longer replicates in terminally differentiated neurons.
3.17.5. Following Dexamethasone (Dex)-induced latency-reactivation, only wtPRV but not PRVtmv+ - inoculated pigs had a memory serum virus neutralizing (SN) antibody response
Following latency-reactivation in TG, PRV and other alpha herpesviruses replicate initially in TG neurons at a low level. Subsequently, progeny viruses travel anterogradely from neuron cell bodies down the axon to nerve endings in the nasal mucosa and replicate there, resulting in nasal virus shedding. Since the pigs were pre-exposed to PRV, there was a B cell response and generation of memory B cells. Consequently, upon virus reactivation in the TG and replication in the nasal epithelium, memory B cells are expected to undergo a recall immune response resulting in a rapid rise in SN antibody titers. Therefore, a drastic increase in the PRV-specific SN antibody titers due to memory immune response following Dex- induced latency reactivation is indirect evidence of latent virus reactivation in the TG, followed by virus replication in the nasal epithelium, and completion of all the steps in between noted above. As shown in Figures 47 and 48, the average SN titers peaked at 50 on 15 dpi in the sera of PRVtmv+-vaccinated/infected pigs, whereas the peak SN titer rose to 235 at 21 dpi in the sera of wt PRV-infected pigs. Subsequently, SN antibody titers dropped to 15 and 120 in PRVtmv+ and wtPRV-infected pigs at 28 days when the pigs stopped shedding the virus and most likely established latency. On 5 dp-Dex, the mean PRV-specific SN antibody titers rose more than nine-fold compared to 0 dp-Dex (from 120 to 1096) in wtPRV-infected pigs. In contrast, the corresponding serum virus neutralization titer in PRVtmv+-vaccinated pigs decreases from 15.1 to 11.7. (Figures 47 and 48). These results are consistent with the finding that wtPRV shed in the nasal secretions following Dex-induced latency reactivation while PRVtmv+ do not. (Figures 42 and 48).
PRVtmv+ establishes latency in the TG neurons and reactivates when induced by dexamethasone (dex) injection but is unable to replicate in the TG neurons, and therefore no nasal virus shedding following the latency -reactivation.
The TK- or gE-deleted viruses replicate in vitro in epithelial cells like the PRV wt virus and with similar virus yields. This is because cellular TK compensates for the viral TK, and the cell-to-cell spread defect phenotype of gE gene deletion does not affect virus replication in cell culture. However, in the neurons in culture or in vivo in the animals, i.e., TG neurons, the TK-deleted virus does not replicate because neurons do not have the thymidine kinase required for virus replication. PRV gE is not required for virus entry into the axon termini of the maxillary branch of the trigeminal nerve in the nasal mucosa and retrograde axonal transport to neuron cell bodies in the TG; however, it is essential for anterograde axonal transport from neuron cell bodies in the TG to nerve endings in the nasal mucosa.
As shown in Figure 49, PRVtmv+ (TK-, gE- and gG-deleted) replicated in the nasal mucosa, established latency in the TG neurons and reactivated following dex treatment. However, following the latency-reactivation PRVtmv+ did not replicate in the TG neurons. Consequently, PRVtmv+ did not shed in the nasal discharge following the dex treatment. - induced reactivation, di early protein (ICPO) gene transcription; however, the reactivated virus (Figure 49). This safety property of PRVtmv+, characterized by “no nasal virus shedding” following reactivation, would be improved further for use in PRV-free countries, including the US and EU.
3.18. PRVtmv+ vaccinated pigs survived a virulent CSFV Brescia challenge and were protected from severe clinical signs, thrombocytopenia, and lymphocytopenia.
The CSFV challenge experiment was performed at Plum Island Foreign Animal Disease Research.
3.18.1. Materials and Methods
Viruses and cells
Cell cultures of swine kidney (SK6) cells, free of bovine viral diarrhea virus (BVDV) which is genetically and serologically related to CSFV, were maintained in Dulbecco's Minimal Essential Media (DMEM) (Gibco, Grand Island, NY) containing 10% fetal calf serum (FCS) (Atlas Biologicals, Fort Collins, CO). A virus derived from an infectious clone-encoding the CSFV Brescia strain (BICv) was use as challenge virus. Titrations of CSFV were performed using SK6 cell cultures in 96-well plates (Costar, Cambridge, MA). Prescence of viral infection was detected after 4 days in culture by immunoproxidase assay using the E2 specific CSFV monoclonal antibody WH303 and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Titers were calculated and expressed as TCID50/ml as described previously with a sensitivity of detection of >1.8 TCID50/ml.
Detection of neutralizing antibodies
Serum neutralization assay (NA) was per-formed with heat-inactivated serum samples (56 °C for 30 min) as described previously (Gavrilov et al., Virology 420 (2011) 135-145). Briefly, two-fold serial dilutions of serum were prepared in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and mixed with equal volumes of BICv containing 102 TCID50. Serum-virus mixtures were incubated for 1 h at 37 °C and then transferred to 96-well flat-bottom tissue culture plates (Coming) followed by additionof SK6 cells (1 x 104 per well). Plates were incubated at 37 °C and 5% CO2 for 4 days. Supernatant was then removed from each well and the cells were fixed with methanol-acetone (50% vol./vol.) solution and air-dried. Plates were stained by immunoperoxidase assay using the Vecstatin ABC Kit, Vector Laboratories, Burlingame, CA following manufacturer's instructions. Neutralizing antibody titers are expressed as the reciprocal of the highest two-fold serum dilution neutralizing BICv (Reed and Muench, 1938). Am. J. Hyg. 27, 493-497
Animal experiments
PRVtmv+ vaccine group pigs (5 animals per group), of 30-401bs female Yorkshire cross-breed pigs, were vaccinated, intranasally (IN) with 4 x 107 PFUs per nostril (total 8 x 107 PFU) and subcutaneously (SC) with filtered (0.2 pm pore size) 4 x 107 PFUs.. An additional group (control group) of pigs were mock-vaccinated. At 28 post-immunization pigs were intranasally (IN) inoculated with 105 TCID50 of CSFV Brescia strain. The presence of clinical signs associated with the disease (anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea, and cough) and body temperature was recorded daily throughout the experiment. Blood samples obtained from the anterior vena cava in EDTA-containing tubes (Vacutainer), were collected at different times post-challenge (as shown in the corresponding figures). Total and differential white blood cell, lymphocytes, and platelet counts were obtained using a Beckman Coulter ACT (Beckman, Coulter, CA). Animal experiments were performed under biosafety level 3 conditions in the animal facilities at Plum Island Animal Disease Center, following a strict protocol approved by the Institutional Animal Care and Use Committee (number 171.12-21-R, approved 12-09-21).3.18.2. Results 3.18.2.1. Control of fever in PRVtmv+ vaccinated pigs following lethal CSFV challenge
After CSFV challenge, the body temperature of PRVtmv+ vaccinated pigs remained below 40C and decreased over time. In contrast, the unvaccinated pigs developed high fever at 4 dpc. (Figure 50)
3.18.2.2. CSFV- Viremia after CSFV challenge
After challenge, PRVtmv+ vaccinated pig had slightly elevated viremia at 4 dpc which decreased at 7dpc. Notably, no viremia was detectable at 14 and 21 dpc, demonstrating a protective effect of the vaccination with PRVtmv+. In contrast, unvaccinated pigs had increased viremia at 4 dpc and 7 dpc to the extend that the pigs were euthanized at 7dpc due to the severity of the CSFV infection. (Figure 51)
3.18.2.3. Survival of vaccinated pigs following lethal CSFV challenge
Pigs in the unvaccinated group were euthanized on 6-7 dpc due to severity of the disease, while 100 percent of the PRVtmv+ vaccinated pigs survived CSFV challenge. (Figure 53)
3.18.2.4. Neutralizing antibody titers in PRVtmv+ vaccinated pigs following vaccination
As shown in the Figure 52, all 5 pigs vaccinated with PRVtmv+ seroconverted with a rise in four-fold or more BICv-specific NA titers (NA) by 28 days post — vaccination.
3.18.2.5. Percent reduction in white blood cells (WBC), lymphocytes and platelet counts following lethal CSFV challenge
After challenge, both groups have reduced WBC, lymphocyts and platelets counts at 4 pdc. After 7 dpc, the lymphocyts and platelets counts stabilized in the vaccinated group while the lymphocytes and platelets counts in the control group remained very low with a decrease of 80 % and 65% of decrease, respectively. (See Figures 54, 55 and 56)
A low WBC count is often related to a decrease in a type of infection-fighting WBC called neutrophils. Given that lymphocytes are immune cells responsible for the adaptive immune response, the stabilization of their counts over time in the PRVtmv+ vaccinated groups shows that the disease is well controlled.
In summary, PRVtmv+-immunized pigs were protected from thrombocytopenia and lymphocytopenia. References
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Meyers, G.; Tautz, N.; Becher, P.; Thiel, H.J.; Kummerer, B.M. Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs. J Virol 1997, 77, 1735, doi:10.1128/JVI.71.2.1735-1735.1997. Reimann, I.; Depner, K.; Trapp, S.; Beer, M. An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus. Virology 2004, 322, 143-157, doi:10.1016/j.virol.2004.01.028. Konig, P.; Biome, S.; Gabriel, C.; Reimann, I.; Beer, M. Innocuousness and safety of classical swine fever marker vaccine candidate CP7_E2alf in non-target and target species. Vaccine 2011, 30, 5-8, doi:10.1016/j.vaccine.2011.10.064. Mettenleiter, T.C.; Schreurs, C.; Zuckermann, F.; Ben-Porat, T. Role of pseudorabies virus glycoprotein gl in virus release from infected cells. J Virol 1987, 61, 2764-2769, doi: 10.1128/JVI.61.9.2764-2769.1987. Mettenleiter, T.C.; Zsak, L.; Kaplan, A.S.; Ben-Porat, T.; Lomniczi, B. Role of a structural glycoprotein of pseudorabies in virus virulence. J Virol 1987, 61, 4030-4032, doi: 10.1128/JVI.61.12.4030-4032.1987. Kit, S. Genetically engineered vaccines for control of Aujeszky's disease (pseudorabies). Vaccine 1990, 8, 420-424, doi: 10.1016/0264-410x(90)90240-m. Volz, D.M.; Lager, K.M.; Mengeling, W.L. Latency of a thymidine kinase-negative pseudorabies vaccine virus detected by the polymerase chain reaction. Arch Virol 1992, 122, 341-348, doi:10.1007/BF01317195. Van de Walle, G.R.; Favoreel, H.W.; Nauwynck, H.J.; Pensaert, M.B. Antibody- induced internalization of viral glycoproteins and gE-gl Fc receptor activity protect pseudorabies virus-infected monocytes from efficient complement-mediated lysis. J Gen Virol 2003, 84, 939-947, doi: 10.1099/vir.0.18663-0. Kritas, S.K.; Pensaert, M.B.; Mettenleiter, T.C. Invasion and spread of single glycoprotein deleted mutants of Aujeszky's disease virus (ADV) in the trigeminal nervous pathway of pigs after intranasal inoculation. Vet Microbiol 1994, 40, 323-334, doi: 10.1016/0378-1135(94)90120-1. Schang, L.M.; Kutish, G.F.; Osorio, F.A. Correlation between precolonization of trigeminal ganglia by attenuated strains of pseudorabies virus and resistance to wildtype virus latency. J Virol 1994, 68, 8470-8476, doi:10.1128/JVI.68.12.8470- 8476.1994. Viejo-Borbolla, A.; Munoz, A.; Tabares, E.; Alcami, A. Glycoprotein G from pseudorabies virus binds to chemokines with high affinity and inhibits their function. J Gen Virol 2010, 91, 23-31, doi: 10.1099/vir.0.011940-0. Firkins, L.D.; Weigel, R.M.; Biehl, L.G.; Hahn, E.C. Field trial to evaluate the immunogenicity of pseudorabies virus vaccines with deletions for glycoproteins G and E. Am J Vet Res 1997, 58, 976-984. Platt, K.B.; Mare, C.J.; Hinz, P.N. Differentiation of vaccine strains and field isolates of pseudorabies (Aujeszky's disease) virus: thermal sensitivity and rabbit virulence markers. Arch Virol 1979, 60, 13-23, doi:10.1007/BF01318093. Fenaux, M.; Halbur, P.G.; Haqshenas, G.; Royer, R.; Thomas, P.; Nawagitgul, P.; Gill, M.; Toth, T.E.; Meng, X.J. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymph nodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions. J Virol 2002, 76, 541-551, doi:10.1128/jvi.76.2.541-551.2002. Chowdhury, ST; Pannhorst, K.; Sangewar, N.; Pavulraj, S.; Wen, X.; Stout, R.W.; Mwangi, W.; Paulsen, D.B. BoHV-1 -Vectored BVDV-2 Subunit Vaccine Induces BVDV Cross-Reactive Cellular Immune Responses and Protects against BVDV-2 Challenge. Vaccines (Basel) 2021, 9, doi:10.3390/vaccines9010046. Pomeranz, L.E.; Reynolds, A.E.; Hengartner, C.J. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev 2005, 69, 462-500, doi:10.1128/MMBR.69.3.462-500.2005. Wang, T.Y.; Yang, Y.L.; Feng, C.; Sun, M.X.; Peng, J.M.; Tian, Z.J.; Tang, Y.D.; Cai, X.H. Pseudorabies Virus UL24 Abrogates Tumor Necrosis Factor Alpha-Induced NF- kappaB Activation by Degrading P65. Viruses 2020, 12, doi:10.3390/vl2010051. Chowdhury, S.I.; Lee, B.J.; Ozkul, A.; Weiss, M.L. Bovine herpesvirus 5 glycoprotein E is important for neuroinvasiveness and neuro virulence in the olfactory pathway of the rabbit. J Virol 2000, 74, 2094-2106, doi:10.1128/jvi.74.5.2094-2106.2000. Pannhorst, K.; Wei, H.; Yezid, H.; He, J.; Chowdhury, S.I. Bovine Herpesvirus 1 UL49.5 Interacts with gM and VP22 To Ensure Virus Cell-to-Cell Spread and Virion Incorporation: Novel Role for VP22 in gM-Independent UL49.5 Virion Incorporation. J Virol 2018, 92, doi:10.1128/JVI.00240-18. Pavulraj, S.; Kamel, M.; Stephanowitz, H.; Liu, F.; Plendl, J.; Osterrieder, N.; Azab, W. Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs. Viruses 2020, 12, doi:10.3390/vl2090999. Anonymous. Manual of Diagnostic Tests for Detection of CSF Availabe online: (accessed virotogi^eu-and-oie-reference-laboraton’/diagnostic-me^bods. 22 October 2021). Grummer, B.; Fischer, S.; Depner, K.; Riebe, R.; Biome, S.; Greiser-Wilke, I. Replication of classical swine fever virus strains and isolates in different porcine cell lines. Disch Tierarztl Wochenschr 2006, 113, 138-142. Nauwynck, H.J. Functional aspects of Aujeszky's disease (pseudorabies) viral proteins with relation to invasion, virulence and immunogenicity. Vet Microbiol 1997, 55, 3-11, doi: 10.1016/s0378- 1135(96)01299-0. Huang, Y.L.; Meyer, D.; Postel, A.; Tsai, K.J.; Liu, H.M.; Yang, C.H.; Huang, Y.C.; Berkley, N.; Deng, M.C.; Wang, F.I., et al. Identification of a Common Conformational Epitope on the Glycoprotein E2 of Classical Swine Fever Virus and Border Disease Virus. Viruses 2021, 13, doi:10.3390/vl3081655. Nielsen, J.; Vincent, I.E.; Botner, A.; Ladekaer-Mikkelsen, A.S.; Allan, G.; Summerfield, A.; McCullough, K.C. Association of lymphopenia with porcine circovirus type 2 induced postweaning multisystemic wasting syndrome (PMWS). Vet Immunol Immunopathol 2003, 92, 97-111, doi:10.1016/s0165-2427(03)00031-x. Darwich, L. ; Segales, J. ; Mateu, E. Pathogenesis of postweaning multisystemic wasting syndrome caused by Porcine circovirus 2: An immune riddle. Arch Virol 2004, 149, 857-874, doi: 10.1007/s00705-003-0280-9. Patterson, A.R.; Ramamoorthy, S.; Madson, D.M.; Meng, X.J.; Halbur, P.G.;
Opriessnig, T. Shedding and infection dynamics of porcine circovirus type 2 (PCV2) after experimental infection. Vet Microbiol 2011, 149, 91-98, doi: 10.1016/j.vetmic.2010.10.020. Sanchez, R.E., Jr.; Meerts, P.; Nauwynck, H.J.; Ellis, J.A.; Pensaert, M.B. Characteristics of porcine circovirus-2 replication in lymphoid organs of pigs inoculated in late gestation or postnatally and possible relation to clinical and pathological outcome of infection. J Vet Diagn Invest 2004, 16, 175-185, doi: 10.1177/104063870401600301. Delva, J.L.; Nauwynck, H.J.; Mettenleiter, T.C.; Favoreel, H.W. The Attenuated Pseudorabies Virus Vaccine Strain Bartha K61: A Brief Review on the Knowledge Gathered During 60 Years of Research. Pathogens 2020, 9, doi: 10.3390/pathogens9110897. Yuan Lin, L.T., Changjian Wang, Shicheng He, Ling Fang, Zicheng Wang, Yating Zhong, Kun Zhang, Daoxin Liu, Qing Yang and Aibing Wang. Serological Investigation and Genetic Characteristics of Pseudorabies Virus in Hunan Province of China From 2016 to 2020. Frontiers in Veterinary Science 2021, 8, doi: 10.3389/fvets.2021.762326. Baekbo, P.; Kristensen, C.S.; Larsen, L.E. Porcine circovirus diseases: a review of PMWS. Transbound Emerg Dis 2012, 59 Suppl 1, 60-67, doi:l 0.1111/j.l 865- 1682.2011.01288.x. Ouyang, T.; Zhang, X.; Liu, X.; Ren, L. Co-Infection of Swine with Porcine Circovirus Type 2 and Other Swine Viruses. Viruses 2019, 77, doi: 10.3390/vl 1020185. Fritzemeier, J.; Teuffert, J.; Greiser-Wilke, I.; Staubach, C.; Schluter, H.; Moennig, V. Epidemiology of classical swine fever in Germany in the 1990s. Vet Microbiol 2000, 77, 29-41, doi:10.1016/s0378-1135(00)00254-6. Leifer, L; Hoffmann, B.; Hoper, D.; Bruun Rasmussen, T.; Biome, S.; Strebelow, G.; Horeth-Bontgen, D.; Staubach, C.; Beer, M. Molecular epidemiology of current classical swine fever virus isolates of wild boar in Germany. J Gen Virol 2010, 97, 2687-2697, doi:10.1099/vir.0.023200-0. Ganges, L.; Crooke, H.R.; Bohorquez, J.A.; Postel, A.; Sakoda, Y.; Becher, P.; Ruggli, N. Classical swine fever virus: the past, present and future. Virus Res 2020, 289, 198151, doi:10.1016/j.virusres.2020.198151.

Claims

WE CLAIM:
1. A pseudorabies virus (PRV) recombinant vector comprising a deletion in the gene encoding glycoprotein gE, a deletion in the gene encoding glycoprotein gG and a deletion in the gene encoding tyrosine kinase (TK), wherein at least one heterologous antigen is inserted in at least one deletion locus.
2. The PRV recombinant vector of claim 1 , wherein the deletion in the gene encoding gG does not affect the transcription of the Us3 gene and the deletion in the gene encoding TK does not affect the transcription of the UL22 and UL24 genes.
3. The PRV recombinant vector of claim 2, wherein a Us3 Poly A sequence of the Us3 gene is repositioned upstream of the gG deletion.
4. The PRV recombinant vector of claim 1 , wherein the deletion in the gene encoding gG disrupts the chemokine binding ability of glycoprotein gG.
5. The PRV recombinant vector of claim 1, wherein the deletion in the gene encoding gG encompasses the sequence coding for amino-terminal amino acid residues 1 to 71.
6. The PRV recombinant vector of claim 2, wherein TAATA boxes of the UL22 and UL24 genes are functional.
7. The PRV recombinant vector of claim 1, wherein the deletion in the gene encoding TK disrupts the tyrosine kinase activity of TK.
8. The PRV recombinant vector of claim 1, wherein the deletion in the gene encoding TK encompasses the sequence coding for amino acid residues 136 to 320.
9. The PRV recombinant vector of claim 1 , wherein the deletion in the gene encoding gE disrupts the Fc binding activity of the complex gE-gl.
10. The PRV recombinant vector of claim 1, wherein the deletion in the gene encoding gE encompasses the complete ORF of gE.
59
11. The PRV recombinant vector of any of claims 1-10, further comprising at least three heterologous antigens inserted therein.
12. The PRV recombinant vector of claim 11, wherein the at least three heterologous antigens are derived from proteins of viruses selected from the group consisting of Porcine Circovirus type 2 (PCV2), Classical Swine Fever Virus (CSFV), and combinations thereof.
13. The PRV recombinant vector of claim 12, wherein at least one PCV2 antigen derived a PCV2 having a genotype selected from the group consisting of PCV2a, PCV2b, PCV2c, PCV2d, PCV2e, and combinations thereof.
14. The PRV recombinant vector of claim 13, wherein the PCV2 antigen is derived from the virus of the PCV2b genotype.
15. The PRV recombinant vector of claim 11, wherein the at least three heterologous antigens are derived from proteins selected from the group consisting of viral envelope glycoproteins, capsid proteins, and combinations thereof.
16. The PRV recombinant vector of claim 15, wherein the capsid protein lacks a nuclear localization signal.
17. The PRV recombinant vector of claim 16, wherein the capsid protein self-assembled as a virus-like particle (VLP).
18. The PRV recombinant vector of claim 11, wherein the at least three heterologous antigens are derived from proteins selected from the group selected from PCV2 Cap, CSFV Ems, CSFV El, CSFV E2, CSFV C, and combinations thereof.
19. The PRV recombinant vector of claim 11, wherein at least one of the at least three heterologous antigens is expressed as a fusion protein with a fusion partner.
20. The PRV recombinant vector of claim 19, wherein the fusion partner is a cytokine that can potentiate a humoral and/or cellular immunity, preferably the cytokine being GM-CSF.
60
21. The PRV recombinant vector of claim 20, wherein the fusion partner is selected from the group consisting of a gD signal sequence, GM-CSF, and combinations thereof.
22. The PRV recombinant vector of any of claims 18-21, wherein the coding sequence of the CSFV E2-derived antigen is inserted into the gE deletion locus, the coding sequence of the PCV2 Cap-derived antigen is inserted into the TK deletion locus, and the coding sequence of the CSFV Ems-GMSCF-derived antigen is inserted into the gG deletion locus.
23. The PRV recombinant vector of any of claims 10-12, wherein at least one of the at least three heterologous antigens is expressed from a heterologous promoter within a gene expression cassette.
24. The PRV recombinant vector of claim 23, wherein the heterologous promoter is selected from the group consisting of a viral promoter and a mammalian promoter.
25. The PRV recombinant vector of claim 24, wherein the promotor is selected from the group consisting of a HCMV promotor, a human elongation factor 1 alpha promotor, a CMV IE promotor and a CAG synthetic promotor.
26. The PRV recombinant vector of any of claims 23-25 expressing a CSFV E2-derived antigen from a gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:2, a PCV2 Cap-derived antigen from a gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:3, and aCSFV Ems-GMSCF-derived antigen from a gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:4.
27. The PRV recombinant vector of claim 26, wherein the gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:2 is inserted in the gE deletion locus, the gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:3 is inserted into the TK deletion locus, and the gene expression cassette having at least 90%, at least 95% or 100% sequence identity with the sequence SEQ ID NO:4 is inserted into the gG deletion locus.
61
28. The PRV recombinant vector of any of claims 1-27, wherein the vector establishes latency and does not replicate in the TG neurons.
29. A composition comprising a carrier and at least one PRV recombinant vector of any of claims 1-28.
30. The composition of claim 29, formulated for administration by an intranasal route.
31. A method for treating a swine having or at risk of having an infection, in particular a porcine infection, by administering at least one PRV recombinant vector of any of claims 1-28 to the swine.
32. The method of claim 31, wherein the porcine infection is a viral porcine infection induced by at least one virus selected from the group consisting of PRV, PCV2, CSFV, SIV, and combinations thereof.
33. The method of claim 31, wherein the porcine infection is caused by PCV2 in combination with at least one pathogen selected from the group consisting of mycoplasma sp, parvovirus and porcine reproductive and respiratory syndrome virus (PRRSV), and combinations thereof.
34. The method of claim 33, wherein the porcine infection is the post-weaning multisystemic wasting syndrome (PMWS).
35. The method of claim 31, wherein administering at least one PRV recombinant vector prevents or reduces the incidence or severity of viral infection in a swine.
36. The method of claim 31, wherein administering at least one PRV recombinant vector induces humoral and/or cellular immunity.
37. The method of claim 36, wherein administering at least one PRV recombinant vector induces humoral immunity against at least one virus selected from the group consisting of PRV, PCV2, CSFV, and combinations thereof.
62
38. The method of claim 36, wherein administering at least one PRV recombinant vector induces cellular immunity against CSFV.
39. The method of claim 31, wherein the swine is a domesticated pig, an experimental pig or a boar.
40. A live attenuated vaccine for protection against at least one porcine infection comprising at least one of the RPV recombinant vector of any of claims 1-28.
41. The vaccine of claim 40, wherein the porcine infection is caused by at least one pathogen selected from the group consisting of PRV, PCV2, CSFV, SIV, mycoplasma sp, parvovirus, PRRSV, and combinations thereof.
42. A vaccine composition comprising the vaccine of claim 40 and a pharmaceutically acceptable vehicle or adjuvant.
43. The vaccine of any of claims 40-42, wherein said vaccine prevents virus shedding.
44. A method of vaccinating a swine against a porcine infection, said method comprising inoculating the swine with the vaccine of any of claims 40-43..
45. The method of claim 44, wherein the PCV2-infected swine is co-infected with at least one pathogen selected from the group consisting of mycoplasma sp, parvovirus, PRRSV, and combinations thereof.
46. The method of claim 45, wherein the vaccinating results in prevention or reduction of the symptoms associated with post-weaning multisystemic wasting syndrome (PMWS).
47. The method of claim 44, wherein the PCV2-infected swine is co-infected with at least one virus selected from the group consisting of SIV, CSFV, PRV, and combinations thereof.
48. The method of claim 47, wherein the vaccinating results in the reduction of the pathogenicity of at least one virus selected from the group consisting of SIV, CSFV, PRV, and combinations thereof.
63
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