Single Radial Haemolysis Antibody Research Articles

Impact of Mixed Equine Influenza Vaccination on Correlate of Protection in Horses.

To evaluate the humoral immune response to mixed Equine Influenza vaccination, a common practice in the field, an experimental study was carried out on 42 unvaccinated thoroughbred weanling foals divided into six groups of seven. Three groups were vaccinated using a non-mixed protocol (Equilis® Prequenza-Te, Proteqflu-Te® or Calvenza-03®) and three other groups were vaccinated using a mix of the three vaccines mentioned previously. Each weanling underwent a primary EI vaccination schedule composed of two primary immunisations (V1 and V2) four weeks apart followed by a third boost immunisation (V3) six months later. Antibody responses were monitored until one-year post-V3 by single radial haemolysis (SRH). The results showed similar antibody responses for all groups using mixed EI vaccination and the group exclusively vaccinated with Equilis® Prequenza-TE, which were significantly higher than the other two groups vaccinated with Proteqflu-TE® and Calvenza-03®. All weanlings (100%) failed to seroconvert after V1 and 21% (9/42) still had low or no SRH antibody titres two weeks post-V2. All weanlings had seroconverted and exceeded the clinical protection threshold one month after V3. The poor response to vaccination was primarily observed in groups exclusively vaccinated with Proteqflu-Te® and Calvenza-03®. A large window of susceptibility (3–4.5-month duration) usually called immunity gap was observed after V2 and prior to V3 for all groups. The SRH antibody level was maintained above the clinical protection threshold for three months post-V3 for the groups exclusively vaccinated with Proteqflu-Te® and Calvenza-03®, and six months to one year for groups using mixed EI vaccination or exclusively vaccinated with Equilis® Prequenza-Te. This study demonstrates for the first time that the mix of EI vaccines during the primary vaccination schedule has no detrimental impact on the correlate of protection against EIV infection.

The Immunity Gap Challenge: Protection against a Recent Florida Clade 2 Equine Influenza Strain.

Vaccination is one of the most effective tools for limiting the impact of equine influenza (EI). The humoral immunity established following a primary vaccination course can decrease significantly between the second (V2) and third immunisations (V3), leaving some horses insufficiently protected for several weeks. This so-called “immunity gap” poses a challenge to all EI vaccines. During this period, the EI infection of vaccinated animals may be followed by marked clinical signs and virus shedding. However, several EI vaccines have been shown to stimulate equine influenza virus (EIV)-specific cell-mediated immunity, which is likely to play a role in protection against EIV infection and/or mitigate the clinical and virological signs of EI. Reducing the interval between V2 and V3 has been shown to be counterproductive to longer-term immunity. Further research is needed to define and address the “immunity gap” in horses. This study aimed to measure the level of protection induced by a whole inactivated, ISCOMatrix adjuvanted, EI and tetanus vaccine (Equilis Prequenza-Te) when challenged during the immunity gap (i.e., immediately before the recommended boost immunisation, more than 5 months after V2) using infection with a recent heterologous Florida Clade 2 (FC2) equine influenza virus (EIV) strain. This vaccine was tested in a Welsh mountain pony model. A group of seven ponies was vaccinated twice, 4 weeks apart. The protective antibody response was measured and ponies were challenged, along with 5 unvaccinated control ponies, by experimental infection with the FC2 A/eq/Northamptonshire/1/13 EIV strain, 158 days (around 5.2 months) after V2 and their clinical signs and virus shedding were monitored. EI serology was measured by single radial haemolysis (SRH) and haemagglutination inhibition (HI). Clinical signs and virus shedding (measured by qRT-PCR and hen’s egg titration) were compared with controls. All vaccinates had detectable, low SRH antibody titres and most had detectable, low HI titres. Significant clinical and virological protection was observed in vaccinates (p < 0.05), supporting the good performance of this vaccine against a recent EIV strain. In this study, the impact of the immunity gap in ponies was limited after primary vaccination with this whole inactivated, ISCOMatrix adjuvanted EI and tetanus vaccine (Equilis Prequenza-Te) when infected several months after V2 with a recent FC2 strain, which is representative of EIV circulating in the EU.

Refinement of the Equine Influenza model: the benefits of individual nebulisation for experimental infection

Equine influenza (EI) is a major respiratory disease of the horse induced by the highly contagious equine influenza virus (EIV). Worldwide, diverse EI vaccines are available commercially. The EIV strain, adjuvant composition and vaccine technology differs greatly. The use of the same EI vaccine throughout the life of a horse is unlikely, due to change of ownership, veterinary practitioner, location and/or vaccine availability. The compatibility of different EI vaccines has rarely been studied but is of great importance as immunisation failure could favour EIV dissemination. Study aims: To determine the impact of mixed EI vaccination on protective immunity to EIV in horses and to measure herd immunity in Hong Kong (HK). Methods: HK has approximately 1,250 race horses with around 450 new horses arriving each year. Imported racehorses must have been vaccinated with an approved EI vaccine, irrespective of their point of origin. All horses are given a new primary course on arrival with a unique ISCOM matrix adjuvanted EI vaccine and are vaccinated twice a year thereafter. EI-specific antibody titres were measured by single radial haemolysis (SRH) assay (A/eq/South Africa/4/03 EIV strain) in 1050+ archived sera collected between 2010 and 2014 from 129+ horses. Objectives: 1) to determine the SRH antibody titre in serums from HK resident horses (i.e. in HK for at least 1.5 year), 2) to evaluate the antibody titre on arrival at HK in relation to their country of origin, 3) to measure the effect of post-arrival mandatory EI primary vaccination and 4) to compare this response with antibody levels measured in HK resident horses. Results: Preliminary results indicates that the average yearly SRH antibody titre for HK resident horses was 113±34mm2 (n1⁄4467), with only 15% of horses below the clinical protection threshold (i.e. 85mm2) and less than 3% of horses below 50mm2. A significant increase was detectable (p-value<0.00004) after each immunisation boost (maximum average titre of 128±42mm2 in October, n1⁄455). The average SRH antibody titre in the week following importation to HK (and prior the mandatory EI vaccination in HK) was 180±40mm2 (n1⁄479), with results in horses originating in Australia significantly higher (207±36mm2, n1⁄410, p-value 1⁄4 0.0281) than horses from New Zealand (171±34mm2, n1⁄442), Great Britain (168±38mm2, n1⁄417) or Ireland (172±31mm2, n1⁄43). SRH antibody titres at the time of importation were maintained for around 75 days (Figure 1) due

Impact of Age at First Immunisation on Equine Influenza Short and Mid‐Term Protective Antibody Levels in Thoroughbred Foals

Reasons for performing studyEvery year, several equine influenza (EI) epidemics are reported worldwide. Equine influenza vaccination is the most efficient method of prevention; however, not all horses develop sufficient immunity after EI immunisation, increasing the risk of infection, infectious virus excretion and the spread of the disease.ObjectivesThis study aimed to better understand poor vaccine response mechanisms during the primary EI vaccination.Study designCohort study.MethodsThe EI humoral immune response was measured in 118 Thoroughbred foals set in 3 different stud farms (SF#1 to SF#3) after the primary course of EI vaccination. All foals were immunised with a recombinant canarypox‐based EI vaccine, following the vaccine manufacturer's recommendations. The age at first vaccination was 4 to 8 months. Sera were tested by single radial haemolysis (SRH) against the A/equine/Jouars/4/2006 EIV strain (Florida clade 2) at the time of the first vaccination (V1), 2 weeks and 3 months after the second immunisation (V2), 2 days and 3 months after the third immunisation (V3).ResultsShort (V2 + 2 weeks) and mid‐term (V3 + 3 months) SRH antibody levels were statistically different between the stud farms (P‐value = 0.0011 and 0.003, respectively), with SF#1&lt;SF#3&lt;SF#2 and SF#1 SRH antibody titres below the protection threshold (i.e. 85 mm2) at all time points studied. SRH antibody levels induced by EI vaccination were related to the age of foals at the first immunisation (SF#1 median age 143 days, SF#2 median age 181 days and SF#3 median age 156 days), but were independent of the presence of maternal derived antibodies (MDA; P‐value 0.41). Given the results in SF#1, a booster immunisation (V4) was brought forward to restore protective levels of antibody.ConclusionIndependently of the presence of MDA, the age of foals at first immunisation plays an important role in the establishment of adequate antibody levels.Ethical animal research: All animal work received ethical approval, and consent was obtained from the owners. Sources of funding: This study was supported by the Basse‐Normandie Region council (France) and the European Regional Development Fund. Competing interests: None declared.

The impact of different equine influenza vaccine products and other factors on equine influenza antibody levels in Thoroughbred racehorses.

More knowledge of equine influenza (EI) vaccine usage in training yards and the factors that influence serological response to vaccination are required to determine evidence-based vaccination strategies. The aim of this study was to ascertain the vaccination history of a population of Thoroughbred racehorses and identify factors that impacted on their antibody titres against EI. Observational field study. The study population consisted of 102 vaccinated Thoroughbred horses in training on a single premises. The vaccination histories recorded in their official passports were analysed. Blood samples for serological testing were collected by the veterinary surgeon one month after booster vaccination with ProteqFlu-Te. Antibodies against EI were measured by single radial haemolysis (SRH). Multivariate statistical analysis was undertaken to determine the predictors of SRH antibody titres. There was a strong correlation between age and number of vaccine doses received. Over 70% of horses received their first vaccine dose between ages 6 and 12 months. On average, horses had received 6 vaccine doses and the mean interval between booster vaccinations was 7.7 months. The majority of horses (95%) received more than one influenza vaccine product while 32% had received 3 vaccine products. Significantly higher antibody levels were observed in females than males and there was a significant association between the number of vaccine products administered and antibody levels. In contrast, a negative association between number of vaccine doses and SRH antibody level was demonstrated. Important predictors of EI antibody titres in racehorses were sex, number of vaccine doses received and number of different vaccine products administered.

Epidemiological and virological investigations of equine influenza outbreaks in Ireland (2010–2012)

Outbreaks of equine influenza (EI) in endemic populations cause disruption and economic loss. To identify (i) factors involved in the spread of EI (ii) virus strains responsible for outbreaks (iii) single radial haemolysis (SRH) antibody levels correlating with protection against current virus strains (iv) evidence of vaccination breakdown. RT-PCR, virus isolation and SRH were carried out on nasopharyngeal swabs and blood samples collected from horses, ponies and donkeys on affected premises. Data relating to 629 samples from 135 equidae were analysed. Outbreaks were sporadic, self limiting and associated with the movement of horses. Vaccination status and age influenced clinical signs of disease while housing and fomites contributed to virus spread. Subclinical infection as defined as a horse which tested positive by one or more of the following; RT-PCR, virus isolation and seroconversion in the absence of clinical signs, was identified in 9% of animals. Of the horses with up to date vaccination records 32% developed clinical signs. Vaccine breakdown occurred among horses vaccinated with all four commercially available vaccines. Analysis of HA1 sequence data generated for 26 viruses indicated that they all belonged to clade 2 of the Florida sublineage. Higher SRH antibody levels were required for both clinical and virological protection than reported in studies where vaccine strains were antigenically and genetically similar to those circulating in the field. The results of this study therefore support the OIE recommendations that vaccines be updated to include representatives of both clades of the Florida sublineage.

Protection, systemic IFNγ, and antibody responses induced by an ISCOM-based vaccine against a recent equine influenza virus in its natural host

In the horse, conventional inactivated or subunit vaccines against equine influenza virus (EIV) induce a short-lived antibody-based immunity to infection. Alternative strategies of vaccination have been subsequently developed to mimic the long-term protection induced by natural infection with the virus. One of these approaches is the use of immune-stimulating complex (ISCOM)-based vaccines. ISCOM vaccines induce a strong antibody response and protection against influenza in horses, humans, and a mouse model. Cell-mediated immunity (CMI) has been demonstrated in humans and mice after ISCOM vaccination, but rarely investigated in the horse. The aim of this study was to evaluate EIV-specific immune responses after intra-muscular vaccination with an ISCOM-EIV vaccine (EQUIP F) containing both equine influenza H7N7 (A/eq/Newmarket/77) and H3N8 (A/eq/Borlänge/91 and A/eq/Kentucky/98) strains. The antibody response was measured by single radial haemolysis (SRH) assay using different H3N8 EIV strains. Stimulation of type-1 immunity was evaluated with a recently developed method that measures EIV-specific IFNgamma synthesis by peripheral blood lymphocytes (PBL). The protective efficacy of this ISCOM-based vaccine against challenge infection with a recent equine influenza (H3N8; A/eq/South Africa/4/03) strain was also evaluated. Vaccinated ponies developed elevated levels of EIV-specific SRH antibody and increased percentage of EIV-specific IFNgamma(+) PBL, whereas these responses were only detected after challenge infection in unvaccinated control ponies. Vaccinates showed minimal signs of disease and did not shed virus when challenged shortly after the second immunisation. In conclusion, evidence of type-1 immunity induced by an ISCOM-based vaccine is described for the first time in horses.

Evidence supporting the inclusion of strains from each of the two co-circulating lineages of H3N8 equine influenza virus in vaccines

Two lineages of antigenically distinct equine influenza A H3N8 subtype viruses, American and European, co-circulate. Experiments were conducted in ponies to investigate the protection induced by vaccines containing virus from one lineage against challenge infection with homologous or heterologous virus. Regression analysis showed that vaccinated ponies with average pre-challenge single radial haemolysis (SRH) antibody levels (i.e. 45–190 mm 2) had a higher probability of becoming infected if they were vaccinated with virus heterologous to the challenge strain than if they were vaccinated with homologous virus. Field studies in Thoroughbred racehorses also showed that SRH antibody levels of ≥150 mm 2 induced by vaccines containing a European lineage strain are protective against infection with a virus from the same lineage, but that the same or higher antibody levels may not be protective against an American lineage virus. In conclusion, vaccines should contain virus strains representative of both H3N8 subtype lineages to maximise protection against infection.

A single radial haemolysis assay for antibody to H5 haemagglutinin

Background: It has been recognised that the haemagglutination–inhibition (HI) test is not sufficiently sensitive to detect human antibody to H5N1 influenza virus and alternative serological tests are needed. A modified single radial haemolysis (SRH) test is described. Methods: Sera from H5N1 human cases in Hong Kong SAR and from H5 vaccine trials were tested by an H5 SRH test which included a virus adsorption step to remove cross-reactive antibody. Test specificity was examined using reference animal antisera. Results: The SRH test recognised antibody in a specific antiserum to A/Hong Kong/489/97 (H5N1) virus, yet rabbit antibody to an A (H10N7) avian virus and human antibody to A (H1N1) or A (H3N2) viruses did not react. H5 SRH antibody induced by human H5N1 virus infection and A/Duck/Singapore/97 (H5N3) vaccination, correlated with antibody detected by microneutralisation techniques. Conclusion: The modified SRH test offers an alternative serological technique for detecting antibody to H5 haemagglutinins, which does not depend on use of live pathogenic H5N1 virus.

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.

Protection against experimental infection with influenza virus A/equine/Miami/63 (H3N8) provided by inactivated whole virus vaccines containing homologous virus.

Thirty-one ponies immunized with inactivated virus vaccine containing A/equine/Miami/63 (H3N8) virus and six seronegative ponies were experimentally challenged with the homologous virus strain. All 6 unvaccinated ponies and 11 out of 31 vaccinated ponies became infected. A clear relationship between pre-challenge antibody, measured by single radial haemolysis (SRH), and protection was demonstrated as judged by virus excretion, febrile responses and antibody responses. Those ponies with SRH antibody levels greater than 74 mm2 were completely protected against challenge infection by the intranasal route.

A single-radial haemolysis technique for measurement of antibody to influenza virus neuraminidase in equine sera

A modified single-radial-haemolysis (SRH) test for measurement of antibody to influenza virus neuraminidase (NA) is described. The test requires treatment of sheep erythrocytes with butanol to increase sensitivity. In comparative assays, SRH was found to be more sensitive than the conventional neuraminidase-inhibition test. The SRH test was reproducible, specifically measured antibody to influenza NA and was easy to use. SRH antibody responses in ponies vaccinated with bivalent equine influenza vaccine were shown to be vaccine dose-related but were lower in magnitude and shorter in duration than comparable anti-haemagglutinin responses.

Interpretation of responses and protective levels of antibody against attenuated influenza A viruses using single radial haemolysis.

Antibody determinations against H3N2 and H1N1 type A influenza viruses were carried out on paired sera obtained from volunteers taking part in influenza virus vaccine studies, using both the haemagglutination-inhibition (HI) and single radial haemolysis (SRH) test. Good correlation between the HI and SRH test was found for both H3N2 and H1N1 antibody and the zone area increases corresponding to significant SRH antibody rises determined for both virus strains. In both H3N2 and H1N1 vaccine studies, intranasal infection of the volunteers with live attenuated viruses was involved and by the measurement of HI and SRH antibodies prior to and following infection, levels of antibody equating with protection against the infecting viruses could be estimated. For the HI test the antibody titres associated with 50% protection were 42 for H1N1, and 44 for H3N2 viruses; for the SRH test, 50% protection was associated with zone areas of 20.0-25.0 mm2 for both H1N1 and H3N2 viruses.

Immune response of calves following the inoculation of Mycoplasma dispar and Mycoplasma bovis

A small but significant reduction in the number of Mycoplasma dispar colonising the respiratory tract after intratracheal challenge was observed in gnotobiotic-calves previously inoculated subcutaneously three times with formalin-killed organisms and oil adjuvant. Injection of M. dispar by the intramuscular route, with oil adjuvant, and 2 weeks later by the intratracheal route, without adjuvant, failed to induce immunity to subsequent intratracheal challenge. Following the subcutaneous injection of killed M. dispar, the amount of antibody detected by single radial haemolysis (SRH) increased markedly with increasing age in groups of calves with average ages of 16 to 155 days when first injected. Most calves aged less than 40 days failed to produce an antibody response to a singel injection of M. dispar. With M. bovis a smaller difference was observed between antibody levels generated in calves of different ages; also, calves less than 40 days old produced a detectable SRH antibody response following a single injection of killed M. bovis. IgG1 and IgG2 antibody to M. dispar and M. bovis were measured by ELISA. IgG1 appeared before IgG2 antibody and this was particularly pronounced in younger calves. Also, for both mycoplasmas IgG2 antibody levels were lower in younger than older calves. The IgG1 response to M. dispar was compared in three groups of calves with average ages of 16, 55 and 155 days and was greatest in the oldest and least in the youngest animals. In contrast, the IgG1 response to M. bovis varied little in calves of different ages. It therefore appears that the immune response of young calves to M. dispar is impaired or defective.