Biomedical subjects

D Hannant

Publications and source records attributed to D Hannant.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

Vaccination against equine influenza: quid novi?

Equine influenza virus is a leading cause of respiratory disease in the horse. Equine influenza vaccines containing inactivated virus were first developed in the 1960s. Despite their intensive use, equine influenza outbreaks still continue to occur and therefore new strategies of vaccination are necessary to improve vaccine efficacy. Numerous methods of vaccination have been evaluated and commercialised in the horse, the most recent being the cold-adapted influenza virus and poxvirus-based vaccines. As a large animal model, the horse is also a useful species in which to evaluate the potential of new generations of influenza vaccine such as live-attenuated influenza virus engineered by reverse genetics. This report details the equine immune responses conferring protection against influenza. It then undertakes a selective review of different strategies of vaccination against equine influenza that have been developed over the last two decades and discusses factors that may influence the efficacy of vaccination. Finally it outlines progress in the development of a novel vaccination strategy against equine influenza using reverse genetics.

Age Factors↗

PubMed2006

A molecular approach to the identification of cytotoxic T-lymphocyte epitopes within equine herpesvirus 1.

Equine herpesvirus 1 (EHV-1) causes respiratory and neurological disease and abortion in horses. Animals with high frequencies of cytotoxic T lymphocytes (CTL) show reduced severity of respiratory disease and frequency of abortion, probably by CTL-mediated control of cell-associated viraemia. This study aimed to identify CTL epitopes restricted by selected major histocompatibility complex (MHC) class I alleles expressed in the equine leukocyte antigen (ELA) A3 haplotype. Effector CTL were induced from EHV-1-primed ponies and thoroughbreds with characterized MHC class I haplotypes and screened against P815 target cells transfected with selected EHV-1 genes and MHC class I genes. Targets that expressed EHV-1 gene 64 and the MHC B2 gene were lysed by effector CTL in a genetically restricted manner. There was no T-cell recognition of targets expressing either the MHC B2 gene and EHV-1 genes 2, 12, 14, 16, 35, 63 or 69, or the MHC C1 gene and EHV-1 genes 12, 14, 16 or 64. A vaccinia virus vector encoding gene 64 (NYVAC-64) was also investigated. Using lymphocytes from ELA-A3 horses, the recombinant NYVAC-64 virus induced effector CTL that lysed EHV-1-infected target cells; the recombinant virus also supplied a functional peptide that was expressed by target cells and recognized in an MHC-restricted fashion by CTL induced with EHV-1. This construct may therefore be used to determine the antigenicity of EHV-1 gene 64 for other MHC haplotypes. These techniques are broadly applicable to the identification of additional CTL target proteins and their presenting MHC alleles, not only for EHV-1, but for other equine viruses.

Alleles↗

PubMed2006

Extended phylogeny of equine arteritis virus: division into new subgroups.

To determine a conclusive phylogeny, equine arteritis viruses from Italy, Austria, Hungary, Sweden, South Africa and other parts of the world were analysed by reverse-transcription polymerase chain reaction amplification and direct sequencing. The nucleotide sequences corresponding to the variable part of the large glycoprotein GP5, specified by open reading frame 5, were compared and added to a previously published phylogenetic tree in which a clear division between 'European' and 'American' type viruses had been established. Adding the sequences determined in this study and new sequences retrieved from GenBank revealed additional diversity and new subgroups.

Animals↗

PubMed2005

Characterisation of CTL and IFN-gamma synthesis in ponies following vaccination with a NYVAC-based construct coding for EHV-1 immediate early gene, followed by challenge infection.

Equine herpesvirus-1 (EHV-1) is a ubiquitous pathogen of horses, which continues to cause respiratory and neurological disease and abortion, despite the widespread use of vaccines. Cell mediated immunity (CMI) is thought to play a major role in protection against infection with EHV-1. The aim of this study was to characterise the virus-specific CMI response in ponies vaccinated with vP1014, a vaccinia-based construct (NYVAC) coding for the immediate early gene (gene 64) of EHV-1. This gene product is a CTL target protein for an equine MHC class I allele expressed on the A3 haplotype. EHV-primed yearling ponies expressing this haplotype were vaccinated once (n = 1), three (n = 1), or four times (n = 2), and one pony was kept as an unvaccinated control. Cytotoxic T lymphocyte (CTL) activity and interferon gamma (IFN-gamma) synthesis were measured before and after vaccination and challenge infection with EHV-1. Multiple immunisations with vP1014 resulted in increased CTL activity and IFN-gamma synthesis specific for EHV-1 compared with unvaccinated or singly vaccinated ponies. The phenotype of EHV-1 specific T-cells synthesising IFN-gamma was also modified by immunisation. In the unvaccinated pony, the predominant population synthesising IFN-gamma after EHV-1 stimulation was CD8alpha+. In contrast, multiply vaccinated ponies demonstrated an increased proportion of CD8alpha- T-cells synthesising IFN-gamma. The results demonstrated that vaccination with a NYVAC-based construct coding for gene 64 stimulated CMI. This immune response alone did not protect against challenge infection. However, the study does illustrate that vaccinia-based vaccines can stimulate CMI in the horse and may therefore contribute to protection against disease caused by EHV-1.

Animals↗

PubMed2005

Equine interferon gamma synthesis in lymphocytes after in vivo infection and in vitro stimulation with EHV-1.

Equine cytotoxic T lymphocyte (CTL) responses to equine herpesvirus-1 (EHV-1) are well characterised but little is known about the cytokine response after infection or vaccination. EHV-1 is common in horses and infects lymphocytes in vivo. This virus was used as a model to measure the synthesis of interferon gamma (IFN-gamma) by equine peripheral blood mononuclear cells (PBMC) after in vivo infection and/or in vitro stimulation with EHV-1. Both flow cytometry and ELISPOT assays were used to quantify equine IFN-gamma using a mouse anti-bovine IFN-gamma monoclonal antibody (clone CC302; shown to cross-react with recombinant equine IFN-gamma) and a rabbit anti-canine IFN-gamma polyclonal antibody. The percentage of PBMC synthesising IFN-gamma after in vitro stimulation with EHV-1 increased with age. In yearlings infected experimentally with EHV-1, PBMC showed two peaks of IFN-gamma synthesis, 11 and 56 days after infection. The IFN-gamma synthesis was principally associated with CD8(+) cells. The patterns of IFN-gamma synthesis detected by intracellular IFN-gamma staining or ELISPOT were compared with CTL data and shown to be similar. These methods were also applied successfully to frozen samples of PBMC. Measurement of equine IFN-gamma using these simple techniques can now be applied to future studies on protective cellular immune responses following virus infection and/or vaccination of horses.

Age Factors↗

PubMed2005

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/Borlänge/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.

Animals↗

PubMed2004

Immune responses and protective efficacy in ponies immunised with an equine influenza ISCOM vaccine containing an 'American lineage' H3N8 virus.

Protective responses generated by vaccination with an immuno-stimulating complex (ISCOM)-based vaccine for equine influenza (EQUIP F), containing a new 'American lineage' H3N8 virus, were studied. Seven ponies in the vaccine group received two intramuscular injections of EQUIP F given 6 weeks apart. Aerosol challenge with an A/eq/Newmarket/1/93 reference strain 4 weeks after booster vaccination resulted in clinical signs of infection and viral shedding in 7 influenza-naive control animals whereas the vaccinated ponies were significantly protected from both clinical signs and virus excretion. Influenza virus-specific IgG responses in serum following immunisation with the ISCOM vaccine were predominantly of the IgGa and IgGb sub-isotypes, a pattern similar to that generated by equine influenza virus infection. However, in contrast to the response following infection, virus-specific antibody responses in nasal washes following immunisation were characterised by the presence of IgG but not IgA.These results demonstrated that an ISCOM-based vaccine containing A/eq/Kentucky/98 provides strong protective immunity against challenge with an 'American lineage' H3N8 reference virus.

Animals↗

PubMed2003

Pre-infection frequencies of equine herpesvirus-1 specific, cytotoxic T lymphocytes correlate with protection against abortion following experimental infection of pregnant mares.

In general, vaccines containing inactivated equine herpesvirus-1 (EHV-1) fail to prevent abortion in pregnant mares following infection with a virulent strain of EHV-1. We have tested the hypothesis that resistance to EHV-1-induced abortion in pregnant mares is associated with high frequencies of EHV-1 specific, major histocompatibility complex (MHC) class I-restricted, cytotoxic T lymphocytes (CTL) in the circulation. To test this theory, three groups of pregnant mares were assembled with varying backgrounds of infection or vaccination in an attempt to mimic the immune status of the general population. Group 1 mares (n=9) were untreated controls selected at random. Group 2 mares (n=5) were vaccinated three times intramuscularly with inactivated EHV-1. Group 3 mares (n=3) had been infected with EHV-1 on four previous occasions. The frequency of CTL in blood leucocytes was measured by limiting dilution analysis at three time points; at the beginning of pregnancy (approximately 28 weeks before infection) in the Group 2 and Group 3 mares (4-7 weeks of gestation) (Group 1 was unavailable for sampling) and then 2 weeks before (30-40 weeks of gestation) and 3 weeks after experimental infection in all the mares. Serum samples were collected to monitor complement fixing (CF) antibody titres. Mares in all three groups were infected experimentally with EHV-1 strain Ab4/8 by the intranasal route after which they were monitored clinically to determine the outcome of pregnancy and samples were collected to determine the duration of nasopharyngeal shedding and cell-associated viraemia. The untreated control mares showed low pre-infection CTL. After experimental infection, they all seroconverted, aborted and demonstrated expected clinical and virological signs. Some vaccinated mares (3/5) had elevated titres of CF antibody prior to their first vaccination. All the vaccinated mares seroconverted after vaccination and exhibited higher CTL frequencies than controls before infection. Four of the five foaled normally. The multiply infected mares had low CF antibody titres prior to infection and showed neither seroconversion nor clinical or virological signs after infection. All multiply infected mares exhibited high frequencies of CTL before infection and they all foaled normally. The CTL frequencies observed differed significantly from the expected frequencies in the control and multiply infected groups at 2 weeks pre-infection (P=0.034) and between the foaling and aborting mares at 2 weeks pre-infection (P=0.005) and 3 weeks post-infection (P=0.015). The results show a positive correlation between the number of virus-specific CTL in the peripheral blood of pregnant mares and their protection against abortion induced by EHV-1 infection. Therefore, as indicated by this study, rational approaches to the development of new vaccines for EHV-1 should stimulate cytotoxic immune responses and develop virus-specific CTL as pre-requisites for protection against abortion.

Abortion, Veterinary↗

PubMed2003

In vitro characterisation of high and low virulence isolates of equine herpesvirus-1 and -4.

Basic in vitro characteristics of high and low virulence isolates of equine herpesviruses-1 and -4 were investigated with particular reference made to the Ab4 and V592 isolates of EHV-1 as both have distinct endotheliotropism and clinical outcomes in pony challenge studies. Additionally, some EHV-4 isolates that showed variations in clinical outcome were included in some experiments. The aim of the study was to identify an in vitro characteristic that would differentiate strains of known virulence. Such a system could then be applied to vaccine and virulence studies as an effective screening tool. Viral growth kinetics in a variety of cell culture systems, plaque size, ability to replicate in fetal endothelium in organ culture, and sensitivity to acyclovir were compared. No reliable marker system that differentiated between higher and lower virulence isolates of EHV-1 and EHV-4 was identified.

Animals↗

PubMed2003

Detection of equine arteritis virus (EAV)-specific cytotoxic CD8+ T lymphocyte precursors from EAV-infected ponies.

Equine arteritis virus (EAV) causes a systemic infection in equids with variable outcome, ranging from subclinical infections to severe disease, and also has the capacity to induce abortion in pregnant mares and persistent infections in stallions. The serum virus-neutralizing antibody response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little is known about cellular immunity against EAV because of a lack of methods for evaluating these immune responses. In the present study, we describe methods for detecting cytotoxic T lymphocyte (CTL) precursors in the peripheral blood of EAV-convalescent ponies using a (51)Cr release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC I but not MHC II and to retain (51)Cr efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV-convalescent ponies that had been incubated with or without live EAV were used as effectors. EAV-induced PBMC cultures showed evidence of expansion and activation of lymphoblasts, with an increase in the CD8(+)/CD4(+) ratio in comparison with mock-induced PBMC. The cytotoxicity induced by EAV-stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8(+) T lymphocytes and could be detected for periods of 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to an understanding of virus-host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection.

Animals↗

PubMed2002

The effect of aging on T cell responses in the horse.

Horses greater than 20 years of age exhibit alterations in their immune responses similar to those observed in other aged individuals. The purpose of this study was to characterize immunosenescence in a population of aged ponies. The peripheral blood mononuclear cells (PBMC) from aged ponies exhibited a decreased proliferative response to various mitogens that was not overcome by the addition of interleukin 2 (IL-2) to the cultures. No difference in overall expression of the IL-2 receptor was seen between young and aged ponies, though CD8(+) cells from aged ponies exhibited increased levels of IL-2 receptor expression. The kinetics of the response to both mitogen and IL-2 did not appear to be affected in the aged PBMCs. These results indicate that the age-related decrease in the proliferative response to mitogens is not due to a failure to produce or respond to IL-2 but probably involves some other process.

Aging↗

PubMed2001

Clinical and virological evaluation of the efficacy of an inactivated EHV1 and EHV4 whole virus vaccine (Duvaxyn EHV1,4). Vaccination/challenge experiments in foals and pregnant mares.

Pregnant mares and young foals were vaccinated with Duvaxyn EHV1,4, an inactivated and adjuvanted vaccine containing both the EHV-1 and 4 antigens. SN and CF antibody titres were induced two weeks after first vaccination. Antibody levels were boosted after second vaccination, however they never reached the levels induced after virus challenge. Young foals were challenged with virulent EHV-1 and EHV-4 field viruses. Pregnant mares were challenged with the highly abortigenic EHV-1 strain Ab4. Vaccinated animals showed a clear reduction in clinical signs and virus excretion compared to unvaccinated control animals. Log transformed antibody levels could be correlated to duration of virus excretion. The incidence of EHV-1 induced abortions was drastically reduced in vaccinated mares. Therefore, although vaccinated animals are not fully protected against disease, Duvaxyn EHV1,4 clearly reduces clinical symptoms, the duration of virus shedding and the quantity of virus shed. It can be concluded that vaccination of foals and pregnant mares with Duvaxyn EHV1,4 significantly reduces the risk of abortions and outbreaks of respiratory disease caused by circulating field viruses.

Abortion, Veterinary↗

PubMed2001

Mercury contamination incident.

BACKGROUND: The aim of this paper is to describe an incident where elemental mercury led to widespread contamination and the exposure of 225 individuals and confirmed toxicity in 19 individuals. The paper describes the incident and difficulties found in trying to assess the risk to individuals and to identify and decontaminate the residences involved. METHODS: All individuals exposed to elemental mercury in the incident were followed up for 15 months. RESULTS: Thirty-seven individuals were found to be 'at risk' and 13 were symptomatic of mercury poisoning. Five patients required chelation therapy. The incident was closed when the risk of poisoning and re-exposure was minimized. CONCLUSION: Incident management depends on early effective communication and collaboration between all agencies involved.

Adolescent↗

PubMed2001

Evaluation of a prototype sub-unit vaccine against equine arteritis virus comprising the entire ectodomain of the virus large envelope glycoprotein (G(L)): induction of virus-neutralizing antibody and assessment of protection in ponies.

An Escherichia coli-expressed recombinant protein (6hisG(L)ecto) comprising the entire ectodomain (aa 18-122) of equine arteritis virus (EAV) glycoprotein G(L), the immunodominant viral antigen, induced higher neutralizing antibody titres than other G(L)-derived polypeptides when compared in an immunization study in ponies. The potential of the recombinant G(L) ectodomain to act as a sub-unit vaccine against EAV was evaluated further in three groups of four ponies vaccinated with doses of 35, 70 or 140 microg of protein. All vaccinated animals developed a virus-neutralizing antibody (VNAb) response with peak titres 1-2 weeks after the administration of a booster on week 5 (VNAb titres of 1.8-3.1), 13 (VNAb titres of 1.4-2.9) or 53 (VNAb titres of 1.2-2.3). Vaccinated and unvaccinated control ponies were infected with EAV at different times post-vaccination to obtain information about the degree of protection relative to the levels of pre-challenge VNAb. Vaccination conferred varying levels of protection, as indicated by reduced or absent pyrexia, viraemia and virus excretion from the nasopharynx. The degree of protection correlated well with the levels of pre-challenge VNAb and, in particular, with levels of virus excretion. These results provide the first evidence that a sub-unit vaccine protects horses against EAV. The use of the sub-unit vaccine in combination with a differential diagnostic test based on other EAV antigens would enable serological discrimination between naturally infected and vaccinated equines.

Amino Acid Sequence↗

PubMed2000

Utilisation of bacteriophage display libraries to identify peptide sequences recognised by equine herpesvirus type 1 specific equine sera.

Three filamentous phage random peptide display libraries were used in biopanning experiments with purified IgG from the serum of a gnotobiotic foal infected with equine herpesvirus-1 (EHV-1) to enrich for epitopes binding to anti-EHV-1 antibodies. The sequences of the amino acids displayed were aligned with protein sequences of EHV-1, thereby identifying a number of potential antibody binding regions. Presumptive epitopes were identified within the proteins encoded by genes 7 (DNA helicase/primase complex protein), 11 (tegument protein), 16 (glycoprotein C), 41 (integral membrane protein), 70 (glycoprotein G), 71 (envelope glycoprotein gp300), and 74 (glycoprotein E). Two groups of sequences, which aligned with either glycoprotein C (gC) or glycoprotein E (gE), identified type-specific epitopes which could be used to distinguish between sera from horses infected with either EHV-1 or EHV-4 in an ELISA using either the phage displaying the peptide or synthetic peptides as antigen. The gC epitope had been previously identified as an immunogenic region by conventional monoclonal antibody screening whereas the gE antibody binding region had not been previously identified. This demonstrates that screening of phage display peptide libraries with post-infection polyclonal sera is a suitable method for identifying diagnostic antigens for viral infections such as EHV-1.

Amino Acid Sequence↗

PubMed2000

Virulence of the V592 isolate of equid herpesvirus-1 in ponies.

The V592 strain of equid herpesvirus-1 (EHV-1), which was originally isolated from a fetus during an abortion epizootic, has proved to be of low virulence in infection studies. Five Welsh Mountain pony mares and one foal were challenged intranasally or by aerosol with this isolate, and monitored clinically and virologically. All six animals shed virus in nasopharyngeal mucus, and viraemia was recorded from day 7 post-infection (PI). Pathological investigations revealed mild rhinitis and bronchiolitis in the mares, with viral antigen expression in degenerating epithelial cells of the nasal mucosa and bronchioles, and in occasional monocytes in the respiratory tract-associated lymph nodes. Viral antigen expression was not detected in vascular endothelium of the mares, although vasculitis was seen to have affected small numbers of blood vessels in the dorsocaudal lung regions of a mare examined on day 10 PI. In the foal, respiratory lesions of a more localized nature included infection of vascular endothelium and associated vasculitis. The foal also had localized encephalitis affecting the olfactory lobes of the brain, with viral antigen expression in degenerating olfactory neurons and microglia. The data suggest that the relatively low virulence of strain V592 is associated with a lower degree of endotheliotropism than that shown by the highly virulent Ab4 and Army 183 isolates, and that this property is influenced by host immunity.

Animals↗

PubMed2000

Immunity to equine influenza: relationship of vaccine-induced antibody in young Thoroughbred racehorses to protection against field infection with influenza A/equine-2 viruses (H3N8).

Field outbreaks of influenza that occurred in vaccinated Thoroughbred racehorses in Newmarket in 1995 and 1996 were investigated by nucleoprotein ELISA and serology. Investigations showed that serum levels of vaccine-induced single radial haemolysis (SRH) antibody correlated closely with protective immunity against equine influenza and were consistent with observations made in previous experimental studies using nebulised aerosol challenge. In the second part of this study, antibody levels stimulated by vaccination were investigated to examine probable protection in high risk groups, such as yearlings and horses in training. Results for yearlings correlated closely with experimentally derived antibody profiles described for several equine influenza vaccines. The horses in training had levels of antibody immediately prior to revaccination, which were higher than those measured in the yearlings. In conclusion, SRH antibody, used in the investigation of outbreaks and surveillance of post vaccination responses, was shown to correlate with and validate experimental vaccination and challenge models currently used in ponies in the licensing of modern vaccines. There may be benefit from serological monitoring of horses following vaccination through identification of susceptible periods to infection and demonstration of poor vaccine responders. This would allow appropriate and timely amendment of vaccination strategies to maximise protective immunity against influenza.

Animals↗