Combined administration in a single injection of a feline multivalent modified live vaccine against FHV, FCV, and FPLV together with a recombinant FeLV vaccine is both safe and efficacious for all four major feline viral pathogens

Abstract

Nobivac Tricat, a lyophilised trivalent modified live attenuated vaccine is routinely used to protect cats against three commonly diagnosed feline viral pathogens namely herpesvirus, calicivirus and panleukopenia virus. The recognition of feline leukaemia virus (FeLV) as an important viral pathogen has prompted the development of an efficacious liquid recombinant subunit FeLV vaccine (p45 envelope protein). Lyophilised Tricat vaccine was dissolved in the liquid FeLV vaccine and no detectable deleterious effect on the titre of any of the live virus components was observed after 2h incubation. In vivo studies where the vaccines were mixed in the same syringe prior to inoculation showed no alteration to the safety profile assessed by repeat and overdose studies. Serological comparisons of the modified live viral antibody titres showed no evidence of reduced responses following administration of the mixed products. Challenge studies using pathogenic herpesvirus and FeLV revealed no difference in the degree of clinical protection. This paper shows that neither safety nor efficacy is adversely affected as a result of mixing the two vaccines.

There is a global consensus that combination vaccines are desirable both in veterinary and human medicine. In particular, in the feline medical field, apart from the obvious practical benefits of administering all the vaccinal antigens that have been judged as being required by an individual cat as a single injection, there are further important health and welfare benefits. For example, in the last few years several reports (Nambiar et al 2001, Vascellari et al 2003) have suggested that the increased incidence of fibrosarcomas in cats at commonly used injection sites may be associated with the repeated inoculation of various injectable products. Reducing the number of injections that a cat will receive should in theory minimise this risk as well as reducing any injection related stress.

Research experience over the years has demonstrated that the formulation of combined vaccines is a much more complex exercise than simply mixing together the different antigens (Brunner et al 2006). Both the physical compatibility and the stability of individual antigens in combination are highly dependent on vaccine excipients. The type of adjuvant and buffer used, the presence or absence of preservatives, the pH and tonicity all play an important role in determining the final combination (Andre 1994). In addition some of these vaccine excipients could potentially inactivate any live attenuated vaccinal components present in the combination. For example, it has been considered that some components of a widely used recombinant subunit feline leukaemia virus (FeLV) vaccine, such as the detergent components of Quil A, may inactivate enveloped viruses such as feline herpesvirus (FHV) (also known as feline rhinotracheitis virus) a component of current live trivalent cat vaccines (Spickler and Roth 2003).

For the purpose of these investigations a trivalent modified live vaccine (Nobivac Tricat) that is routinely used in practice against feline calicivirus (FCV), FHV and feline panleukopenia virus (FPLV) infections was tested for compatibility with the aforementioned recombinant p45 FeLV envelope protein vaccine (Nobivac FeLV). Although the in vitro potency tests that are currently used before release of monovalent vaccines provide useful information regarding the feasibility of these vaccinal combinations, these may not predict the occurrence of immune interference when they are administered to animals (Sesardic et al 1999). Therefore, as part of this series of investigations additional in vivo studies were performed to compare the immunogenicity of the individual vaccinal antigens in groups of cats given extreme combinations of either high or low titre Nobivac Tricat with or without Nobivac FeLV vaccine. The immunogenicity of these two vaccines was also assessed in maternally derived antibody (MDA) positive kittens for all the live attenuated vaccinal viruses.

One of the biggest challenges in the development of combination vaccines is ensuring that the combination remains as efficacious as its individual component antigens. Therefore, for this exercise, challenge studies were also undertaken against FHV and FeLV as serum antibodies against these pathogens are not usually regarded as predictive in respect of clinical protection.

Finally, the safety profile of this combination was assessed in overdose and repeated dose animal studies by examining the vaccinated cats for any local or general reactogenicity to the individual or combined vaccines. These studies were used to support a variation to the authorised summary of product characteristics to allow concurrent and simultaneous use of these vaccines.

Materials and Methods

Vaccines

Several commercial batches of the Nobivac Tricat and Nobivac FeLV (Intervet UK) were used in each of the experiments below.

Cells and viruses

Crandell–Rees feline kidney II (CrFK) cells were used to assay the titres of the live attenuated viral vaccines (FHV, FCV and FPLV) as they have been detailed elsewhere (Dawson et al 2001). In brief, the CrFK cells were cultured at 37°C in M6B8 culture medium supplemented with 10% foetal calf serum and antibiotics (Invitrogen–Gibco). Both the FPL and FCV fractions were assayed at 37°C and the FHV at 34°C.

The vaccinal FPLV, FHV and FCV strains were used as the viral control antigens in the titration assays. The Glasgow FeLV-A and the FHV SGE strains were used as the challenge viral stocks.

In vitro compatibility

Each live viral vaccine component (FCV, FHV, and FPLV) was individually reconstituted with Nobivac FeLV and titrated on CrFK cells either immediately after reconstitution (T=0) or after the vaccine suspensions were held at room temperature for different times (30, 60, and 120 min). The viral components were also individually reconstituted in Nobivac Solvent and titrated concurrently at the different time points as mentioned above, for comparison. The titre for each virus was calculated in log₁₀TCID₅₀ ml⁻¹ (Reed and Muench 1938).

In vivo compatibility

All kittens used for the following four in vivo studies (experiments 1–4) were obtained from an accredited breeding establishment and were shown to be negative before vaccination for the presence of FHV, FCV, FPLV using standard virus isolation methods (Coutts et al 1994) and FeLV using a p27 ELISA kit (ViraChek; Synbiotics). They were also negative for Bordetella bronchiseptica (Jacobs et al 1993). It was important to confirm the absence of this pathogen as B bronchiseptica produces clinical signs which can mimic FHV infection (Hoskins 1999). Antibodies to the various vaccine components were quantified by serum neutralisation assays (FCV-F9 strain, Johnson and Povey 1983; FHV-G2620A strain, Dawson et al 2001 and haemaglutination inhibition assay (HAI), FPLV-Bristol strain; Dawson et al 2001). Heparinised blood samples were submitted to the Feline Advisory Unit, Glasgow University to detect the presence of FeLV antibodies (neutralising and p27) and antigen.

Experiment 1: 10× overdose and repeat dose safety study

Twelve 9-week-old kittens, seronegative for FCV, FHV, FPLV and FeLV were each given a subcutaneous inoculation of Nobivac Tricat (10× maximum release dose) combined with two doses of Nobivac FeLV immediately before use and clinically observed for 14 days thereafter for adverse reactions. Three weeks post vaccination each cat was given a second inoculation of Nobivac Tricat (1× maximum release dose) combined with one dose of Nobivac FeLV. Cats were clinically monitored for a further 3 weeks at time of feeding for signs of adverse reactions. A third vaccination, comprising Nobivac Tricat (1× maximum release dose) combined with one dose of Nobivac FeLV was given at 3 weeks post second vaccination and the cats were then clinically observed for a further 3-week period post this third vaccination.

Experiment 2: vaccination with Nobivac Tricat+Nobivac FeLV in MDA positive kittens followed by assessment of the immune responses to the live attenuated vaccinal viruses

Twenty-seven 9-week-old kittens, that were virus negative for FCV, FHV, FPLV and FeLV, were used in this study that comprised three equally sized groups. All kittens were MDA positive for FHV, FCV and FPLV. In group 1, nine cats were vaccinated subcutaneously twice in accordance with the recommended schedule (9 and 12 weeks of age) with minimum release titres of Nobivac Tricat reconstituted with Nobivac FeLV. In group 2, nine cats were vaccinated as above but with the Nobivac Tricat vaccine and the Nobivac FeLV vaccine given at the same time but at different sites. In group 3, nine cats were vaccinated as above but with Nobivac Tricat only.

All the cats were bled on the day of, but prior to, vaccination to determine the antibody status to all vaccinal components and then at secondary vaccination, and then again 3 weeks post secondary vaccination to compare antibody responses against all vaccinal components between the groups.

Antibodies against FCV and FHV were measured by a TCID₅₀ serum neutralisation test; and for FPLV by HAI test as described in the previous section.

Experiment 3: vaccination with Nobivac FeLV and minimum release doses of Nobivac Tricat followed by challenge with FHV

Twenty-five 9-week-old kittens, seronegative and virus negative for FCV, FHV, FPLV and FeLV, were used in this study that comprised three groups. In group 1a, 10 cats were vaccinated subcutaneously twice in accordance with the recommended schedule (9 and 12 weeks of age) with minimum titres of Nobivac Tricat reconstituted with Nobivac FeLV. In group 2a, 10 cats were vaccinated as above but only with the Nobivac Tricat vaccine. In group 3a, five cats were kept as negative controls and they did not receive any vaccination.

All cats in groups 1a and 2a were bled at times of vaccination, but prior to the event and 2 weeks post secondary vaccination, to quantify the antibody responses to the live viral components. All three groups were challenged with pathogenic FHV (SGE strain) 4 weeks post second vaccination and monitored over a 21-day period, after challenge, for clinical signs associated with FHV infection. Pharyngeal swabs were also taken to determine the viral excretion. For each clinical sign a clinical score was assigned according to an in-house clinical scoring system (Table 1) and comparison of these scores was used to determine if the subunit FeLV had a detrimental effect on the FHV component of Nobivac Tricat.

Table 1.

Daily clinical scoring system used for FHV challenge trial

Clinical sign	Score
Pyrexia (°C)
39.7–40.0	1
40.1–40.5	2
40.6–41.0	3
>41.0	4
Nasal discharge
Serous (marked)	1
Mild mucopurulent	2
Marked mucopurulent	3
Sneeze
2–5 in 10 min	1
6–20 in 10 min	2
>20 in 10 min	3
Hyperpnoea	2
Dyspnoea	4
Malaise/depression/eating	1
Malaise/depression/reduced appetite	2
Anorexia	3
Dehydration	4
Respiratory rate
31–40	1
41–50	2
51–60	3
61+	4
Ocular
Lachrymation	1
Marked mucopurulent	4
Mild conjunctivitis	2
Marked conjunctivitis	4

Experiment 4: vaccination with Nobivac FeLV and maximum release dose of Nobivac Tricat followed by challenge with FeLV

In the fourth part of the in vivo studies, 22 9-week-old kittens, seronegative and virus negative for FCV, FHV, FPLV and FeLV were divided into three groups. In the first group (1b) eight cats were vaccinated subcutaneously with maximum titres of Nobivac Tricat reconstituted with Nobivac FeLV. In group 2b, eight cats were vaccinated subcutaneously with Nobivac FeLV, while in group 3b, six cats were vaccinated subcutaneously with Nobivac Tricat (maximum release titres).

All cats were vaccinated as above at 9 and 12 weeks of age and bled at times of vaccination, but prior to the event to measure antibody responses to the live viral components. At 15 weeks of age, all cats were challenged by the oronasal route with 1.0 ml wild type FeLV-A containing 2×10⁶ focus forming units (ffu). Subsequently, all cats were bled at 3-week intervals for 12 weeks to detect viraemia/antigenaemia and neutralising antibody against FeLV.

Statistical analysis

Where appropriate, the data were analysed statistically with the Mann–Whitney U non-parametric test and/or the Wilcoxon rank sum test using Minitab software (USA).

Results

In vitro compatibility

The viral titres (log₁₀TCID₅₀) of Nobivac Tricat components (minimum titres) after incubation with Nobivac FeLV or Nobivac Solvent as illustrated in Table 2 were comparable at all the tested times.

Table 2.

Overview of the titres recovered for each of Nobivac Tricat's components following their reconstitution with Nobivac FeLV for different mixing times

Component	Time (min) post reconstitution	Nobivac Tricat+Nobivac FeLV	Nobivac Tricat+Nobivac Solvent
FCV	0	4.7	4.9
	30	5.1	4.5
	60	4.9	4.3
	120	4.9	4.7
FHV	0	7.4	6.6
	30	7.3	6.8
	60	7.3	6.8
	120	7.3	6.8
FPLV	0	5.4	5.5
	30	5.4	5.4
60	5.5	5.5
	120	5.5	5.6

Results are expressed in TCID₅₀ ml⁻¹.

The results show that there was no adverse effect on any of the three viral components of Nobivac Tricat when combined with Nobivac FeLV in so far as the titres were not significantly altered (ie, ±0.3 log₁₀) even after a 2h incubation period.

In vivo compatibility

Experiment 1: 10× overdose and repeat dose safety study

Following primary vaccination with 10× overdose vaccine, some cats exhibited a slight temperature rise within 24h post vaccination. This resolved by day 2. The main observation was mild localised transient swelling at site of inoculation in 11/12 cats which was probably due to the FeLV component (as this was also observed in subsequent clinical trials including FeLV alone). This, however, was not unexpected, as it is described in the UK summary of product characteristics of this FeLV vaccine. All cats remained healthy throughout second and third vaccinations and no significant temperature increases were observed in any cat post third vaccination. Swellings were again recorded at site of inoculation post third vaccination, although most of these swellings were given a clinical score of 1 (maximum up to 5) indicative of the mildest reaction. Only two cats had clinical scores of 3 (swellings) for a short duration post third vaccination.

Experiment 2: vaccination with Nobivac Tricat+Nobivac FeLV in MDA positive kittens followed by assessment of the immune responses to the live attenuated vaccinal viruses

It is very common in practice that the initial vaccination of kittens is performed in the presence of specific MDA to the vaccinal antigens. It was, therefore, decided to assess the compatibility of these products initially in cats that were MDA positive for FHV, FCV and FPLV. It should be noted that all the cats at the time of first vaccination were FeLV antibody negative. In this study, a comparison between the serological responses of the cats in each group to the same viral component was the main criteria used to assess compatibility. As is shown in Fig 1, no significant differences were observed in the mean antibody responses of all three groups to FPLV and FCV. Most cats, however, showed a decline in FCV antibody 3 weeks post primary vaccination probably due to high MDA levels at time of vaccination. These responses were not, however, sufficient to mask the boosting effect of the secondary vaccination. These results also indicate that the recombinant FeLV vaccine had no detrimental effect on the cats' responses to FCV and FPLV.

Fig 1.

Schematic representation of the kinetics of mean antibody responses (log₁₀) to FHV, FCV and FPLV in MDA positive 8–9-week-old kittens following vaccination. As can be seen the magnitude of the antibody responses to all the vaccinal antigens in all three groups was similar at the end of the recommended primary vaccination scheme (experiment 2).

No significant difference was observed between the response of groups 1 and 3 to FHV, indicating that the subunit FeLV vaccine had no detrimental effect on any of the Tricat components when combined in the same syringe. Interestingly, the antibody response when both vaccines were given at different sites was lower than Tricat alone. This result appears anomalous when compared to the other responses as it would be expected that if the combination of two vaccines in the same syringe showed no incompatibility, then the same vaccines given at different sites should also reveal no incompatibilities. Nonetheless, because in practice it is more convenient to give all vaccines in one syringe, no further investigation of this observation took place and all future studies were conducted on the combined product.

No adverse effects or signs of ill health were observed either at time of vaccination or throughout the observation period post each vaccination, attesting further to the safety profile of this combination of vaccines.

Experiment 3: vaccination with Nobivac FeLV and minimum release doses of Nobivac Tricat followed by challenge with FHV

In this experiment the effect of the combination with FeLV vaccine on the efficacy of the FHV fraction of the Tricat vaccine was tested using a batch of Nobivac Tricat with minimum titres. It was assumed that if this minimum titre combination is efficacious against challenge with a virulent FHV strain then all commercial batches would be at least as efficacious.

Three weeks following the completion of their secondary vaccination schedule all cats were bled and antibodies to the three modified live viral (MLV) vaccines in Tricat were assessed. As shown in Fig 2 the mean antibody responses in the two vaccinated groups for each of the vaccinal antigens were comparable. All the negative control cats were seronegative at that sampling point for all these antigens.

Fig 2.

Group mean antibody responses to FHV, FCV and FPLV in MDA negative kittens 3 weeks following the completion of their secondary vaccination scheme. Titres are expressed in log₁₀ and were similar between the different vaccinated groups for all the vaccinal antigens. The control groups remained seronegative throughout the course of this experiment (experiment 3).

Subsequently, all the cats were exposed to a pathogenic FHV strain (intranasally) and began showing characteristic signs of feline infectious rhinotracheitis. The control cats exhibited more severe signs (mean score=15.8) after challenge compared to the Tricat alone (mean score=4.7) or to the Tricat+FeLV (mean score=7.3) vaccinated groups of cats. The results in both cases were statistically significant (P<0.05). However, no statistically significant difference was observed between both vaccinated groups (Nobivac Tricat and Nobivac Tricat+FeLV groups).

One of the critical issues after viral infection is the duration of viral shedding and an added benefit of a successful vaccination could be the reduction of the virus shedding. Therefore, in this experiment the levels of FHV excretion following challenge with a field FHV strain were also monitored. As shown in Fig 3, although virus was isolated from all cats in each group for a similar period, there was a statistically significant difference observed in the mean peak (>1.5 log₁₀) of excretion (vaccinates: 6 days; controls: 8 days) and in the mean titre excreted on these days (vaccinates: 10^3.3TCID₅₀ ml⁻¹; controls: 10^4.4TCID₅₀ ml⁻¹). Thus the results indicate that vaccination has reduced the time of peak excretion as well as the titre of excreted virus as compared to the non-vaccinated control group. There was no difference observed in either of these two parameters (duration of excretion and virus titre) between the Tricat and the Tricat+FeLV vaccinated groups.

Fig 3.

FHV isolation results (mean titres per group) after challenge with wild type FHV (experiment 3).

Experiment 4: vaccination with maximum release dose of Nobivac Tricat+Nobivac FeLV followed by challenge with FeLV

The main focus of the previous experiments has been on the safety of this vaccine combination and on the efficacy of the live vaccinal components. Although it was not anticipated that this combination of vaccines was likely to have any adverse effect on the efficacy of the purified recombinant FeLV vaccine, the theoretical issues of antigen competition and masking of antigenic epitopes were addressed herein. Therefore, in this particular experiment maximum release titres of the live trivalent vaccine were mixed with normal FeLV batches and were administered to healthy kittens in accordance with the recommended vaccination schedule. Additional control groups of cats that were vaccinated either with Tricat or FeLV vaccines alone were also included and 3 weeks post last vaccination were challenged with a virulent FeLV-A strain by the oronasal route. Following challenge all cats were bled at 3-week intervals up to 12-weeks post challenge to test for antigenaemia (FeLV p27 antigen) and virus isolation as an indicator of FeLV infection. As shown in Table 3, six out of the eight cats in both FeLV vaccinated groups (2b and 3b) were protected from the challenge as they did not show viraemia at any testing point. In contrast, all the control, Nobivac Tricat-vaccinated cats became persistently viraemic demonstrating the high infectivity of the challenge model. It should be noted that all the protected cats in the FeLV vaccinated groups had developed FeLV neutralising antibodies by the end of the experiment. Therefore, as expected, the combination of the Tricat with the FeLV vaccine did not have any deleterious effect on the efficacy of the FeLV vaccine.

Table 3.

Comparison of the FeLV viraemia results between vaccinated and non-vaccinated cats post FeLV challenge (experiment 4)

Vaccine	Animal ID	Viraemia pre challenge	Weeks post challenge
			3	6	9	12
Nobivac Tricat/Nobivac FeLV (group 1b)	19G	Neg	−	−	−	−
	819	Neg	−	−	−	−
	94P	Neg	+	+	+	+
	X88	Neg	−	−	−	−
	X91	Neg	−	−	−	−
	X92	Neg	+	+	+	+
	X99	Neg	−	−	−	−
	Y02	Neg	−	−	−	−
Nobivac FeLV (group 2b)	18Y	Neg	−	−	−	−
	19A	Neg	−	−	−	−
	19E	Neg	+	+	+	+
	822	Neg	−	−	−	−
	X89	Neg	−	−	−	−
	X93	Neg	−	−	−	−
	Y00	Neg	+	+	+	+
	Y04	Neg	−	−	−	−
Nobivac Tricat (group 3b)	18Z	Neg	+	+	+	+
	821	Neg	+	+	+	+
	X85	Neg	−	+	+	+
	X95	Neg	+	+	+	+
	X96	Neg	+	+	+	+
	Y03	Neg	+	+	+	+

Discussion

The increasing complexity of recommended cat immunisation schedules has resulted in the need to consider combining several vaccines together. However, quite often, in practice, there is confusion over which vaccines can or cannot be combined and, therefore, more research is needed to ensure comparable safety and immunogenicity of any proposed combinations. In this paper, we attempted to determine the compatibility of two vaccines that together protect cats against four major feline viral diseases. The effect of Nobivac Tricat (FHV, FCV, and FPLV) on Nobivac FeLV, and vice versa, was investigated to ensure that efficacy and safety are not compromised by combining both products. At the time of writing the two vaccines have received formal approval from the UK licensing authorities to be combined in the same syringe just prior to administration and given as a single inoculation.

In order to avoid unnecessary animal trials the products were initially tested in vitro for evidence of any detrimental effect of the subunit FeLV vaccine on the viability of the live FPLV, FHV or FCV components. Using susceptible cell cultures it was shown that the FeLV vaccine did not adversely affect any of the individual components of the live trivalent vaccine even when incubated for up to 2h. This long incubation period prior to testing was designed deliberately in order to represent extreme situations where in practice vaccines may be left after reconstitution for some time at room temperature prior to use, although this practice is not recommended.

In terms of safety, cats that received both products together did not develop any adverse side effects on any occasion apart from mild transient local swelling at the site of inoculation which was also noticeable in the vaccinated cats with FeLV alone. Even when the combination of vaccines was given in overdose (10 high release titre doses together) or in repeat doses as stated in the safety requirements of the European Pharmacopoeia for live veterinary vaccines, the combination was well tolerated. In terms of immunogenicity when both vaccines were given together at the same site, cats' antibody responses to FCV, FHV and FPLV were comparable and in some cases even improved following use of the combination although any observed improvement was not found to be statistically significant.

Following stimulation of B cells by vaccinal antigens, quantification of specific antibodies can be used as a reliable indicator of the potency of some vaccines (Roitt et al 1993). However, additional tests and/or challenge experiments are needed for some other viral vaccines (such as FHV) where the T cell responses have been shown to be crucial to the development of protective immunity (Leutenegger et al 2000). In the case of FHV it is known that antibody responses can be variable and do not necessarily correlate well with protection (Gaskell and Willoughby 1999) and it was, therefore, considered necessary to perform a challenge experiment in order to determine if the excipients of the FeLV vaccines had any detrimental effect on this large enveloped DNA vaccinal virus. Results of this study showed that cats that were vaccinated with either a normal FeLV dose combined with minimum release titres of Nobivac Tricat, or with Tricat alone, were equally better protected than the negative control cats after exposure to a pathogenic FHV strain. Vaccination with Nobivac Tricat also reduced the shedding of the field FHV. This may be of particular importance in catteries where the spread of this disease could occur rapidly and with life-threatening consequences.

It has been conjectured that combination of MLV vaccines with subunit vaccines such as this FeLV vaccine could potentially also have a deleterious effect on the efficacy of the subunit vaccine. Antigen presenting cells (APCs) capture and degrade antigens for presentation as peptides to immune effector cells including CD4+ and CD8+ T cells. When multivalent vaccines are delivered, the APCs will preferentially process and present certain peptides over others, at least in part depending on the affinity of the interaction of the peptides with the major histocompatibility complex (MHC) on which they are presented. It is, therefore, possible that when an APC is processing and presenting multiple vaccines, high affinity binding of a peptide from one vaccine could interfere with presentation of peptides derived from the other vaccines (Pastoret et al 1997). The competition between peptides derived from the co-administered vaccines could, therefore, lead to an incomplete or qualitatively different overall T cell response to certain vaccines. Furthermore, the immunosuppressive properties of some infectious agents such as parvoviruses (Mastro et al 1986) present in this MLV vaccine, might possibly adversely affect the efficacy of the FeLV subunit vaccine. In this study we evaluated the effectiveness of the FeLV vaccine after combining it with maximum release titres of Tricat for maximum MLV stimulus. After a very potent FeLV challenge where 100% of the unvaccinated control cats became p27 antigenaemic, it was shown that 75% of the cats in both of the vaccinated groups were protected thus suggesting that the aforementioned theoretical issues have little, if any, practical effect.

Although there are recent examples of vaccines that contain all these antigens in one vial (Poulet et al 2003), an advantage of this particular combination is that it gives the practitioner the flexibility to decide if he needs to combine an FeLV vaccine with all three (FHV+FCV+FPLV) or less MLV antigens that are routinely used for the vaccination of cats.

These results are in agreement with previous studies (Gueguen et al 2000) where the efficacy of subunit FeLV vaccines was found not to be compromised after co-administration with similar feline MLV vaccines. However, herein we report further studies where the efficacy of a similar combination with regards to the MLV vaccine was also tested at different doses (10× overdose, maximum, minimum, repeat doses) either in seronegative or in MDA positive cats and most importantly after challenge experiments with FHV and FeLV. Therefore, the conclusion which can be drawn from these studies is that Nobivac Tricat (FCV+FHV+FPLV) can be reconstituted with Nobivac FeLV and inoculated subcutaneously into cats as a single dose preparation without compromising the efficacy or the safety profiles of either of these individual vaccines.