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.

Diagnosis and prevention of equine infectious diseases: present status, potential, and challenges for the future.

The frequent transfers of horses, whether on a permanent or temporary basis, make strict control of infectious diseases essential. Such control needs a reliable and rapid means to accurately diagnose the relevant diseases. Indirect diagnosis based on antibody detection remains certainly the best method to secure the epidemiologic surveillance of the diseases at regional, national, or even world level, while direct diagnosis is the only way to diagnose a new outbreak. New diagnostic methods resulting from advances in biochemistry, molecular biology, and immunology are now available. As far as antibody detection is concerned, the new methods are mainly based on immunoassays, especially ELISAs. Regarding the identification of the pathogens, while isolation is still of importance, much progress has been made with immunocapture tests including capture ELISA based on monoclonal antibodies. DNA probes and amplification tests such as PCR or RT-PCR are representing a real breakthrough. Factors common to all of these tests are specificity, sensitivity, rapid implementation, and quick results. Such tests are, however, often still at the development stage. They absolutely need to be validated under multicentric evaluations prior to being used on a larger scale. At the same time there is an obvious need for the standardization of the reagents used. The technical and economic impact of a false (either positive or negative) diagnosis justifies such an harmonization which could effectively be achieved worldwide under the aegis of the Office International des Epizooties (OIE), which is itself the primary source of disease information. Vaccines are also essential for the control of equine infectious diseases. Most vaccines used in the prevention of viral or bacterial diseases are inactivated adjuvanted vaccines, which may cause unacceptable side effects. Also, their efficacy can sometimes be questioned. Subunit vaccines, when available, represent significant advances especially with regards to safety. Greater progress is expected from the use of new technologies taking advantage of recent developments in molecular biology (recombinant DNA technology) and in immunology (immunomodulators). Significant results have been obtained with subunit vaccines or with live vectored vaccines using recombinant DNA technology. Good results are on the way to be achieved with genetic (or naked-DNA) vaccines. It is therefore possible to expect the availability of a new generation of vaccines in the rather short term. Such vaccines will not only be safer and more efficacious, but they will also make it possible to differentiate vaccinated from infected animals, which will contribute to better control of the infection. Whatever the quality of the vaccines of the future may be, vaccination alone will never be sufficient to control infectious diseases. It is therefore essential to keep on making the animal owners and their veterinarians aware of the importance of the management and the hygiene in the diseases control and to organize them under "Common Codes of Practice."

Experimental rabies infection and oral vaccination in vampire bats (Desmodus rotundus).

A rabies virus variant isolated from a vampire bat (Desmodus rotundus) and characterized by genome sequencing was used for the standardization of an experimental infection in this species. The parenteral administration of 10(6) MICLD50 of this variant was capable of inducing death from rabies in 89% of animals. The mean duration of post-challenge survival was 12 days. None of the experimental rabid vampire bats showed aggressive behaviour. A vaccinia-rabies glycoprotein recombinant virus vaccine was administered orally to vampire bats on days -120, -90, -30 or -18 pre-challenge, on the same day of challenge, or on day +5 post-challenge. A significant protection was noticed only in animals vaccinated on days -18 or -30 pre-challenge. A longer period of incubation was observed in animals vaccinated 5 days post-challenge.

Evaluation of parenteral vaccination methods with glycoproteins against Aujeszky's disease in pigs.

A comparative evaluation of vaccination methods with glycoproteins for the induction of immune responses and protection of the pig against Aujeszky's disease virus (ADV) was performed. Different vaccination routes (intradermal (i.d.) versus intramuscular (i.m.)), inoculation sites (the neck versus the back) and number of inoculation points (2 versus 6) per site were compared. Body weight (BW) changes and viral excretion after challenge were compared with virus-neutralizing titers, antigen-specific IgG and IgA responses in serum and virus-specific lymphoproliferative responses in peripheral blood during the immunisation period. According to BW changes better protection was obtained with six-point than two-point i.d. injections. i.d. vaccination in the back at six points gave similar results as i.m. vaccination in the neck but appeared inferior in the reduction of virus excretion. Regarding the immunological parameters, the virus-specific IgA response in serum gave the best indication for protection. It can be concluded that according to BW changes, six-point i.d. immunisation in the back and i.m. immunisation in the neck provided the best protection and that six-point i.d. injections resulted in a better vaccination than two-point i.d. injections.

Field use of a vaccinia-rabies recombinant vaccine for the control of sylvatic rabies in Europe and North America.

During recent years, most research on the control of sylvatic rabies has concentrated on developing methods of oral vaccination of wild rabies vectors. To improve both the safety and the stability of the vaccine used, a recombinant vaccinia virus, which expresses the immunising glycoprotein of rabies virus (VRG), has been developed and tested extensively in the laboratory as well as in the field. From 1989 to 1995, approximately 8.5 million VRG vaccine doses were dispersed in Western Europe to vaccinate red foxes (Vulpes vulpes), and in the United States of America (USA) to vaccinate raccoons (Procyon lotor) and coyotes (Canis latrans). In Europe, the use of VRG has led to the elimination of sylvatic rabies from large areas of land, which have consequently been freed from the need for vaccination. Nevertheless, despite very good examples of cross-border cooperation, reinfections have occurred in some regions, due to the difficulty of co-ordinating vaccination plans among neighbouring countries. In the USA, preliminary data from field trails indicate a significant reduction in the incidence of rabies in vaccinated areas.

Stability of recombinant vaccinia-rabies vaccine in veterinary use.

Wildlife vaccination depends on vaccines which can be orally administered by a baiting system. Therefore only two possibilities exist: either the use of attenuated strains of viruses, or recombinant vector viruses. As far as rabies is concerned, the choice of the recombinant vaccinia-rabies virus was made because it was safer and more stable. An in vitro stability study of the recombinant product compared to wild rabies virus at different temperatures (4 degrees C, 20 degrees C, 37 degrees C, 45 degrees C) showed that the recombinant virus was more stable. The stability of the recombinant virus was also tested under field conditions; besides natural freezing and thawing cycles, the virus titre remained unchanged in the bait for a month. Taking into account the fact that all baits are eaten by wild animals within this period, one can assume that the vaccine is efficacious for all baiting animals in field conditions. The stability of the recombinant vaccinia-rabies vaccine is of considerable interest in such uncontrolled conditions.

A blocking ELISA using a monoclonal antibody for the serological detection of Mycoplasma hyopneumoniae.

A blocking ELISA was developed by using a monoclonal antibody (4082-05-344-18) which specifically detected an epitope on the Mycoplasma hyopneumoniae 40 kDa membrane protein without cross-reacting with M flocculare or M hyorhinis. The results obtained with sera from specific pathogen-free pigs inoculated with M flocculare or M hyorhinis confirmed the specificity of the assay. An immunoblotting procedure was used to characterise the antibody response of pigs experimentally infected with M hyopneumoniae. Antibodies to the 40 kDa antigen were detected two weeks after infection and remained as major markers for at least 20 weeks. Cross-reacting antibodies to this antigen were not detected in convalescent sera from piglets infected with M flocculare or M hyorhinis. Sera from experimentally infected pigs were compared by means of the blocking ELISA and an indirect ELISA. The kinetics of ELISA antibodies after experimental inoculation were also studied. The detection of antibody was rather more stable for a longer time with the blocking ELISA than with the indirect ELISA. In an evaluation of more than 1000 sera from the field there was excellent agreement between the two methods.

[Biotechnology and animal health]. / Biotechnologie et santé animale.

The development of the first vaccines for use in animals, by Louis Pasteur at the end of the 19th Century, was an initial step in applying biotechnology to animal health. However, it is only much more recently that decisive progress has been made in finding applications for biotechnology, in both detecting and preventing infectious and parasitic diseases. This progress has shown the way to developing a range of procedures, the application of which will benefit the health of domestic and wild animals, enhance the well-being of companion animals, develop the performance of sporting animals and improve the productivity of farm animals, while also serving to protect human health. Such progress results from the increasingly rapid application of knowledge gained in the material and life sciences, all of which contribute to the multidisciplinary nature of biotechnology. Similarly, reagents and diagnostic techniques have been made more specific, sensitive, reproducible, rapid and robust by updating them through recent discoveries in immunology, biochemistry and molecular biology (monoclonal antibodies, nucleic probes, deoxyribonucleic acid amplification and many more). The development of new vaccines which combine efficacy, duration of protection, innocuity, stability, multivalence and ease of use (subunit vaccines, recombinant vaccines, synthetic vaccines and anti-idiotype vaccines) has resulted from recent progress in immunology, immunochemistry, molecular biology and biochemistry. Finally, the availability of new anti-infective, anti-parasitic agents and immunomodulatory therapeutic agents (capable of stimulating the specific and non-specific defence mechanisms of the body) demonstrates that biotechnology is continuing to find new applications in the field of animal health. New diagnostic techniques, vaccines and therapeutic substances are the most immediate applications of knowledge which may, in the future, extend to the development of transgenic animals of revised genetic potential, which will be more resistant to diseases and more productive. The ultimate aim of biotechnology applied to animal health and animal production is to protect human health, preserve the environment and ensure the health and well-being of animals.

Protection of cats against feline leukemia virus by vaccination with a canarypox virus recombinant, ALVAC-FL.

Two ALVAC (canarypox virus)-based recombinant viruses expressing the feline leukemia virus (FeLV) subgroup A env and gag genes were assessed for their protective efficacy in cats. Both recombinant viruses contained the entire gag gene. ALVAC-FL also expressed the entire envelope glycoprotein, while ALVAC-FL(dl IS) expressed an env-specific gene product deleted of the putative immunosuppressive region. Although only 50% of the cats vaccinated with ALVAC-FL(dl IS) were protected against persistent viremia after oronasal exposure to a homologous FeLV isolate, all cats administered ALVAC-FL resisted the challenge exposure. Significantly, protection was afforded in the absence of detectable FeLV-neutralizing antibodies. These results represent the first effective vaccination of cats against FeLV with a poxvirus-based recombinant vector and have implications that are relevant not only to FeLV vaccine development but also to developing vaccines against other retroviruses, including human immunodeficiency virus.

Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants.

Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.

Large-scale eradication of rabies using recombinant vaccinia-rabies vaccine.

Rabies infection of domestic and wild animals is a serious problem throughout the world. The major disease vector in Europe is the red fox (Vulpes vulpes) and rabies control has focused on vaccinating and/or culling foxes. Culling has not been effective, and the distribution of five vaccine baits is the only appropriate method for the vaccination of wild foxes. Although some European countries have conducted field vaccination campaigns using attenuated rabies virus strains, their use has not been extensively approved because they retain pathogenicity for rodents and can revert to virulence. These strains cannot be used in North America because they are pathogenic for the striped skunk (Mephitis mephitis) and are ineffective in the raccoon (Procyon lotor). We have constructed a recombinant vaccinia virus, VVTGgRAB, expressing the surface glycoprotein (G) of rabies virus (ERA strain). The recombinant was a highly effective vaccine in experimental animals, in captive foxes and in raccoons. We report here the results of a large-scale campaign of fox vaccination in a 2,200 km2 region of southern Belgium, an area in which rabies is prevalent. After distribution, 81% of foxes inspected were positive for tetracycline, a biomarker included in the vaccine bait and, other than one rabid fox detected close to the periphery of the treated area, no case of rabies, either in foxes or in domestic livestock, has been reported in the area.

Newcastle disease virus (NDV) vaccine based on immunization with avian cells expressing the NDV hemagglutinin-neuraminidase glycoprotein.

Newcastle disease virus (NDV) is a paramyxovirus that bears two envelope glycoproteins at the virion surface. These proteins, fusion and hemagglutinin-neuraminidase (HN), are involved in the immune response against NDV infection. Recombinant cells constitutively expressing at their surface the HN protein from the velogenic Texas strain were generated by introducing the HN gene with a helper-free AEV-based vector. These recombinant cells were used to immunize chickens by various protocols, and birds were subsequently challenged with a lethal NDV injection. Both NDV protection and serologic response were observed.

Recombinant vaccinia virus expression of the bovine leukaemia virus envelope gene and protection of immunized sheep against infection.

The bovine leukaemia virus (BLV) envelope gene encoding extracellular glycoprotein gp51 and transmembrane glycoprotein gp30 was cloned into the HA locus of vaccinia virus (Copenhagen strain), downstream of the vaccinia virus early-late promoter, H6, or a triple promoter element consisting of the promoter for the vaccinia virus H6 gene, the promoter for the cowpox virus A-type inclusion (ATI) gene and the promoter for the vaccinia virus HA gene. Inoculation of rabbits or sheep with the recombinant vaccinia virus coding for gp51 and gp30 or an uncleaved env precursor induced neutralizing antibodies to BLV. These antibodies competed with monoclonal antibodies directed against gp51 epitopes F, G, and H previously shown to be of crucial importance for BLV infection. Seven out of eight sheep vaccinated with the vaccinia recombinants resisted a drastic challenge (1.5 x 10(3) sheep infectious doses) with BLV-infected lymphocytes. These results show that vaccination with BLV env vaccinia recombinants protects sheep against infection with extremely high doses of BLV-infected heterologous lymphocytes.

Efficacy studies on a canarypox-rabies recombinant virus.

Recombinant avipox viruses have been developed expressing the rabies glycoprotein gene. A fowlpox-rabies recombinant has previously been shown to be protective against live rabies virus challenge in a number of non-avian species. This report describes the development of a canarypox-rabies recombinant. A comparison is made of the protective efficacy of this recombinant with other pox-rabies recombinants.

Identification and production of pestivirus proteins for diagnostic and vaccination purposes.

Using a panel of monoclonal antibodies (MAbs) previously characterized by seroneutralization, immunofluorescence and radioimmunoprecipitation, we have identified Pestivirus proteins useful for diagnostic purposes from the cytopathic Osloss isolate of bovine viral diarrhea virus (BVDV). Proteins that should be useful for vaccination have also been analysed. Cell-free translation of RNA from glycoprotein-coding cDNA fragments produced, when synthesized in the presence of canine pancreatic microsomes, two glycosylated proteins that were independently recognized and immunoprecipitated by two distinct classes of neutralizing MAbs. A similar in vitro procedure was carried out on nonstructural protein-coding sequences and allowed to identify a viral translation product that specifically reacted with MAbs directed against the 80 kDA protein of a number of Pestivirus strains. Its positioning within the polyprotein encoded by the viral genome was refined by epitope scanning using synthetic hexameric peptides. This viral antigen was further expressed in E. coli, produced as inclusion bodies and used successfully as an ELISA antigen in both competitive and indirect assays for the detection of BVD antibodies in cattle sera.

Protection of chickens with a recombinant fowlpox virus expressing the Newcastle disease virus hemagglutinin-neuraminidase gene.

A recombinant fowlpox virus expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) strain Texas was generated. Immunoprecipitation with chicken anti-NDV serum confirmed authentic expression of the HN protein. Protection of chickens from infection with NDV was observed when birds were immunized with the recombinant HN fowlpox virus by the intramuscular route after one or two inoculations. Vaccination by the ocular route with a mixture of fowlpox recombinants expressing the fusion and HN proteins did not show added protection over that seen with the individual viruses.

Efficacy of a baiting system for vaccinating foxes against rabies with vaccinia-rabies recombinant virus.

The efficacy of a vaccinia-rabies recombinant virus (10(8) TCID50) contained in a machine-made baiting system has been tested in 22 captive young foxes which were divided into three experimental groups of six and a control group of four foxes. Each fox in groups 1, 2 and 3 were fed one, two and three vaccine-baits, respectively, on successive days. The four unvaccinated foxes were housed separately. As shown by the incorporation of a tetracycline biomarker into their bones, all the baited foxes ingested at least one bait. Thirty days after baiting seroconversion to rabies was observed in 15 (83 per cent) of the foxes and seroconversion to vaccinia in 14 (78 per cent). Sixteen of the 18 (89 per cent) baited foxes resisted a rabies challenge 30 days after baiting. One cub was protected against rabies despite the absence of detectable anti-rabies antibody. The results demonstrate that the bait-sachet system permits a good release of the virus suspension into the mouth.

Vaccination against pseudorabies with glycoprotein gI+ or glycoprotein gI- vaccine.

Subunit pseudorabies vaccines that contained only purified glycoproteins of either of 2 strains of pseudorabies virus (PRV) were prepared and subsequently tested for safety and efficacy. The strains of virus used for vaccine production differed in at least 2 properties. One strain (Kojnok) was virulent for pigs and was believed to code for the entire complement of viral glycoproteins. The other (Kaplan) was a deletion mutant that was unable to code for structural viral glycoproteins gI and gp63. Purified glycoproteins were dispersed in an oil-in-water emulsion and were administered IM to pigs. Both vaccines were found to be safe and effective immunogens. Neither caused any local or general reactions, as verified by examination of the injection site (local safety) and by vaccination of pregnant sows in PRV-infected and noninfected herds. Sows vaccinated with the gI+ or gI- vaccine protected their pigs at levels of 93 and 92%, respectively, against a severe challenge exposure that killed 98% of pigs born from nonvaccinated sows. Vaccinated pigs were tested for active immunity by intranasal challenge exposure with the NIA 3 strain. Protection was quantitated by measuring the relative daily weight difference, expressed in percent per day, between vaccinated and control pigs during the first week after challenge exposure (delta G7); the estimated differences were 2.25 and 2.13% for gI+ and gI- vaccines, respectively. The absence of gI and gp63 did not affect the efficacy of this type of subunit glycoprotein vaccines.(ABSTRACT TRUNCATED AT 250 WORDS)