Vaccine 33 (2015) 1375–1381
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Canine distemper virus DNA vaccination of mink can overcome interference by maternal antibodies Trine Hammer Jensen a,b,∗ , Line Nielsen a , Bent Aasted a , Cino Pertoldi b , Merete Blixenkrone-Møller c a Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegårdsvej 15, DK-1870 Frederiksberg C, Denmark b Department of Chemistry and Bioscience, Faculty of Engineering and Sciences, Aalborg University/Aalborg Zoo, Frederik Bajers Vej 7H, DK-9220 Aalborg, Denmark1 c Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 København N, Denmark
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 22 November 2014 Accepted 9 January 2015 Available online 28 January 2015 Keywords: Canine distemper virus Morbillivirus Neonatal Mink Maternal antibodies DNA vaccination
a b s t r a c t Canine distemper virus (CDV) is highly contagious and can cause severe disease against which conventional live vaccines are ineffective in the presence of maternal antibodies. Vaccination in the presences of maternal antibodies was challenged by vaccination of 5 days old and 3 weeks old mink kits with CDV DNA vaccines. Virus neutralising (VN) antibody responses were induced in mink kits vaccinated with a plasmid encoding the haemaglutinin protein (H) of CDV (n = 5, pCDV-H) or a combination of the H, fusion (F) and nucleoprotein (N) of CDV (n = 5, pCDV-HFN). These DNA vaccinated kits were protected against virulent experimental infection with field strains of CDV. The pCDV-H was more efficient in inducing protective immunity in the presence of maternal antibodies compared to the pCDV-HFN. The results show that DNA vaccination with the pCDV-H or pCDV-HFN (n = 4) only given once at 5 days of age induces virus specific immune response in neonatal mink and protection against virulent CDV exposure later in life. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Canine distemper virus (CDV) belongs to the morbillivirus genus along with measles virus (MeV), rinderpest virus, phocine distemper virus, peste-des-petits-ruminants virus and cetacean morbillivirus [1]. CDV can cause high morbidity and mortality and affects an expanding number of carnivores worldwide even jumping species barriers [2]. Disease caused by CDV can effectively be prevented by vaccination with modified live vaccines (MLV) [3] or recombinant vaccines [4–6]. However, in the window of susceptibility when a young animal still have maternal antibodies that can interfere with vaccination but are too low to protect the animal against virulent CDV exposure, conventional vaccine strategies are
Abbreviations: MLV, modified live vaccine; VN, virus neutralising; pCDV-H, plasmid encoding haemaglutinin protein of CDV. ∗ Corresponding author at: Department of Chemistry and Bioscience, Frederik Bajers Vej 7H, 9220 Aalborg, Denmark. Tel.: +45 40 17 51 94. E-mail address: [email protected] (T.H. Jensen). 1 Present address. http://dx.doi.org/10.1016/j.vaccine.2015.01.029 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
inefficient [7]. CDV DNA vaccines have demonstrated some potential to induce CDV virus neutralising (VN) antibody titres after boost with MLV in young dogs with maternal antibodies [8]. Only few studies address MeV DNA vaccination in the presence of surrogate maternal antibodies [9,10]. Maternal antibodies have a different iso-subtype compared to antibodies of adults and thus the outcome of vaccination might not be the same [11]. In general, immaturity of the immune system of neonates and maternal antibodies interfere with many vaccine strategies [11]. At least human infants are believed to be more competent to mount a Tcell than a humoral immune response [9]. MeV DNA vaccination stimulated T-cell immune response in new born macaques [12]. However, in juvenile macaques significant levels of VN antibodies, T-cell immune response and protective immunity was induced by MeV DNA vaccination [13,14]. Immune responses to early vaccination are dependent on many factors including the specific vaccine (type, dosage, interval etc.), the host and age at vaccination and the titres of maternal antibodies but even a small priming of the immune system is important to prevent early life disease [15]. This study is the first to investigate the protective effect of DNA vaccination of neonatal mink (Neovison
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vison), a natural host of CDV, followed by virulent inoculation with a Danish field strain of CDV. A combination of plasmids encoding the haemaglutinin (H), fusion (F) and nucleoprotein (N) of CDV and the H protein alone was used. The mink kits were vaccinated once, twice or four times with 2 weeks intervals as early as 5 days of age in the presence of maternal antibodies. 2. Materials and methods 2.1. Mink Pregnant mink (Neovison) were purchased from two Danish mink farms (GT Mink and J.O.D. Hansen, Denmark). Ten pregnant females were vaccinated with an attenuated live CDV vaccine (Endivac, United Vaccines, WI, USA) and 8 mink were not vaccinated. The care and use of the animals were in accordance with the guidelines and approved by the Animal Experiments Inspectorate, (license 2005/561-999, http://www.foedevarestyrelsen.dk/ english/Animal/Pages/The-Animal-Experiments-Inspectorate. aspx). Three expression plasmids were used for immunisations: (1) plasmid vector pV1J containing an insert of the H gene (1815 base pairs, pCDV-H); (2) pV1J with an insert of the N gene (1573 base pairs, pCDV-N), the H and N genes originated from the Onderstepoort strain of CDV [16–18]; (3) plasmid vector pVR1012 with an insert of the F gene (2204 base pairs) of the Dog/DK91B strain of CDV (pCDV-F) [19]. DNA vaccines encoding the proteins from the Dog/DK91B and Onderstepoort strain of CDV previously induced protection equally well against inoculation with CDV wild type strains [19]. Purified plasmids were suspended in PBS to a concentration of 1 mg DNA/ml. Mink were immunised either with the pCDV-H alone or a combination of the pCDV-H, pCDV-N and pCDV-F (pCDV-HFN, Fig. 1, details in supplemental file). The mink in experiment A were inoculated with CDV wild type strain, Mink/DK2004 (6.5 × 104 TCID50 ) at 3 months of age under anaesthesia. Spleen and lymph nodes from CDV infected Danish farmed mink from 2004 was used for passage of Mink/DK2004 twice before used in this experiment (data not shown). Mink/DK2004 was genetically similar to the CDV mink strains from 2000 to 2002 [20] (data not shown). The mink in experiments B and C was inoculated with a well described Danish field strain of CDV, Dog/DK91B (2.2 × 106 TCID50 ) at the age of 5 and 9 months, respectively [21,22] The inoculation dose was administered partly intraperitoneally (20% suspension) and to the conjunctival and nasal mucosa (10% suspension, details in the supplemental file). In each of the three experiments, 5 non-vaccinated mink were inoculated with non-infectious organ homogenate (mockinoculation). This non-infectious organ homogenate originated from non-vaccinated CDV free farmed mink and the organ homogenate was tested for CDV by reverse transcriptase (RT)-PCR prior to use (data not shown). 2.2. Blood, swabs and tissues analysed for VN antibodies and CDV Blood samples were taken at various time points before and at the time of vaccination and after inoculation with CDV (Fig. 1). Blood samples were taken from the tail vein up to 4 weeks of age; from 6 weeks of age, blood was taken from V. cephalica and at time of euthanasia, blood was taken from anaesthetised mink by cardiac puncture followed by an intracardial barbiturate injection. VN antibody titres were determined in Vero cells using a TCID50 format microtitre assay as described by Blixenkrone-Møller et al. [23] with few modifications. Viraemia (experiments B and C) was
detected by isolation of peripheral blood mononuclear lymphocytes (PBMCs) from heparinised blood followed by intracellular CDV antigen by immunofluorescence assay (IFA) [24]. Lymphocyte counts, percentage of IFN-␥ producing peripheral blood leukocytes (PBLs, experiment A), and intracellular CDV antigen in PBLs (experiment A) and detection of viral RNA in lung and brain by RT-PCR was determined as described previously [17,25]. The mixed conjunctival/nasal swabs taken 14 days after CDV inoculation were titrated by use of Vero cells expressing canine signalling lymphocyte activation molecule (Vero-DST, donated by Yanagi) as described earlier [25]. 2.3. Isolation, stimulation and CDV antigen detection of lymph node lymphocytes Lymph node cells were isolated from the jejunal lymph node in experiment C. Cytospin were made with 100,000 lymphocytes on each slide for antigen detection by IFA [24]. Cell cultures were prepared with 5 × 106 cells to determine PBLs production of IFN-␥ and IL-4 [17]. The lymphocytes (106 cells per well) were stimulated with 50 g phytohaemagglutinin/ml (PHA-L, Sigma) for 40 h in round bottom 96-well microtitre plates. After 40 h a sample of both stimulated and unstimulated lymphocytes were incubated with 0.3 Cu/well methyl-(3 H)’thymidine (Amersham Biosciences, Germany) for 6 h. The stimulation index was calculated as the ratio of the counts of the antigen-containing culture to medium control and values higher than 2.5 were considered significant. All the statistical analysis was conducted by using the software PAST ver.2.17c [26]. No correction for large number of tests conducted was applied, as the tests conducted were not independent to each other [27]. Student’s t-test was used for evaluation of the percentage of IFN-␥ producing PBLs. 3. Results 3.1. Virus neutralising antibodies induced by early life CDV DNA vaccination Mink vaccinated with pCDV-HFN in the presence of maternal antibodies at 3 weeks of age had significantly higher antibody titre levels than those vaccinated with pCDV-H (p < 0.0001) whereas no significant differences were observed at weeks 5 and 7 (p > 0.05, Fig. 2A). At 7 weeks of age, the maternal antibodies of the mink vaccinated with pEmpty had declined to undetectable levels. However, CDV specific VN antibodies were detectable in the mink vaccinated in the presence of maternal antibodies even in the mink vaccinated once (Fig. 2A). The CDV VN antibodies induced in the vaccinated mink persisted until CDV inoculation at 3 months of age. At this time the CDV VN antibody titre of the mink DNA vaccinated in the presence of maternal antibodies was not significantly different from the CDV VN antibody titre of the mink vaccinated in the absence of maternal antibodies (p > 0.05). CDV VN antibody titres were boosted after CDV inoculation in the vaccinated mink (Fig. 2A). The CDV VN antibody titres increased more rapidly in the mink vaccinated with pCDV-H compared to the mink vaccinated with the pCDV-HFN plasmid in the presence of maternal antibodies 6, 14 and 21 days after CDV inoculation (p < 0.0001; Fig. 2A). Experiment B: Vaccination with the pCDV-HFN of 4 weeks old mink induced CDV VN antibody titres almost identical in mink vaccinated with and without maternal antibodies after the second vaccination (at the age of 8 weeks, p > 0.05, Fig. 2B). No differences were found between the mink given pEmpty and PBS in the development of CDV VN antibodies or in the following CDV infection so these 2 groups were merged to simplify interpretation of the results
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Fig. 1. Experimental design. Vaccination (vacc.) of mink kits with DNA plasmids encoding the H, F and N proteins of CDV or the H protein alone at the age 5 days and 3 weeks in the presence and absence of maternal antibodies (experiment A). Vaccination of mink kits with pCDV-HFN at the age of 4, 6, 8 and 10 weeks and challenge inoculation at the age of 20 weeks (experiment B) and 36 weeks (experiment C). The figure is not drawn to scale and the broken lines indicate the differences in number of weeks between vaccination and challenge inoculation.
of experiment B and C. After CDV inoculation the VN antibody titres were boosted to equal levels in mink vaccinated in presence or absence of maternal antibodies (p > 0.05, Fig. 2B). 3.2. Early life CDV DNA vaccination induced a memory T-cell immune response (experiment A) The percentage of IFN-␥ producing PBLs were significantly higher in the pCDV-HFN mink vaccinated in presence of maternal antibodies compared to the other pCDV-HFN vaccinated mink 9 and 14 days after CDV inoculation (p < 0.05, Fig. 3A). The mink vaccinated with pCDV-H in absence maternal antibodies had increased IFN-␥ producing PBLs 9 days after CDV inoculation peaking 14 days after inoculation together with the other pCDV-H vaccinated mink (Fig. 3B). The pEmpty or non-vaccinated vaccinated mink had a significant increase in the percentage of IFN-␥ producing PBLs (p < 0.05) 28 days after CDV inoculation (Fig. 3). 3.3. CDV DNA vaccination early in life protect against virulent CDV inoculation An index of protection was calculated to compare the groups of DNA vaccinated mink with respect to the efficacy of the DNA vaccines in inducing protection against CDV infection (Table 1). Following parameters were included in the index: viraemia, lymphopenia, virus excretion and viral RNA in the respiratory tract and central nervous system (CNS). Each of the parameters was scored with equivalent weights according to the presence and severity of each parameter. A score of 3 represented severe lymphopenia and/or viraemia and/or the finding of viral RNA in the lungs or the brain and/or viral isolation from oculo-nasal swabs. A score of 0 was given to negative values. A score of 1 and 2 was used for intermediate values; 1 representing the lowest levels of viraemia and lymphopenia. The index of protection was calculated by adding the scores of mink within a group of mink vaccinated with the same DNA vaccine divided by the total number of animal observations
within the respective group of mink. All groups of DNA vaccinated kits were substantially protected compared to the pEmpty/nonvaccinated mink (Table 1). Severe parameters of CDV infection in the pEmpty/non-vaccinated mink resulted in an index of protection more than 10 times higher than the index of the most efficiently protected mink. Vaccination with pCDV-H in the presence and absence of maternal antibodies conferred similar levels of protection against CDV inoculation (Table 1). The mink vaccinated with the pCDV-HFN without maternal antibodies had higher levels of viraemia, lymphopenia and spread of virus to the lungs and hence a higher index of protection compared to the mink vaccinated with the H gene alone (Table 1). The mink only vaccinated once at 5 days of age were equally protected to mink vaccinated twice, although results must be carefully interpreted with the low number of mink (n = 2) vaccinated only once. The mink vaccinated 4 times (experiment B) with the pCDV-HFN in the absence and presence of maternal antibodies were protected equally against viraemia and lymphopenia after CDV inoculation (Table 1).
3.4. Long-term protective immunity following early life vaccination with the pCDV-HFN (experiment C) The mink DNA vaccinated 4 times early in life and CDV inoculated at the age of 9 months had CDV VN antibody titres about 1:100 at the age of 9 months (Fig. 4A). No differences were detected in the mink vaccinated in the presence and absences of maternal antibodies so these groups were merged to simplify interpretation. All mink vaccinated with a combination of pCDV-HFN were protected against lymphopenia, viraemia, lymph node infection and immune suppression as measured by lymphocyte stimulation capacity in contrast to the non-vaccinated mink measured 5 and 11 days after CDV inoculation (Fig. 4B). The percentages of IFN-␥ and IL-4 producing PBLs were almost absent in the non-vaccinated mink 5 days
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Fig. 2. CDV specific virus neutralising antibody titres induced by DNA vaccination and inoculation with a wild type strain of CDV. (A) Mink were vaccinated at the age of 5 days and 3 weeks with pCDV-HFN or pCDV-H alone, in the presence (n = 5) and absence (n = 5) of maternal antibodies. Four mink were vaccinated only at 5 days of age. Three mink were given pEmpty in the presence of maternal antibodies. (B) Mink were vaccinated with pCDV-HFN in the presence (n = 8) or absence (n = 6) of maternal antibodies and 6 mink were given either pEmpty or PBS in the presence of maternal antibodies. Serum was collected prior to vaccination at the age of 4, 6, 8 and 10 weeks, prior to CDV inoculation at the age 20 weeks and at the indicated days after CDV inoculation. The horizontal dotted line indicate log10 = 2 that equals a neutralising titre of 100. Values shown are geometric means + standard deviations.
after CDV inoculation (Fig. 4C and D). The DNA vaccinated mink were protected against this decline in cytokine production. 4. Discussion The results of these vaccine experiments show that neonatal CDV DNA vaccination induces CDV VN antibodies and protect mink against virulent CDV inoculation later in life irrespective of the presence of maternal antibodies at time of vaccination. Mink vaccinated only once or twice already at 5 days and 3 weeks of age were not as robustly protected as the mink vaccinated 4 times. Vaccination only at 5 days of age with either CDV-H or CDV-HFN induced CDV VN antibody response and the mink were protected against virulent CDV equally to the mink vaccinated twice (Fig. 2A and Table 1). Also the percentage of IFN-␥ producing PBLs of the mink vaccinated once was similar to the mink vaccinated twice
(Fig. 3). This indicates a distinct capability of the immune system of neonate mink. In contrast to the immaturity of neonate macaques in which vaccination with MeV H did not prime any response and vaccination with MeV H and F primed only a T-cell immune response [12]. Vaccination as early in life as possible is of practical clinical importance for protection of the susceptible offspring against distemper. In addition, early life vaccination will reduce the stress imposed to the mink by handling and decrease the cost and labour associated with several handlings. Previous studies showed four vaccinations induced an effective immune response in young mink vaccinated with pCDV-H [25], in adult mink vaccinated with pCDV-HFN [16,19] and in young dogs vaccinated with a combination of CDV H, F, N genes [28]. In this study, 4 DNA vaccinations induced the most robust protection (Table 1). Two CDV DNA vaccinations of adult mink has previously induced humoral and cellular immunity and partly
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Fig. 3. Percentage of IFN-␥ producing PBLs of mink vaccinated with pCDV-HFN (A) or pCDV-H alone (B). The mink given empty plasmid or left non-vaccinated (pEmpty/nonvacc.) and the mock inoculated mink (mock) were the same in (A) and (B). Table 1 Protective capacity of DNA vaccination with the H, F and N genes of CDV or the H gene alone.
Each box represents one mink. The results from each individual animal are shown in the same sequence from left to right. Black represents the highest score (3) indicating a severe viraemia (>25% PBLs positive for staining with a monoclonal antibody for the CDV N protein by FACS analysis), severe lymphopenia defined as
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Canine distemper virus DNA vaccination of mink can overcome interference by maternal antibodies Trine Hammer Jensen a,b,∗ , Line Nielsen a , Bent Aasted a , Cino Pertoldi b , Merete Blixenkrone-Møller c a Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegårdsvej 15, DK-1870 Frederiksberg C, Denmark b Department of Chemistry and Bioscience, Faculty of Engineering and Sciences, Aalborg University/Aalborg Zoo, Frederik Bajers Vej 7H, DK-9220 Aalborg, Denmark1 c Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 København N, Denmark
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 22 November 2014 Accepted 9 January 2015 Available online 28 January 2015 Keywords: Canine distemper virus Morbillivirus Neonatal Mink Maternal antibodies DNA vaccination
a b s t r a c t Canine distemper virus (CDV) is highly contagious and can cause severe disease against which conventional live vaccines are ineffective in the presence of maternal antibodies. Vaccination in the presences of maternal antibodies was challenged by vaccination of 5 days old and 3 weeks old mink kits with CDV DNA vaccines. Virus neutralising (VN) antibody responses were induced in mink kits vaccinated with a plasmid encoding the haemaglutinin protein (H) of CDV (n = 5, pCDV-H) or a combination of the H, fusion (F) and nucleoprotein (N) of CDV (n = 5, pCDV-HFN). These DNA vaccinated kits were protected against virulent experimental infection with field strains of CDV. The pCDV-H was more efficient in inducing protective immunity in the presence of maternal antibodies compared to the pCDV-HFN. The results show that DNA vaccination with the pCDV-H or pCDV-HFN (n = 4) only given once at 5 days of age induces virus specific immune response in neonatal mink and protection against virulent CDV exposure later in life. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Canine distemper virus (CDV) belongs to the morbillivirus genus along with measles virus (MeV), rinderpest virus, phocine distemper virus, peste-des-petits-ruminants virus and cetacean morbillivirus [1]. CDV can cause high morbidity and mortality and affects an expanding number of carnivores worldwide even jumping species barriers [2]. Disease caused by CDV can effectively be prevented by vaccination with modified live vaccines (MLV) [3] or recombinant vaccines [4–6]. However, in the window of susceptibility when a young animal still have maternal antibodies that can interfere with vaccination but are too low to protect the animal against virulent CDV exposure, conventional vaccine strategies are
Abbreviations: MLV, modified live vaccine; VN, virus neutralising; pCDV-H, plasmid encoding haemaglutinin protein of CDV. ∗ Corresponding author at: Department of Chemistry and Bioscience, Frederik Bajers Vej 7H, 9220 Aalborg, Denmark. Tel.: +45 40 17 51 94. E-mail address: [email protected] (T.H. Jensen). 1 Present address. http://dx.doi.org/10.1016/j.vaccine.2015.01.029 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
inefficient [7]. CDV DNA vaccines have demonstrated some potential to induce CDV virus neutralising (VN) antibody titres after boost with MLV in young dogs with maternal antibodies [8]. Only few studies address MeV DNA vaccination in the presence of surrogate maternal antibodies [9,10]. Maternal antibodies have a different iso-subtype compared to antibodies of adults and thus the outcome of vaccination might not be the same [11]. In general, immaturity of the immune system of neonates and maternal antibodies interfere with many vaccine strategies [11]. At least human infants are believed to be more competent to mount a Tcell than a humoral immune response [9]. MeV DNA vaccination stimulated T-cell immune response in new born macaques [12]. However, in juvenile macaques significant levels of VN antibodies, T-cell immune response and protective immunity was induced by MeV DNA vaccination [13,14]. Immune responses to early vaccination are dependent on many factors including the specific vaccine (type, dosage, interval etc.), the host and age at vaccination and the titres of maternal antibodies but even a small priming of the immune system is important to prevent early life disease [15]. This study is the first to investigate the protective effect of DNA vaccination of neonatal mink (Neovison
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vison), a natural host of CDV, followed by virulent inoculation with a Danish field strain of CDV. A combination of plasmids encoding the haemaglutinin (H), fusion (F) and nucleoprotein (N) of CDV and the H protein alone was used. The mink kits were vaccinated once, twice or four times with 2 weeks intervals as early as 5 days of age in the presence of maternal antibodies. 2. Materials and methods 2.1. Mink Pregnant mink (Neovison) were purchased from two Danish mink farms (GT Mink and J.O.D. Hansen, Denmark). Ten pregnant females were vaccinated with an attenuated live CDV vaccine (Endivac, United Vaccines, WI, USA) and 8 mink were not vaccinated. The care and use of the animals were in accordance with the guidelines and approved by the Animal Experiments Inspectorate, (license 2005/561-999, http://www.foedevarestyrelsen.dk/ english/Animal/Pages/The-Animal-Experiments-Inspectorate. aspx). Three expression plasmids were used for immunisations: (1) plasmid vector pV1J containing an insert of the H gene (1815 base pairs, pCDV-H); (2) pV1J with an insert of the N gene (1573 base pairs, pCDV-N), the H and N genes originated from the Onderstepoort strain of CDV [16–18]; (3) plasmid vector pVR1012 with an insert of the F gene (2204 base pairs) of the Dog/DK91B strain of CDV (pCDV-F) [19]. DNA vaccines encoding the proteins from the Dog/DK91B and Onderstepoort strain of CDV previously induced protection equally well against inoculation with CDV wild type strains [19]. Purified plasmids were suspended in PBS to a concentration of 1 mg DNA/ml. Mink were immunised either with the pCDV-H alone or a combination of the pCDV-H, pCDV-N and pCDV-F (pCDV-HFN, Fig. 1, details in supplemental file). The mink in experiment A were inoculated with CDV wild type strain, Mink/DK2004 (6.5 × 104 TCID50 ) at 3 months of age under anaesthesia. Spleen and lymph nodes from CDV infected Danish farmed mink from 2004 was used for passage of Mink/DK2004 twice before used in this experiment (data not shown). Mink/DK2004 was genetically similar to the CDV mink strains from 2000 to 2002 [20] (data not shown). The mink in experiments B and C was inoculated with a well described Danish field strain of CDV, Dog/DK91B (2.2 × 106 TCID50 ) at the age of 5 and 9 months, respectively [21,22] The inoculation dose was administered partly intraperitoneally (20% suspension) and to the conjunctival and nasal mucosa (10% suspension, details in the supplemental file). In each of the three experiments, 5 non-vaccinated mink were inoculated with non-infectious organ homogenate (mockinoculation). This non-infectious organ homogenate originated from non-vaccinated CDV free farmed mink and the organ homogenate was tested for CDV by reverse transcriptase (RT)-PCR prior to use (data not shown). 2.2. Blood, swabs and tissues analysed for VN antibodies and CDV Blood samples were taken at various time points before and at the time of vaccination and after inoculation with CDV (Fig. 1). Blood samples were taken from the tail vein up to 4 weeks of age; from 6 weeks of age, blood was taken from V. cephalica and at time of euthanasia, blood was taken from anaesthetised mink by cardiac puncture followed by an intracardial barbiturate injection. VN antibody titres were determined in Vero cells using a TCID50 format microtitre assay as described by Blixenkrone-Møller et al. [23] with few modifications. Viraemia (experiments B and C) was
detected by isolation of peripheral blood mononuclear lymphocytes (PBMCs) from heparinised blood followed by intracellular CDV antigen by immunofluorescence assay (IFA) [24]. Lymphocyte counts, percentage of IFN-␥ producing peripheral blood leukocytes (PBLs, experiment A), and intracellular CDV antigen in PBLs (experiment A) and detection of viral RNA in lung and brain by RT-PCR was determined as described previously [17,25]. The mixed conjunctival/nasal swabs taken 14 days after CDV inoculation were titrated by use of Vero cells expressing canine signalling lymphocyte activation molecule (Vero-DST, donated by Yanagi) as described earlier [25]. 2.3. Isolation, stimulation and CDV antigen detection of lymph node lymphocytes Lymph node cells were isolated from the jejunal lymph node in experiment C. Cytospin were made with 100,000 lymphocytes on each slide for antigen detection by IFA [24]. Cell cultures were prepared with 5 × 106 cells to determine PBLs production of IFN-␥ and IL-4 [17]. The lymphocytes (106 cells per well) were stimulated with 50 g phytohaemagglutinin/ml (PHA-L, Sigma) for 40 h in round bottom 96-well microtitre plates. After 40 h a sample of both stimulated and unstimulated lymphocytes were incubated with 0.3 Cu/well methyl-(3 H)’thymidine (Amersham Biosciences, Germany) for 6 h. The stimulation index was calculated as the ratio of the counts of the antigen-containing culture to medium control and values higher than 2.5 were considered significant. All the statistical analysis was conducted by using the software PAST ver.2.17c [26]. No correction for large number of tests conducted was applied, as the tests conducted were not independent to each other [27]. Student’s t-test was used for evaluation of the percentage of IFN-␥ producing PBLs. 3. Results 3.1. Virus neutralising antibodies induced by early life CDV DNA vaccination Mink vaccinated with pCDV-HFN in the presence of maternal antibodies at 3 weeks of age had significantly higher antibody titre levels than those vaccinated with pCDV-H (p < 0.0001) whereas no significant differences were observed at weeks 5 and 7 (p > 0.05, Fig. 2A). At 7 weeks of age, the maternal antibodies of the mink vaccinated with pEmpty had declined to undetectable levels. However, CDV specific VN antibodies were detectable in the mink vaccinated in the presence of maternal antibodies even in the mink vaccinated once (Fig. 2A). The CDV VN antibodies induced in the vaccinated mink persisted until CDV inoculation at 3 months of age. At this time the CDV VN antibody titre of the mink DNA vaccinated in the presence of maternal antibodies was not significantly different from the CDV VN antibody titre of the mink vaccinated in the absence of maternal antibodies (p > 0.05). CDV VN antibody titres were boosted after CDV inoculation in the vaccinated mink (Fig. 2A). The CDV VN antibody titres increased more rapidly in the mink vaccinated with pCDV-H compared to the mink vaccinated with the pCDV-HFN plasmid in the presence of maternal antibodies 6, 14 and 21 days after CDV inoculation (p < 0.0001; Fig. 2A). Experiment B: Vaccination with the pCDV-HFN of 4 weeks old mink induced CDV VN antibody titres almost identical in mink vaccinated with and without maternal antibodies after the second vaccination (at the age of 8 weeks, p > 0.05, Fig. 2B). No differences were found between the mink given pEmpty and PBS in the development of CDV VN antibodies or in the following CDV infection so these 2 groups were merged to simplify interpretation of the results
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Fig. 1. Experimental design. Vaccination (vacc.) of mink kits with DNA plasmids encoding the H, F and N proteins of CDV or the H protein alone at the age 5 days and 3 weeks in the presence and absence of maternal antibodies (experiment A). Vaccination of mink kits with pCDV-HFN at the age of 4, 6, 8 and 10 weeks and challenge inoculation at the age of 20 weeks (experiment B) and 36 weeks (experiment C). The figure is not drawn to scale and the broken lines indicate the differences in number of weeks between vaccination and challenge inoculation.
of experiment B and C. After CDV inoculation the VN antibody titres were boosted to equal levels in mink vaccinated in presence or absence of maternal antibodies (p > 0.05, Fig. 2B). 3.2. Early life CDV DNA vaccination induced a memory T-cell immune response (experiment A) The percentage of IFN-␥ producing PBLs were significantly higher in the pCDV-HFN mink vaccinated in presence of maternal antibodies compared to the other pCDV-HFN vaccinated mink 9 and 14 days after CDV inoculation (p < 0.05, Fig. 3A). The mink vaccinated with pCDV-H in absence maternal antibodies had increased IFN-␥ producing PBLs 9 days after CDV inoculation peaking 14 days after inoculation together with the other pCDV-H vaccinated mink (Fig. 3B). The pEmpty or non-vaccinated vaccinated mink had a significant increase in the percentage of IFN-␥ producing PBLs (p < 0.05) 28 days after CDV inoculation (Fig. 3). 3.3. CDV DNA vaccination early in life protect against virulent CDV inoculation An index of protection was calculated to compare the groups of DNA vaccinated mink with respect to the efficacy of the DNA vaccines in inducing protection against CDV infection (Table 1). Following parameters were included in the index: viraemia, lymphopenia, virus excretion and viral RNA in the respiratory tract and central nervous system (CNS). Each of the parameters was scored with equivalent weights according to the presence and severity of each parameter. A score of 3 represented severe lymphopenia and/or viraemia and/or the finding of viral RNA in the lungs or the brain and/or viral isolation from oculo-nasal swabs. A score of 0 was given to negative values. A score of 1 and 2 was used for intermediate values; 1 representing the lowest levels of viraemia and lymphopenia. The index of protection was calculated by adding the scores of mink within a group of mink vaccinated with the same DNA vaccine divided by the total number of animal observations
within the respective group of mink. All groups of DNA vaccinated kits were substantially protected compared to the pEmpty/nonvaccinated mink (Table 1). Severe parameters of CDV infection in the pEmpty/non-vaccinated mink resulted in an index of protection more than 10 times higher than the index of the most efficiently protected mink. Vaccination with pCDV-H in the presence and absence of maternal antibodies conferred similar levels of protection against CDV inoculation (Table 1). The mink vaccinated with the pCDV-HFN without maternal antibodies had higher levels of viraemia, lymphopenia and spread of virus to the lungs and hence a higher index of protection compared to the mink vaccinated with the H gene alone (Table 1). The mink only vaccinated once at 5 days of age were equally protected to mink vaccinated twice, although results must be carefully interpreted with the low number of mink (n = 2) vaccinated only once. The mink vaccinated 4 times (experiment B) with the pCDV-HFN in the absence and presence of maternal antibodies were protected equally against viraemia and lymphopenia after CDV inoculation (Table 1).
3.4. Long-term protective immunity following early life vaccination with the pCDV-HFN (experiment C) The mink DNA vaccinated 4 times early in life and CDV inoculated at the age of 9 months had CDV VN antibody titres about 1:100 at the age of 9 months (Fig. 4A). No differences were detected in the mink vaccinated in the presence and absences of maternal antibodies so these groups were merged to simplify interpretation. All mink vaccinated with a combination of pCDV-HFN were protected against lymphopenia, viraemia, lymph node infection and immune suppression as measured by lymphocyte stimulation capacity in contrast to the non-vaccinated mink measured 5 and 11 days after CDV inoculation (Fig. 4B). The percentages of IFN-␥ and IL-4 producing PBLs were almost absent in the non-vaccinated mink 5 days
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Fig. 2. CDV specific virus neutralising antibody titres induced by DNA vaccination and inoculation with a wild type strain of CDV. (A) Mink were vaccinated at the age of 5 days and 3 weeks with pCDV-HFN or pCDV-H alone, in the presence (n = 5) and absence (n = 5) of maternal antibodies. Four mink were vaccinated only at 5 days of age. Three mink were given pEmpty in the presence of maternal antibodies. (B) Mink were vaccinated with pCDV-HFN in the presence (n = 8) or absence (n = 6) of maternal antibodies and 6 mink were given either pEmpty or PBS in the presence of maternal antibodies. Serum was collected prior to vaccination at the age of 4, 6, 8 and 10 weeks, prior to CDV inoculation at the age 20 weeks and at the indicated days after CDV inoculation. The horizontal dotted line indicate log10 = 2 that equals a neutralising titre of 100. Values shown are geometric means + standard deviations.
after CDV inoculation (Fig. 4C and D). The DNA vaccinated mink were protected against this decline in cytokine production. 4. Discussion The results of these vaccine experiments show that neonatal CDV DNA vaccination induces CDV VN antibodies and protect mink against virulent CDV inoculation later in life irrespective of the presence of maternal antibodies at time of vaccination. Mink vaccinated only once or twice already at 5 days and 3 weeks of age were not as robustly protected as the mink vaccinated 4 times. Vaccination only at 5 days of age with either CDV-H or CDV-HFN induced CDV VN antibody response and the mink were protected against virulent CDV equally to the mink vaccinated twice (Fig. 2A and Table 1). Also the percentage of IFN-␥ producing PBLs of the mink vaccinated once was similar to the mink vaccinated twice
(Fig. 3). This indicates a distinct capability of the immune system of neonate mink. In contrast to the immaturity of neonate macaques in which vaccination with MeV H did not prime any response and vaccination with MeV H and F primed only a T-cell immune response [12]. Vaccination as early in life as possible is of practical clinical importance for protection of the susceptible offspring against distemper. In addition, early life vaccination will reduce the stress imposed to the mink by handling and decrease the cost and labour associated with several handlings. Previous studies showed four vaccinations induced an effective immune response in young mink vaccinated with pCDV-H [25], in adult mink vaccinated with pCDV-HFN [16,19] and in young dogs vaccinated with a combination of CDV H, F, N genes [28]. In this study, 4 DNA vaccinations induced the most robust protection (Table 1). Two CDV DNA vaccinations of adult mink has previously induced humoral and cellular immunity and partly
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Fig. 3. Percentage of IFN-␥ producing PBLs of mink vaccinated with pCDV-HFN (A) or pCDV-H alone (B). The mink given empty plasmid or left non-vaccinated (pEmpty/nonvacc.) and the mock inoculated mink (mock) were the same in (A) and (B). Table 1 Protective capacity of DNA vaccination with the H, F and N genes of CDV or the H gene alone.
Each box represents one mink. The results from each individual animal are shown in the same sequence from left to right. Black represents the highest score (3) indicating a severe viraemia (>25% PBLs positive for staining with a monoclonal antibody for the CDV N protein by FACS analysis), severe lymphopenia defined as
