Feline calicivirus (FCV) is a common feline pathogen with a potential for antigenic diversity. This study aimed to evaluate and characterize the protective efficacy of the FCV-F9 valency of a tetravalent vaccine, Leucofeligen, against challenge with an unrelated strain. Ten 9-week-old kittens were vaccinated while 10 remained as unvaccinated controls. The vaccinated cats received Leucofeligen twice subcutaneously with a 3-week interval. Four weeks after the second vaccination, all cats were challenged with virulent heterologous FCV and followed up for 21 days, monitoring their general condition, clinical signs, and immunological responses. During the vaccination phase, rectal temperatures and body weights were indistinguishable between the two groups. Only vaccinated cats showed FCV-specific seroconversion (both total and neutralizing antibodies). In the first week after challenge, the vaccinated cats had an 82.6% reduction in median clinical score compared to controls. Leucofeligen was thus shown to provide a significant clinical protection to kittens challenged with heterologous virulent FCV. This protection was similar whether the cats had neutralizing antibody or not, indicating a key role for cellular immunity in the overall protection. This also suggests that previously reported seroneutralisation studies may underestimate the level of cross-protection against field strains obtained with this modified live FCV-F9 vaccine.
Feline calicivirus (FCV) is a common pathogen of cats normally infecting the oral cavity and upper respiratory tract. The initial infection generally results in acute clinical signs such as fever and lingual or oral ulceration, in addition to sneezing, rhinitis, and conjunctivitis [
FCV has a single-stranded positive-sense RNA genome of ~7.7 kb. The replication of FCV, like other RNA viruses in general, results in a high proportion of genomic as well as antigenic variants. Indeed the overall identity of FCV isolates collected worldwide was reported to be approximately 80% for the variable and immunodominant regions C to E of the capsid gene [
A tetravalent vaccine, Leucofeligen, was developed in our laboratory containing live attenuated FCV, feline herpesvirus (FHV-1, also known as feline rhinotracheitis virus) and feline panleukopenia virus (FPV), as well as the recombinant p45 antigen of feline leukaemia virus (FeLV).
When developing this vaccine, we decided to evaluate the protective efficacy of the FCV valency against an unrelated virulent heterologous challenge strain and also to attempt to characterize the nature of the protective immune response.
The study was carried out in accordance with the Good Laboratory Practice guidelines, and additionally in accordance with the recommendations issued in the European Pharmacopoeia [
Twenty specific-pathogen-free (SPF) European kittens, 9 weeks old, were randomly assigned to 2 groups: control (unvaccinated, hereafter designated group C) and vaccinated (hereafter designated group V). Cats were acclimatized for 6 days to the animal housing conditions (12 h light/dark cycle, 18 ± 3°C, 55 ± 10% humidity, with free access to water). Each group was housed in a separate airspace in the animal housing facility.
Group V cats were vaccinated twice at a 3-week interval (day 0 and day 21) by subcutaneous injection (1 mL) according to the recommendations of the manufacturer. In order to better assess the local tolerance of the injections, the first injection was given half way between the shoulder and hip on the right side, and left side was used for the second injection. Group C cats did not receive any injections.
Four weeks after the second vaccination, on day 49, equivalent to postchallenge time 0 (= pct0), all cats were challenged with a virulent heterologous strain of calicivirus (FCV-255). Cats were first anesthetized and then inoculated intranasally with
Leucofeligen was granted a pan-European marketing authorization (centralised procedure) in 2009. It is presented as a freeze-dried fraction containing the live attenuated viruses, that is, FCV (F9), FHV-1 (F2), and FPV (LR72), and a liquid fraction containing the recombinant FeLV-envelope antigen p45 (derived from the gp70 of FeLV) with aluminium hydroxide and QA-21 adjuvants. The calici valency in the freeze-dried fraction was formulated at the minimum accepted titre for this vaccine. The vaccine vials were stored at
In the vaccination phase, cats were monitored daily for general health status (food intake, appearance of feces, and behaviour/depression). The animals were weighed weekly. In the postchallenge phase, clinical examinations were performed daily, and the clinical status was evaluated according to a scoring system based on that specified in the pharmacopoeia monograph [
Scoring system for the parameters concerning general health conditions and clinical signs.
Symptoms | Description | Notation |
---|---|---|
Rectal temperature (°C) | 37.1–39.4 |
0 |
| ||
Body weight* | Gain or loss of <3% |
0 |
| ||
Ulcers (oral and/or nasal) | Absence |
0 |
| ||
Nasal discharge | Absence |
0 |
| ||
Ocular discharge | Absence |
0 |
Blood samples were collected from the animals in uncoated tubes for serological assessment on days 0, 21, 35, 49 (= pct0), 56 (= pct7), 63 (= pct14), and 70 (= pct21).
The serological assessments involved assaying IgG anticalicivirus antibody (Ab) and anti-calicivirus neutralizing antibody (NAb) titres.
Titres of IgG against calicivirus were assessed using an immunofluorescent antibody assay. Briefly, 50 µL of two-fold dilutions of each serum (from 1/64 to 1/8192) was added to a 96-well plate containing acetone-fixed CRFK cells infected with FCV-F9. 50 µL of a positive serum and 50 µL of a negative serum were diluted in the same way and used as controls. They were incubated for 1 hour at 37°C and revealed with a fluorescein-conjugated antifeline IgG antibody and a solution of Evans Blue. The positivity threshold was 1/128.
Titres of NAb were determined to the homologous FCV-F9. Briefly, 50 µL of each serum was diluted with L15/McCoy’s medium to provide 6 2-fold dilution steps between 1/8 and 1/256. 200 µL per dilution was incubated for 1 hour with 200 µL of FCV-F9 suspension at a concentration of approximately 100 TCID50 to allow viral neutralisation. 50 µL of each mixture was then added to 6 plates of a 96-well plate containing 70% confluent CRFK cells. After 6 days of incubation, the characteristic cytopathic effect was assessed. The titre was determined by the Spearman and Karber method [
All statistical analyses were performed using the S-PLUS 6.2 software package (Insightful, Paris). For the comparison of rectal temperatures, body weights, and clinical scores between group C and group V, Student’s
This work was performed under the supervision of the Ethical Committee of Virbac, and in accordance with the requirements of the official European Pharmacopoeia [
During the vaccination phase (day 0 to 49), all cats remained in normal health with a steady increase in their body weights and with normal body (rectal) temperatures. No abnormal general or local reactions were noted in relation to the vaccinations administered.
No cat in either group displayed any clinical signs during the vaccination phase.
The time course of the Ab responses induced by FCV-F9 vaccination (day 0–49, vaccination phase) is shown in Figure
Time courses of homologous anti-FCVF9-NAb responses in cats after vaccination with FCV-F9 (group mean,
Time courses of anti-FCV-F9-IgG responses in cats after vaccination with FCV-F9 (group mean,
In contrast to the NAb responses, anti-FCV-IgG responses were elicited in a uniform and strong manner in all vaccinated cats by day 35 and they remained high on day 49 (Figure
At the start of the challenge phase (pct0), there was no difference in the body weights of the two groups (
Time points of the body weights (group mean,
During the same time period, hyperthermia (>39.4°C) was observed for every cat in group C on at least 2 of the days, with 4 of the 10 cats presenting peaks of at least 40°C and one of them sustaining a rectal temperature of >40°C for 4 days. In contrast, in group V transient peaks, lasting no more than 1 day, were observed in 7 of the 10 cats. 3 of those were only 39.5°C, and none of the group V cats reached 40°C. Mean rectal temperatures were significantly different between the two groups on days 3 to 5 after challenge (
Daily rectal temperatures of the cats (group mean,
During the first week after FCV challenge (pct0 to pct7) a significant difference was observed between the groups regarding the development of clinical signs.
Nine of the 10 group C cats developed oral and/or nasal ulcers: 4 cats had the maximum score of 3 (corresponding to large and/or numerous ulcers—see Table
In general, group V cats developed substantially reduced clinical signs and for a substantially shorter duration compared with the control cats (Figure
Cumulative clinical scores (CS) using the scoring system as defined in Table
Cat ID group V | Cumulative CS Week 1 (pct0 to pct7) | NAb at challenge | Cat ID group C | Cumulative CS Week 1 (pct0 to pct7) | NAb at challenge |
---|---|---|---|---|---|
1 | 12 | (−) | 1 | 10 | (−) |
2 | 3 | (−) | 2 | 11 | (−) |
3 | 0 | + | 3 | 13 | (−) |
4 | 1 | + | 4 | 8 | (−) |
5 | 1 | (−) | 5 | 8 | (−) |
6 | 4 | (−) | 6 | 29 | (−) |
7 | 1 | + | 7 | 12 | (−) |
8 | 4 | + | 8 | 7 | (−) |
9 | 6 | + | 9 | 16 | (−) |
10 | 1 | (−) | 10 | 18 | (−) |
| |||||
Median | 2.0 | Median | 11.5 | ||
| |||||
Q25%; Q75% | 1.0; 4.0 | Q25%; Q75% | 8.0; 16.0 | ||
| |||||
Mean |
3.3 |
Mean |
13.2 |
Cumulative clinical scores (weekly median,
After the first week following challenge, and entirely as expected, most cats improved rapidly. However, cumulative scores for ulceration remained significantly different between pct8 and pct21 (
Following the heterologous FCV-challenge on day 49 (= pct0), the mean homologous NAb titres (Figure
The stimulation of anti-FCV-IgG responses (Figure
Due to the variable nature of FCV, the ability to cross-protect against unrelated strains is a key requirement for an efficacious vaccine [
The choice of FCV-255 as a heterologous challenge strain was legitimate due to the dissimilarity of the two virus strains. It seems that the maximum dissimilarity detected within the calicivirus pool is in the order of 30% [
The level of clinical protection achieved in this study was very encouraging. Current guidelines [
The signs which are most visible for the pet owner (nasal discharge, ocular discharge, and weight loss) were completely prevented during this time. Likewise severe fever likely to result in noticeable lethargy and malaise was also completely prevented. In this context, it can be noted that, in the monograph for FHV, severe hyperthermia (40°C or higher) is given a higher score, allowing the severity of this sign to be reflected in the final scores [
Ulcers are noticeable to the owner only when widespread and severe. Therefore the reduction in both the duration and the severity of the ulceration also means that it is unlikely that an owner would be aware of these ulcers in a vaccinated cat in the great majority of cases. Complete prevention of ulceration is not expected in such a study with direct administration of high doses of pathogenic virus. Indeed we can assume that the levels of mucosal IgA required to block such doses of virus are unlikely to be achievable, meaning that some cytopathic effect is inevitable. Nevertheless, a reduction in the severity and duration of ulceration and fever is probably the two parameters which most benefit the welfare of the animal, one of the main reasons to use FCV vaccines.
Following the serological responses in addition to the clinical responses was also very interesting in this study. IgG antibodies levels rose to high titres during the challenge phase in all cats in this study, regardless of the severity of the clinical signs, and therefore appear to be simply a marker of exposure to the virus and do not indicate useful information about the level of protection achieved.
During the vaccination phase (day 0–49) of the present study, the titres of anti-FCV-NAb produced by the vaccinated cats were neither high nor long lasting. This is very much in line with previous published work, where studies based on seroneutralisation required use of altered and intense protocols of exposure to the vaccine strains to induce sufficiently high titres to permit cross-neutralisation assays to be performed [
At the time of the heterologous challenge, cats in our study with detectable levels of NAb were protected against severe disease. However, by contrast, half the vaccinated cats had no detectable NAb, and the lack of NAb was not correlated with susceptibility to the infection.
As a result, we can conclude that high NAb titres appear to function as a marker that an active immune response has been produced but are probably not the key protective agent against this virus. The lack of NAb titres in healed (and presumably therefore immune) control cats strongly supports this conclusion.
Such a result is perhaps not entirely surprising. Indeed in terms of antibody protection, circulating antibody has a minimal benefit to offer in terms of protection against a mucosal virus, where perhaps levels of IgA mucosal antibody would be more interesting if it was possible to assay these accurately. More importantly, when using modified live vaccines, it is widely accepted that cell-mediated immunity is likely to be a major factor in the protection induced by the vaccine [
In a study focussed on a bivalent inactivated vaccine [
This finding on the relevance of NAb titres also confirms the need to perform confirmatory challenge studies in order to be able to draw useful conclusions on the efficacy of an FCV vaccine. It would also be interesting to look further at specific cell-mediated immunity parameters in future studies.
Our study demonstrated that the combination vaccine, Leucofeligen, could provide a protective efficacy (reduction of clinical score) of 82.6% during the first week after challenge in vaccinated kittens challenged with virulent heterologous FCV. The signs most visible for the pet owner (nasal discharge, ocular discharge, and weight loss) were completely prevented during this time, as was severe fever likely to result in noticeable lethargy or malaise. High titres of neutralising antibody seem to indicate protection, but the absence of NAb does not indicate susceptibility, indicating a role for cell-mediated immunity with this vaccine. It therefore appears that, when modified live F9 vaccines are used,
The paper relates to the performance of a vaccine which is manufactured by Virbac. All the authors of this paper were employed by Virbac, which also funded the study.