Single Radial Hemolysis Assays Research Articles

Equine influenza serological methods.

Serologic tests for equine influenza virus (EIV) antibodies are used for many purposes, including retrospective diagnosis, subtyping of virus isolates, antigenic comparison of different virus strains, and measurement of immune responses to EIV vaccines. The hemagglutination-inhibition (HI), single radial hemolysis (SRH), and serum micro-neutralization tests are the most widely used for these purposes and are described here. The presence of inhibitors of hemagglutination in equine serum complicates interpretation of HI assay results, and there are alternative protocols (receptor-destroying enzyme, periodate, trypsin-periodate) for their removal. With the EIV H3N8 strains in particular, equine antibody titers may be magnified by pretreating the HI test antigen with Tween-80 and ether. The SRH assay offers stronger correlations between serum antibody titers and protection from disease. Other tests are sometimes used for specialized purposes such as the neuraminidase-inhibition assay for subtyping, or ELISA for measuring different specific antibody isotypes, and are not described here.

Immunogenicity and safety of an inactivated 2012/2013 trivalent influenza vaccine produced in mammalian cell culture (Optaflu®)

Background: The present study aimed to evaluate immunogenicity and safety of the 2012/2013 seasonal influenza vaccine (Optaflu®) after the World Health Organization recommended two new strains for the composition.Results: Twenty-one days post-vaccination geometric mean titers (GMTs) against A(H1N1), A(H3N2) and the B strain were 528, 935, and 201 for adults and 272, 681, and 101 for elderly subjects, respectively. The proportion of subjects with a HI titer of ≥ 40 against the three strains A(H1N1), A(H3N2) and B was 98%, 100%, and 98% in adults and 100%, 100%, and 85% in elderly subjects, respectively. Optaflu® met the CHMP criteria of the Committee for Medicinal Products for Human Use (CPMP/BWP/214/96). Pre-vaccination titers indicated seroprotection against the A(H1N1), the A(H3N2) and the B strain in 56%, 86%, and 54% of the adults and in 61%, 85%, and 40% of the elderly with highest titers against the A(H3N2) strain. In the safety analysis injection site pain (37%) and myalgia (31%) were the most common local and systemic reactions. No serious adverse events were recorded.Conclusion: The 2012/2013 seasonal influenza vaccine Optaflu® showed good immunogenicity and an acceptable safety profile in both adults and elderly.Methods: In this trial, 126 subjects (63 adults ≥18 to ≤60 y, 63 elderly ≥61 y) were vaccinated with a single dose Optaflu® containing each of the three virus strains recommended for the 2012/2013 season (A/California/7/2009(H1N1)-like strain, A/Victoria/361/2011(H3N2)-like strain, and B/Wisconsin/1/2010-like strain). Immunogenicity was assessed by hemagglutinin inhibition (HI) and single radial hemolysis (SRH) assays on day 22, the safety profile was investigated throughout the whole study period.

A phase II study of an investigational tetravalent influenza vaccine formulation combining MF59®

An investigational tetravalent vaccine combining pre-pandemic, MF59®-adjuvanted A/H5N1 vaccine with non-adjuvanted, trivalent, seasonal influenza vaccine has been developed, which has the potential to be used for pre-pandemic priming and to improve levels of compliance and coverage. It is important to determine whether the safety and immunogenicity of the combination vaccine is equivalent to that of the two separate vaccines when administered concomitantly. Healthy adults (n = 601) were randomly assigned to three vaccination groups to receive either: (1) tetravalent vaccine and placebo concomitantly (in separate arms) on Day 1, followed by A/H5N1 vaccine on Day 22; (2) A/H5N1 vaccine and placebo concomitantly on Day 1, followed by tetravalent vaccine on Day 22; or (3) A/H5N1 and seasonal vaccines concomitantly on Day 1, followed by A/H5N1 vaccine on Day 22. Antibody responses were measured using single radial hemolysis (SRH), haemagglutination inhibition (HI), and microneutralization (MN) assays on Days 1, 22, and 43. Solicited adverse reactions were recorded for seven days after vaccination. Spontaneous adverse events were recorded throughout the study. The tetravalent vaccine elicited antibody titers equivalent to those for separate A/H5N1 and seasonal vaccines, and sufficient to meet the European licensure criteria against A/H5N1 and all three seasonal strains. Local and systemic reactions were mainly mild to moderate. No vaccine-related serious adverse events occurred. These findings demonstrate that MF59-adjuvanted A/H5N1 and seasonal influenza vaccines had an acceptable safety profile and could be effectively administered as a tetravalent formulation, supporting the possibility of integrating pre-pandemic priming into seasonal influenza vaccination programs.

Equine Influenza and Equine Herpesvirus Vaccination: Evaluation of Immune Response and Safety of Concurrent Administration of Equip® FT and Equip® EHV 1,4

AimsThe concurrent administration of EHV1,4 and EIV vaccines is not currently recommended. This study aimed to evaluate safety and immunogenicity of concurrent administration of Equip® FT and Equip® EHV1,4 (treatment group T04) as compared with vaccination with Equip® FT (treatment group T02) or Equip® EHV1,4 (treatment group T03) alone.MethodsEach treatment group included 10 ponies vaccinated on study Days 0 and 42. Immune responses to vaccine antigens were measured on Day 56. Tetanus antibody titres were determined with the toxin binding inhibition test (ToBI). Immunity to equine influenza virus (EIV) induced by vaccination was measured by single radial haemolysis (SRH; antibody response) and IFN gamma assay (a marker of cell‐mediated immunity; CMI). Immune responses to Herpesvirus 1,4 were measured by complement fixation (CF) and IFN gamma assay.ResultsConcurrent administration of EIV and EHV1,4 vaccines was well tolerated. Concurrent administration was proven to be efficacious against tetanus with similar serological immune response of treatment group T04 as compared with T02. Mean SRH results for ponies that received both vaccines were above 85 mm2 for A/eq/Borlange/91 and A/eq/Kentucky/98 antigens, a threshold associated with clinical protection against closely related EIV strains. The highest levels of IFN gamma response were measured in group T02. Although concurrent vaccination seemed to have an effect on both equine herpesvirus (EHV) serology and EHV CMI, EHV results obtained during this study were not conclusive and may have been influenced by a natural EHV‐4 infection occurring prior to study start.Conclusion and practical relevanceConcurrent administration of Equip® FT and Equip® EHV1,4 was well tolerated and induced mean serum antibody levels consistent with clinical protection for tetanus and EIV. However, it was not possible to gather relevant information about the EHV immune response under the conditions of this study.Ethical animal researchThe study was conducted under the Animal Health Trust Home Office Project License and with approval of the Zaventem Ethics Review Assessment team (Zoetis). Sources of funding: The study was sponsored by Pfizer/Zoetis Animal Health. Competing interests: Equip® FT and Equip® EHV1,4 are owned by Zoetis, who sponsored this study. Romain Paillot reports no conflict of interest. All other authors are employed by the study sponsor.

Assessment of Antigen-Specific and Cross-Reactive Antibody Responses to an MF59-Adjuvanted A/H5N1 Prepandemic Influenza Vaccine in Adult and Elderly Subjects

Preparedness against an A/H5N1 influenza pandemic requires well-tolerated, effective vaccines which provide both vaccine strain-specific and heterologous, cross-clade protection. This study was conducted to assess the immunogenicity and safety profile of an MF59-adjuvanted, prepandemic influenza vaccine containing A/turkey/Turkey/01/2005 (H5N1) strain viral antigen. A total of 343 participants, 194 adults (18 to 60 years) and 149 elderly individuals (≥61 years), received two doses of the investigational vaccine given 3 weeks apart. Homologous and heterologous antibody responses were analyzed by hemagglutination inhibition (HI), single radial hemolysis (SRH), and microneutralization (MN) assays 3 weeks after administration of the first vaccine dose and 3 weeks and 6 months after the second dose. Immunogenicity was assessed according to European licensure criteria for pandemic influenza vaccines. After two vaccine doses, all three European licensure criteria were met for adult and elderly subjects against the homologous vaccine strain, A/turkey/Turkey/1/2005, when analyzed by HI and SRH assays. Cross-reactive antibody responses were observed by HI and SRH analyses against the heterologous H5N1 strains, A/Indonesia/5/2005 and A/Vietnam/1194/2004, in adult and elderly subjects. Solicited local and systemic reactions were mostly mild to moderate in severity and occurred less frequently in the elderly than in adult vaccinees. In both adult and elderly subjects, MF59-adjuvanted vaccine containing 7.5 μg of A/Turkey strain influenza virus antigen was highly immunogenic, well tolerated, and able to elicit cross-clade, heterologous antibody responses against A/Indonesia and A/Vietnam strains 6 weeks after the first vaccination.

Cell culture (Vero cell) derived whole-virus non-adjuvanted H5N1 influenza vaccine induces long-lasting cross-reactive memory immune response: Homologous or heterologous booster response following two dose or single dose priming

BackgroundInfluenza pandemic preparedness involves priming of the population with pre-pandemic vaccines. Such vaccines should be well tolerated and induce a long-lasting immunological memory that can effectively be boosted with a single dose of pandemic vaccine once available. The presented studies assessed different prime-boost regimens with a Vero cell-derived whole virus non-adjuvanted H5N1 vaccine. MethodsIn one study, 281 healthy adult (18–59 years) and 280 elderly (≥60 years) subjects received two vaccinations, 21 days apart, with Vero cell-derived whole virus non-adjuvanted H5N1 vaccine (7.5μg HA antigen A/Vietnam/1203/2004) followed by a 6, 12–15, or 24 month booster (7.5 or 3.75μg A/Indonesia/05/2005 or A/Vietnam/1203/2004). In the other study, 230 healthy adults (18–59 years) received single dose priming (7.5μg A/Vietnam/1203/2004) followed by a 12 month booster (7.5 or 3.75μg A/Indonesia/05/2005). Antibody responses were assessed by microneutralization (MN) and single radial hemolysis (SRH) assay. Vaccine safety was assessed throughout. ResultsTwo dose priming was equally immunogenic in adults and the elderly: >72% of subjects in each population achieved MN titers ≥1:20 after the second vaccination. Booster vaccinations at 6, 12–15, and 24 months induced substantial antibody increases to both strains: after a 7.5μg A/Indonesia/05/2005 booster, 93–95% of adults and 72–84% of the elderly achieved MN titers≥1:20 against this strain. Homologous and heterologous booster responses were higher in the 7.5μg dose group than in the 3.75μg dose group. Booster responses following single dose priming were similar; a 7.5μg booster dose induced homologous MN titers ≥1:20 in 93% of subjects. ConclusionsA Vero cell derived whole virus non-adjuvanted H5N1 influenza vaccine is well tolerated and induces long-lasting cross-clade immunological memory that can be effectively boosted 1–2 years after two dose or single dose priming, supporting its suitability for pre-pandemic vaccination.

Homologous and heterologous antibody responses to a one-year booster dose of an MF59®: Adjuvanted A/H5N1 pre-pandemic influenza vaccine in pediatric subjects

Background: Primary immunization with two doses of MF59®-adjuvanted A/H5N1 influenza vaccine has been shown to be highly immunogenic and well tolerated in children and adolescents. Assessment of long-term antibody persistence after priming, and the effects of a one-year booster dose in children and adolescents was needed.Objectives This study assessed homologous and heterologous antibody responses to a one-year booster dose of MF59-adjuvanted A/H5N1 influenza vaccine in previously primed children.Subjects and methods: Twelve months after primary vaccination, toddlers, children and adolescents received a single booster dose of the same A/H5N1 vaccine. Paired sera were collected before and three weeks after booster vaccination. Homologous antibody responses against the A/Vietnam/1194/2004 vaccine strain were measured by hemagglutination inhibition (HI), single radial hemolysis (SRH) and microneutralization (MN) assays. Heterologous antibody responses against A/Indonesia/5/2005 and A/Anhui/1/2005 strains were assessed by MN assay only.Results: Twelve months after primary vaccination, persistent, homologous, seroprotective HI antibody titers (≥ 40) were observed in 46%, 26% and 30% of toddlers, children and adolescents; following booster vaccination, seroprotection rates increased to 99%, 98% and 91%, respectively. All toddlers and children, and 99% of adolescents achieved MN antibody titers ≥ 40. Cross-reactive A/H5N1 antibodies were detected in 94–98% of subjects after booster vaccination.Conclusions Two priming doses of MF59-adjuvanted A/H5N1 vaccine resulted in homologous and heterologous antibody responses which persisted for up to one year after immunization. A one-year booster dose was highly immunogenic, generating high homologous and cross-reactive A/H5N1 antibody titers.

The use of equine influenza pseudotypes for serological screening

Standard assays used for influenza serology present certain practical issues, such as inter-laboratory variability, complex protocols and the necessity for handling certain virus strains in high biological containment facilities. In an attempt to address this, avian and human influenza HA pseudotyped retroviruses have been successfully employed in antibody neutralization assays. In this study we generated an equine influenza pseudotyped lentivirus for serological screening. This was achieved by co-transfection of HEK293T cells with plasmids expressing the haemagglutinin (HA) protein of an H3N8 subtype equine influenza virus strain, HIV gag-pol and firefly luciferase reporter genes and harvesting virus from supernatant. In order to produce infective pseudotype particles it was necessary to additionally co-transfect a plasmid encoding the TMPRSS2 endoprotease to cleave the HA. High titre pseudotype virus (PV) was then used in PV antibody neutralization assays (PVNAs) to successfully distinguish between vaccinated and non-vaccinated equines. The sera were also screened by single radial haemolysis (SRH) assay. There was a 65% correlation between the results of the two assays, with the PVNA assay appearing slightly more sensitive. Future work will extend the testing of the PVNA with a larger number of serum samples to assess sensitivity/specificity, inter/intra-laboratory variability and to define a protective titre.

Thymosin-alpha 1 (Zadaxin™) enhances the immunogenicity of an adjuvated pandemic H1N1v influenza vaccine (Focetria™) in hemodialyzed patients: A pilot study

Although influenza vaccination is widely recommended for immunosuppressed people, the same immune dysfunction that can increase the risk of contracting influenza might also compromise vaccine effectiveness, especially during pandemics. Clinical data have highlighted the role of adjuvants in improving vaccine efficacy. As uremic patients are especially vulnerable to infections, it is recommended that they should be vaccinated yearly against influenza. This paper presents the results of a pilot clinical trial, conducted in hemodialyzed patients with an adjuvated pandemic H1N1v influenza vaccine alone and combined with Thymosin-alpha 1. Subjects were subdivided into 3 treatment groups receiving: the adjuvated pandemic influenza vaccine (Focetria) only (first treatment group), and the Vaccine+Thymosin alpha 1 (Zadaxin) at a dose of 3.2 and 6.4 mg (second and third treatment groups respectively). The immunoresponse was assessed on days 0, 21, 42, 84 and 168 after vaccine administration by means of Hemagglutination Inhibition (HI), Microneutralization (MN) and Single Radial Hemolysis (SRH) assays. The CHMP regards HI as the gold standard test to evaluate the immune response to influenza vaccines before influenza vaccines are licensed. The CHMP criteria are slightly different in adults (18-60-year-old subjects) and the elderly (>60 years old). Indeed, 40% of seroconversion, 70% of subjects seroprotected 21 days after vaccination, and a 2.5-fold increase in GMR (Geometric Mean Ratio) are required in adults, while in the elderly, the corresponding threshold values are: 30%, 60% and a 2-fold increase. All these criteria must be met for the licensing of a pandemic influenza vaccine. Safety evaluation was performed by means of Adverse Event (AE) recording, laboratory assays (hematology and chemistry), electrocardiogram, and assessment of vital signs. Three populations were considered: Intention-To-Treat (ITT) (94 patients), Per Protocol (PP) (82 patients), and Safety population (99 patients). With regard to the Geometric Mean Titer (GMT) and the Geometric Mean Ratio (GMR) of HI on Day 21 in the ITT population, both "Vaccine+Thymosin alpha 1" groups presented better results than the "Vaccine only" group. A large proportion of ITT patients in the two Vaccine+Thymosin alpha 1 groups achieved seroconversion by Day 21. On Day 42, the decrease in the GMT of HI was greater in the Vaccine+Thymosin alpha 1 groups than in the vaccine only group. Similar results were obtained in the PP population. The CHMP criteria were fully met in the groups treated with Vaccine+Thymosin alpha 1. No AE was found to be related to Thymosin alpha 1 nor to the Focetria vaccine. Although further studies in larger hemodialyzed populations are necessary, it can be concluded that Thymosin alpha 1 enhanced the immunogenicity of the pandemic influenza vaccine used. Moreover, it proved safe and well tolerated, and did not affect hematology or blood-chemistry values.

Evaluation of a virosomal H5N1 vaccine formulated with Matrix M™ adjuvant in a phase I clinical trial

The avian influenza H5 virus epizootic continues to cause zoonosis with human fatalities, highlighting the continued need for pandemic preparedness against this subtype. This study evaluated the tolerability and immunogenicity of a Matrix M™ adjuvanted virosomal H5N1 vaccine in a phase I clinical trial. Sixty healthy adults were vaccinated intramuscularly with two doses of influenza H5N1 (NIBRG-14) virosomal vaccine alone (30 μg haemagglutinin (HA)) or 1.5, 7.5 or 30 μg HA formulated with 50 μg Matrix M™ adjuvant. The antibody response was analysed by haemagglutination inhibition (HI), microneutralisation (MN) and single radial haemolysis (SRH) assays. The vaccine was well tolerated in all groups but injection site pain was more frequently observed in the Matrix M™ adjuvanted groups. The vaccine elicited homologous and heterologous H5N1-specific antibody responses and the Matrix M™ adjuvanted formulations met all the EU regulatory criteria. In conclusion, Matrix M™ adjuvant was well tolerated and augmented the antibody response allowing considerable dose sparing down to 1.5 μg HA.

Immunogenicity and Safety of MF59-Adjuvanted H5N1 Influenza Vaccine From Infancy to Adolescence

This study evaluated the immunogenicity, safety, and tolerability of a MF59-adjuvanted H5N1 vaccine in a population 6 months through 17 years of age. Healthy subjects 6 to <36 months, 3 to <9 months, and 9 to <18 years of age were assigned randomly to receive 2 doses of either a MF59-adjuvanted H5N1 vaccine (7.5 μg/dose) or a MF59-adjuvanted trivalent seasonal influenza control vaccine (15 μg/dose for each antigen). Immunogenicity against the A/Vietnam/1194/2004-like vaccine strain was measured before and 3 weeks after the 2-dose primary series, through hemagglutination inhibition (HI), single radial hemolysis (SRH), and microneutralization. Local and systemic reactions were recorded. A total of 335 subjects received the H5N1 vaccine, and 137 subjects received the seasonal vaccine. Rates of seroprotection (HI titer of ≥40) against the H5N1 vaccine antigen were 97% for children 6 to 36 months and 3 to 9 years of age and 89% for older children. All subjects seroconverted in the SRH assay. Microneutralization titers of ≥40 were achieved by 99% of subjects, and ≥98% of subjects, respectively. Local reactions, particularly injection site pain in older children, were common, generally mild to moderate in nature, and transient and resolved spontaneously. Up to 5% of participants. There were no vaccine-related serious adverse events in either group. In this pediatric population, MF59-adjuvanted H5N1 vaccine was highly immunogenic, had a good safety profile, reactogenicity comparable with that of an adjuvanted seasonal influenza control vaccine.

Safety and immunogenicity of an inactivated thimerosal‐free influenza vaccine in infants and children

Objective Few prospective studies of inactivated split virion influenza vaccine have been conducted in infants and children. Our objective was to evaluate the safety, reactogenicity and immunogenicity of a thimerosal‐free inactivated influenza vaccine (Fluvax®; CSL Limited, Parkville, Australia) in children aged 6 months to <9 years. Methods A prospective, open‐label, phase III clinical trial was conducted in 298 healthy children previously unvaccinated with influenza, commencing in the Southern Hemisphere 2005 autumn. Participants were divided into two groups (Group A: ≥6 months to <3 years; Group B: ≥3 years to <9 years), and received two doses of the 2005 vaccine, and one dose of the 2006 vaccine one year later (Group A: 0·25 ml per dose; Group B: 0·5 ml per dose). Vaccine safety and reactogenicity was evaluated for 30 days after each dose. Immunogenicity was assessed using hemagglutination inhibition and single radial hemolysis assays. Results There were no withdrawals due to adverse events (AEs). The majority of solicited local and systemic AEs were of mild severity. A maximum intensity of severe was reported for injection site pain and fever by only 3·0% and 3·4% of participants, respectively. The vaccine was immunogenic for all antigens, with ≥95% of both younger and older children achieving seroprotection after dose 2. Conclusions This thimerosal‐free inactivated influenza vaccine had a favorable safety profile and was immunogenic in children aged ≥6 months and <9 years. Primary and booster vaccination produced consistently immunogenic responses including in children under 3 years of age receiving 0·25 ml doses of vaccine.

MF59®-Adjuvanted H5N1 Vaccine Induces Immunologic Memory and Heterotypic Antibody Responses in Non-Elderly and Elderly Adults

BackgroundPathogenic avian influenza virus (H5N1) has the potential to cause a major global pandemic in humans. Safe and effective vaccines that induce immunologic memory and broad heterotypic response are needed.Methods and FindingsHealthy adults aged 18–60 and >60 years (n = 313 and n = 173, respectively) were randomized (1∶1) to receive two primary and one booster injection of 7.5 μg or 15 μg doses of a subunit MF59-adjuvanted H5N1 (A/Vietnam/1194/2004) (clade 1) vaccine. Safety was monitored until 6 months after booster. Immunogenicity was assessed by hemagglutination inhibition (HI), single radial hemolysis (SRH) and microneutralization assays (MN). Mild injection-site pain was the most common adverse reaction. No serious adverse events relating to the vaccine were reported. The humoral immune responses to 7.5 μg and 15 μg doses were comparable. The rates for seroprotection (HI>40; SRH>25mm2; MN ≥40) after the primary vaccination ranged 72–87%. Six months after primary vaccination with the 7.5 μg dose, 18% and 21% of non-elderly and elderly adults were seroprotected; rates increased to 90% and 84%, respectively, after the booster vaccination. In the 15 μg group, seroprotection rates among non-elderly and elderly adults increased from 25% and 62% after primary vaccination to 92% and 88% after booster vaccination, respectively. A heterologous immune response to the H5N1/turkey/Turkey/05 strain was elicited after second and booster vaccinations.ConclusionsBoth formulations of MF59-adjuvanted influenza H5N1 vaccine were well tolerated. The European Union requirement for licensure for pre-pandemic vaccines was met by the lower dose tested. The presence of cross-reactive antibodies to a clade 2 heterologous strain demonstrates that this vaccine may be appropriate for pre-pandemic programs.Trial RegistrationClinicalTrials.gov NCT00311480

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.

Pro-inflammatory and antiviral cytokine expression in vaccinated and unvaccinated horses exposed to equine influenza virus

Most studies of the cytokine response to influenza virus infection have been carried out in human, porcine and murine models, however the data available on equine cytokines is limited. An experimental challenge study was undertaken in unvaccinated naïve horses and horses vaccinated with a commercial inactivated influenza vaccine. The humoral antibody response to vaccination and virus challenge was measured by single radial haemolysis (SRH) assay and clinical signs of influenza and viral shedding were monitored post-challenge. Levels of three equine pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α and the antiviral cytokine interferon (IFN)-α were examined by quantitative RT-PCR of mRNA. Vaccination provided significant clinical and virological protection and resulted in a significant reduction of IFN-α and IL-6 expression on day 2 post-challenge. The patterns of cytokine expression observed in control animals suffering from influenza after challenge are comparable to those reported in studies of other species.

Antibody and IFN-γ 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-γ) 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-γ protein synthesis in vaccinated ponies following challenge infection with EIV compared with control ponies.

The relationship between single radial hemolysis, hemagglutination inhibition, and virus neutralization assays used to detect antibodies specific for equine influenza viruses

Antibodies specific for equine influenza viruses are usually quantified using single radial hemolysis (SRH), hemagglutination inhibition (HI) or virus neutralization (VN). Neutralizing antibodies are thought to provide optimum protection to challenged animals. The purpose of this study was to determine the extent to which SRH and HI assays detect antibodies which neutralize equine influenza viruses. Acute and convalescent sera from 41 horses were analyzed using VN, SRH and HI assays. These horses were present in a population of Thoroughbred racehorses during an epidemic of upper respiratory tract disease associated with influenza A/equine/Saskatoon/1/91 (H3N8) infections. Concentrations of antibodies binding to influenza A/equine/Kentucky/1/81 (H3N8), A/equine/Miami/1/63 (H3N8), and A/equine/Prague/1/56 (H7N7) were determined. Results of the VN assay were compared with results from the SRH and HI assays for acute antibody levels, changes in antibody concentrations between acute and convalescent sampling, and the occurrence of seroconversion. The correlation between assays for pre-exposure antibody levels ranged from 88% to 96%. The correlation between assays for change in antibody concentration ranged from 83% to 90% for the H3N8 viruses. This study shows that antibody concentrations specific for equine influenza virus, measured using SRH and HI assays, are highly correlated with concentrations detected using a VN assay.

The effect of changing single radial hemolysis assay method when quantifying influenza A antibodies in serum

Single radial hemolysis (SRH) is a simple method for measuring concentrations of antibody specific for influenza A viruses. There are many differences in reported assay methods and these changes may significantly affect results. This study evaluated the effect of changing erythrocyte concentrations, influenza virus concentrations used to sensitize erythrocytes, incubation times, and incubation temperatures. These factors were shown to significantly affect results of the SRH assay. A method for standardizing the assay and expressing results as relative antibody concentrations is described. Standardized relative antibody concentrations show significantly less variability than do hemolysis zone measurements when the SRH assay method is changed. This paper demonstrates that use of this method would allow for more consistent results to be obtained within laboratories. Use of this method with quantified, internationally standardized sera would allow results of the single radial hemolysis assay to be directly compared between laboratories.

Use of monoclonal anti-haemagglutinin antibodies for the ?In vitro? selection of a sequential influenza virus antigenic variant

A sequential antigenic variant of the A/Texas/77 (H3N2) influenza virus was obtained in vitro using a monoclonal antibody against the haemagglutinin (HA) of the antigenic variant V18 previously selected in vitro from the parental Texas virus. The sequential antigenic variant, designated DV1, the V18 antigenic variant and the parental A/Texas/77 viruses were used to evaluate the frequency of anti-haemagglutinin antibodies in human sera in single radial haemolysis assays. Twenty six of 100 children's sera, which contained antibodies to the parental A/Texas/77 virus, failed to react with the V18 antigenic variant. A higher proportion of sera (42%) failed to react with the DV1 antigenic variant with alterations in two different antigenic determinants with respect to the parental virus. The results are discussed in relation to the mechanism of antigenic drift and to the in vivo reaction of antigenic variants selected in vitro.

Gene responsible for dificient activity of the ? subunit of C8, the eighth component of complement, is located on mouse chromosome 4

Sera from 73 strains of mice were tested for hemolytic activity through the classical and the alternative pathways (CP and AP) in a single radial hemolysis assay. Sera from 16 out of 45 laboratory inbred strains had no lytic activity, and Ouchterlony analysis with anti-C5 serum showed them to be C5-deficient. Sera from 2 out of 28 strains derived from wild mice also had no lytic activity, but the C5 molecule was detectable in both. The hemolytic activity of sera from these strains can be restored by the addition of partially purified human C8 or human serum deficient in C8 alpha-gamma, leading us to conclude that strains M.MOL-MSM (MSM) and Mae are deficient in the beta subunit of C8. Typing of (DBA/2J X MSM)F1 hybrids and of progeny of a backcross to MSM showed that this C8 deficiency is controlled by a single recessive gene, designated C8b; the allele with hemolytic activity is C8b1; and the allele with no activity C8b0. Because of synteny homologies in mouse and human, we looked for and found close linkage between C8b and Pgm-2. Typing of recombinant mice for Mup-1 mapped the C8b locus 2.3 centimorgans (cM) telomeric to Pgm-2 on mouse chromosome 4.