Onset of Protection of FeLV Vaccine Containing Purified P45 FeLV-Envelope Antigen after One Single Injection of Primary Vaccination in Cats

Animals and Housing

Twenty-five specific-pathogen free European kittens (63-67 days old; average body weight of 1.02 ± 0.18 kg on day 0, ranging from 0.7 to 1.5 kg) were allocated into two groups: group A (vaccinated with Leucogen®), group B (control, not vaccinated). The cats were acclimated for 5 days to the animal housing conditions before vaccination. The animals were fed with a complete diet for growing kittens, and food and water were provided ad libitum. On the day of the challenge strain inoculation, feed and water were withdrawn prior to sedation and returned when all cats had recovered from sedation.

Vaccine and Vaccination

Leucogen® has been granted a European marketing authorisation (centralised procedure). It is presented as a liquid fraction containing the recombinant FeLV-envelope antigen p45 (derived from FeLV gp70) with aluminium hydroxide gel and Quillaja saponaria QA-21 adjuvants. The vaccine vials were stored at temperature between + 2°C and + 8°C before administration.

The cats were allocated into two groups according to age, weight, sex and avoiding having a group consisting of animals from the same litter. Cats assigned to group A received each one dose of Leucogen® vaccine administered subcutaneously halfway between the right shoulder and the right hip (on the flank). The injection site was clipped and disinfected prior to vaccination. Cats assigned to group B were not vaccinated.

Challenge

Three weeks after vaccination, at day 21 (Week Post Challenge 0), all cats were inoculated with a virulent strain of Feline Leukaemia virus, FeLV subgroup A (FeLV-A 13.04). The origin of this challenge strain seed is cells infected with FeLV-A, provided by Cambridge Biosciences Corporation. They were anesthetized, and 1 ml of a viral suspension was inoculated per cat by oro-nasal route (0.25 ml in each nostril and 0.5 ml in the mouth). They were then observed for 15 weeks, until day 126 (Week Post Challenge 15). This FeLV

subgroup A strain is the dominant subgroup found in cats. The oronasal route is selected to abide by the European Pharmacopoeia and also to try to be as close as possible to the natural infection. The transmission by FeLV-infected cats occurs through viral shedding mainly in saliva and mainly through friendly contact.

Monitoring and Samples

During the whole study, cats were observed daily for any abnormal behaviour, clinical sign, aspect of faeces, food intake, and general health status. Furthermore, a complete clinical examination, including weighing and rectal temperature measurement, was carried out on a weekly basis. Additional clinical examinations were conducted when clinical findings were observed. Blood samples of at least 2 mL were collected at day 0 before vaccine administration and then at day 14. On the day of challenge (WPC0), blood collection was carried out prior to virus inoculation. After the challenge, one blood sample was collected on a weekly basis from day 42 (WPC3) till day 126 (WPC15). Serum was collected by these samples and stored at kept frozen (≤ 15°C) until analysis.

Laboratory Analyses and Scoring

Titration of Antibodies against FeLV p45 Protein: Antibodies against FeLV p45 protein were dosed using ELISA test in serum samples collected during the vaccinal phase. Plates were firstly coated with the FeLV p45 protein, identical to that contained in Leucogen® vaccine (0.5 µg/well in carbonate buffer). After blocking of non-specific sites with 1% BSA TNE buffer, several dilutions of each serum (three fold serial dilutions in 0.1% BSA TNE buffer: 1/150th to 1/36450th) were added to the plate. Positive and negative sera were diluted the same way and used as controls. After incubation, the antibodies specifically bound to the p45 protein were revealed with a specific peroxidase conjugated antibody (ABTS coloration). The titre corresponding to the first dilution with an optical density at 405 nm inferior to 0.5 was recorded. The positivity threshold was set up at 1/450.

Detection of FeLV p27 Antigen Protein: Detection of FeLV p27 protein in serum samples collected during the challenge phase was performed using the commercial IDEXX ELISA FeLV Antigen test kit (Référence IDEXX du kit: 99-07896). This test kit uses 12-well strips coated with anti-p27 antibodies. Samples were added in the wells previously coated and incubated with anti-FeLV antibodies conjugated to HorseRadisch PerOxidase (HRPO). If present in the sample, the FeLV antigen binded with both the antibody coated in the well and the HPRO/antibody, conjugate. After washing the wells, the substrate and the chromogen were added. The change in blue colour of the patch corresponding to “Ag FeLV” indicates the presence of FeLV-derived P27 antigens in the sample tested. The test is valid if the possible appearance of background colour doesn’t prevent the reading, the patch corresponding to the positive control turns in blue colour and the patch corresponding to the negative control is not (or very slightly) coloured. In the case of a valid test, the result is negative if only the patch corresponding to the positive control turns in blue colour. In case of a valid test, the result is positive if the patch corresponding to the positive control and the patch corresponding to “Ag FeLV” turn in blue colour.

Statistical Tests: The statistical analysis is performed using SAS 9.3 software. The proportion of cats persistently infected in vaccinated and control groups is compared using Fisher’s Exact Test. A p value < 0.05 is considered significant.

Vaccination Phase (day 0 to day 21)

Clinical Monitoring and Safety: During the vaccinal phase, the cats remained in good health, with a normal increase of their body weight and a rectal temperature within the physiological range. No specific clinical findings were observed during or shortly after vaccination. No unexpected general or local reactions were reported after vaccination. On a single occasion (day 28), a pin head-sized subcutaneous swelling at the injection site (right thoracic wall) was observed in one out of 15 vaccinated animals. The injection site swelling was movable and not painful. This transient finding may be considered to be due to the vaccination and represents a not unexpected finding.

Immune Response: Prior to vaccination, all cats were free from antibodies against FeLV p45 protein. The control cats remained seronegative from day 0 to day 21. After vaccination with Leucogen®, 4 cats out of 15 seroconverted to the vaccine protein from day 14, with antibody titres ranging from 1/450 to 1/1350. 3 additional cats seroconverted from day 21, with antibody titres of 1/450.

Post-Challenge Phase (day 21 to day 126)

Clinical Monitoring: After inoculation of the challenge strain, the cats remained in good health, with the body weight increase expected for cats at this age and a rectal temperature within the physiological range. During this 15- week period, enlarged superficial lymph nodes (lymph node mandibularis, and popliteus superficialis) were recorded after the inoculation in both groups. In the control group, 9 out of 10 (90%) not vaccinated cats showed enlarged peripheral lymph nodes for at least 7 weeks, consecutive or not. In the vaccinated group, peripheral lymph nodes appeared enlarged in only 8 out of 15 cats (53%). The challenge with virulent FeLV led to the occurrence of enlarged peripheral lymph nodes at a higher incidence in the control group (9/10 cat i.e. 90%) compared to the vaccinated group (8/15 cats i.e. 53%). FeLV infection related clinical signs (oedematous swelling at the head, moderately impaired general condition, enlarged superficial lymph nodes, pale mucosa, no feed consumption) were observed in one control animal from D56 to D70. Similar findings were not found in the animals of the vaccinated group.

P27 Antigenemia Follow-Up: The number and percentage of animals with circulating P27 viral antigen in the blood at each time point and the persistent infection status at the end of the study are summarised in Table 1. All kittens were negative for FeLV p27 antigen prior to inoculation of the challenge strain (WPC0). Three weeks later, on WPC3, 80% of the control cats (8/10) and 33% of the vaccinated cats (5/15) were positive for FeLV p27 antigen. During the rest of the post-challenge phase, the percentage of positive animals ranged from 70 to 90% in the control group whereas it dropped to 27% in the vaccinated group from WPC4 and remained stable until the end of the study.

Persistent Viraemia Status: According to the European Pharmacopoeia Monograph n°1321 (Feline Leukemia Vaccine Inactivated), a cat is considered as persistently infected if it shows positive P27 antigenaemia either during three consecutive weeks or on five occasions, consecutive or not, between the third and the fifteenth week post-challenge. The persistent infection status was determined for each cat (Table 1). Following administration of the virulent FeLV strain, 9 control cats out of 10 (90%) and 4 Leucogen® vaccinated cats out of 15 (27%) met the criteria for a persistent infection. According to the European Monograph, a FeLV challenge was valid when at least 80% of control animals revealed a persistent infection. The validity of this experimental infection model used for assessing FeLV vaccine efficacy is confirmed by the results obtained in the control group, as 90% of the control cats developed a persistent infection. After one injection of Leucogen® vaccine, 11 vaccinated cats out of 15 (73%) did not develop a FeLV persistent viremia. Statistical analyses showed that the proportion of cats persistently infected was significantly lower in the vaccinated group than in the control group (p=0.0036). The status of cats regarding persistent infection post challenge were presented in Figure 1.

Vaccine Efficacy Assessment: Persistent viraemia preventable fraction was calculated as follows: (% of controls with a persistent infection - % of vaccinates with a persistent infection) / % of controls with a persistent infection. The vaccine efficacy measured as the persistent viraemia preventable fraction for Leucogen® vaccine was equal to 70%.

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a: (-) absence of circulating FeLV p27 protein in the blood; (+) detection of circulating FeLV p27 protein in the blood b: cat showing antibody response post-vaccination c Number (percentage) of cats positive for FeLV p27 antigen or positive for persistent infection Table 1: Post-Challenge FeLV p27 antigenaemia status and persistent infection status of cats in each group.