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Onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleucopenia, feline calicivirus and feline herpesvirus
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The Veterinary Journal The Veterinary Journal 182 (2009) 86–93 www.elsevier.com/locate/tvjl
Onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleucopenia, feline calicivirus and feline herpesvirus D. Jas a, C. Aeberle´ a, V. Lacombe a, A.L. Guiot b, H. Poulet a,* a
Research and Development, Merial SAS, 29 Avenue Tony Garnier, 69007 Lyon, France b Conseils en Pharmacie et Biologie, Sante´, 69110 Ste Foy les Lyon, France Accepted 28 May 2008
Abstract The induction of a quick onset of immunity against feline parvovirus (FPV), feline herpesvirus (FHV) and feline calicivirus (FCV) is critical both in young kittens after the decline of maternal antibodies and in cats at high risk of exposure. The onset of immunity for the core components was evaluated in 8–9 week old specific pathogen free kittens by challenge 1 week after vaccination with a combined modified live (FPV, FHV) and inactivated (FCV) vaccine. The protection obtained 1 week after vaccination was compared to that obtained when the challenge was performed 3–4 weeks after vaccination. The protocol consisted of a single injection for vaccination against FPV and two injections 4 weeks apart for FHV and FCV. At 1 week after vaccination, the kittens showed no FPV-induced clinical signs or leukopenia following challenge, and after FCV and FHV challenges the clinical score was significantly lower in vaccinated animals than in controls. Interestingly, the relative efficacy of the vaccination was comparable whether the animals were challenged 1 week or 3–4 weeks after vaccination, indicating that the onset of protection occurred within 7 days of vaccination. Following the 1-week challenge, excretion of FPV, FHV and FCV was significantly reduced in vaccinated cats compared to control kittens, confirming the onset of immunity within 7 days of vaccination. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Feline parvovirus; Feline calicivirus; Feline herpesvirus; Vaccine; Challenge
Introduction Feline parvovirus (FPV), feline herpesvirus (FHV) and feline calicivirus (FCV) all cause major infectious diseases in cats. FPV-induced panleucopenia is often fatal, especially in young kittens; it is highly contagious and particularly resistant in the environment. FHV and FCV infections are both major causes of upper-respiratory tract disease in cats. FHV-induced disease is generally self-limiting, although it may occasionally be severe and evolve towards a chronic upper-respiratory disease, after which most cats become lifelong latently infected carriers and undergo periodic episodes of virus reactivation, particu*
Corresponding author. Tel.: +33 4 72 72 34 24; fax: +33 4 72 72 29 63. E-mail address: [email protected] (H. Poulet).
1090-0233/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2008.05.025
larly after stress (Gaskell et al., 2007). FCV is characterised by a strong antigenic and clinical diversity. Recently, outbreaks of a severe systemic disease with high mortality have been reported in North America and Europe (Coyne et al., 2006). Because FPV, FCV and FHV infections are highly prevalent, easily transmitted and/or result in severe diseases, vaccination of all cats was recommended by the Advisory Board on Cat Diseases (European Advisory Board on Cat Diseases ABCD Guidelines on FPV, June 2006; FHV, October 2006; FCV, March 2007). Although the duration of immunity for feline vaccines has been frequently discussed (Scott and Geissinger, 1999; Gaskell et al., 2006; Schultz, 2006), the onset of immunity after vaccination is poorly documented and immediate efficacy of vaccines is most often evaluated 3–4 weeks after the
D. Jas et al. / The Veterinary Journal 182 (2009) 86–93
last vaccination. However, pet owners are often anxious to know when immunity is effective, especially with young kittens that are particularly susceptible to various pathogens. Induction of a rapid onset of immunity may also be critical for cats in high-risk environments such as boarding facilities, shelters, or catteries. In this study, the onset of immunity for the core components of a feline vaccine was evaluated by challenge 1 week following vaccination with a combination product (Purevax RCPCh-FeLV, Merial). Materials and methods Animals and vaccination Specific pathogen free (SPF) 8–9 week old kittens were purchased from Charles River Laboratories, France. For each trial, kittens were randomly allocated to two groups according to age, sex and litter. One group was vaccinated and the other was retained as a control group. For the FPV trials, kittens were vaccinated once with the combination vaccine on day zero (D0). For FHV and FCV, kittens were vaccinated twice at 4-week interval with the same vaccine. All vaccines were administered via the subcutaneous route in the interscapular area. The animal experiments and associated procedures were reviewed and approved by the Merial Ethical Committee.
Infectious challenges For the challenges, kittens were anaesthetised with ketamine (Imalgene 500, Merial) or tiletamin and zolazepam (Zoletil 50, Virbac) intramuscularly. In the FPV trials, all kittens (vaccinates and controls) received 1 mL of the CU4 FPV strain containing 108.1–108.4 CCID50 (cell culture 50% infective dose) per animal by the intraperitoneal route. In the FHV trials, all kittens (vaccinates and controls) received 0.25 mL in each nostril and each eye of the C27 FHV strain (i.e. 105.0–105.4 CCID50/animal). In the FCV trials, all kittens (vaccinates and controls) received 0.25 mL in each nostril and 0.5 mL per os of the FCV100869 strain (i.e. approximately 105.6–106.6 CCID50/animal). The animals were challenged 1 week (FPV, FHV, and FCV), 3 weeks (FPV), or 4 weeks (FHV and FCV) after vaccination.
Vaccines The vaccine used in the different trials was a non adjuvanted vaccine, consisting of a freeze-dried pellet containing FHV (live FHV F2 strain), FCV antigens (inactivated FCV G1 and 431 strains), Chlamydophila felis (905 live strain) and FPV (PLI IV live strain), reconstituted with a diluent containing the recombinant canarypox virus vCP97 expressing protective immunogens of feline leukaemia virus (FeLV) (i.e. the same components as in Purevax RCPCh-FeLV, Merial). In addition, a recombinant canarypox-rabies vaccine (vCP65) was injected simultaneously with the RCPCh-FeLV vaccine. The vaccines used in the six trials were formulated with the minimum antigen content for all components (103.2 or 103.5 CCID50 of FPV per dose for the 3-week challenge trial and the 1-week challenge trial, respectively; 104.9 CCID50 of FHV F2 per dose; 2.7 or 2.1 ELISA units of FCV G1/431 antigen per dose for the 4-week challenge trial and the 1-week challenge trial, respectively).
Clinical observations The animals were observed on a daily basis for 14 days after FPV, FHV and FCV challenges. General condition (apathy, depression, prostration), rectal temperature, and specific symptoms were recorded on a daily basis. Kittens were weighed twice a week. A clinical score was cal-
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culated according to FHV and FCV European Pharmacopeia monographs (scoring for FCV: 2 for depression, 1 for hyperthermia P39.5 °C, 1 for few and small oral ulcers, 3 for large or numerous ulcers, 1 for slight nasal discharge, 2 for copious nasal discharge, 1 for ocular discharge, 2 for weight loss. For FHV: 2 for depression, 1 for hyperthermia P39.5 °C, 2 for hyperthermia P40 °C, 3 for glossitis, 1 for slight nasal discharge, 2 for copious nasal discharge, 2 for cough, 1 for sneezing, 1 for slight ocular discharge, 2 for serious ocular discharge, 2 for conjunctivitis, 5 for weight loss P5%).
Serology Serum was collected at the time of vaccination, before challenge, and at the end of the follow-up period. FPV and FHV antibodies were measured by seroneutralisation or by enzyme-linked immunosorbent assay (ELISA). An ELISA test was used for the titration of FCV antibodies. Antibodies against FHV, FPV and FCV, measured by blocking ELISA using specific monoclonal antibodies, were titrated according to the technique described by Poulet (2007). Neutralising antibodies against FPV or FHV were measured by conventional method on Crandell-Reese feline kidney (CrFK) cells. FPV was detected by haemagglutination and FHV by the presence of a specific cytopathic effect.
Haematology Blood samples in EDTA tubes were collected 4, 6, 8, 10 and 12 days after FPV challenge for leukocyte counts. Baseline values were established using blood samples collected 8 and 4 days before challenge in both FPV trials. Counting was done with a MS9 cell counter.
Virus shedding In the FPV 1-week challenge trial, rectal swabs were collected on all kittens the day of challenge and on 2, 4, 6, 8, 10, 12 and 14 days after challenge. Samples were stored at 20 °C before testing. FPV was quantified by PCR. In FHV experiments, nasal swabs were collected from each animal just before challenge, then 2, 4, 8, 11 and 14 days post-challenge (dpc) in the 4-week challenge trial, or 2, 4, 6, 8, 10 and 14 dpc in the 1week challenge trial. Samples were stored at 70 °C in F15 medium enriched with antibiotics (3 mL of medium/swab) until viral isolation. FHV was isolated and titrated on CrFK cells. In the 1-week challenge trial, FHV DNA copies were measured by PCR. In FCV trials, pharyngeal swabs were collected on each animal just before challenge and on 2, 4, 6, 8, 10 or 11 and 14 dpc. Samples were stored at 70 °C in F15 medium enriched with antibiotics (3 mL of medium/swab) until viral isolation. FCV was isolated and titrated on CrFK cells.
Post-mortem examination In FPV trials, after euthanasia or death, the animals were necropsied with minimal delay. Samples from the small intestine, mesenteric lymph nodes and organs were collected in formaldehyde (10%) for histological examination.
Analysis of the results Statistical analyses were carried out using Statgraphics software. Statistical significance was based on two-tailed tests of the null hypothesis resulting in P-values of 0.05 or less. In the FPV trials, the change in leukocyte number was compared to the baseline value (mean of the two leukocyte cell counts before challenge) for each cat and each sample time after challenge. The quantification threshold of the PCR technique was 4.2 log10 copies of FPV DNA/sample. This baseline value was assigned to the samples in which FPV DNA could be detected but not quantified and for samples which were lacking due to the death of cats after challenge. The detection threshold of the technique was 2.5 log10 copies/sample. This
D. Jas et al. / The Veterinary Journal 182 (2009) 86–93
value was assigned to the samples in which FPV DNA could not be detected. The area under the curve (AUC) of the viral excretion measured by PCR analysis on rectal swabs was calculated in order to take into account the intensity and the duration of the excretion. The AUC of the viral excretion was compared between vaccinated and control groups after challenge by the Wilcoxon test. In the FHV and FCV trials, the global clinical scores of the two groups were compared using Student’s t test, one-way ANOVA test or a Wilcoxon test. The AUC of infectious titres or DNA copies was calculated for each cat after challenge. AUCs of the vaccinated and control groups were compared using Student’s t test or a Wilcoxon test. In addition, the relative efficacy of vaccination for each group was calculated according to the following formula: 100 (mean clinical score for control cats mean clinical score for vaccinated cats)/(mean clinical score for control cats).
35 Mean WBC count (1000/µL)
88
30 25 20 15 10 5
FPV challenge, 3 weeks after V1
0 0
2
Results
8
10
12
10
12
Controls
Table 1 Clinical signs after FPV challenge performed 1 or 3 weeks after one vaccine injection Challenge 3 weeks post-vaccination
Challenge 1 week post-vaccination
Controls
Vaccinates
Controls
Vaccinates
1/6 3/6 1/6 4/6 4/6 5/6
0/6 0/6 0/6 0/6 0/6 0/6
2/5 2/5 1/5 3/5 5/5 4/5
0/5 0/5 0/5 0/5 0/5 1/5
1/6 0/6 0/6 1/6 4/6
0/6 0/6 0/6 0/6 2/6
1/5 3/5 1/5 4/5 5/5
0/5 0/5 1/5 0/5 0/5
Mean WBC count (1000/µL)
30
In controls, FPV challenges induced severe clinical signs, including apathy, depression or prostration, hyperthermia, weight loss, anorexia, severe vomiting, diarrhoea and/or dehydration, with a rate of mortality of 60% (1-week challenge) or 67% (4-week challenge). In both trials, all vaccinates remained healthy. Only a slight and transient dehydration was recorded in two of the vaccinated kittens in the 3-week challenge trial. In the 1-week challenge trial, one vaccinated cat had a slight and transient hyperthermia (maximal temperature = 39.7 °C), and another vaccinate had once moderate vomiting and soft stools, but no liquid diarrhoea was observed in the vaccinated cats (Table 1). White blood cell (WBC) counts dropped in all control cats. In most cases, leukocyte counts dropped by >75% of their initial count. Some control cats died before the drop in WBC count and the surviving controls showed a decrease of 54–73%. After challenge, no leucopenia was observed in the vaccinates, and leukocyte counts remained >50% of the baseline value at the critical stage of the challenge (4–8 days post challenge). A transient and slight decrease in WBC counts was noted 10 dpc in the 3-week challenge trial, where two vaccinates had leukocyte counts slightly below 50% of their initial count (Fig. 1).
Apathy Exhaustion Prostration Death Weight loss Hyperthermia (P39.5 °C) Hypothermia (637 °C) Anorexia Vomiting Diarrhoea (liquid faeces) Dehydration
6
Vaccinates
FPV trials
Clinical sign
4
Days post-challenge
25 20 15 10 5
FPV challenge, 1 week after V1
0 0
2
4
6
8
Days post-challenge Vaccinates
Controls
Fig. 1. WBC count after the FPV challenges 3 weeks and 1 week postvaccination.
All cats were seronegative before vaccination, and control cats were seronegative at the time of challenge. Vaccination induced a strong seroconversion with high neutralising antibody titres 3 weeks after immunisation (average P4.1 log10). In the 1-week challenge trial, 7 days after vaccination, all vaccinates had high ELISA antibody titres (average 2.4 log10). In both studies, antibody titres at the time of challenge were above the protective threshold (1.2 log10). At necropsy, congestion of the jejunum, ileum and colon, enlarged spleen and mesenteric lymph nodes were noticed in the controls. Typical lesions of lymphoid hypoplasia of the thymus, severe acute necrotising and haemorrhagic enteritis, and a severe acute lymphoid depletion of the mesenteric lymph nodes were observed after histological examination in controls. No lesions were recorded in vaccinates. On the day of challenge, all vaccinates still shed low amounts of vaccine virus detectable by PCR (average <4.5 log10 DNA copies). Excretion in vaccinated animals remained low after challenge (average <5.1 log10 2 dpc) and below the quantification threshold (<4.2 log10) from 6 to 14 dpc. On the day of challenge, all but two controls were negative (<2.5 log10 viral DNA copies). These two cats had a very low viral excretion measured by PCR (<4.2 log10).
D. Jas et al. / The Veterinary Journal 182 (2009) 86–93
Between 4 and 6 days after challenge, high amounts of FPV were found in rectal swabs of all controls (peak of excretion at 8.9 log10 DNA copies on average, 6 dpc). Virus shedding decreased between day 6 and 14 after challenge in the two surviving controls (Fig. 2). Virus shedding was significantly higher in controls (AUC = 85.7) than in vaccinates (AUC = 61.1) (Wilcoxon’s test; P = 0.01).
Mean DNA copies/sample (log10)
10.00
FPV challenge. 1 week after V1 9.00
FHV trials After challenge, all control cats displayed clinical signs of rhinotracheitis (weight loss (P5%), hyperthermia, depression, sneezing, copious ocular discharge, abundant and sustained nasal discharge, glossitis, and/or conjunctivitis) and had high clinical scores. All vaccinates remained healthy, and none lost weight during the critical period (up to 12 dpc). Vaccinated cats showed mild and transient clinical signs, such as a slight nasal discharge, sneezing, ocular discharge, and/or conjunctivitis. Slight hyperthermia (39.5 °C) and transient conjunctivitis were observed in a few vaccinated cats. The mean global clinical score was significantly lower in the vaccinated group than in the controls: 7.7 vs. 26.7 (3.5) in the 4-week challenge trial (Student’s t test, P = 7.10 5 ), and 15.3 vs. 40.8 (2.7) in the 1-week challenge trial (Student’s t test, P = 1.0 10 4) (Fig. 3). The relative efficacy was 62.5% in the 1-week FHV challenge trial and 71.2% in the 4-week FHV challenge trial. All vaccinates and controls were seronegative on day 0 in both FHV trials. In the 4-week challenge trial, after the first vaccine injection, only 2/11 vaccinates seroconverted. The second vaccine injection induced neutralising antibodies in 7/11 vaccinates (average 0.6 log10). All control cats were seronegative at the time of challenge (average 60.2 log10).
AUC: vaccinated = 61.1 controls = 85.7
8.00 7.00 6.00 5.00 4.00 3.00 2.00 0
2
4
6 8 10 Days post-challenge Vaccinates
12
89
14
Controls
Fig. 2. Virus shedding measured by quantitative PCR in rectal swabs after FPV challenge, 1 week post-vaccination. 10
Global score :
Mean clinical score
FHV challenge, 4 weeks after V2
vaccinated = 7.7 controls = 26.7
8 6 4 2 0
General condition
Weight
Hyperthermia
Glossitis
Nasal discharge
Vaccinates
Cough
Sneezing
Conjunctivitis
Controls
14
Global score :
FHV challenge, 1 week after V2
vaccinated = 15.3 controls = 40.8
12 Mean clinical score
Ocular discharge
10 8 6 4 2 0
General condition
Weight
Hyperthermia
Glossitis
Nasal discharge
Vaccinates
Cough
Sneezing
Controls
Fig. 3. Clinical scores after FHV challenge 4 weeks and 1 week post-vaccination.
Ocular discharge
Conjunctivitis
90
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In the 1-week challenge trial, all vaccinates developed FHV ELISA antibodies after the first injection (average 1.6 log10) or the second injection (average 2.3 log10), whereas controls remained seronegative (average <0.9 log10). No virus was isolated from any cat prior to challenge. After challenge, viral excretion was observed in both groups with a peak 4 dpc in both FHV trials. In the 4-week challenge trial, viral excretion decreased more rapidly in the vaccinated group than in the control group. Two days after challenge, 8/11 control cats (72.7%) and 7/11 vaccinated cats (63.6%) shed virus. From 4 to 8 dpc, all controls and vaccinates shed virus. Eleven dpc, viral excretion was detected in only 4/11 vaccinates (36.4%), whereas 8/11 controls (72.7%) excreted virus. Virus shedding was on average twice as high in controls as in vaccinates for the entire duration of the study. Although the duration of excretion was usually reduced in vaccinates, the difference between groups was not statistically significant (AUC vaccinated group 48.7 vs. control group 53.2; Student’s t test, P = 0.2). In the 1-week challenge trial, virus isolation and PCR method gave consistent results. FHV excretion began earlier and lasted longer in controls than in vaccinates. On the basis of the infectious titres, controls excreted 10 times more virus on average after challenge during the entire monitoring period. Viral shedding (PCR results) was significantly reduced in the vaccinated group (AUC vacci-
nated group = 78.0 vs. control group = 85.2, Student’s t test, P = 5 10 4). FCV trials After challenge, all control cats displayed typical clinical signs such as oronasal ulcers (most often large and numerous), nasal discharge, hyperthermia, and/or weight loss (P5%). Ocular discharge and depression were occasionally observed. Cutaneous ulcerations (footpads) were recorded in 2/10 controls in the 1-week challenge trial. Vaccinates presented only mild clinical signs limited to a few oronasal ulcers of small size, and a slight nasal discharge. Occasional weight loss was observed in one vaccinated cat in the 4-week challenge trial. Slight and transient hyperthermia (39.5–39.7 °C) was recorded in 3/10 vaccinates in the 1-week challenge trial. None of the vaccinated cats had cutaneous ulceration (Fig. 4). In both trials, the mean global score of the control group was significantly higher than that of the vaccinated group (27.8 in controls vs. 6 in vaccinates in the 4-week challenge trial and 24.0 in controls vs. 4.9 in vaccinates in the 1-week challenge trial) (one-way ANOVA test, P = 0.01 in the 4-week challenge trial; Wilcoxon test, P = 0.0017 in the 1-week challenge trial). The relative efficacy was 79.6% in the 1-week FCV challenge trial and 78.4% in the 4-week FCV challenge trial.
20
Global score : vaccinated = 6.0 controls = 27.8
Mean clinical score
FCV challenge, 4 weeks after V2 16 12 8 4 0
General condition Weight loss
Hyperthermia Oronasal ulceration Nasal discharge Vaccinates
Ocular discharge
Controls
20 Global score : vaccinated = 4.9 controls = 24.0
FCV challenge, 1 week after V2 Mean clinical score
16
12
8
4
0 General condition
Weight loss
Hyperthermia Oronasal ulceration Nasal discharge Ocular discharge Vaccinates
Controls
Fig. 4. Clinical scores after FCV challenge 4 weeks and 1 week post-vaccination.
D. Jas et al. / The Veterinary Journal 182 (2009) 86–93
All vaccinates developed FCV antibodies after the second vaccine injection (mean = 2.2 log10 at D56 in the 4week challenge trial and mean = 2.3 log10 on D35 in the 1-week challenge trial), whereas controls remained seronegative (mean <0.5 log10 at D56). None of the cats shed FCV before challenge but all control cats excreted FCV, with a peak on day 4, after challenge. Virus shedding was lower and shorter in the vaccinated group. In the 4-week challenge trial, 4 dpc, three vaccinates and all controls excreted FCV. Controls shed on average about twice as much virus as vaccinates after challenge. Viral shedding was significantly reduced in the vaccinated group (AUC vaccinated group 22.0 vs. control group 27.0; one-way ANOVA test, P = 0.007). In the 1-week challenge trial, controls excreted on average about twice as much virus as the vaccinates after challenge. Viral shedding was significantly reduced in the vaccinated group (AUC vaccinated group 33.9 vs. control group 39.3; Wilcoxon test, P = 7.3 10 3). Table 2 summarises the overall results of efficacy studies for FPV, FHV and FCV. Discussion The objective of these trials was to evaluate the onset of immunity in kittens following vaccination against feline parvovirus, herpesvirus and calicivirus. Onset of immunity was evaluated by challenge, 1 week after vaccination with a RCPCh-FeLV combination vaccine formulated at minimum dose. The protection obtained 1 week after vaccination was compared with that obtained when the animals were challenged 3 or 4 weeks after vaccination. The RCPCh-FeLV vaccine is a non-adjuvanted vaccine combining live and inactivated components and the parvovirus and herpesvirus components are live, attenuated strains. Live attenuated FPV vaccines are preferred because of their quick onset of immunity and greater efficacy in the presence of maternal antibodies. The efficacy of modified live FHV vaccines is similar to that of inactivated vaccines (Povey and Wilson, 1978), but live vaccines do not require
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an adjuvant. The use of FPV and FHV vaccine strains was made possible after demonstration of their safety and the irreversibility of their attenuation. Unlike those genetically stable DNA viruses, FCV is a RNA virus with a high rate of mutation and genetic variability (Kreutz et al., 1998; Glenn et al., 1999; Coyne et al., 2007), and modified live strains have been shown to occasionally generate pathogenic variants (Dawson et al., 1993; Radford et al., 1997; Radford et al., 2001). Therefore, for safety reasons, an inactivated FCV vaccine is preferred. The FCV antigen was designed to work in the absence of adjuvant. In addition, to improve the cross-protection, the vaccine contains a combination of inactivated FCV antigens derived from two strains with broad cross-reactivity (Poulet et al., 2005). A rapid antibody response against FPV was observed as soon as 7 days after the first vaccination. For FHV and FCV, two injections of vaccine were typically required to induce a seroconversion, whether or not the vaccine was attenuated or inactivated. FHV neutralising antibody titres induced by vaccination were low, as is usually the case with live FHV vaccines (Gaskell et al., 2007). By measuring antibody titres with a sensitive ELISA, it was possible to detect seroconversion after the first vaccine injection in most of the cats. To demonstrate vaccine efficacy, cats were challenged with virulent strains of FPV, FHV or FCV. Highly virulent strains were used to ensure that all control animals would exhibit clinical signs and validate the challenge. Unlike FPV and FHV viruses, FCV is characterised by a strong antigenic diversity (Seal et al., 1993). To be representative of field challenge, the efficacy of the vaccine was tested against a recent heterologous antigenic variant. This variant was poorly neutralised by FCVG1 and FCV431 specific antisera (titre <1.2 log10), and could therefore be considered a stringent test for the vaccine. While controls displayed typical clinical signs after FPV, FHV and FCV challenges performed 1 or 3–4 weeks after vaccination, all vaccinates remained in excellent general condition. Vaccinates were completely protected from clinical disease after FPV challenge, and no severe
Table 2 Comparison of vaccine efficacy against FPV, FHV or FCV challenges performed 1 or 3–4 weeks after vaccination Component of the vaccine
Challenge
Criterion
Controls
Vaccinates
P value
FPV
3 weeks post-vaccination
Death WBC <75% baseline values Death WBC <75% baseline values Virus shedding (AUC)
4/6 2/6 3/5 4/5 85.7
0/6 0/6 0/5 0/5 61.1
– – – – 0.01
Clinical score Virus shedding (AUC) Clinical score Virus shedding (AUC)
26.7 48.7 40.8 85.2
7.7 53.2 15.3 78
<0.0001 0.22 0.0001 0.0005
Clinical score Virus shedding (AUC) Clinical score Virus shedding (AUC)
27.8 27.0 24.0 39.3
6.0 22.0 4.9 33.9
0.01 0.007 0.0017 0.007
1 week post-vaccination
FHV
4 weeks post-vaccination 1 week post-vaccination
FCV
4 weeks post-vaccination 1 week post-vaccination
92
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leucopenia was recorded, even when the challenge was performed 7 days after a single administration of vaccine. Following FCV and FHV challenges, vaccinated cats showed less severe and more transient clinical signs than controls. A significant decrease in clinical scores was observed in vaccinates after FHV (reduction by a factor of 2.7–3.5) and FCV (reduction by a factor of 4.6–4.9) challenges. Interestingly, the efficacy of the vaccine against a challenge performed 1 week after vaccination was as good as that at 3–4 weeks after vaccination, showing that onset of immunity occurred within a week of vaccination. Vaccine-induced protection was also demonstrated in the reduction of viral shedding. As early as 7 days after vaccination, virus excretion was significantly reduced in intensity and duration in vaccinates after FPV, FHV and FCV challenges. Virus excretion remained low (below the quantification threshold by PCR) and transient in all vaccinates after FPV challenge. Vaccination against FPV reduced excretion titres by several log10. Although all vaccinates excreted virus after FCV and FHV challenges, the amount and duration of virus shedding were significantly reduced compared to controls. Reduced excretion is expected to lower the infectious pressure in cat collectives, and thereby contribute to the efficacy of vaccination. Our results confirmed that FPV is a strong immunogen, requiring only one vaccine injection to induce strong protection. The immunogenicity of FPV attenuated vaccines is explained, at least in part, by the strong replication of the FPV vaccine strain within the vaccinated host. FCV and FHV vaccines are less immunogenic and, regardless of the technology used (attenuated or inactivated components), two vaccine injections are required to induce a protective immune response. In addition, FCV or FHV vaccines do not prevent infection but rather reduce the severity of clinical symptoms and occasionally virus shedding (Radford et al., 2006; Gaskell et al., 2007). It is often assumed that inactivated vaccines have a longer onset of immunity than live attenuated vaccines but our study has shown that a non-adjuvanted inactivated FCV vaccine could induce a significant reduction of clinical signs and excretion with a rapid onset of immunity. The induction of a quick onset of immunity against FPV, FHV and FCV is particularly important in young kittens, especially when maternal antibodies have declined and leave the kitten without protection at 8–12 weeks of age. It is also critical for cats at high risk of exposure, for example in boarding facilities, catteries or shelters. In these conditions, some veterinarians recommend the use of intranasal (IN) vaccines and IN administration of FHV or FCV modified live vaccines has been shown to induce a rapid onset of immunity. A single vaccine administration moderately reduced clinical signs after a FHV challenge 4 days after vaccination (Lappin et al., 2006). IN vaccines may however occasionally induce an upper respiratory tract infection associated with shedding of vaccine virus (Gaskell et al., 2007). Live FCV vaccine strains share the same genetic instability as wild-type strains and may occasion-
ally generate new antigenic variants. Therefore, the benefit/risk ratio of IN live attenuated vaccines should be carefully evaluated. There are currently no IN FHV, FCV and/or FPV vaccines available in Europe. An earlier onset of immunity in young kittens may result if the course of vaccination is started at a younger age. However, maternal antibodies against FPV or FCV may persist for up to 16 weeks and interfere with vaccination (Poulet, 2007). A modified protocol starting at 6 weeks of age and consisting of three vaccine injections at 6, 9 and 12 weeks has been tested in the field (Dawson et al., 2001). With the early vaccination scheme, a few cats responded to the first vaccine injection, but overall, there was no difference between early and conventional vaccination schemes in the rate of response at 12 and 15 weeks of age. In kittens expected to have high maternal antibody titres, it is recommended that the last vaccine injection is given after 16 weeks of age (European Advisory Board on Cat Diseases ABCD Guidelines on FPV June 2006; ABCD Guidelines on FCV March 2007). Conclusions This study showed that the onset of immunity occurred within 1 week following one vaccine injection for FPV and within 1 week following two vaccine injections for FHV and FCV. Because the two injections can be given at an interval of 3–4 weeks, cats can be protected within 1 month. Conflict of interests statement The author and co-authors of this article are employees of Merial, the company marketing Purevax RCPCh-FeLV. References Coyne, K.P., Jones, B.R., Kipar, A., Chantrey, J., Porter, C.J., Barber, P.J., Dawson, S., Gaskell, R.M., Radford, A.D., 2006. Lethal outbreak of disease associated with feline calicivirus infection in cats. Veterinary Record 158, 544–550. Coyne, K.P., Gaskell, R.M., Dawson, S., Porter, C.J., Radford, A.D., 2007. Evolutionary mechanisms of persistence and diversification of a calicivirus within endemically infected natural host populations. Journal of Virology 81, 1961–1971. Dawson, S., Mc Ardle, F., Bennett, D., Carter, S.D., Bennett, M., Ryvar, R., Gaskell, R.M., 1993. Investigation of vaccine reactions and breakdowns after feline calicivirus vaccination. Veterinary Record 132, 346–350. Dawson, S., Willoughby, K., Gaskell, R.M., Wood, G., Chalmers, W.S., 2001. A field trial to assess the effect of vaccination against feline herpesvirus, feline calicivirus and feline panleucopenia virus in 6-weekold kittens. Journal of Feline Medicine and Surgery 3, 17–22. European Advisory Board on Cat Diseases (June 2006) ABCD guidelines on Feline Panleucopenia Virus.. pp. 1–15. European Advisory Board on Cat Diseases (October 2006) ABCD Guidelines on Feline Herpesvirus-1. . pp. 1–17. European Advisory Board on Cat Diseases (March 2007) ABCD Guidelines on Feline Calicivirus. . pp. 1–23.
D. Jas et al. / The Veterinary Journal 182 (2009) 86–93 Gaskell, R.M., Dawson, S., Radford, A.D., 2006. Duration of immunity (DOI)—the regulatory issues. Veterinary Microbiology 117, 80–85. Gaskell, R., Dawson, S., Radford, A., Thiry, E., 2007. Feline herpesvirus. Veterinary Research 38, 337–354. Glenn, M., Radford, A.D., Turner, P.C., Carter, M., Lowery, D., DeSilver, D.A., Meanger, J., Baulch-Brown, C., Bennett, M., Gaskell, R.M., 1999. Nucleotide sequence of UK and Australian isolates of feline calicivirus (FCV) and phylogenetic analysis of FCVs. Veterinary Microbiology 67, 175–193. Kreutz, L.C., Johnson, R.P., Seal, B.S., 1998. Phenotypic and genotypic variation of feline calicivirus during persistent infection of cats. Veterinary Microbiology 59, 229–236. Lappin, M.R., Sebring, R.W., Porter, M., Radecki, S.J., Veir, J., 2006. Effects of a single dose of an intranasal feline herpesvirus 1, calicivirus, and panleucopenia vaccine on clinical signs and virus shedding after challenge with virulent feline herpesvirus 1. Journal of Feline Medicine and Surgery 8, 158–163. Poulet, H., Brunet, S., Leroy, V., Chappuis, G., 2005. Immunisation with a combination of two complementary feline calicivirus strains induces a broad cross-protection against heterologous challenges. Veterinary Microbiology 106, 17–31. Poulet, H., 2007. Alternative early life vaccination programs for companion animals. Journal of Comparative Pathology 137 (Suppl. 1), S67–S71.
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Povey, R.C., Wilson, M.R., 1978. A comparison of inactivated feline viral rhinotracheitis and feline caliciviral disease vaccines with live-modified viral vaccines. Feline Practice 8, 35–42. Radford, A.D., Bennett, M., Mc Ardle, F., Dawson, S., Turner, P.C., Glenn, M.A., Gaskell, R.M., 1997. The use of sequence analysis of a feline calicivirus (FCV) hypervariable region in the epidemiological investigation of FCV related disease and vaccine failures. Vaccine 15, 1451–1458. Radford, A.D., Sommerville, L., Ryvar, R., Cox, M.B., Johnson, D.R., Dawson, S., Gaskell, R.M., 2001. Endemic infection of a cat colony with a feline calicivirus closely related to an isolate used in live attenuated vaccines. Vaccine 19, 4358–4362. Radford, A.D., Dawson, S., Coyne, K.P., Porter, C.J., Gaskell, R.M., 2006. The challenge for the next generation of feline calicivirus vaccines. Veterinary Microbiology 117, 14–18. Schultz, R.D., 2006. Duration of immunity for canine and feline vaccines: a review. Veterinary Microbiology 117, 75–79. Scott, F.W., Geissinger, C.M., 1999. Long-term immunity in cats vaccinated with an inactivated trivalent vaccine. American Journal of Veterinary Research 60, 652–658. Seal, B.S., Ridpath, J.F., Mengeling, W.L., 1993. Analysis of feline calicivirus capsid protein genes: identification of variable antigenic determinant regions of the protein. Journal of General Virology 74, 2519–2524.
The Veterinary Journal The Veterinary Journal 182 (2009) 86–93 www.elsevier.com/locate/tvjl
Onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleucopenia, feline calicivirus and feline herpesvirus D. Jas a, C. Aeberle´ a, V. Lacombe a, A.L. Guiot b, H. Poulet a,* a
Research and Development, Merial SAS, 29 Avenue Tony Garnier, 69007 Lyon, France b Conseils en Pharmacie et Biologie, Sante´, 69110 Ste Foy les Lyon, France Accepted 28 May 2008
Abstract The induction of a quick onset of immunity against feline parvovirus (FPV), feline herpesvirus (FHV) and feline calicivirus (FCV) is critical both in young kittens after the decline of maternal antibodies and in cats at high risk of exposure. The onset of immunity for the core components was evaluated in 8–9 week old specific pathogen free kittens by challenge 1 week after vaccination with a combined modified live (FPV, FHV) and inactivated (FCV) vaccine. The protection obtained 1 week after vaccination was compared to that obtained when the challenge was performed 3–4 weeks after vaccination. The protocol consisted of a single injection for vaccination against FPV and two injections 4 weeks apart for FHV and FCV. At 1 week after vaccination, the kittens showed no FPV-induced clinical signs or leukopenia following challenge, and after FCV and FHV challenges the clinical score was significantly lower in vaccinated animals than in controls. Interestingly, the relative efficacy of the vaccination was comparable whether the animals were challenged 1 week or 3–4 weeks after vaccination, indicating that the onset of protection occurred within 7 days of vaccination. Following the 1-week challenge, excretion of FPV, FHV and FCV was significantly reduced in vaccinated cats compared to control kittens, confirming the onset of immunity within 7 days of vaccination. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Feline parvovirus; Feline calicivirus; Feline herpesvirus; Vaccine; Challenge
Introduction Feline parvovirus (FPV), feline herpesvirus (FHV) and feline calicivirus (FCV) all cause major infectious diseases in cats. FPV-induced panleucopenia is often fatal, especially in young kittens; it is highly contagious and particularly resistant in the environment. FHV and FCV infections are both major causes of upper-respiratory tract disease in cats. FHV-induced disease is generally self-limiting, although it may occasionally be severe and evolve towards a chronic upper-respiratory disease, after which most cats become lifelong latently infected carriers and undergo periodic episodes of virus reactivation, particu*
Corresponding author. Tel.: +33 4 72 72 34 24; fax: +33 4 72 72 29 63. E-mail address: [email protected] (H. Poulet).
1090-0233/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2008.05.025
larly after stress (Gaskell et al., 2007). FCV is characterised by a strong antigenic and clinical diversity. Recently, outbreaks of a severe systemic disease with high mortality have been reported in North America and Europe (Coyne et al., 2006). Because FPV, FCV and FHV infections are highly prevalent, easily transmitted and/or result in severe diseases, vaccination of all cats was recommended by the Advisory Board on Cat Diseases (European Advisory Board on Cat Diseases ABCD Guidelines on FPV, June 2006; FHV, October 2006; FCV, March 2007). Although the duration of immunity for feline vaccines has been frequently discussed (Scott and Geissinger, 1999; Gaskell et al., 2006; Schultz, 2006), the onset of immunity after vaccination is poorly documented and immediate efficacy of vaccines is most often evaluated 3–4 weeks after the
D. Jas et al. / The Veterinary Journal 182 (2009) 86–93
last vaccination. However, pet owners are often anxious to know when immunity is effective, especially with young kittens that are particularly susceptible to various pathogens. Induction of a rapid onset of immunity may also be critical for cats in high-risk environments such as boarding facilities, shelters, or catteries. In this study, the onset of immunity for the core components of a feline vaccine was evaluated by challenge 1 week following vaccination with a combination product (Purevax RCPCh-FeLV, Merial). Materials and methods Animals and vaccination Specific pathogen free (SPF) 8–9 week old kittens were purchased from Charles River Laboratories, France. For each trial, kittens were randomly allocated to two groups according to age, sex and litter. One group was vaccinated and the other was retained as a control group. For the FPV trials, kittens were vaccinated once with the combination vaccine on day zero (D0). For FHV and FCV, kittens were vaccinated twice at 4-week interval with the same vaccine. All vaccines were administered via the subcutaneous route in the interscapular area. The animal experiments and associated procedures were reviewed and approved by the Merial Ethical Committee.
Infectious challenges For the challenges, kittens were anaesthetised with ketamine (Imalgene 500, Merial) or tiletamin and zolazepam (Zoletil 50, Virbac) intramuscularly. In the FPV trials, all kittens (vaccinates and controls) received 1 mL of the CU4 FPV strain containing 108.1–108.4 CCID50 (cell culture 50% infective dose) per animal by the intraperitoneal route. In the FHV trials, all kittens (vaccinates and controls) received 0.25 mL in each nostril and each eye of the C27 FHV strain (i.e. 105.0–105.4 CCID50/animal). In the FCV trials, all kittens (vaccinates and controls) received 0.25 mL in each nostril and 0.5 mL per os of the FCV100869 strain (i.e. approximately 105.6–106.6 CCID50/animal). The animals were challenged 1 week (FPV, FHV, and FCV), 3 weeks (FPV), or 4 weeks (FHV and FCV) after vaccination.
Vaccines The vaccine used in the different trials was a non adjuvanted vaccine, consisting of a freeze-dried pellet containing FHV (live FHV F2 strain), FCV antigens (inactivated FCV G1 and 431 strains), Chlamydophila felis (905 live strain) and FPV (PLI IV live strain), reconstituted with a diluent containing the recombinant canarypox virus vCP97 expressing protective immunogens of feline leukaemia virus (FeLV) (i.e. the same components as in Purevax RCPCh-FeLV, Merial). In addition, a recombinant canarypox-rabies vaccine (vCP65) was injected simultaneously with the RCPCh-FeLV vaccine. The vaccines used in the six trials were formulated with the minimum antigen content for all components (103.2 or 103.5 CCID50 of FPV per dose for the 3-week challenge trial and the 1-week challenge trial, respectively; 104.9 CCID50 of FHV F2 per dose; 2.7 or 2.1 ELISA units of FCV G1/431 antigen per dose for the 4-week challenge trial and the 1-week challenge trial, respectively).
Clinical observations The animals were observed on a daily basis for 14 days after FPV, FHV and FCV challenges. General condition (apathy, depression, prostration), rectal temperature, and specific symptoms were recorded on a daily basis. Kittens were weighed twice a week. A clinical score was cal-
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culated according to FHV and FCV European Pharmacopeia monographs (scoring for FCV: 2 for depression, 1 for hyperthermia P39.5 °C, 1 for few and small oral ulcers, 3 for large or numerous ulcers, 1 for slight nasal discharge, 2 for copious nasal discharge, 1 for ocular discharge, 2 for weight loss. For FHV: 2 for depression, 1 for hyperthermia P39.5 °C, 2 for hyperthermia P40 °C, 3 for glossitis, 1 for slight nasal discharge, 2 for copious nasal discharge, 2 for cough, 1 for sneezing, 1 for slight ocular discharge, 2 for serious ocular discharge, 2 for conjunctivitis, 5 for weight loss P5%).
Serology Serum was collected at the time of vaccination, before challenge, and at the end of the follow-up period. FPV and FHV antibodies were measured by seroneutralisation or by enzyme-linked immunosorbent assay (ELISA). An ELISA test was used for the titration of FCV antibodies. Antibodies against FHV, FPV and FCV, measured by blocking ELISA using specific monoclonal antibodies, were titrated according to the technique described by Poulet (2007). Neutralising antibodies against FPV or FHV were measured by conventional method on Crandell-Reese feline kidney (CrFK) cells. FPV was detected by haemagglutination and FHV by the presence of a specific cytopathic effect.
Haematology Blood samples in EDTA tubes were collected 4, 6, 8, 10 and 12 days after FPV challenge for leukocyte counts. Baseline values were established using blood samples collected 8 and 4 days before challenge in both FPV trials. Counting was done with a MS9 cell counter.
Virus shedding In the FPV 1-week challenge trial, rectal swabs were collected on all kittens the day of challenge and on 2, 4, 6, 8, 10, 12 and 14 days after challenge. Samples were stored at 20 °C before testing. FPV was quantified by PCR. In FHV experiments, nasal swabs were collected from each animal just before challenge, then 2, 4, 8, 11 and 14 days post-challenge (dpc) in the 4-week challenge trial, or 2, 4, 6, 8, 10 and 14 dpc in the 1week challenge trial. Samples were stored at 70 °C in F15 medium enriched with antibiotics (3 mL of medium/swab) until viral isolation. FHV was isolated and titrated on CrFK cells. In the 1-week challenge trial, FHV DNA copies were measured by PCR. In FCV trials, pharyngeal swabs were collected on each animal just before challenge and on 2, 4, 6, 8, 10 or 11 and 14 dpc. Samples were stored at 70 °C in F15 medium enriched with antibiotics (3 mL of medium/swab) until viral isolation. FCV was isolated and titrated on CrFK cells.
Post-mortem examination In FPV trials, after euthanasia or death, the animals were necropsied with minimal delay. Samples from the small intestine, mesenteric lymph nodes and organs were collected in formaldehyde (10%) for histological examination.
Analysis of the results Statistical analyses were carried out using Statgraphics software. Statistical significance was based on two-tailed tests of the null hypothesis resulting in P-values of 0.05 or less. In the FPV trials, the change in leukocyte number was compared to the baseline value (mean of the two leukocyte cell counts before challenge) for each cat and each sample time after challenge. The quantification threshold of the PCR technique was 4.2 log10 copies of FPV DNA/sample. This baseline value was assigned to the samples in which FPV DNA could be detected but not quantified and for samples which were lacking due to the death of cats after challenge. The detection threshold of the technique was 2.5 log10 copies/sample. This
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value was assigned to the samples in which FPV DNA could not be detected. The area under the curve (AUC) of the viral excretion measured by PCR analysis on rectal swabs was calculated in order to take into account the intensity and the duration of the excretion. The AUC of the viral excretion was compared between vaccinated and control groups after challenge by the Wilcoxon test. In the FHV and FCV trials, the global clinical scores of the two groups were compared using Student’s t test, one-way ANOVA test or a Wilcoxon test. The AUC of infectious titres or DNA copies was calculated for each cat after challenge. AUCs of the vaccinated and control groups were compared using Student’s t test or a Wilcoxon test. In addition, the relative efficacy of vaccination for each group was calculated according to the following formula: 100 (mean clinical score for control cats mean clinical score for vaccinated cats)/(mean clinical score for control cats).
35 Mean WBC count (1000/µL)
88
30 25 20 15 10 5
FPV challenge, 3 weeks after V1
0 0
2
Results
8
10
12
10
12
Controls
Table 1 Clinical signs after FPV challenge performed 1 or 3 weeks after one vaccine injection Challenge 3 weeks post-vaccination
Challenge 1 week post-vaccination
Controls
Vaccinates
Controls
Vaccinates
1/6 3/6 1/6 4/6 4/6 5/6
0/6 0/6 0/6 0/6 0/6 0/6
2/5 2/5 1/5 3/5 5/5 4/5
0/5 0/5 0/5 0/5 0/5 1/5
1/6 0/6 0/6 1/6 4/6
0/6 0/6 0/6 0/6 2/6
1/5 3/5 1/5 4/5 5/5
0/5 0/5 1/5 0/5 0/5
Mean WBC count (1000/µL)
30
In controls, FPV challenges induced severe clinical signs, including apathy, depression or prostration, hyperthermia, weight loss, anorexia, severe vomiting, diarrhoea and/or dehydration, with a rate of mortality of 60% (1-week challenge) or 67% (4-week challenge). In both trials, all vaccinates remained healthy. Only a slight and transient dehydration was recorded in two of the vaccinated kittens in the 3-week challenge trial. In the 1-week challenge trial, one vaccinated cat had a slight and transient hyperthermia (maximal temperature = 39.7 °C), and another vaccinate had once moderate vomiting and soft stools, but no liquid diarrhoea was observed in the vaccinated cats (Table 1). White blood cell (WBC) counts dropped in all control cats. In most cases, leukocyte counts dropped by >75% of their initial count. Some control cats died before the drop in WBC count and the surviving controls showed a decrease of 54–73%. After challenge, no leucopenia was observed in the vaccinates, and leukocyte counts remained >50% of the baseline value at the critical stage of the challenge (4–8 days post challenge). A transient and slight decrease in WBC counts was noted 10 dpc in the 3-week challenge trial, where two vaccinates had leukocyte counts slightly below 50% of their initial count (Fig. 1).
Apathy Exhaustion Prostration Death Weight loss Hyperthermia (P39.5 °C) Hypothermia (637 °C) Anorexia Vomiting Diarrhoea (liquid faeces) Dehydration
6
Vaccinates
FPV trials
Clinical sign
4
Days post-challenge
25 20 15 10 5
FPV challenge, 1 week after V1
0 0
2
4
6
8
Days post-challenge Vaccinates
Controls
Fig. 1. WBC count after the FPV challenges 3 weeks and 1 week postvaccination.
All cats were seronegative before vaccination, and control cats were seronegative at the time of challenge. Vaccination induced a strong seroconversion with high neutralising antibody titres 3 weeks after immunisation (average P4.1 log10). In the 1-week challenge trial, 7 days after vaccination, all vaccinates had high ELISA antibody titres (average 2.4 log10). In both studies, antibody titres at the time of challenge were above the protective threshold (1.2 log10). At necropsy, congestion of the jejunum, ileum and colon, enlarged spleen and mesenteric lymph nodes were noticed in the controls. Typical lesions of lymphoid hypoplasia of the thymus, severe acute necrotising and haemorrhagic enteritis, and a severe acute lymphoid depletion of the mesenteric lymph nodes were observed after histological examination in controls. No lesions were recorded in vaccinates. On the day of challenge, all vaccinates still shed low amounts of vaccine virus detectable by PCR (average <4.5 log10 DNA copies). Excretion in vaccinated animals remained low after challenge (average <5.1 log10 2 dpc) and below the quantification threshold (<4.2 log10) from 6 to 14 dpc. On the day of challenge, all but two controls were negative (<2.5 log10 viral DNA copies). These two cats had a very low viral excretion measured by PCR (<4.2 log10).
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Between 4 and 6 days after challenge, high amounts of FPV were found in rectal swabs of all controls (peak of excretion at 8.9 log10 DNA copies on average, 6 dpc). Virus shedding decreased between day 6 and 14 after challenge in the two surviving controls (Fig. 2). Virus shedding was significantly higher in controls (AUC = 85.7) than in vaccinates (AUC = 61.1) (Wilcoxon’s test; P = 0.01).
Mean DNA copies/sample (log10)
10.00
FPV challenge. 1 week after V1 9.00
FHV trials After challenge, all control cats displayed clinical signs of rhinotracheitis (weight loss (P5%), hyperthermia, depression, sneezing, copious ocular discharge, abundant and sustained nasal discharge, glossitis, and/or conjunctivitis) and had high clinical scores. All vaccinates remained healthy, and none lost weight during the critical period (up to 12 dpc). Vaccinated cats showed mild and transient clinical signs, such as a slight nasal discharge, sneezing, ocular discharge, and/or conjunctivitis. Slight hyperthermia (39.5 °C) and transient conjunctivitis were observed in a few vaccinated cats. The mean global clinical score was significantly lower in the vaccinated group than in the controls: 7.7 vs. 26.7 (3.5) in the 4-week challenge trial (Student’s t test, P = 7.10 5 ), and 15.3 vs. 40.8 (2.7) in the 1-week challenge trial (Student’s t test, P = 1.0 10 4) (Fig. 3). The relative efficacy was 62.5% in the 1-week FHV challenge trial and 71.2% in the 4-week FHV challenge trial. All vaccinates and controls were seronegative on day 0 in both FHV trials. In the 4-week challenge trial, after the first vaccine injection, only 2/11 vaccinates seroconverted. The second vaccine injection induced neutralising antibodies in 7/11 vaccinates (average 0.6 log10). All control cats were seronegative at the time of challenge (average 60.2 log10).
AUC: vaccinated = 61.1 controls = 85.7
8.00 7.00 6.00 5.00 4.00 3.00 2.00 0
2
4
6 8 10 Days post-challenge Vaccinates
12
89
14
Controls
Fig. 2. Virus shedding measured by quantitative PCR in rectal swabs after FPV challenge, 1 week post-vaccination. 10
Global score :
Mean clinical score
FHV challenge, 4 weeks after V2
vaccinated = 7.7 controls = 26.7
8 6 4 2 0
General condition
Weight
Hyperthermia
Glossitis
Nasal discharge
Vaccinates
Cough
Sneezing
Conjunctivitis
Controls
14
Global score :
FHV challenge, 1 week after V2
vaccinated = 15.3 controls = 40.8
12 Mean clinical score
Ocular discharge
10 8 6 4 2 0
General condition
Weight
Hyperthermia
Glossitis
Nasal discharge
Vaccinates
Cough
Sneezing
Controls
Fig. 3. Clinical scores after FHV challenge 4 weeks and 1 week post-vaccination.
Ocular discharge
Conjunctivitis
90
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In the 1-week challenge trial, all vaccinates developed FHV ELISA antibodies after the first injection (average 1.6 log10) or the second injection (average 2.3 log10), whereas controls remained seronegative (average <0.9 log10). No virus was isolated from any cat prior to challenge. After challenge, viral excretion was observed in both groups with a peak 4 dpc in both FHV trials. In the 4-week challenge trial, viral excretion decreased more rapidly in the vaccinated group than in the control group. Two days after challenge, 8/11 control cats (72.7%) and 7/11 vaccinated cats (63.6%) shed virus. From 4 to 8 dpc, all controls and vaccinates shed virus. Eleven dpc, viral excretion was detected in only 4/11 vaccinates (36.4%), whereas 8/11 controls (72.7%) excreted virus. Virus shedding was on average twice as high in controls as in vaccinates for the entire duration of the study. Although the duration of excretion was usually reduced in vaccinates, the difference between groups was not statistically significant (AUC vaccinated group 48.7 vs. control group 53.2; Student’s t test, P = 0.2). In the 1-week challenge trial, virus isolation and PCR method gave consistent results. FHV excretion began earlier and lasted longer in controls than in vaccinates. On the basis of the infectious titres, controls excreted 10 times more virus on average after challenge during the entire monitoring period. Viral shedding (PCR results) was significantly reduced in the vaccinated group (AUC vacci-
nated group = 78.0 vs. control group = 85.2, Student’s t test, P = 5 10 4). FCV trials After challenge, all control cats displayed typical clinical signs such as oronasal ulcers (most often large and numerous), nasal discharge, hyperthermia, and/or weight loss (P5%). Ocular discharge and depression were occasionally observed. Cutaneous ulcerations (footpads) were recorded in 2/10 controls in the 1-week challenge trial. Vaccinates presented only mild clinical signs limited to a few oronasal ulcers of small size, and a slight nasal discharge. Occasional weight loss was observed in one vaccinated cat in the 4-week challenge trial. Slight and transient hyperthermia (39.5–39.7 °C) was recorded in 3/10 vaccinates in the 1-week challenge trial. None of the vaccinated cats had cutaneous ulceration (Fig. 4). In both trials, the mean global score of the control group was significantly higher than that of the vaccinated group (27.8 in controls vs. 6 in vaccinates in the 4-week challenge trial and 24.0 in controls vs. 4.9 in vaccinates in the 1-week challenge trial) (one-way ANOVA test, P = 0.01 in the 4-week challenge trial; Wilcoxon test, P = 0.0017 in the 1-week challenge trial). The relative efficacy was 79.6% in the 1-week FCV challenge trial and 78.4% in the 4-week FCV challenge trial.
20
Global score : vaccinated = 6.0 controls = 27.8
Mean clinical score
FCV challenge, 4 weeks after V2 16 12 8 4 0
General condition Weight loss
Hyperthermia Oronasal ulceration Nasal discharge Vaccinates
Ocular discharge
Controls
20 Global score : vaccinated = 4.9 controls = 24.0
FCV challenge, 1 week after V2 Mean clinical score
16
12
8
4
0 General condition
Weight loss
Hyperthermia Oronasal ulceration Nasal discharge Ocular discharge Vaccinates
Controls
Fig. 4. Clinical scores after FCV challenge 4 weeks and 1 week post-vaccination.
D. Jas et al. / The Veterinary Journal 182 (2009) 86–93
All vaccinates developed FCV antibodies after the second vaccine injection (mean = 2.2 log10 at D56 in the 4week challenge trial and mean = 2.3 log10 on D35 in the 1-week challenge trial), whereas controls remained seronegative (mean <0.5 log10 at D56). None of the cats shed FCV before challenge but all control cats excreted FCV, with a peak on day 4, after challenge. Virus shedding was lower and shorter in the vaccinated group. In the 4-week challenge trial, 4 dpc, three vaccinates and all controls excreted FCV. Controls shed on average about twice as much virus as vaccinates after challenge. Viral shedding was significantly reduced in the vaccinated group (AUC vaccinated group 22.0 vs. control group 27.0; one-way ANOVA test, P = 0.007). In the 1-week challenge trial, controls excreted on average about twice as much virus as the vaccinates after challenge. Viral shedding was significantly reduced in the vaccinated group (AUC vaccinated group 33.9 vs. control group 39.3; Wilcoxon test, P = 7.3 10 3). Table 2 summarises the overall results of efficacy studies for FPV, FHV and FCV. Discussion The objective of these trials was to evaluate the onset of immunity in kittens following vaccination against feline parvovirus, herpesvirus and calicivirus. Onset of immunity was evaluated by challenge, 1 week after vaccination with a RCPCh-FeLV combination vaccine formulated at minimum dose. The protection obtained 1 week after vaccination was compared with that obtained when the animals were challenged 3 or 4 weeks after vaccination. The RCPCh-FeLV vaccine is a non-adjuvanted vaccine combining live and inactivated components and the parvovirus and herpesvirus components are live, attenuated strains. Live attenuated FPV vaccines are preferred because of their quick onset of immunity and greater efficacy in the presence of maternal antibodies. The efficacy of modified live FHV vaccines is similar to that of inactivated vaccines (Povey and Wilson, 1978), but live vaccines do not require
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an adjuvant. The use of FPV and FHV vaccine strains was made possible after demonstration of their safety and the irreversibility of their attenuation. Unlike those genetically stable DNA viruses, FCV is a RNA virus with a high rate of mutation and genetic variability (Kreutz et al., 1998; Glenn et al., 1999; Coyne et al., 2007), and modified live strains have been shown to occasionally generate pathogenic variants (Dawson et al., 1993; Radford et al., 1997; Radford et al., 2001). Therefore, for safety reasons, an inactivated FCV vaccine is preferred. The FCV antigen was designed to work in the absence of adjuvant. In addition, to improve the cross-protection, the vaccine contains a combination of inactivated FCV antigens derived from two strains with broad cross-reactivity (Poulet et al., 2005). A rapid antibody response against FPV was observed as soon as 7 days after the first vaccination. For FHV and FCV, two injections of vaccine were typically required to induce a seroconversion, whether or not the vaccine was attenuated or inactivated. FHV neutralising antibody titres induced by vaccination were low, as is usually the case with live FHV vaccines (Gaskell et al., 2007). By measuring antibody titres with a sensitive ELISA, it was possible to detect seroconversion after the first vaccine injection in most of the cats. To demonstrate vaccine efficacy, cats were challenged with virulent strains of FPV, FHV or FCV. Highly virulent strains were used to ensure that all control animals would exhibit clinical signs and validate the challenge. Unlike FPV and FHV viruses, FCV is characterised by a strong antigenic diversity (Seal et al., 1993). To be representative of field challenge, the efficacy of the vaccine was tested against a recent heterologous antigenic variant. This variant was poorly neutralised by FCVG1 and FCV431 specific antisera (titre <1.2 log10), and could therefore be considered a stringent test for the vaccine. While controls displayed typical clinical signs after FPV, FHV and FCV challenges performed 1 or 3–4 weeks after vaccination, all vaccinates remained in excellent general condition. Vaccinates were completely protected from clinical disease after FPV challenge, and no severe
Table 2 Comparison of vaccine efficacy against FPV, FHV or FCV challenges performed 1 or 3–4 weeks after vaccination Component of the vaccine
Challenge
Criterion
Controls
Vaccinates
P value
FPV
3 weeks post-vaccination
Death WBC <75% baseline values Death WBC <75% baseline values Virus shedding (AUC)
4/6 2/6 3/5 4/5 85.7
0/6 0/6 0/5 0/5 61.1
– – – – 0.01
Clinical score Virus shedding (AUC) Clinical score Virus shedding (AUC)
26.7 48.7 40.8 85.2
7.7 53.2 15.3 78
<0.0001 0.22 0.0001 0.0005
Clinical score Virus shedding (AUC) Clinical score Virus shedding (AUC)
27.8 27.0 24.0 39.3
6.0 22.0 4.9 33.9
0.01 0.007 0.0017 0.007
1 week post-vaccination
FHV
4 weeks post-vaccination 1 week post-vaccination
FCV
4 weeks post-vaccination 1 week post-vaccination
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leucopenia was recorded, even when the challenge was performed 7 days after a single administration of vaccine. Following FCV and FHV challenges, vaccinated cats showed less severe and more transient clinical signs than controls. A significant decrease in clinical scores was observed in vaccinates after FHV (reduction by a factor of 2.7–3.5) and FCV (reduction by a factor of 4.6–4.9) challenges. Interestingly, the efficacy of the vaccine against a challenge performed 1 week after vaccination was as good as that at 3–4 weeks after vaccination, showing that onset of immunity occurred within a week of vaccination. Vaccine-induced protection was also demonstrated in the reduction of viral shedding. As early as 7 days after vaccination, virus excretion was significantly reduced in intensity and duration in vaccinates after FPV, FHV and FCV challenges. Virus excretion remained low (below the quantification threshold by PCR) and transient in all vaccinates after FPV challenge. Vaccination against FPV reduced excretion titres by several log10. Although all vaccinates excreted virus after FCV and FHV challenges, the amount and duration of virus shedding were significantly reduced compared to controls. Reduced excretion is expected to lower the infectious pressure in cat collectives, and thereby contribute to the efficacy of vaccination. Our results confirmed that FPV is a strong immunogen, requiring only one vaccine injection to induce strong protection. The immunogenicity of FPV attenuated vaccines is explained, at least in part, by the strong replication of the FPV vaccine strain within the vaccinated host. FCV and FHV vaccines are less immunogenic and, regardless of the technology used (attenuated or inactivated components), two vaccine injections are required to induce a protective immune response. In addition, FCV or FHV vaccines do not prevent infection but rather reduce the severity of clinical symptoms and occasionally virus shedding (Radford et al., 2006; Gaskell et al., 2007). It is often assumed that inactivated vaccines have a longer onset of immunity than live attenuated vaccines but our study has shown that a non-adjuvanted inactivated FCV vaccine could induce a significant reduction of clinical signs and excretion with a rapid onset of immunity. The induction of a quick onset of immunity against FPV, FHV and FCV is particularly important in young kittens, especially when maternal antibodies have declined and leave the kitten without protection at 8–12 weeks of age. It is also critical for cats at high risk of exposure, for example in boarding facilities, catteries or shelters. In these conditions, some veterinarians recommend the use of intranasal (IN) vaccines and IN administration of FHV or FCV modified live vaccines has been shown to induce a rapid onset of immunity. A single vaccine administration moderately reduced clinical signs after a FHV challenge 4 days after vaccination (Lappin et al., 2006). IN vaccines may however occasionally induce an upper respiratory tract infection associated with shedding of vaccine virus (Gaskell et al., 2007). Live FCV vaccine strains share the same genetic instability as wild-type strains and may occasion-
ally generate new antigenic variants. Therefore, the benefit/risk ratio of IN live attenuated vaccines should be carefully evaluated. There are currently no IN FHV, FCV and/or FPV vaccines available in Europe. An earlier onset of immunity in young kittens may result if the course of vaccination is started at a younger age. However, maternal antibodies against FPV or FCV may persist for up to 16 weeks and interfere with vaccination (Poulet, 2007). A modified protocol starting at 6 weeks of age and consisting of three vaccine injections at 6, 9 and 12 weeks has been tested in the field (Dawson et al., 2001). With the early vaccination scheme, a few cats responded to the first vaccine injection, but overall, there was no difference between early and conventional vaccination schemes in the rate of response at 12 and 15 weeks of age. In kittens expected to have high maternal antibody titres, it is recommended that the last vaccine injection is given after 16 weeks of age (European Advisory Board on Cat Diseases ABCD Guidelines on FPV June 2006; ABCD Guidelines on FCV March 2007). Conclusions This study showed that the onset of immunity occurred within 1 week following one vaccine injection for FPV and within 1 week following two vaccine injections for FHV and FCV. Because the two injections can be given at an interval of 3–4 weeks, cats can be protected within 1 month. Conflict of interests statement The author and co-authors of this article are employees of Merial, the company marketing Purevax RCPCh-FeLV. References Coyne, K.P., Jones, B.R., Kipar, A., Chantrey, J., Porter, C.J., Barber, P.J., Dawson, S., Gaskell, R.M., Radford, A.D., 2006. Lethal outbreak of disease associated with feline calicivirus infection in cats. Veterinary Record 158, 544–550. Coyne, K.P., Gaskell, R.M., Dawson, S., Porter, C.J., Radford, A.D., 2007. Evolutionary mechanisms of persistence and diversification of a calicivirus within endemically infected natural host populations. Journal of Virology 81, 1961–1971. Dawson, S., Mc Ardle, F., Bennett, D., Carter, S.D., Bennett, M., Ryvar, R., Gaskell, R.M., 1993. Investigation of vaccine reactions and breakdowns after feline calicivirus vaccination. Veterinary Record 132, 346–350. Dawson, S., Willoughby, K., Gaskell, R.M., Wood, G., Chalmers, W.S., 2001. A field trial to assess the effect of vaccination against feline herpesvirus, feline calicivirus and feline panleucopenia virus in 6-weekold kittens. Journal of Feline Medicine and Surgery 3, 17–22. European Advisory Board on Cat Diseases (June 2006) ABCD guidelines on Feline Panleucopenia Virus.
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