Jules Maarten Minke

Recombinant canarypoxvirus vaccine carrying the prM/E genes of West Nile virus protects horses against a West Nile virus-mosquito challenge.

An ALVAC (canarypoxvirus)-based recombinant (vCP2017) expressing the prM and E genes derived from a 1999 New York isolate of West Nile virus (WNV) was constructed and assessed for its protective efficacy in horses in two different experiments. In the first trial, a dose titration study was conducted to evaluate both serum neutralising antibody responses to WNV and duration of immunity. In the second trial the onset of protection was determined. Twenty-eight adult horses received two doses of vCP2017 administered intramuscularly at 5-week intervals and sixteen horses comprised age-matched non-vaccinated controls. Individual sera were taken periodically and tested for neutralising antibodies against WNV. Horses were challenged by allowing WNV-infected Aedes albopictus mosquitoes to feed on them two weeks (second trial) or one year (first trial) after the second vaccination. After challenge, horses were monitored for clinical signs of disease, and blood samples were collected for detection of WNV viremia and antibody. In both trials, all vaccinated horses developed neutralising antibodies against WNV. None of the vaccinated or control horses developed clinical signs of WNV disease upon challenge. None of the nine horses challenged 2 weeks after primary vaccination and only one of the ten vaccinated horses challenged 1 year after vaccination developed detectable viremia after challenge, whereas more than 80% of the controls became infected. Results from these studies demonstrated that a primary course of two doses of vCP2017 provides both antibody response and an early immunity in horses against WNV viremia.

Efficacy of a recombinant equine influenza vaccine against challenge with an American lineage H3N8 influenza virus responsible for the 2003 outbreak in the United Kingdom

Fifteen influenza-naive Welsh mountain ponies were randomly assigned to three groups of five. A single dose of a recombinant ALVAC vaccine was administered intramuscularly to five of the ponies, two doses, administered five weeks apart, were administered to five, and the other five served as unvaccinated, challenge controls. Two weeks after the completion of the vaccination programme, the ponies were all challenged by exposure to an aerosol of influenza virus A/eq/Newmarket/5/03. Their clinical signs were scored daily for 14 days according to a standardised scoring protocol, and nasal swabs were taken daily for 10 days to monitor the excretion of virus. The challenge produced severe clinical signs of influenza (fever, coughing, nasal discharge and dyspnoea) in all five control ponies, but the vaccinated ponies developed only mild disease, consisting of a serous nasal discharge lasting for only one day. The excretion of virus was almost completely suppressed in the vaccinated ponies, but the control ponies shed the virus for up to seven days after the challenge.

A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC-WNV vaccine (RECOMBITEK WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-gamma(+) producing cells against WNV in vaccinated horses. Prior exposure to ALVAC-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-gamma producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine.

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.

Rabies DNA vaccine in the horse: strategies to improve serological responses

In order for DNA vaccines to become a practical alternative to conventional vaccines their ability to induce antibody responses in large mammals needs to be improved. We used DNA vaccination against rabies in the horse as a model to test the potential of two different strategies to enhance antibody responses in a large mammalian species. The administration of the DNA vaccine in the presence of aluminum phosphate improved both the onset and the intensity of serological responses but was not potent enough to achieve seroconversion in all vaccinated ponies. However, when the DNA vaccine was formulated with the cationic lipid DMRIE-DOPE instead of aluminum phosphate, a very strong impact on both onset and intensity of serological responses was observed. This latter strategy ensured excellent seroconversion in all vaccinated ponies after a primary course of two injections, demonstrating a clear improvement of the homogeneity of the induced responses. These data indicate that rabies DNA vaccination is feasible in horses and further suggests that properly formulated DNA vaccines can generate immune responses in large veterinary species at a level comparable to the responses achieved with conventional vaccines.

Vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge

None of the currently available distemper vaccines provides a satisfactory solution for the immunization of very young carnivores in the face of maternal-derived immunity. Since mucosal immunization with replication-competent adenovirus-based vaccines has been proven effective in the face of passive immunity against the vector, it has the potential to provide a solution for the vaccination of young puppies born to canine distemper virus (CDV)-immune dams. We report the engineering and the characterization of two replication-competent canine adenovirus type 2 (CAV2)-based vaccines expressing, respectively, the CDV hemagglutinin (HA) and fusion (F) antigens. We first demonstrated that the intranasal vaccination with a mixture of both recombinant CAV2s provides an excellent level of protection in seronegative puppies, confirming the value of replication-competent adenovirus-based vectors for mucosal vaccination. In contrast, intranasal immunization with the same vaccine of puppies born to CDV- and CAV2-immune dams, failed to activate specific and protective immune responses. We hypothesized that an active CAV2 infection occurred while puppies were in close contact with the vaccinated dams in the breeding units and that the resulting active mucosal immunity interfered with the intranasal administration of CAV2-based CDV vaccine. However, when puppies born to CDV- and CAV2-immune dams were vaccinated subcutaneously with the CAV2-based CDV vaccine, significant seroconversion and solid protective immunity were triggered despite pre-existing systemic immunity to the vector. This latter result is surprising and suggests that subcutaneous vaccination with a replication-competent recombinant CAV2 may be an efficient strategy to overcome both passive and active adenovirus specific immunity in the dog. From a practical point of view, this could pave the way for an original strategy to vaccinate young puppies in the face of maternal-derived immunity.

Comparison of antibody responses after vaccination with two inactivated rabies vaccines.

Thirty laboratory dogs were randomly assigned to two groups (A and B) of 15 dogs and subcutaneously vaccinated with a single dose of one of two commercially available monovalent inactivated rabies vaccines: RABISIN (Merial, France) (group A) and NOBIVAC Rabies (Intervet International) (group B). Rabies antibodies were measured over a period of 4 months using the fluorescent antibody virus neutralization (FAVN) test. The two vaccines performed differently in terms of magnitude and persistence of rabies antibodies titers in dogs. Two weeks after vaccination, average rabies antibody titers peaked at 2.53 IU/mL (range, 0.17-13.77 IU/mL) and 1.26 IU/mL (range, 0.50-4.56 IU/mL) in groups A and B dogs, respectively. The average FAVN antibody titres against rabies on D28, D56, D84, D112 and D120 were significantly higher in group A than in group B. Although all dogs from group B serologically responded to vaccination, the proportion of dogs with antibody titres >or=0.5 IU/mL dropped significantly after D28 and was statistically significantly lower on D56, D84 and D112 compared to group A dogs. In conclusion, in the context of international trade, the choice of the vaccine and the timing of blood tests are critical factors in achieving successful serological test results after rabies vaccination. RABISIN induces high and sustained antibody titres against rabies, increasing the flexibility for the time of blood sampling after primo-vaccination.

Onset and duration of protective immunity against clinical disease and renal carriage in dogs provided by a bi-valent inactivated leptospirosis vaccine.

Abstract Protection against clinical disease and prevention of the renal carrier state remain the key objectives of vaccination against leptospirosis in the dog. In the present paper, groups of dogs were vaccinated twice with a commercial bacterin (EURICAN® L) containing Leptospira interrogans serovars icterohaemorrhagiae and canicola and challenged with heterologous representatives of both serovars at 2 weeks (onset of immunity) or 14 months (duration of immunity) after the second vaccination. Control dogs were not vaccinated against leptospirosis and kept with the vaccinated dogs. The challenges, irrespective of the serovar, reliably produced clinical signs consistent with Leptospira infection in the control pups with up to 60% mortality. As expected clinical disease in the adult controls was less severe, but we were able to induce morbidity and mortality as well. Under these extreme challenge conditions, clinical signs in the vaccinated dogs were rare, and when observed, mild and transient in nature. Following experimental infection, 100% of the control pups and 83% of the adult controls became renal carriers. Despite the heavy challenges, none of the 18 vaccinated puppies (onset of immunity studies) and only 2 out of the 16 vaccinated adult dogs (duration of immunity studies) developed a renal carrier state. These results show that a primary course of two doses of EURICAN® L provided quick onset and long-term protection against both clinical leptospirosis and the renal carrier stage. This vaccine should provide veterinarians with a powerful tool to prevent clinical disease in dogs and zoonotic transmission of leptospirosis to humans.

Humoral and cell-mediated immune responses of old horses following recombinant canarypox virus vaccination and subsequent challenge infection

Equine influenza virus is a leading cause of respiratory disease in the horse population; however, the susceptibility of old horses to EIV infection remains unknown. While advanced age in horses (>20 years) is associated with age-related changes in immune function, there are no specific recommendations regarding the vaccination of older horses even though a well-characterized effect of aging is a reduced antibody response to standard vaccination. Therefore, we evaluated the immunological and physiological response of aged horses to a live non-replicating canarypox-vectored EIV vaccine and subsequent challenge infection. Vaccination of the aged horses induced EIV-specific IgGb and HI antibodies. No specific increase in cell-mediated immune (CMI) response was induced by the vaccine as determined by EIV-specific lymphoproliferation and the detection of EIV-specific IFNγ(+) CD5(+)T cells, IFNγ, IL-2, IL-4 and IL-13 mRNA expression. Non-vaccinated aged horses exhibited clinical signs of the disease (coughing, nasal discharge, dyspnea, depression, anorexia) as well as increased rectal temperature and viral shedding following challenge. Concomitant with the febrile episodes, we also observed increased production of pro-inflammatory cytokine mRNA production in vivo using RT-PCR. Naïve horses were included in this study for vaccine and challenge controls only. As expected, the canarypox-vectored EIV vaccine stimulated significant CMI and humoral immune responses and provided significant protection against clinical signs of disease and reduced virus shedding in naive horses. Here, we show that aged horses remain susceptible to infection with equine influenza virus despite the presence of circulating antibodies and CMI responses to EIV and vaccination with a canarypox-vectored EIV vaccine provides protection from clinical disease.

Protection provided by a recombinant ALVAC(®)-WNV vaccine expressing the prM/E genes of a lineage 1 strain of WNV against a virulent challenge with a lineage 2 strain.

The emergence of lineage 2 strains of WNV in Europe as a cause of clinical disease and mortality in horses raised the question whether the existing WNV vaccines, all based on lineage 1 strains, protect against circulating lineage 2 strains of WNV. In the present paper we have determined the level of cross protection provided by the recombinant ALVAC(®)-WNV vaccine in a severe challenge model that produces clinical signs of WNV type 2 disease. Ten horses were vaccinated twice at 4 weeks interval with one dose of the ALVAC-WNV vaccine formulated at the minimum protective dose. A further 10 horses served as controls. Two weeks after the second vaccination, all horses were challenged intrathecally with a recent neurovirulent lineage 2 strain of WNV. The challenge produced viraemia in 10 out of 10 and encephalitis in 9 out of 10 control horses. Three horses had to be euthanized for humane reasons. In contrast, none of the vaccinated horses developed WNV disease and only 1 vaccinated horse became viraemic at a single time point at low titre. The prevalence of WNV disease and viraemia were significantly lower in the vaccinated horses than in the control horses (P<0.0001 for both). Based on these results, the ALVAC-WNV vaccine will provide veterinarians with an effective tool to control infections caused by lineage 1 and 2 strains of WNV.