Anti-SU Antibody Responses in Client-Owned Cats Following Vaccination against Feline Leukaemia Virus with Two Inactivated Whole-Virus Vaccines (Fel-O-Vax® Lv-K and Fel-O-Vax® 5).

A field study undertaken in Australia compared the antibody responses induced in client-owned cats that had been vaccinated using two inactivated whole feline leukaemia virus (FeLV) vaccines, the monovalent vaccine Fel-O-Vax® Lv-K and the polyvalent vaccine Fel-O-Vax® 5. Serum samples from 428 FeLV-uninfected cats (118 FeLV-vaccinated and 310 FeLV-unvaccinated) were tested for anti-FeLV neutralising antibodies (NAb) using a live virus neutralisation assay to identify 378 FeLV-unexposed (NAb-negative) and 50 FeLV-exposed (NAb-positive; abortive infections) cats, following by anti-surface unit (SU) FeLV-A and FeLV-B antibody ELISA testing. An additional 42 FeLV-infected cats (28 presumptively regressively infected, 14 presumptively progressively infected) were also tested for anti-SU antibodies. NAb-positive cats displayed significantly higher anti-SU antibody ELISA responses compared to NAb-negative cats (p < 0.001). FeLV-unexposed cats (NAb-negative) that had been vaccinated less than 18 months after a previous FeLV vaccination using the monovalent vaccine (Fel-O-Vax® Lv-K) displayed higher anti-SU antibody ELISA responses than a comparable group vaccinated with the polyvalent vaccine (Fel-O-Vax® 5) (p < 0.001 for both anti-FeLV-A and FeLV-B SU antibody responses). This difference in anti-SU antibody responses between cats vaccinated with the monovalent or polyvalent vaccine, however, was not observed in cats that had been naturally exposed to FeLV (NAb-positive) (p = 0.33). It was postulated that vaccination with Fel-O-Vax® 5 primed the humoral response prior to FeLV exposure, such that antibody production increased when the animal was challenged, while vaccination with Fel-O-Vax® Lv-K induced an immediate preparatory antibody response that did not quantitatively increase after FeLV exposure. These results raise questions about the comparable vaccine efficacy of the different FeLV vaccine formulations and correlates of protection.

The Diagnosis of Feline Leukaemia Virus (FeLV) Infection in Owned and Group-Housed Rescue Cats in Australia.

A field study was undertaken to (i) measure the prevalence of feline leukaemia virus (FeLV) exposure and FeLV infection in a cross-section of healthy Australian pet cats; and (ii) investigate the outcomes following natural FeLV exposure in two Australian rescue facilities. Group 1 (n = 440) consisted of healthy client-owned cats with outdoor access, predominantly from eastern Australia. Groups 2 (n = 38) and 3 (n = 51) consisted of a mixture of healthy and sick cats, group-housed in two separate rescue facilities in Sydney, Australia, tested following identification of index cases of FeLV infection in cats sourced from these facilities. Diagnostic testing for FeLV exposure/infection included p27 antigen testing using three different point-of-care FeLV kits and a laboratory-based ELISA, real-time polymerase chain reaction (qPCR) testing to detect FeLV proviral DNA in leukocytes, real-time reverse-transcription PCR (qRT-PCR) testing to detect FeLV RNA in plasma, and neutralising antibody (NAb) testing. Cats were classified as FeLV-uninfected (FeLV-unexposed and presumptively FeLV-abortive infections) or FeLV-infected (presumptively regressive and presumptively progressive infections). In Group 1, 370 FeLV-unexposed cats (370/440, 84%), 47 abortive infections (47/440, 11%), nine regressive infections (9/440, 2%), and two progressive infections (2/440, 0.5%) were identified, and 12 FeLV-uninfected cats (12/440, 3%) were unclassifiable as FeLV-unexposed or abortive infections due to insufficient samples available for NAb testing. In Groups 2 and 3, 31 FeLV-unexposed cats (31/89, 35%), eight abortive infections (8/89, 9%), 22 regressive infections (22/89; 25%), and 19 progressive infections (19/89; 21%) were discovered, and nine FeLV-uninfected cats (9/89; 10%) were unclassifiable due to insufficient samples available for NAb testing. One of the presumptively progressively-infected cats in Group 3 was likely a focal FeLV infection. Two other presumptively progressively-infected cats in Group 3 may have been classified as regressive infections with repeated testing, highlighting the difficulties associated with FeLV diagnosis when sampling cats at a single time point, even with results from a panel of FeLV tests. These results serve as a reminder to Australian veterinarians that the threat of FeLV to the general pet cat population remains high, thus vigilant FeLV testing, separate housing for FeLV-infected cats, and FeLV vaccination of at-risk cats is important, particularly in group-housed cats in shelters and rescue facilities, where outbreaks of FeLV infection can occur.

Diagnosing feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infection: an update for clinicians.

With the commercial release in Australia in 2004 of a vaccine against feline immunodeficiency virus (FIV; Fel-O-Vax FIV®), the landscape for FIV diagnostics shifted substantially. Point-of-care (PoC) antibody detection kits, which had been the mainstay for diagnosing FIV infection since the early 1990s, were no longer considered accurate to use in FIV-vaccinated cats, because of the production of vaccine-induced antibodies that were considered indistinguishable from those produced in natural FIV infections. Consequently, attention shifted to alternative diagnostic methods such as nucleic acid detection. However, over the past 5 years we have published a series of studies emphasising that FIV PoC test kits vary in their methodology, resulting in differing accuracy in FIV-vaccinated cats. Importantly, we demonstrated that two commercially available FIV antibody test kits (Witness™ and Anigen Rapid™) were able to accurately distinguish between FIV-vaccinated and FIV-infected cats, concluding that testing with either kit offers an alternative to PCR testing. This review summarises pertinent findings from our work published in a variety of peer-reviewed research journals to inform veterinarians (particularly veterinarians in Australia, New Zealand and Japan, where the FIV vaccine is currently commercially available) about how the approach to the diagnosis of FIV infection has shifted. Included in this review is our work investigating the performance of three commercially available FIV PoC test kits in FIV-vaccinated cats and our recommendations for the diagnosis of FIV infection; the effect of primary FIV vaccination (three FIV vaccines, 4 weeks apart) on PoC test kit performance; our recommendations regarding annual testing of FIV-vaccinated cats to detect 'vaccine breakthroughs'; and the potential off-label use of saliva for the diagnosis of FIV infection using some FIV PoC test kits. We also investigated the accuracy of the same three brands of test kits for feline leukaemia virus (FeLV) diagnosis, using both blood and saliva as diagnostic specimens. Based on these results, we discuss our recommendations for confirmatory testing when veterinarians are presented with a positive FeLV PoC test kit result. Finally, we conclude with our results from the largest and most recent FIV and FeLV seroprevalence study conducted in Australia to date.

Efficacy of an inactivated FeLV vaccine compared to a recombinant FeLV vaccine in minimum age cats following virulent FeLV challenge.

The aim of the study was to determine the efficacy of an inactivated feline leukemia virus (FeLV) vaccine (Versifel(®) FeLV, Zoetis.) compared to a recombinant FeLV vaccine (Purevax(®) FeLV, Merial Animal Health) in young cats, exposed under laboratory conditions to a highly virulent challenge model. The study was designed to be consistent with the general immunogenicity requirements of the European Pharmacopoeia 6.0 Monograph 01/2008:1321-Feline Leukaemia Vaccine (Inactivated) with the exception that commercial-strength vaccines were assessed. Fifty seronegative cats (8-9 weeks old) were vaccinated subcutaneously on two occasions, three weeks apart, with either placebo (treatment group T01), Versifel FeLV Vaccine (treatment group T02), or Purevax FeLV Vaccine (treatment group T03) according to the manufacturer's directions. Cats were challenged three weeks after the second vaccination with a virulent FeLV isolate (61E strain). Persistent FeLV antigenemia was determined from 3 to 15 weeks postchallenge. Bone marrow samples were tested for the presence of FeLV proviral DNA to determine FeLV latent infection. At week 15 after challenge with the virulent FeLV 61E strain, the Versifel FeLV Vaccine conferred 89.5% protection against FeLV persistent antigenemia and 94.7% protection against FeLV proviral DNA integration in bone marrow cells. In comparison, the Purevax FeLV Vaccine conferred 20% protection against FeLV persistent antigenemia and 35% protection against FeLV proviral DNA integration in bone marrow cells following challenge. The data from this study show that the Versifel FeLV Vaccine was efficacious in preventing both FeLV persistent p27 antigenemia and FeLV proviral DNA integration in bone marrow cells of cats challenged with this particular challenge model under laboratory conditions and provided better protection than Purevax FeLV in this experimental challenge model with highly virulent FeLV.

A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine.

We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

Difficulties in demonstrating long term immunity in FeLV vaccinated cats due to increasing age-related resistance to infection.

BACKGROUND: Feline leukaemia virus (FeLV) is a pathogen causing fatal illness in cats worldwide, and as such there is a high demand for products to protect against disease. The duration of immunity provided by an inactivated FeLV vaccine, Versifel FeLV, when administered to cats of the target age was determined. Kittens received two vaccinations when aged 7 to 9 weeks old, and were subsequently challenged up to 36 months later with the FeLV-A Glasgow isolate. RESULTS: In all studies, all of the younger aged control kittens showed persistent FeLV p27 antigenaemia confirming that the challenge virus was severe and efficacious. In contrast, the control cats did not show the required level of persistent antigenaemia, with a maximum of 45% cats affected in the middle duration study and only 10% in the longer study. However, apart from one animal in the short duration study, all of the cats vaccinated with Versifel FeLV were negative for persistent antigenaemia and can be considered treatment successes. CONCLUSION: In conclusion, we have shown that although age-related resistance to infection with a virulent FeLV challenge is evident from as early as 10 months of age, vaccination with Versifel FeLV may aid in the protection of cats from FeLV related disease up to three years after primary vaccination as kittens.

Antibody response to vaccines for rhinotracheitis, caliciviral disease, panleukopenia, feline leukemia, and rabies in tigers (Panthera tigris) and lions (Panthera leo).

This article presents the results of a study of captive tigers (Panthera tigris) and lions (Panthera leo) vaccinated with a recombinant vaccine against feline leukemia virus; an inactivated adjuvanted vaccine against rabies virus; and a multivalent modified live vaccine against feline herpesvirus, calicivirus, and panleukopenia virus. The aim of the study was to assess the immune response and safety of the vaccines and to compare the effects of the administration of single (1 ml) and double (2 ml) doses. The animals were separated into two groups and received either single or double doses of vaccines, followed by blood collection for serologic response for 400 days. No serious adverse event was observed, with the exception of abortion in one lioness, potentially caused by the incorrect use of the feline panleukopenia virus modified live vaccine. There was no significant difference between single and double doses for all vaccines. The recombinant vaccine against feline leukemia virus did not induce any serologic response. The vaccines against rabies and feline herpesvirus induced a significant immune response in the tigers and lions. The vaccine against calicivirus did not induce a significant increase in antibody titers in either tigers or lions. The vaccine against feline panleukopenia virus induced a significant immune response in tigers but not in lions. This report demonstrates the value of antibody titer determination after vaccination of nondomestic felids.

[FeLV infection in the cat: clinically relevant aspects]. / Die FeLV-Infektion der Katze: Praxis-relevante Aspekte.

The feline leukemia virus (FeLV) is a retrovirus of the domestic cat that was described almost 50 years ago. The FeLV-infection may lead to fatal diseases in domestic and small wild cats. The use of efficacious diagnostics assays and vaccines led to a reduction of the FeLV prevalence; however, FeLV still poses a problem for the cat presented with the infection. This article aims to describe recent developments in diagnostics and findings in the infection pathogenesis that are clinically relevant.

Protection against feline leukemia virus challenge for at least 2 years after vaccination with an inactivated feline leukemia virus vaccine.

Twelve cats were vaccinated at 8 and 11 weeks of age with a commercially available inactivated FeLV vaccine (Nobivac FeLV, Intervet/Schering-Plough Animal Health). Eleven cats served as age-matched, placebo-vaccinated controls. All cats were kept in isolation for 2 years after vaccination and were then challenged with virulent FeLV to evaluate vaccine efficacy and duration of immunity. Cats were monitored for 12 weeks after challenge for development of persistent viremia using a commercial FeLV p27 ELISA. Persistent viremia developed in all 11 (100%) of the control cats, whereas 10 of 12 (83%) vaccinated cats were fully protected from persistent viremia following challenge. The results demonstrate that the vaccine used in this study protects cats from persistent FeLV viremia for at least 2 years after vaccination.

Feline leukaemia. ABCD guidelines on prevention and management.

OVERVIEW: Feline leukaemia virus (FeLV) is a retrovirus that may induce depression of the immune system, anaemia and/or lymphoma. Over the past 25 years, the prevalence of FeLV infection has decreased considerably, thanks both to reliable tests for the identification of viraemic carriers and to effective vaccines. INFECTION: Transmission between cats occurs mainly through friendly contacts, but also through biting. In large groups of non-vaccinated cats, around 30-40% will develop persistent viraemia, 30-40% show transient viraemia and 20-30% seroconvert. Young kittens are especially susceptible to FeLV infection. DISEASE SIGNS: The most common signs of persistent FeLV viraemia are immune suppression, anaemia and lymphoma. Less common signs are immune-mediated disease, chronic enteritis, reproductive disorders and peripheral neuropathies. Most persistently viraemic cats die within 2-3 years. DIAGNOSIS: In low-prevalence areas there may be a risk of false-positive results; a doubtful positive test result in a healthy cat should therefore be confirmed, preferably by PCR for provirus. Asymptomatic FeLV-positive cats should be retested. DISEASE MANAGEMENT: Supportive therapy and good nursing care are required. Secondary infections should be treated promptly. Cats infected with FeLV should remain indoors. Vaccination against common pathogens should be maintained. Inactivated vaccines are recommended. The virus does not survive for long outside the host. VACCINATION RECOMMENDATIONS: All cats with an uncertain FeLV status should be tested prior to vaccination. All healthy cats at potential risk of exposure should be vaccinated against FeLV. Kittens should be vaccinated at 8-9 weeks of age, with a second vaccination at 12 weeks, followed by a booster 1 year later. The ABCD suggests that, in cats older than 3-4 years of age, a booster every 2-3 years suffices, in view of the significantly lower susceptibility of older cats.

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.

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.

How molecular methods change our views of FeLV infection and vaccination.

FeLV was discovered 40 years ago and vaccines have been commercially available for almost two decades. So far, most FeLV pathogenesis and vaccine studies were conducted assaying parameters, such as virus isolation and antigen detection. Accordingly, regressive infection was characterized by transient or undetectable viremia, while persistent viremia is typically observed in cats with progressive infection. Using real-time polymerase chain reaction assays, the spectrum of host response categories to FeLV infection was recently refined by investigating proviral and plasma viral RNA loads. Cats believed to be immune to FeLV infection were found to turn provirus-positive after virus exposure. Moreover, efficacious FeLV vaccines were found unable to prevent provirus-integration and minimal viral replication. Remarkably, no difference was found in initial proviral and plasma viral RNA loads between cats with different infection outcomes. Only subsequently, the infection outcome is associated with FeLV loads. FeLV provirus was found to persist for years; reoccurrence of viremia and disease development was observed in some cats. Thus, aviremic provirus-positive cats are FeLV carriers and, following reactivation, may act as an infection source. However, integrated viral DNA may also be essential for solid protection and long-lasting maintenance of protective immunity. In conclusion, real-time TaqMan PCR and RT-PCR assays are highly sensitive and specific. They yield a more sensitive measure for FeLV exposure than antigen detection, virus isolation or immunofluoresence assays. We recommend the use of real-time PCR assays to identify FeLV exposed cats, particularly in catteries, and investigate obscure clinical cases that may be FeLV-associated. The use of sensitive molecular methods will contribute to a more in-depth understanding of the FeLV pathogenesis.

Protection from challenge following administration of a canarypox virus-vectored recombinant feline leukemia virus vaccine in cats previously vaccinated with a killed virus vaccine.

OBJECTIVE: To compare protection against FeLV challenge obtained following administration of 2 doses of an adjuvanted, chemically inactivated, whole FeLV (FeLV-k) vaccine with protection obtained following administration of 1 dose of an FeLV-k vaccine followed by 1 dose of a canarypox virus-vectored recombinant FeLV (rCP-FeLV) vaccine. DESIGN: Prospective study. ANIMALS: Thirty-two 9-week-old domestic shorthair cats. PROCEDURE: Cats received 2 doses of the FeLV-k vaccine SC, 21 days apart (n = 11); 1 dose of the FeLV-k vaccine SC and, 21 days later, 1 dose of the rCP-FeLV vaccine transdermally (11); or 2 doses of physiologic saline (0.9% NaCl) solution (control; 10). Four weeks after the second vaccine dose, all cats were challenged with FeLV by means of oronasal administration. Blood samples were collected at weekly intervals beginning 21 days after challenge, and serum was tested for FeLV antigen. RESULTS: All 10 control cats became persistently infected (ie, FeLV antigen detected in > or = 3 consecutive serum samples) following FeLV challenge, whereas only 1 of 11 cats that received 2 doses of the FeLV-k vaccine and none of the 11 cats that received 1 dose of the FeLV-k vaccine and 1 dose of the rCP-FeLV vaccine did. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that protection against FeLV challenge obtained following SC administration of a single dose of an FeLV-k vaccine followed, 21 days later, by transdermal administration of a single dose of an rCP-FeLV vaccine was similar to that obtained following SC administration of 2 doses of the FeLV-k vaccine 21 days apart.

Feline B7.1 and B7.2 proteins produced from swinepox virus vectors are natively processed and biologically active: potential for use as nonchemical adjuvants.

Costimulatory ligands, B7.1 and B7.2, have been incorporated into viral and DNA vectors as potential nonchemical adjuvants to enhance CTL and humoral immune responses against viral pathogens. In addition, soluble B7 proteins, minus their transmembrane and cytoplasmic domains, have been shown to block the down regulation of T-cell activation through blockade of B7/CTLA-4 interactions in mouse tumor models. Recently, we developed swinepox virus (SPV) vectors for delivery of feline leukemia antigens for vaccine use in cats [Winslow, B.J., Cochran, M.D., Holzenburg, A., Sun, J., Junker, D.E., Collisson, E.W., 2003. Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin. Virus Res. 98, 1-15]. To explore the use of feline B7.1 and B7.2 ligands as nonchemical adjuvants, SPV vectors containing full-length feline B7.1 and B7.2 ligands were constructed and analyzed. Full-length feline B7.1 and B7.2 produced from SPV vectors were natively processed and costimulated Jurkat cells to produce IL-2, in vitro. In addition, we explored the feasibility of utilizing SPV as a novel expression vector to produce soluble forms of feline B7.1 (sB7.1) and B7.2 (sB7.2) in tissue culture. The transmembrane and cytoplasmic regions of the B7.1 and B7.2 genes were replaced with a poly-histidine tag and purified via a two-step chromatography procedure. Receptor binding and costimulation activity was measured. Although feline sB7.1-his and sB7.2-his proteins bound to the human homolog receptors, CTLA-4 and CD28, both soluble ligands possessed greater affinity for CTLA-4, compared to CD28. However, both retained the ability to partially block CD28-mediated costimulation in vitro. Results from these studies establish the use of SPV as a mammalian expression vector and suggest that full-length-vectored and purified soluble feline B7 ligands may be valuable, nonchemical immune-modulators.

Comparison of the safety and efficacy of a recombinant feline leukemia virus (FeLV) vaccine delivered transdermally and an inactivated FeLV vaccine delivered subcutaneously.

The efficacy of a new recombinant FeLV vaccine (rFeLV), delivered transdermally via a needle-free delivery device was compared to that of an inactivated FeLV vaccine (FeLV-k), administered subcutaneously, with a conventional needle and syringe. Kittens were immunized with either rFeLV (0.25 ml, transdermal) or FeLV-k (1 ml, subcutaneous); or they were sham-vaccinated with physiologic saline (0.25 ml, transdermal). Two vaccinations were administered 21 days apart. Injection sites were monitored for any acute or subacute reactions relative to vaccine administration. Four weeks following the final vaccination, all cats were subject to oro-nasal FeLV challenge. Blood was collected for determination of FeLV antigenemia (p27) at weekly intervals beginning three weeks post-challenge. All of the vaccinated cats from both groups resisted FeLV challenge; and 90% of the control cats developed persistent FeLV antigenemia in response to challenge. No acute or persistent injection site reactions were observed. The rFeLV, delivered transdermally, provides protection against persistent FeLV antigenemia following a robust challenge that is equivalent to that of FeLV-k.

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.