Equine Influenza Virus Vaccine Research Articles

Evaluation of the ability of canarypox-vectored equine influenza virus vaccines to induce humoral immune responses against canine influenza viruses in dogs

To evaluate canarypox-vectored equine influenza virus (EIV) vaccines expressing hemagglutinins of A/equine/Kentucky/94 (vCP1529) and A2/equine/Ohio /03 (vCP2242) for induction of antibody responses against canine influenza virus (CIV) in dogs. 35 dogs. Dogs were randomly allocated into 4 groups; group 1 (n = 8) and group 2 (9) were inoculated SC on days 0 and 28 with 1.0 mL (approx 10(5.7) TCID(50)) of vCP1529 and vCP2242, respectively. Dogs in group 3 (n = 9) were inoculated twice with 0.25 mL (approx 10(5.7) TCID(50)) of vCP2242 via the transdermal route. The 9 dogs of group 4 were control animals. All dogs were examined for adverse reactions. Sera, collected on days -1, 7, 13, 21, 28, 35, and 42, were tested by hemagglutination inhibition (HI) and virus neutralization (VN) assays for antibodies against CIV antigens A/Canine/FL/43/04-PR and A/Canine/NY/115809/05, respectively. Inoculations were tolerated well. The HI and VN antibodies were detected by 7 days after primary inoculation. Most dogs of groups 1 and 2 and all dogs of group 3 had detectable antibodies by 14 days after initial inoculation. The second inoculation induced an anamnestic response, yielding geometric mean HI titers of 139, 276, and 1,505 and VN titers of 335, 937, and 3,288 by day 42 (14 days after booster inoculation) in groups 1, 2, and 3, respectively. Canarypox-vectored EIV vaccines induce biologically important antibodies and may substantially impact CIV transmission within a community and be of great value in protecting dogs against CIV-induced disease.

Formulation with CpG ODN enhances antibody responses to an equine influenza virus vaccine

Previous studies have shown that protection against equine influenza virus (EIV) is partially mediated by virus-specific IgGa and IgGb. In this study we tested whether addition of a CpG ODN formulation to a commercial killed virus vaccine would enhance EIV-specific IgGa and IgGb antibody responses, and improve protection against an experimental EIV challenge. Thirty naïve horses were assigned to one of three groups and vaccinated as follows: 10 were given vaccine (Encevac TC4, Intervet Inc.) alone, 10 were given vaccine plus 0.25 mg CpG ODN 2007 formulated with 30% Emulsigen (CpG/Em), and 10 controls were given saline. All horses were challenged with live virus 12 weeks after the final vaccination. Antibody responses were tested by single radial hemolysis (SRH) and ELISA, and protection was evaluated by determination of temperature, coughing, and clinical scores. Killed virus vaccine combined with CpG/Em induced significantly greater serologic responses than did the vaccine alone. All antibody isotypes tested increased after the addition of CpG/Em, although no shift in relative antibody isotypes concentrations was detected. Vaccination significantly improved protection against challenge but the differences between the two vaccine groups were not statistically significant. This study is the first demonstration that CpG/Em enhances antigen-specific antibody responses in horses and supports its potential to be used as an adjuvant for vaccines against equine infections.

Wave microcarrier cultivation of MDCK cells for influenza virus production in serum containing and serum-free media

A process for equine influenza virus vaccine production using a microcarrier system (Cytodex 1) in a 2 L Wave bioreactor is described. Growth of Madin Darby canine kidney (MDCK) cells in serum containing GMEM medium (SC) is compared to growth in serum-free Ex-Cell MDCK medium (SF) without washing steps and medium exchange before infection. Cultivations with microcarrier concentrations of 2 and 4 g/L for both media are shown. Metabolic data from carbon and amino acid metabolism are discussed. Additionally, in roller bottle experiments the influence of multiplicity of infection (moi) and trypsin concentration on the HA value was investigated. Analysis of HA and TCID 50 at 37 °C showed a stable HA of maximum 2.6 log HA/100 μL for 2 weeks. Peak TCID 50 titers of 10 7.7 viruses/mL were achieved 20 h post infection, but infectivity was below detection limit after 150 h. Cell attachment onto microcarriers under serum-free conditions was improved by Ca 2+ addition and by cell harvesting without trypsin using only an EDTA/PBS solution. For the wave cultivation maximum virus titers of 2.3–2.6 log HA units/100 μL were reached from infection with a moi of 0.05. However, in SF medium pH dropped to less than pH 6.8 which resulted in lower HA titers of 1.7 log HA units/100 μL. For the higher microcarrier concentration (4 g/L) medium exchange steps (500 mL) were needed for both media. Omission of the washing step and medium exchange before infection in SF medium clearly simplified the influenza production process; however, for higher virus yields a better pH control of the wave bioreactor would be required. Higher cell densities (2.8 × 10 6 cells/mL for 2 g/L microcarrier) and better attachment compared to stirred tank bioreactors showed, that the wave bioreactor is a good alternative to stirred tank processes for expanding production capacities in case of a pandemic.

A new modified live equine influenza virus vaccine: phenotypic stability, restricted spread and efficacy against heterologous virus challenge.

Flu Avert IN vaccine is a new, live attenuated virus vaccine for equine influenza. We tested this vaccine in vivo to ascertain 1) its safety and stability when subjected to serial horse to horse passage, 2) whether it spread spontaneously from horse to horse and 3) its ability to protect against heterologous equine influenza challenge viruses of epidemiological relevance. For the stability study, the vaccine was administered to 5 ponies. Nasal swabs were collected and pooled fluids administered directly to 4 successive groups of naïve ponies by intranasal inoculation. Viruses isolated from the last group retained the vaccine's full attenuation phenotype, with no reversion to the wild-type virus phenotype or production of clinical influenza disease. The vaccine virus spread spontaneously to only 1 of 13 nonvaccinated horses/ponies when these were comingled with 39 vaccinates in the same field. For the heterologous protection study, a challenge model system was utilised in which vaccinated or naïve control horses and ponies were exposed to the challenge virus by inhalation of virus-containing aerosols. Challenge viruses included influenza A/equine-2/Kentucky/98, a recent representative of the 'American' lineage of equine-2 influenza viruses; and A/equine-2/Saskatoon/90, representative of the 'Eurasian' lineage. Clinical signs among challenged animals were recorded daily using a standardised scoring protocol. With both challenge viruses, control animals reliably contracted clinical signs of influenza, whereas vaccinated animals were reliably protected from clinical disease. These results demonstrate that Flu Avert IN vaccine is safe and phenotypically stable, has low spontaneous transmissibility and is effective in protecting horses against challenge viruses representative of those in circulation worldwide.

Safety, efficacy, and immunogenicity of a modified-live equine influenza virus vaccine in ponies after induction of exercise-induced immunosuppression.

To determine safety, efficacy, and immunogenicity of an intranasal cold-adapted modified-live equine influenza virus vaccine administered to ponies following induction of exercise-induced immunosuppression. Prospective study. Fifteen 9- to 15-month old ponies that had not had influenza. Five ponies were vaccinated after 5 days of strenuous exercise on a high-speed treadmill, 5 were vaccinated without undergoing exercise, and 5 were not vaccinated or exercised and served as controls. Three months later, all ponies were challenged by nebulization of homologous equine influenza virus. Clinical and hematologic responses and viral shedding were monitored, and serum and nasal secretions were collected for determination of influenza-virus-specific antibody isotype responses. Exercise caused immunosuppression, as indicated by depression of lymphocyte proliferation in response to pokeweed mitogen. Vaccination did not result in adverse clinical effects, and none of the vaccinated ponies developed clinical signs of infection following challenge exposure. In contrast, challenge exposure caused marked clinical signs of respiratory tract disease in 4 control ponies. Vaccinated and control ponies shed virus after challenge exposure. Antibody responses to vaccination were restricted to serum IgGa and IgGb responses in both vaccination groups. After challenge exposure, ponies in all groups generated serum IgGa and IgGb and nasal IgA responses. Patterns of serum hemagglutination inhibition titers were similar to patterns of IgGa and IgGb responses. Results suggested that administration of this MLV vaccine to ponies with exercise-induced immunosuppression was safe and that administration of a single dose to ponies provided clinical protection 3 months later.

Local and systemic isotype-specific antibody responses to equine influenza virus infection versus conventional vaccination

Inactivated alum-adjuvanted conventional equine influenza virus vaccines are of poor efficacy and offer limited short-term protection against infection. In sharp contrast, natural infection with equine influenza virus confers long-term protective immunity. In order to identify the protective immune responses to equine influenza virus, the influenza virus-specific IgA, IgGa, IgGb, IgGc and IgG(T) antibody responses in nasal secretions and serum induced by natural infection and a commercial vaccine were studied by ELISA. Two groups of four influenza-naive ponies were established. In the natural infection group, ponies received 10 8.5 EID 50 of A/equine/Ky/1/81 by intranasal instillation, were allowed to recover, and then were rechallenged 100 days later. All four ponies exhibited clinical signs of influenza virus infection and viral shedding following primary infection, but were completely protected from challenge infection. Antibody responses to primary infection were characterized by nasal IgA and serum IgGa and IgGb responses. Ponies in the conventional vaccine group received a commercially available vaccine by intramuscular injection followed by a booster injection 3 weeks later. Challenge infection 100 days after vaccination resulted in clinical signs of infection and viral shedding. Antibody responses to vaccination were restricted to serum IgG(T) responses only. These results demonstrate that the protective immunity generated by natural equine influenza virus infection is associated with a mucosal IgA immune response and humoral IgGa and IgGb sub-isotype responses, and that this pattern of response is not generated by conventional vaccines.

Identification of two antigenically and genetically distinct lineages of H3N8 equine influenza virus in Sweden.

Four Swedish strains of equine H3N8 influenza virus isolated from outbreaks during the last 4 years were characterized. Antigenic typing using monoclonal antibodies raised against a variety of H3N8 strains showed that the viruses are heterogeneous, the 1993 isolate being closely related to the 1991 Swedish isolate TAB/91 and the other three isolates from 1994 and 1996 being more closely related to each other. This pattern is reflected in the phylogenetic data calculated from nucleotide sequencing of the haemagglutinin genes. H3N8 equine influenza can be seen to be evolving in two distinct lineages, one European and one American. The 1993 isolate is closely related to the European lineage and is the most recent Swedish strain of this lineage to be isolated. The 1994 and 1996 isolates fit into the American lineage, which contains recent isolates from the United States and also Britain. These results indicate that American-type H3N8 viruses have become endemic in Sweden and, in light of the antigenic differences which can be observed between viruses belonging to the two lineages, we believe that equine influenza virus vaccines should be updated with an American-type virus strain.