Efficacy of a canarypox-vectored recombinant vaccine expressing the hemagglutinin gene of equine influenza H3N8 virus in the protection of ponies from viral challenge

OBJECTIVE: To determine onset and duration of immunity provided by a 2- or 3-dose series of a new canarypox-vectored recombinant vaccine for equine influenza virus (rCP-EIV vaccine) expressing the hemagglutinin genes of influenza H3N8 virus strains A/eq/Kentucky/94 and A/eq/Newmarket/2/93 in ponies. ANIMALS: Forty-nine 1- to 3-year-old male Welsh Mountain Ponies that were seronegative for equine influenza virus. PROCEDURES: Vaccinated and control ponies were challenged with aerosolized influenza virus A/eq/Sussex/89 (H3N8), representative of the Eurasian lineage of circulating influenza viruses. In trial 1, control ponies and ponies that received rCP-EIV vaccine were challenged 2 weeks after completion of the 2-dose primary vaccination program. In trial 2, ponies were challenged 5 months after 2 doses of rCP-EIV vaccine or 1 year after the first boosting dose of rCP-EIV vaccine, administered 5 months after completion of the primary vaccination program. After challenge, ponies were observed daily for clinical signs of influenza and nasal swab specimens were taken to monitor virus excretion. RESULTS: The challenge reliably produced severe clinical signs consistent with influenza infection in the control ponies, and virus was shed for up to 7 days. The vaccination protocol provided clinical and virologic protection to vaccinates at 2 weeks and 5 months after completion of the primary vaccination program and at 12 months after the first booster. CONCLUSION AND CLINICAL RELEVANCE: The rCP-EIV vaccine provided protection of ponies to viral challenge. Of particular importance was the protection at 5 months after the second dose, indicating that this vaccine closes an immunity gap between the second and third vaccination.     

Replacement of internal protein genes, with the exception of the matrix, in equine 1 viruses by equine 2 influenza virus genes during evolution in nature.

To establish the evolutionary association between the equine 1 H7 HA and M genes, phylogenetic analyses of the six internal gene segments of equine 1 influenza viruses (H7N7 subtype) were performed using partial nucleotide sequences. The results demonstrated that five internal genes (PBI, PB2, PA, NP and NS) of equine 1 viruses isolated after 1964 were replaced by those of equine 2 H3N8 viruses. However, the M gene was maintained during the evolution of these equine 1 viruses. These findings suggest a functional association between equine H7 HA and M gene products, most likely M2 protein.     

ISCOM-matrix-based equine influenza (EIV) vaccine stimulates cell-mediated immunity in the horse

The humoral immune response induced by ISCOM-matrix (Immuno Stimulating COMplex-Matrix)-adjuvanted equine influenza virus (EIV) vaccine is well documented in horses. ISCOM-matrix adjuvanted vaccines against human influenza are strong inducers of cell-mediated immunity (CMI), including T cell proliferation and virus-specific cytotoxic T cell. In the horse, the CMI response to equine influenza vaccination is less well characterised. An ISCOM-based vaccine has been shown to induce interferon gamma (IFN-γ) synthesis, a CMI marker, in the horse, but this has not been shown for the ISCOM-matrix vaccine, which is a different formulation. The objective of this study was to measure EIV-specific IFN-γ synthesis after vaccination with an ISCOM-matrix-adjuvanted EIV vaccine. Equilis Prequenza is a commercialised inactivated EIV vaccine containing purified haemagglutinin (HA) and neuraminidase (NA) subunits adjuvanted with ISCOM-matrix. Six influenza-naïve Welsh mountain ponies were vaccinated twice with Equilis Prequenza at an interval of four weeks. Six control ponies received a placebo of physiological water. EIV-specific IFN-γ synthesis by peripheral blood lymphocytes and the antibody response to a panel of representative EIV isolates were measured prior to and after both injections. Immunisation with the ISCOM-matrix-based EIV vaccine stimulated significant EIV-specific IFN-γ synthesis and EIV-specific single radial haemolysis (SRH) antibody. In conclusion, EIV vaccine adjuvanted with ISCOM-matrix stimulates both antibody and a cellular immune response in the horse.     

Use of DNA and recombinant canarypox viral (ALVAC) vectors for equine herpes virus vaccination

In this study, experimental canarypox virus (ALVAC) and plasmid DNA recombinant vaccines expressing the gB, gC and gD glycoproteins of EHV-1 were assessed for their ability to protect conventional ponies against a respiratory challenge with EHV-1. In addition, potential means of enhancing serological responses in horses to ALVAC and DNA vaccination were explored. These included co-administration of the antigen with conventional adjuvants, complexation with DMRIE-DOPE and co-expression of the antigen along with equine GM-CSF. Groups of EHV primed ponies were vaccinated twice intra-muscularly with one dose of the appropriate test vaccine at an interval of 5 weeks. Two to 3 weeks after the second vaccination, ponies were infected intra-nasally with the virulent Ab4 strain of EHV-1 after which they were observed clinically and sampled for virological investigations. The results demonstrated that DNA and ALVAC vaccination markedly reduced virus excretion after challenge in terms of duration and magnitude, but failed to protect against cell-associated viremia. Noteworthy was the almost complete absence of virus excretion in the group of ponies vaccinated with ALVAC-EHV in the presence of Carbopol adjuvant or DNA plasmid formulated with aluminium phosphate. The administration of the DNA vaccine in the presence of GM-CSF and formulated in DMRIE-DOPE and of the ALVAC vaccine in the presence of Carbopol adjuvant significantly improved virus neutralising antibody responses to EHV-1. These findings indicate that DNA and ALVAC vaccination is a promising approach for the immunological control of EHV-1 infection, but that more research is needed to identify the immunodominant protective antigens of EHV-1 and their interaction with the equine immune system.     

Antibody responses after vaccination against equine influenza in the Republic of Korea in 2013

In this study, antibody responses after equine influenza vaccination were investigated among 1,098 horses in Korea using the hemagglutination inhibition (HI) assay. The equine influenza viruses, A/equine/South Africa/4/03 (H3N8) and A/equine/Wildeshausen/1/08 (H3N8), were used as antigens in the HI assay. The mean seropositive rates were 91.7% (geometric mean antibody levels (GMT), 56.8) and 93.6% (GMT, 105.2) for A/equine/South Africa/4/03 and A/equine/Wildeshausen/1/08, respectively. Yearlings and two-year-olds in training exhibited lower positive rates (68.1% (GMT, 14) and 61.7% (GMT, 11.9), respectively, with different antigens) than average. Horses two years old or younger may require more attention in vaccination against equine influenza according to the vaccination regime, because they could be a target of the equine influenza virus.     

DNA vaccination against influenza viruses: a review with emphasis on equine and swine influenza

The influenza virus vaccines that are commercially-available for humans, horses and pigs in the United States are inactivated, whole-virus or subunit vaccines. While these vaccines may decrease the incidence and severity of clinical disease, they do not consistently provide complete protection from virus infection. DNA vaccines are a novel alternative to conventional vaccination strategies, and offer many of the potential benefits of live virus vaccines without their risks. In particular, because immunogens are synthesized de novo within DNA transfected cells, antigen can be presented by MHC class I and II molecules, resulting in stimulation of both humoral and cellular immune responses. Influenza virus has been used extensively as a model pathogen in DNA vaccine studies in mice, chickens, ferrets, pigs, horses and non-human primates, and clinical trials of DNA-based influenza virus vaccines are underway in humans. Our studies have focused on gene gun delivery of DNA vaccines against equine and swine influenza viruses in mice, ponies and pigs, including studies employing co-administration of interleukin-6 DNA as an approach for modulating and adjuvanting influenza virus hemagglutinin-specific immune responses. The results indicate that gene gun administration of plasmids encoding hemagglutinin genes from influenza viruses is an effective method for priming and/or inducing virus-specific immune responses, and for providing partial to complete protection from challenge infection in mice, horses and pigs. In addition, studies of interleukin-6 DNA co-administration in mice clearly demonstrate the potential for this approach to enhance vaccine efficacy and protection.     

Antibody and IFN-gamma responses induced by a recombinant canarypox vaccine and challenge infection with equine influenza virus

In horses, equine influenza virus (EIV) is a leading cause of respiratory disease. Conventional inactivated vaccines induce a short-lived immune response. By comparison, natural infection confers a long-term immunity to re-infection. An aim of new equine influenza vaccines is to more closely mimic natural infection in order to achieve a better quality of immunity. A new live recombinant vaccine derived from the canarypox virus vector and expressing haemagglutinin genes of EIV (subtype H3N8) has been developed. Stimulation of the immune system was studied after immunisation with this canarypox-based vaccine and challenge infection by exposure to a nebulised aerosol of EIV. The humoral immune response was evaluated by measuring serum antibody levels using the single radial haemolysis (SRH) assay. The cellular immune response was assessed by the measurement of interferon gamma (IFN-gamma) synthesis in peripheral blood mononuclear cells (PBMC). Clinical signs of the disease (temperature, coughing, nasal discharge, dyspnoea, depression and anorexia) and virus excretion were monitored after challenge infection. Clinical signs and virus shedding were significantly reduced in vaccinates compared with unvaccinated controls. EIV-specific immunity was stimulated by vaccination with a recombinant vaccine as serological responses were detected after immunisation. This study also provided the first evidence for increased IFN-gamma protein synthesis in vaccinated ponies following challenge infection with EIV compared with control ponies.     

Comparison of two modern vaccines and previous influenza infection against challenge with an equine influenza virus from the Australian 2007 outbreak

During 2007, large outbreaks of equine influenza (EI) caused by Florida sublineage Clade 1 viruses affected horse populations in Japan and Australia. The likely protection that would be provided by two modern vaccines commercially available in the European Union (an ISCOM-based and a canarypox-based vaccine) at the time of the outbreaks was determined. Vaccinated ponies were challenged with a representative outbreak isolate (A/eq/Sydney/2888-8/07) and levels of protection were compared. A group of ponies infected 18 months previously with a phylogenetically-related isolate from 2003 (A/eq/South Africa/4/03) was also challenged with the 2007 outbreak virus. After experimental infection with A/eq/Sydney/2888-8/07, unvaccinated control ponies all showed clinical signs of infection together with virus shedding. Protection achieved by both vaccination or long-term immunity induced by previous exposure to equine influenza virus (EIV) was characterised by minor signs of disease and reduced virus shedding when compared with unvaccinated control ponies. The three different methods of virus titration in embryonated hens’ eggs, EIV NP-ELISA and quantitative RT-PCR were used to monitor EIV shedding and results were compared. Though the majority of previously infected ponies had low antibody levels at the time of challenge, they demonstrated good clinical protection and limited virus shedding. In summary, we demonstrate that vaccination with current EIV vaccines would partially protect against infection with A/eq/Sydney/2888-8/07-like strains and would help to limit the spread of disease in our vaccinated horse population.     

The use of a systemic prime/mucosal boost strategy with an equine influenza ISCOM vaccine to induce protective immunity in horses

In horses, natural infection confers long lasting protective immunity characterised by mucosal IgA and humoral IgGa and IgGb responses. In order to investigate the potential of locally administered vaccine to induce a protective IgA response, responses generated by vaccination with an immunostimulating complex (ISCOM)-based vaccine for equine influenza (EQUIP F) containing A/eq/Newmarket/77 (H7N7), A/eq/Borl?nge/91 (H3N8) and A/eq/Kentucky/98 (H3N8) using a systemic prime/mucosal boost strategy were studied. Seven ponies in the vaccine group received EQUIP F vaccine intranasally 6 weeks after an initial intramuscular immunisation. Following intranasal boosting a transient increase in virus-specific IgA was detected in nasal wash secretions. Aerosol challenge with the A/eq/Newmarket/1/93 reference strain 4 weeks after the intranasal booster resulted in clinical signs of infection and viral shedding in seven of seven influenza-naive control animals whereas the seven vaccinated ponies had statistically significantly reduced clinical signs and duration of virus excretion. Furthermore, following this challenge, significantly enhanced levels of virus-specific IgA were detected in the nasal washes from vaccinated ponies compared with the unvaccinated control animals. These data indicate that the intranasal administration of EQUIP F vaccine primes the mucosal system for an enhanced IgA response following exposure to live influenza virus.     

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

OBJECTIVE: 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. DESIGN: Prospective study. ANIMALS: Fifteen 9- to 15-month old ponies that had not had influenza. PROCEDURE: 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. RESULTS: 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. CONCLUSIONS AND CLINICAL RELEVANCE: 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.     

Equine influenza vaccine containing older H3N8 strains offers protection against A/eq/South Africa/4/03 (H3N8) strain in a short-term vaccine efficacy study

REASONS FOR PERFORMING STUDY: Surveillance of equine influenza viruses has suggested that strains included in currently licensed vaccines are a poor match for those predominantly circulating in the field. OBJECTIVE: To assess the ability of Duvaxyn IE-T Plus to provide cross protection against the newly evolved South Africa/4/03 (H3N8) strain of equine influenza virus. METHODS: The vaccine efficacy was evaluated by challenge infection with influenza strain A/eq/South Africa/4/03 (H3N8) 2 weeks after a primary course of 2 vaccinations with Duvaxyn IE-T Plus given at a 4-week interval. The outcome of challenge in vaccinated ponies was compared with that in unvaccinated animals. RESULTS: At the time of challenge, all vaccinated ponies had high levels of antibody to Newmarket/1/93, Newmarket/2/93 and South Africa/4/03 strains measured by single radial haemolysis. After challenge infection, there were statistically significantly decreased clinical scores and virus shedding was significantly lower in the vaccinated ponies compared to unvaccinated controls. CONCLUSION: Two doses of Duvaxyn IE-T Plus provides good clinical and virological protection against challenge with a variant virus 2 weeks after the 2 doses of vaccine. POTENTIAL RELEVANCE: When variant strains of equine influenza virus first emerge, booster immunisations with currently available vaccines may limit infection provided sufficiently high antibody levels are achieved, suggesting that vaccination in the face of an outbreak may be beneficial.     

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 na?ve 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 na?ve 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.     

Molecular changes associated with adaptation of equine influenza H3N8 virus in embryonated chicken eggs

Embryonated chicken eggs (ECEs) are routinely used to isolate equine influenza virus. Propagation of the virus in ECEs results in selection of variants. In the present study, we determined nucleotide sequences of entire coding regions of parent A/equine/Tottori/1/07 (H3N8) and its derivatives that have different passage histories in ECE. After 12 passages, nucleotide sequence analysis predicted 3 amino acid substitutions in hemagglutinin (HA; 2 in HA1 and 1 in HA2). The two amino acid substitutions in HA1 were located in the vicinity of the cell receptor-binding site. Three other amino acid substitutions were predicted in internal proteins, 1 in the M1, 1 in the NP and 1 in the PA. This is the first report showing mutations in the internal protein genes of equine influenza virus associated with adaptation to ECE.     

马流感病毒血凝素基因甲病毒复制子重组表达质粒的构建及其免疫效力评价

为评价马流感病毒(EIV)HA基因核酸免疫效果,本研究以甲病毒复制子载体pSFV1CS分别构建了表达EIV H3N8亚型的美洲型和欧洲型HA基因的重组真核表达质粒。并将其转染293T细胞,经间接免疫荧光鉴定表明HA基因获得表达;以重组质粒免疫的BALB/c鼠能够检测到特异性抗体产生,而且HI抗体水平持续升高,同时小鼠体内IFN-γ、IL-4分泌水平也有所升高。攻毒后小鼠表现轻度临床症状,但病毒分离和RT-PCR均未检测到病毒。上述结果表明,该重组质粒pSFV1CS-EIV-HA具有良好的免疫原性并且可以诱导免疫动物产生较高免疫应答的能力。     

Effects of DNA dose,route of vaccination,and coadministration of porcine interleukin-6 DNA on results of DNA vaccination against influenza virus infection in pigs

OBJECTIVE: To examine the effects of DNA dose, site of vaccination, and coadministration of a cytokine DNA adjuvant on efficacy of H1-subtype swine influenza virus hemagglutinin (HA) DNA vaccination of pigs. ANIMALS: 24 eight-week-old mixed-breed pigs. PROCEDURE: 2 doses of DNA were administered 27 days apart by use of a particle-mediated delivery system (gene gun). Different doses of HA DNA and different sites of DNA administration (skin, tongue) were studied, as was coadministration of porcine interleukin-6 (pIL-6) DNA as an adjuvant. Concentrations of virus-specific serum and nasal mucosal antibodies were measured throughout the experiment, and protective immunity was assessed after intranasal challenge with homologous H1N1 swine influenza virus. RESULTS: Increasing the dose of HA DNA, but not coadministration of pIL6 DNA, significantly enhanced virus-specific serum antibody responses. Pigs that received DNA on the ventral surface of the tongue stopped shedding virus 1 day sooner than pigs vaccinated in the skin of the ventral portion of the abdomen, but none of the vaccinated pigs developed detectable virus-specific antibodies in nasal secretions prior to challenge, nor were they protected from challenge exposure. Vaccinated pigs developed high virus-specific antibody concentrations after exposure to the challenge virus. CONCLUSIONS AND CLINICAL RELEVANCE: Co-administration of pIL-6 DNA did not significantly enhance immune responses to HA DNA vaccination or protection from challenge exposure. However, HA DNA vaccination of pigs, with or without coadministration of pIL-6 DNA, induced strong priming of the humoral immune system.     

Replicon particle vaccine protects swine against influenza

An alphavirus derived replicon particle (RP) vaccine expressing the cluster IV H3N2 swine influenza virus (SIV) hemagglutinin (HA) gene induced protective immunity against homologous influenza virus challenge. However, pigs with maternal antibody had no protective immunity against challenge after vaccination with RP vaccines expressing HA gene alone or in combination with nucleoprotein gene.     

Responses of cattle, sheep and poultry to a recombinant vaccinia virus expressing a swine influenza haemagglutinin

Groups of cattle, sheep and poultry were inoculated with a recombinant vaccinia virus expressing the haemagglutinin of the swine influenza virus A/NJ/11/76. No adverse clinical responses were recorded and none of the animals developed a viraemia when inoculated with the recombinant or wild-type vaccinia virus. Recombinant virus reisolated from lesions in cattle was stable, maintaining its thymidine kinase negative phenotype and ability to express the swine influenza haemagglutinin. Antibodies to the influenza haemagglutinin were detected in cattle, sheep and poultry inoculated with the recombinant virus. While no animals inoculated with wild-type virus developed these antibodies, there was no detectable spread of either recombinant or wild-type virus from the inoculation sites or to in-contact uninoculated animals. The results indicate that recombinant vaccinia viruses can induce immune responses in cattle, sheep and poultry demonstrating their potential as vaccine vectors in a variety of important veterinary species.     

Equine influenza vaccine efficacy: the significance of antigenic variation

To investigate the level of cross-protection induced by equine influenza H3N8 vaccines derived from different lineages, two studies have been carried out with ponies vaccinated with 'American-like' and 'European-like' vaccines and experimentally challenged with a European-like strain. The results demonstrated that equine influenza vaccines clearly protect against challenge with homologous virus if serum antibody titres are sufficiently high. On the other hand, protection is incomplete even when animals vaccinated with heterologous strains have comparative antibody levels. Nevertheless, the protection afforded by heterologous viruses can be improved by stimulating high levels of antibody. It would be advisable to update equine influenza vaccine strains regularly so that they contain similar strains to variants that are circulating in the field.     

表达绿色荧光蛋白的MVA重组毒株的构建及筛选

为了构建及筛选表达绿色荧光蛋白（GFP）的改良型痘苗病毒安卡拉（MVA）重组毒株,并对其进行鉴定,本研究基于同源重组原理设计引物,通过PCR扩增获取含有MVA两侧同源臂的外源基因GFP片段,将GFP基因片段转染到感染了MVA的CEF细胞中使之同源重组到MVA ORF086-087位点（基因组中70303-70304 bp之间）,利用倒置荧光显微镜观察并标记表达GFP的单个噬斑,筛选获取重组毒株,应用倒置荧光技术、PCR及Western blotting对该重组毒株进行鉴定。结果显示,经过3轮噬斑筛选,在倒置荧光显微镜下可观察到大量表达GFP的单个噬斑,PCR扩增检测结果表明目的基因已成功整合到重组毒株MVA-GFP中。Western blotting结果表明,GFP在感染的细胞内成功表达。本研究利用基因工程技术成功获得表达GFP的重组毒株MVA-GFP,可为进一步将其他抗原基因插入GFP位点中筛选无标记的重组毒株及疫苗研究提供材料。