A vectored equine herpesvirus type 1 (EHV-1) vaccine elicits protective immune responses against EHV-1 and H3N8 equine influenza virus

Vaccination is commonly used to control equine respiratory pathogens such as equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV). Here, we describe the generation and characterization of a recombinant EHV-1 modified live virus vaccine (MLV) based on a recent abortogenic EHV-1 strain, NY03. The immunogenicity and efficacy of the MLV was tested in horses in an EHV-1 vaccination/challenge experiment using the highly virulent neurovirulent EHV-1 strain OH03. Induction of a robust EHV-1-specific immune response was observed. Upon challenge infection, vaccinated horses were partially protected against disease as demonstrated by a significant reduction in clinical signs, nasal shedding and viremia levels. In addition, the NY03-based MLV was used to express the EIV H3 protein and immunogenicity was tested in horses. Expression of H3 was readily detected in NY03-H3-infected cells in vitro. Vaccination of horses resulted in the induction of a robust serological immune responses against two recent but genetically distinct EIV representatives, VA05 and NY-99, which were above the threshold predicted to be protective against development of clinical disease.     

Virus-like particle vaccine protects against H3N2 canine influenza virus in dog

In the present study, virus-like particles (VLPs) were evaluated as a candidate veterinary vaccine against canine influenza virus (CIV) subtype H3N2. Specific pathogen-free (SPF) beagle dogs received a single injection of a VLP vaccine containing hemagglutinin (HA) and M1 protein of CIV H3N2 (H3 HA VLP). The vaccine was tested at 3 different doses with an adjuvant and 1 dose without an adjuvant. To evaluate the immunogenicity and protective efficacy of the H3 HA VLP vaccine, we performed hemagglutination inhibition tests to determine serological immune responses and conducted challenge studies using SPF beagle dogs. The addition of Montanide ISA 25 adjuvant significantly increased the immunogenicity of the H3 HA VLP vaccine. The experimental infection study showed that a single dose of H3 HA VLP vaccine induced protection against wild-type virus challenge in dogs. These results provide support for continued development of the VLP as an animal vaccine against influenza virus.     

Avian-origin H3N2 canine influenza virus circulating in farmed dogs in Guangdong,China

Since 2006, more and more cases of the infectious H3N2 canine influenza virus (CIV) in pet dogs have been reported in southern China. However, little is known about the prevalence situation of H3N2 CIV infections in farmed dogs in China. This is the first systematic epidemiological surveillance of CIV in different dog populations in southern China. Two virus strains A/Canine/Guangdong/1/2011(H3N2) and A/canine/Guangdong/5/2011(H3N2) were isolated from canine nasal swabs collected at one dog farm in Guangzhou and the other farm in Shenzhen. Sequence and phylogenetic analysis of eight gene segments of these viruses revealed that they were most similar to the newly isolated canine H3N2 viruses in dogs and cats from Korea and China, which originated from avian strain. This indicates that H3N2 CIV may be a common pathogen for pet and farmed dog populations in southern China at present. Serological surveillance has shown that the infection rate of this avian-origin canine influenza in farmed dogs and in pet dogs were 12.22% and 5.3%, respectively; as determined by the ELISA. The data also suggested that transmission occurred, most probably by close contact, between H3N2 CIV infected dogs in different dog populations in recently years. As H3N2 outbreaks among dogs continue in the Guangdong province (located very close to Hong Kong), the areas where is densely populated and with frequent animal trade, there is a continued risk for pets H3N2 CIV infections and for mutations or genetic reassortment leading to new virus strains with increased transmissibility among dogs. Further in-depth study is required as the H3N2 CIV has been established in different dog populations and posed potential threat to public health.     

Avian-origin H3N2 canine influenza virus circulating in farmed dogs in Guangdong,China

Since 2006, more and more cases of the infectious H3N2 canine influenza virus (CIV) in pet dogs have been reported in Southern China. However, little is known about the prevalence situation of H3N2 CIV infections in farmed dogs in China. This is the first systematic epidemiological surveillance of CIV in different dog populations in Southern China. Two virus strains A/Canine/Guangdong/1/2011(H3N2) and A/canine/Guangdong/5/2011(H3N2) were isolated from canine nasal swabs collected at one dog farm in Guangzhou and the other farm in Shenzhen. Sequence and phylogenetic analysis of eight gene segments of these viruses revealed that they were most similar to the newly isolated canine H3N2 viruses in dogs and cats from Korea and China, which originated from avian strain. This indicates that H3N2 CIV may be a common pathogen for pet and farmed dog populations in Southern China at present. Serological surveillance has shown that the infection rate of this avian-origin canine influenza in farmed dogs and in pet dogs were 12.22% and 5.3%, respectively; as determined by the ELISA. The data also suggested that transmission occurred, most probably by close contact, between H3N2 CIV infected dogs in different dog populations in recently years. As H3N2 outbreaks among dogs continue in the Guangdong Province (located very close to Hong Kong), the areas where is densely populated and with frequent animal trade, there is a continued risk for pet H3N2 CIV infections and for mutations or genetic reassortment leading to new virus strains with increased transmissibility among dogs. Further in-depth study is required as the H3N2 CIV has been established in different dog populations and posed potential threat to public health.     

A universal epitope-based influenza vaccine and its efficacy against H5N1

Previous studies have shown that a recombinant vaccine expressing four highly conserved influenza virus epitopes has a potential for a broad spectrum, cross-reactive vaccine; it induced protection against H1, H2 and H3 influenza strains. Here, we report on the evaluation of an epitope-based vaccine in which six conserved epitopes, common to many influenza virus strains are expressed within a recombinant flagellin that serves as both a carrier and adjuvant. In an HLA-A2.1 transgenic mice model, this vaccine induced both humoral and cellular responses and conferred some protection against lethal challenge with the highly pathogenic H5N1 avian influenza strain. Hence, it is expected to protect against future strains as well. The data presented, demonstrate the feasibility of using an array of peptides for vaccination, which might pave the way to an advantageous universal influenza virus vaccine that does not require frequent updates and/or annual immunizations.     

Use of apathogenic vaccinia virus MVA expressing EHV-1 gC as basis of a combined recombinant MVA/DNA vaccination scheme

The nonreplicating chicken adapted vaccinia virus strain MVA was used in a combined vaccine scheme. Using the equine herpesvirus type 1 (EHV-1) encoded complement-receptor glycoprotein C as antigen, only poor antibody response was induced by exclusive vaccination with DNA plasmids. The administration of recombinant MVA followed by plasmid immunization elicited both humoral and cellular immune responses in hamster comparable to EHV-1 full virus vaccines.Our results indicate that recombinant constructs based on MVA represent a safe and efficient way to overcome problems of poor immunogenicity of certain antigens upon intramuscular DNA vaccination, thus replacing sophisticated adjuvants or application methods, which are not readily applicable in routine practice.     

Transmission of avian influenza virus (H3N2) to dogs

In South Korea, where avian influenza virus subtypes H3N2, H5N1, H6N1, and H9N2 circulate or have been detected, 3 genetically similar canine influenza virus (H3N2) strains of avian origin (A/canine/Korea/01/2007, A/canine/Korea/02/2007, and A/canine/Korea/03/2007) were isolated from dogs exhibiting severe respiratory disease. To determine whether the novel canine influenza virus of avian origin was transmitted among dogs, we experimentally infected beagles with this influenza virus (H3N2) isolate. The beagles shed virus through nasal excretion, seroconverted, and became ill with severe necrotizing tracheobronchitis and bronchioalveolitis with accompanying clinical signs (e.g., high fever). Consistent with histologic observation of lung lesions, large amounts of avian influenza virus binding receptor (SAalpha 2,3-gal) were identified in canine tracheal, bronchial, and bronchiolar epithelial cells, which suggests potential for direct transmission of avian influenza virus (H3N2) from poultry to dogs. Our data provide evidence that dogs may play a role in interspecies transmission and spread of influenza virus.     

Comparison of the efficacy of inactivated combination and modified-live virus vaccines against challenge infection with neuropathogenic equine herpesvirus type 1 (EHV-1)

Equine herpesvirus type 1 (EHV-1) is a ubiquitous alphaherpesvirus of horses which causes rhinopneumonitis, abortion and myeloencephalopathy. To test the efficacy of commercial vaccines in protection against neurological EHV-1 challenge, groups of five horses were immunized with modified-live virus or an inactivated vaccine, or received placebo. Horses were challenged by aerosol with a recent virus isolate obtained from a case of paralytic EHV-1. The duration of fever decreased significantly in the modified-live virus vaccine group. Three animals in each of the inactivate and control groups showed alterations in neurological status. When compared to the inactivated vaccine, the modified-live virus vaccine induced significantly lower virus-neutralizing antibodies over the course of the study. The modified-live virus vaccine resulted in low EHV-1-specific IgG(T)/IgGa and IgG(T)/IgGb ratios, suggesting a bias towards a cytotoxic immune response. Virus shedding from the nasopharynx was almost undetectable in the modified-live virus group, and was significantly lower when compared to that in the other groups. Normalized lymphocyte viral genome copies were similar for the three groups, although animals vaccinated with the modified-live virus vaccine were qPCR-positive on fewer days when compared to those of the other groups. Based on data from neurological signs, rectal temperatures, virus isolation from nasal swabs and immune response specificity, we concluded that protection induced by the modified-live virus vaccine is superior to that induced by the inactivated combination vaccine.     

New assays to measure equine influenza virus-specific Type 1 immunity in horses

Equine influenza virus (EIV) is a leading cause of respiratory disease in horses. Equine influenza infection induces a long-term immunity to re-infection. Recent strategies of vaccination aim to mimic this immunity by stimulating both antibody and cellular immune responses. Cell-mediated immunity (CMI) to influenza is well defined in man, but little has been done to characterise the responses in the horse. Additionally, the development of reliable assays for the measurement of equine CMI has lagged behind serological methods and vaccine development. In this study, two methods of measuring EIV-specific T lymphocyte responses have been developed. An EIV 'bulk' cytotoxic T lymphocytes (CTL) assay using equine dermal fibroblasts as target cells has been adapted from a method used in the 1980s. This method was also complemented with a new EIV-specific IFNgamma synthesis assay. When compared with the measurement of EIV-specific IFNgamma synthesis previously described, this method required the amplification of EIV-specific lymphocytes by culture and was sensitive enough to detect stimulation of EIV-specific T lymphocytes induced by experimental infection with EIV or vaccination with recombinant canarypox viruses coding for EIV-HA molecules. This study provides the tools to characterise the stimulation of CMI by the new generation of vaccines against equine influenza.     

Vaccination with viral vectors expressing NP,M1 and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice

Seasonal influenza virus infections cause significant morbidity and mortality every year. Annual influenza virus vaccines are effective but only when well matched with circulating strains. Therefore, there is an urgent need for better vaccines that induce broad protection against drifted seasonal and emerging pandemic influenza viruses. One approach to design such vaccines is based on targeting conserved regions of the influenza virus hemagglutinin. Sequential vaccination with chimeric hemagglutinin constructs can refocus antibody responses towards the conserved immunosubdominant stalk domain of the hemagglutinin, rather than the variable immunodominant head. A complementary approach for a universal influenza A virus vaccine is to induce T-cell responses to conserved internal influenza virus antigens. For this purpose, replication deficient recombinant viral vectors based on Chimpanzee Adenovirus Oxford 1 and Modified Vaccinia Ankara virus are used to express the viral nucleoprotein and the matrix protein 1. In this study, we combined these two strategies and evaluated the efficacy of viral vectors expressing both chimeric hemagglutinin and nucleoprotein plus matrix protein 1 in a mouse model against challenge with group 2 influenza viruses including H3N2, H7N9 and H10N8. We found that vectored vaccines expressing both sets of antigens provided enhanced protection against H3N2 virus challenge when compared to vaccination with viral vectors expressing only one set of antigens. Vaccine induced antibody responses against divergent group 2 hemagglutinins, nucleoprotein and matrix protein 1 as well as robust T-cell responses to the nucleoprotein and matrix protein 1 were detected. Of note, it was observed that while antibodies to the H3 stalk were already boosted to high levels after two vaccinations with chimeric hemagglutinins (cHAs), three exposures were required to induce strong reactivity across subtypes. Overall, these results show that a combinations of different universal influenza virus vaccine strategies can induce broad antibody and T-cell responses and can provide increased protection against influenza.     

Prime–boost vaccination with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in Pekin ducks challenged with Asian H5N1 HPAI

The efficacy of different vaccination schedules was evaluated in 17-day-old Pekin ducks using an experimental inactivated whole virus vaccine based on the H5N9 A/chicken/Italy/22A/98 isolate (H5N9-It) and/or a fowlpox recombinant (vFP-H5) expressing a synthetic HA gene from an Asian H5N1 isolate (A/chicken/Indonesia/7/2003). Full protection against clinical signs and shedding was induced by the different vaccination schemes. However, the broadest antibody response and the lowest antibody increase after challenge were observed in the group of ducks whose immune system was primed with the fowlpox vectored vaccine and boosted with the inactivated vaccine, suggesting that this prime-boost strategy induced optimal immunity against H5N1 and minimal viral replication after challenge in ducks. In addition, this prime-boost vaccination scheme was shown to be immunogenic in 1-day-old ducklings.     

Genetic and subunit vaccines based on the stem domain of the equine influenza hemagglutinin provide homosubtypic protection against heterologous strains

H3N8 influenza virus strains have been associated with infectious disease in equine populations throughout the world. Although current vaccines for equine influenza stimulate a protective humoral immune response against the surface glycoproteins, disease in vaccinated horses has been frequently reported, probably due to poor induction of cross-reactive antibodies against non-matching strains. This work describes the performance of a recombinant protein vaccine expressed in prokaryotic cells (ΔHAp) and of a genetic vaccine (ΔHAe), both based on the conserved stem region of influenza hemagglutinin (HA) derived from A/equine/Argentina/1/93 (H3N8) virus.Sera from mice inoculated with these immunogens in different combinations and regimes presented reactivity in vitro against highly divergent influenza virus strains belonging to phylogenetic groups 1 and 2 (H1 and H3 subtypes, respectively), and conferred robust protection against a lethal challenge with both the homologous equine strain (100%) and the homosubtypic human strain A/Victoria/3/75 (H3N2) (70–100%). Animals vaccinated with the same antigens but challenged with the human strain A/PR/8/34 (H1N1), belonging to the phylogenetic group 1, were not protected (0–33%). Combination of protein and DNA immunogens showed higher reactivity to non-homologous strains than protein alone, although all vaccines were permissive for lung infection.     

The first safe inactivated equine influenza vaccine formulation adjuvanted with ISCOM-Matrix that closes the immunity gap

Equine influenza is a contagious diseases caused by equine influenza viruses which belong to the orthomyxovirus family. Outbreaks of equine influenza cause severe economic loses to the horse industry and consequently competition horses are required to be regularly vaccinated against equine influenza. Currently available inactivated vaccines are only able to induce protection against clinical disease and virus excretion after a primary vaccination course consisting of three vaccine applications at 4–6 and 22–26 weeks apart, respectively. It has been suggested that these vaccines induce no adequate protection in horses at 22–26 weeks (5 months) in the primary vaccination course (immediately prior to the last booster), despite various alternative vaccination regimens proposed. In this paper we describe the efficacy and safety profile, tested in an experimental setting according to European legislation of a novel inactivated equine influenza vaccine formulation (Prequenza). This formulation consists besides influenza antigen, of second generation ISCOM-Matrix as an adjuvant. The vaccine aims at the induction of protection from the onset of immunity, i.e. after the first two vaccine applications, until the first booster given 5 months later, against challenge with a virulent equine influenza strain. The protection against A/equine/Kentucky/95 (H3N8) was evidenced by a reduction of clinical signs of influenza, a reduction of virus excretion and a reduction of fever. The vaccine was shown to be safe in pregnant mares, foals and is used safely since 2 years as a commercial vaccine in Europe.     

The immune response of a recombinant fowlpox virus coexpressing the HA gene of the H5N1 highly pathogenic avian influenza virus and chicken interleukin 6 gene in ducks

Ducks have played an important role in the emergence of H5N1 subtype of highly pathogenic avian influenza (HPAI), and the development of an effective vaccine against HPAI in ducks is a top priority. It has been shown that a recombinant fowlpox virus (FPV)-vectored vaccine can provide protection against HPAI in ducks. In this study, a recombinant fowlpox virus (rFPV-AIH5AIL6) coexpressing the haemagglutinin (HA) gene of the H5N1 subtype of the avian influenza virus (AIV) and chicken interleukin 6 gene was constructed and tested in Gaoyou and cherry valley ducks to evaluate the immune response in ducks. These animal studies demonstrated that rFPV-AIH5AIL6 induced a higher anti-AIV HI antibody response, an enhanced lymphocyte proliferation response, an elevated immune protection, and a reduction in virus shedding compared to a recombinant fowlpox virus expressing the HA gene alone (rFPV-SYHA). These data indicate that rFPV-AIH5AIL6 may be a potential vaccine against the H5 subtype of avian influenza in ducks and chicken interleukin 6 may be an effective adjuvant for increasing the immunogenicity of FPV-vectored AIV vaccines in ducks.     

Canine H3N8 influenza vaccines partially protect mice against the canine H3N2 strain currently circulating in the United States

Influenza A viruses infect many species and cross-species transmission occurs occasionally. An equine H3N8 influenza virus began circulating in dogs in 1999 and an avian H3N2 influenza virus began circulating in dogs in 2006. The canine H3N8 (cH3N8) viral strain has become endemic in parts of the United States and there is a commercially available vaccine against this strain. The canine H3N2 (cH3N2) strain did not circulate widely in the United States until 2015. Here, we used a mouse model to determine if the cH3N8 and cH3N2 strains are antigenically related and if a commercially available cH3N8 vaccine protects animals against the cH3N2 outbreak strain. We find that the cH3N8 vaccine elicits antibodies that react to internal viral proteins and the hemagglutinin stalk region of cH3N2 viruses. These antibodies do not provide sterilizing immunity against cH3N2 infection, but these antibodies limit cH3N2 replication in the lung.     

Influenza A virus (H3N8) in dogs with respiratory disease, Florida

In 2004, canine influenza virus subtype H3N8 emerged in greyhounds in the United States. Subsequent serologic evidence indicated virus circulation in dog breeds other than greyhounds, but the virus had not been isolated from affected animals. In 2005, we conducted virologic investigation of 7 nongreyhound dogs that died from respiratory disease in Florida and isolated influenza subtype H3N8 virus. Antigenic and genetic analysis of A/canine/Jacksonville/2005 (H3N8) and A/canine/Miami/2005 (H3N8) found similarity to earlier isolates from greyhounds, which indicates that canine influenza viruses are not restricted to greyhounds. The hemagglutinin contained 5 conserved amino acid differences that distinguish canine from equine lineages. The antigenic homogeneity of the canine viruses suggests that measurable antigenic drift has not yet occurred. Continued surveillance and antigenic analyses should monitor possible emergence of antigenic variants of canine influenza virus.     

Protective efficacy of an inactivated Eurasian avian-like H1N1 swine influenza vaccine against homologous H1N1 and heterologous H1N1 and H1N2 viruses in mice

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses are prevalent in pigs in Europe and Asia, but occasionally cause human infection, which raises concern about their pandemic potential. Here, we produced a whole-virus inactivated vaccine with an EA H1N1 strain (A/swine/Guangxi/18/2011, SW/GX/18/11) and evaluated its efficacy against homologous H1N1 and heterologous H1N1 and H1N2 influenza viruses in mice. A strong humoral immune response, which we measured by hemagglutination inhibition (HI) and virus neutralization (VN), was induced in the vaccine-inoculated mice upon challenge. The inactivated SW/GX/18/11 vaccine provided complete protection against challenge with homologous SW/GX/18/11 virus in mice and provided effective protection against challenge with heterologous H1N1 and H1N2 viruses with distinctive genomic combinations. Our findings suggest that this EA H1N1 vaccine can provide protection against both homologous H1N1 and heterologous H1N1 or H1N2 virus infection. As such, it is an excellent vaccine candidate to prevent H1N1 swine influenza.     

Duration of immunity induced by an equine influenza and tetanus combination vaccine formulation adjuvanted with ISCOM-Matrix

Equine influenza is a contagious disease caused by equine influenza virus which belongs to the orthomyxovirus family. Outbreaks of equine influenza cause severe economic loses to the horse industry and consequently horses in competition are required to be regularly vaccinated against equine influenza. Unlike the existing inactivated vaccines, Equilis Prequenza Te is the only one able to induce protection against clinical disease and virus excretion after a primary vaccination course consisting of two vaccine applications 4–6 weeks apart until the recommended time of the third vaccination. In this paper we describe the duration of immunity profile, tested in an experimental setting according to European legislation, of this inactivated equine influenza and tetanus combination vaccine. In addition to influenza antigen, the formulation contains a second generation ISCOM (the so called ISCOMatrix) as an adjuvant. The vaccine aims at the induction of protection from the primary vaccination course until the time of annual revaccination 12 months later, against challenge with a virulent equine influenza strain. The protection against A/equine/Kentucky/95 (H3N8) at the time of annual revaccination was evidenced by a significant reduction of clinical signs of influenza, a significant reduction of virus excretion and a significant reduction of fever. The effect of the annual revaccination on the duration of immunity against influenza and tetanus was also studied by serology. For tetanus, as a consequence of the 24 months duration of immunity, an alternating annual vaccination schedule consisting of Prequenza and Prequenza Te is proposed after the first three doses of Prequenza Te.     

Immunoinformatic comparison of T-cell epitopes contained in novel swine-origin influenza A (H1N1) virus with epitopes in 2008–2009 conventional influenza vaccine

In March 2009 a novel swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the Western United States. Vaccination with conventional influenza vaccine (CIV) does not result in cross-reactive antibodies, however, the disproportionate number of cases (37%) occurring among persons younger than 50 years old suggested that adaptive immune memory might be responsible for the relative lack of virulence in older, healthy adults. Using EpiMatrix, a T-cell epitope prediction and comparison tool, we compared the sequences of the three hemagglutinin (HA) and neuraminidase (NA) proteins contained in 2008–2009 CIV to their counterparts in A/California/04/2009 (H1N1) looking for cross-conserved T-cell epitope sequences. We found greater than 50% conservation of T helper and CTL epitopes between novel S-OIV and CIV HA for selected HLA. Conservation was lower among NA epitopes. Sixteen promiscuous helper T-cell epitopes are contained in the S-OIV H1N1 HA sequence, of which nine (56%) were 100% conserved in the 2008–2009 influenza vaccine strain; 81% were either identical or had one conservative amino acid substitution. Fifty percent of predicted CTL epitopes found in S-OIV H1N1 HA were also found in CIV HA sequences. Based on historical performance, we expect these epitope predictions to be 93–99% accurate. This in silico analysis supports the proposition that T-cell response to cross-reactive T-cell epitopes, due to vaccination or exposure, may have the capacity to attenuate the course of S-OIV H1N1 induced disease—in the absence of cross-reactive antibody response. The value of the CIV or live-attenuated influenza vaccine containing the 2008–2009 vaccine strains, as defense against H1N1, could be further tested by evaluating human immune responses to the conserved T-cell epitopes using PBMC from individuals infected with H1N1 and from CIV vaccinees.