Paramyxoviridae expression system
11730805 · 2023-08-22
Assignee
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2760/18443
CHEMISTRY; METALLURGY
C12N2750/14034
CHEMISTRY; METALLURGY
C12N2760/16034
CHEMISTRY; METALLURGY
C12N2770/20034
CHEMISTRY; METALLURGY
C12N2750/14022
CHEMISTRY; METALLURGY
C12N2760/16022
CHEMISTRY; METALLURGY
C12N2770/20022
CHEMISTRY; METALLURGY
C12N2760/18421
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to the field of (vector) vaccines, and especially to an enhanced arrangement of nucleotide sequences for expressing a Paramyxoviridae virus containing an exogenous gene of interest. The present invention further concerns related expression cassettes and vectors, which are suitable to express genes of interest, especially antigen encoding sequences. The viral vectors of the present invention are useful for producing an immunogenic composition or vaccine.
Claims
1. A Paramyxoviridae virus vector comprising an expression cassette inserted between the P gene and the M gene, wherein said expression cassette comprises a first nucleotide sequence, wherein said first nucleotide sequence is a heterologous or exogenous nucleotide sequence of interest, and a second nucleotide sequence flanking the 5′ end of the first nucleotide sequence, wherein said second nucleotide sequence is the 5′ non-coding region of an N gene of a Paramyxoviridae virus, and a third nucleotide sequence flanking the 3′ end of the first nucleotide sequence, wherein said third nucleotide sequence comprises or consists of the 3′ non-coding region of an H gene of a Paramyxoviridae virus.
2. The Paramyxoviridae virus vector of claim 1, wherein said expression cassette further comprises an intergenic sequence of a Paramyxoviridae virus flanking the 5′ end of said second nucleotide sequence.
3. The Paramyxoviridae virus vector of claim 2, wherein said third nucleotide sequence further comprises a Kozak sequence between the first nucleotide sequence and the 3′ non-coding region of an H gene of said Paramyxoviridae virus.
4. The Paramyxoviridae virus vector of claim 3, wherein said nucleotide sequences are RNA sequences, and/or wherein said first nucleotide sequence is operably linked to the gene start (GS) sequence included in said third nucleotide sequence and/or to the genome promoter of a Paramyxoviridae virus.
5. The Paramyxoviridae virus vector of claim 4, wherein said Paramyxoviridae virus is a virus of the genus Morbillivirus, and wherein the virus of the genus Morbillivirus is selected from the group consisting of canine distemper virus (CDV) feline morbillivirus (FeMV), and peste-des-petits-ruminants virus (PPRV).
6. The Paramyxoviridae virus vector of claim 5, wherein said Paramyxoviridae virus is a CDV, and wherein the 5′ non-coding region of a gene of a CDV is selected from the group consisting of the 5′ non-coding region of an N gene of a CDV, wherein the 5′ non-coding region of an N gene of a CDV consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:1, and/or wherein the 3′ non-coding region of a gene of a CDV is selected from the group consisting of the 3′ non-coding region of an H gene of a CDV, wherein the 3′ non-coding region of an H gene of a CDV consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:7.
7. The Paramyxoviridae virus vector of claim 6, wherein said Paramyxoviridae virus is a CDV, and wherein the 5′ non-coding region of a gene of a CDV is selected from the group consisting of the 5′ non-coding region of an N gene of a CDV, wherein the 5′ non-coding region of an N gene of a CDV consists of or comprises an RNA sequence selected from the group consisting of 95%, 96%, 97%, 98%, 99% and 100% identical with the sequence of SEQ ID NO:1, and/or wherein the 3′ non-coding region of a gene of a CDV is selected from the group consisting of the 3′ non-coding region of an H gene of a CDV, wherein the 3′ non-coding region of an H gene of a CDV consists of or comprises an RNA sequence selected from the group consisting of 95%, 96%, 97%, 98%, 99% and 100% identical with the sequence of SEQ ID NO:7.
8. A mammalian host cell containing the Paramyxoviridae virus vector of claim 6.
9. A vaccine or pharmaceutical composition comprising a. the Paramyxoviridae virus vector of claim 6, and b. recombinant protein expressed by said vector and/or a virus like particle comprising a plurality of a recombinant protein expressed by said vector, and c. pharmaceutical- or veterinary-acceptable carrier or excipient, said carrier is suitable for oral, intradermal, intramuscular or intranasal application, and d. optionally said vaccine further comprises an adjuvant; and wherein said recombinant protein expressed by the vector is a parvovirus VP2 antigen or an influenza virus envelope protein, wherein said envelope protein is optionally hemagglutinin.
10. The vaccine or pharmaceutical composition of claim 9, wherein the vaccine or pharmaceutical composition comprising: a. the CDV vector of claim 5, and b. a polypeptide or recombinant protein comprising or consisting of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:36, and c. a pharmaceutical- or veterinary-acceptable carrier or excipient, said carrier is suitable for oral, intradermal, intramuscular or intranasal application, d. and optionally an adjuvant; and wherein the vaccine or pharmaceutical composition for use in a method of reducing or preventing the clinical signs or disease caused by an infection with at least one pathogen in an animal, and wherein said infection with at least one pathogen is an infection with CDV and/or an infection with swine influenza virus, wherein the swine influenza virus is a subtype H3 influenza virus, and wherein said subtype H3 influenza virus is a swine influenza virus of the subtype H3N2 or H3N1.
11. The vaccine or pharmaceutical composition according to claim 9, wherein the parvovirus VP2 antigen is CPV VP2 protein or the influenza virus envelope protein is-H3.
12. The vaccine or pharmaceutical composition of claim 9, wherein the vaccine or pharmaceutical composition comprising: a. the CDV vector of claim 5, and b. a polypeptide or recombinant protein comprising or consisting of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:35, c. a pharmaceutical- or veterinary-acceptable carrier or excipient, said carrier is suitable for oral, intradermal, intramuscular or intranasal application, d. and optionally an adjuvant; and wherein the vaccine or pharmaceutical composition for use in a method of reducing or preventing the clinical signs or disease caused by an infection with at least one pathogen in an animal, and wherein said infection with at least one pathogen is an infection with CDV and/or CPV.
13. A kit comprising: a) a syringe or a dispenser capable of administering a vaccine to said animal; and b) the vaccine or pharmaceutical composition of claim 12, and optionally an instruction leaflet.
14. A nucleic acid molecule which encodes the expression cassette of claim 1 and wherein said nucleic acid molecule is a DNA molecule.
15. A DNA construct comprising the DNA molecule according to claim 14.
16. A mammalian host cell containing the nucleic acid molecule of claim 14.
17. An immunogenic composition comprising the nucleic acid molecule of claim 14.
18. A mammalian host cell containing the expression cassette of claim 1.
19. An immunogenic composition comprising the Paramyxoviridae virus vector of claim 1.
20. A method for the preparation of the Paramyxoviridae virus vector of claim 6, wherein said method comprises the steps of: a. providing a host cell expressing a heterologous RNA polymerase; b. transfecting the host cell with the DNA construct of claim 14, and wherein the DNA molecule is transcribed by the heterologous RNA polymerase, and c. isolating the viruses produced by the cells.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
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EXAMPLES
(10) The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
(11) A) CPV-VP2 Expression by CDV Vectors
(12) In an in vitro study using a CDV backbone derived from Lederle vaccine strain with an insert of the sequence of SEQ ID NO:17 between the P gene and the M gene (the vector thus comprising the sequence of SEQ ID NO:21), it was possible to show intracellular expression of canine parvovirus (type 2b) VP2 protein in CDV associated fluorescent focuses. Respective results were also achieved for a corresponding CDV vector (i.e. only differing in the sequence encoding the CPV VP2) encoding a canine parvovirus type 2c (CPV-2c) VP2 protein. Results obtained for both vectors by immunofluorescence indicate strong expression of VP2 protein of CPV in all CDV infected syncytia. VP2, in contrast to CDV antigens, accumulated in nuclei and partially in the cytoplasm of infected cells, which varies during the course of syncytia development and might indicate that CPV-VP2 has transient nucleus trafficking, although no putative nuclear localization sequence has been detected by a routine search within the VP2 gene, nor published in the scientific literature.
(13) B) Genetic Stability
(14) Both CDV vectors of Example 1 A) were evaluated by serial passages involving freeze-thaw cycles at the end of each passage in a Vero cell line (i.e. the CDV production cell line). For assessing the genetic stability of the recombinants, 20 serial cell passages on Vero cells were performed. Sequencing of recombinant clones revealed full genetic stability of the inserted—CPV VP2 sequence and flanking regions. The sequence (incl. mutations) coding for both proteins (at amino acid level) remained unchanged. Additionally, using immunofluoresecence (IF) and western blotting (WB), it was possible to demonstrate that strong VP2 expression was present in the cells infected with CDV-VP2 recombinants during the all passage levels.
(15) C) In Vitro Growth
(16) Both CDV vectors described in Example 1 A) and B) were grown in Vero cells. The peak titres of both recombinants in the roller bottle system were achieved at 144 hours post infection and the titres dramatically increased after freezeing/thawing cycle indicating the cellular association of the virus (similar to parental CDV virus). When compared with parental classical CDV-MLVs, the generated CDV recombinant vaccine candidates have very similar end-titres, acceptable for bio-processing.
(17) In conclusion, both CDV vectors showed good growth kinetics on production in Vero cells, reaching peak titres at 6 days post infection in roller bottles. Both vectors showed strong expression characteristics of transgenes (VP2) as determined by IF and WB, qualifying them as a dual CDV-CPV vaccine candidate. In addition, both viruses were tested for genetic stability for 20 cell passages on Vero cells. Both viruses remained fully genetically stable, indicating that are susceptible for vaccine bio-processing.
Example 2
(18) In Vivo Efficacy Study in Swine:
(19) For the purpose of this experiment a group of 6 piglets were vaccinated with 1×10.sup.5 TCID50 of the recombinant CDV vector encoding CPV-2b VP2 (CDV-VP2-2b) of Example 1 at study day (SD) 0 and boosted with 2×10.sup.4 TCID50 at SD21. Both applications were performed intramuscularly (IM) in the neck (2 mL volume each dose).
(20) Four animals served as negative control and were inoculated with Vero cell homogenates (2 mL) in the neck. Three animals served as sentinels and did not receive any treatment.
(21) Inclusion Criteria: Pigs clinically healthy and normally developed for age
(22) Exclusion Criteria: Pigs with injuries, congenital abnormalities or clinical signs of disease which interfered with the study as assessed by the Study Director or designee Pigs with a weak body condition (weight <5 kg)
(23) All study animals were housed in facilities appropriate for their age and were kept under same controlled conditions. The room had either plain flooring or alternatively partly-perforated flooring suitable for the age of the animals with four pens of approximately 20 m.sup.2 each. Before vaccination, the sentinel animals were removed from the group, kept in a separate pen, and were regrouped 12 h after vaccination.
(24) Rooms were under negative pressure (−75 Pa) for biosafety reasons. Room temperature was according to the requirements of the age of the animals. Ventilation rate was approximately 9/h. Animals had 12 h light at >80 lux and 12 h of no light. Environmental enrichment for the animals was provided. Water and feed was available ad libitum in appropriate quality and was managed in accordance with the pigs' requirements at their age and standard procedures.
(25) Laboratory results showed that the CDV vectors replicated in limited fashion in swine host, targeting lymphatic cells. The virus was detected mostly in lymph nodes, spleen or tonsils.
(26) Furthermore, serum virus neutralization tests were performed to measure neutralizing antibodies to CDV and CPV, respectively, in the tests animals.
(27) In the following, the protocol of the serum neutralization test for CDV is given:
(28) Cell Preparation 1 Day Before Neutralization Test
(29) Trypsinize highly confluent Vero cells and resuspend them in an appropriate volume of MEM Earle's media containing 5% FCS Count cells in suspension and adjust to 7×10.sup.5 cells/10 mL per 96-well plate One plates is sufficient for 4 samples. Seed 100 μL of cell suspension/well into all wells of 96-well plates Incubate plates at 37° C. in CO.sub.2 incubator for 16-24 h
Limited Dilution of Serum Samples Pre-dilute samples 1:4 with sterile PBS Inactivate prediluted sera at 56° C. for 30 minutes Add 60 μL of MEM Earle's media supplemented with 1% 100× Pen/Strep to each well of an empty 96-well plate from column 1-11 Add an additional 36 μL of MEM Earle's media supplemented with 1% 100× Pen/Strep to column 1 (A1-H1) Add 24 μL of heat-inactivated serum sample to column 1 (corresponds to 1:5 dilution) (For each serum sample add 24 μL to 3 wells of column 1 to test serum in triplicates) Perform twofold serial dilutions of sera by transferring 60 μL from column to column Repeat until column 11 (corresponding to 1:5120 dilution) Discard 60 μL from the last column
Dilution of CDV-Virus Dilute CDV-virus to 200 TCID.sub.50/100 μL in MEM Earle's media supplemented with 1% 100× Pen/Strep, calculate 6 mL per 96-well plate.
Incubation of Serum Samples with CDV-Virus Add 60 μL of diluted virus to all wells of column 1-11 of the 96-well plates containing sera dilutions. Start adding virus in column 11. Gently agitate plates and incubate for 2 hours at 37° C., 5% CO.sub.2
Incubation of Serum Sample +/−CDV-Virus on v.d.s Cells
(30) Remove 60 μL media from 1 day old v.d.s cells cells in 96-well plates After completion of the 2 hours incubation time transfer 100 μL of each well from the serum-virus mixture to the corresponding wells of the v.d.s cell plates column 1-11 Add 100 μL of MEM Earle's media containing 1% 100× Pen/Strep to all wells of column 12 (=cell only control) Incubate plates for 3 days at 37° C., 5% CO.sub.2 Read results by light microscopy observing the virus-induced cytopathic effect.
(31) The results for the serum neutralization test for CDV are presented in
(32) The serum neutralization test regarding CPV was performed respectively, the results of which are presented in
(33) Importantly, all sentinel animals remained CDV and CPV sero-negative until the end of the study, indicating that the recombinant CDV viruses were not spread from vaccinated animals to non-vaccinated animals upon vaccination.
Example 3
(34) Immunogenicity in H3N2-MDA-Positive Piglets:
(35) In this study, an Hemagglutinin (HA)-specific IgG ELISA and an IFNγ-ELISpot assay were employed for investigating the humoral and cellular immune response of swine influenza maternally derived antibody (MDA)-positive animals vaccinated with a CDV vector encoding H3 of SwIV.
(36) The vaccination of the animals was performed in accordance with Example 2 with the differences that: the insert included in the CDV backbone had the sequence of SEQ ID NO:19 (thus the vector comprising the sequence of SEQ ID NO:22 encoding H3 of SwIV), the vector (named CDV-H3 in the following) was administered to five piglets being H3N2-MDA-positive (MDA+ animals)) and one piglet considered to be (almost) H3N2 seronegative (or having low MDA, respectively), and three piglets served as negative control.
(37) A) HA-Specific IgG ELISA
(38) In the following, the protocol of the ELISA as used is provided:
(39) Flat-bottom 96-well plates (Nunc MaxiSorp #44-2404-21) are coated with 1 μg/mL recombinant influenza hemagglutinin protein (Trenzyme) in carbonate buffer, pH 9.6 overnight at 4° C. On the next day the coating antigen is discarded and plates are washed 5 times with wash buffer (50 mM Tris/0,14M NaCl/0,05% Tween20, pH 8.0) and subsequently incubated. 1 h at room temperature with 200 μL per well of blocking buffer (50 mM Tris/0,14M NaCl/1% BSA, pH 8.0). Sample dilutions are prepared in Sample/Conjugate Diluent (50 mM Tris/0,14M NaCl/0,05% Tween20/1% BSA). Then 100 μL/well of samples and controls (in duplicate) are dispensed into designated wells, covered plates are incubated at RT for 1 hour. Plates are washed 5 times with wash buffer. Afterwards 100 μl of the 1:100.00 diluted HRP-conjugated goat α-Pig IgG-H+L detection antibody (Southern Biotech, Cat #6050-05) is added into each well, and the covered plates are incubated for 1 hour at RT in the dark. After the washing step (5 times with wash buffer), 100 μL/well of the TMB substrate solution (POD, ready to use; Sigma #T4444) is added and the plates are incubated for 5-15 min at RT in the dark. The reaction is stopped by adding 50 μL/well of stop solution (2N sulfuric acid) and the absorbance measured at 450 nm at the ELISA reader (Synergy 2, Biotek).
(40) Using this ELISA, it was seen by means of samples taken from the animals on study day 45 (SD45) that the animals were medium responsive towards the vaccination (animals 1 and 5, see
(41) B) IFNγ-ELISpot
(42) Additionally, cellular immune response from CDV-H3 vaccinated animals was measured using ELISPOT.
(43) In the following, the protocol of the ELISpot assay as used is provided:
(44) ELISpot plates (96 wells) are coated with purified anti-IFN-γ antibody for at least 12 h. PBMC are thawed, washed twice in PBS (no calcium, no magnesium), counted using trypan blue, and adjusted in RPMI medium to be dispensed to 3×10.sub.5 cells/well. After extensive washing of the plate containing the coating antibody with PBS, plate is blocked at least for 1 hour at 37° C. and 5% CO2. Cell culture medium is added containing 3 μg/ml of the polyclonal activator ConA (positive control of IFNγ release) or with different concentrations of the antigen, followed by seeding of the PBMCs. Wells containing only medium or unstimulated cells serve as negative controls. After stimulation for 48 h, plates are washed first two times with water then with PBS/0.01% Tween20. Plates are incubated at room temperature for 1,5 h using a detection biotinylated anti-IFNγ antibody, diluted in PBS/0.01% Tween20/0,1% BSA. Subsequently, streptavidin-alkaline phosphatase enzyme diluted in PBS/0.01% Tween20/0,1% BSA is added to the plates (in the dark, for 45 min incubation). Finally, NBT and 5-Bromo-4-Chloro-3-Indolyl Phosphate are used as substrate of the alkaline phosphatase (development phase takes place also in the dark). These substrate systems produce an insoluble NBT diformazan end product that is blue to purple in color and can be measured in a plate reader. After extensive washing with running tap water, plates are left overnight to ensure complete drying. Spot counting is performed using the C.T.L. ELISpot reader.
(45) In the assay, recombinant H3 protein was used to stimulate isolated PBMC from the animals vaccinated with CDV-H3 recombinant. As shown in
Example 4
(46) Vaccine Efficacy Study
(47) Porcine epidemic diarrhea (PED) is a highly contagious swine disease that can have tremendous economic impact. While all age classes of pigs are susceptible to infection, severe clinical signs and mortality are mainly seen in suckling piglets. The causative agent is PED virus (PEDV), an enveloped, single positive-stranded RNA-virus of the genus Alphacoronavirus within the Coronaviridae virus family. In Europe, PEDV first occurred in the late 1970ies in England. Afterwards it spread through whole Europe causing sporadic outbreaks. In the late 1990ies, PEDV had disappeared from the European pig farms as evidenced by very low seroprevalence and non-existent disease reporting. Outbreaks and endemic infections were still reported from Asia where the disease has high impact on the productivity of industrialized pig farms. Starting from 2005, PED cases were again reported from Europe, i.e. Italy. After the introduction of an apparently highly virulent PEDV into the United States in 2013, cases were also reported from Central Europe, including Germany and neighboring countries. The latter cases were caused by related but distinct PEDV strains (so-called S-INDEL strains). In Germany, cases were reported starting from May 2014 with high morbidity and variable lethality in suckling pigs.
(48) This study, in which a CDV backbone derived from Lederle vaccine strain with an insert of the sequence of SEQ ID NO:38 (encoding a PEDV Spike protein) between the P gene and the M gene (the vector thus comprising the sequence of SEQ ID NO:41) was tested as vector vaccine (named hereinafter “CDV_PEDV-Spike vaccine” or “CDV PEDV-Spike vector vaccine”, respectively), included six sows and their offspring.
(49) All animals were checked for PEDV by RT-qPCR targeting the S-gene, and PEDV-specific antibodies. Only negative animals were enrolled in the study.
(50) Three treatment groups (see below) received randomly assigned animals: Group 1 (negative control): Two sows (designated #1 and #2), unvaccinated Group 2 (positive control): Two sows (designated #3 and #4), unvaccinated Group 3 (CDV_PEDV-Spike): Two sows (designated #5 and #6), vaccinated with CDV_PEDV-Spike vector vaccine.
(51) The vaccination of the two sows of group 3 was done according to the following scheme, wherein the stock titer of the CDV_PEDV-Spike vaccine, defined by endpoint titration, was 7,94×10.sup.4 TCID50/ml:
(52) 9 weeks prior to expected farrowing date: each of the two sows received 4 ml of the vaccine intranasally (2 ml in each nostril);
(53) 6 weeks prior to expected farrowing date: each of the two sows received 4 ml of the vaccine intranasally (2 ml in each nostril);
(54) 3 weeks prior to expected farrowing date: each of the two sows received 4 ml of the respective vaccine intranasally (2 ml in each nostril) and additionally 2 ml intramuscularly.
(55) Piglets born to sows of group 1 (13 piglets of sow #1 and 12 piglets of sow #2) were orally mock-inoculated. Piglets born to sows of group 2 (12 piglets of sow #3 and 14 piglets of sow #4), and group 3 (5 piglets of sow #5 and 15 piglets of sow #6) were orally challenged with a PEDV field strain (named “PEDV EU” hereinafter) at an age of 4 days of life.
(56) For inoculation of piglets of groups 2 and 3, cell culture adapted PEDV EU was used. The titer was 2.15×10.sup.5 TCID50/ml. Piglets of groups 2 and 3 were orally inoculated. In this case, each piglet received 1 ml of a 1:10 diluted viral stock (titer 2.15×10.sup.4 TCID50) using 2 ml syringes.
(57) Piglets of group 1 were orally mock-inoculated using 1 ml cell culture medium in 2 ml syringes.
(58) During the whole trial, rectal swabs (COPAN plain swabs without medium) were taken at the day of inoculation and on day 1 to 10 post inoculation (pi) as well as day 14, 17 and 20/21 pi of all animals for RT-qPCR analyses. Additional rectal swabs were taken from 4 piglets of each sow prior to inoculation and two days post challenge for bacteriological examination. Moreover, clinical signs indicative for PED were recorded daily using the established standardized cumulative score system (see below). Blood samples were taken at the day of inoculation and day 14 and 20/21 pi (end of trial) or the day of euthanasia or death of the respective animal.
(59) Clinical Monitoring
(60) The established cumulative clinical score was used for daily monitoring for clinical signs indicative for PED (see table below).
(61) TABLE-US-00001 TABLE 1 Cumulative clinical score for clinical signs indicative for PED General Feed intake/ Gastrointestinal Score behaviour suckling symptoms 0 Agile, attentive, Greedy suckling, Physiological no abnormalities good filled stomach, feces intake of piglet feed 1 Slight depression Slow suckling, Pasty feces, hardly interested vomiting in piglet feed 2 Depression, Reluctant feed intake, Watery feces, isolaton hardly interested in reddened anal from group, suckling/piglet feed, region, vomiting vocalisation sunken flanks (moaning) 3 Lateral position, Total anorexia, Watery feces with signs of severe decreasing of milk blood or fibrin added, dehydration, low production of sow highly reddened anal body temperature region, vomiting
Sample Preparation and Nucleic Acid Extraction
(62) Rectal swabs were submerged in 1 ml Dulbecco's Modified Eagle Medium and incubated for 1 hour at room temperature. Viral RNA was extracted using either the QIAmp ViralRNA Mini Kit (Qiagen) or the NucleoMagVet-Kit in combination with the KingFisher extraction platform. The RNA was stored at −20° degree until further use.
(63) Blood samples were centrifuged at 2031×g for 20 min at room temperature to obtain serum. The resulting serum was aliquoted and stored at −20° C.
(64) Virus Detection
(65) To detect PEDV shedding, RT-qPCR-systems targeting the S-gene of PEDV were used as previously described (Stadler et al., BMC Vet Res. 11:142 (2015)). Samples taken at days 0 to 7 dpi and at 10 and 20/21 dpi were tested for PEDV-genome. The amount of genome copies/μl was calculated using an in-house standard.
(66) Antibody Detection
(67) A commercial indirect ELISA (INgezim PEDV, INGENASA, Madrid, Spain) was performed with all sera according to the producer's manual.
(68) Bacteriology
(69) Fecal swabs of four piglets per litter were taken at 0 and 2 dpi for differential bacteriology.
(70) Statistics
(71) Shapiro-Wilk test was used for normality testing and a Mann-Whitney rank sum test was conducted as implemented in the software package. Statistical significance was tested using SigmaPlot software.
(72) Results
(73) Antibody Detection in Serum:
(74) All piglets of the CDV group showed positive results in the ELISA (detecting antibodies against PEDV Spike protein) prior to challenge inoculation due to antibody positive colostrum intake, while all animals of the positive and negative control group showed clearly negative results.
(75) At 14 dpi all but three piglets in the positive control group seroconverted, while all animals in the vaccine group showed still high amounts of PEDV specific IgG in serum samples.
(76) At the end of the study all piglets of the CDV group and of the positive control group showed strongly positive results in the ELISA. None of the animals in the negative control seroconverted during the whole trial.
(77) In a further study it was also seen that respective antibody results were likewise achieved when the mother sows were only vaccinated twice via the intranasal route.
(78) Bacteriology:
(79) Fecal swabs taken at 0 and 2 dpi did not show any pathogenic bacteria. The bacterial flora did not undergo significant changes upon infection.
(80) Clinical Signs:
(81) Piglets of the positive control group (group 2) clearly showed clinical signs indicative for PEDV over 7 days starting with vomiting 24 hpi followed by diarrhea. 8 of 26 of the piglets had to be euthanized due to severe dehydration and clinical score values over 6 (humane endpoint). First clinical signs indicative for PEDV were detectable at 36 hpi.
(82) In total, the clinical signs of the CDV vector vaccinated and PEDV challenged piglets (group 3) were better regarding the general behavior and only 2 of 20 (10%) of the pigs of group 3 had to be euthanized due to severe dehydration and clinical score values over 6 (as compared to 31% of the piglets of group 2).
(83) Animals in the negative control stayed healthy during the whole trial.
(84) Shedding of Virus
(85) A clear difference in virus shedding could be detected between the challenged groups. At 1 dpi all challenged piglets were positive for virus genome in rectal swabs, but animals in the CDV-PEDV vaccinated group showed significantly lower PEDV genome copy numbers (mean CT value 32,79), then in challenge group (mean CT value 26,65).
(86) Also, while for the next five days pi, the genome load in rectal swabs of the CDV group was quite similar to the positive control, beginning at 7 dpi the detectable amount of virus genome declined below the cutoff level in piglets protected by the vaccinated sows, while all animals in the positive control group still shed PEDV.
(87) No PEDV genome could be detected in swabs of the negative control group.
(88) In conclusion, the outcome of the study was that piglets born to sows vaccinated with the CDV PEDV-Spike recombinant vaccine showed a reduction of clinical signs, as compared to the positive control, and in particular, a great improvement was seen with regard to the mortality/letality of the piglets. Furthermore, virus shedding after the PEDV challenge was significantly reduced.
(89) All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the following claims.
(90) The following clauses are also disclosed herein: 1. An expression cassette for insertion between two adjacent essential genes (1; 2) of a Paramyxoviridae virus such that the first gene (1) is located in 3′ direction and the second gene (2) is located in 5′ direction of the expression cassette, wherein said expression cassette comprises a first nucleotide sequence, wherein said first nucleotide sequence is a nucleotide sequence of interest, and a second nucleotide sequence flanking the 5′ end of the first nucleotide sequence, wherein said second nucleotide sequence is the 5′ non-coding region of a gene, wherein said gene is selected from the group consisting of the essential genes of a Paramyxoviridae virus excluding the first gene (1), and a third nucleotide sequence flanking the 3′ end of the first nucleotide sequence, wherein said third nucleotide sequence comprises or consists of the 3′ non-coding region of a gene, wherein said gene is selected from the group consisting of the essential genes of a Paramyxoviridae virus excluding the second gene (2). 2. The expression cassette of clause 1, wherein said expression cassette consists of said first nucleotide sequence, and said second nucleotide sequence, and said third nucleotide sequence, and a further nucleotide sequence flanking the 5′ end of said second nucleotide sequence or flanking the 3′ end of said third nucleotide sequence, wherein said further nucleotide sequence is an intergenic sequence of a Paramyxoviridae virus. 3. The expression cassette of clause 1 or 2, wherein said third nucleotide sequence consists of the 3′ non-coding region of a gene selected from the group consisting of the essential genes of a Paramyxoviridae virus excluding the second gene (2), and a sequence flanking the 5′ end of said 3′ non-coding region, wherein said sequence flanking the 5′ end of said 3′ non-coding region encodes a consensus sequence for initiation or enhancing of translation, and wherein said consensus sequence for initiation or enhancing of translation is optionally a Kozak sequence. 4. The expression cassette of any one of clauses 1 to 3, wherein said two adjacent genes (1; 2) of a Paramyxoviridae virus are selected from the group consisting of the essential genes of a Paramyxoviridae virus, and/or
(91) wherein said essential genes of a Paramyxoviridae virus are the N, P, M, F, H and L gene of a Paramyxoviridae virus, or the N, P, M, F, HN and L gene of a Paramyxoviridae virus, or the N, P, M, F, G, and L gene of a Paramyxoviridae virus. 5. The expression cassette of any one of clauses 1 to 4 for insertion between the P gene and the M gene of a Paramyxoviridae virus, wherein said group consisting of the essential genes of a Paramyxoviridae virus excluding the first gene (1) is the group consisting of the essential genes of a Paramyxoviridae virus excluding the P gene of a Paramyxoviridae virus, and wherein said gene is optionally selected from the group consisting of the N, M, F, H and L gene of a Paramyxoviridae virus, and said group consisting of the essential genes of a Paramyxoviridae virus excluding the second gene (2) is the group consisting of the essential genes of a Paramyxoviridae virus excluding the M gene of a Paramyxoviridae virus, and wherein said gene is optionally selected from the group consisting of the H, P, F, L and N gene of a Paramyxoviridae virus. 6. The expression cassette of any one of clauses 1 to 5, wherein said second nucleotide sequence is the 5′ non-coding region of a gene selected from the essential genes of a Paramyxoviridae virus located in 3′ direction of the expression cassette, excluding the 5′ non-coding region of the first gene (1), and/or said third nucleotide sequence comprises or consists of the 3′ non-coding region of an essential gene of a Paramyxoviridae virus located in 5′ direction of the expression cassette, excluding the 3′ non-coding region of the second gene (2). 7. The expression cassette of any one of clauses 1 to 6, wherein said first nucleotide sequence is operably linked to the gene start (GS) sequence included in said third nucleotide sequence and/or to the genome promoter of a Paramyxoviridae virus. 8. The expression cassette of any one of clauses 1 to 7, wherein said nucleotide sequences are RNA sequences. 9. A Paramyxoviridae virus vector, comprising the expression cassette of any one of clauses 1 to 8. 10. The expression cassette of any one of clauses 1 to 8 or the Paramyxoviridae virus vector of clause 9, wherein said 5′ non-coding region is the 5′ non-coding region of an N gene of a Paramyxoviridae virus, and/or said 3′ non-coding region is the 3′ non-coding region of an H gene of a Paramyxoviridae virus, and/or wherein said expression cassette is inserted between a P gene and an M gene of a Paramyxoviridae virus. 11. A Paramyxoviridae virus vector, comprising an RNA sequence inserted between two adjacent essential genes (1; 2) of a Paramyxoviridae virus such that the first gene (1) is located in 3′ direction and the second gene (2) is located in 5′ direction of said inserted RNA sequence, and wherein said inserted RNA sequence comprises or consists of a first RNA sequence, wherein said first RNA sequence is a nucleotide sequence of interest, and a second RNA sequence flanking the 5′ end of the first RNA sequence, wherein said second RNA sequence is the 5′ non-coding region of a gene, wherein said gene is selected from the group consisting of the essential genes of a Paramyxoviridae virus excluding the first gene (1), and a third RNA sequence flanking the 3′ end of the first RNA sequence, wherein said third RNA sequence comprises or consists of the 3′ non-coding region of a gene, wherein said gene is selected from the group consisting of the essential genes of a Paramyxoviridae virus excluding the second gene (2). 12. The Paramyxoviridae virus vector of clause 11, wherein said third RNA sequence consists of the 3′ non-coding region of a gene selected from the group consisting of the essential genes of a Paramyxoviridae virus excluding the second gene (2), and a sequence flanking the 5′ end of said 3′ non-coding region, wherein said sequence flanking the 5′ end of said 3′ non-coding region encodes a consensus sequence for initiation or enhancing of translation, and wherein said consensus sequence for initiation or enhancing of translation is optionally a Kozak sequence. 13. The Paramyxoviridae virus vector of clause 11 or 12, further comprising a fourth RNA sequence flanking the 5′ end of the second RNA sequence, wherein said fourth RNA sequence is an intergenic sequence of a Paramyxoviridae virus, and/or a fifth RNA sequence flanking the 3′ end of the fourth RNA sequence, wherein said fifth RNA sequence is an intergenic sequence of a Paramyxoviridae virus. 14. The Paramyxoviridae virus vector of any one of clauses 11 to 13, wherein said two adjacent genes (1; 2) of a Paramyxoviridae virus are selected from the group consisting of the essential genes of a Paramyxoviridae virus, and/or
(92) wherein said essential genes of a Paramyxoviridae virus are the N, P, M, F, H and L gene of a Paramyxoviridae virus, or the N, P, M, F, HN and L gene of a Paramyxoviridae virus, or the N, P, M, F, G, and L gene of a Paramyxoviridae virus. 15. The Paramyxoviridae virus vector of any one of clauses 11 to 14, wherein said two adjacent essential genes (1; 2) of a Paramyxoviridae virus are the P gene and the M gene of a Paramyxoviridae virus, and wherein said group consisting of the essential genes of a Paramyxoviridae virus excluding the first gene (1) is the group consisting of the essential genes of a Paramyxoviridae virus excluding the P gene of a Paramyxoviridae virus, and wherein said gene is optionally selected from the group consisting of the N, M, F, H and L gene of a Paramyxoviridae virus, and said group consisting of the essential genes of a Paramyxoviridae virus excluding the second gene (2) is the group consisting of the essential genes of a Paramyxoviridae virus excluding the M gene of a Paramyxoviridae virus, and wherein said gene is optionally selected from the group consisting of the H, P, F, L and N gene of a Paramyxoviridae virus. 16. The Paramyxoviridae virus vector of any one of clauses 11 to 15, wherein said second nucleotide sequence is the 5′ non-coding region of a gene selected from the essential genes of a Paramyxoviridae virus located in 3′ direction of the expression cassette, excluding the 5′ non-coding region of the first gene (1), and/or said third nucleotide sequence comprises or consists of the 3′ non-coding region of an essential gene of a Paramyxoviridae virus located in 5′ direction of the expression cassette, excluding the 3′ non-coding region of the second gene (2). 17. The Paramyxoviridae virus vector of any one of clauses 11 to 16, wherein said 5′ non-coding region is a 5′ non-coding region of an N gene of a Paramyxoviridae virus, and/or said 3′ non-coding region is a 3′ non-coding region of an H gene of a Paramyxoviridae virus. 18. The Paramyxoviridae virus vector of any one of clauses 11 to 17, wherein said first RNA sequence is operably linked to the gene start (GS) sequence included in said third RNA sequence and/or to the genome promoter of a Paramyxoviridae virus. 19. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said Paramyxoviridae virus is a virus of the genus Morbillivirus, and wherein the virus of the genus Morbillivirus is preferably selected from the group consisting of canine distemper virus (CDV), feline morbillivirus (FeMV), and peste-des-petits-ruminants virus (PPRV), and wherein the virus of the genus Morbillivirus is most preferably a canine distemper virus (CDV). 20. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said Paramyxoviridae virus is a CDV, and wherein said 5′ non-coding region of a gene of a CDV is selected from the group consisting of the 5′ non-coding region of an N gene of a CDV, wherein the 5′ non-coding region of an N gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:1, the 5′ non-coding region of a P gene of a CDV, wherein the 5′ non-coding region of a P gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:2, the 5′ non-coding region of an M gene of a CDV, wherein the 5′ non-coding region of an M gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:3, the 5′ non-coding region of an F gene of a CDV, wherein the 5′ non-coding region of an F gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:4, the 5′ non-coding region of an H gene of a CDV, wherein the 5′ non-coding region of an H gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:5, and the 5′ non-coding region of an L gene of a CDV, wherein the 5′ non-coding region of an L gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:6. 21. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said Paramyxoviridae virus is a CDV, and wherein said 3′ non-coding region of a gene of a CDV is selected from the group consisting of the 3′ non-coding region of an H gene of a CDV, wherein the 3′ non-coding region of an H gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:7, the 3′ non-coding region of an N gene of a CDV, wherein the 3′ non-coding region of an N gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:8, the 3′ non-coding region of a P gene of a CDV, wherein the 3′ non-coding region of a P gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:9, the 3′ non-coding region of an M gene of a CDV, wherein the 3′ non-coding region of an M gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:10, the 3′ non-coding region of an F gene of a CDV, wherein the 3′ non-coding region of an F gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:11, and the 3′ non-coding region of an L gene of a CDV, wherein the 3′ non-coding region of an L gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:12. 22. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said second nucleotide sequence or said second RNA sequence is the 5′ non-coding region of an N gene of a CDV, and wherein said 5′ non-coding region of an N gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:1. 23. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said 3′ non-coding region of a gene selected from the group consisting of the essential genes of a Paramyxoviridae virus excluding the second gene (2) is the 3′ non-coding region of an H gene of a CDV, and wherein said 3′ non-coding region of an H gene of a CDV preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:7, and/or said sequence flanking the 5′ end of said 3′ non-coding region sequence encodes a Kozak sequence being 5 to 8 nucleotides in length, and wherein the Kozak sequence preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:13. 24. The expression cassette of any one of clauses 2 to 8, and 19 to 23 or the Paramyxoviridae virus vector of any one of clauses 9, and 13 to 23, wherein said intergenic sequence of a Paramyxoviridae virus is an intergenic sequence of a CDV, and wherein said intergenic sequence of a CDV preferably consists of or comprises an RNA sequence being at least 66% identical with the sequence of SEQ ID NO:14. 25. The expression cassette or Paramyxoviridae virus vector of any one of the preceding clauses, wherein said nucleotide sequence of interest is a gene of interest or an antigen encoding sequence, and/or wherein said nucleotide sequence of interest is non-naturally occurring and/or recombinant. 26. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said nucleotide sequence of interest is recombinant and/or heterologous and/or exogenous. 27. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said nucleotide sequence of interest encodes an antigen from a disease-causing agent, wherein the disease-causing agent is preferably a disease-causing agent capable of infecting a companion animal, such as a canine or feline and/or any other domestic or wild carnivore, or capable of infecting a food producing animal such as swine or cattle. 28. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein the Paramyxoviridae virus is a Paramyxoviridae virus capable of infecting an animal of a first biological family, and the nucleotide sequence of interest encodes an antigen from a disease-causing agent capable of infecting an animal of said first biological family, and wherein said disease-causing agent is preferably different from said Paramyxoviridae virus, and wherein said animal of said first biological family is preferably selected from the group consisting of an animal of the family canidae, an animal of the family felidae and an animal of the family suidae, and wherein said animal of said first biological family is most preferably a canine, feline or swine such as a dog, cat or pig. 29. The expression cassette or Paramyxoviridae virus vector of clause 28, wherein said Paramyxoviridae virus capable of infecting an animal of a first biological family is a CDV and said disease-causing agent capable of infecting an animal of said first biological family is a Canine Parvovirus (CPV), or wherein said Paramyxoviridae virus capable of infecting an animal of a first biological family is a La Piedad Michoacán Mexico virus (LPMV) and said disease-causing agent capable of infecting an animal of said first biological family is a a swine influenza virus (SwIV) or a porcine epidemic diarrhea virus (PEDV). 30. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said nucleotide sequence of interest encodes an antigen from a canine parvovirus (CPV), feline parvovirus (FPV), swine influenza virus (SwIV) or porcine epidemic diarrhea virus (PEDV). 31. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said nucleotide sequence of interest encodes a Protoparvovirus capsid protein, and wherein said Protoparvovirus capsid protein is preferably selected from the group consisting of Carnivore protoparvovirus I (CPV or FPV) capsid protein, Primate protoparvovirus 1 capsid protein, Rodent protoparvovirus 1 capsid protein, Rodent protoparvovirus 2 capsid protein, Ungulate parvovirus 1 (PP capsid protein; or an influenza virus envelope protein, wherein said envelope protein is optionally hemagglutinin and/or wherein said influenza virus is optionally selected from the group consisting of influenza A virus, influenza B virus and influenza C virus, and wherein the influenza A virus is preferably selected from the group of the influenza viruses H3N2, H3N1, H1N1, H1N2, H2N1, H2N3 and H911; or a coronavirus Spike (S) protein, and wherein said coronavirus S protein is preferably selected from the group consisting of Alpaca coronavirus S protein, Alphacoronavirus 1 S protein, Human coronavirus 229E S protein, Human Coronavirus NL63 S protein, Porcine epidemic diarrhea virus (PEDV) S protein, Human coronavirus OC43 S protein, Human coronavirus HKU1 S protein, Murine coronavirus S protein, Severe acute respiratory syndrome-related coronavirus (SARS-CoV) S protein, Middle East respiratory syndrome-related coronavirus (MERS-CoV) S protein and Avian infectious bronchitis virus (IBV) S protein. 32. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said nucleotide sequence of interest encodes a Canine Parvovirus (CPV) VP2 protein, and wherein said CPV VP2 protein preferably comprises or consists of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:35; or an H3-subtype hemagglutinin (H3), in particular H3 of a swine influenza virus, and wherein said H3 preferably comprises or consists of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:36; or a porcine epidemic diarrhea virus (PEDV) spike (S) protein, and wherein said PEDV S protein preferably comprises or consists of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:45. 33. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said nucleotide sequence of interest encodes a Canine Parvovirus (CPV) VP2 protein, and wherein said sequence encoding a CPV VP2 protein preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:15; or an H3-subtype hemagglutinin (H3), preferably H3 of a swine influenza virus, and wherein said sequence encoding H3 preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:16; or a porcine epidemic diarrhea virus (PEDV) spike (S) protein, and wherein said sequence encoding a PEDV S protein preferably consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:37 or SEQ ID NO:38. 34. The expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, wherein said expression cassette consists of or said Paramyxoviridae virus vector comprises a polynucleotide having an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:17 or SEQ ID NO:18; or a polynucleotide having an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:19 or SEQ ID NO:20; or a polynucleotide having an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:39 or SEQ ID NO:40. 35. The Paramyxoviridae virus vector of any one of the preceding clauses comprising an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:21; or an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:22; or an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:41. 36. The Paramyxoviridae virus vector of any one of clauses 13 to 35, further comprising a sixth RNA sequence flanking the 5′ end of the fourth RNA sequence, wherein said sixth RNA sequence consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:23, and/or a seventh RNA sequence flanking the 3′ end of the fifth RNA sequence, wherein said seventh RNA sequence consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:24. 37. A CDV vector comprising a heterologous nucleotide sequence of interest, wherein said heterologous nucleotide sequence of interest encodes a Canine Parvovirus (CPV) VP2 protein. 38. The CDV vector of clause 37, wherein said heterologous nucleotide sequence of interest encoding a CPV VP2 protein consists of or comprises an RNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:15. 39. The CDV vector of clause 37 or 38, wherein said CPV VP2 protein comprises or consists of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:35. 40. The CDV vector of any one of clauses 37 to 39, wherein said heterologous nucleotide sequence of interest is located between a P gene and an M gene of a CDV; and/or said heterologous nucleotide sequence of interest is operably linked to a gene start (GS) sequence located in 3′ direction of said heterologous RNA sequence and/or to the genome promoter of a CDV. 41. The CDV vector of clause 40, wherein said GS sequence is included in an exogenous 3′ non-coding region of a gene of a CDV, and wherein said exogenous 3′ non-coding region of a gene of a CDV preferably flanks the 3′ end of the heterologous nucleotide sequence of interest, and/or wherein said heterologous nucleotide sequence of interest is an RNA sequence of interest. 42. A nucleic acid molecule which encodes the expression cassette or the Paramyxoviridae virus vector of any one of the preceding clauses, and wherein said nucleic acid molecule is preferably a DNA molecule. 43. A DNA molecule, in particular the DNA molecule of clause 42, wherein said molecule comprises (i) a DNA sequence encoding a polypeptide of interest, (ii) a DNA sequence flanking the 3′ end of the sequence of (i) and being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:25, (iii) a DNA sequence flanking the 5′ end of the sequence of (i) and being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:26, and (iv) a DNA sequence flanking the 5′ end of the sequence of (iii) and being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:27. 44. The DNA molecule of clause 43, further comprising (v) a DNA sequence flanking the 5′ end of the sequence of (ii) and being at least 66% identical with the sequence of SEQ ID NO:28, and/or (vi) a DNA sequence flanking the 3′ end of the sequence of (iv) and being at least 66% identical with the sequence of SEQ ID NO:28. 45. The DNA molecule of clause 44, further comprising (vii) a DNA sequence flanking the 3′ end of the sequence of (v) and being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:29, and/or (viii) a DNA sequence flanking the 5′ end of the sequence of (vi) and being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:30. 46. The DNA molecule of any one of clauses 42 to 45, wherein the sequence of (i) is a DNA sequence encoding a Canine Parvovirus (CPV) VP2 protein, and wherein said sequence is preferably a DNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:31, or a DNA sequence encoding an H3-subtype hemagglutinin (H3), preferably H3 of a swine influenza virus, and wherein said sequence is preferably a DNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:32; or a DNA sequence encoding a PEDV S protein, and wherein said sequence is preferably a DNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:42 or SEQ ID NO:43. 47. The DNA molecule of any one of clauses 42 to 46, wherein said DNA molecule comprises a DNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:33; or a DNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:34; or a DNA sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:44. 48. A mammalian host cell containing the expression cassette or Paramyxoviridae virus vector or DNA acid molecule according to any one of the preceding clauses. 49. The expression cassette or Paramyxoviridae virus vector or nucleic acid molecule according to any one of the preceding clauses for use as a medicament, preferably as a vaccine. 50. A DNA construct comprising a DNA molecule according to any one of clauses 42 to 47. 51. An RNA transcript of the DNA construct of clause 50. 52. A cell transfected with the DNA construct of clause 50. 53. A cell transfected with the RNA transcript of clause 51. 54. A method for the preparation of an infectious Paramyxoviridae virus containing a heterologous gene, in particular for preparing the Paramyxoviridae virus vector of any one of clauses 9 to 41, wherein said method comprises the steps of: a. providing a host cell expressing a heterologous RNA polymerase; b. transfecting the host cell with the DNA construct of clause 50, and wherein the DNA molecule is transcribed by the heterologous RNA polymerase, and c. isolating the viruses produced by the cells. 55. Use of the vector of any one of clauses 9 to 41 or of the cell according to any one of clauses 48, 52 and 53 for the manufacture of an immunogenic composition or a vaccine. 56. An immunogenic composition comprising the vector according to any one of clauses 9 to 41, wherein said vector is optionally an infectious and/or attenuated virus or said vector is optionally an attenuated and/or modified live virus, and optionally a recombinant protein expressed by said vector and/or a virus like particle comprising a plurality of a recombinant protein expressed by said vector, and optionally a pharmaceutical- or veterinary-acceptable carrier or excipient, wherein said carrier is preferably suitable for oral, intradermal, intramuscular or intranasal application. 57. The immunogenic composition of clause 56, wherein said recombinant protein expressed by the vector is a parvovirus VP2 antigen such as CPV VP2 protein or an influenza virus envelope protein, wherein said envelope protein is optionally hemagglutinin such as H3. 58. The immunogenic composition of clause 56 or 57, comprising or consisting of the CDV vector of any one of clauses 37 to 41, and wherein said vector is preferably the vector of any one of clauses 19 to 36, and optionally a polypeptide or recombinant protein comprising or consisting of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:35 or SEQ ID NO:36, and optionally a pharmaceutical- or veterinary-acceptable carrier or excipient, wherein said carrier is preferably suitable for oral, intradermal, intramuscular or intranasal application. 59. The immunogenic composition of clause 56 or 57, comprising or consisting of the vector of any one of clauses 19 to 36, and wherein said vector is preferably the CDV vector of any one of clauses 37 to 41, and optionally a recombinant protein expressed by said vector, wherein said recombinant protein comprises or consists of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:35 or SEQ ID NO:36, and optionally a pharmaceutical- or veterinary-acceptable carrier or excipient, wherein said carrier is preferably suitable for oral, intradermal, intramuscular or intranasal application. 60. The immunogenic composition of clause 56, wherein said recombinant protein expressed by said vector is a coronavirus S protein, and wherein said coronavirus S protein is optionally a PEDV S protein. 61. The immunogenic composition of clause 56 or 60, wherein said recombinant protein expressed by said vector is a PEDV S protein comprising or consisting of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:45. 62. A vaccine or pharmaceutical composition comprising a. the vector according to any one of clauses 9 to 41, and b. a recombinant protein expressed by said vector and/or a virus like particle comprising a plurality of a recombinant protein expressed by said vector, and c. a pharmaceutical- or veterinary-acceptable carrier or excipient, preferably said carrier is suitable for oral, intradermal, intramuscular or intranasal application, and d. optionally said vaccine further comprises an adjuvant. 63. The vaccine or pharmaceutical composition according to clause 62, wherein said recombinant protein expressed by the vector is a parvovirus VP2 antigen such as CPV VP2 protein or an influenza virus envelope protein, wherein said envelope protein is optionally hemagglutinin such as H3. 64. The vaccine or pharmaceutical composition of clause 62 or 63, comprising or consisting of a. the CDV vector of any one of clauses 37 to 41, and wherein said vector is preferably the vector of any one of clauses 19 to 36, and b. a polypeptide or recombinant protein comprising or consisting of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:35 or SEQ ID NO:36, and c. a pharmaceutical- or veterinary-acceptable carrier or excipient, preferably said carrier is suitable for oral, intradermal, intramuscular or intranasal application, d. and optionally an adjuvant. 65. The vaccine or pharmaceutical composition of clause 62 or 63, comprising or consisting of a. the CDV vector of any one of clauses 19 to 36, and wherein said vector is preferably the vector of any one of clauses 37 to 41, and b. a recombinant protein expressed by said vector, wherein said recombinant protein comprises or consists of an amino acid sequence being comprising or consisting of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:35 or SEQ ID NO:36, and c. a pharmaceutical- or veterinary-acceptable carrier or excipient, preferably said carrier is suitable for oral, intradermal, intramuscular or intranasal application, d. and optionally an adjuvant. 66. The vaccine or pharmaceutical composition of clause 62, wherein said recombinant protein expressed by said vector is a coronavirus S protein, and wherein said coronavirus S protein is optionally a PEDV S protein. 67. The vaccine or pharmaceutical composition of clause 62 or 66, wherein said recombinant protein expressed by said vector is a PEDV S protein comprising or consisting of an amino acid sequence being at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with the sequence of SEQ ID NO:45. 68. The immunogenic composition according to any one of clauses 56 to 61 or the vaccine or pharmaceutical composition according to any one of clauses 62 to 67, wherein said vector is the vector of clause 27. 69. The immunogenic composition according to any one of clauses 56 to 61 or the vaccine or pharmaceutical composition according to any one of clauses 62 to 67, wherein said vector is the vector of clause 28. 70. The immunogenic composition according to any one of clauses 56 to 61 or the vaccine or pharmaceutical composition according to any one of clauses 62 to 67, wherein said vector is the vector of clause 29. 71. A method for the preparation of an immunogenic composition or a vaccine for reducing the incidence or the severity of one or more clinical signs associated with or caused by an infection, comprising the following steps: a. infecting a mammalian host cell with the vector according to any one of clauses 9 to 41, b. cultivating the infected cells under suitable conditions, c. collecting infected cell cultures, d. optionally purifying the collected infected cell cultures of step c), e. optionally mixing said collected infected cell culture with a pharmaceutically acceptable carrier. 72. The method according to clause 71, wherein said immunogenic composition or said vaccine reduces, in particular in an animal, the severity of one or more clinical signs associated with or caused by an infection with canine distemper virus (CDV) and/or canine parvovirus (CPV) or an infection with an influenza virus, wherein said influenza virus is optionally selected from the group consisting of influenza A virus, influenza B virus and influenza C virus, and wherein the influenza A virus is preferably selected from the group of the influenza viruses H3N2, H3N1, H1N1, H1N2, H2N1, H2N3 and H911, or an infection with a coronavirus, wherein said coronavirus is optionally selected from the group consisting of Alpaca coronavirus, Alphacoronavirus 1, Human coronavirus 229E, Human Coronavirus NL63, Porcine epidemic diarrhea virus (PEDV), Human coronavirus OC43, Human coronavirus HKU1, Murine coronavirus, Severe acute respiratory syndrome-related coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV) and Avian infectious bronchitis virus (IBV). 73. The immunogenic composition according to any one of clauses 56 to 61 or the vaccine or pharmaceutical composition according to any one of clauses 62 to 67 for use in a method of reducing or preventing the clinical signs or disease caused by an infection with at least one pathogen in an animal or for use in a method of treating or preventing an infection with at least one pathogen in an animal, preferably said animal is a companion animal, such as a canine or feline and/or any other domestic or wild carnivore, or a food producing animal such as swine. 74. The immunogenic composition according to any one of clauses 56 to 61 or the vaccine or pharmaceutical composition according to any one of clauses 62 to 67 for use according to clause 73, wherein said infection with at least one pathogen is an infection with CDV and/or CPV or an infection with swine influenza virus, wherein the swine influenza virus is optionally a subtype H3 influenza virus, and wherein said subtype H3 influenza virus is preferably a swine influenza virus of the subtype H3N2 or H3N1 or an infection with PEDV. 75. The immunogenic composition according to any one of clauses 56 to 61 or the vaccine or pharmaceutical composition according to any one of clauses 62 to 67 for use in a method for inducing an immune response against CPV and CDV in an animal, preferably in a canine, or inducing an immune response against swine influenza virus in a pig, wherein the swine influenza virus is optionally a subtype H3 influenza virus, and wherein said subtype H3 influenza virus is preferably a swine influenza virus of the subtype H3N2 or H3N1, or inducing an immune response against PEDV in a pig, in particular in a preferably pregnant sow. 76. The immunogenic composition according to any one of clauses 56, 60 and 61 or the vaccine or pharmaceutical composition according to any one of clauses 62, 66 and 67 for use in a method of reducing or preventing the clinical signs or disease caused by an infection with a PEDV in a piglet, wherein the piglet is to be suckled by a sow to which the immunogenic composition has been adminstered. 77. The immunogenic composition for use according to clause 76, wherein said sow to which the immunogenic composition has been administered is a sow to which the immunogenic composition has been administered while said sow has been pregnant, in particular with said piglet. 78. The immunogenic composition according to any one of clauses 56 to 61 or the vaccine or pharmaceutical composition according to any one of clauses 62 to 67 for use according to any of clauses 73 to 77, wherein said immunogenic composition or said vaccine or pharmaceutical composition is to be administered mucosally, preferably intranasally. 79. The immunogenic composition according to any one of clauses 56 to 61 or the vaccine or pharmaceutical composition according to any one of clauses 62 to 67 for use according to any one of clauses 75 to 77, wherein said immunogenic composition or said vaccine or pharmaceutical composition is to be administered mucosally, preferably intranasally, to said sow. 80. A method of immunizing an animal such as a companion animal, such as a canine or feline and/or any other domestic or wild carnivore, or a food producing animal including swine against a clinical disease caused by at least one pathogen in said animal, said method comprising the step of administering to the animal the immunogenic composition according to any one of clauses 56 to 61 or the vaccine or pharmaceutical composition according to any one of clauses 62 to 67, wherein said immunogenic composition or vaccine fails to cause clinical signs of infection but is capable of inducing an immune response that immunizes the animal against pathogenic forms of said at least one pathogen. 81. The method of clause 80, wherein said at least one pathogen is CDV or CPV or SwIV or PEDV, or wherein said at least one pathogen is CDV and CPV. 82. A method for inducing the production of antibodies specific for PEDV in a sow, wherein said method comprises administering the immunogenic composition according to any one of clauses 56, 60 and 61 or the vaccine or pharmaceutical composition according to any one of clauses 62, 66 and 67 to said sow. 83. A method of reducing or preventing the clinical signs or disease caused by an infection with a PEDV in a piglet, wherein said method comprises administering the immunogenic composition according to any one of clauses 56, 60, and 61 or the vaccine or pharmaceutical composition according to any one of clauses 62, 66 and 67 to a sow, and allowing said piglet to be suckled by said sow. 84. The method of clause 83, wherein said sow is a sow being pregnant, in particular with said pig. 85. The method of clause 83 or 84, comprising the steps of administering the immunogenic composition according to any one of clauses 56, 60, and 61 or the vaccine or pharmaceutical composition according to any one of clauses 62, 66 and 67 to a sow being pregnant with said piglet, allowing said sow to give birth to said piglet, and allowing said piglet to be suckled by said sow. 86. The method of any one of clauses 82 to 85, wherein said immunogenic composition or said vaccine or pharmaceutical composition is administered mucosally, preferably intranasally, to said sow. 87. A kit for inducing an immune response against at least one pathogen in an animal or for vaccinating an animal, preferably a companion animal, such as a canine or feline and/or any other domestic or wild carnivore, or food producing animal such as swine or cattle, against a disease associated with and/or reducing the incidence or the severity of one or more clinical signs associated with or caused by at least one pathogen in an animal, comprising: a) a syringe or a dispenser capable of administering a vaccine to said animal; and b) the immunogenic composition according to any one of clauses 56 to 61 or the vaccine according to any one of clauses 62 to 67, and c) optionally an instruction leaflet, and wherein said at least one pathogen is preferably CDV and/or CPV or wherein said at least one pathogen is optionally SwIV or PEDV. 88. The method of clause 71 or 72, the immunogenic composition or the vaccine or pharmaceutical composition for use according to any one of clauses 73 to 79, the method according to any one of clause 80 to 86 or the kit according to clause 87, wherein said immunogenic composition is the immunogenic composition of clause 68, and wherein said at least one pathogen is said disease-causing agent of which the antigen encoded by the nucleotide sequence of interest is from. 89. The method of clause 71 or 72, the immunogenic composition or the vaccine or pharmaceutical composition for use according to any one of clauses 73 to 79, the method according to any one of clause 80 to 86 or the kit according to clause 87, wherein said immunogenic composition is the immunogenic composition of clause 69, and wherein said at least one pathogen are said Paramyxoviridae virus and said disease-causing agent of which the antigen encoded by the nucleotide sequence of interest is from. 90. The method of clause 71 or 72, the immunogenic composition or the vaccine or pharmaceutical composition for use according to any one of clauses 73 to 79, the method according to any one of clauses 80 to 86 or the kit according to clause 87, wherein said immunogenic composition is the immunogenic composition of clause 70, and wherein said at least one pathogen are said Paramyxoviridae virus and said disease-causing agent of which the antigen encoded by the nucleotide sequence of interest is from. 91. The method of clause 71 or 72, the immunogenic composition or the vaccine or pharmaceutical composition for use according to any one of clauses 73 to 79, the method according to any one of clauses 80 to 86 or the kit according to clause 87, wherein said immunogenic composition is the immunogenic composition of clause 70, and wherein said at least one pathogen are CDV and CPV. 92. The method of any one of clauses 72, 80, 81, and 88 to 91, wherein said animal is a canine, and wherein said canine is preferably a dog. 93. The method of any one of clauses 72, 80, 81, and 88 to 91, wherein said animal is a feline, and wherein said feline is preferably a cat. 94. The immunogenic composition or the vaccine or pharmaceutical composition for use according to any one of clauses 73 to 75, and 88 to 91, wherein said animal is a canine, and wherein said canine is preferably a dog. 95. The immunogenic composition or the vaccine or pharmaceutical composition for use according to any one of clauses 73 to 75, and 88 to 91, wherein said animal is a feline, and wherein said feline is preferably a cat. 96. The kit according to any one of clauses 87, and 88 to 91, wherein said animal is a canine, and wherein said canine is preferably a dog. 97. The kit according to any one of clauses 87, and 88 to 91, wherein said animal is a feline, and wherein said feline is preferably a cat.
REFERENCES
(93) The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 1. Parks C L, Wang H P, Kovacs G R, Vasilakis N, Kowalski J, Nowak R M, Lerch R A, Walpita P, Sidhu M S, Udem S A. 2002. Expression of a foreign gene by recombinant canine distemper virus recovered from cloned DNAs. Virus Res; 83(1-2):131-47. 2. von Messling V, Milosevic D, Devaux P, Cattaneo R. 2004. Canine distemper virus and measles virus fusion glycoprotein trimers: partial membrane-proximal ectodomain cleavage enhances function. J Virol. 78(15):7894-903. 3. Wang X, Feng N, Ge J, Shuai L, Peng L, Gao Y, Yang S, Xia X, Bu Z. 2012 Recombinant canine distemper virus serves as bivalent live vaccine against rabies and canine distemper. Vaccine 30(34):5067-72. doi: 10.1016/j.vaccine.2012.06.001. Epub 2012 Jun. 12. 4. Glover S, Anderson C, Piontkowski M, Terry N (2012) Canine Parvovirus (CPV) type 2b vaccine protect puppies with maternal antibodies to CPV when challenged with virulent CPV 2 C virus. International Journal of Applied Research in Veterinary Medicine 10: 217-224 5. Taguchi M, Namikawa K, Maruo T, Orito K, Lynch J, Sahara H. Antibody titers for canine parvovirus type-2, canine distemper virus, and canine adenovirus type-1 in adult household dogs. The Canadian Veterinary Journal. 2011; 52(9):983-986.