Canine adenovirus vaccine constructs

Abstract

Recombinant replication-competent canine adenovirus (CAV) vaccine compositions engineered to contain one or more heterologous open reading frames (ORFs) encoding heterologous antigenic proteins, such as antigenic proteins from canine distemper virus (CDV) and / or canine parainfluenza virus (CPiV), are described. The heterologous ORFs are inserted at the site of a complete or partial deletion of an E3 region of the CAV. Immunogenic compositions that include one or more of the recombinant CAV vaccines are also described. The recombinant CAVs and immunogenic compositions thereof can be used to elicit an immune response against CAV, CDV and / or CPiV and / or to prevent or treat a CAV, CDV and / or CPiV infection.

Description

[0001] 7158-105382-02

[0002] CANINE ADENOVIRUS VACCINE CONSTRUCTS CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 779,122, filed March 27, 2025, which is herein incorporated by reference in its entirety.

[0004] FIELD

[0005] This disclosure concerns the assembly and properties of synthetic canine adenovirus (CAV) genomes that express heterologous antigens from other pathogens, such as from canine distemper virus (CDV) or canine parainfluenza virus (CPiV), and methods of use, to induce immunity and neutralizing antibodies that vaccinate and protect canines and other veterinary species.

[0006] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0007] The electronic sequence listing, submitted herewith as an XML file named 7158-105382-02. xml (585,127 bytes), created on March 18, 2026, is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Many viruses can cause acute and severe illness in domestic dogs, including canine distemper virus (CDV), canine parainfluenza virus (CPiV), and canine adenovirus (CAV). CDV is an RNA virus that belongs to the Paramyxoviridae family. CDV infection generally causes gastrointestinal, respiratory and / or neurological systems. Distemper is most commonly observed in dogs 3 to 6 months of age, but can also be found in older dogs, even in vaccinated dog populations (Sykes, Small Animal Critical Care Medicine 2015:504-508). CPiV is also a member of the Paramyxoviridae family. CPiV is a highly contagious virus that is one cause of canine infectious respiratory disease complex (CIRDC), also known as “kennel cough.” CAV is a DNA virus that is a member of the family Adenoviridae. There are two types of canine adenoviruses - CAV-1 and CAV-2. Like CPiV, CAV-2 is known to cause CIRDC. CAV-1 , also known as infectious canine hepatitis virus (ICHV), causes canine hepatitis. As detailed hereafter, a need exists for improved vaccines for the prevention of CDV, CPIV and CAV in canines and other veterinary species.

[0010] SUMMARY

[0011] Disclosed herein are synthetic replication competent canine adenovirus (CAV) vaccines that have been engineered to express one or more heterologous CDV or CPiV proteins within their genomes, which confers distinct replication, antigen expression and immune inducing properties, and which can be administered via multiple routes and elicit protective immunity.

[0012] The synthetic CAVs and immunogenic compositions thereof can be used, for example, to elicit a protective immune response against CAV-1, CAV-2, CPiV and / or CDV. In some instances, the immune7158-105382-02

[0013] response is elicited by a single virus genome composition or in other cases a mixture of two or more different virus compositions.

[0014] Provided herein are recombinant, replication-competent CAVs having a genome that includes a complete or partial deletion of an E3 region and a first heterologous open reading frame (ORF) encoding a first antigenic protein inserted at the site of the complete or partial deletion of the E3 region. In some aspects, the first antigenic protein is from a pathogen that infects a veterinary species (such as a canine). In some examples, the first antigenic protein is a protein from a CDV, such as a CDV hemagglutinin (CDVH) protein or a CDV nucleoprotein (CDVNP), or a fragment thereof. In other examples, the first antigenic protein is a protein from a CPiV, such as a CPiV fusion (CPiVF) protein or a CPiV hemagglutinin-neuraminidase (CPiVHN) protein, or a fragment thereof.

[0015] Also provided are immunogenic compositions that include one or more recombinant CAVs disclosed herein, and a pharmaceutically acceptable carrier. In some aspects, the immunogenic composition further includes an adjuvant. In some aspects, tire immunogenic composition further includes one or more additional vaccines, such as a canine parvovirus vaccine, a rabies virus vaccine, an influenza virus vaccine, a canine coronavirus vaccine, a Leptospirosis vaccine and / or a Bordetella bmnchiseptica vaccine. In specific examples, tire iimnunogenic compositions are formulated for oral and / or mucosal administration.

[0016] Further provided are methods for eliciting an immune response in a subject by administering an effective amount of a recombinant CAV or immunogenic composition disclosed herein. In some aspects, the subject is a canine. In particular instances, the immune response is protective against CAV, CDV, and / or CPiV.

[0017] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS FIG.1: Exemplary nucleotide sequence alignment illustrating the similarity and conservation of the CAV1 (canine Adi), CAV2 (canine Ad2), and human adenovirus type 5 (human Ad5) genomes, with CAV2 used as the reference sequence. The alignment highlights sequence divergence between human Ad5 and both CAV1 and CAV2, while CAV1 and CAV2 exhibit a higher degree of conservation. The most notable difference between the CAV 1 and CAV2 genomes is a deletion within the E3-ORF2 gene in CAV1 relative to CAV2, though their overall genome architecture and gene organization remain conserved. Despite sequence-level differences, the functional arrangement of gene units across the CAV1 and CAV2 genomes remains structurally consistent, supporting their shared evolutionary origin and functional similarities.

[0019] FIG.2: Exemplary sequence alignment of the E3 region from human adenovirus 5 (Ad5), human adenovirus 34 (Ad34), canine adenovirus 1 (CAV1), and three strains of canine adenovirus 2 (CAV2),7158-105382-02

[0020] including the Toronto strain, G1 strain, and SH-R2 isolate. The alignment is shown relative to the hAd5 sequence. There is significant sequence divergence (white shading of boxes is not conserved, grey is conserved) evident between the canine adenovirus strains and the human adenoviruses in this genomic region. However, a conserved TATA box and promoter element embedded within the pVIII coding sequence is highly conserved and preserved across all strains. These findings suggest that, although not previously characterized, canine adenoviruses likely utilize an embedded internal promoter within tire protein VIII gene to initiate the transcription of downstream E3 open reading frames.

[0021] FIG.3: Comparison of the pVIII and E3-ORF1 junctions in hAd5 (top), CAV1 (middle), and CAV2 (bottom). In hAd5, tire initiation codon (Met) for E3-ORF1, encoding tire 12.5 kDa protein, occurs immediately downstream of tire pVIII stop codon, with no overlap between reading frames. In contrast, in CAV1 and CAV2, E3-ORF1 has predicted overlapping coding sequences with tire C-terminus of pVIII: potential alternative start codons, including methionine residues (MAM), are predicted within an alternative reading frame of tire pVIII coding sequence. Arrows indicate tire predicted initiation codons for E3-ORF1 in each species, highlighting that in CAV1 and CAV2 the predicted E3-ORF1 start codon overlaps the pVIII coding sequence, whereas in hAd5 tire E3-ORF1 start codon is located immediately downstream of the pVIII stop codon. The C-terminal amino acid sequence of pVIII is conserved across all three species as "DGYD" (SEQ ID NO: 117); however, the underlying nucleotide sequence is degenerate, allowing for these alternative start codons in CAV1 and CAV2. This overlap could result in N-terminal extensions, in-frame fusions, or aberrant localization (e.g., if signal peptides are inadvertently translated), which are undesirable for expression of a therapeutic ORF and are an important consideration for engineering ectopic gene placements at this junction.

[0022] FIG.4: Exemplary sequence design for engineering synonymous pVIII mutations to drive the optimal expression and translation of a therapeutic ORF, in this example, a vaccine antigen CDV-H, by mutating and deleting E3-ORF1 coding sequences. The figure highlights genetic modifications introduced to enable faithful CDV-H protein expression while preserving the integrity of the pVIII amino acid sequence. The endogenous E3-ORF1 sequences were replaced with the CDV-H coding sequence, and synonymous T-to-C mutations were introduced to ablate alternative ATG start codons in the alternative reading frame of pVIII. These mutations preserved the conserved DGYD (SEQ ID NO: 117) sequence of pVIII while abolishing upstream ATG start codons in a different frame that could result in the incorporation of unintended amino acid sequences in a heterologous antigen engineered in place of E3-ORF1. Additionally, the natural E3-ORF1 lacked a Kozak consensus sequence for optimal translation. To address this, the E3 GAGAGC sequence was deleted and a G nucleotide was engineered after the pVIII stop codon at tire -1 position of the CDV-H ATG, creating an optimized Kozak sequence to enhance translation initiation and protein expression. This engineered Kozak sequence is absent in the corresponding Ad5 sequence. Alternative designs without tire Kozak sequence are also feasible for achieving heterologous antigen expression.

[0023] FIG.5: Alignment of tire CAV2 (top), CAV1 (middle), and PCMN-1507 (bottom) E3-ORF2 regions at the non-coding junction witir the U exon. PCMN-1507 is an exemplary recombinant CAV7158-105382-02

[0024] vaccine genome expressing CDV-H (canine distemper virus hemagglutinin) in place of E3-ORF1 coding sequences, with E3-ORF2 sequences mostly deleted, except for non-coding sequences between E3-ORF1 and the start of E3-ORF2. Conserved predicted regulatory elements and motifs are annotated and their spatial arrangements visualized. The endogenous CAV1 and CAV2 non-coding sequences at the U exon junction contain a conserved polyadenylation (polyA) motif (AAATAAACT) and a 5' splice site upstream of the U exon on the negative strand. These elements are preserved in the PCMN-1507 vaccine design, as they may play a role in the transcription and translation of CDV-H or other heterologous ORFs that replace endogenous CAV E3 ORFs, and / or in the splicing and translation of late proteins expressed from the major late promoter and the U exon. Additionally, a non-coding remnant normally found between E3-ORF1 and E3-ORF2 is preserved, which includes a predicted polyA consensus sequence (ATT AAA) and splicing motifs that may contribute to the generation of L5 fiber transcripts and die post-transcriptional regulation of CAV genes or heterologous ORFs. While diis design represents one option, alternative designs could employ different consensus motifs for polyA signals and splice sites, or delete the E3-ORF1 / E3-ORF2 junction sequences entirely.

[0025] FIG. 6: An illustration of die annotated genes in die CAV2 reference genome, which is 31,232 base pairs in length. Nomenclature of protein coding genes largely follows accepted terminology of the human adenoviruses. Annotations are predicted by genome sequence analysis. The genomic organization, transcriptional and functional gene modules are substantially similar between CAV1 and CAV2, even if sequence variation exists. In this example, to facilitate the construction and engineering of synthetic recombinant CAV vaccine strains, the CAV2 genome was divided into four genomic modules. These exemplary genomic modules are designated as El, Core, E3, and E4, as indicated in the CAV genome map.

[0026] 1. El Module: Spans from the left-hand internal terminal repeat (ITR) through the El genes and the TATA box for pIX. The E1A and E1B gene products encoded within this module are essential for viral replication.

[0027] 2. Core Module: Contains the early / intermediate genes, pIX and IV A2; the major late promoter (MLP) and a transcriptional unit that drives the expression of CAV capsid and late proteins, including: 52 / 55k, pllla, penton, pVII, pV, pX, pVI, hexon, endoproteinase, 100k, 22k, and 33k on the positive strand. On the negative strand, the E2 transcriptional unit drives the expression of the viral DNA replication proteins, including E2A DNA binding protein (DBP), pTP and E2B DNA polymerase..

[0028] 3. E3 Module: Includes the MLP transcribed late protein pVIII, the E3 protein coding genes (E3-ORF1 and E3-ORF2) and the CAV fiber gene on the positive strand, and the U exon on die negative strand.

[0029] 4. E4 Module: Includes the right ITR and the E4 transcriptional unit and protein coding genes are on the negative strand. The E4 genes play important functions in usurping host cell signaling padiways and function to facilitate viral replication in human Ads and likely canine Ads. In CAV there are five annotated ORFs, which include from right to left, E4-ORF1, E4-ORF2, E4-ORF3, E4-ORF4 and E4-ORF5.7158-105382-02

[0030] FIG. 7: An exemplary schematic showing an overview of the CAV El, Core, E3, and E4 plasmids that were constructed, engineered and assembled to create recombinant CAV vaccines. The CAV El , Core, E3, and E4 genomic regions (from FIG. 6) were generated from geneblocks (gblock) and / or using the CAV2 reference virus genome as a source, as indicated. The genomic regions and modules defined in FIG. 6 were cloned into plasmid backbones with features designed to enable downstream genetic engineering and systematic assembly of recombinant CAV genomes using scarless (CAVSLIC) and recombination based cloning techniques (CAVsembly). The El, E3, and E4 genomic modules were inserted into Entry plasmids such that they are compatible with Gateway cloning, sequence and ligation-independent cloning (SLIC), and Gibson assembly. The E4 module was constructed using three primer extension PCR products from the CAV2 ATCC reference virus genome and modified to have silent nucleotide substitutions in E4 coding sequences that eliminate endogenous Pad and Bglll sites and facilitate flexible modular assembly. The CAV Core genomic region was cloned into a plasmid containing an ampicillin resistance gene that was engineered to have unique Pad and Bglll sites on the left and right sides, respectively, for scarless cloning of El and E3 / E4 modules. The core plasmid serves as an exemplary central backbone for assembling the complete CAV genome by facilitating the seamless integration of the El, E3, and E4 modules through Gateway®, SLIC, or Gibson assembly. Together, these fom modular plasmids enable an exemplary precise assembly and engineering of CAV-based vaccine strains while preserving essential genomic functions for replication and antigen expression.

[0031] FIG. 8: Exemplary schematic showing El, E3 and E4 modules assembled with core modules via scarless cloning methods to create recombinant CAV genomes. The CAV El, E3 and E4 modules are linearized by PCR for Gibson assembly. The larger size of the core module means that there is a higher risk of PCR induced errors, thus linearization via engineered Pacl / Bglll restriction enzymes is more efficient. Breaking the CAV genome into functional genomic plasmid modules facilitates the engineering of individual module genetic modifications, and combinations thereof, when assembled together to generate recombinant CAV genomes.

[0032] FIG. 9: An exemplary schematic illustration of the scarless assembly of CAV genomic modules for the creation of multivalent, replication-competent CAV-based vaccines. CAVSLIC of El , E3 and E4 modules with the core module can be performed in any order, and in two, three or four steps, which is dictated by combinatorial complexity as well as efficiency. The assembly reaction with larger nucleic acid molecules is lower when more than three fragments are included. Thus, two- or three-fragment SLIC / Gibson assemblies were typically used. As such, it is often desirable to assemble an El and core module first into an El-core macromodule intermediary plasmid, which is subsequently assembled with E3 and / or E4 modules in another Gibson / SLIC reaction. The generic seamless assembly process depicted can be Gibson assembly, SLIC, In-Fusion, or restriction enzyme and ligation cloning. In this illustration, a Pad digested Core plasmid is assembled with PCR linearized El modules, which are Gibson / SLIC assembled, transformed into bacteria, selected and purified. The El-Core macromodule plasmid is then digested with Bglll and assembled in a 3-fragment Gibson / SLIC assembly reaction with PCR-linearized E3 and E4 genomic modules. In this example, the E3 module has been genetically modified to replace7158-105382-02

[0033] E3-ORFs with heterologous antigens from CDV. The final plasmid includes a fully assembled, replication-competent, recombinant CAV genome capable of expressing heterologous antigens.

[0034] FIG. 10: An exemplary schematic illustrating the scarless assembly of recombinant CAV genome vaccines using CAVSLIC plasmid modules. Similar to the example illustrated in FIG. 9, an El / core micromodule plasmid is first created and then assembled with an E4 module in a second Gibson reaction to generate an El-core-E4 macromodule plasmid, which is then Bglll digested and assembled with an E3 module in a third Gibson reaction. Assembly can be performed using Gibson cloning, although other ligation-independent techniques, such as SLIC, may also be employed.

[0035] FIG. 11: Map of a CAV El module plasmid (El-269) utilized in the construction and assembly of synthetic engineered viral vaccine compositions described herein. The CAV El entry module includes the CAV El genomic region, extending from the right-hand ITR region to include the El A and E1B genes, as well as the TATA box associated with protein IX. In addition to tire CAV El genomic components, tire plasmid backbone contains several other features labeled within tire map. These include an I-Scel restriction site flanking the left ITR as well as a kanamycin resistance gene for bacterial selection.

[0036] FIG. 12: Plasmid map of a CAV core capsid and DNA replication protein module (Core-059), used for the assembly and engineering of recombinant and synthetic CAV vaccine viruses and viral genomes described herein. The Core-059 plasmid module includes tire CAV2 core genomic region spanning from the protein IX gene to the L433k gene. The plasmid backbone features a low-copy pl5A bacterial origin of replication, transcriptional terminator sequences and an ampicillin resistance gene that enable viral genome plasmid propagation and selection in selective media. There is also an I-Scel expression cassette that includes a mammalian RSV viral promoter that drives the 1-Scel restriction enzyme upon plasmid transfection into mammalian (including canine) cells. The mammalian promoter and expression of the I-Scel restriction enzyme specifically in mammalian cells cuts the CAV vaccine plasmid backbone at the I-Scel cleavage sites that flank the left and right assembled CAV genome ITRs linearizing and releasing the CAV genome so that it can be recognized by CAV E2-TP and undergo E2B mediated Type I and II viral genome replication and virion production. Exemplary key elements that facilitate seamless genome assembly include: (1) a Pad site - enables linearization of the plasmid for the efficient insertion of El genomic modules; and (2) a Bglll site - facilitating the seamless cloning of both El and E4 genomic modules.

[0037] FIG. 13: Map of the CAV E3-522 entry plasmid comprising a CAV E3 genomic module. This module includes pVIII, through the E3-ORF1 and E3-ORF2 early region immunomodulatory proteins and the fiber gene on the positive strand, and the U-exon on tire negative strand. This parent E3 plasmid was modified for inserting heterologous antigen genes that are driven by the predicted identification of a pVIII embedded promoter and wherein the heterologous antigens wholly or partially replace the CAV E3 ORFs. The deletion and replacement of the E3 ORFs not only facilitates heterologous antigen expression from native viral architecture, but creates tire necessary genomic ‘space’ for exogenous payloads and stimulation of a more robust immune response through deletion of E3 ORFs, which are generally7158-105382-02

[0038] associated with host immune suppression in other Ad species, a feature that is not desirable for a virus vaccine composition. The CAV E3-522 plasmid contains the wild-type CAV E3-ORF1 and E3-ORF2 genes and serves as the parental plasmid for subsequent genetic modifications. These modifications involved replacing the endogenous E3 genes with sequences engineered to express heterologous antigens from CDV and CPiV. This genetic engineering enabled the development of replication-competent, multivalent CAV-based therapeutic vaccines. The plasmid backbone includes a kanamycin resistance gene for bacterial selection.

[0039] FIG. 14: Plasmid map of the CAV E4-099 genomic module, which includes the CAV E4 genomic region. This region includes the right-hand ITR and the E4 transcriptional unit, encoding the E4-ORF1, E4-ORF2, E4-ORF3, E4-ORF4, and E4-ORF5 early viral genes. These genes play key roles in productive CAV replication by supporting viral transcription, DNA replication, and host cell modulation. The E4 plasmid backbone includes a kanamycin resistance gene for bacterial selection and an I-Scel restriction site, which enables linearization and release of tire viral genome upon transfection into mammalian cells. The E4 module was constructed using three primer extension PCR products amplified from the CAV2 genome. Additionally, two silent base-pair modifications were engineered within tire E4 coding region to eliminate Bglll and Pact restriction sites while preserving the encoded amino acid sequences.

[0040] FIG. 15: Canine distemper virus (CDV) is an enveloped RNA morbillivirus belonging to the Paramyxoviridae family, which infects a wide array of terrestrial carnivores, with a predominant impact on canines, as shown in the exemplary drawing. The genome of CDV consists of 15,690 nucleotides and encodes six structural proteins: the nucleocapsid protein (NP), phosphoprotein (P) , matrix protein (M), fusion protein (F), hemagglutinin protein (H), and large protein (L). Additionally, there are two non-structural proteins, V and C, which are associated with the immunosuppression of the host organism.

[0041] FIG. 16: An exemplary schematic illustration depicting the stepwise assembly of CAV plasmid modules to create the PCMN-1507 multivalent CAV-CDVH vaccine genome. This assembly process involved the sequential construction of modular plasmids encoding distinct CAV genomic regions, culminating in a fully assembled vaccine genome capable of expressing the CDVH protein.

[0042] 1. Core-059 and El assembly: The assembly process began with Pact digestion of the Core-059 plasmid, followed by Gibson assembly with the El genomic region derived from the El-269 plasmid. This step generated an intermediate macromodule designated ASMM-149.

[0043] 2. E3 module: The E3-575 plasmid contains the E3 genomic module in which E3-ORF1 and E3-ORF2 protein coding sequences were deleted and replaced to express the CDVH protein coding gene. This engineered modification leverages the native viral transcriptional architecture to drive CDVH expression.

[0044] 3. Recombinant whole genome assembly: ASMM 149 was digested with Bglll and assembled with PCR linearized E3-575 and E4-099 genomic modules, generating the PCMN-1507 viral genome vaccine plasmid.7158-105382-02

[0045] FIG. 17: Map of the E3-575 genomic module plasmid, which includes a modified pVIII sequence and in which the E3-ORF1 and E3-ORF2 genes have been wholly or partially deleted and replaced with the CDVH protein coding sequence.

[0046] FIG. 18: Plasmid map of the intermediate ASMM-149 macromodule, assembled through Pad digestion and Gibson assembly of the El-269 plasmid and the Core -059 plasmid. The ASMM-149 plasmid contains the CAV El genomic region, essential for viral replication, seamlessly integrated with the Core-059 backbone, which encompasses the core capsid and DNA replication proteins. This plasmid facilitates further downstream assembly steps, including the integration of modified E3 and E4 genomic modules to create replication-competent, multivalent CAV vaccine strains.

[0047] FIG. 19: Plasmid map of the fully assembled PCMN-1507 multivalent vaccine genome, which includes El, Core, genetically modified E3-CDVH, and E4 genomic modules. Hie PCMN-1507 construct serves as a replication-competent multivalent vaccine platform, leveraging the CAV2 backbone for stable and robust heterologous antigen expression. The El and E4 genomic regions were engineered to be flanked by I-Scel restriction enzyme sites, enabling precise cleavage for viral genome release. The plasmid backbone contains an RSV promoter, which becomes activated exclusively in mammalian cells. This promoter drives the expression of the I-Scel restriction enzyme, facilitating the linearization of the viral genome at the left and right ITRs. Upon cleavage, the viral genome is released, allowing for efficient DNA replication and amplification in canine cells, resulting in the production of replication-competent viral particles.

[0048] FIG.20: An exemplary schematic representation of the PCMN-1507 (CAV-CDVH) viral genome following I-Scel restriction enzyme digestion, which excises the plasmid backbone sequences and generates the replication-competent viral genome. This excised form represents the functional viral genome that undergoes DNA replication and amplification upon transfection into mammalian cells. The genome retains all essential CAV2 elements, including El, Core, E3-CDVH, and E4 genomic regions, ensuring proper transcription, replication, and expression of the CDVH heterologous antigen within the viral backbone.

[0049] FIG.21: An exemplary schematic illustration depicting the stepwise assembly of CAV plasmid modules to create the PCMN-1508 multivalent CAV-CDVN vaccine genome. This assembly process involved the sequential construction of modular plasmids encoding distinct CAV genomic regions, culminating in a fully assembled vaccine genome capable of expressing the CDVN protein coding gene.

[0050] 1. Core-059 and El assembly: The assembly process began with Pad digestion of the Core-059 plasmid, followed by Gibson assembly with the El genomic region derived from the El-269 plasmid. This step generated an intermediate macromodule designated ASMM-149.

[0051] 2. E3 module integration: Tire E3-581 plasmid contains the E3 genomic module, in which tire E3-ORF1 and E3-ORF2 protein coding sequences were deleted and replaced with the CDVN protein coding gene. This engineered modification leverages the native viral architecture to drive CDVN expression.7158-105382-02

[0052] 3. Recombinant whole genome assembly: ASMM-149 was digested with Bglll and assembled with PCR linearized E3-581 and E4-099 genomic modules, generating the PCMN-1508 viral genome.

[0053] FIG. 22: Map of the E3-581 genomic module plasmid, which includes a modified pVIII sequence and where the E3-ORF1 and E3-ORF2 protein coding sequences have been wholly or partially deleted and replaced with the CDVNP protein coding sequence.

[0054] FIG. 23: Plasmid map of the fully assembled PCMN-1508 multivalent vaccine genome, which includes El, Core, genetically modified E3-CDVNP, and E4 genomic modules. The PCMN-1508 construct serves as a replication-competent multivalent vaccine platform, leveraging the CAV2 backbone for stable and robust heterologous antigen expression.

[0055] FIG. 24: Canine parainfluenza virus (CPiV), like CDV, is a negative-sense, single stranded RNA virus, as illustrated in the exemplary drawing. CPiV is a highly contagious respiratory virus that affects dogs. It is one of the main causes of infectious tracheobronchitis, commonly known as "kennel cough." FIG. 25: An exemplary CAVSLIC and Gibson assembly of tire ASMM-150 macromodule plasmid.

[0056] FIG. 26: An exemplary CAVSLIC assembly of the PCMN-1556 CAV2 CPiV multivalent vaccine genome composition.

[0057] FIG. 27: Map of the E3-595 genomic module plasmid, which includes a modified pVIII sequence. The E3-ORF1 and E3-ORF2 genes were wholly or partially deleted and replaced to express two heterologous CPiV antigenic proteins, CPiV F and CPiVHN, which are operably linked, translated and cleaved via a P2A sequence.

[0058] FIG. 28: Plasmid map of the intermediate ASMM-150 macromodule, which was assembled by Bglll digestion of ASMM-149 and Gibson assembly with the E4-099 CAV genomic module.

[0059] FIG. 29: Plasmid map of the pCMN-1556 CPiV F+HN assembled CAV2 viral vaccine genome.

[0060] FIG. 30: An exemplary schematic of CAVSLIC assembly of the PCMN-1557 CAV2 CPiV F vaccine genome composition.

[0061] FIG. 31: Map of the E3-596 genomic module plasmid, which includes a modified pVIII sequence. The E3-ORF1 and E3-ORF2 genes were wholly or partially deleted and replaced to express CPIV F.

[0062] FIG. 32: Plasmid map of the PCMN-1557 CPIV F assembled CAV2 viral vaccine genome. FIG. 33: Schematic showing CAVSLIC assembly of the PCMN-1558 CAV2 CPiV HN vaccine genome composition.

[0063] FIG. 34: Map of the E3-597 genomic module plasmid, which includes a modified pVIII sequence. The E3-ORF1 and E3-ORF2 genes were wholly or partially deleted and replaced to express CPiV HN.

[0064] FIG.35: Map of the PCMN-1558 CAV CPiVHN viral genome vaccine plasmid.

[0065] FIG.36: An exemplary schematic illustration of a general workflow and method of producing and evaluating CAV vaccine composition replication and payload expression in canine cells.7158-105382-02

[0066] FIG. 37: Exemplary images showing the replication and cytopathic effects of the PCMN-1507 CAV2-CDVH viral genome in MDCK cells following transfection with the assembled viral genome plasmid. This plasmid encodes all of the components required for CAV replication, with the CDVH antigen expressed in place of the endogenous E3-ORF1 and E3-ORF2 genes. The CAV2 life cycle typically spans 48 to 72 hours, and these brightfield images were taken at four days and nine days posttransfection. (Top) The black circles highlight individual plaques forming in the MDCK cell monolayer 4 days post-transfection. These plaques represent regions of viral replication and cell lysis, indicating successful viral replication from the transfected plasmid genome. (Bottom) By day nine, the virus has undergone multiple rounds of amplification, leading to complete monolayer destruction and a pronounced cytopathic effect (CPE). These results demonstrate that tire PCMN-1507 plasmid is replication-competent, with viral growth kinetics comparable to those observed with the wild-type CAV2 genome, including an intact E3 region.

[0067] FIG.38: Exemplary images showing the replication and cytopathic effects of the PCMN-1508 viral genome in MDCK cells following transfection with tire assembled viral genome plasmid. The brightfield microscopy images were taken at 4 and 11 days post-transfection of MDCK cells with the complete CAV vaccine genome plasmid. (Top) At 4 days post-trausfection, small clusters (plaque-like) of virus transduced / infected expressing cells are seen. (Bottom) The CPE and cell death (change in refractive index and darker patches) resulting from productive viral replication and spread is apparent in complete destruction of the MDCK monolayer between 4 and 11 days. These data demonstrate successful and robust exponential viral replication of the CDVNP expressing CAV2 vaccine composition.

[0068] FIG.39: Exemplary images showing the replication and cytopathic effects of the PCMN-1556 viral genome in MDCK cells following transfection with the assembled viral genome plasmid. The brightfield microscopy images were taken at 4 and 9 days post-transfection of MDCK cells with the complete CAV vaccine genome plasmid. (Top) At 4 days post-transfection, small clusters (plaque-like) of virus transduced / infected expressing cells are seen. (Bottom) The CPE and cell death (change in refractive index and darker patches) resulting from productive viral replication and spread is apparent in complete destruction of the MDCK monolayer between 4 and 9 days.

[0069] FIG. 40: Exemplary images showing the replication and cytopathic effects of the PCMN-1557 viral genome in MDCK cells following transfection with the assembled viral genome plasmid. The brightfield microscopy images were taken at 4 and 9 days post-transfection of MDCK cells with the complete CAV vaccine genome plasmid. (Top) At 4 days post-transfection, small clusters (plaque-like) of virus transduced / infected expressing cells are seen. (Bottom) The CPE and cell death (change in refractive index and darker patches) resulting from productive viral replication and spread is apparent in complete destruction of the MDCK monolayer between 4 and 9 days.

[0070] FIG. 41: Exemplary images showing the replication and cytopathic effects of the PCMN-1558 viral genome in MDCK cells following transfection with the assembled viral genome plasmid. The brightfield microscopy images were taken at 4 and 9 days post-transfection of MDCK cells with the7158-105382-02

[0071] complete CAV vaccine genome plasmid. (Top) At 4 days post-transfection, small clusters (plaque-like) of virus transduced / infected expressing cells are seen. (Bottom) The CPE and cell death (change in refractive index and darker patches) resulting from productive viral replication and spread is apparent in complete destruction of the MDCK monolayer between 4 and 9 days.

[0072] FIG.42: Exemplary immunofluorescence images showing CDVH expression in MDCK cells following infection with the PCMN-1507 vaccine composition. Uninfected MDCK cells served as a negative control (top left), while MDCK cells transfected with a pCDNA-CDVH expression plasmid construct were used as a positive control (bottom left). Cells were fixed 12 hours post-infection and stained with a monoclonal antibody specific to CDV envelope proteins, including CDV-H, detected with fluorescent secondary antibodies. Brightfield images are shown on the left of each panel; immunofluorescence images on the right, where positive signal appears as bright cells against a dark background. PCMN-1507-infected cells (top right) show strong CDV-H expression across the monolayer, comparable to expression levels achieved through plasmid-based transfection (bottom right), confirming functional CDV-H antigen expression from the recombinant CAV vaccine platform.

[0073] FIG.43: Immunofluorescence images demonstrating expression of CDV antigens in MDCK cells following infection with either PCMN-1507 (CDV-H, center) or PCMN-1508 (CDV-N, right). Uninfected MDCK cells served as a negative control (left). Cells were fixed and stained with antibodies specific for CDV-H and CDV-N antigens, detected with fluorescent secondary antibodies. Positive signal appears as bright cells against a dark background. Both PCMN-1507 and PCMN-1508 show strong antigen expression across the monolayer, while uninfected cells show no signal above background, confirming the specificity and functional expression of heterologous CDV antigens by the recombinant CAV vaccine constructs.

[0074] FIG.44: Immunofluorescence images showing CPiV-HN antigen expression in MDCK cells following infection with PCMN-1556, PCMN-1557, or PCMN-1558 CAV vaccine viruses, or a control virus (PCMN-1366, mCherry). Uninfected cells were included as an additional negative control. Cells were fixed 12 hours post-infection and stained with antibodies specific to CPiV-HN, detected with fluorescent secondary antibodies (bottom row). Nuclei were counterstained with DAPI (top row).

[0075] Positive immunofluorescence signal appears as bright cells against a dark background. The results confirm that PCMN-1556 and PCMN-1558 express the CPiV-HN antigen, while PCMN-1557 (CPIF only), PCMN-1366 (mCherry control), and uninfected cells show no signal above background, validating the specificity and functionality of the recombinant CAV vaccine compositions.

[0076] FIG.45: Exemplary immunofluorescence images demonstrating CPiV antigen expression in MDCK cells infected with either PCMN-1556, PCMN-1557, or PCMN-1558 CAV vaccine compositions. Uninfected cells served as a negative control. Cells were fixed 12 hours post- infection and stained with anti-CPiV antibodies that were detected with fluorescent secondary antibodies (bottom panel), with nuclei counterstained with DAPI (top panel). Positive immunofluorescence signal appears as bright cells against a dark background. The results confirm that tire PCMN-1556, PCMN-1557, and7158-105382-02

[0077] PCMN-1558 CAV vaccine compositions express CPiV antigen payloads, confirming the functional expression of recombinant CPIV proteins by recombinant CAV virus vaccine constructs.

[0078] FIGS. 46A-46C: Graphs showing serum neutralization titers (SNT) of canines vaccinated with recombinant CAVs expressing CDV F, H and NP proteins. Canines in Group T1 were vaccinated with PCMN-1506 (CAV-CDV F), PCMN-1507 (CAV-CDV H), and PCMN-1508 (CAV-CDV NP); canines in Group T2 were vaccinated with PCMN-1508 (CAV-CDV NP) and PCMN-1561 (CAV-CDV F-2A-H); canines in Group T3 were unvaccinated. Canines in Groups T1 and T2 received two oral doses of vaccine 21 days apart. Animals in all groups were challenged with CDV intracranially (IC) 21 days after the second dose of vaccine. Shown are neutralization titers specific for CAV (FIG. 46A), ICHV (FIG.

[0079] 46B) and CDV (FIG. 46C). SNT were quantified 0, 7 and 14 days after the first vaccination (0DPV1, 7DPV1, 14DPV1) and the second vaccination (0DPV2, 7DPV2, 14DPV2), one day prior to challenge (-1DPC), and 7 days post-challenge (7DPC).

[0080] FIG.47: Graph showing CAV-specific SNT of canines vaccinated with recombinant CAVs expressing CDV F and CDV H. Canines were vaccinated orally with two doses of PCMN-1506 (CAV-CDV F) and PCMN-1507 (CAV-CDV H) 21 days apart. Control animals were unvaccinated. All animals were challenged IV with ICHV 21 days after the second vaccination. Shown axe neutralization titers at -1, 7 and 14 days after the first vaccination (-1DPV1, 7DPV1, 14DPV1) and the second vaccination (-1DPV2, 7DPV2, 14DPV2), one day prior to challenge (-1DPC), and 7, 14 and 21 days post-challenge (7DPC, 14DPC, 21DPC).

[0081] FIGS. 48A-48B: Graphs showing SNT and nasal titers of canines vaccinated with recombinant CAVs expressing CPiV F and CPiV HN. Canines in Group T1 were vaccinated with PCMN-1557 (CAV-CPiV F) and PCMN-1558 (CAV-CPiV HN); canines in Group T2 were vaccinated with PCMN-1556 (CAV-CPiV F-2A-HN); and canines in Group T3 were unvaccinated. Canines in Groups T1 and T2 received two oral doses of vaccine 21 days apart. Animals in all groups were challenged with CPiV intranasally (IN) 21 days after the second dose of vaccine. Shown are neutralization titers specific for CPiV (FIG. 48A) and nasal titers of CPiV (FIG. 48B). SNT were quantified at -1, 7 and 14 days after the first vaccination (-1 DPV1 , 7DPV1 , 14DPV1) and the second vaccination (-1 DPV2, 7DPV2, 14DPV2), one day prior to challenge (-1DPC), and 14 days post-challenge (14DPC). Nasal titers were measured one day prior to challenge (-1DPC) or 2 to 10 days post-challenge (2DPC to 10DPC).

[0082] FIGS. 49A-49F: Graphs showing SNT and nasal titers of canines vaccinated with various doses (104to IO55) of recombinant CAVs expressing CDV F, CDV H, CDV HN, CPiV F, and CPiV HN, and subsequently challenged with CPiV. Animals were vaccinated with PCMN-1506 (CAV-CDV F), PCMN-1507 (CAV-CDV H), PCMN-1556 (CAV-CPiV F-2A-HN), and an approved canine parvovirus vaccine (MLV CPV). Animals in Group 1 and Group 2 were vaccinated twice, 21 days apart. Animals in Group 3 received a single vaccination and control animals (Group 4) were unvaccinated. All animals were challenged with CPiV 6 months after the second vaccination. Shown are CPiV SNT (FIG. 49 A), CPiV nasal titers (FIG. 49B), CAV SNT (FIG.49C), ICHV SNT (FIG. 49D), CPV SNT (FIG. 49E) and CDV SNT (FIG. 49F). SNT were quantified one day prior to tire first and second vaccinations (-1DPV1,7158-105382-02

[0083] -1DPV2); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); 7 days, 21 days, 28 days, 8 weeks, 12 weeks, 16 weeks, and 20 weeks after the second vaccination (7DPV2, 21DPV2, 28DPV2, 8WPV1, 12WPV2, 16WPV2, 20WPV2); and one day prior to challenge (-1DPC). Nasal titers were measured one day prior to challenge (-1DPC) and 2 to 10 days post-challenge (2DPC to 10DPC).

[0084] FIGS. 50A-50B: Graphs showing CDV SNT of canines one year post-vaccination. Canines were vaccinated as described in FIGS. 49A-49F. After approximately one year, the study groups were subdivided to receive either a booster vaccination or to be challenged IC with CDV. Control animals were subdivided and challenged IC or IV / IN. FIG. 50A shows SNT of canines from one day prior to the first and second vaccinations (-1DPV1, -1DPV2); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); and 7 days, 21 days, and 28 days after the second vaccination (7DPV2, 21DPV2, 28DPV2). FIG. 50B shows SNT of boosted animals only at 0, 21 and 43 days after the boost vaccination (0DPV, 21DPV and 43 DPV).

[0085] FIGS. 51A-51C: Graphs showing SNT and nasal titers of canines vaccinated with recombinant CAVs expressing CPiVF and / or CPiVHN. Animals were either un vaccinated (Control) or vaccinated orally with PCMN-1557 (CAV-CPiVF) alone, PCMN-1558 (CAV-CPiVHN) alone, PCMN-1557 + PCMN-1558, or PCMN-1556 (CAV-CPiVF-2A-HN) alone, at a dose of IO55. Animals received two doses of vaccine 21 days apart. All animals groups were challenged IN with CPiV (target dose of 1036FAID50) 21 days after the final vaccination. FIG. 51A shows CAV SNT at one day prior to tire first and second vaccinations (-1DPV1, -1DPV2); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); 7 and 14 days after the second vaccination (7DPV2, 14DPV2); one day prior to challenge (-1DPC); and 14 days after challenge (14DPC). FIG. 51B shows CPiV SNT at the same timepoints as FIG. 51 A. FIG. 51C shows CPiV nasal titers one day prior to challenge and 2 to 10 days post-challenge.

[0086] FIGS. 52A-52B: Graphs showing CAV and CDV SNT in canines vaccinated with PCMN-1506 (CAV-CDVF) alone, PCMN-1507 (CAV-CDVH) alone, or the combination of PCMN-1506 and PCMN-1507. Animals received two doses of vaccine 21 days apart at a target dose of 1050FAID5O, followed by IC challenge with CDV (target dose of IO28FA IDs, ) 21 days after the second vaccination. FIG. 52A shows CAV SNT at one day prior to the first and second vaccinations (-1DPV1 , -1DPV2); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); 7 and 14 days after the second vaccination (7DPV2, 14DPV2); one day prior to challenge (-1DPC); and 21 days after challenge (21DPC). FIG. 52B shows CDV SNT at one day prior to the first and second vaccinations (-1DPV1, -1DPV2); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); 7 and 14 days after the second vaccination (7DPV2, 14DPV2); and one day prior to challenge (-1DPC).

[0087] FIGS. 53A-53C: Graphs showing CPiV nasal titer, CPiV SNT and CAV SNT in canines vaccinated with CAV-CPiV-HN. Animals were vaccinated with 1050, ID43or IO36of modified live CAV2-CPiV-HN virus + IO96CFU B. bronchiseptica. Placebo control animals were administered 1096CFU of B. bronchiseptica. Animals received two doses of vaccine 21 days apart, followed by intranasal challenge with CPiV (target dose of 1036FAID50) 21 days after tire final vaccination. FIG. 53A shows CPiV nasal titers one day prior to challenge and 1 to 10 days post-challenge. FIG. 53B shows CPiV SNT7158-105382-02

[0088] one day prior to the first vaccination (-1DPV1); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); one day prior to challenge (-1DPC); and 7 and 14 days after challenge (7PDC, 14DPC). FIG.

[0089] 53C shows CAV SNT at the same timepoints as FIG. 53B.

[0090] SEQUENCES

[0091] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0092] SEQ ID NO 1 is tlie nucleotide sequence of tire PCMN-1507 genome.

[0093] SEQ ID NO 2 is tire nucleotide sequence of tire PCMN-1507 genome plasmid.

[0094] SEQ ID NO 3 is tire nucleotide sequence of tire PCMN-1508 genome.

[0095] SEQ ID NO 4 is the nucleotide sequence of the PCMN-1508 genome plasmid.

[0096] SEQ ID NO 5 is tire nucleotide sequence of tire PCMN-1556 genome.

[0097] SEQ ID NO 6 is the nucleotide sequence of the PCMN-1556 genome plasmid.

[0098] SEQ ID NO 7 is the nucleotide sequence of the PCMN-1557 genome.

[0099] SEQ ID NO 8 is the nucleotide sequence of the PCMN-1557 genome plasmid.

[0100] SEQ ID NO 9 is the nucleotide sequence of the PCMN-1558 genome.

[0101] SEQ ID NO 10 is the nucleotide sequence of the PCMN-1558 genome plasmid.

[0102] SEQ ID NO 11 is the nucleotide sequence of the El-269 CAV El entry plasmid.

[0103] SEQ ID NO 12 is the nucleotide sequence of the Core-059 plasmid.

[0104] SEQ ID NO 13 is the nucleotide sequence of the E3-522 wildtype plasmid.

[0105] SEQ ID NO 14 is the nucleotide sequence of the E3-575 CDVH plasmid.

[0106] SEQ ID NO 15 is the nucleotide sequence of the E3-581 CDVNP plasmid.

[0107] SEQ ID NO 16 is the nucleotide sequence of the E3-595 CPiVF-P2A-CPiVHN plasmid. SEQ ID NO 17 is the nucleotide sequence of the E3-596 CPiVF plasmid.

[0108] SEQ ID NO 18 is the nucleotide sequence of the E3-597 CPiVHN plasmid.

[0109] SEQ ID NO 19 is the nucleotide sequence of the E4-099 plasmid.

[0110] SEQ ID NO 20 is the nucleotide sequence of the ASMM-149 El-Core macromodule plasmid.

[0111] SEQ ID NO 21 is the nucleotide sequence of the ASMM-150 macromodule plasmid.

[0112] SEQ ID NOs: 22-61 are primer sequences.

[0113] SEQ ID NOs: 62-63 are modified E4 region nucleic acid fragments.

[0114] SEQ ID NO: 64 is an amino acid sequence of CDVH.

[0115] SEQ ID NO: 65 is a nucleic acid sequence encoding CDVH.

[0116] SEQ ID NO: 66 is a nucleic acid sequence of the PCMN-1507 E3 region.

[0117] SEQ ID NO: 67 is an amino acid sequence of CDVNP.

[0118] SEQ ID NO: 68 is a nucleic acid sequence encoding CDVNP.7158-105382-02

[0119] SEQ ID NO: 69 is a nucleic acid sequence of the PCMN-1508 E3 region.

[0120] SEQ ID NO: 70 is an amino acid sequence of CPiVF and P2A.

[0121] SEQ ID NO: 71 is a nucleic acid sequence encoding CPiVF and P2A.

[0122] SEQ ID NO: 72 is an amino acid sequence of CPiVHN and the final proline of P2A. SEQ ID NO: 73 is a nucleic acid sequence encoding CPiVHN and the final proline of P2A.

[0123] SEQ ID NO: 74 is the amino acid sequence of a P2A cleavage product.

[0124] SEQ ID NO: 75 is a nucleic acid sequence of the PCMN-1556 E3 region.

[0125] SEQ ID NO: 76 is an amino acid sequence of CPiVF.

[0126] SEQ ID NO: 77 is a nucleic acid sequence encoding CPiVF.

[0127] SEQ ID NO: 78 is a nucleic acid sequence of the PCMN-1557 E3 region.

[0128] SEQ ID NO: 79 is an amino acid sequence of CPiVHN.

[0129] SEQ ID NO: 80 is a nucleic acid sequence encoding CPiVHN.

[0130] SEQ ID NO: 81 is a nucleic acid sequence of the PCMN-1558 E3 region.

[0131] SEQ ID NOs: 82-89 are 2A peptide sequences.

[0132] SEQ ID NO: 90 is a peptide sequence (DGYD).

[0133] SEQ ID NO: 91 is a primer sequence.

[0134] DETAILED DESCRIPTION

[0135] I. Abbreviations

[0136] Ad adenovirus

[0137] CAV canine adenovirus

[0138] CDV canine distemper virus

[0139] CDVF canine distemper virus fusion protein

[0140] CDVH canine distemper virus hemagglutinin protein

[0141] CDVNP canine distemper virus nucleoprotein

[0142] CPE cytopathic effect

[0143] CPiV canine parainfluenza virus

[0144] CPiVF canine parainfluenza virus fusion protein

[0145] CPiVHN canine parainfluenza virus hemagglutinin-neuraminidase protein CPV canine parvovirus

[0146] DPC day(s) post-challenge

[0147] DPV day(s) post-vaccination

[0148] FAID50 50% fluorescent antibody infective dose

[0149] IC intracranial

[0150] ICHV infectious canine hepatitis virus

[0151] IN intranasal

[0152] IV intravenous

[0153] ITR inverted terminal repeat7158-105382-02

[0154] MDCK Madin-Darby canine kidney

[0155] MLV modified live virus

[0156] ORF open reading frame

[0157] OV oncolytic virus

[0158] SLIC sequence- and ligation-independent cloning

[0159] SNT serum neutralization titer

[0160] SSR site-specific recombination

[0161] WPV week(s) post- vaccination

[0162] IL Summary of Terms

[0163] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin 's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both tire singular' as well as plural, unless the context clearly indicates otherwise. For example, the term “a module” includes singular' or plural modules and can be considered equivalent to tire phrase “at least one module.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular' mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, tire present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0164] 2A peptide: A type of self-cleaving peptide encoded by some RNA viruses, such as picomaviruses. 2A peptides function by making the ribosome skip the synthesis of a peptide bond at the C -terminus of a 2A element, leading to separation between the end of the 2A sequence and the downstream peptide (Kim et al., PLoS One 6(4):el 8556, 2011). The "cleavage" occurs between the glycine and proline residues found on the C-terminus of the 2A peptide. Exemplary 2A peptides include, but are not limited to, the 2A peptides encoded by Thosea asigna virus (TaV; T2A), equine rhinitis A virus (ERAV; E2A), porcine teschovirus-1 (PTV1; P2A) and foot and mouth disease virus (FMDV; F2A), having the following sequences:

[0165] P2A: ATNFSLLKQAGDVEENPGP (SEQ ID NO: 82)

[0166] F2A: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 83)

[0167] E2A: QCTNYALLKLAGDVESNPGP (SEQ ID NO: 84)

[0168] T2A: EGRGSLLTCGDVEENPGP (SEQ ID NO: 85)

[0169] In some examples, the 2A peptide is modified to include Gly-Ser-Gly at the N-terminus to improve cleavage efficiency. The sequences of modified P2A, F2A, E2A and T2A are provided below:

[0170] Modified P2A: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 86)7158-105382-02

[0171] Modified F2A: GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 87)

[0172] Modified E2A: GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 88)

[0173] Modified T2A: GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 89)

[0174] Adjuvant: A component of an immunogenic composition used to enhance antigenicity. In some aspects, an adjuvant can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and / or causes influx of macrophages). In some aspects, the adjuvant used in a disclosed immunogenic composition is a combination of lecithin and carbomer homopolymer (such as the ADJUPLEX™ adjuvant available from Advanced BioAdjuvants, LLC; see also Wegmann, Clin Vaccine Immunol 22(9): 1004-1012, 2015). Additional adjuvants for use in the disclosed immunogenic compositions include the QS21 purified plant extract, Matrix M, AS01, MF59, ALFQ, Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, MF59, AS03, aluminum hydroxide, aluminum phosphate, Quil A, AS04, MPLA, CpG ODN 1018, CpG ODN 7909, IC31, Imiquimod (R837), Resiquimod, 3M-052, 2’,3’-cGAMP, 3',3’-cGAMP, c-di-GMP, c-di-AMP, CARBOPOL, CARBOPOL® 934, CARBOPOL® 941 NF, Mn2+, MnJ, MnARK, and / or nanoMn adjuvants.

[0175] Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants also include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL and toll-like receptor (TLR) agonists, such as TLR-9 agonists. Additional examples of adjuvants that can be used are provided in e.g., Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).

[0176] Administration: To provide, prescribe, or give a subject an agent, such as a therapeutic agent (e.g., a recombinant virus), by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, mucosal, intraductal, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.

[0177] Antigenic protein: A protein that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.

[0178] Canine adenovirus (CAV): A type of virus with a double-stranded linear DNA genome belonging to the genus Mastadenovirus, family Adenoviridae. There are two types of canine adenoviruses - CAV-1 and CAV-2, which cause canine hepatitis and infectious tracheobronchitis, respectively, in canines.

[0179] Canine distemper virus (CDV): A single-stranded RNA virus of tire family Paramyxoviridae. CDV is a highly contagious and serious disease that affects the respiratory, gastrointestinal and nervous7158-105382-02

[0180] systems of puppies and dogs. CDV is also found in wildlife, including foxes, wolves, coyotes, raccoons, skunks, mink and ferrets. Infected animals typically develop a watery or pus-like discharge from their eyes, which can be followed by fever, nasal discharge, coughing, lethargy, reduced appetite, and vomiting. If the virus spreads to the nervous system, animals can exhibit circling behavior, head tilt, muscle twitches, convulsions, seizures and / or paralysis. CDV infection can also lead to a thickening or hardening of the footpads. Immunogenic proteins of CDV include, but are not limited to, the CDV fusion protein (CDVF), the CDV hemagglutinin protein (CDVH) and the CDV nucleoprotein (CDVNP).

[0181] Exemplary amino acid sequences for tire CDVH and CDVNP proteins are set forth herein as SEQ ID NO: 64 and SEQ ID NO: 67, respectively.

[0182] Canine parainfluenza virus (CPiV): An enveloped, non-segmented, negative-sense RNA virus of the Paramyxoviridae family. CPiV is a highly contagious virus that causes canine infectious respiratory disease complex (CIRDC). Immunogenic proteins of CPiV include, but are not limited to, tire CPiV fusion protein (CPiVF) and the CPiV hemagglutinin-neuraminidase protein (CPiVHN). Exemplary amino acid sequences for tire CPiVF and CPiVHN proteins are set forth herein as SEQ ID NO: 76 and SEQ ID NO: 79, respectively.

[0183] CAV core genomic module: A portion of the CAV genome that includes tire early / intermediate genes, pIX and IV A2, the major late promoter (MLP) and transcriptional unit that drives the expression of CAV capsid and late proteins, including: 52 / 55k, pllla, penton, pVII, pV, pX, pVI, protease, hexon, 100k, 22k (putative), and 33k (putative) on the positive strand. On the negative strand, the CAV E2 transcriptional unit drives the expression of tire viral DNA replication proteins, including E2A DBP, pTP and E2B DNA polymerase.

[0184] CAV core plasmid module: A nucleic acid molecule corresponding to a portion of a CAV genome that extends 5’ from the early protein IX to the putative L4-33K gene on the positive strand and from the 3’ direction includes the E2 transcriptional unit and E2 encoded proteins E2A, E2B polymerase and terminal protein genes on the negative strand (see FIG. 6). This region of the genome includes the following ORFs: IX, IVa2, DNA pol, pTP, 52K, pllla, penton, pVII, V, pX, pVI, protease, hexon, protease, DNA binding protein (DBP), 100K, 22K (putative) and 33K (putative). Most of the genes in this module are responsible for viral DNA replication and assembly into a protein capsid.

[0185] CAV El genomic module: A portion of the CAV genome that spans from the left-hand internal terminal repeat (ITR) through the E1B-55K gene and the TATA box for pIX. The El A and E1B gene products encoded within this module are essential for viral replication. These genes can be deleted for the assembly and production of recombinant non-replicating CAV vectors.

[0186] CAV El plasmid module: A nucleic acid molecule corresponding to a portion of a CAV genome that includes the left-hand inverted terminal repeat (ITR), and the El A, ElB-19k and ElB-55k ORFs and El A and E1B transcriptional regulatory sequences and units (see FIG. 6). The El genes and promoters may be partially or wholly deleted, mutated, or replaced with heterologous open reading frames (ORFs) to generate replication-incompetent viral vectors for gene delivery, gene therapy and vaccines.7158-105382-02

[0187] CAV E3 genomic module: A portion of the CAV genome that includes the MLP transcribed late protein pVIII, the E3 transcriptional unit and protein coding genes (E3-ORF1 and E3-ORF2), and the fiber gene on the positive strand, and the U exon on the negative strand.

[0188] CAV E3 plasmid module: A nucleic acid molecule corresponding to a portion of a CAV genome that includes pVIII, the E3 promoter and genes, the U-exon, and the fiber gene (see FIG. 6). This region includes the following ORFs: pVIII, E3-ORF1, E3-ORF2, U exon, and Fiber. pVIII is predicted to be transcribed from the major late promoter. An E3 promoter element is predicted to be embedded within the pVIII coding sequence. Also predicted are E3-ORF1 alternative ATG-methionine start codons at the end of pVIII gene in an alternative reading frame. The E3 genes can be partially or completely deleted to expand packaging capacity, regulate host immune responses and / or encode heterologous genes and are not essential for virus replication in most adenoviruses.

[0189] CAV E4 genomic module: A portion of tire CAV genome that includes the right ITR and the E4 transcriptional unit and protein coding genes on the negative strand. The E4 genes have important functions in usurping host cell signaling pathways and function to facilitate viral replication in human Ads and likely canine Ads. In CAV, there are five predicted and annotated ORFs, which include from right to left, E4-ORF1, E4-ORF2, E4-ORF3, E4-ORF4 and E4-ORF5.

[0190] CAV E4 plasmid module: A nucleic acid molecule corresponding to a portion of a CAV genome that includes the right hand ITR, E4-ORF1, E4-ORF2, E4-ORF3, E4-ORF4, and E4-ORF5 (see FIG. 6).

[0191] CAVsembly: A ‘Multisite Gateway’ based genome assembly method. In some aspects of this assembly method, the CAV core module is cloned into a plasmid with ampicillin resistance and made into a Gateway-compatible destination (DEST) plasmid whereby the CAV core genome module is flanked on the left and / or right with multisite Gateway cassettes that have site-specific recombination sequences (att sites) and ccdB counterselection cassettes. The El, E3, and E4 genome modules are cloned into specialized Multisite Gateway compatible entry plasmids that can be combined with, for example, a “dual-DEST” core module in a standard Multisite Gateway LR reaction. The resultant reaction contains fully assembled CAV genomes. Assembled CAV genomes can then be transformed into bacteria and correct assemblies selected by virtue of ampicillin resistance and successful recombination and replacement of ccdB with CAV genome modules. The virus genome can also be transfected into canine cells as only correctly assembled CAV genomes are replication-competent.

[0192] CAVSLIC: An assembly method of CAV genome module plasmids that uses scarless and sequence ligation independent cloning (SLIC) based assembly of El, E3 and E4 modules to create a complete CAV genome in two or three steps. In some aspects of this assembly method, the CAV core genome module plasmid is engineered to have unique restriction sites engineered at the left and right hand sides. The core module is linearized by restriction enzyme digestion and assembled with a PCR linearized E3 and E4 module via SLIC / Gibson, generating a macromodule plasmid. Following transformation and selection of core-E3-E4 macromodule plasmids, the El module is added by7158-105382-02

[0193] linearizing it with restriction enzyme digestion and then adding an El module using PCR and then assembly via Gibson / SLIC. This generates fully assembled viral genomes.

[0194] Degenerate variant: A polynucleotide encoding a peptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0195] Deletion: An adenovirus genome that has a “deletion” of an adenovirus ORF (such as an E3-ORF1 or E3-ORF2 deletion) refers to an adenovirus having a complete deletion of the ORF, or a partial deletion of the ORF that results in the absence of expression of a protein sequence encoded by the ORF. In some aspects herein, E3-ORF1 and / or E3-ORF2 are completely deleted. In other aspects, E3-ORF1 and / or E3-ORF2 are partially deleted, such as 50%, 60%, 70%, 80%, 90%, 95% or 99% deleted.

[0196] Effective amount: A quantity of a specific substance, such as a disclosed recombinant CAV or immunogenic composition, sufficient to achieve a desired effect in a subject being treated, such as a protective immune response. An “effective amount” or a “therapeutically effective amount” can be the amount necessary to inhibit CAV, CDV and / or CPiV replication or heat a CAV, CDV and / or CPiV infection in a subject. An “effective amount” or a “prophylactically effective amount” can be the amount necessary to inhibit or prevent establishment of an infection, such infection by CAV, CDV and / or CPiV. It is understood that to obtain a protective immune response against a virus of interest, multiple administrations of a disclosed recombinant CAV or immunogenic composition thereof can be required, and / or administration of a disclosed immunogenic composition as the “prime” in a prime boost protocol wherein the boost immunogen can be different from the prime immunogenic composition. Accordingly, an effective amount of a disclosed recombinant CAV / immunogenic composition can be tire amount of the recombinant CAV or immunogenic composition thereof sufficient to elicit a priming immune response in a subject that can be subsequently boosted with the same or a different immunogen to elicit a protective immune response.

[0197] In one example, a desired response is to elicit an immune response that inhibits or prevents CDV infection. The CDV infected cells do not need to be completely eliminated or prevented for the recombinant CAV / immunogenic composition to be effective. For example, administration of an effective amount of an immunogen or immunogenic composition can elicit an immune response that decreases the number of CDV infected cells (or prevents the infection of cells) by, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable CDV infected cells), as compared to the number of CDV infected cells in tire absence of the immunization.

[0198] In another example, tire desired response is to elicit an immune response that inhibits or prevents CPiV infection. The CPiV infected cells do not need to be completely eliminated or prevented for the recombinant CAV / immunogenic composition to be effective. For example, administration of an effective amount of an immunogen or immunogenic composition can elicit an immune response that decreases the number of CPiV infected cells (or prevents the infection of cells) by, for example, at least7158-105382-02

[0199] 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable CPiV infected cells), as compared to the number of CPiV infected cells in the absence of the immunization.

[0200] In another example, a desired response is to elicit an immune response that inhibits or prevents CAV infection. The CAV infected cells do not need to be completely eliminated or prevented for the recombinant CAV / immunogenic composition to be effective. For example, administration of an effective amount of an immunogen or immunogenic composition can elicit an immune response that decreases the number of CAV infected cells (or prevents the infection of cells) by, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable CAV infected cells), as compared to tire number of CAV infected cells in tire absence of the immunization.

[0201] Embedded gene promoter: A regulatory DNA sequence capable of initiating transcription that is located within the coding region of another gene. This overlapping genetic architecture enables tire regulation of an alternative transcriptional unit while existing within the sequence of a distinct, independently transcribed gene. Such embedded promoters may be naturally occurring, facilitating the expression of alternative or overlapping hanscripts, and may be exploited or synthetically engineered to enable co-regulation of heterologous genes with applications in gene therapy, vaccines, recombinant protein expression, and viral vector systems.

[0202] Fluorescent antibody infective dose (FAID): A quantitative virological assay used to determine the amount of infectious virus particles required to produce a measurable infection in a defined proportion of target cells, using fluorescently labeled antibodies that detect viral antigens. This method is commonly used for viral titration, vaccine development, and antiviral drug testing.

[0203] Frame-dependent synonymous mutation: A nucleotide substitution occurring within a single open reading frame (ORF) where the mutation is synonymous, preserving the amino acid sequence in that frame, while simultaneously inducing a non-synonymous mutation in an overlapping ORF, resulting in an amino acid substitution or functional modification in the alternate reading frame. Such mutations leverage the degeneracy of the genetic code to engineer precise genetic modifications in overlapping coding regions, allowing for targeted alteration of one protein sequence while maintaining functional integrity of another in dual-coding sequences.

[0204] Fusion protein: A protein containing amino acid sequence from at least two different (heterologous) proteins or peptides. Fusion proteins can be generated, for example, by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, tire nucleic acid sequences must be in die same reading frame and contain no internal stop codons. Fusion proteins, particularly short fusion proteins, can also be generated by chemical synthesis.

[0205] Gateway® cloning: A cloning method based on die site-specific recombination system used by phage to integrate its DNA into bacterial chromosomes. This method relies on specific recombination (“att”) sites, which can be 25-242 base pairs. In this method, a gene of interest is cloned into an “entry7158-105382-02

[0206] vector” using the BP reaction. The gene of interest is then subcloned from the entry vector into a “destination vector” using the LR reaction. The BP clonase enzyme reaction recombines attB sites (flanking the gene of interest) with attP sites (in the donor vector), which generates attL sites. The LR reaction is a recombination reaction between attL and attR sites. See, for example, Reece-Hoyes and Walhout (Cold Spring Harb Proloc. January 2, 2018, pdb.top094912), Katzen (Expert Opin. Drug Discov. 2, 571-589, 2007) and Hartley (Curr Protoc Protein Sci, Chapter 5, unit 5.17, February 2003).

[0207] Gibson assembly: A sequence-independent, seamless DNA assembly method that enables the joining of one or more linear DNA fragments in a single reaction. The process is based on the coordinated use of enzymatic activities, including exonuclease, DNA polymerase, and DNA ligase, to facilitate the precise and scarless assembly of DNA molecules. A 5’ exonuclease digests DNA ends, creating single-stranded complementary overhangs. Overlapping regions hybridize via complementary base pairing. A high-fidelity DNA polymerase extends and synthesizes missing nucleotides. A DNA ligase enzyme covalently seals the assembled DNA fragments into a contiguous molecule. Gibson assembly is described in Gibson el al. (Nat Methods 6:343-345, 2009).

[0208] Heterologous: A protein or polypeptide (or nucleic acid molecule) derived from a different source or species.

[0209] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In some aspects, the response is specific for a particular antigen (an “antigenspecific response”), such as a CAV, CDV or CPiV antigen. In some aspects, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In other aspects, the response is a B cell response, and results in the production of specific antibodies (such as neutralizing antibodies). “Priming an immune response” refers to treatment of a subject with a “prime” immunogen / immunogenic composition to induce an immune response that is subsequently “boosted” with a boost immunogen / immunogenic composition. Together, the prime and boost immunizations produce the desired immune response in the subject.

[0210] Immunize: To render a subject protected from infection by a particular infectious agent, such as CAV, CDV or CPiV. Immunization does not require 100% protection. In some examples, immunization provides at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% protection against infection compared to infection in the absence of immunization.

[0211] Immunogenic composition: A composition that includes an immunogen (antigen) or a nucleic acid molecule or vector encoding an immunogen (such as a CAV, CDV or CPiV antigen), that elicits a measurable CTL response against the immunogen, and / or elicits a measurable B cell response (such as production of antibodies) against the immunogen, when administered to a subject. It further refers to isolated nucleic acids encoding an immunogen, such as a nucleic acid that can be used to express the immunogen (and thus be used to elicit an immune response against this immunogen). For in vivo use, the immunogenic composition can include tire protein or nucleic acid molecule in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant. Similarly, an immunogenic protein or an antigenic protein is a protein that elicits a measurable immune response such as a CTL7158-105382-02

[0212] response and / or an antibody response. In some examples herein, the antigenic protein is a CAV protein (such as a CAV fiber protein), a CDV protein (such as CDVH or CDVNP), or a CPiV protein (such as CPiVF or CPiVHN).

[0213] Isolated: An “isolated” biological component (such as a nucleic acid molecule, protein, virus or cell) has been substantially separated or purified away from other biological components in the cell or tissue of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been “isolated” include those purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.

[0214] Live replication competent virus vaccine: A genetically engineered or attenuated virus that retains the ability to undergo full replication within host cells, in the absence of providing ectopically expressed viral genes, and mimicking the infection dynamics, kinetics and viral particles produced by a wild-type virus while being designed to induce a robust and protective immune response without causing disease. These vaccines enable viral genome replication, transcription, and translation, leading to antigen expression of virally encoded and any genetically engineered heterologous encoded proteins from other pathogens, immune system activation, and durable immunity through both humoral and cellular responses. Replication-competent viral vaccines enhance antigen presentation and elicit stronger immune memory compared to inactivated or subunit vaccines.

[0215] This differs from a non-replicating viral vector, which is genetically modified to lack essential replication genes, thereby providing payload capacity to express heterologous gene expression cassettes that may impair ability to undergo full viral replication analogous to wild type virus in host cells. Nonreplicating vectors require higher doses or booster administrations to achieve sustained immunity, whereas replication-competent vaccines more closely mimic natural infection dynamics, providing enhanced immunogenicity through self-amplifying antigen expression.

[0216] Replication-competent live viral vaccines may be naturally attenuated, recombinant, or codon-modified to balance safety, immunogenicity, and transmission control, making them suitable for mucosal delivery, vector-based vaccine platforms, and infectious disease prevention strategies.

[0217] Macromodule: A nucleic acid molecule that contains at least two CAV genome modules, such as the El module combined with the core module (El-core macromodule), or the El module combined with both the core module and the E4 module (El-core-E4 macromodule).

[0218] Modification: A change in the sequence of a nucleic acid or protein sequence. Amino acid sequence modifications include, for example, substitutions, insertions, deletions, or combinations thereof. Insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Deletions are characterized by the removal of one or more amino acid residues from tire protein sequence (or tire removal of one or more nucleotides / ribonucleotides from a nucleic acid sequence). In some aspects herein, the modification (such as a substitution, insertion or deletion) results in a change in function, such as a reduction or enhancement of a particular activity of a7158-105382-02

[0219] protein. Substitutional modifications are those in which at least one residue has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final mutant sequence. These modifications can be prepared by modification of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the modification. Techniques for making insertion, deletion and substitution mutations at predetermined sites in DNA having a known sequence are well-known. A “modified” protein, nucleic acid or virus is one that has one or more modifications as outlined above.

[0220] Modular assembly: A method or system for constructing a nucleic acid sequence (e.g., a viral genome) from discrete, interchangeable, and pre-defined CAV genomic plasmids (“modules”) that are designed to be functionally and physically compatible with one another. Each module comprises a distinct CAV genomic region — that can be independently modified, inserted, or exchanged, to assemble a recombinant genome without requiring redesign of tire entire genome.

[0221] Module: One of several parts that is made separately but can be joined with other parts to build a structure. In tire context of tire present disclosure, a CAV “genomic module” is a defined portion of the CAV genome (see FIG. 6) and a CAV “plasmid module” is a nucleic acid molecule that corresponds to a portion of a CAV genome that is engineered and designed with defined boundaries and standardized interfaces (e.g., overhangs, recombination sites, homology arms, restriction sites) to enable seamless integration and / or recombination with other modules using a common framework to assemble a whole genome structure. The CAV plasmid modules disclosed herein include the El module, the core module, the E3 module and the E4 module. The joining of all four plasmid modules produces a complete CAV genome.

[0222] Open reading frame (ORF): A span of DNA sequence that begins with a start codon and ends with a stop codon. In the context of the present disclosure, an open reading frame (ORF) sequence refers to a contiguous nucleotide sequence within a viral genome that initiates with a start codon (typically AUG), extends uninterrupted by in-frame stop codons, and encodes a functional viral polypeptide. ORF sequences in viral genomes may be arranged as monocistronic or polycistronic transcripts, with some ORFs overlapping within alternative reading frames, allowing for the compact encoding of multiple viral proteins from a constrained genomic region.

[0223] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in tire same reading frame.

[0224] Overlapping open reading frame: A nucleotide sequence within a genetic element that is encoded in a different reading frame relative to a primary coding sequence and is capable of directing the translation of a distinct polypeptide. Such alternative ORFs may be located within the same nucleic acid7158-105382-02

[0225] strand but shifted by +1 or -1 nucleotides relative to the primary ORF, or may exist as dual-coding sequences where a single genomic region encodes functionally distinct proteins depending on the frame utilized during translation. These overlapping reading frames may be endogenous viral ORFs or may be engineered to allow for co-expression of multiple or individual proteins within a compact complex genetic region. The presence of an alternative ORF can contribute to regulatory complexity, functional diversity, and optimization of genetic constructs for use in therapeutics, synthetic biology, vaccine development, and recombinant expression systems.

[0226] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press (2013), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents (e.g., recombinant viruses).

[0227] In general, the nature of tire carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or tire like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0228] Polyadenylation sequence (poly A): The poly (A) consensus sequence refers to a conserved nucleotide motif found near the 3’ untranslated region (3’ UTR) of mRNAs. This sequence is recognized by the cleavage and polyadenylation machinery, leading to the addition of a poly(A) tail, which is important for mRNA stability, nuclear export, and translation efficiency. Poly(A) consensus motifs include the canonical sequence AAUAAA and the non-canonical sequences AUUAAA (most common alternative), AGUAAA, UAUAAA, AAGAAA, and AACAAA.

[0229] Polypeptide, peptide or protein: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein. These terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

[0230] A conservative substitution in a polypeptide is a substitution of one amino acid residue in a protein sequence for a different amino acid residue having similar biochemical properties. Typically, conservative substitutions have little to no impact on the activity of a resulting polypeptide. For example,7158-105382-02

[0231] a protein or peptide including one or more conservative substitutions (for example no more than 1, 2, 3, 4 or 5 substitutions) retains the structure and function of the wild-type protein or peptide. A polypeptide can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that polypeptide using, for example, standard procedures such as site-directed mutagenesis or PCR. In one example, such variants can be readily selected by testing antibody crossreactivity or its ability to induce an immune response. Examples of conservative substitutions are shown below.

[0232] Original Residue Conservative Substitutions

[0233] Ala Ser

[0234] Arg Lys

[0235] Asn Gin, His

[0236] Asp Glu

[0237] Cys Ser

[0238] Gin Asn

[0239] Glu Asp

[0240] His Asn; Gin

[0241] He Leu, Vai

[0242] Leu He ; Vai

[0243] Lys Arg ; Gin ; Glu

[0244] Met Leu ; He

[0245] Phe Met; Leu; Tyr

[0246] Ser Thr

[0247] Thr Ser

[0248] Tip Tyr

[0249] Tyr Tip ; Phe

[0250] Vai He ; Leu

[0251] Conservative substitutions generally maintain (a) the structure of tire polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) tire charge or hydrophobicity of the molecule at tire target site, or (c) the bulk of the side chain.

[0252] The substitutions that in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, seryl or threonyl, is substituted for (or by) a hydrophobic residue, for example, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.7158-105382-02

[0253] Promoter: A region of DNA that directs / initiates transcription of a nucleic acid (e.g., a gene). A promoter includes necessary nucleic acid sequences near the start site of transcription. Typically, promoters are located near the genes they transcribe. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an “inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor or tetracycline). A “tissue-specific promoter” is a promoter that is only active in a particular tissue or cell (e.g., a musclespecific promoter or a liver- specific promoter).

[0254] Recombinant: When used with reference, e.g., to a cell, virus, nucleic acid, protein, or vector, indicates that tire cell, virus, nucleic acid, protein or vector, has been modified by tire introduction of a heterologous nucleic acid or protein or tire alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of tire cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0255] Scarless cloning: Cloning methods that do not leave recombination sites or other heterologous nucleotides in tire final product. Scarless cloning is also called seamless cloning. Scarless cloning methods include, for example, sequence- and ligation-independent cloning (SLIC) and Gibson assembly.

[0256] Self-cleaving peptides: Peptides that induce the ribosome to skip the synthesis of a peptide bond at the C-terminus, leading to separation of tire peptide sequence and a downstream polypeptide. Virally encoded 2A peptides are a type of self-cleaving peptide. Virally encoded 2A peptides include, for example, 2A peptides from porcine teschovirus-1 (PTV1), foot and mouth disease virus (FMDV), equine rhinitis A virus (ERAV) and Thosea asigna vims (TaV).

[0257] Sequence- and ligation-independent cloning (SLIC): A scarless cloning method that allows for assembly of one or more linear nucleic acid fragments (e.g., assembly of a linearized vector and one or more inserts). The fragments to be joined are linearized via restriction enzyme digestion, PCR or gene synthesis and are treated with T4 polymerase. The 3' to 5' exonuclease activity of T4 polymerase produces 3' overhangs on each DNA fragment, allowing for annealing of complementary DNA sequences. In the case of SLIC assembly of plasmid sequences, plasmids are transformed into bacteria where DNA molecules are repaired and ligated and propagated. SLIC is described in Li and Elledge (2012, SLIC: A Method for Sequence- and Ligation-Independent Cloning. In: Peccoud J. (eds) Gene Synthesis. Methods in Molecular Biology (Methods and Protocols), vol 852. Humana Press).

[0258] Sequence identity: The identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); tire higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high7158-105382-02

[0259] degree of sequence identity / siinilarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species which are more closely related (such as human and mouse sequences), compared to species more distantly related (such as human and C. elegans sequences).

[0260] Methods of alignment of sequences for comparison are well-known. Various programs and alignment algorithms are described in: Smith & Waterman, A dv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988;

[0261] Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0262] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

[0263] Splicing motifs: In the context of tire present disclosure, splicing sequences and regulatory motifs refer to conserved or semi-conserved nucleotide sequences within a nucleic acid molecule that mediate the processing of precursor mRNA (pre-mRNA) into mature mRNA through splicing. These sequences include canonical splice donor, splice acceptor, and branch point sites, which are recognized by the spliceosome to facilitate exon-exon ligation and intron removal.

[0264] A splicing consensus sequence refers to the highly conserved nucleotide pattern found at essential splicing junctions, such as the GT-AG rule for most introns, wherein the 5' splice donor sequence (typically GU in RNA) and the 3' splice acceptor sequence (typically AG in RNA) define exonintron boundaries. Additionally, the branch point sequence (BPS), polypyrimidine tract (PPT), and exon splicing enhancers (ESEs) contribute to splicing efficiency and alternative splicing regulation.

[0265] Degeneracy within splicing sequences refers to the allowance for nucleotide variability at certain positions while maintaining functional recognition by the splicing machinery. This degeneracy enables alternative splicing, exon skipping, and regulatory flexibility while preserving transcript fidelity.

[0266] Regulatory motifs such as splicing silencers (ESS, ISS) and splicing enhancers (ESE, ISE) further modulate exon recognition, affecting gene expression and protein diversity.

[0267] These splicing sequences and regulatory motifs may be engineered or optimized for applications in gene therapy, viral vector design, synthetic biology, and recombinant protein production, enabling precise control over transcript processing, exon inclusion, and alternative splicing events.

[0268] Subject: Living multi-cellular vertebrate organisms, a category that includes human and nonhuman mammals. In some aspects, the subject is a veterinary species, such as a canine. In other aspects, the subject is human. In yet other aspects, the subject is a coyote, fox, wolf, raccoon, skunk, mink or ferret.7158-105382-02

[0269] Synthetic: Produced by artificial means in a laboratory, for example a synthetic nucleic acid or protein can be chemically synthesized in a laboratory.

[0270] TATA Box: A highly conserved cis-regulatory DNA element found in eukaryotic and viral core promoters, typically positioned 25 to 35 nucleotides upstream of the transcription start site (TSS). The TATA box is characterized by the consensus sequence TATAWAWR (where W = A / T and R = A / G) and serves as a binding site for the TATA-binding protein (TBP), a key component of the transcription factor IID (TFIID) complex, which facilitates RNA polymerase II recruitment and transcription initiation. This sequence element plays an important role in gene expression regulation, influencing transcription efficiency, promoter strength, and chromatin accessibility. Variations in the TATA box sequence can modulate transcriptional output, contributing to differential gene expression patterns. The TATA box may be naturally occurring or synthetically engineered to enhance promoter activity, enable tissuespecific expression, or integrate regulatory elements in recombinant DNA constructs, with applications in gene therapy, vaccine development, synthetic biology, and biopharmaceutical production.

[0271] Translational read-through: In the context of the present disclosure, translational read-through of multiple open reading frames (ORFs) refers to a molecular mechanism wherein ribosomal decoding continues beyond a canonical stop codon, enabling translation of downstream overlapping or distinct ORFs within a single mRNA transcript. This read-through process may be naturally occurring or engineered through sequence modifications, including stop codon suppression, RNA structural elements, programmed ribosomal frameshifting, or alternative translational signals. The ability to bypass termination codons and extend translation across multiple ORFs allows for the coordinated expression of functionally linked polypeptides, production of polyproteins, or regulatory modulation of gene expression. Such translational control mechanisms are applicable to viral genome engineering, synthetic biology, recombinant protein expression systems, and gene therapy platforms, where precise modulation of translational termination and read-through can enhance therapeutic efficacy or enable novel genetic architectures.

[0272] Unit dosage form: A physically discrete unit, such as a capsule, tablet, or solution, that is suitable as a unitary dosage for a subject, each unit containing a predetermined quantity of one or more active ingredient(s) calculated to produce a therapeutic or prophylactic effect, in association with at least one pharmaceutically acceptable diluent or carrier, or combination thereof.

[0273] Vaccine: A preparation of immunogenic material capable of stimulating an immune response, administered to a subject for the prevention, amelioration, or treatment of infectious or other types of disease. Vaccines may elicit both prophylactic (preventative or protective) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response.

[0274] Veterinary species: A group of species that typically includes, for example, dogs, cats, hamsters, ferrets, guinea pigs, mice, rats, rabbits, birds, reptiles, horses, cows, sheep, pigs and goats.7158-105382-02

[0275] III. Canine Diseases and Current Vaccination Regimens

[0276] Vaccination plays a crucial role in safeguarding dogs from severe, highly transmissible, and potentially life-threatening diseases. Canine adenoviruses (Type 1 and Type 2 - CAV1 and CAV2), canine parainfluenza virus (CPiV) and the canine distemper virus (CDV) are significant pathogens in dogs, capable of causing fatal diseases and life-threatening symptoms in unvaccinated animals.

[0277] Consequently, the development of effective and improved vaccines that protect animals from adenovirus, parainfluenza and distemper viruses is essential not only for tire individual health of the dog but also for controlling the spread of these diseases within the canine population and potentially zoonotic transmission. Thus, puppies require vaccinations to protect them from CAV1, CAV2, and canine distemper virus (CDV), among other pathogens, at approximately 6 to 8 weeks of age. However, many of these vaccines are associated with significant side effects, necessitate invasive and painful injections, and typically require boosters administered every 2 to 4 weeks until the puppy reaches around 16 weeks of age. This process is costly and invasive, and the method of administration along with tire number of required boosters often results in poor patient compliance, unvaccinated populations of puppies, and, frequently, fatal diseases.

[0278] Furthermore, it is advisable for adult dogs to receive booster vaccinations according to their veterinarian’s recommendations, which may occur annually or every three years, contingent upon the specific vaccine administered. Nonetheless, pre-existing immunity against the initial vector or vehicle can often hinder effective viral antigen expression and reduce booster efficacy. Therefore, there is also a necessity for different types of vaccine vectors and vehicles that can address these challenges.

[0279] Additionally, many major viral pathogens of dogs (including CAV1, CAV2, and CDV) are transmitted to dogs by oral or respiratory routes. Thus, a vaccine that elicits potent mucosal immunity, for example anti-viral IgA immunity, may offer better protection than a vaccine in which the primary immunity is driven by systemic IgG. Licensed vaccines for canine distemper virus, canine parvovirus (CPV), and canine adenoviruses Type 1 and Type 2 are generally administered parenterally, and fail to elicit potent mucosal immunity to combat viruses like C AV 1 , C AV2, or CDV. There are no existing licensed vaccines that are delivered orally (and thus should elicit a mucosal immune response) for CAV 1 , CAV2, or CDV. Furthermore, the use of an oral vaccine obviates the need for painful and invasive needle-based administration. Thus, there is a need for improved vaccines that elicit mucosal immunity and provide vaccine administration conveniences.

[0280] To develop live vaccines that can be administered via multiple routes and that induce protective immunity against life threatening diseases, the present disclosure describes tire engineering and assembly of recombinant modified canine adenovirus genomes that are replication competent and express heterologous CDV or CPiV antigens from the CAV transcriptional architecture.

[0281] A. Canine Adenovirus Disease

[0282] Adenoviridae is a family of dsDNA viruses (~ 26-38 kb) encapsulated within 80-100 nm icosahedron protein capsids that infect vertebrates, including humans, simians, mice, fish, amphibians, reptiles, mammals, and birds.7158-105382-02

[0283] There are two canine adenovirus subtypes, Type 1 (CAV1) and Type 2 (CAV2), with variants arising in each subtype depending on geographical location and genetic differences. Both Type 1 and Type 2 canine adenoviruses are significant pathogens in dogs and cause distinct disease and symptoms.

[0284] CAV1 is the etiological agent responsible for a disease referred to as infectious canine hepatitis (ICH). CAV1 primarily affects the liver, kidneys, and blood vessels, leading to infectious canine hepatitis. It can cause symptoms ranging from mild to severe and can be fatal, especially in young or unvaccinated dogs. Symptoms of ICH can include fever, lethargy, loss of appetite, abdominal pain, vomiting, diarrhea jaundice (yellowing of the skin and eyes), and in severe cases, bleeding disorders. The virus is transmitted through direct contact with infected bodily fluids, such as urine, saliva, and feces, or contaminated objects. Vaccination is necessary to prevent CAV1 infection and ICH.

[0285] CAV2 is the etiological agent responsible for Canine Infectious Respiratory Disease Complex (CIRDC), often informally referred to generically as kennel cough. CAV2 primarily affects tire respiratory system and causes a highly contagious respiratory disease. Symptoms of CAV2 infection include a dry, hacking cough, retching, nasal discharge, fever, and lethargy. While it is generally less severe than CAV1, it can lead to complications such as pneumonia, especially in young or immunocompromised dogs.

[0286] Canine adenovirus pathologies and disease are preventable through vaccination, which is considered a core component of canine vaccination protocols.

[0287] CAV1 recombinant inactivated vaccine does not protect against CAV2. Therefore, CAV2 is often used as the vaccinating agent for both Type 1 and Type 2 canine adenovirus associated diseases as CAV2 induces neutralizing antibodies and cross-protective immunity against CAV1.

[0288] An advantage of CAV2-based vaccines is that in the event of recombination, and depending on administration route, there is also far less risk of adverse effects and fatal disease than with a CAV1-based vaccine, especially in the case of live replication competent vaccines. As such, CAV2-based virus compositions are exemplified herein; however, given the conservation of the genomic placements and deletions of E3 sequences in CAV1, corresponding CAV1 vaccine compositions can also be engineered.

[0289] B. Canine Distemper Virus Disease

[0290] Distemper is a fatal disease in young canines that is caused by infection with canine distemper virus (CDV). CDV is an enveloped RNA morbillivirus (FIG. 15) belonging to the Paramyxoviridae family, which infects a wide array of terrestrial carnivores, with a predominant impact on canines. The genome of CDV consists of 15,690 nucleotides and encodes six structural proteins: the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H), and large protein (L). The H and F viral envelope glycoproteins play crucial roles in the entry of the virus into host cells. Specifically, the H protein facilitates tire binding of tire virus to the cell membrane, while the F protein mediates the fusion of the viral and cell membranes, enabling the entry of tire viral genome into the cytoplasm.

[0291] CDV is a highly contagious pathogen that leads to canine distemper, a condition characterized by the loss of normal temperament. The disease manifests with symptoms including fever, pneumonia,7158-105382-02

[0292] leukopenia, and neurological deficits. Distemper is most commonly observed in dogs 3 to 6 months of age, but can also be found in older dogs, even in vaccinated dog populations (Sykes, Small Animal Critical Care Medicine 2015:504-508).

[0293] In addition to dogs, CDV infection is also a major threat to other members of the Canidae family (such as foxes, wolves, dingoes, and coyotes), the Felidae family (such as lions, tigers, and leopards), the Procyonidae family (e.g., raccoons), the Ursidae family (bears), the Mustelidae family (ferrets and minks), the Hyaenidae family, among others. Significant outbreaks of distemper have also affected marine mammals, including seals. CDV can also cross species barriers and infect non-human primates, such as rhesus monkeys and cynomolgus macaques, which raises emerging concerns regarding the potential zoonotic transmission risk of CDV to humans. As such, effective vaccine strategies are critical to counter the threat of CDV not only in dogs, but cross species infections and pandemics.

[0294] Several strains of CDV have been identified, which can vary in their virulence and geographical distribution. Onderstepoort and Snyder Hill are two of the best characterized clinical isolates and strains of CDV that have been used as tire genomic basis for the development of vaccines against CDV proteins and antigens. Onderstepoort was isolated in South Africa and is associated with milder symptoms compared to Snyder Hill, which was isolated in the United States.

[0295] Some viral protein epitopes are highly conserved across different CDV strains, including Onderstepoort and Snyder Hill, which means that vaccinating dogs against a protein from an individual strain can induce cross protective immunity and neutralizing antibodies that protect animals form different CDV strains and variants. This is advantageous for vaccine design, as it allows for broader protection.

[0296] Commercially available CDV vaccines are offered in various formulations, primarily as modified live virus (MLV) vaccines or recombinant vaccines. A non-inclusive list of commercial vaccines includes Nobivac® Canine 1-DAPPv; Recombitek® C4 (a non-replicating canarypox vector, which minimizes the risk of vaccine-induced disease); Galaxy® D (a modified-live virus vaccine specifically targeting CDV); and Duramune® Adult (a multivalent modified live virus vaccine that provides protection against CDV, canine adenovirus, and parvovirus). The selection of an appropriate vaccine is influenced by various factors, including the animal's age, health status, and the presence of maternal antibodies in puppies.

[0297] Current CDV vaccines are administered parenterally, despite the route of inoculation and infection in naive dogs being oronasal. Mucosal delivery of vaccines has been shown extensively to elicit IgA antibodies. These IgA antibodies protect a subject from pathogen challenge at mucosal surfaces. There are currently no licensed or approved CDV vaccines that are administered orally.

[0298] Additionally, with the exception of canarypox vectored vaccines (for example the Recombitek® C4 product), all licensed CDV vaccines are attenuated, replicating strains of CDV, also known as modified live virus (MLV) vaccines. It is possible that these MLV vaccines, given their ability to replicate in an immunized subject, can revert to a virulent form through mutation. Alternatively, these MLV vaccines can be under attenuated in some subjects. In both cases, these MLV vaccines may cause unintended disease, and in the latter case, several reports exist suggesting that tire Rockborn strain found in many7158-105382-02

[0299] approved vaccines (for example the Duramune line of vaccines) may cause vaccine-induced distemper disease in some subjects (see Anis et al., Vet Microbiol 219:154-160, 2018). Thus, several opportunities to improve on the current CDV vaccine field exist, including vaccines intended to elicit mucosal immunity, and vaccines with a low to non-existent ability to revert to a virulent form, for example by vectoring the protective antigens for CDV using a vector vaccine technology. Indeed, a vectored vaccine that is delivered orally or intranasally is expected to address both of these challenges in the CDV vaccine field.

[0300] C. Canine Parainfluenza Virus

[0301] Canine parainfluenza virus (CPiV), like CDV, is a negative-sense, single stranded RNA virus (FIG. 24). CPiV is a highly contagious respiratory virus that affects dogs. It is one of tire main causes of infectious tracheobronchitis, commonly known as "kennel cough." CPiV primarily targets the respiratory system, leading to symptoms such as coughing, sneezing, nasal discharge, and fever. While it typically results in mild to moderate illness, it can be more severe in puppies, elderly dogs, or those with compromised immune systems. CPiV is usually spread through direct contact with infected dogs, airborne droplets, or contaminated surfaces. Vaccination is available and is often included in combination vaccines to help prevent the spread of tire virus.

[0302] Such combination vaccines include tire following:

[0303] 1. DHPP vaccine: This combination vaccine protects against Distemper, Hepatitis (adenovirus), Parainfluenza, and Parvovirus. The parainfluenza component helps reduce the severity and spread of the virus.

[0304] 2. DA2PP vaccine: Similar to the DHPP vaccine, this vaccine includes protection against Distemper, Adenovirus type 2 (which also covers hepatitis), Parainfluenza, and Parvovirus. 3. Bordetella combination vaccine: This vaccine combines protection against Bordetella bronchiseptica, another major cause of kennel cough, with CPiV and sometimes adenovirus. It can be administered intranasally, orally, or as an injection.

[0305] These vaccines are generally recommended for all dogs, especially those frequently in contact with other dogs, such as in boarding facilities, dog parks, or training classes. The vaccination schedule typically starts in puppies at 6-8 weeks of age, with boosters given every 3-4 weeks until 16 weeks of age, followed by regular annual or triennial boosters.

[0306] Interestingly, currently licensed CPiV vaccines elicit low serum neutralizing antibody responses, and do not completely eliminate the shedding of CPiV virus upon experimental challenge. Thus, several gaps remain amongst currently licensed CPiV or CPiV-containing vaccines, namely the elicitation of potent antibody responses, as well as vaccines that can completely prevent the shed of CPiV infection. A vaccine that elicits potent mucosal immunity is expected to solve both challenges, and as described above for CDV, a vaccine delivered via a mucosal route (for example, either orally or intranasally) and capable of replicating and expressing protective antigens in situ is expected to elicit such desired, potent mucosal immunity.7158-105382-02

[0307] D. Unmet Needs in Canine Core Vaccines

[0308] Typically, puppies are vaccinated at around 6 weeks of age. The main core vaccine includes CAV2, CDV, PPV-2b (canine parvovirus), and sometimes CPiV. Typically, the vaccine is comprised of the combined lyophilized virus(es) and is given subcutaneously (sub-Q) or intramuscularly (IM).

[0309] However, a core ‘live’ multivalent vaccine that could be delivered by the buccal route is an unmet need, that has the potential to overcome passive immunity and maternal antibodies, induce mucosal immunity, have a lower cost and be more readily adapted by veterinarians and patients resulting in far more effective immunity than current regimens.

[0310] Most core multivalent canine vaccines are delivered via injection. This can cause pain at the site of injection, swelling and infection. It can also induce adverse psychological association with the veterinarian in young animals. This can lead to poor adoption by owner and failure to complete vaccine series, resulting in ineffective immunity and local epidemics, or at worse pandemics.

[0311] Furthermore, CDV is not only a pathogen in companion animals but also infects wild animals in the Canidae family (such as, foxes, wolves, dingoes, and coyotes), Felidae (such as lions, tigers, and leopards), Procyonidae (e.g., raccoons), Ursidae (bears), Mustelidae (ferrets and minks), Hyaenidae, among others. Significant outbreaks of distemper have also affected marine mammals, including seals. CDV can also cross species barriers, raising emerging concerns regarding the potential zoonotic transmission risk of CDV to humans. As such, effective vaccine strategies are critical to counter the threat of CDV not only in dogs, but cross species infections and pandemics.

[0312] Oral rabies vaccination has led to the virtual elimination of fox-mediated rabies in Europe. As such, an orally administered and cost-effective vaccine that protects wild animals against CDV and CAVs would provide several advantages over traditional parentally administered vaccine as described in the sections above, which is not practicable.

[0313] There is currently no orally available vaccine for CDV. The existing vaccines for CDV are primarily injectable modified live virus (MLV) vaccines. The oral administration of live modified vaccines or recombinant CDV Canarypox-vectored vaccines does not confer protection against distemper.

[0314] While CPiV is specifically adapted to infecting canines, there is limited evidence indicating it may also have the potential to infect other animal species, particularly those closely related to canids. However, CPiV is not known to cause significant illness in non-canine species.

[0315] The virus is highly contagious among dogs, especially in environments where many dogs are in close contact, such as kennels, shelters, or dog parks. Orally administered CPiV vaccines remain unavailable, and as described elsewhere, an oral route of administration is likely preferential over a parenteral route of administration, as tire former should elicit potent mucosal immunity. Thus, there remains a gap in the field, namely the development of oral CPiV (and CDV) vaccines.

[0316] E. Live Versus Recombinant Vaccines

[0317] Live vaccines stimulate the immune system by mimicking a natural infection triggering a strong and effective immune response without causing the full-blown disease. Live vaccines offer numerous7158-105382-02

[0318] advantages, including more durable and long-lasting immunity with fewer doses in comparison to inactivated, recombinant, RNA or subunit vaccines. The amplification of the virus means that lower doses of inoculum are also needed, which can offset particle toxicity and reduce cost of goods. The replication of the virus also stimulates a broad, robust, and more enduring long-term immune response, thereby diminishing the necessity for booster doses. Furthermore, live vaccines typically induce immunity more rapidly than inactivated vaccines, a crucial factor when addressing the needs of young animals with naive immune systems. These vaccines elicit both humoral (antibody-mediated) and cellular (T-cell mediated) immunity, thereby ensuring comprehensive protection.

[0319] The presence of maternal antibodies is found in puppies consuming milk and colostrum from their mother. Thus, there remains a period early in the life of the puppy in which it is difficult to immunize with a traditional MLV vaccine as the maternal antibodies directed against CDV neutralize the MLV CDV vaccine, preventing the elicitation of CDV immunity. The "critical susceptibility period" is defined as the timeframe during which maternally derived antibodies no longer confer protective immunity but remain capable of interfering with tire immunological response elicited by standard vaccinations. Given that CDV is a fatal disease in young animals, tire provision of protection during this critical stage, which can effectively overcome maternal antibody neutralization, is an urgent challenge that must be addressed.

[0320] Current CDV vaccines use either live attenuated CDV or non-CDV viruses expressing CDV antigens - these cannot be given via the oral / buccal route. A CAV2 virus encoding CDV antigens would function as a 2-in-l vaccine which would reduce cost of goods and could be given via tire oral / buccal route.

[0321] A replication-competent live virus that induces mild symptoms yet possesses the capability to infect and disseminate within wildlife populations, could potentially induce widespread herd immunity and mitigate the risk of zoonotic diseases or epidemics. This would also confer benefits for puppies housed collectively in kennels.

[0322] Another critical unmet need is the development of single agents, vectors, or vaccines that can induce immunity against multiple pathogens. Current regimens primarily consist of combinations of individual agents, which increases costs substantially, raises the risk of recombination, complicates formulation and manufacturing processes, and poses safety concerns, among other issues. For example, current canine core vaccines may include four or more separate MLV fractions, each requiring a separate manufacturing cell line, production process, and set of analytical methods. The cost of vaccines can be a concern for some pet owners, especially if multiple vaccines are needed, and thus a single viral vaccine vector platform, that relies on a common manufacturing process, agnostic to that antigen(s) against which it protects, can reduce tire cost of vaccines.

[0323] F. Replication Competent Adenovirus Vaccines

[0324] Adenoviruses (Ad) are not only pathogens but can be engineered to be life-saving therapies, such as gene therapy, vaccines and oncolytic virus cancer therapies. The understanding of adenovirus biology, and their engineering and therapeutic applications is almost exclusively based upon human subgroup C7158-105382-02

[0325] adenovirus, particularly Adenovirus Type 5 (Ad5) and Adenovirus Type 2 (Ad2). However, human Ad5 does not undergo productive replication and secondary infection in canines, which occludes their development as live replicating adenovirus vaccines in canines. Furthermore, the genomes and capsid proteins of human adenoviruses have significantly diverged from canine adenoviruses, and they do not induce protective immunity against canine adenoviruses.

[0326] In contrast, the development of replication competent canine adenoviruses that express antigens from CDV and CPIV, and undergo robust replication in the natural and desired host, canines, could induce protection not only against C AV 1 / 2 but also CDV and CPi V.

[0327] Live replication competent CAV vaccines would offer significant advantages in inducing robust and long-lasting immunity, particularly through the stimulation of mucosal immunity, which is closely tied to the replication of tire vaccine virus. This capability is especially important for vaccines administered via the oral or intranasal routes, as these pathways are essential for overcoming the limitations posed by maternal antibodies. Maternal antibodies can neutralize vaccine agents, rendering them ineffective during a critical period in a puppy’s early life, leading to susceptibility to severe diseases such as CDV.

[0328] Currently, there is no orally available vaccine for CDV, and existing MLV vaccines have proven ineffective in this regard. However, adenovirus vectors, particularly those that replicate, have shown promise in inducing mucosal immunity and circumventing the challenges posed by maternal antibodies. The replication of these viruses is strongly correlated with their immunogenicity, making live replicating CAV vaccines crucial for effective protection.

[0329] A major challenge in the development of such vaccines lies in the limited packaging capacity of the CAV genome. Typically, the genome can accommodate up to 105% of its original size, beyond which deletions are necessary to create space for additional antigens. The insertion of promoters and other regulatory sequences required for antigen expression can negatively impact virus replication, underscoring the importance of identifying insertion sites that are compatible with virus replication.

[0330] Historically, CAV vaccines have been based on CAV2, as the original CAV1 vaccines, which were inactivated strains, failed to induce cross-protective immunity against CAV2. Furthermore, live CAV1 vaccines were associated with vaccine-related diseases due to the virus’s virulence. Consequently, CAV2 has emerged as the preferred strain for canine adenovirus vaccines, given its ability to induce immunity against both CAV1 and CAV2. However, previous CAV2 vaccines have often been inactivated and non-replicating, which limits their effectiveness.

[0331] Previous attempts at generating a commercial CAV vaccine expressing heterologous antigens, such as those from CDV, have been unsuccessful due to genetic instability (loss of transgenes) and / or inability to generate high titer replication competent viruses. Described herein are novel CAV genome compositions and heterologous ORF placements that overcome these challenges and enable the production of high titer replication competent CAV vaccines that protect canines against one or more pathogens, including, CDV and CPiV, and which can be administered via multiple routes, including oral.7158-105382-02

[0332] The development and engineering of CAV vaccine compositions can address critical unmet needs and has applications for treating both human and animal diseases. The present disclosure provides the assembly, genetic engineering and development of synthetic canine adenovirus genomes and vaccine compositions that can be administered via multiple routes, replicate robustly and induce protective immunity against multiple canine pathogens, such as CAV1 / 2, CDV and CPiV.

[0333] G. Challenges to Engineering Live Multivalent Replication Competent Canine Vaccines

[0334] Engineering Ad vectors requires not only die ability to systematically manipulate and assemble a 32kb viral genome, but also an understanding of adenovirus transcription and functions. The CAV genome is only 31,232 kb compared to human Ad5 at 36 kb. Although the genomic organization and transcriptional units are conserved, there is only 53% identity between die human Ad5 and canine CAV2 reference genome sequences. The E3 genomic region and ORFs encoded therein are the most divergent, with almost no overlap widi respect to die sequence or number of genes encoded. In general, the packaging capacity of the Ad capsid is approximately 105% of the native genome size, which does not provide a great deal of space for encoding heterologous payloads.

[0335] Due to the paucity of tools to study CAV and to engineer diem systematically, the functional roles of the CAV genes are not as well characterized or understood. Early protein differences, and dieir functional role and requirement for productive viral replication are not well established.

[0336] A key requirement to enable the use of CAV as a vaccine vector is the ability to systematically engineer and assay novel CAV vectors, vaccines and oncolytic viruses.

[0337] IV. Engineering and Design Considerations for Assembly of Live Replication Competent Multivalent Canine Adenovirus (CAV) Vaccines that Express CDV or CPiV Antigens and Induce Protective Immunity

[0338] The development and engineering of CAV vaccine compositions address critical unmet needs and has important applications for treating both human and animal diseases. The present disclosure describes the assembly, genetic engineering and development of synthetic canine adenovirus genomes and vaccine compositions that can be administered via multiple routes, replicate robustly and induce protective immunity against multiple canine pathogens, such as CAV1 / 2, CDV and CPiV.

[0339] Engineering and Design considerations for Assembly and Expression of heterologous Antigens in Recombinant Canine Adenovirus Vaccines

[0340] A major challenge in the development of live replication competent CAV vaccines that express heterologous genes is the limited packaging capacity of the CAV genome. For example, the CDV H gene is 1824 bp. As such, to engineer multivalent vaccines that express antigenic proteins, such as CDVH, it is desirable to create the genomic space through deletion of CAV genes, while still maintaining CAV viral replication for a live vaccine composition.7158-105382-02

[0341] The CAV genome is only 31,232 kb compared to human Ad5 at 36 kb. Although the genomic organization and transcriptional units are conserved, there is only 53% identity between the human Ad5 and canine CAV2 reference genome sequences. The annotation of CAV genes in the NCBI database is largely hypothetical, relying on computational predictions rather than experimental validation. The E3 genomic region and predicted ORFs encoded therein are the most divergent, with almost no overlap with respect to the sequence or number of genes encoded (see FIG. 1).

[0342] The CAV E3 genes are not well characterized but it is demonstrated herein that they can be deleted and still maintain virus replication similar to wild type virus kinetics. Previous studies have inserted ectopic promoters in the CAV E3 region to drive the expression of heterologous genes.

[0343] However, it has been previously shown in human Ad5 that although deletions of E3 genes can be tolerated and maintain Ad replication, placing ectopic promoters to express payloads in this genomic location can result in a severe defect in viral replication and / or select for loss of the transgene and payload (WO 2022 / 010949). Placing a strong unregulated ectopic promoter in the E3 genomic region results in defects in late capsid protein expression, which can be attributed to unregulated transcriptional interference with major late promoter encoded L4 and L5 transcripts. These data are also corroborated by Suzuki et al. (Clin Cancer Res 7(1): 120-126, 2001) in a non-replicating Ad5 vector. In HEK293-E4 cells, Suzuki et al. found that an exogenous gene employing the EFla promoter, placed in the E3 region, led to greatly reduced virus particle yield. Given the conserved viral genome organization between Ads across species, placing promoters in the CAV E3 region is also expected to lead to a loss in virus replication and likely select for genome instability and loss of transgene expression.

[0344] As such, disclosed herein is the engineering and expression of the pVIII and E3 genomic regions that are compatible with viral replication and that harness the native CAV viral transcriptional architecture to drive the expression of heterologous antigens while deleting E3 genes that are shown herein to be dispensable for CAV replication.

[0345] FIG. 2 presents a sequence alignment of the pVIII sequence from human adenovirus 5 (Ad5), human adenovirus 34 (Ad34), canine adenovirus 1 (CAV1), and three strains of canine adenovirus 2 (CAV2), specifically the Toronto strain, G1 strain, and SHR2 isolate. The alignment is displayed relative to the Ad5 reference sequence, with the consensus sequence shown on the top line. The alignment reveals sequence divergence between the canine adenovirus strains and the human adenoviruses. However, despite these differences, a conserved TATA box and promoter element are predicted to be preserved in CAV genomes, including both CAV1 and CAV2 isolates. While this promoter activity in CAV1 and CAV2 has not been previously characterized or demonstrated, tire conservation of key regulatory elements strongly suggests its functional relevance. As such, the present disclosure tested and engineered heterologous ORFs in tire E3 region without inserting ectopic promoter or regulatory sequences that might interfere with viral replication but instead harness a putative conserved native viral promoter architecture identified and predicted herein.7158-105382-02

[0346] Genetic modifications, design considerations and engineering of recombinant CAV E3 genomic module placements for live replication competent CAV vaccines and heterologous antigenic protein expression

[0347] The present disclosure provides a novel method for harnessing a predicted embedded promoter (FIG. 2) within the pVIII gene of canine adenoviruses (CAV1 and CAV2) to drive the expression of downstream open reading frames (ORFs), replacing the endogenous viral E3 genes. This approach facilitates the efficient expression of heterologous antigens or therapeutic proteins while maintaining the structural and functional integrity of the viral genome. In addition, harnessing the native CAV transcriptional architecture would overcome the need to also insert sequences for ectopic promoters, poly A elements and other non-coding sequences necessary for tire transcription and / or translation of heterologous antigens, which reduces the genomic payload size, which is limited in CAV genome. Genomic payloads that exceed a critical threshold, approximately 3 kb, can result in defective viruses, which are unable to package tire recombinant genome within the capsid.

[0348] In Ad5, the ATG-methionine for the first E3 ORF, tire 12.5 kilodalton (kDa) protein, occurs immediately after the pVIII stop codon. In contrast, in CAV1 and CAV2, alternative start codons are predicted within an alternative reading frame at the C-terminal region of pVIII (FIG. 3). The C-terminal amino acid sequence of pVIII is conserved across Ad species strains as DGYD (SEQ ID NO: 90), despite underlying nucleotide degeneracy. In an alternative reading frame, this degeneracy in CAVs can result in alternative start codons that could lead to additional amino acid sequences being appended to the N terminal of engineered transgenes introduced in place of the native E3 ORFs after the pVIII stop codon. To overcome these challenges, the disclosed recombinant CAV vaccines include the following modifications:

[0349] 1. Frame-dependent synonymous mutations:

[0350] Frame-dependent T to C synonymous mutations were introduced within the pVIII coding region to preserve the conserved DGYD (SEQ ID NO: 90) amino acid sequence while abolishing potential alternative start codons and methionine residues in the alternative pVIII reading frame (see FIG. 4). Specifically, T-to-C substitutions were made to eliminate potential alternative methionine codons for E3 encoded heterologous ORFs without altering the pVIII protein sequence. These modifications ensure for example, that CDVH protein coding sequences and translation are initiated at the engineered ATG-start codon after the pVIII stop codon. Furthermore, they preserve the pVIII protein coding sequence, which might otherwise result in defective virus replication and low titers. While this represents one aspect of the disclosure, expression of heterologous proteins can still be transcribed and expressed in the absence of these modifications.

[0351] 2. Kozak sequence:

[0352] To optimize translation efficiency of heterologous encoded ORFs, which replace E3 protein coding sequences, an exemplary optimal Kozak sequence motif (FIG. 4) was engineered by inserting a G nucleotide immediately after the pVIII stop codon, at the -1 position relative to, in this example, the CDVH start codon (ATG). This modification created a Kozak consensus sequence, which is predicted to7158-105382-02

[0353] enhance ribosomal recognition and translation efficiency. Notably, a Kozak consensus sequence is absent in Ad5 E3 12.5k, further highlighting its unique design for CAV-based platforms. Alternative designs without the Kozak sequence can also achieve heterologous ORF expression.

[0354] 3. Replacement of endogenous E3-ORF1 protein coding sequences:

[0355] The endogenous E3-ORF1 and E3-ORF2 protein coding sequences were deleted and replaced with, for example, the CDVH coding sequence, leveraging the predicted native viral transcriptional architecture to drive heterologous antigen protein expression. This ensures that the heterologous antigen is expressed in a manner consistent with the virus’s natural transcriptional architecture.

[0356] The prediction and exploitation of a predicted TATA box and embedded promoter element within the pVIII gene, or alternative native promoter in CAV genome, provide an efficient mechanism to express heterologous antigens inserted in place of E3-ORF1 and E3-ORF2, and minimize tire size of the genomic payload to be inserted to the protein coding sequences. This innovation enables the generation of replication-competent CAV-based vaccines and therapeutics, expressing heterologous antigens proteins.

[0357] This discovery has important implications for the design of recombinant CAV-based vaccines. The prediction and exploitation of a native, conserved promoter enables the efficient expression of heterologous antigens from the E3 region without disrupting essential transcriptional regulatory signals. Accordingly, the engineering of CAV-based vaccine platforms can leverage this endogenous promoter activity to drive the expression of therapeutic antigens, such as those for canine distemper virus (CDV) and canine parainfluenza virus (CPiV), within the E3 genomic context.

[0358] Thus, tire present disclosure provides a robust exemplary design for engineering considerations for the development of replication competent multivalent adenoviral vaccines, leveraging the natural viral architecture while optimizing heterologous gene expression for therapeutic and prophylactic applications.

[0359] Engineering of Heterologous Antigen Expression and the E3-ORF1, E3-ORF2 and U exon noncoding sequence junctions for development of Replication-Competent multivalent C V-Based Vaccines

[0360] The present disclosure relates to the design and engineering of live, replication-competent canine adenovirus (CAV)-based multivalent vaccines, leveraging the E3 genomic region as a platform for heterologous antigen expression. This approach involves the replacement of endogenous E3 open reading frames (ORFs) with heterologous antigenic proteins, while preserving key non-coding sequences important for viral replication and transgene expression.

[0361] Replacement of E3-ORF1 and additional deletions of E3-ORF2 for increased cargo capacity

[0362] As illustrated in FIG. 5, and by way of an example, the PCMN-1507 CAV2-CDVH synthetic engineered viral genome was constructed by replacing the endogenous E3-ORF1 protein coding sequences with the CDVH heterologous gene at the engineered pVIII / E3 junction, as described above.7158-105382-02

[0363] This replacement ablates the E3-ORF1 region while deleting the majority of the E3-ORF2 coding sequence, thereby creating additional cargo capacity for the insertion of immunogenic CDV and CPiV dual protein P2A fusions, without compromising viral replication or genome packaging.

[0364] In some aspects, while the coding sequences of E3-ORF2 were largely deleted, non-coding sequences at the junction between the end of E3-ORF1 and start of the E3-ORF2 coding sequence were retained as they may have regulatory motifs that function in transgene expression and viral replication. These sequences include:

[0365] 1. Polyadenylation (Poly- A) signals:

[0366] Conserved poly-A motif sequences between the endogenous E3-ORF1 and E3-ORF2 and another that is after the stop codon of E3-ORF2 and the U Exon.

[0367] 2. Splicing motifs:

[0368] Predicted splicing motif sequences between E3-ORF1 and E3-ORF2 and after the stop codon of E3-ORF2 and the U Exon on the negative strand.

[0369] While the PCMN-1507 CDVH sequence composition represents one aspect, alternative designs can be employed. These include:

[0370] 1. Different poly-A and splicing motifs:

[0371] Other consensus sequences for polyadenylation and splicing may be substituted to enhance transgene expression without affecting viral replication.

[0372] 2. Further deletion of non-coding sequences:

[0373] In some aspects, the E3-ORF 1 and E3-ORF2 junction sequences are completely or partially removed.

[0374] The present disclosure leverages the endogenous CAV architecture to drive tire expression of heterologous antigens while ensuring efficient viral replication and transgene expression. By replacing endogenous E3 coding sequences with heterologous immunogenic proteins while preserving key noncoding motifs, this platform enables the development of live, replication-competent CAV-based vaccines expressing protective antigens, such as CDVH, in a genetically stable and biologically active form.

[0375] CAV Assembly and CAVSLIC System for Modular Assembly of replication competent CAV genomes that express heterologous antigenic proteins

[0376] A key requirement to enable the use of CAV as a vaccine is the ability to systematically engineer CAV genomes to incorporate and test the design considerations contemplated above. However, there is a paucity of reagents to do so relative to hAd5. Thus, the present disclosure provides an exemplary platform to engineer and assemble novel replication competent CAV vaccines that express multiple pay loads and / or reporters.

[0377] The present disclosure describes systems for the modular assembly of canine adenovirus (CAV) genomes, facilitating the construction of synthetic, replication-competent CAV vaccine strains. These systems, referred to as CAV assembly (“CAVsembly”) and CAVSLIC (sequence- and ligation-7158-105382-02

[0378] independent cloning), enable the precise and efficient assembly of full-length CAV genomes from plasmid-based genomic modules through scarless assembly techniques.

[0379] Modular CAV Genome Organization

[0380] To facilitate genome manipulation and synthetic vaccine design, the CAV2 genome, which is 31,232 base pairs in length and encodes approximately 32 known genes, was divided into four distinct functional and transcriptional genome modules (FIG. 6). These modules were cloned into individual plasmids to generate corresponding CAV plasmid modules, each representing a specific CAV genomic region. The modular design reflects the natural genomic organization of Ads across species, despite sequence-level differences (FIGS. 1 and 6). Assembly of the CAV2 genome is exemplified herein, but the same methods and systems can be applied to both CAV1 and other CAV strains, which have a conserved viral genome module architecture and organization (FIG. 1).

[0381] The four CAV plasmid modules include:

[0382] 1. El plasmid module:

[0383] This module includes the CAV2 genomic region extending from the left-hand internal terminal repeat (ITR) through tire E1B-55K gene and the TATA box for pIX. The E1A and E1B gene products encoded within this module are essential for viral replication, playing critical roles in transcriptional activation and cell cycle modulation following viral infection.

[0384] 2. Core plasmid module:

[0385] This module encompasses the core genomic region, spanning from the pIX gene through the putative late 33K gene. It includes the capsid proteins, such as hexon, penton base, and fiber, which are essential for the formation of the viral capsid that encapsulates the viral genome. This module also contains genes encoding DNA replication machinery, ensuring efficient genome replication and viral assembly.

[0386] 3. E3 plasmid module:

[0387] This module spans the region from the late pVIII gene through to the fiber gene, encompassing E3-ORF1 , E3-ORF2 and the U exon. In the present disclosure, the E3 module serves as a platform for the expression of heterologous antigens, replacing endogenous E3-ORF1 and E3-ORF2 sequences with therapeutic transgenes.

[0388] 4. E4 plasmid module:

[0389] This module includes the E4-ORF5 gene through the right-hand ITR.

[0390] CAVSLIC System and Scarless Assembly

[0391] The CAVSLIC system utilizes scarless assembly strategies, such as Gibson assembly, In-Fusion cloning or SLIC, for modular genome assembly. However, assembly is not limited to these methods; restriction enzyme digestion and ligation dependent cloning could also be used, but would not be sequence independent.7158-105382-02

[0392] Assembly generally begins by linearizing the destination core module plasmid through restriction enzyme digestion, such as with PacI or Bglll (FIG. 8). Linearization via Pad or Bglll enables serial assembly of El or E3 / E4 fragments at the 5’ or 3’ ends, respectively.

[0393] The El, E3, and E4 plasmid modules are then linearized with either restriction enzymes or PCR. Single strand overhangs are generated upon treatment of core and E1 / E3 / E4 PCR fragments with an exonuclease, which are then assembled through hybridization of complementary sequences encoded within genome module plasmids or PCR primers. In SLIC and In Fusion, DNA repair and ligation occurs upon transformation of plasmid sequences into bacteria. In Gibson assembly, coordinated activities of an exonuclease, DNA polymerase and DNA mix perform the reaction in a single tube, one step reaction.

[0394] CAVSLIC of El, E3 and E4 modules with the core module can be performed in any order, and in two, three or four steps, which is dictated by combinatorial complexity of engineering goals and modifications as well as efficiency. The assembly reaction with larger nucleic acid molecules is lower when using more than three fragments. Thus, two- or three-fragment SLIC / Gibson assemblies are used herein. As such, it can be desirable to assemble an El and core module first into an El-core macromodule intermediary plasmid, which is subsequently assembled with E3 and / or E4 modules in another Gibson / SLIC reaction. The generic seamless assembly process depicted can be Gibson assembly, SLIC, In-Fusion, or restriction enzyme and ligation cloning. In FIG. 9, a PacI digested Core plasmid is assembled with PCR linearized El modules, which are Gibson / SLIC assembled, transformed into bacteria, selected and purified. The El -Core macromodule plasmid is then digested with Bglll and assembled in a 3-fragment Gibson / SLIC assembly reaction with PCR-linearized E3 and E4 genomic modules. In FIG. 9, another assembly process example is shown, whereby an El / core macromodule plasmid is first created then assembled with an E4 module in a second Gibson reaction to generate an El-core-4 macromolecule plasmid, which is then Bglll digested and assembled with an E3 module in a third Gibson reaction.

[0395] This stepwise approach is advantageous for iterative genome modification and the generation of diverse vaccine candidates.

[0396] Applications and Advantages

[0397] The CAVsembly and CAVSLIC systems enable the efficient assembly of recombinant CAV genomes, facilitating the engineering of vaccines. The modular approach offers several advantages:

[0398] 1. Genomic Integrity and Stability:

[0399] Scarless assembly ensures that the native genome architecture remains intact, minimizing the risk of recombination events and genomic instability.

[0400] 2. Customizable Vaccine Platforms:

[0401] By using modular assembly, one can easily swap transgenes, optimize expression cassettes, or introduce therapeutic payloads, creating tailored vaccine platforms for different indications.

[0402] 3. Rapid Iterative Development:7158-105382-02

[0403] The ability to assemble intermediate macromodules facilitates the rapid testing of vaccine candidates, enabling the identification of optimal designs before final genome assembly.

[0404] The CAV Assembly and CAVSLIC systems described herein provide an efficient, flexible, and scalable platform for the assembly of synthetic CAV genomes, enabling the development of live, replication-competent CAV-based vaccines. By dividing the genome into functional plasmid modules, this system allows for the precise engineering of viral strains, ensuring efficient transgene expression, genome stability, and high-yield viral production.

[0405] Previous attempts at generating a commercial CAV vaccine expressing heterologous antigens, such as those from CDV, have been unsuccessful due to genetic instability (loss of transgenes) and / or inability to generate high titer replication competent viruses. Described herein are novel CAV genome compositions and heterologous ORF placements that use the viral transcriptional architecture and deletions of CAV genes to overcome these challenges and enable the production of high titer replication competent CAV vaccines drat protect canines against one or more pathogens, including CDV and CPiV, and which can be administered via multiple routes, including oral.

[0406] Currently, there is no orally available vaccine for CDV, and existing MLV vaccines have proven ineffective in this regard. However, adenovirus vectors, particularly those that replicate, have shown promise in inducing mucosal immunity and circumventing the challenges posed by maternal antibodies. The replication of these viruses is strongly correlated with tlreir immunogenicity, making live replicating CAV vaccines crucial for effective protection.

[0407] Live replication competent CAV vaccines offer significant advantages in inducing robust and long-lasting immunity, particularly through the stimulation of mucosal immunity, which is closely tied to the replication of the vaccine virus. This capability is especially important for vaccines administered via the oral or intranasal routes, as these pathways are essential for overcoming the limitations posed by maternal antibodies. Maternal antibodies can neutralize vaccine agents, rendering them ineffective during a critical period in a puppy’s early life, leading to susceptibility to severe diseases such as CDV.

[0408] V. Recombinant Canine Adenovirus Vaccine Compositions

[0409] Described herein are recombinant CAVs that encode one or more (such as one, two or three) heterologous antigenic proteins, such as antigenic proteins from one or more viruses that cause disease in canines or other veterinary species, and immunogenic compositions thereof. The recombinant CAVs and immunogenic compositions can be used, for example, to immunize canines against viral diseases.

[0410] Provided herein are recombinant, replication-competent CAVs having a genome that includes a complete or partial deletion of an E3 region, and a first heterologous open reading frame (ORF) encoding a first antigenic protein inserted at the site of the partial or completion deletion of die E3 region. In some aspects, the first antigenic protein is from a pathogen that infects a veterinary species.

[0411] In some aspects, the complete or partial deletion of the E3 region includes a partial deletion of an E3-ORF1 coding sequence. In some examples, die partial deletion of the E3-ORF1 coding sequence excludes a 5’ region of the E3-ORF1 diat overlaps with a 3’ region of the pVIII ORF. In particular7158-105382-02

[0412] examples, the 5’ region of the E3-ORF1 that overlaps with the 3’ region of the pVIII ORF includes one or more nucleotide substitutions, wherein the one or more nucleotide substitutions eliminate one or two alternative start codons, and wherein the one or more nucleotide substitutions do not result in any amino acid substitutions in the pVIII protein.

[0413] In some aspects, the first heterologous ORF includes a start codon and / or a Kozak consensus sequence.

[0414] In some aspects, the complete or partial deletion of the E3 region further includes a complete or partial deletion of an E3-ORF2 coding sequence.

[0415] In some aspects, the E3 region includes a U exon ORF coding sequence and a splice junction for the fiber ORF.

[0416] In some aspects, the E3 region of the recombinant CAV genome does not include a heterologous promoter.

[0417] In some aspects, first antigenic protein is a protein from canine distemper virus (CDV). In some examples, tire fust antigenic protein is the CDV hemagglutinin protein (CDVH) or the CDV nucleoprotein (CDVNP). In particular examples, the amino acid sequence of CDVH is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 64; and / or the amino acid sequence of CDVNP is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 67.

[0418] In some aspects in which the first antigenic protein is a CDV protein, tire genome of the recombinant CAV includes a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 3. In some examples, the nucleotide sequence of the genome of the recombinant CAV consists or consists essentially of SEQ ID NO: 1 or SEQ ID NO: 3.

[0419] In other aspects, the first antigenic protein is a protein from the canine parainfluenza virus (CPiV). In some examples, the first antigenic protein is the CPiV fusion protein (CPiVF) or the CPiV hemagglutinin-neuraminidase protein (CPiVHN). In particular examples, the amino acid sequence of CPiVF is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 76; and / or the amino acid sequence of CPiVHN is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 79.

[0420] In some aspects in which the first antigenic protein is a CPiV protein, the genome of the recombinant CAV includes a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 7 or SEQ ID NO: 9. In some examples, tire nucleotide sequence of the genome of the recombinant CAV consists or consists essentially of SEQ ID NO: 7 or SEQ ID NO: 9.7158-105382-02

[0421] In some aspects, the genome of the recombinant CAV further includes a second heterologous ORF encoding a second antigenic protein inserted at the site of the complete or partial deletion of the E3 region, wherein the second antigenic protein is a protein from a pathogen that infects canines.

[0422] In some examples, the first antigenic protein and the second antigenic protein are both CPiV proteins. In specific examples, the first antigenic protein and the second antigenic protein are CPiVF and CPiVHN. In particular examples, the amino acid sequence of CPiVF is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 76; and / or the amino acid sequence of CPiVHN is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 79.

[0423] In some examples, the first heterologous ORF and the second heterologous ORF are operably linked by a self-cleaving peptide coding sequence. In specific examples, the self-cleaving peptide is a 2A peptide, such as P2A, T2A, F2A or E2A peptide, or a modified version thereof. In particular instances, the amino acid sequence of the 2A peptide (or variant thereof) includes or consists of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88 or SEQ ID NO: 89.

[0424] In some aspects, tire genome of tire recombinant CAV includes a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5. In some examples, the nucleotide sequence of the genome of the recombinant CAV consists or consists essentially of SEQ ID NO: 5.

[0425] Also provided herein are immunogenic compositions that include one or more of the recombinant CAVs disclosed herein. In some aspects, the immunogenic compositions further include a pharmaceutically acceptable carrier, an adjuvant, or both. In some examples, the immunogenic composition is formulated for oral and / or mucosal administration.

[0426] In some aspects, the immunogenic composition includes at least two, at least three, at least four, or at least five different recombinant CAVs disclosed herein. In some examples, the immunogenic composition includes a first recombinant CAV encoding a CDVH protein and a second recombinant CAV encoding a CDVNP ; a first recombinant CAV encoding a CPiVF protein and a second recombinant CAV encoding a CPiVHN protein; a first recombinant CAV encoding a CDVH protein, a second recombinant CAV encoding a CDVNP, a third recombinant CAV encoding a CPiVF protein, and a fourth recombinant CAV encoding a CPiVHN protein; or a first recombinant CAV encoding a CDVH protein, a second recombinant CAV encoding a CDVNP, and a third recombinant CAV encoding a CPiVF protein and a CPiV HN protein.

[0427] In some aspects, the immunogenic composition includes one or more additional canine vaccines. In specific examples, the immunogenic composition includes a canine parvovirus vaccine (such as a modified live virus CPV vaccine), a rabies virus vaccine, an influenza virus vaccine, a canine coronavirus vaccine, a Leptospirosis vaccine and / or a Bordetella bronchiseptica vaccine.

[0428] In some aspects, the immunogenic composition includes a 50% fluorescent antibody infective dose (FAID50) of about 103to about 107of each recombinant CAV. In some examples, tire immunogenic7158-105382-02

[0429] composition includes about 103, about 104, about 105, about 106, or about 107FAID50 of each recombinant CAV. In specific examples, the immunogenic composition includes about 103to about 106, about 103to about 105, about 104to about 105FAID50 of each recombinant CAV.

[0430] Further provided are methods of eliciting an immune response in a subject by administering to the subject an effective amount of a recombinant CAV or immunogenic composition disclosed herein. In some aspects, the subject is a canine subject. In some aspects, the effective amount of the recombinant CAV is about 103to about 107FAID50. In some examples, the effective dose is about 103, about 104, about 105, about 106, or about 107FAID50. hi specific examples, the effective dose is about 103to about 106, about 103to about 105, about 104to about 105FAID50. In some aspects, the immune response includes the production of neutralizing antibodies against CAV, CDV, CPiV, or any combination thereof. In some examples, the recombinant CAV or the immunogenic composition is administered orally and / or mucosally. In some examples, the recombinant CAV or the immunogenic composition is administered in a single dose. In other examples, the recombinant CAV or the immunogenic composition is administered in multiple doses.

[0431] VI. Immunogenic Compositions

[0432] Immunogenic compositions that include one or more recombinant CAVs disclosed herein, and a pharmaceutically acceptable carrier are provided herein. Such compositions can be administered to subjects (such as a canine subject) by a variety of administration modes, for example, oral, intranasal, onto the tonsils, inhalation, intravenous, intramuscular, subcutaneous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 22ndEdition, Loyd V. Allen el al., editors, Pharmaceutical Press (2012). Pharmaceutically acceptable carriers include materials that are not biologically or otherwise undesirable, e.g., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the immunogenic composition in which it is contained. If administered to a subject, the carrier is optionally selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.

[0433] A recombinant CAV described herein can be formulated with pharmaceutically acceptable carriers to help retain biological activity while also promoting increased stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides, or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. In one aspect, the pharmaceutically acceptable carrier is phosphate buffer saline solution. In one aspect, the pharmaceutically acceptable carrier is water. Aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations can be combined with a sterile solution prior to administration for either single or multiple dosing.7158-105382-02

[0434] Formulations suitable for oral administration can include (a) liquid solutions, such as an effective amount of the recombinant CAV suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets (including orally dissolving tables, also known as ODT), each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, com starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising tire active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and tire like containing, in addition to the active ingredient, carriers known in the art.

[0435] The recombinant CAV (or multiple recombinant CAV), alone or in combination with other suitable components, can be made into aerosol formulations (e.g., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and tire like.

[0436] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection soludons, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the provided methods, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0437] Formulated compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually <1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

[0438] The immunogenic compositions of tire disclosure can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and tire like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0439] The immunogenic composition may further include an adjuvant to enhance an immune response of tire host. Exemplary adjuvants include, for example, a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; water-in-oil emulsions, for example, in which7158-105382-02

[0440] antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity; AIPO4, alhydrogel; Lipid-A or derivatives or variants thereof; oil-emulsions; saponins; neutral liposomes; and liposomes containing cytokines, non-ionic block copolymers, and / or chemokines. In some aspects, the adjuvant includes a combination of lecithin and carbomer homopolymer (such as the ADJUPLEX™ adjuvant available from Advanced BioAdjuvants, LLC; see also Wegmann, Clin Vaccine Immunol 22(9): 1004-1012, 2015). Additional adjuvants for use in the disclosed immunogenic compositions include the QS21 purified plant extract, Matrix M, AS01, MF59, and ALFQ adjuvants. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants also include biological molecules, such as costimulatory molecules, for example adjuvants that include IL-2, RANTES, GM-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL or toll-like receptor (TLR) agonists, such as TLR-9 agonists. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), can also be used as an adjuvant (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product (such as a recombinant CAV). In some aspects, an adjuvant is not required and is thus not administered with the immunogenic composition.

[0441] In some aspects, the composition is a sterile composition. The immunogenic composition typically contains an effective amount of a disclosed immunogen (e.g., recombinant CAV) and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected as an amount which elicits an immune response without significant, adverse side effects. In some examples, the dose of recombinant CAV is about 1 x 103to about 1 x 107infectious particles, such as about 1 x 103, about 2.5 x 103, about 5 x 103, about 1 x 104, about 2.5 x 104, about 5 x 104, about 1 x 105, about 2.5 x 105, about 5 x 105, about 1 x 106, about 2.5 x 106, about 5 x 107or about 1 x 107infectious particles.

[0442] In some aspects, the composition is provided in unit dosage form for use to elicit an immune response in a subject, for example, to prevent CAV, CDV and / or CPiV infection in the subject, such as a canine subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and / or a metering mechanism for administering the unit dose or multiples thereof. In some examples, the unit dosage is about 1 x 103to about 1 x 107infectious particles, such as about 1 x 103, about 2.5 x 103, about 5 x 103, about 1 x 104, about 2.5 x 104, about 5 x 104, about 1 x 105, about 2.5 x 105, about 5 x 105, about 1 x 106, about 2.5 x 106, about 5 x 107or about 1 x 107infectious particles.

[0443] In some aspects, the immunogenic compositions include at least two, at least three or at least four different recombinant CAVs. In some examples, the immunogenic composition includes a first recombinant CAV encoding a CDVH protein and a second recombinant CAV encoding a CDVNP ; a first recombinant CAV encoding a CPiVF protein and a second recombinant CAV encoding a CPiVHN7158-105382-02

[0444] protein; a first recombinant CAV encoding a CDVH protein, a second recombinant CAV encoding a CDVNP, a third recombinant CAV encoding a CPiVF protein, and a fourth recombinant CAV encoding a CPiVHN protein; or a first recombinant CAV encoding a CDVH protein, a second recombinant CAV encoding a CDVNP, and a third recombinant CAV encoding a CPiVF protein and a CPiV HN protein In some aspects, the immunogenic composition further includes one or more additional canine vaccines. In specific examples, the immunogenic composition includes a canine parvovirus vaccine, a rabies virus vaccine, an influenza virus vaccine, a canine coronavirus vaccine, a Leptospirosis vaccine and / or a Bordetella bronchiseptica vaccine

[0445] VII. Methods of Eliciting an Immune Response

[0446] The disclosed recombinant CAVs and immunogenic compositions including one or more recombinant CAVs, can be used in methods of inducing an immune response to CAV, CDV and / or CPiV to prevent, inhibit (including inhibiting transmission), and / or treat a CAV, CDV and / or CPiV infection. In some aspects, tire immune response includes the production of neutralizing antibodies.

[0447] Provided herein are methods of eliciting an immune response (such as an immune response against CAV, CDV and / or CPiV) in a subject. In some aspects, the method includes administering to the subject an effective amount of a recombinant CAV (expressing CDV and / or CPiV antigens) or an immunogenic composition disclosed herein. In some examples, the recombinant CAV or immunogenic composition is administered orally (such as by using enteric-coated tablets), intravenously, intranasally and / or intraperitoneally. In some aspects, the subject is a canine subject.

[0448] When inhibiting, treating, or preventing CAV, CDV and / or CPiV infection, the methods can be used either to avoid infection in a CAV, CDV and / or CPiV seronegative subject (e.g., by inducing an immune response that protects against CAV, CDV and / or CPiV infection), or to treat an existing infection in a CAV, CDV and / or CPiV seropositive subject.

[0449] To identify subjects for prophylaxis or treatment according to the methods of the disclosure, accepted screening methods can be employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods to detect and / or characterize CAV, CDV and / or CPiV infection. These and other methods allow for selection of a subject in need of prophylaxis or treatment using the methods and immunogenic compositions of the disclosure. In accordance with these methods and principles, a composition can be administered according to the teachings herein, or other conventional methods, as an independent prophylaxis or treatment program, or as a follow-up, adjunct or coordinate treatment regimen to other treatments.

[0450] The disclosed recombinant CAVs and immunogenic compositions thereof can be used in coordinate (or prime-boost) immunization protocols or combinatorial formulations. In certain aspects, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate7158-105382-02

[0451] immunogens or formulations, each directed toward eliciting an anti-CAV, anti-CDV and / or anti-CPiV immune response, such as an immune response to CAV fiber, CDVF, CDVH, CDVNP, CPiVF and / or CPiVHN. Separate immunogenic compositions that elicit the anti-CAV, anti-CDV and / or anti-CPiV immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate immunization protocol. In some aspects, the immunogenic composition further includes one or more of a canine parvovirus vaccine, a rabies virus vaccine, an influenza virus vaccine, a canine coronavirus vaccine, a Leptospirosis vaccine and / or a Bordetella bronchiseptica vaccine.

[0452] In one aspect, a suitable immunization regimen includes at least two separate inoculations with one or more immunogenic compositions with a second inoculation being administered more than about two, about three to eight, or about four weeks following tire first inoculation. A third inoculation can be administered several months after the second inoculation, and in specific aspects, more than about five months after tire first inoculation, more than about six months to about two years after tire first inoculation, or about eight months to about one year- after tire first inoculation. Periodic inoculations beyond the third may also be used to enhance tire subject's “immune memory.” The adequacy of tire vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, the T cell populations can be monitored by conventional methods. In addition, the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of CAV, CDV and / or CPiV infection, improvement in disease state (e.g., reduction in viral load), or reduction in transmission frequency. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, a dose of a disclosed recombinant CAV can be increased or the route of administration can be changed.

[0453] In one non-limiting example, a boost dose is administered about 28 days after the prime dose. It is contemplated that there can be several boosts, and that each boost can be a different immunogen. It is also contemplated in some examples that the boost may be the same immunogen as another boost, or the prime.

[0454] The prime and the boost can be administered as a single dose or multiple doses, for example, two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks, or months. Multiple boosts can also be given, such one to five, or more. Different dosages can be used in a series of sequential inoculations. For example, a relatively large dose in a primary inoculation and then a boost with relatively smaller doses. The immune response against the selected antigenic surface can be elicited by one or more inoculations of a subject.

[0455] In several aspects, a disclosed immunogen can be administered to the subject simultaneously with tire administration of an adjuvant. In other aspects, the immunogen can be administered to tire7158-105382-02

[0456] subject after the administration of an adjuvant and within a sufficient amount of time to elicit the immune response. In other aspects, no adjuvant is administered.

[0457] CAV, CDV and / or CPiV infection does not need to be completely inhibited for the methods to be effective. For example, elicitation of an immune response to CAV, CDV and / or CPiV can reduce or inhibit CAV, CDV and / or CPiV infection by, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable CAV, CDV and / or CPiV infected cells), as compared to CAV, CDV and / or CPiV infection in the absence of immunization. In additional examples, CAV, CDV and / or CPiV replication can be reduced or inhibited by the disclosed methods. CAV, CDV and / or CPiV replication does not need to be completely eliminated for tire method to be effective. For example, tire immune response elicited using one or more of the disclosed recombinant CAV can reduce CAV, CDV and / or CPiV replication by, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable CAV, CDV and / or CPiV replication), as compared to CAV, CDV and / or CPiV replication in the absence of the immune response.

[0458] Following immunization of a subject (such as a canine subject), serum can be collected from tire subject at appropriate time points, frozen, and stored for neutralization testing. Methods to assay for neutralization activity, include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry based assays, single-cycle infection assays, and pseudovirus neutralization assays.

[0459] EXAMPLES

[0460] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0461] Example 1: CAV genomic modules and plasmids for assembly of CAV viral genome vaccines The CAV genomes for vaccines were engineered and assembled using El , core, E3 and E4 CAV2 plasmid modules using CAVSLIC. The El module spans from the left-hand ITR through E1B-55k; the core module spans from protein IX through the putative 33k gene; the E3 module spans from pVIII through Fiber; and the E4 module spans from E4-ORF5 through the right-hand ITR (FIG. 6). The four plasmid modules can be assembled in two, three or four steps, and in different orders, using scarless assembly methods, examples of which are shown (FIGS. 8-10), but are not limited to these examples.

[0462] The CAV Core, El, E3 and E4 plasmid modules were cloned as depicted in FIG. 7. Primers used for module construction and genome assembly are shown in Table 1. The CAV core plasmid module was constructed via Gibson assembly by combining (1) a 571 bp gBlock™ gene fragment (Integrated DNA Technologies), (2) CAV2 VR-800 (ATCC) DNA digested with BstZ17I / PshAI, and (3) a plasmid backbone with a pl5A origin of replication and ampicillin resistance, amplified by PCR using primers nl and n2. The El, E3 and E4 plasmid modules were constructed using PCR amplification of7158-105382-02

[0463] pCAV2 ATCC VR-800 DNA reference genome as the template using primers ml and m2 (El module), ol and o2 (E3 module), and pl, p2, p3, p4, p5 and p6 (E4 module), and each module was Gibson assembled with an Entry plasmid backbone having a pMBl origin of replication and kanamycin resistance, amplified with PCR primers m3 and m4 (El module), o3 and o4 (E3 module), and p7 and p8 (E4 module). The E4 module was engineered with two silent nucleotide mutations (shown in bold underline below) to eliminate Bglll and Pad restriction sites, while maintaining amino acid sequence. This facilitated linearization of plasmid modules by restriction enzyme digestion and assembly of El, E3, and E4 plasmids with core plasmid modules by scarless Gibson and SLIC based assembly methods.

[0464] E4 region 1 (SEQ ID NO : 62)

[0465] agcacccgctctattacagacctcacccacacagcacagtt

[0466] E4 region 2 (SEQ ID NO: 63)

[0467] acgtcagaaatttctttaatcaaagtgcctttaaaatgtgc

[0468] Table 1. PCR primers for CAV plasmid module construction*

[0469] Primer Sequence SEQ ID NO: ml gcagattaccctgttatccctaCATCATCAATAATATACAGGACAAAGAGGTGTG 22 G

[0470] m2 agttgggctgtaaatacacagcCACGTACCGCGCCCCTTTTATAC 23 m3 GCTGTGTATTTACAGCCCAAC 24 m4 TAGGGATAACAGGGTAATCTGC 25 nl cacgtaccgcgccccttttaATT A AGT A ATCTT AC AG AC A AGCTGT G AC 26 n2 caacaaatactgtcaaggactcgagtccggcacagactgagcagatcTATAAACGCAGAAAGG 27 CCCAC

[0471] 01 ATGTCTAAAGAAATACCAACCCC 28 o2 CCGCTCGCGTGTATGAAAAATAAAG 29 o3 gggttggtatttctttagacatCACAACTTTTGTATACAAAGTTGGC 30 o4 tttttcatacacgcgagcggTACAACTTTGTATAATAAAGTTGAACGAG 31 pl ccaactttgtataataaagttgTAAGGCTGCCGCCTTCAG 32 P2 tgtgggtgaggTCT GT A AT AG AGCGGGT GC 33 p3 ctattacagacCTCACCCACACAGCACAG 34P4 aaaggcactttgATTAAAGAAATTTCTGACGTTGTTAATAATCAC 35 p5 tttctttaatcAAAGTGCCTTTAAAATGTGCAAGAG 36 p6 gctgattaccctgttatccctaC ATC AT C A AT A AT AT AC AGG AC A A AG AGG 37 P7 CTTTTTTATAATGCCAACTTTGTATAATAAAGTTG 38 p8 TAGGGATAACAGGGTAATCAGC 39

[0472]

[0473] 7158-105382-02

[0474] *Upper case letters indicate overlap with template DNA, lower case letters indicate overlap with neighboring regions for Gibson assembly

[0475] The El plasmid used to assemble virus vaccine compositions described in the following Examples is designated El-269 and has the features shown in FIG. 11. The sequence of the El-269 plasmid is set forth herein as SEQ ID NO: 11.

[0476] The Core plasmid used to assemble virus vaccine compositions described herein is designated Core-059 and has tire features shown in FIG. 12. The sequence of the Core-059 plasmid is set forth herein as SEQ ID NO: 12.

[0477] The parental wildtype CAV2 E3 plasmid that was used to assemble wildtype E3 containing CAV viruses and that was subsequently modified in the following Examples to delete or partially delete E3-encoded ORFs and replace them with heterologous antigens, is designated E3-522 and has the features shown in FIG. 13. The sequence of the E3-522 plasmid is set forth herein as SEQ ID NO: 13.

[0478] The E4 module plasmid used to assemble CAV vaccine strains described herein is designated E4-099 and has the features shown in FIG. 14. The sequence of the E4-099 plasmid is set forth herein as SEQ ID NO: 19.

[0479] Bacterial stains: E. coli DHIOb competent cells were used to transform and amplify the enty, destination and whole virus genome constructs.

[0480] Canine cell-lines: Madin-Darby Canine Kidney (MDCK) cells (such as from ATCC) were used as the host cell line for construct transfection and canine adenovirus production.

[0481] Example 2: Construction of PCMN-1507 using CAVSLIC

[0482] Canine distemper virus (CDV) is an enveloped RNA morbillivirus (FIG. 15) belonging to the Paramyxoviridae family

[0483] This example describes the production of the recombinant CAV PCMN-1507, which contains a heterologous ORF encoding the full-length canine distemper virus hemagglutinin (H) protein (CDVH) that is transcribed and expressed from the native viral E3 transcriptional architecture and replaces the E3-ORF1 and E3-ORF2 protein coding sequences. (AE3-ORF1[CDVH], AE3-ORF2). The vector sequence of PCMN-1507 plasmid is set forth herein as SEQ ID NO: 1.

[0484] The PCMN-1507 genome plasmid (SEQ ID NO: 2) that contains the recombinant CAV2 viral genome vaccine composition was cloned and assembled using the CAV SLIC plasmid modules and methodologies described in Example 1 and shown in FIGS. 7-10 (and the parental plasmid maps are shown in FIGS. 11-14).

[0485] The CDV haemagglutinin protein H (CDVH) is encoded by a DNA of 1821 bp. The CDVH amino acid sequence is as described in GenBank accession ACZ56433 (SEQ ID NO: 64), from recombinant CDV strain Snyder Hill. The CDVH gene (SEQ ID NO: 65) was PCR amplified from a gBlock™ gene fragment that was codon-optimized for Canis familiaris using the Integrated DNA Technologies DNA codon optimization tool.7158-105382-02

[0486] CDVH amino acid sequence (SEQ ID NO: 64) MLSYQDKVGAFYKDNARANPSKLSLVTEEHGGRRPPYLLFVLLVLLVGILALLAITGVRFHKVS TSNMEFSRLLKEDMEKSEAVHHQVIDVLTPLFKIIGDEIGLRLPQKLNEIKQFILQKTNFFNPNREF DFRDLHWCINPPSKVKVNFTNYCETIGIRKSIASAANPILLSALSGGRSDIFPPYRCSGATTSVGKV FPLSVSLSMSLISRTSVIINMLTAISDGVYGKTYLLVPDDIEREFDTQEIRVFEIGFIKRWLNDMPLL QTTNYMVLPENSKAKVCTIAVGELTLASLCVEESTVLLYHDSRGSQDGILVVTLGIFGATSMDHI EEVIPVAHPSMEKIHITNHRGFIKDSIATWMVPALASEKQEEQKGCLESACQRKTYPMCNQTSW EPFGGGQLPSYGRLTLPLDASVDLQLNISFTYGPVILNGDGMDYYESPLLNSGWLTIPPKNGTILG LINKASRGDQFTVIPHVLTFAPRESGGNCYLPIQTSQIIDRDVLIESNLVVLPTQSFRYVIATYDISR NDHAIVYYVYDPFRTIFYTYPFRLTTKGRPDFLRIECFVWDDNLWCHQFYRYEANIANSTTSVEN LVRIRFSCNRSNP CDVH DNA sequence (SEQ ID NO: 65) atgcttagctatcaggacaaagtcggcgcattctataaagacaatgcacgggcgaacccctctaaactttctctcgtgaccgaggaacatgggggccgg aggccgccgtatcttttgttcgtcctcctcgtgttgctcgttggaatcctggctctcctcgctataactggcgtccggttccataaggtcagcacctccaatat ggaattttcaaggctcctgaaagaggacatggaaaagagcgaggcggttcatcaccaagtcatcgacgttctgacgcccctcttcaaaatcatcgggga tgagatcggactgaggcttccccaaaaactcaatgaaatcaagcagttcattttgcagaaaaccaacttctttaatcctaaccgagagttcgattttagagac cttcactggtgtatcaacccaccatctaaagtgaaggtcaactttacgaactattgcgaaactatcggtattaggaaatcaattgcttctgcagcgaatccga tcctgcttagcgctcttagcgggggtcgcagtgatatattcccgccataccggtgtagcggtgcgacaacgtccgtcgggaaggtgttccccctgtctgtt agtctttcaatgtccctgattagcaggacatcagttataataaacatgctcactgctataagcgatggagtgtatggtaagacgtacttgctggtccccgatg acatcgaacgggaattcgacactcaggagatacgcgtctttgaaattggttttataaaacgatggctgaatgacatgccgttgcttcaaaccactaattacat ggtgctgcctgaaaactctaaggccaaagtgtgcaccatagctgttggtgaattgactctggccagcctttgtgtggaagaatcaacggttctcctttatca cgactctcgcggtagtcaggatggcattctcgtggtcaccctcggcatatttggtgccacgtctatggaccatattgaggaagtcatcccagtggctcatcc atcaatggagaaaatacacattacgaatcaccgcggttttattaaagacagcattgcaacttggatggtgccggctctcgcaagcgaaaaacaggaaga acagaagggttgcctggaaagtgcttgtcaaagaaagacttatcccatgtgcaatcaaactagctgggaacccttcggcggaggtcagcttccaagctat ggcaggctgacgcttcccttggatgctagcgtcgaccttcaactcaatatttcattcacgtacggacctgttatacttaatggcgatggtatggactattacg aaagtccgcttctgaacagcggatggcttaccattccaccaaagaacggtaccattctggggttgataaacaaagcgtccaggggcgaccagttcactgt catcccgcatgtcttgacgtttgcaccgcgcgaatctggggggaactgttatttgccaattcaaacgtcacaaataatcgacagagatgtcttgatagaaag caacctcgttgtcctccctacacaatcattcagatacgtcatagctacttacgatatatcccgcaacgatcatgcaattgtttattatgtctatgatccattcaga actatattttacacctacccgttcagactgactactaaggggagaccggatttcttgcggattgaatgctttgtttgggatgacaacctgtggtgtcaccagtt ttatcgatacgaagccaatatagccaatagcacgacttctgttgagaacttggtgcgcattcggttttcatgcaaccgctcaaatcct Construction of PCMN-1507 is illustrated in FIG. 16. PCR primers used for inserting CDVH DNA (CDVH) into the PCMN-1507 E3 module are shown in Table 2. An E3 plasmid module originally derived fromE3-522 was first modified to remove E3-ORF2 sequences using Gibson assembly with DNA amplified using primer-extension PCR with primers al and a2. Donor sequence (CDVH DNA) was then inserted into E3-ORF1 using Gibson assembly with DNA amplified using primer-extension PCR with primers bl and b2, which was combined with the E3-ORF2-deleted shuttle vector plasmid amplified by PCR with primers cl and c2. Included in this insert is the upstream pVIII-E3-ORFl DNA sequence that was modified to introduce mutations that ablate alternative E3-ORF1 and CDVH start codons while maintaining the pVIII coding sequence in the alternative reading frame. The sequence of the modified E3 region of PCMN-1507 (SEQ ID NO: 66) is shown below.7158-105382-02

[0487] Table 2. PCR primers for insertion of donor DNA into entry plasmid

[0488] Primer Sequence SEQ ID NO: al cgccgCTTGTCAAATAAACTTACCTAATTTTTGCTAAGACGTCTG 46 a2 acaagCGGCGGCGATGAAGAAGC 47 bl tctgtcgacggctacgactgagATGCTTAGCTATCAGGACAAAG 48 b2 gctgtaaaacaagtttaataatattatctaAGGATTTGAGCGGTTGC 49 cl TAGATAATATTATTAAACTTGTTTTACAGCTACCACC 50 c2 ctcagtcgtagccgTCGACAGAGTTGGTTACAATGTC 51

[0489]

[0490] PCMN-1507 E3 region (SEQ ID NO: 66)

[0491] Modified sequence (bold), CDVH insertion (italics), location of genome deletion (underline) ccagaggtataccttaaccctttttcaggaccaccggacacctttcctgatcagttcattcctaactacgacattgtaaccaactctgtcgacggctacgact gag tgcttagctatcaggacaaagtcggcgcattctataaagacaatgcacgggcgaacccctctaaactttctctcgtgaccgaggaacatggg ggccggaggccgccgtatcttttgttcgtcctcctcgtgttgctcgttggaatcctggctctcctcgctataactggcgtccggttccataaggtcagcac ctccaatatggaattttcaaggctcctgaaagaggacatggaaaagagcgaggcggttcatcaccaagtcatcgacgttctgacgcccctcttcaa aatcatcggggatgagatcggactgaggcttccccaaaaactcaatgaaatcaagcagttcattttgcagaaaaccaacttctttaatcctaaccga gagttcgattttagagaccttcactggtgtatcaacccaccatctaaagtgariggtcaactttacgaactattgcgaaactatcggtattaggaaatca attgcttctgcagcgaatccgatcctgcttagcgctcttagcgggggtcgcagtgatatattcccgccataccggtgtagcggtgcgacaacgtccgtc gggaaggtgttccccctgtctgttagtctttcaatgtccctgattagcaggacatcagttataataaacatgctcactgctataagcgatggagtgtatg gtaagacgtacttgctggtccccgatgacatcgaacgggaattcgacactcaggagatacgcgtcttgaaattggttttataaaacgatggctgaat gacatgccgttgcttcaaaccactaattacatggtgctgcctgaaaactctaaggccaaagtgtgcaccatagctgttggtgaattgactctggccag cctttgtgtggaagaatcaacggttctcctttatcacgactctcgcggtagtcaggatggcattctcgtggtcaccctcggcatatttggtgccacgtcta tggaccatattgaggaagtcatcccagtggctcatccatcaatggagaaaatacacattacgaatcaccgcggttttattaaagacagcattgcaac ttggatggtgccggctctcgcaagcgaaaaacaggaagaacagaagggttgcctggaaagtgcttgtcaaagaaagacttatcccatgtgcaatc aaactagctgggaacccttcggcggaggtcagcttccaagctatggcaggctgacgcttcccttggatgctagcgtcgaccttcaactcaatatttca ttcacgtacggacctgttatacttaatggcgatggtatggactattacgaaagtccgcttctgaacagcggatggcttaccattccaccaaagaacgg taccattctggggttgataaacaaagcgtccaggggcgaccagttcactgtcatcccgcatgtcttgacgtttgcaccgcgcgaatctggggggaac tgttatttgccaattcaaacgtcacaaataatcgacagagatgtcttgatagaaagcaacctcgttgtcctccctacacaatcattcagatacgtcata gctacttacgatatatcccgcaacgatcatgcaattgtttattatgtctatgatccattcagaactatattttacacctacccgttcagactgactactaag gggagaccggatttcttgcggattgaatgctttgtttgggatgacaacctgtggtgtcaccagttttatcgatacgaagccaatatagccaatagcacg acttctgttgagaacttggtgcgcattcggttttcatgcaaccgctcaaatccftagataatattattaaactgtttacagctaccaccataatgcgctcag cttcttcatcgccgccgcttgtcaaataaacttacctaatttttgctaagacgtctgggtcctgcgtttct

[0492] The final E3-CDVH module plasmid used to construct the whole genome vaccine compositions is designated E3-575 CDVH (see FIG. 17) and has the sequence set forth as SEQ ID NO: 14.

[0493] CAVSLIC was used to construct the PCMN-1507 whole viral genome vaccine composition that expresses CDVH from the endogenous viral E3 transcriptional architecture in place of the CAV E3-ORFs. The core module plasmid, Core-059 (SEQ ID NO: 12), was digested with PacI and Gibson assembled with the linear product of the El-269 plasmid (SEQ ID NO: 11) amplified with primers rl and r2. This plasmid macromodule is designated ASMM-149 (SEQ ID NO: 20) and has the features shown in FIG. 18.

[0494] The ASMM-149 macromodule plasmid (SEQ ID NO: 20) was then digested with Bglll and Gibson assembled with the linear products of the E3-575 CDVH plasmid module (SEQ ID NO: 14) amplified with primers si and s2, and the E4-099 plasmid module (SEQ ID NO: 19) amplified with7158-105382-02

[0495] primers tl and t2. See Table 3 for the primer sequences. The left column of Table 3 shows the logic for assembly with what is a universal set of primers for assembly of CAVSLIC El, E3 and E4 modules to core and with each other in exemplary two or three step assembly schemes, and not particular to only this example.

[0496] Table 3. Primers for amplification of El, E3 and E4 modules

[0497] Primer Primer Sequence SEQ ID Description NO:

[0498] El to core Dest El_For (rl) agcttgtctgtaagattacttaCTTTTTTATAATGCCAACT 40 plasmid AA TTGTACAAAAAAGCAG

[0499] El to pIX-core El Rev (r2) AB CACGTACCGCGCCCCTTTTATAC 41 E3 to core 33k cgagtccggcacagactgagcaATGTCTAAAGAAATAC 42 E3 For (si) AE

[0500] assembly CAACCCCTTATATGTGGAG

[0501] E3 to E4 tttcctgaaggcggcagccttaCCGCTCGCGTGTATGAA 43 E3 Rev (s2) AF

[0502] no Bglll AAATAAAG agctttatttttcatacacgcgagcgGTAAGGCTGCCGCCT 44 E4 to E3 E4 For (tl) AF

[0503] TCAG

[0504] E4 to core Dest ggtgggcctttctgcgtttataaCTTATAATGCCAACTTTG 45 E4 Rev (t2) AD

[0505] plasmid TACAAGAAAGCTG

[0506] E4 to core 33k E4 to core For TCCGGCACAGACTGAGCAGATCTAAGGCTG 91 CCGCCTTCAG

[0507] with Bglll (t3) AC

[0508] E4 to core Dest ggtgggcctttctgcgtttataaCTTATAATGCCAACTTTG 45 E4 Rev (t2) AD

[0509] plasmid TACAAGAAAGCTG

[0510]

[0511] Final constructs were selected using ampicillin resistance. The PCMN-1507 genome sequence is set forth herein as SEQ ID NO: 1. A map of the PCMN-1507 virus genome plasmid is shown in FIG. 19. A linear version of the PCMN-1507 genome is shown in FIG. 20.

[0512] E. coli DHIOb cells were used to transform and amplify tire donor shuttle and assembled virus genome plasmids.

[0513] MDCK canine cells (such as from ATCC) were used as the host cell line for viral plasmid genome construct transfection and virus propagation. Transfection was done using Lipofectamine 3000 using 2 pg DNA per 6-well. Upon transfection into mammalian cells, tire RSV promoter drives expression of the I-Scel enzyme which releases and linearizes the CAV genome at the left and right ITRs so the Ad genome can be replicated (FIG. 20). Thus, there is no need to cut, isolate and purify the CAV genome DNA for transfection into canine cells and viral production. Virus successfully grown in MDCK cells with MEM10 media was transferred to freshly plated MDCK cells for virus propagation.7158-105382-02

[0514] Example 3: Construction of PCMN-1508 using CAVSLIC

[0515] This example describes the production of the recombinant CAV PCMN-1508, which contains a heterologous ORF encoding the full-length canine distemper virus nucleoprotein (CDVNP) inserted into E3-ORF1, and a deletion of E3-ORF2 (AE3-ORF1 [CDVNP], AE3-ORF2). The vector sequence of PCMN-1508 is set forth herein as SEQ ID NO: 3.

[0516] Core, El, E3 and E4 modules were constructed as described in Examples 1 and 2 (and shown in FIGS. 7-10).

[0517] The CDV nucleoprotein (CDVNP) sequence is encoded by a DNA of 1569bp. The CDVNP amino acid sequence is as described in GenBank accession ACZ56427 (SEQ ID NO: 67), from recombinant CDV strain Snyder Hill. The CDVNP gene (SEQ ID NO: 68) was PCR amplified from a gBlock™ gene fragment that was codon-optimized for Canis familiaris using the Integrated DNA Technologies DNA codon optimization tool.

[0518] CDVNP amino acid sequence (SEQ ID NO: 67) MASLLKSLTLFKRTRDQPPLASGSGGAIRGIKHVIIVLIPGDSSIVTRSRLLDRLVRLVGDPEINGP KLTGILISILSLFVESPGQLIQRIIDDPDVSIKLVEVIPSINSVCGLTFASRGASLDSEADEFFKIVDEG SKAQGQLGWLENKDIVDIEVDDAEQFNILLASILAQIWILLAKAVTAPDTAADSEMRRWIKYTQ QRRVVGEFRMNKIWLDIVRNRIAEDLSLRRFMVALILDIKRSPGNKPRIAEMICDIDNYIVEAGLA SFILTIKFGIETMYPALGLHEFSGELTTIESLMMLYQQMGETAPYMVILENSVQNKFSAGSYPLL WSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSTLAAELGITKEEAQLVS EIASKTTEDRTIRATGPKQSQITFLHSERSEVANQQPPTINKRSENQGGDKYPIHFSDERLPGYTPD VNSSEWSESRYDTRIIQDDGNDDDRKSMEAIAKMRMLTKMLSQPGTSEDSSPVYNDRELLN CDVNP DNA sequence (SEQ ID NO: 68) atggcatccttgttgaaatcactgaccctgtttaaaaggacccgagatcaacctcctctggcgtccgggtccggtggtgctatccgagggattaagcatgt gattatcgttcttatccctggggattccagcatcgtgacaaggtctcggcttctggatcgccttgtgcggcttgttggagacccggagataaatggacccaa gttgacgggcatcctcatctcaatcctttctctctttgtcgaaagtccagggcagttgatccagaggataatagacgacccggacgtgtctatcaagcttgtg gaagttatcccgtcaataaattctgtgtgtggacttaccttcgccagtcgcggagcctcattggactcagaggccgatgagttctttaagatcgttgacgag gggtcaaaagcacagggtcaactgggctggctcgagaacaaggacatagtggatatcgaggttgacgatgcggaacagtttaacattttgctcgcatca atactggctcaaatatggattctgctcgccaaggcggttactgcacccgacacagcagcagactctgagatgcgacggtggatcaagtatacacaacaa aggagagtggttggagagttccgaatgaacaagatttggttggacatcgtgcggaaccgaatagcggaggacctgagcttgcggaggtttatggttgca cttattcttgacatcaaacggtctccgggcaacaagccacggattgcggaaatgatttgcgacatcgataactacatagttgaagccggactggcttccttt atactgactataaaatttgggattgaaactatgtaccctgccctcgggctccacgagttttctggtgagctcaccacaatagagtccctgatgatgttgtatca gcagatgggtgagacggcaccatacatggtcatactcgaaaacagtgtgcaaaacaaattctcagccggcagttatccgcttctttggtcctacgctatgg gtgttggcgtcgaattggaaaatagtatgggtggcctgaacttcggacggtcttatttcgatcccgcttacttccgactgggtcaagagatggtccgcaga agtgctgggaaagtttctagtacactggccgccgaattgggaattaccaaagaagaggctcaacttgtctcagagatcgctagcaagacaacagaggat aggacaataagggctacggggcctaagcaatcccagattacattcctgcatagtgaacggagcgaagttgcgaatcagcaaccgccgacaataaataa acggtccgagaatcaagggggtgacaagtatccgatccattttagtgatgaacgcttgcctgggtacacacctgatgttaatagctccgagtggagcgaa tcccgctacgacacacgcattatacaggatgacggcaatgatgatgatcgaaagagtatggaggctattgcgaaaatgaggatgttgactaaaatgctttc ccaacctggaacttccgaggactcttcccctgtgtacaacgacagggagctcctcaac

[0519] Construction of PCMN-1508 is illustrated in FIG. 21. PCR primers used for inserting donor DNA (CDV NP) into the PCMN-1507 E3 module are shown in Table 4. An E3 containing plasmid module originally derived from E3-522 (SEQ ID NO: 13) was first modified to remove E3-ORF2 sequences using Gibson assembly with DNA amplified using primer-extension PCR with primers al and a2. Donor sequence (CDVNP DNA) was then inserted into E3-ORF1 using Gibson assembly with DNA7158-105382-02

[0520] amplified using primer-extension PCR with primers bl and b2, which was combined with the E3-ORF2-deleted shuttle vector plasmid amplified by PCR with primers cl and c2. Included in this insert is the upstream pVIII-E3-ORFl DNA sequence that was modified to introduce mutations that ablate alternative E3-ORF1 and CDV NP start codons while maintaining the pVIII coding sequence in the alternative reading frame. The sequence of the modified E3 region of PCMN-1508 (SEQ ID NO: 69) is shown below.

[0521] Table 4. PCR primers for insertion of donor DNA into shuttle vector

[0522] Primer Sequence SEQ ID NO: al cgccgCTTGTCAAATAAACTTACCTAATTTTTGCTAAGACGTCTG 46 a2 acaagCGGCGGCGATGAAGAAGC 47 bl tctgtcgacggctacgactgagATGGCATCCTTGTTGAAATCAC 52 b2 gctgtaaaacaagtttaataatattatctaGTTGAGGAGCTCCCTGTC 53 cl TAGATAATATTATTAAACTTGTTTTACAGCTACCACC 50 c2 ctcagtcgtagccgTCGACAGAGTTGGTTACAATGTC 51

[0523]

[0524] PCMN-1508 E3 region (SEQ ID NO: 69)

[0525] Modified sequence (bold), CDVNP insertion (italics), location of genome deletion (underline) ccagaggtataccttaaccctttttcaggaccaccggacacctttcctgatcagttcattcctaactacgacattgtaaccaactctgtcgacggctacgact gagatggcatccttgttgaaatcactgaccctgtttaaaaggacccgagatcaacctcctctggcgtccgggtccggtggtgctatccgagggattaa gcatgtgattatcgttcttatccctggggattccagcatcgtgacaaggtctcggcttctggatcgccttgtgcggcttgttggagacccggagataaat ggacccaagtgacgggcatcctcatctcaatcctttctctctttgtcgaaagtccagggcagttgatccagaggataatagacgacccggacgtgtc tatcaagcttgtggaagttatcccgtcaataaattctgtgtgtggacttaccttcgccagtcgcggagcctcattggactcagaggccgatgagttctta agatcgtgacgaggggtcaaaagcacagggtcaactgggctggctcgagaacaaggacatagtggatatcgaggttgacgatgcggaacagtt taacattttgctcgcatcaatactggctcaaatatggattctgctcgccaaggcggttactgcacccgacacagcagcagactctgagatgcgacgg tggatcaagtatacacaacaaaggagagtggttggagagttccgaatgaacaagatttggttggacatcgtgcggaaccgaatagcggaggacc tgagcttgcggaggtttatggttgcacttattcttgacatcaaacggtctccgggcaacaagccacggangcggaaatgatttgcgacatcgataact acatagttgaagccggactggcttcctttatactgactataaaatttgggattgaaactatgtaccctgccctcgggctccacgagttttctggtgagctc accacaatagagtccctgatgatgttgtatcagcagatgggtgagacggcaccatacatggtcatactcgaaaacagtgtgcaaaacaaattctca gccggcagttatccgcttctttggtcctacgctatgggtgttggcgtcgaattggaaaatagtatgggtggcctgaacttcggacggtcttatttcgatcc cgcttacttccgactgggtcaagagatggtccgcagaagtgctgggaaagtttctagtacactggccgccgaattgggaattaccaaagaagagg ctcaacttgtctcagagatcgctagcaagacaacagaggataggacaataagggctacggggcctaagcaatcccagattacattcctgcatagt gaacggagcgaagttgcgaatcagcaaccgccgacaataaataaacggtccgagaatcaagggggtgacaagtatccgatccattttagtgatg aacgcttgcctgggtacacacctgatgttaatagctccgagtggagcgaatcccgctacgacacacgcattatacaggatgacggcaatgatgatg atcgaaagagtatggaggctatgcgaaaatgaggatgttgactaaaatgctttcccaacctggaacttccgaggactcttcccctgtgtacaacga cagggagctcctcaactagataatattattaaacttgttttacagctaccaccataatgcgcttcagcttcttcatcgccgccgcttgtcaaataaacttaccta atttttgctaagacgtctgggtcctgcgtttct

[0526] The sequence of the E3-581 CDV-NP plasmid (see map FIG. 22) used to construct the PCMN- 1508 vaccine genome construct is set forth herein as SEQ ID NO: 15.

[0527] CAVSLIC was used to construct the PCMN-1508 whole viral genome vaccine composition that expresses CDV NP from the endogenous viral E3 transcriptional architecture in place of the CAV E3- ORFs. As in Examples 1 and 2, the core module plasmid, Core-059 (SEQ ID NO: 12), was digested with7158-105382-02

[0528] PacI and Gibson assembled with the linear product of the El-269 plasmid (SEQ ID NO: 11) amplified with primers rl and r2 to create the plasmid macromodule ASMM-149 (SEQ ID NO: 20).

[0529] The ASMM-149 macromodule plasmid was then digested with Bglll and Gibson assembled with the linear products of the E3-581 CDVNP plasmid module (SEQ ID NO: 15) amplified with primers si and s2, and the E4-099 plasmid module (SEQ ID NO: 19) amplified with primers tl and t2. See Table 3 for the rl, r2, si, s2, tl and t2 primer sequences. Final constructs were selected using ampicillin resistance. A map of the PCMN-1508 virus genome plasmid is shown in FIG. 23.

[0530] E. coli DHIOb cells were used to transform and amplify tire donor shuttle and assembled virus genome plasmids. MDCK canine cells (such as from ATCC) were used as tire host cell line for viral plasmid genome construct transfection and virus propagation. Transfection was done using Lipofectamine 3000 using 2 pg DNA per 6-well. Virus successfully grown in MDCK cells with MEM10 media was transferred to freshly plated MDCK cells for virus propagation.

[0531] Example 4: Construction of PCMN-1556 using CAVSLIC

[0532] Canine parainfluenza virus (CPiV), like CDV, is a negative-sense, single stranded RNA virus (FIG. 24). This example describes the production of the recombinant CAV PCMN-1556, which contains a deletion of E3-ORF1 and E3-ORF2, a first heterologous ORF encoding the fusion protein of canine parainfluenza virus (CPiVF) and a second heterologous ORF encoding the hemagglutinin-neuraminidase (HN) protein of CPiV (CPiVHN), separated by a P2A coding sequence, inserted into E3-ORF1 (AE3- ORFl[CPiVF-P2A-CPiVHN], AE3-ORF2). The vector sequence of PCMN-1556 is set forth herein as SEQ ID NO: 5.

[0533] Core, El, E3 and E4 modules were constructed as described in Examples 1 and 2.

[0534] The CPiVF and CPiVHN protein sequences are encoded by DNA sequences of 1653 bp and 1695 bp, respectively. The amino acid sequences are based on the D008 challenge strain obtained from the USDA. Alignment with public NCBI sequences shows the CPiVF amino acid sequence matches the amino acid sequence described in GenBank accession AFE48526 (SEQ ID NO: 76, corresponding to residues 1-551 of SEQ ID NO: 70), from the fusion protein of mammalian rubulavirus 5 (GenBank accession JQ743328). The CPiVF gene (SEQ ID NO: 77, corresponding to nucleotides 1-1653 of SEQ ID NO: 71) was PCR amplified from a gBlock™ gene fragment that was codon-optimized for Canis familiaris using tire Integrated DNA Technologies DNA codon optimization tool.

[0535] CPiVF + P2A amino acid sequence (SEQ ID NO: 70; P2A sequence shown in bold) MGTIIQFLVVSCLLAGAGSLDPAALMQIGVIPTNVRQLMYYTEASSAFIVVKLMPTIDSPISGCNIT SISSYNATVTKLLQPIGENLETIRNQLIPTRRRRRFAGVVIGLAALGVATAAQVTAAVALVKANK NAAAILNLKNAIQKTNTAVADVVQATQSLGTAVQAVQDHINSVVSPAITAANCKAQDAIIGSILN LYLTELTTIFHNQITNPALSPITIQALRILLGSTLPTVVEKSFNTQISAAELLSSGLLTGQIVGLDLTY MQMV1K1ELPTLTVQPATQ11DLATISAF1NNQEVMAQLPTRV1VTGSLIQAYPASQCT1TPNTVYC RYNDAQVLSDDTMACLQGNLTRCTFSPVVGSFLTRFMLFDGIVYANCRSMLCKCMQPAAVILQ PSSSPVTVIDMYKCVSLQLDNLRFTITQLANVTYNSTIKLETSQILPIDPLDISQNLAAVNKSLSDA LQHLAQSDTYLSAITSATTTSVLSIMAICLGSLGLILIILLSVVVWKLLTIVTANRNRMENFVYHNS AFHHSRSDLSEKNQPATLGTRGSGATNFSLLKQAGDVEENPG7158-105382-02

[0536] CPiVF + P2A DNA sequence (SEQ ID NO: 71; P2A coding sequence shown in bold) atggggacaattattcagttcttggtggtgtcttgcctgctggcaggagcggggtccctggatccagccgccctcatgcagataggtgtgattcccaccaa cgtcagacagctcatgtattataccgaagccagctcagcatttatcgtcgtgaaattgatgcctacgattgacagcccaatatctggttgtaatattacatcaa taagttcttataatgctacagtcacgaaacttctccagcctataggcgaaaatctcgaaacgattaggaatcagcttatacccacaagaagaagaagacga tttgctggcgtcgttatcggtcttgccgcactgggtgtcgctactgcagcccaagtgacagcggctgtcgcactcgtgaaggctaacaagaacgctgcag ctattctcaacctgaaaaatgcgatacagaaaactaacaccgctgtcgcagatgttgtccaggcgacacaatctctgggtacggcagttcaagctgttcag gaccacatcaatagcgttgtgtctccggccattaccgccgcgaactgtaaggcacaggatgcaattataggaagtatccttaacctctacctcactgaact cacgacaatcttccataaccagataacgaacccagctctgtcaccgataacaattcaggcgctccgaatcttgcttggctcaacccttccgacggttgtgg aaaagtcttttaacacgcagataagtgctgcagaactgcttagctctggtttgcttacaggtcaaatagtcggactggatctgacctatatgcagatggtgat taaaatcgagctgcctaccctgactgtccagccagcaacccaaattatcgaccttgcaactataagcgcctttatcaacaaccaagaagtcatggctcagt tgcccaccagagtcattgtgactggctccctgatacaggcttaccctgcaagtcagtgtacaattacgccgaacacggtgtattgcagatacaacgacgc acaagttctctctgatgatacaatggcatgtctgcaaggaaatctgacaaggtgtacgttcagtccggtcgtggggtctttcctcacccgcttcatgttgtttg atggcatagtgtatgcgaattgtcggtcaatgctctgcaagtgtatgcagcctgctgcggttatactccagccctccagcagcccggtgactgtgatcgat atgtataagtgcgtttcacttcagcttgacaacctgaggttcacaataacccaattggcgaacgtgacttataattctacgattaaacttgagactagccagat ccttccgatcgatcctctggatataagccaaaacctggcggcagtgaataaaagtctcagtgatgccttgcagcacctggcccaatcagacacgtatcttt ccgctataacctcagccacgacaacaagtgttctttccataatggcaatctgtcttggtagtctgggtctcatactcattatactgcttagcgttgtggtctgga agctgcttacgattgtcaccgcaaatcgcaatcgaatggagaactttgtctatcacaacagcgcattccaccatagtcgatccgacttgagcgaaaaaaac cagcctgctactctcgggactcgcggaagcggagctactaacttcagcctgctgaagcaggctggtgacgtcgaggagaatcctggc Alignment with public NCBI sequences showed that the CPiVHN amino acid sequence was most similar to GenBank accession AFE48527 (SEQ ID NO: 79, corresponding to residues 2-566 of SEQ ID NO: 72), from the hemagglutinin-neuraminidase protein of mammalian rubulavirus 5 (GenBank accession JQ743328) with differences in amino acids 288 and 447. The CPiVHN gene (SEQ ID NO: 80, corresponding to nucleotides 4-1698 of SEQ ID NO: 73) was PCR amplified from a gBlock™ gene fragment that was codon-optimized for Canis familiaris using the Integrated DNA Technologies DNA codon optimization tool.

[0537] CPiVHN amino acid sequence + final proline of P2A (SEQ ID NO: 72) PMVAEDAPVRGTCRVLFRTTTLIFLCTLLALSISILYESLITQKQIMSHAGSTGSNSRLGSITDLLN NILSVANQIIYNSAVALPLQLDTLESTLLTAIKSLQTSDKLEQNCSWGAALINNNRYINGINQFYFS IAEGRNLTLGPLLNIPSFIPTATTPEGCTRIPSFSLTKTHWCYTHNVILNGCQDHVSSNQFVSMGIIE PTSAGFPSFRTLKTLYLSDGVNRKSCSISTVPGGCMMYCFVSTQPERDDYFSTAPPEQRIIIMYYN DTIVERIINPPGVLDVWATLNPGTGSGVYYLGWVLFPIYGGVIKDTSLWNNQANKYFIPQMVAA LCSQNQATQVQNAKSSYYSSWFGNRMIQSGILACPLQQDLTNECLVLPFSNDQVLMGAEGRLY MYGDSVYYYQRSNSWWPMTMLYKVTITFTNGQPSAISAQNVPTQQVPRPGTGDCSATNRCPGF CLKGVYADAWLLTNPSSTSTFGSEATFTGSYLNAATQRINPTMYIANNTQIISSQQFGSSGQEAA YSHTTCFRDTGSVMVYCIYIIELSSSLLGQFQIVPFIRQVTLS CPiVHN DNA sequence + final codon of P2A (SEQ ID NO: 73) ccaatggtcgctgaggatgcgccagttaggggaacctgtcgcgtgctcttccgaaccaccacgctcatattcctctgcaccttgcttgcgctttcaatctca atcttgtatgagtcacttataacacagaaacaaattatgtctcatgctgggtctacaggatcaaactccaggctgggtagtatcactgatctcttgaataatatt cttagtgtggcgaaccagattatctataactcagcggttgcgctccctctccagttggatactctggagagcaccctgttgactgctattaagtctcttcaaac cagtgacaagctcgagcagaactgtagctggggcgcggctctgattaataacaacagatatattaacggcataaatcaattttatttctccattgccgaagg tagaaacctgaccctgggacctttgctcaatatccctagcttcataccaacggcgaccacccctgagggctgtactcgcattccatcctttagtcttaccaa aacacactggtgctacacccataatgttatccttaatggatgccaagatcacgtctcatcaaaccagttcgtctctatgggtataatcgaacccaccagcgc tggcttcccatcatttcgaacattgaagacgctttatctttccgatggtgtcaaccgaaagtcctgctctatttctactgtgccgggagggtgcatgatgtact gctttgtttccacgcagcctgaacgagacgattattttagtacggctccgccggagcaacggataataatcatgtactataacgatacgatagtggaaaga ataatcaatcctcctggtgttctcgacgtgtgggcgacgttgaatcctggaaccggatctggcgtctattatcttggatgggttctgtttcctatttacggcgg agttattaaagacacaagcttgtggaataatcaagctaataaatatttcattccacaaatggtcgcagccttgtgttcccaaaaccaggcgacccaagttca7158-105382-02 aaacgcaaaaagttcctattacagttcatggttcgggaatcgcatgattcaatcaggcattctggcctgcccgttgcagcaggacttgacgaacgaatgtct ggtccttcctttttccaacgaccaagttctgatgggggctgagggtagactttacatgtacggagattccgtttactactaccagcggagcaattcttggtgg ccgatgaccatgttgtataaagtcacaatcactttcactaatgggcagccgagcgccatttccgctcagaatgtccccacccaacaagttcctcgaccagg tacaggcgattgtagtgcgactaacagatgccctggtttttgccttaagggcgtttacgctgacgcatggttgttgacaaatccatctagtacgagtacttttg ggagcgaggccacattcacgggctcatacctgaatgctgcaacgcaacgcataaaccctacaatgtatatcgccaacaacacgcaaataattagtagcc aacagtttggatcatctgggcaggaggcagcctacagtcatacgacgtgtttccgggacaccgggtctgtgatggtgtattgtatctacataatagagttga gtagttcactcttggggcagttccagatcgtcccattcattaggcaagtgacgctctct

[0538] Construction of PCMN-1556 is illustrated in FIGS. 25 and 26. PCR primers used for inserting donor DNA into tine PCMN-1556 E3 module are shown in Table 5. The E3 module was first modified to remove E3-ORF2 using Gibson assembly with DNA amplified using primer-extension PCR with primers al and a2. CPiVF and CPiVHN DNA sequences were then inserted into E3-ORF1 using Gibson assembly with DNA amplified using primer-extension PCR with primers bl and b2 and PCR with primers cl and c2, which was combined with the E3-ORF2-deleted shuttle vector plasmid amplified by PCR with primers dl and d2. The P2A sequence was introduced in primers b2 and cl. Included in this insert is the upstream pVIII-E3-ORF 1 DNA sequence that was modified to remove potential E3-ORF1 start codons while maintaining the pVIII coding sequence. CPiVF and CPiVHN are linked via a P2A self-cleaving peptide. The efficiency of P2A cleavage will determine the amount of linked and unlinked CPiVF and CPiVHN. Cleaved P2A results in the residual peptide GSGATNFSLLKQAGDVEENPG (SEQ ID NO: 74) on the C-terminus of CPiVF, and a residual proline on the N-terminus of CPiVHN (residue 1 of SEQ ID NO: 72). The sequence of the modified E3 region of PCMN-1556 (SEQ ID NO: 75) is shown below.

[0539] Table 5. PCR primers for insertion of donor DNA

[0540] Primer Sequence SEQ ID NO: al cgccgCTTGTCAAATAAACTTACCTAATTTTTGCTAAGACGTCTG 46 a2 acaagCGGCGGCGATGAAGAAGC 47 bl tctgtcgacggctacgactgagATGGGGACAATTATTCAGTTCTTG 54 b2 caccagcctgcttcagcaggctgaagttagtagctccgcttccGCG AGT CCCG AG AGT AG 55 cl cctgctgaagcaggctggtgacgtcgaggagaatcctggcccaATGGTCGCTGAGGATGC 56 c2 acaagtttaataatattatctaAGAGAGCGTCACTTGCCTAATG 51 dl TAGATAATATTATTAAACTTGTTTTACAGCTACCACC 57 d2 ctcagtcgtagccgTCGACAGAGTTGGTTACAATGTC 58

[0541]

[0542] PCMN-1556 E3 region (SEQ ID NO: 75)

[0543] Modified sequence (bold), CPiVF and CPiVHN insertions (italics), P2A insertion (bold italics), location of genome deletion (underline) ccagaggtataccttaaccctttttcaggaccaccggacacctttcctgatcagttcattcctaactacgacattgtaaccaactctgtcgacggctacgact gagatggggacaattattcagttcttggtggtgtcttgcctgctggcaggagcggggtccctggatccagccgccctcatgcagataggtgtgattccc accaacgtcagacagctcatgtatataccgaagccagctcagcatttatcgtcgtgaaattgatgcctacgattgacagcccaatatctggttgtaat7158-105382-02 attacatcaataagttctataatgctacagtcacgaaacrtctccagcctataggcgaaaatctcgaaacgattaggaatcagcttatacccacaag aagaagaagacgatttgctggcgtcgttatcggtcttgccgcactgggtgtcgctactgcagcccaagtgacagcggctgtcgcactcgtgaaggct aacaagaacgctgcagctattctcaacctgaaaaatgcgatacagaaaactaacaccgctgtcgcagatgttgtccaggcgacacaatctctggg tacggcagttcaagctgttcaggaccacatcaatagcgttgtgtctccggccanaccgccgcgaactgtaaggcacaggatgcaattataggaagt atccttaacctctacctcactgaactcacgacaatcttccataaccagataacgaacccagctctgtcaccgataacaattcaggcgctccgaatctt gctggctcaacccttccgacggttgtggaaaagtctttaacacgcagataagtgctgcagaactgctagctctggmgcttacaggtcaaatagtc ggactggatctgacctatatgcagatggtgattaaaatcgagctgcctaccctgactgtccagccagcaacccaaatatcgaccttgcaactataa gcgcctttatcaacaaccaagaagtcatggctcagttgcccaccagagtcattgtgactggctccctgatacaggcttaccctgcaagtcagtgtaca atacgccgaacacggtgtatgcagatacaacgacgcacaagttctctctgatgatacaatggcatgtctgcaaggaaatctgacaaggtgtacgt tcagtccggtcgtggggtctttcctcacccgcttcatgttgtttgatggcatagtgtatgcgaattgtcggtcaatgctctgcaagtgtatgcagcctgctg cggttatactccagccctccagcagcccggtgactgtgatcgatatgtataagtgcgtttcacttcagcttgacaacctgaggtcacaataacccaat tggcgaacgtgacttataattctacgattaaactgagactagccagatccttccgatcgatcctctggatataagccaaaacctggcggcagtgaat aaaagtctcagtgatgcctgcagcacctggcccaatcagacacgtatctttccgctataacctcagccacgacaacaagtgttctttccataatggc aatctgtcttggtagtctgggtctcatactcanatactgcttagcgtgtggtctggaagctgcttacgatgtcaccgcaaatcgcaatcgaatggaga acmgtctatcacaacagcgcattccaccatagtcgatccgacttgagcgaaaaaaaccagcctgctactctcgggactcgcggaagcggagcta ctaactcagcctgctgaagcaggctggtgacgtcgaggagaatcctggcccaatggtcgctgaggatgcgccagttaggggaacctgtcgcgtg ctcttccgaaccaccacgctcatattcctctgcaccttgctgcgcttcaatctcaatcttgtatgagtcacttataacacagaaacaaattatgtctcat gctgggtctacaggatcaaactccaggctgggtagtatcactgatctcttgaataatattcttagtgtggcgaaccagattatctataactcagcggttg cgctccctctccagttggatactctggagagcaccctgttgactgctattaagtctcttcaaaccagtgacaagctcgagcagaactgtagctggggc gcggctctgattaataacaacagatatataacggcataaatcaatmatttctccattgccgaaggtagaaacctgaccctgggaccmgctcaata tccctagcttcataccaacggcgaccacccctgagggctgtactcgcattccatcctttagtcttaccaaaacacactggtgctacacccataatgtat ccttaatggatgccaagatcacgtctcatcaaaccagtcgtctctatgggtataatcgaacccaccagcgctggctcccatcattcgaacatgaa gacgctttatcttccgatggtgtcaaccgaaagtcctgctctattctactgtgccgggagggtgcatgatgtactgctttgttccacgcagcctgaac gagacgattattttagtacggctccgccggagcaacggataataatcatgtactataacgatacgatagtggaaagaataatcaatcctcctggtgtt ctcgacgtgtgggcgacgtgaatcctggaaccggatctggcgtctattatcttggatgggttctgtttcctamacggcggagttattaaagacacaa gcttgtggaataatcaagctaataaatattcattccacaaatggtcgcagccttgtgttcccaaaaccaggcgacccaagttcaaaacgcaaaaag ttcctattacagttcatggttcgggaatcgcatgattcaatcaggcattctggcctgcccgttgcagcaggacttgacgaacgaatgtctggtccttcctt tttccaacgaccaagttctgatgggggctgagggtagactttacatgtacggagattccgtttactactaccagcggagcaattcttggtggccgatga ccatgtfgtataaagtcacaatcactttcactaatgggcagccgagcgccatttccgctcagcuitgtccccacccaacaagttcctcgaccaggtaca ggcgattgtagtgcgactaacagatgccctggtttttgccttaagggcgmacgctgacgcatggttgttgacaaatccatctagtacgagtacttttgg gagcgaggccacatcacgggctcatacctgaatgctgcaacgcaacgcataaaccctacaatgtatatcgccaacaacacgcaaataattagta gccaacagtttggatcatctgggcaggaggcagcctacagtcatacgacgtgtttccgggacaccgggtctgtgatggtgtattgtatctacataata gagtlgagtagtlcaclcllggggcagtlccagalcgtcccallcallaggcaagtgacgctctcttagataatatiattaaacttgtttacagciaccacc ataatgcgcttcagcttcttcatcgccgccgcttgtcaaataaacttacctaatttttgctaagacgtctgggtcctgcgtttct

[0544] The CPiVF-P2A-CPiVHN E3 plasmid is designated E3-595 (FIG. 27) and has the sequence of SEQID NO: 16.

[0545] CAVSLIC was used to construct the PCMN-1556 whole viral genome vaccine composition that expresses CPiVF and CPIVHN from the native viral architecture in place of the CAV E3-ORFs.

[0546] The ASMM-149 El-core macromodule plasmid (SEQ ID NO: 20) was digested with Bglll and Gibson assembled with the linear product of E4-099 (SEQ ID NO: 19) amplified with primers t3 and t2 to create the El-core-E4 macromodule plasmid ASMM-150 (SEQ ID NO: 21; FIG. 28). The ASMM-150 plasmid was then digested with Bglll and Gibson assembled with tire lineal' product of tire CPiVF E3-596 plasmid (SEQ ID NO: 17) amplified with si and s2 primers. See Table 3 for the rl, r2, si, s2, t2 and t3 primer sequences. Final constructs were selected using ampicillin resistance. A map of the PCMN-1556 virus genome plasmid is shown in FIG. 29.

[0547] E. coli DHIOb cells were used to transform and amplify the shuttle vector and assembled virus genome. MDCK cells (such as from ATCC) were used as tire host cell line for construct transfection and virus production. Transfection was done using Lipofectamine 3000 using 2 pg DNA per 6-well. Virus7158-105382-02

[0548] successfully grown in MDCK cells with MEM10 media was transferred to freshly plated MDCK cells for virus propagation.

[0549] Example 5: Construction of PCMN-1557 using CAVSLIC

[0550] This example describes the production of the recombinant CAV PCMN-1557, which contains a heterologous ORF encoding the fusion protein of canine parainfluenza virus (CPiVF) inserted into E3-ORF1, and a deletion of E3-ORF2 (AE3-ORF1 [CPiVF], AE3-ORF2). The vector sequence of PCMN-1557 is set forth herein as SEQ ID NO: 7.

[0551] Core, El, E3 and E4 modules were constructed as described in Examples 1 and 2.

[0552] The CPiVF sequence is encoded by a DNA of 1653 bp. The amino acid sequence is based on the D008 challenge strain obtained from the USDA. Alignment with public NCBI sequences shows the CPiVF amino acid sequence matches the amino acid sequence described in GenBank accession AFE48526 (SEQ ID NO: 76), from fusion protein of mammalian rubulavirus 5 (GenBank accession JQ743328). The CPiVF gene (SEQ ID NO: 77) was PCR amplified from a gBlock™ gene fragment that was codon-optimized for Canis farniliaris using the Integrated DNA Technologies DNA codon optimization tool.

[0553] CPiVF amino acid sequence (SEQ ID NO: 76) MGTIIQFLVVSCLLAGAGSLDPAALMQIGVIPTNVRQLMYYTEASSAFIVVKLMPTIDSPISGCNIT SISSYNATVTKLLQPIGENLETIRNQLIPTRRRRRFAGVVIGLAALGVATAAQVTAAVALVKANK NAAAILNLKNAIQKTNTAVADVVQATQSLGTAVQAVQDHINSVVSPAITAANCKAQDAIIGSILN LYLTELTTIFHNQITNPALSPITIQALRILLGSTLPTVVEKSFNTQISAAELLSSGLLTGQIVGLDLTY MQMVIKIELPTLTVQPATQIIDLATISAFINNQEVMAQLPTRVIVTGSLIQAYPASQCTITPNTVYC RYNDAQVLSDDTMACLQGNLTRCTFSPVVGSFLTRFMLFDGIVYANCRSMLCKCMQPAAVILQ PSSSPVTVIDMYKCVSLQLDNLRFTITQLANVTYNSTIKLETSQILPIDPLDISQNLAAVNKSLSDA LQHLAQSDTYLSAITSATTTSVLSIMAICLGSLGLILIILLSVVVWKLLTIVTANRNRMENFVYHNS AFHHSRSDLSEKNQPATLGTR CPiVF DNA sequence (SEQ ID NO: 77) atggggacaattattcagttcttggtggtgtcttgcctgctggcaggagcggggtccctggatccagccgccctcatgcagataggtgtgattcccaccaa cgtcagacagctcatgtattataccgaagccagctcagcatttatcgtcgtgaaattgatgcctacgattgacagcccaatatctggttgtaatattacatcaa taagttcttataatgctacagtcacgaaacttctccagcctataggcgaaaatctcgaaacgattaggaatcagcttatacccacaagaagaagaagacga tttgctggcgtcgttatcggtcttgccgcactgggtgtcgctactgcagcccaagtgacagcggctgtcgcactcgtgaaggctaacaagaacgctgcag ctattctcaacctgaaaaatgcgatacagaaaactaacaccgctgtcgcagatgttgtccaggcgacacaatctctgggtacggcagttcaagctgttcag gaccacatcaatagcgttgtgtctccggccattaccgccgcgaactgtaaggcacaggatgcaattataggaagtatccttaacctctacctcactgaact cacgacaatcttccataaccagataacgaacccagctctgtcaccgataacaattcaggcgctccgaatcttgcttggctcaacccttccgacggttgtgg aaaagtcttttaacacgcagataagtgctgcagaactgcttagctctggtttgcttacaggtcaaatagtcggactggatctgacctatatgcagatggtgat taaaatcgagctgcctaccctgactgtccagccagcaacccaaattatcgaccttgcaactataagcgcctttatcaacaaccaagaagtcatggctcagt tgcccaccagagtcattgtgactggctccctgatacaggcttaccctgcaagtcagtgtacaattacgccgaacacggtgtattgcagatacaacgacgc acaagttctctctgatgatacaatggcatgtctgcaaggaaatctgacaaggtgtacgttcagtccggtcgtggggtctttcctcacccgcttcatgttgtttg atggcatagtgtatgcgaattgtcggtcaatgctctgcaagtgtatgcagcctgctgcggttatactccagccctccagcagcccggtgactgtgatcgat atgtataagtgcgtttcacttcagcttgacaacctgaggttcacaataacccaattggcgaacgtgacttataattctacgattaaacttgagactagccagat ccttccgatcgatcctctggatataagccaaaacctggcggcagtgaataaaagtctcagtgatgccttgcagcacctggcccaatcagacacgtatcttt ccgctataacctcagccacgacaacaagtgttctttccataatggcaatctgtcttggtagtctgggtctcatactcattatactgcttagcgttgtggtctgga agctgcttacgattgtcaccgcaaatcgcaatcgaatggagaactttgtctatcacaacagcgcattccaccatagtcgatccgacttgagcgaaaaaaac cagcctgctactctcgggactcgc7158-105382-02

[0554] Construction of PCMN-1557 is illustrated in FIG. 30. PCR primers used for inserting donor DNA into the PCMN-1557 E3 module are shown in Table 6. The E3 module was first modified to remove E3-ORF2 using Gibson assembly with DNA amplified using primer-extension PCR with primers al and a2. The CPiVF DNA sequence was then inserted into E3-ORF1 using Gibson assembly with DNA amplified using primer-extension PCR with primers bl and b2, which was combined with the E3-ORF2-deleted shuttle vector plasmid amplified by PCR with primers cl and c2. Included in this insert is the upstream pVIII-E3-ORFl DNA sequence that was modified to remove potential E3-ORF1 start codons while maintaining the pVIII coding sequence. The sequence of the modified E3 region of PCMN-1557 is shown below (SEQ ID NO: 78).

[0555] Table 6. PCR primers for insertion of donor DNA

[0556] Primer Sequence SEQ ID NO: al cgccgCTTGTCAAATAAACTTACCTAATTTTTGCTAAGACGTCTG 46 a2 acaagCGGCGGCGATGAAGAAGC 47 bl tctgtcgacggctacgactgagATGGGGACAATTATTCAGTTCTTG 54 b2 acaagtttaataatattatctaGCGAGTCCCGAGAGTAG 59 cl TAGATAATATTATTAAACTTGTTTTACAGCTACCACC 50 c2 ctcagtcgtagccgTCGACAGAGTTGGTTACAATGTC 51

[0557]

[0558] PCMN-1557 E3 region (SEQ ID NO: 78)

[0559] Modified sequence (bold), CPiVF insertion (italics), location of genome deletion (underline) ccagaggtataccttaaccctttttcaggaccaccggacacctttcctgatcagttcattcctaactacgacattgtaaccaactctgtcgacggctacgact gagatggggacaattattcagttcttggtggtgtcttgcctgctggcaggagcggggtccctggatccagccgccctcatgcagataggtgtgattccc accaacgtcagacagctcatgtattataccgaagccagctcagcatttatcgtcgtgaaattgatgcctacgattgacagcccaatatctggttgtaat attacatcaataagttcttataatgctacagtcacgaaacttctccagcctataggcgaaaatctcgaaacgattaggaatcagcttatacccacaag aagaagaagacgatttgctggcgtcgttatcggtcttgccgcactgggtgtcgctactgcagcccaagtgacagcggctgtcgcactcgtgaaggct aacaagaacgctgcagctattctcaacctgaaaaatgcgatacagaaaactaacaccgctgtcgcagatgttgtccaggcgacacaatctctggg tacggcagttcaagctgttcaggaccacatcaatagcgttgtgtctccggccattaccgccgcgaactgtaaggcacaggatgcaattataggaagt atccttaacctctacctcactgaactcacgacaatcttccataaccagataacgaacccagctctgtcaccgataacaattcaggcgctccgaatctt gcttggctcaacccttccgacggttgtggaaaagtcttttaacacgcagataagtgctgcagaactgcttagctctggtttgcttacaggtcaaatagtc ggactggatctgacctatatgcagatggtgattaaaatcgagctgcctaccctgactgtccagccagcaacccaaattatcgaccttgcaactataa gcgcctttatcaacaaccaagaagtcatggctcagttgcccaccagagtcattgtgactggctccctgatacaggcttaccctgcaagtcagtgtaca attacgccgaacacggtgtattgcagatacaacgacgcacaagttctctctgatgatacaatggcatgtctgcaaggaaatctgacaaggtgtacgt tcagtccggtcgtggggtctttcctcacccgcttcatgttgtttgatggcatagtgtatgcgaattgtcggtcaatgctctgcaagtgtatgcagcctgctg cggttatactccagccctccagcagcccggtgactgtgatcgatatgtataagtgcgtttcacttcagcttgacaacctgaggttcacaataacccaat tggcgaacgtgacttataattctacgattaaacttgagactagccagatccttccgatcgatcctctggatataagccaaaacctggcggcagtgaat aaaagtctcagtgatgccttgcagcacctggcccaatcagacacgtatctttccgctataacctcagccacgacaacaagtgtctttccataatggc aatctgtcttggtagtctgggtctcatactcattatactgcttagcgttgtggtctggaagctgctacgattgtcaccgcaaatcgcaatcgaatggaga actttgtctatcacaacagcgcattccaccatagtcgatccgacttgagcgaaaaaaaccagcctgctactctcgggactcgctagataatattattaa acttgttttacagctaccaccataatgcgcttcagcttcttcatcgccgccgcttgtcaaataaacttacctaatttttgctaagacgtctgggtcctgcgtttct The CPiVF E3 plasmid is designated E3-596 and has the features shown in FIG. 31. The sequence of the E3-596 plasmid is set forth herein as SEQ ID NO: 17.7158-105382-02

[0560] CAVSLIC was used to construct the pCMN-1557 whole viral genome vaccine composition that expresses CPiVF from the endogenous viral E3 transcriptional architecture in place of the CAV E3-ORFs. The ASMM-150 plasmid was then digested with Bglll and Gibson assembled with the linear product of the CPiVF E3-596 plasmid (SEQ ID NO: 17) amplified with si and s2 primers. See Table 3 for the primer sequences. Final constructs were selected using ampicillin resistance. A map of the PCMN-1557 vaccine construct is shown in FIG. 32.

[0561] E. coli DHIOb cells were used to transform and amplify the shuttle vector and assembled virus genome. MDCK cells (such as from ATCC) were used as the host cell line for construct transfection and virus production. Transfection was done using Lipofectamine 3000 using 2 pg DNA per 6-well. Virus successfully grown in MDCK cells with MEM10 media was transferred to freshly plated MDCK cells for virus propagation.

[0562] Example 6: Construction of PCMN-1558 using CAVSLIC

[0563] This example describes the production of the recombinant CAV PCMN-1558, which contains a heterologous ORF encoding the hemagglutinin-neuraminidase (HN) protein of CPiV (CPiVHN) inserted into E3-ORF1, and a deletion of E3-ORF2 (AE3-ORF1 [CPiVHN], AE3-ORF2). The vector sequence of PCMN-1558 is set forth herein as SEQ ID NO: 9.

[0564] Core, El, and E4 plasmid modules were constructed as described in Examples 1 and 2 (see also FIGS. 7-8). The parent CAV E3 plasmid module was cloned and constructed as described in Example 1 (see also FIGS. 7 and 13), and then modified to encode the CPIVHN gene by replacing the endogenous CAV E3 ORFs using the same E3 genomic placements and general cloning strategy as described for CDVH in FIGS. 4-5 and Example 2 (although the heterologous ORF primer specific sequences differ).

[0565] The CPiVHN protein is encoded by a DNA of 1695 bp. The amino acid sequence is based on the D008 challenge strain obtained from the USDA. Alignment with public NCBI sequences shows the CPiVHN amino acid sequence is most similar to GenBank accession AFE48527 from hemagglutininneuraminidase protein of mammalian rubulavirus 5 (GenBank accession JQ743328), with differences in amino acids 288 and 447 (SEQ ID NO: 79). The CPiVHN gene (SEQ ID NO: 80) was PCR amplified from a gBlock™ gene fragment that was codon-optimized for Canis familiaris using the Integrated DNA Technologies DNA codon optimization tool.

[0566] CPiVHN amino acid sequence (SEQ ID NO: 79) MVAEDAPVRGTCRVLFRTTTLIFLCTLLALSISILYESLITQKQIMSHAGSTGSNSRLGSITDLLNNI LSVANQIIYNSAVALPLQLDTLESTLLTAIKSLQTSDKLEQNCSWGAALINNNRYINGINQFYFSIA EGRNLTLGPLLNIPSFIPTATTPEGCTRIPSFSLTKTHWCYTHNVILNGCQDHVSSNQFVSMGIIEP TSAGFPSFRTLKTLYLSDGVNRKSCSISTVPGGCMMYCFVSTQPERDDYFSTAPPEQRIIIMYYND TIVERIINPPGVLDVWATLNPGTGSGVYYLGWVLFPIYGGVIKDTSLWNNQANKYFIPQMVAAL CSQNQATQVQNAKSSYYSSWFGNRMIQSGILACPLQQDLTNECLVLPFSNDQVLMGAEGRLYM YGDSVYYYQRSNSWWPMTMLYKVTITFTNGQPSAISAQNVPTQQVPRPGTGDCSATNRCPGFC LKGVYADAWLLTNPSSTSTFGSEATFTGSYLNAATQRINPTMYIANNTQIISSQQFGSSGQEAAY SHTTCFRDTGSVMVYCIYIIELSSSLLGQFQIVPFIRQVTLS7158-105382-02

[0567] CPiVHN DNA sequence (SEQ ID NO: 80) atggtcgctgaggatgcgccagttaggggaacctgtcgcgtgctcttccgaaccaccacgctcatattcctctgcaccttgcttgcgctttcaatctcaatct tgtatgagtcacttataacacagaaacaaattatgtctcatgctgggtctacaggatcaaactccaggctgggtagtatcactgatctcttgaataatattctta gtgtggcgaaccagattatctataactcagcggttgcgctccctctccagttggatactctggagagcaccctgttgactgctattaagtctcttcaaaccag tgacaagctcgagcagaactgtagctggggcgcggctctgattaataacaacagatatattaacggcataaatcaattttatttctccattgccgaaggtag aaacctgaccctgggacctttgctcaatatccctagcttcataccaacggcgaccacccctgagggctgtactcgcattccatcctttagtcttaccaaaac acactggtgctacacccataatgttatccttaatggatgccaagatcacgtctcatcaaaccagttcgtctctatgggtataatcgaacccaccagcgctgg cttcccatcatttcgaacattgaagacgctttatctttccgatggtgtcaaccgaaagtcctgctctatttctactgtgccgggagggtgcatgatgtactgctt tgtttccacgcagcctgaacgagacgattattttagtacggctccgccggagcaacggataataatcatgtactataacgatacgatagtggaaagaataa tcaatcctcctggtgttctcgacgtgtgggcgacgttgaatcctggaaccggatctggcgtctattatcttggatgggttctgtttcctatttacggcggagtta ttaaagacacaagcttgtggaataatcaagctaataaatatttcattccacaaatggtcgcagccttgtgttcccaaaaccaggcgacccaagttcaaaacg caaaaagttcctattacagttcatggttcgggaatcgcatgattcaatcaggcattctggcctgcccgttgcagcaggacttgacgaacgaatgtctggtcc ttcctttttccaacgaccaagttctgatgggggctgagggtagactttacatgtacggagattccgtttactactaccagcggagcaattcttggtggccgat gaccatgttgtataaagtcacaatcactttcactaatgggcagccgagcgccatttccgctcagaatgtccccacccaacaagttcctcgaccaggtacag gcgattgtagtgcgactaacagatgccctggtttttgccttaagggcgtttacgctgacgcatggttgttgacaaatccatctagtacgagtacttttgggag cgaggccacattcacgggctcatacctgaatgctgcaacgcaacgcataaaccctacaatgtatatcgccaacaacacgcaaataattagtagccaaca gtttggatcatctgggcaggaggcagcctacagtcatacgacgtgtttccgggacaccgggtctgtgatggtgtattgtatctacataatagagttgagtag ttcactcttggggcagttccagatcgtcccattcattaggcaagtgacgctctct

[0568] Construction of PCMN-1558 is illustrated in FIG. 33. PCR primers used for inserting donor DNA into the PCMN-1558 E3 module are shown in Table 7. The E3 module was first modified to remove E3-ORF2 using Gibson assembly with DNA amplified using primer-extension PCR with primers al and a2. The CPiVHN DNA sequence was then inserted into E3-ORF1 location using Gibson assembly with DNA amplified using primer-extension PCR with primers bl and b2, which was combined with the E3-ORF2-deleted shuttle vector plasmid amplified by PCR with primers cl and c2. Included in this insert is the upstream pVIII-E3-ORFl DNA sequence that was modified to remove potential E3-ORF1 start codons while maintaining the pVIII coding sequence. The sequence of the modified E3 region of PCMN-1558 is shown below (SEQ ID NO: 81).

[0569] Table 7. PCR primers for insertion of donor DNA

[0570] Primer Sequence SEQ ID NO: al cgccgCTTGTCAAATAAACTTACCTAATTTTTGCTAAGACGTCTG 46 a2 acaagCGGCGGCGATGAAGAAGC 47 bl tctgtcgacggctacgactgagATGGTCGCTGAGGATGC 60 b2 acaagtttaataatattatctaAGAGAGCGTCACTTGCCTAATG 61 cl TAGATAATATTATTAAACTTGTTTTACAGCTACCACC 50 c2 ctcagtcgtagccgTCGACAGAGTTGGTTACAATGTC 51

[0571]

[0572] PCMN-1558 E3 region (SEQ ID NO: 81)

[0573] Modified sequence (bold), CPiVHN insertion ( italics) . location of genome deletion (underline) ccagaggtataccttaaccctttttcaggaccaccggacacctttcctgatcagttcattcctaactacgacattgtaaccaactctgtcgacggctacgact gaggtggtcgctgaggatgcgccagttaggggaacctgtcgcgtgctcttccgaaccaccacgctcatattcctctgcaccttgcttgcgctttcaatct caatcttgtatgagtcacttataacacagaaacaaattatgtctcatgctgggtctacaggatcaaactccaggctgggtagtatcactgatctcttga ataatattcttagtgtggcgaaccagattatctataactcagcggttgcgctccctctccagttggatactctggagagcaccctgttgactgctattaag7158-105382-02 tctcttcaaaccagtgacaagctcgagcagaactgtagctggggcgcggctctgattaataacaacagatatattaacggcataaatcaatmatttc tccattgccgaaggtagaaacctgaccctgggaccmgctcaatatccctagcttcataccaacggcgaccacccctgagggctgtactcgcattcc atcctttagtcttaccaaaacacactggtgctacacccataatgttatcctaatggatgccaagatcacgtctcatcaaaccagttcgtctctatgggta taatcgaacccaccagcgctggcttcccatcatttcgaacattgaagacgcmatctttccgatggtgtcaaccgaaagtcctgctctamctactgtg ccgggagggtgcatgatgtactgctttgtttccacgcagcctgaacgagacgattattttagtacggctccgccggagcaacggataataatcatgta ctataacgatacgatagtggaaagaataatcaatcctcctggtgttctcgacgtgtgggcgacgttgaatcctggaaccggatctggcgtctattatct tggatgggttctgtttcctatttacggcggagttataaagacacaagcttgtggaataatcaagctaataaatatttcattccacaaatggtcgcagcc ttgtgttcccaaaaccaggcgacccaagttcaaaacgcaaaaagttcctattacagttcatggttcgggaatcgcatgattcaatcaggcattctggc ctgcccgtgcagcaggactgacgaacgaMgtctggtccttcctttttccaacgaccaagttctgatgggggctgagggtagactttacatgtacgg agattccgmactactaccagcggagcaattcttggtggccgatgaccatgttgtataaagtcacaatcactttcactaatgggcagccgagcgccat ttccgctcagaatgtccccacccaacaagttcctcgaccaggtacaggcgatgtagtgcgactaacagatgccctggtttttgccttaagggcgttta cgctgacgcatggttgttgacaaatccatctagtacgagtactmgggagcgaggccacattcacgggctcatacctgaatgctgcaacgcaacgc ataaaccctacaatgtatatcgccaacaacacgcaaataattagtagccaacagtttggatcatctgggcaggaggcagcctacagtcatacgac gtgtttccgggacaccgggtctgtgatggtgtatgtatctacataatagagttgagtagttcactcttggggcagttccagatcgtcccattcattaggc fltfgtgflcgcmmrtagataatattattaaacttgttttacagctaccaccataatgcgcttcagcttcttcatcgccgccgcttgtcaaataaacttacctaattt ttgctaagacgtctgggtcctgcgtttct

[0574] The CPiVHN E3 plasmid is designated E3-597 and has the features shown in FIG. 34. The sequence of the E3-597 plasmid is set forth herein as SEQ ID NO: 18.

[0575] CAVSLIC was used to construct the pCMN-1558 whole viral genome vaccine composition that expresses CPiVF from the endogenous viral E3 transcriptional architecture in place of the CAV E3- ORFs.

[0576] The ASMM-150 plasmid was then digested with Bglll and Gibson assembled with the linear product of the CPiVHN E3-597 plasmid (SEQ ID NO: 18) amplified with si and s2 primers. See Table 3 for the primer sequences. Final constructs were selected using ampicillin resistance. A map of the PCMN-1558 vaccine construct is shown in FIG. 35.

[0577] E. coli DHIOb cells were used to transform and amplify the shuttle vector and assembled virus genome. MDCK cells (such as from ATCC) were used as the host cell line for construct transfection and virus production. Transfection was done using Lipofectamine 3000 using 2 (1g DNA per 6-well. Virus successfully grown in MDCK cells with MEM10 media was transferred to freshly plated MDCK cells for virus propagation.

[0578] Example 7. Transfection and propagation of recombinant CAV virus vaccine compositions in canine cells

[0579] Viral genome plasmids described in the above examples were propagated in E. coli, DNA isolated and transfected into canine cells to propagate and produce CAV virus vaccines. Upon transfection into mammalian cells, the RSV promoter in tire viral plasmid backbone drives expression of the I-Scel enzyme which releases and linearizes tire CAV genome from the bacterial plasmid sequences at the left and right ITRs so the CAV genome can be replicated. Thus, there is no need to cut, isolate and purify the CAV genome for transfection into canine cells and viral production. Virus successfully grown in MDCK cells with MEM10 media was transferred to freshly plated MDCK cells for virus propagation.

[0580] The following workflow and schema were used to produce and characterize the replication and production of CAV virus vaccine compositions from the examples above (FIG. 36).7158-105382-02

[0581] Transfection (per well of 6-well plate, using Lipofectamine3000)

[0582] 1. Plate 500,000 MDCK cells per well of 6-well plate in 2.5 mL MEM+10%FBS (heat inactivated).

[0583] Zero to 4 hours after plating, add DNA for transfection.

[0584] 2. Tube 1:

[0585] a. Add 2000 ng plasmid DNA to 1.5 ml tube

[0586] b. Add 100 (1L Opti-MEM (or other media without serum)

[0587] c. Add 8 |1L P3000 reagent (from Lipofectamine 3000 Transfection Reagent kit) 3. Tube 2:

[0588] a. Add 100 )1L Opti-MEM (or other media without serum)

[0589] b. Add 12 |1L Lipofectamine 3000 reagent (from Lipofectamine 3000 Transfection Reagent kit)

[0590] 4. Thoroughly vortex tubes #1 and #2. Briefly spin tubes in microfuge.

[0591] 5. Transfer 100 |1L from tube #2 to tube #1. Thoroughly vortex tube #1. Briefly spin tube #1 in microfuge. Let tube #1 sit for 10-20 minutes.

[0592] 6. To well of 6-well plate, dropwise add contents of tube #1 to the well.

[0593] 7. Return cells to incubator.

[0594] 8. Exchange ~1.5 mL media with -1.5 mL fresh MEM+10%FBS every 2-3 days.

[0595] 9. Plaques should be visible starting from day 3 to day 5. Cells are typically wiped out between days 7 to 14. Continue exchanging media every 2-3 days.

[0596] 10. Harvest 6-well, using small cell scraper, transferring media and cells into a 15 mL conical tube. Virus expansion (10 cm)

[0597] 1. Split MDCK cells to one 10 cm plate in lOmL DMEM+10%FBS.

[0598] 2. When cells are -90% confluent, change media to MEM+10%FBS.

[0599] 3. Freeze-thaw tire 15 ml conical tube (containing tire 6-well lysate) 3 times. Dry-ice / 95%etlianol bath and 37°C water bath work well for tire freeze-thaw process. Freezing in a -80°C freezer also works for the freeze step. Be careful to avoid ethanol / water near the cap of the tube. Spin lysate 1000g for 5 minutes to pellet tire cell debris.

[0600] 4. Transfer 1.25 ml lysate (half of 6-well lysate) to 10 cm plate of MDCK cells.

[0601] 5. If cells take longer than 3 days for full cytopathic effect, exchange half of media with fresh MEM+10%FBS every 2-3 days.

[0602] 6. Harvest 10 cm plate, using small cell scraper, transferring media and cells into a 50 mL conical tube.

[0603] Virus expansion (2x15 cm)

[0604] 1. Split MDCK cells to two 15 cm plates in 16 mL DMEM+10%FBS.

[0605] 2. When cells are -90% confluent, change media to MEM+10%FBS.7158-105382-02

[0606] 3. Freeze-thaw the 50 ml conical tube (containing the 10 cm lysate) 3 times. Dry-ice / 95%ethanol bath and 37°C water bath work well for the freeze-thaw process. Freezing in a -80°C freezer also works for the freeze step. Be careful to avoid ethanol / water near the cap of the tube. Spin lysate 1000g for 5 minutes to pellet the cell debris.

[0607] 4. Transfer 2.5 mL lysate (of 10 cm lysate) to two 15 cm plates of MDCK cells.

[0608] 5. If cells take longer than 3 days for full cytopathic effect, exchange half of media with fresh MEM+10%FBS every 2-3 days.

[0609] 6. Harvest 15 cm plates, using large cell scraper, transferring media and cells from both plates into a 50 mL conical tube.

[0610] Virus expansion (15x15 cm)

[0611] 1. Split MDCK cells to 15x15 cm plates in 16 mL DMEM+10%FBS.

[0612] 2. When cells are -90% confluent, change media to MEM+10%FBS.

[0613] 3. Freeze-thaw the 50 ml conical tube (containing the 2x15 cm lysate) 3 times. Dry-ice / 95%ethanol bath and 37°C water bath work well for the freeze-thaw process. Freezing in a -80°C freezer also works for tire freeze step. Be careful to avoid ethanol / water near tire cap of the tube. Spin lysate 1000g for 5 minutes to pellet the cell debris.

[0614] 4. Transfer 2 mL lysate (of 2x15 cm lysate) to fifteen 15 cm plates of MDCK cells.

[0615] 5. If cells take longer than 3 days for full cytopathic effect, exchange half of media with fresh MEM+10%FBS every 2-3 days.

[0616] 6. Har vest 15 cm plates, using large cell scraper, transferring media and cells from 15 plates into a 250 mL conical tube. If cytopathic effect is sporadic across plate, harvest free cells, without the scraper, and replace media with fresh MEM+10%FBS, spinning down cells (200g for 5 minutes) to be combined with additional harvests at later timepoints.

[0617] 7. For non CSCL2 purified virus preparation, freeze the 15x15 cm harvest.

[0618] Materials:

[0619] Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific cat#L3000015)

[0620] Opti-MEM (Gibco cat#51985-034)

[0621] DMEM (Corning cat#10-013-CV)

[0622] MEM (Corning cat#10-010-CM)

[0623] NucleoBond XTra Midi (Macherey-Nagel cat#740410)

[0624] NEB lOBeta competent cells (NEB cat#C3019H)

[0625] Example 8: Productive Exponential Replication and Propagation of Recombinant CAV Vaccine Virus Constructs

[0626] This Example describes an exemplary method to assess whether a recombinant CAV is replication competent. The method includes transfecting canine cells with whole virus genome plasmids (such as those described in the Examples above) so that only a small percentage of cells are transfected.7158-105382-02

[0627] Alternatively, if virus supernatants from cells or purified virus is used, cells should be infected at low multiplicities of infection, such that only a small percentage of cells are initially transduced and secondary infection and spread can be observed by spread to surrounding cells in monolayer over several days. Characteristic ‘plaques’ in cell monolayers are a result of initial virus infection and spread to neighboring cells. The CAV viral life cycle is approximately 48-72 hours and at low MOIs / transfection percentages isolated individual plaques / CPE can be observed in cell monolayers at 2-4 days postinfection. Brightfield images are taken to capture primary, secondary, tertiary infection over an extended time course. Successful replication, cell lysis and virus production are evident if individual virus infected plaques ‘grow’ and spread over time to undergo secondary and tertiary infection that result in widespread cytopathic effects (CPE) and complete disruption and death of MDCK monolayers on successive days.

[0628] Experimental Design and Infection Protocol

[0629] To assess tire ability of the CAV vaccine constructs to undergo productive infection, MDCK cells are transfected with individual vaccine constructs such that a small percentage of cells are initially transduced. This allows for die observation of isolated plaques and subsequent rounds of viral replication and spread in a monolayer.

[0630] The CAV life cycle is known to range between 48 to 72 hours, providing a timeframe for monitoring viral replication and the progression of infection across die cell monolayer.

[0631] Brightfield Imaging and Cytopathic Effects (CPE)

[0632] Brightfield images are captured at various time points post-transfection to evaluate cytopathic effects (CPE), including:

[0633] 1. Primary Replication and Plaque Formation:

[0634] Characteristic plaque-like clearing is observed in the MDCK cell monolayer. These plaques represent localized regions of cell lysis, indicative of initial viral infection and cytotoxicity. The morphology of infected cells shows rounding, detachment, and cellular debris, consistent with CAV-induced cytopathic effects.

[0635] 2. Secondary and Tertiary Infection:

[0636] If the CAV vaccine construct undergoes productive infection and replication, the virus spreads from initially infected cells to adjacent cells, resulting in the expansion of plaques and progressive monolayer clearing. Over successive days, the cytopathic effect spreads across the entire monolayer, demonstrating robust viral replication and propagation.

[0637] 3. Non-Productive Infection:

[0638] In contrast, viruses capable of infecting cells but unable to undergo productive replication fail to spread beyond the initial infection sites. In these cases, early-stage plaques remain localized, and the monolayer remains intact throughout tire extended time course. The ability to observe progressive plaque formation, cytopathic spread, and monolayer clearance provides a quantifiable and reproducible method7158-105382-02

[0639] for assessing the functional integrity of recombinant CAV vaccine candidates, ensuring their suitability for clinical and veterinary applications.

[0640] Example 9. Productive Exponential Replication and Propagation of Recombinant CAV CDVH Vaccine Virus Constructs

[0641] MDCK cells were transfected with PCMN-1507 using the method described in Example 8. B rightfield images were taken on successive days. At 4 days post transfection, the successful replication and propagation of the initially transduced MDCK cells that received CAV-based CDVH vaccine constructs ware characterized by tire following hallmarks (FIG. 37):

[0642] 1. Plaque Formation:

[0643] Distinct plaques were observed within 4 days post-transfection, confirming viral replication and cytopathic effects consistent with productive infection. These are highlighted with circles in FIG. 37.

[0644] 2. Progressive Spread of Infection:

[0645] Infection with replication-competent CAV virus particles led to secondary and tertiary rounds of infection, resulting in extensive monolayer disruption and widespread cytopathic effects across the culture. At 9 days post initial transfection, the CAV vaccine particles produced initially had spread to and killed the entire MDCK monolayer.

[0646] Implications for Vaccine Development

[0647] These findings demonstrate that the CAV-based vaccine compositions described herein are replication-competent, capable of amplifying in infected cells, and able to propagate efficiently across cell cultures. This productive replication is critical for ensuring robust antigen expression, immune stimulation, and effective vaccine performance.

[0648] Example 10: Productive Exponential Replication and Propagation of Recombinant CAV CDVNP Vaccine Virus Constructs

[0649] MDCK cells were transfected with PCMN-1508 using the method described in Example 8. B rightfield images were taken in successive days. At 4 days post transfection, the successful replication and propagation of the initially transduced MDCK cells that received CAV-based CDVNP vaccine constructs were characterized by the following hallmarks (FIG. 38):

[0650] 1. Plaque Formation:

[0651] Distinct plaques were observed within 4 days post-transfection, confirming viral replication and cytopathic effects consistent with productive infection.

[0652] 2. Progressive Spread of Infection:

[0653] Infection with replication-competent CAV vaccine particles led to secondary and tertiary rounds of infection, resulting in extensive monolayer disruption and widespread cytopathic effects across tire culture. At 11 days post initial transfection, tire CAV vaccine particles produced initially had spread to and killed the entire MDCK monolayer.7158-105382-02

[0654] Implications for Vaccine Development

[0655] These findings demonstrate that the CAV-based vaccine compositions described herein are replication-competent, capable of amplifying in infected cells, and able to propagate efficiently across cell cultures. This productive replication is critical for ensuring robust antigen expression, immune stimulation, and effective vaccine performance.

[0656] The ability to observe progressive plaque formation, cytopathic spread, and monolayer clearance provides a quantifiable and reproducible method for assessing the functional integrity of recombinant CAV vaccine candidates, ensuring their suitability for clinical and veterinary applications.

[0657] Example 11: Productive Exponential Replication and Propagation of Recombinant CAV CPiV Vaccine Virus Constructs

[0658] MDCK cells were transfected with PCMN-1556 (FIG. 39), PCMN-1557(FIG. 40) or PCMN-1558 (FIG. 41) using the method described in Example 8. Brightfield images were taken on day 4 and day 9:

[0659] 1. Plaque Formation:

[0660] Distinct plaques were observed within 4 days post-transfection, confirming viral replication and cytopathic effects consistent with productive infection.

[0661] 2. Progressive Spread of Infection:

[0662] Infection with replication-competent CAV vaccine particles led to secondary and tertiary rounds of infection, resulting in extensive monolayer disruption and widespread cytopathic effects across the culture. At 9 days post initial transfection, the CAV CPiV vaccine particles produced initially had spread to and killed the entire MDCK monolayer.

[0663] Implications for Vaccine Development

[0664] These findings demonstrate that the CAV-based vaccine compositions described herein are replication-competent, capable of amplifying in infected cells, and able to propagate efficiently across cell cultures. This productive replication is critical for ensuring robust antigen expression, immune stimulation, and effective vaccine performance.

[0665] The ability to observe progressive plaque formation, cytopathic spread, and monolayer clearance provides a quantifiable and reproducible method for assessing the functional integrity of recombinant CAV vaccine candidates, ensuring their suitability for clinical and veterinary applications.

[0666] Example 12: Recombinant CAV vaccines express CDV payloads from the modified E3 module in infected cells

[0667] Immunofluorescence microscopy was performed to evaluate CDV antigen payload expression by recombinant CAV vaccine compositions (FIG.42). MDCK cells were either uninfected (negative control), transfected with a pcDNA CDVH expression plasmid (positive control) or infected with PCMN-7158-105382-02

[0668] 1508. Cells were fixed with paraformaldehyde, permeabilized and blocked with 5% normal goat serum. Cells were incubated overnight with primary monoclonal antibody at a 1 :40 dilution against CDV envelope proteins (VMRD, catalog: 1C42H11).

[0669] Cells were washed and then stained for 1 hour at room temperature with secondary antibodies, AlexaFluor 488 Goat anti-Mouse IgG (Thermo) 1 :400, and washed. Nuclei were counterstained with DAPI. Cells were imaged using brightfield and fluorescence microscopy.

[0670] A second immunofluorescence experiment was also performed on MDCK cells infected with 10-fold dilutions of either PCMN-1507 or PCMN-1508. Uninfected cell were used as a negative control. Cells infected with PCMN-1507 were fixed with 3.7% formaldehyde and then permeabilized with 0.1% Triton X-100 and blocked with 5% FBS. Cells infected with PCMN-1508 were fixed with methanol / acetone (50 / 50% mix). CDV H and NP were detected with undiluted FITC-conjugated goat antiserum (VMRD, catalog: CJ-F-CDV-10ML). Expression of CDVH and CDVNP was detected in MDCK cells infected with PCMN-1507 or PCMN-1508, respectively (FIG.43). The results of the fluorescence microscopy studies demonstrate payload expression of CDV antigens by vaccine compositions.

[0671] Example 13: Recombinant CAV vaccines express CPiV payloads in infected cells

[0672] MDCK cells were infected with PCMN-1556, PCMN-1557 or PCMN-1558 virus supernatants. PCMN-1366, which expresses mCherry instead of CPiV antigens, was used as a negative control for background staining.

[0673] Cells were fixed with paraformaldehyde 12 hours post infection, permeabilized with 0.1% Triton X-100, blocked with 5% normal goat serum and incubated overnight with primary antibodies. Cells were washed and then stained for 1 hour at room temperature with fluorescent secondary antibodies and washed. Nuclei were counterstained with DAPI. Cells were imaged using fluorescence microscopy.

[0674] Antibodies used to detect CPiV antigens:

[0675] CPIV8-4 mAb hybridoma supernatant Lot 2898-093-19Augl4 (Elanco 070720) against CPiV HN at 1 : 160 dilution. Anti-CPI 1 : 1000 CPI Capture Antibody chicken antiserum Lot 2626-42-11 Iun08 (Elanco 070720).

[0676] As shown in FIG. 44, expression of the CPiV HN is clearly demonstrated and specific to PCMN-1556 and PCMN-1558 virus infected cells, but not PCMN-1557 or PCMN-1366 infected cells or noninfected cells.

[0677] Since CPiVHN is expressed as a P2A fusion with CPiVF as the first heterologous antigen, detection of CPiVHN in PCMN-1556 infected cells demonstrates that CPiVF is also expressed and that the P2A fusion performs as designed and is validated by these data.

[0678] The anti-CPI antibody staining has high background staining (FIG. 45). However, as shown in FIG. 45, the intensity of staining is higher in PCMN-1556, PCMN-1557 and PCMN-1558 infected cells, which express CPI antigens, versus control infected and uninfected cells.7158-105382-02

[0679] Example 14: Vaccination with recombinant CAVs expressing CDV F, H and NP proteins is protective against CDV challenge

[0680] This example describes a study to evaluate whether vaccination with combinations of PCMN-1506 (CAV-CDV F), PCMN-1507 (CAV-CDV H), PCMN-1508 (CAV-CDV N) and / or PCMN-1561 (CAV-CDV F-2A-H) can protect canine subjects against challenge with CDV. PCMN-1506 contains a heterologous ORF encoding the full-length canine distemper virus fusion (F) protein inserted into E3-ORF1, and a deletion of E3-ORF2 (AE3-ORF1[CDVF], AE3-ORF2). PCMN-1561 contains a first heterologous ORF encoding CDVF and a second heterologous ORF encoding CDVH, separated by a P2A coding sequence, inserted into E3-ORF1, and a deletion of E3-ORF2 (AE3-ORF1[CDVF-P2A-CDVH], AE3-ORF2). PCMN-1507 and PCMN-1508 are described in Examples 2 and 3, respectively. The following groups were used in this study:

[0681] Number Target CAV-CDV

[0682] Group Vaccine Route Challenge / route of dogs Dosage (FAIDso)

[0683] T1 6 PCMN-1506 > IO60Oral liquid, up to CDV / IC PCMN-1507 1.4 mL

[0684] PCMN-1508

[0685] T2 6 PCMN-1561 > 1060Oral liquid, up to CDV / IC PCMN-1508 1.4 mL

[0686] T3 3 None None None CDV / IC

[0687]

[0688] Canine subjects in Groups T1 and T2 received two doses of vaccine 21 days apart. All groups were challenged intracranially (IC) with CDV at a targeted lethal target dose of 1025FAIDso 21 days after the second vaccination (DPV2). Serum neutralization titers (SNT) against CAV (CAV2), CDV and ICHV (CAV1) were measured at various timepoints following the first and second vaccinations. The results are shown in FIGS. 46A-46C. As expected, unvaccinated canines in Group T3 did not have detectable SNT against CAV, CDV or ICHV. Animals in Groups T1 and T2 exhibited increasing SNT against CAV, ICHV and CDV over time, although Group T2 showed a delay in developing SNT against CDV.

[0689] In addition, all canines vaccinated with the combination of PCMN-1506, PCMN-1507 and PCMN-1508 were fully protected against challenge with a lethal dose of CDV. Only 2 of 6 animals vaccinated with the combination of PCMN-1508 and PCMN-1561 survived challenge; tlie remaining four animals showed signs of illness and were euthanized between 7 and 9 days post challenge (DPC).

[0690] These data demonstrate that the combination of recombinant CAV expressing monovalent CDV F, H and NP was protective against CDV challenge. Furthermore, the CAV vector elicited neutralizing antibodies against both CAV1 (ICHV) and CAV2.7158-105382-02

[0691] Example 15: Vaccination with recombinant CAVs expressing CDVF and CDVH protects against ICHV challenge

[0692] This example describes a study to evaluate whether vaccination of canines with a combination of PCMN-1506 (CAV-CDV F) and PCMN-1507 (CAV-CDV H) can protect canine subjects against challenge with ICHV (CAV1). The following groups were used in this study:

[0693] Number Target CAV-CDV

[0694] Group Vaccine Route Challenge route of dogs Dosage (FAID50)

[0695] 1 5 PCMN-1506 > IO60Oral, up to 2.0 IV PCMN-1507 mL

[0696] 2 3 None None None IV

[0697]

[0698] Canine subjects in Group 1 received two doses of vaccine 21 days apart. Animals in both groups were challenged intravenously (IV) with ICHV (target dose 10sFAID5o) at day 27 after the second vaccination. Serology was performed weekly post- vaccination.

[0699] All canines in Group 1 survived challenge with ICHV. In contrast, all unvaccinated animals showed signs of illness and were euthanized at 4 or 5 DPC. As shown in FIG. 47, all animals in Group 1 showed increasing levels of SNT against CAV over time.

[0700] Example 16: Vaccination with recombinant CAVs expressing CPiV F and / or CPiV HN

[0701] This example describes a study to evaluate the efficacy of PCMN-1557 (CAV-CPiV F), PCMN-1558 (CAV-CPiV HN), and PCMN-1556 (CAV-CPiV F-2A-HN) in canines. The following groups were used in this study:

[0702] Number of Target CAV-CDV

[0703] Group Vaccine Route / Volume Challenge / route dogs Dosage (FAID50)

[0704] T1 10 PCMN-1557 6.47 logic, Oral liquid CPiV / IN (6M, 4F) PCMN-1558 6.25 logic. 1.0 mL

[0705] T2 10 PCMN-1556 6.39 logw Oral liquid CPiV / IN (5M, 5F) 1.0 mL

[0706] T3 10 None None None CPiV / IN (7M, 3 F)

[0707]

[0708] Canine subjects in Groups T1 and T2 received two doses of vaccine 21 days apart. All groups were challenged intranasally (IN) with CPiV (target dose IO36FAID50) 21 days after the second vaccination. SNT against CPiV were measured at various timepoints following the first and second vaccinations. As shown in FIG. 48A, vaccination with either the combination of PCMN-1557 and PCTM-1558, or with PCMN-1556 alone, led to an increase in SNT over time. Nasal titers of CPiV were also measured up to 10 days post challenge (DPC). As shown in FIG. 48B, the T1 and T2 groups did not7158-105382-02

[0709] have detectable CPiV titers above baseline, while CPiV titers in unvaccinated animals peaked at approximately 4 DPC.

[0710] These data demonstrate that immunization with either the combination of monovalent vaccines (CAV-CPiV F and CAV-CPiV HN) or the divalent vaccine (CAV-CPiV F-2A-HN) was highly effective for producing neutralizing antibodies and eliminating virus shedding after CPiV challenge.

[0711] Example 17: Post- vaccination immune response

[0712] This example describes a study to monitor the immune response post-vaccination and to determine if canines respond to annual revaccination. This study also evaluated lower doses of recombinant CAV vaccines (104to IO55) and compared vaccination with a single dose to vaccination with two doses. The following groups were used in this study:

[0713] Number Target Dosage

[0714] Group Vaccine 1stDose 2ndDose of dogs (FAID50)

[0715] 1 10 PCMN-1506 (CAV-CDVF) IO40Study Study Day PCMN-1507 (CAV-CDVH) IO40Day O 21 PCMN-1556 (CAV-CPiV F-2A-HN) 1040

[0716] MLV CPV* IO5'5

[0717] 2 10 PCMN-1506 (CAV-CDVF) 1050Study Study Day PCMN-1507 (CAV-CDVH) 1050Day O 21 PCMN-1556 (CAV-CPiV F-2A-HN) IO50

[0718] MLV CPV IO55

[0719] 3 4 PCMN-1506 (CAV-CDVF) IO50Study None PCMN-1507 (CAV-CDVH) IO50Day O

[0720] PCMN-1556 (CAV-CPiV F-2A-HN) IO50

[0721] MLV CPV IO55

[0722] 4 6 Control None None None

[0723]

[0724] * MLV CPV is an approved canine parvovirus vaccine

[0725] Canines in groups 1 and 2 received two doses of vaccine 21 days apart. Canines in group 3 received only one dose of vaccine. On Study Day 201 animals were challenged with CPiV at a target dose of IO36FAID50.

[0726] As shown in FIG. 49A, animals hr all vaccinated groups produced neutralizing antibodies against CPiV. Animals vaccinated once or twice at a dose of 10shad higher levels of CPiV neutralizing antibodies compared to animals vaccinated twice at a dose of 104. Also, a single vaccination at a dose of 105resulted in the greatest reduction in virus shedding, although all vaccinated groups exhibited a reduction in virus shedding (FIG. 49B). In addition, all vaccinated groups produced neutralizing antibodies against CAV (FIG.49C), ICHV (FIG.49D), CPV (FIG. 49E) and CDV (FIG. 49F).7158-105382-02

[0727] Example 18: CDV challenge / vaccine booster at one year

[0728] After approximately one year (Study Day 369-370), the animal groups from Example 17 were sub-divided to receive a booster vaccination or to be challenged IC with CDV. Control dogs were sub-divided and challenged IC or IV / IN.

[0729] No. Target Booster CDV Group Treatment

[0730] Dogs Dosage Vaccination Challenge

[0731] 5 PCMN-1506 (CAV-CDVF) IO40SD 369 NA PCMN-1507 (CAV-CDVH) IO40

[0732] 1

[0733] PCMN-1556 (CAV-CPiV F-2A-HN) IO40

[0734] 5 NA SD 370 MLV CPV 105 S

[0735] PCMN-1506 (CAV-CDVF) IO50

[0736] 5 SD 369 NA PCMN-1507 (CAV-CDVH) IO50

[0737] 2

[0738] PCMN-1556 (CAV-CPiV F-2A-HN) IO50

[0739] 4 NA SD 370 MLV CPV io5-5

[0740] PCMN-1506 (CAV-CDVF) 1050

[0741] PCMN-1507 (CAV-CDVH) 1050

[0742] 3 4 SD 369 NA PCMN-1556 (CAV-CPiV F-2A-HN) IO50

[0743] MLV CPV io5-5

[0744] 3 NA IC SD 370 4 Control None

[0745] 3 NA IV SD 370

[0746]

[0747] Canines were vaccinated as described in Example 18. After approximately one year, the study groups were subdivided to receive either a booster vaccination or to be challenged IC with CDV. Control animals were subdivided and challenged IC or IV / IN. FIG. 50A shows SNT of canines from one day prior to tire first and second vaccinations (-1DPV1, -1DPV2); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); and 7 days, 21 days, and 28 days after the second vaccination (7DPV2, 21DPV2, 28DPV2). FIG. 50B shows SNT of boosted animals only at 0, 21 and 43 days after tire boost vaccination (ODPV, 21DPV and 43 DPV).

[0748] Example 19: Comparison of monovalent and divalent constructs for CPiV vaccination

[0749] This example describes a study to evaluate protection against CPiV using monovalent and divalent vaccines. The following groups were used in this study:7158-105382-02

[0750] Target Vaccination Number Challenge Group Vaccine Dosage Route,

[0751] of dogs Route (FAID50) Volume

[0752] T1 8 PCMN-1557 (CAV-CPiVF) IO5.5Oral, 1.0 mL IN T2 8 PCMN-1558 (CAV-CPiVHN) IO5.5Oral ,1.0 mL IN PCMN-1557 (CAV-CPiVF)

[0753] T3 8 IO5.5Oral, 1.0 mL IN PCMN-1558 (CAV-CPiVHN)

[0754] T4 8 PCMN-1556 (CAV-CPiV F-2A-HN) None Oral, 1.0 mL None T5 8 Control None None IN

[0755]

[0756] Animals were either unvaccinated (Control) or vaccinated orally with PCMN-1557 (CAV-CPiVF) alone, PCMN-1558 (CAV-CPiVHN) alone, PCMN-1557 + PCMN-1558, or PCMN-1556 (CAV-CPiVF-2A-HN) alone, at a dose of 1055. Animals received two doses of vaccine 21 days apart. All animal groups were challenged IN with CPiV (target dose of IO36FAID50) 21 days after the final vaccination. FIG. 51A shows CAV SNT at one day prior to the first and second vaccinations (-1DPV1, -1DPV2); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); 7 and 14 days after the second vaccination (7DPV2, 14DPV2); one day prior to challenge (-1DPC); and 14 days after challenge (14DPC). FIG. 51B shows CPiV SNT at the same timepoints as FIG. 51 A. FIG. 51C shows CPiV nasal titers one day prior to challenge and 2 to 10 days post-challenge.

[0757] Example 20: Comparison of monovalent and divalent constructs for CDV vaccination

[0758] This example describes a study to evaluate protection against CDV using monovalent vaccines. The following groups were used in this study:

[0759] Target Vaccination Number Challenge Group Vaccine Dosage Route,

[0760] of dogs Route (FAID50) Volume

[0761] T1 11 PCMN-1507 (CAV-CDVH) IO50Oral ,1.0 mL IC T2 5 Control None None IC

[0762]

[0763] Animals received two doses of vaccine 21 days apart at a target dose of 105oFAID5o, followed by IC challenge with CDV (target dose of 1028FAID50) 21 days after the second vaccination. FIG. 52A shows CAV SNT at one day prior to the first and second vaccinations (-1DPV1, -1DPV2); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); 7 and 14 days after the second vaccination (7DPV2, 14DPV2); one day prior to challenge (-1DPC); and 21 days after challenge (21DPC). FIG. 52B shows CDV SNT at one day prior to the first and second vaccinations (-1DPV1, -1DPV2); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); 7 and 14 days after the second vaccination (7DPV2, 14DPV2); and one day prior to challenge (-1 DPC).7158-105382-02

[0764] Example 21: Minimum effective dose of CAV-CPiV-HN

[0765] This example describes a study to determine the minimum effective dose of the CAV-CPiV-HN vaccine.

[0766] Number Target Dosage Dose Vaccination Challenge

[0767] Group

[0768] of dogs (FAIDso)* Volume Day Day

[0769] 1 10 IO500.5 mL 0 21

[0770] 2 10 io4-30.5 mL 0 21

[0771] 3 10 io360.5 mL 0 21

[0772] 4 5 None None 0 21

[0773]

[0774] *Titers were measured by IFA of CPiV HN rather than CAV

[0775] Animals were vaccinated with IO50, IO43or IO36of modified live CAV2-CPiV-HN virus + IO96CFU B. bronchiseptica. Placebo control animals were administered IO96CFU of B. bronchiseptica. Animals received two doses of vaccine 21 days apart, followed by intranasal challenge with CPiV (target dose of 1036FAID50) 21 days after the final vaccination. FIG. 53A shows CPiV nasal titers one day prior to challenge and 1 to 10 days post-challenge. FIG. 53B shows CPiV SNT one day prior to the first vaccination (-1DPV1); 7 and 14 days after the first vaccination (7DPV1, 14DPV1); one day prior to challenge (-1DPC); and 7 and 14 days after challenge (7PDC, 14DPC). FIG. 53C shows CAV SNT at the same timepoints as FIG. 53B.

[0776] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

7158-105382-02CLAIMS1. A recombinant canine adenovirus (CAV), wherein the genome of the recombinant CAV comprises:a complete or partial deletion of an E3 region; anda first heterologous open reading frame (ORF) encoding a first antigenic protein inserted at the site of the complete or partial deletion of the E3 region,wherein the first antigenic protein is from a pathogen that infects a veterinary species, and wherein the recombinant CAV is replication-competent.

2. The recombinant CAV of claim 1, wherein the complete or partial deletion of the E3 region comprises a partial deletion of an E3-ORF1 coding sequence.

3. The recombinant CAV of claim 2, wherein the partial deletion of the E3-ORF1 coding sequence excludes a 5’ region of the E3-ORF1 that overlaps with a 3’ region of the pVIII ORF.

4. The recombinant CAV of claim 3, wherein the 5’ region of tire E3-ORF1 that overlaps with the 3’ region of the pVIII ORF comprises one or more nucleotide substitutions, wherein the one or more nucleotide substitutions eliminate one or two alternative start codons, and wherein the one or more nucleotide substitutions do not result in any amino acid substitutions in the pVIII protein.

5. The recombinant CAV of any one of claims 1-4, wherein the first heterologous ORF comprises a start codon and / or a Kozak consensus sequence.

6. The recombinant CAV of any one of claims 1-5, wherein the complete or partial deletion of the E3 region further comprises a complete or partial deletion of an E3-ORF2 coding sequence.

7. The recombinant CAV of any one of claims 1-6, wherein the E3 region comprises a U exon ORF coding sequence and a splice junction for the fiber ORF.

8. The recombinant CAV of any one of claims 1-7, wherein the E3 region of the recombinant CAV genome does not comprise a heterologous promoter.

9. The recombinant CAV of any one of claims 1-8, wherein the first antigenic protein is a protein from a canine distemper virus (CDV).

10. The recombinant CAV of claim 9, wherein the first antigenic protein is:a CDV hemagglutinin (CDVH) protein or a fragment thereof; or7158-105382-02a CDV nucleoprotein (CDVNP), or a fragment thereof.

11. The recombinant CAV of claim 10, wherein:the amino acid sequence of the CDVH protein is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 64; orthe amino acid sequence of the CDVNP is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 67.

12. The recombinant CAV of any one of claims 1-11, wherein tire nucleotide sequence of the genome of the recombinant CAV is at least 80% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

13. The recombinant CAV of claim 12, wherein the nucleotide sequence of the genome of the recombinant CAV comprises SEQ ID NO: 1 or SEQ ID NO: 3.

14. The recombinant CAV of claim 12 or claim 13, wherein the nucleotide sequence of the genome of the recombinant CAV consists or consists essentially of SEQ ID NO: 1 or SEQ ID NO: 3.

15. The recombinant CAV of any one of claims 1-8, wherein the first antigenic protein is a protein from a canine parainfluenza virus (CPiV).

16. The recombinant CAV of claim 15, wherein the first antigenic protein is:a CPiV fusion (CPiVF) protein or a fragment thereof; ora CPiV hemagglutinin-neuraminidase (CPiVHN) protein, or a fragment thereof.

17. The recombinant CAV of claim 16, wherein:the amino acid sequence of the CPiVF protein is least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 76; orthe amino acid sequence of the CPiVHN protein is least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 79.

18. The recombinant CAV of any one of claims 1-8 and 15-17, wherein the nucleotide sequence of the genome of the recombinant CAV is at least 80% identical to SEQ ID NO: 7 or SEQ ID NO: 9.

19. The recombinant CAV of claim 18, wherein the nucleotide sequence of the genome of the recombinant CAV comprises SEQ ID NO: 7 or SEQ ID NO: 9.7158-105382-0220. The recombinant CAV of claim 18 or claim 19, wherein the nucleotide sequence of the genome of the recombinant CAV consists or consists essentially of SEQ ID NO: 7 or SEQ ID NO: 9.

21. The recombinant CAV of any one of claims 1-20, further comprising a second heterologous ORF encoding a second antigenic protein inserted at the site of the complete or partial deletion of the E3 region, wherein the second antigenic protein is a protein from a pathogen that infects canines.

22. The recombinant CAV of claim 21, wherein the first antigenic protein and the second antigenic protein are both CPiV proteins.

23. The recombinant CAV of claim 21 or claim 22, wherein the first antigenic protein and the second antigenic protein are CPiVF and CPiVHN.

24. The recombinant CAV of any one of claims 21-23, wherein the first heterologous ORF and tire second heterologous ORF are operably linked by a self-cleaving peptide coding sequence.

25. The recombinant CAV of claim 24, wherein tire self-cleaving peptide is P2A.

26. The recombinant CAV of any one of claims 21-25, wherein the nucleotide sequence of the genome of the recombinant CAV is at least 80% identical to SEQ ID NO: 5.

27. The recombinant CAV of claim 26, wherein the nucleotide sequence of the genome of the recombinant CAV comprises SEQ ID NO: 5.

28. The recombinant CAV of claim 26 or claim 27, wherein the nucleotide sequence of the genome of the recombinant CAV consists or consists essentially of SEQ ID NO: 5.

29. An immunogenic composition comprising the recombinant CAV of any one of claims 1-28 and a pharmaceutically acceptable carrier.

30. The immunogenic composition of claim 29, wherein the composition comprises: a first recombinant CAV encoding a CDVH protein and a second recombinant CAV encoding a CDVNP;a first recombinant CAV encoding a CPiVF protein and a second recombinant CAV encoding a CPiVHN protein;7158-105382-02a first recombinant CAV encoding a CDVH protein, a second recombinant CAV encoding a CDVNP, a third recombinant CAV encoding a CPiVF protein, and a fourth recombinant CAV encoding a CPiVHN protein; ora first recombinant CAV encoding a CDVH protein, a second recombinant CAV encoding a CDVNP, and a third recombinant CAV encoding a CPiVF protein and a CPiV HN protein.

31. The immunogenic composition of claim 29 or claim 30, further comprising a canine parvovirus vaccine, a rabies virus vaccine, an influenza virus vaccine, a canine coronavirus vaccine, a Leptospirosis vaccine and / or a Bordetella bronchiseptica vaccine.

32. The immunogenic composition of any one of claims 29-31, further comprising an adjuvant.

33. The immunogenic composition of any one of claims 29-32, which is formulated for oral and / or mucosal administration.

34. A method of eliciting an immune response in a subject, comprising administering to tire subject an effective amount of the recombinant CAV of any one of claims 1-28 or the immunogenic composition of any one of claims 29-33.

35. The method of claim 34, wherein tire immune response comprises the production of antibodies against CAV, CDV, CPiV, or any combination thereof.

36. The method of claim 34 or claim 35, wherein the recombinant CAV or the immunogenic composition is administered orally and / or mucosally.

37. The method of any one of claims 34-36, wherein the subject is a canine subject.

38. The method of any one of claims 34-37, wherein the recombinant CAV or the immunogenic composition is administered in a single dose.

39. The method of any one of claims 34-37, wherein the recombinant CAV or the immunogenic composition is administered in multiple doses.

40. An immunogenic composition, comprising a pharmaceutically acceptable carrier and a first recombinant canine adenovirus (CAV), wherein the genome of the first recombinant CAV comprises:a complete or partial deletion of an E3 region; and7158-105382-02a heterologous open reading frame (ORF) encoding an antigenic protein inserted at the site of the complete or partial deletion of the E3 region,wherein the antigenic protein is a canine distemper virus (CDV) hemagglutinin (CDVH) protein, a CDV nucleoprotein (CDVNP), a canine parainfluenza virus (CPiV) fusion (CPiVF) protein, or a CPiV hemagglutinin-neuraminidase (CPiVHN) protein,wherein the recombinant CAV is replication-competent41. The immunogenic composition of claim 40, wherein the composition further comprises a second recombinant CAV, a third recombinant CAV and / or a fourth recombinant CAV, wherein the composition comprises:a first recombinant CAV encoding a CDVH protein and a second recombinant CAV encoding a CDVNP protein;a first recombinant CAV encoding a CPiVF protein and a second recombinant CAV encoding a CPiVHN protein;a first recombinant CAV encoding a CDVH protein, a second recombinant CAV encoding a CDVNP protein, a third recombinant CAV encoding a CPiVF protein, and a fourth recombinant CAV encoding a CPiVHN protein; ora first recombinant CAV encoding a CDVH protein, a second recombinant CAV encoding a CDVNP protein, and a third recombinant CAV encoding a CPiVF protein and a CPiV HN protein.

42. The immunogenic composition of claim 40 or claim 41, further comprising a canine parvovirus vaccine, a rabies virus vaccine, an influenza virus vaccine, a canine coronavirus vaccine, a Leptospirosis vaccine and / or a Bordetella bronchiseptica vaccine.

43. An immunogenic composition, comprising:(i) a pharmaceutically acceptable carrier;(ii) a recombinant canine adenovirus (CAV), wherein the genome of the recombinant CAV comprises:a complete or partial deletion of an E3 region; anda heterologous open reading frame (ORF) encoding an antigenic protein inserted at the site of the complete or partial deletion of the E3 region,wherein the antigenic protein is a CPiV hemagglutinin-neuraminidase (CPiVHN) protein, andwherein the recombinant CAV is replication-competent; and(iii) a Bordetella bronchiseptica vaccine.

44. An immunogenic composition, comprising:(i) a pharmaceutically acceptable carrier;7158-105382-02(ii) a recombinant canine adenovirus (CAV), wherein the genome of the recombinant CAV comprises:a complete or partial deletion of an E3 region; anda heterologous open reading frame (ORF) encoding an antigenic protein inserted at the site of the complete or partial deletion of the E3 region,wherein the antigenic protein is a canine distemper virus (CDV) hemagglutinin (CDVH) protein, andwherein the recombinant CAV is replication-competent; and(iii) a canine parvovirus (CPV) vaccine.

45. The immunogenic composition of any one of claims 40-44, further comprising an adjuvant.

46. The immunogenic composition of any one of claims 40-44, which is formulated for oral and / or mucosal administration.

47. A method of eliciting an immune response in a subject, comprising administering to tire subject an effective amount of recombinant canine adenovirus (CAV), wherein the genome of the recombinant CAV comprises:a complete or partial deletion of an E3 region; anda first heterologous open reading frame (ORF) encoding a first antigenic protein inserted at the site of the complete or partial deletion of the E3 region,wherein the first antigenic protein is from a pathogen that infects a veterinary species, and wherein the recombinant CAV is replication-competent.

48. A method of eliciting an immune response in a subject, comprising administering to the subject an effective amount of an immunogenic composition comprising a pharmaceutically acceptable carrier and a recombinant canine adenovirus (CAV), wherein the genome of the recombinant CAV comprises:a complete or partial deletion of an E3 region; anda first heterologous open reading frame (ORF) encoding a first antigenic protein inserted at the site of the complete or partial deletion of the E3 region,wherein the first antigenic protein is from a pathogen that infects a veterinary species, and wherein the recombinant CAV is replication-competent.

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