Multivariate analysis of the immune response to different rabies vaccines.

In a previous study, we proposed as an alternative to the use of animals in infectious challenge studies, a new approach describing the vaccine-induced immune response through the multivariate analysis of a defined set of immune parameters characterizing the B and T immune responses. This multivariate analysis, i.e. immune fingerprint, was evaluated first to assess the impact of minor changes in well characterized vaccines. The approach showed promising results in the assessment of the compatibility between two licensed vaccines. In the present study, the immune fingerprint was used to compare adjuvants with the various immunological parameters of the immune fingerprint as well as to assess the ability of this approach to discriminate different Rabies vaccine formulations in dogs. RABISIN® was the reference vaccine, adjuvanted with aluminum hydroxide. An exploratory factor analysis was used to analyse the covariance structure of the immunological data. Significant differences were observed between groups. RABISIN and a linear polyacrylate (SPA09) adjuvanted vaccine performed better than chitosan adjuvanted ones, both for humoral and cell immune responses. This study showed that the immune fingerprint approach can be used to screen vaccine formulations. It provides additional information compared to classical vaccination and infectious challenge efficacy study.

Differential association between circulating testosterone and infection risk by several viruses in natural cat populations: a behavioural-mediated effect?

Testosterone is involved in the development and expression of physiological, morphological and behavioural traits. High levels are often associated with high infection risk and/or intensity, suggesting a trade-off between sexual traits and immunity. Classically invoked mechanisms are immunological or behavioural, i.e., testosterone increases susceptibility or resistance to parasites via an impact on immunity or modulates behaviours involved in parasite transmission. However, studies report contrasted patterns. Given its modes of action and the diversity of host-parasite interactions, testosterone should not act similarly on all interactions. To reduce host and context diversity, we studied 3 viruses in the same cat population: the aggressively transmitted Feline Immunodeficiency virus (FIV), and the Feline Calicivirus (FCV) and Herpesvirus (FHV) both transmitted during friendly contacts. Testosterone had a strong effect on the probability of being positive to FIV whereas its effect was significantly weaker on FCV and FHV. These findings demonstrate that testosterone can be differentially associated with parasites of the same type (viruses). The difference we observed was consistent with a behavioural-mediated effect (increased aggressiveness), supporting the idea that the testosterone effect on infection risk is at least partially driven by behavioural mechanisms in our system. Further investigations (e.g., individual immunity measures) are required to confirm this hypothesis.

Three-year duration of immunity in cats vaccinated with a canarypox-vectored recombinant rabies virus vaccine.

Despite the availability of efficacious vaccines for animals and humans, rabies is still a major zoonosis. Prevention of rabies in dogs and cats is key for reducing the risk of transmission of this deadly disease to humans. Most veterinary vaccines are adjuvanted inactivated vaccines and have been shown to provide one to four-year duration of immunity. In response to debates about the safety of adjuvanted vaccines in cats, a non-adjuvanted feline rabies vaccine with one-year duration of immunity claim was specifically developed using the canarypoxvirus vector technology. The objective of this study was to validate a vaccination program based on primary vaccination, revaccination one year later and boosters every three years. Seronegative cats were vaccinated at 12 weeks of age and received a booster vaccination one year later. This vaccination regimen induced a strong and sustained antibody response, and all vaccinated animals were protected against virulent rabies challenge carried out 3 years after vaccination. These results validated 3-year duration of immunity after a complete basic vaccination program consisting in primary vaccination from 12 weeks of age followed by revaccination one year later with a non-adjuvanted canarypox-vectored vaccine.

Time-course analysis of main markers of primary infection in cats with the feline immunodeficiency virus.

Studies of the response of the immune system to feline immunodeficiency virus (FIV) during primary infection have shown that a subpopulation of CD8(+) T-cells with an activated phenotype and reduced expression of the CD8ß chain (denoted CD8ß(low) T cells) expands to reach up to 80% of the total CD8(+) T cell count. The expansion of this subpopulation is considered to be a signature of FIV and an indicator of immune system alteration. We use a simple mathematical formalism to study the relationships over time between the dose of infection, the size of the CD8ß(low) population, and the circulating viral load in cats infected with FIV. Viremia profiles are described using a combination of two exponential laws, whereas the CD8ß(low) percentage (out of the total CD8(+) population) is represented by a Gompertz law including an expansion phase and a saturation phase. Model parameters are estimated with a population approach using data from 102 experimentally infected cats. We examine the dose of infection as a potential covariate of parameters. We find that the rates of increase of viral load and of CD8ß(low) percentage are both correlated with the dose of infection. Cats that develop strong acute viremia also show the largest degree of CD8ß(low) expansion. The two simple models are robust tools for analysing the time course of CD8ß(low) percentage and circulating viral load in FIV-infected cats and may be useful for generating new insights on the disease and on the design of therapeutic strategies, potentially applicable to HIV infection.

When cats' ways of life interact with their viruses: a study in 15 natural populations of owned and unowned cats (Felis silvestris catus).

In natural populations, virus circulation is influenced by host behavior and physiological characteristics. Cat populations exhibit a great variability in social and spatial structure, the existence of different ways of life within a same population may also result in different epidemiological patterns. To test this hypothesis, we used a logistic regression to analyze the risk factors of Feline immunodeficiency virus (FIV), feline herpes virus (FHV), feline calicivirus (FCV), and feline parvovirus (FPV) infection in owned (fed and sheltered) and unowned (neither fed nor sheltered, unsocialized) cats living in a rural environment in the North Eastern part of France. A serological survey was carried out in 492 non-vaccinated and non-sterilized individuals from 15 populations living in the same area. The prevalence of feline leukemia virus (FeLV) was also studied, but too few were infected to analyze the risk factors of this virus. For each virus, the epidemiological pattern was different in owned and unowned cats. Unowned cats were more frequently infected by directly transmitted viruses like FIV, FHV and FCV (21.22%, 67.66%, 86.52% in unowned cats vs 9.55%, 53.88%, 77.18% in owned cats, respectively), a difference that may be explained by a more solitary and more aggressive behavior in unowned adults, and/or possibly by a higher sensitivity related to a more stressful life. On the contrary, owned cats were more frequently infected with FPV (36.41% in owned cats vs 15.61% in unowned cats), possibly as a result of their concentration around human settlements. The present study showed that owned and unowned cats living in a same area have behavioral and physiological characteristics sufficiently different to influence virus circulation. Pooling different types of cats in a single sample without taking it into account could give a wrong picture of the epidemiology of their viruses. The conclusion of this work can be extended to any epidemiological studies led in wildlife species with flexible behavior as any variations in social or spatial structure, between or within populations, could result in different virus circulation.

Onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleucopenia, feline calicivirus and feline herpesvirus.

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.

Ability of antibodies to two new caliciviral vaccine strains to neutralise feline calicivirus isolates from the UK.

This study examined a panel of 110 UK field isolates of feline calicivirus (FCV) for susceptibility to cross-neutralisation by a panel of eight antisera raised in cats infected with FCV strains F9, 255, FCVG1 and FCV431. The pairs of antisera raised against F9 or 255, neutralised 20 and 21 per cent or 37 and 56 per cent of field strains of virus respectively. In contrast, the pairs of antisera raised against the newer vaccine strains FCVG1 or FCV431 neutralised 29 and 70 per cent or 67 and 87 per cent of field strains respectively. Antisera raised against the two newer strains, namely FCVG1 and FCV431, neutralised a greater proportion of field strains of calicivirus than antisera raised against the older FCV vaccine strains F9 and 255.

Efficacy of a bivalent inactivated non-adjuvanted feline calicivirus vaccine: relation between in vitro cross-neutralization and heterologous protection in vivo.

Feline calicivirus (FCV) is a major pathogen of the cat characterized by a strong genomic, antigenic and clinical diversity. Despite vaccination, FCV infection is highly prevalent, and for a few years, outbreaks of virulent systemic disease (VSD) have been reported in North America and Europe. An inactivated non-adjuvanted bivalent vaccine was recently developed by combining antigens derived from two broadly cross-reactive FCV strains. The antigenic relatedness between the vaccine strains and other antigenic variants was demonstrated by cross-neutralization studies in vitro. This study showed that vaccine-induced protection against heterologous challenges was correlated to in vitro cross-neutralization, and it validated the use of cross-neutralization tests to select vaccine FCV strains. This correlation applies also for the highly virulent strains causing VSD (VS-FCV).

Protection of newborn animals through maternal immunization.

Providing protective immunity to neonatal animals in early life is associated with numerous challenges regarding vaccine safety and efficacy. A much simpler approach is maternal vaccination, either before or during pregnancy, to provide the neonate with passively transferred immunity. In humans, the medical, societal and legal risks of immunizing pregnant women are important considerations in undertaking this approach. By contrast, maternal vaccination has been successfully employed in the animal health industry for decades. These veterinary vaccines have proven to be safe and efficient. Although only passively transferred antibodies have been extensively studied, other immunological mechanisms may be equally important in providing maternally derived immunity.

Alternative early life vaccination programs for companion animals.

An experimental challenge study of multicomponent vaccination of kittens is reported. Seven-to-nine week old, specific pathogen-free kittens received two injections (4 weeks apart) of non-adjuvanted, multicomponent vaccine formulated at the minimum protective dose. Kittens were challenged at 4 weeks or 1 year post-vaccination with individual infectious agents. Vaccination induced complete protection against challenge from feline parvovirus on both occasions, but at 1 year, the protection against feline herpesvirus, feline calicivirus and Chlamydophila felis was not as strong as 4 weeks after vaccination. This demonstration of a decline in protective immunity at the normal time of administration of the first booster vaccine suggests that earlier administration of this booster (at 4-6 months of age) may provide better protection. The effect of maternally derived antibody (MDA) on kitten vaccination was determined by conducting an identical experiment but with kittens born to queens vaccinated during pregnancy. Serum antibody titres to specific vaccine components were measured in these kittens on day 0 (time of first vaccination), day 28 (time of second vaccination) and day 42. There was heterogeneity in transfer of MDA to kittens within a litter, and between litters. MDA may neutralize the serological response of kittens on the first, and occasionally the second, occasion of vaccination when vaccination is performed at 8 and 12 weeks of age. This finding underpins recent recommendations that the final vaccination in the primary series be administered at 16 weeks of age.

A kinetic study of histopathological changes in the subcutis of cats injected with non-adjuvanted and adjuvanted multi-component vaccines.

The aim of this study was to investigate the subcutaneous tissue response to administration of a single dose of multi-component vaccine in the cat. Three groups of 15 cats were injected with one of three vaccine products with saline as a negative control. Cats in group A received non-adjuvanted vaccine; cats in group B received vaccine with a lipid-based adjuvant; whilst those in group C were vaccinated with a product adjuvanted with an alum-Quil A mixture. The vaccine and saline injection sites were sampled on days 7, 21 and 62 post-vaccination. Biopsies of these vaccine sites were examined qualitatively and scored semi-quantitatively for a series of parameters related to aspects of the inflammatory and tissue repair responses. These data were analysed statistically, including by principal component analysis. At all three time points of the experiment, there was significantly less inflammation associated with administration of non-adjuvanted vaccine (p=0.000). Although there was evidence of tissue repair by day 62 in all groups, those cats receiving adjuvanted vaccines had evidence of residual adjuvant material accumulated within macrophages at this late time point. The severity of tissue reactions may vary significantly in response to vaccines which include adjuvants or are non-adjuvanted.

Highly pathogenic avian influenza H5N1 virus in cats and other carnivores.

The Asian lineage highly pathogenic avian influenza (HPAI) H5N1 virus is a known pathogen of birds. Only recently, the virus has been reported to cause sporadic fatal disease in carnivores, and its zoonotic potential has been dominating the popular media. Attention to felids was drawn by two outbreaks with high mortality in tigers, leopards and other exotic felids in Thailand. Subsequently, domestic cats were found naturally infected and experimentally susceptible to H5N1 virus. A high susceptibility of the dog to H3N8 equine influenza A virus had been reported earlier, and recently also HPAI H5N1 virus has been identified as a canine pathogen. The ferret, hamster and mouse are suitable as experimental animals; importantly, these species are also kept as pets. Experimental intratracheal and oral infection of cats with an HPAI H5N1 virus isolate from a human case resulted in lethal disease; furthermore, cats have been infected by the feeding of infected chickens. Spread of the infection from experimentally infected to in-contact cats has been reported. The epidemiological role of the cat and other pet animal species in transmitting HPAI H5N1 virus to humans needs continuous consideration and attention.

Toward a detailed characterization of feline immunodeficiency virus-specific T cell immune responses and mediated immune disorders.

Infection of domestic cats with feline immunodeficiency virus (FIV) is associated with the development of an acquired immunodeficiency syndrome (AIDS). The pathogenesis of FIV is not fully understood but it has been reported that the immune system is progressively impaired during disease progression. As a result, anti-FIV specific immune response will usually not clear the virus and the acute stage is followed by a chronic asymptomatic phase. The overall objective of this study was to characterized FIV-induced immune cellular responses and -mediated immune disorder following the first weeks post-infection. Using both cytokine ELISpot and intracellular staining assays, FIV-specific T cells were monitored at 6, 9 and 12 weeks post-infection. We demonstrated that both IFNgamma(+) and, CD4 and CD8 TNFalpha(+) T cells specifically respond to FIV antigens. These responses were found to reach a peak at 9 weeks post-infection. It was further shown that the TNFalpha(+)CD8(+) responding T cells were contained within a CD8beta(low)CD62L(-) T cell subpopulation, expanded in FIV-infected cats. This T cell subpopulation which present features of activated CD8 T cells was further shown to be susceptible to spontaneous apoptosis following a short-term in vitro culture. Moreover, it was observed that cell death by apoptosis of this T cell subset was increased following FIV antigen-recognition. Therefore, FIV might alter immune homeostasis in inducing chronic activation of TNFalpha(+)CD8(+) T cells which eventually will die following antigen contact while deleting CD4(+) T cells. Interestingly, this study confirmed the strong similarity between FIV and HIV pathogenesis.

Efficacy of a canarypox virus-vectored vaccine against feline leukaemia.

Canarypox virus recombinant vaccines have a unique efficacy and safety profile for the vaccinated host because the canarypox virus is non-replicative in mammalian hosts. After the vaccination of a mammalian species, recombinant canarypox viruses express the inserted genes but cannot multiply in the host. They stimulate a strong immune response in the absence of any virus amplification in the host or any viral spread into the environment. A new canarypox-based recombinant vaccine is the canarypox-feline leukaemia virus (FeLV) vaccine (EURIFEL FeLV; Merial) that expresses the FeLV env and gag protective genes. This paper describes experiments which demonstrate that it is effective against any oronasal FeLV challenge. The protection was shown to be solid against an oronasal challenge one year after the initial vaccination, and was effective against a very severe 'in-contact' challenge. Furthermore, the canarypox virus-FeLV vaccine was effective without an adjuvant.

Seroprevalence of canine herpesvirus-1 in the Belgian dog population in 2000.

Canine herpesvirus-1 (CHV-1) is known to be associated with fertility and fecundity disorders as well as neonatal mortality in puppies of less than 3 weeks of age. The virus is presumed to be enzootic in dogs all over the world and recent studies in several European countries suggest a high seroprevalence among the dog population. In the year 2000, a total of 647 Belgian canine sera from 102 privately owned patients and 545 breeding dogs were analysed with an enzyme-linked immunosorbent assay (ELISA). Furthermore 77 of the samples were submitted to two serum neutralization (SN) tests for comparison. An overall CHV-1 seroprevalence of 45.75% was observed in the Belgian dog population. No significant differences could be observed based on breeding status, reason for consultation or sex. The correlation between the ELISA and both SN tests appeared to be moderate with a significantly greater sensitivity of the ELISA. This study also demonstrated that the CHV-1 seroprevalence in the Belgian dog population is similar to that in other recently investigated European countries and that the incidence in breeding units is not necessarily higher than in non-breeding dogs.

Protection of puppies against canine herpesvirus by vaccination of the dams.

Six bitches free of canine herpesvirus 1 (CHV-1) were vaccinated against the virus; a first injection was given 10 days after the presumed date of mating and a second six weeks later. Six similar bitches were left unvaccinated as controls, and all the pups were challenged oronasally with a virulent strain of CHV-1 at three days of age. All the vaccinated bitches seroconverted and had high antibody titres when the puppies were challenged, but the control bitches remained seronegative. In the control group, 62 per cent (18 of 29) of the pups died of CHV-1-induced disease; most of them showed typical clinical signs and macroscopic lesions, and CHV-1 infection was confirmed by the isolation of the virus or by PCR. None of the puppies in the vaccinated group died of CHV-1 infection. The efficacy of the vaccine was confirmed in CHV-1-positive breeding units. The rate of pregnancy tended to be higher in vaccinated bitches and the mortality of pups before weaning was significantly reduced in the litters born to vaccinated bitches.

Comparison between acute oral/respiratory and chronic stomatitis/gingivitis isolates of feline calicivirus: pathogenicity, antigenic profile and cross-neutralisation studies.

Feline calicivirus (FCV) is a major oral and respiratory pathogen of cats, able to induce subclinical infection as well as acute disease. It is also characterized by a high degree of antigenic variation. This work sought to address the question of the existence of distinct biotypes of FCV. Eight French, 6 British and 9 American FCV isolates, responsible for acute oral/respiratory disease or chronic gingivitis/stomatitis, were compared for their pathogenicity, antigenic profiles and serological relationships. Antigenic profiles were assessed by an indirect immunofluorescence assay with a large panel of characterized monoclonal antibodies. Cross-neutralisation assays were performed with specific cat antisera collected at 30 days p.i., then analysed by calculation of antigenic bilateral relatedness and dominance. Whatever their pathogenic origin, all the isolates induced an acute upper-respiratory tract infection in oronasally infected SPF kittens. Their antigenic profiles were different and did not correlate with their geographical or pathological origin. Cross-neutralisation studies and calculation of the mean bilateral relatedness allowed us to distinguish chronic original isolates from acute original ones. This study did not confirm the existence of FCV biotypes but showed that the chronic carrier state is related to the emergence of antigenically distant viruses.