Single Radial Haemolysis Research Articles

First Reported Circulation of Equine Influenza H3N8 Florida Clade 1 Virus in Horses in Italy.

Equine influenza (EI) is a highly contagious viral disease of equids characterized by pyrexia and respiratory signs. Like other influenza A viruses, antigenic drift or shift could lead to a vaccine-induced immunity breakdown if vaccine strains are not updated. The aim of this study was to genetically characterize EIV strains circulating in Italy, detected in PCR-positive samples collected from suspected cases, especially in the absence of formal active surveillance. Between February and April 2019, blood samples and nasal swabs collected from each of the 20 symptomatic horses from North and Central Italy were submitted to the National Reference Centre for Equine Diseases in Italy to confirm preliminary analysis performed by other laboratories. None of the sera analysed using haemagglutination inhibition and single radial haemolysis presented a predominant serological reactivity pattern for any antigen employed. All nasal swabs were positive with IAV RRT-PCR. Only one strain, isolated in an embryonated chicken egg from a sample collected from a horse of a stable located in Brescia, Lombardy, was identified as H3N8 Florida lineage clade 1 (FC1). In the constructed phylogenetic trees, this strain is located within the FC1, together with the virus isolated in France in 2018 (MK501761). This study reports the first detection of H3N8 FC1 in Italy, highlighting the importance of monitoring circulating EIV strains to verify the vaccine composition appropriateness for maximum efficacy.

Immunostimulating Effect of Inactivated Parapoxvirus Ovis on the Serological Response to Equine Influenza Booster Vaccination.

Equine influenza virus (EIV) is responsible for recurring outbreaks that are detrimental to the equine industry. Vaccination is key for prevention, but the effectiveness and duration of protection provided by existing vaccines is often insufficient. In order to improve vaccine efficacy, we evaluated the benefit of immune stimulation with inactivated Parapoxvirus ovis (iPPVO) on the antibody response induced by a vaccine boost against EIV. A whole inactivated ISCOMatrix-adjuvanted equine influenza vaccine was administered alone (n = 10) or combined with iPPVO injections at D0, D2 and D4 post vaccination (n = 10) to adult horses that required a vaccine boost 6 months after the last immunization, as now recommended by the WOAH. Antibody levels were measured with the single radial haemolysis (SRH) assay at 1, 3 and 6 months post-vaccination. Results revealed that horses that received iPPVO had higher antibody levels than the control group injected with the EI vaccine alone. Although the vaccine used contains only a clade 1 and European lineage strain, the increase in protective antibodies was also observed against a clade 2 strain. Thus, immune stimulation with iPPVO, a substance already marketed as an immunostimulant, could be used to improve vaccination protocols in horses and potentially other species.

Seroprevalence of Equine Influenza and Its Associated Risk Factors in Northwest Nigeria.

Equine influenza (EI) is a fast-spreading respiratory disease of equids caused by equine influenza A virus (EIV), often resulting in high morbidity and a huge economic impact on the equine industry globally. In this cross-sectional study to determine the seroprevalence of EI and its associated risk factors, sera from 830 horses bled on a single occasion in Northwest Nigeria between October 2019 and January 2020 were screened for antibodies to A/equine/Richmond/1/2007 (H3N8) using the single radial haemolysis (SRH) assay. Antibodies were detected in 71.3% (592/830, 95% CI: 68-74%) of horses (SRH area ≥ 0.5 mm2). Although there were statistically significant univariable associations between seropositivity and age, sex, breed, purpose and coat colour, only age remained significant when included with each of the other variables in bivariable analyses. There was a clear trend for increasing odds of seropositivity with increasing age: OR 1.6, 95% CI: 1.05-2.40 (p = 0.03) for 5-14-year-olds and OR 8.13, 95% CI: 2.75-24.1 (p < 0.001) for ≥15-year-olds compared to horses <5 years old. The mean SRH value was 78.2 mm2 (median = 88 mm2, interquartile range = 0-121 mm2) with only 9% of the horses having an SRH value > 150 mm2, considered sufficient to protect against clinical disease and virus shedding. Comparative screening of a subset of the horses (n = 118) with a 2019 H3N8 virus (A/equine/Worcestershire/2019) revealed a significantly greater seropositivity (p = 0.0001) than A/equine/Richmond/1/2007 consistent with exposure of the population during a widespread outbreak of EI in the region in 2019. In conclusion, there was an insufficient level of protection against EI in the region and introduction of a vaccination programme with vaccines containing recently circulating virus is recommended to mitigate against further outbreaks of EI in Nigeria.

Seroprevalence of Infectious Respiratory Agents in Thoroughbred Race Horses at the Seoul Race Park, Republic of Korea

Infectious respiratory disease is one of the most frequent causes of lost days in training and reduced performance of Thoroughbred racehorses. Equine influenza virus (EIV), Equine herpesvirus-1 (EHV-1) and -4 (EHV-4), equine rhinitis virus A (ERAV) and B (ERBV), and Streptococcus equi subspecies equi (S. equi) are important infectious agents of the respiratory tract of horses. The purpose of this study was to determine the seroprevalence of EHV-1, EHV-4, ERAV, ERBV, and S. equi and to measure EIV antibody levels of Thoroughbred racehorses at Seoul Race Park (SRP), Republic of Korea. All horses had previously been vaccinated against EIV and S. equi, but not against any of the other pathogens that were tested. A total of 94 serum samples, which were collected from race participants at the SRP were tested using the single radial haemolysis (SRH) test for EIV (H3N8), the complement-fixation (CF) test for EHV-1, EHV-4, ERAV, ERBV and an indirect enzyme-linked immunosorbent assay (iELISA) for S. equi. Serum samples from seventy eight out of 94 horses (83%) generated zones of over 85 mm2 in the SRH test, which classified them as clinically protected against EIV (H3N8). The most sero-prevalent agent detected was EHV-4 (30.9%, 29/94), followed by EHV-1 (9.6%, 9/94), S. equi (2.1%, 2/94), ERAV (1.1%, 1/94) and ERBV (1.1%, 1/94). All horses showed no visual clinical signs. The present study showed that the seroprevalence of infectious respiratory agents was relatively low and provides evidence of low risk of respiratory infectious agents in Thoroughbred race horses at SRP.

The role of cell-mediated immunity against influenza and its implications for vaccine evaluation

Influenza vaccines remain the most effective tools to prevent flu and its complications. Trivalent or quadrivalent inactivated influenza vaccines primarily elicit antibodies towards haemagglutinin and neuraminidase. These vaccines fail to induce high protective efficacy, in particular in older adults and immunocompromised individuals and require annual updates to keep up with evolving influenza strains (antigenic drift). Vaccine efficacy declines when there is a mismatch between its content and circulating strains. Current correlates of protection are merely based on serological parameters determined by haemagglutination inhibition or single radial haemolysis assays. However, there is ample evidence showing that these serological correlates of protection can both over- or underestimate the protective efficacy of influenza vaccines. Next-generation universal influenza vaccines that induce cross-reactive cellular immune responses (CD4+ and/or CD8+ T-cell responses) against conserved epitopes may overcome some of the shortcomings of the current inactivated vaccines by eliciting broader protection that lasts for several influenza seasons and potentially enhances pandemic preparedness. Assessment of cellular immune responses in clinical trials that evaluate the immunogenicity of these new generation vaccines is thus of utmost importance. Moreover, studies are needed to examine whether these cross-reactive cellular immune responses can be considered as new or complementary correlates of protection in the evaluation of traditional and next-generation influenza vaccines. An overview of the assays that can be applied to measure cell-mediated immune responses to influenza with their strengths and weaknesses is provided here.

Seroprevalence, associated risk factors and clinico-pathological studies of buffalopox disease in various regions of Punjab province, Pakistan.

Buffalopox (BPX) is a highly contagious disease that causes high morbidity and production losses in buffaloes. During this study, seroprevalence, effect of various associated risk factors, and pathological studies of BPX were recorded in the Punjab province. A total of 97 blood samples and 63 scabs were collected from clinically pox suspected buffaloes. Serum was harvested to perform single radial hemolysis to assess the seroprevalence, and scabs were subjected to PCR for BPX virus confirmation. Results revealed that, animal demographics and environmental associated factors showed significant effect (p⟨0.05,1⟨R2⟩0) on BPX occurrence. The overall BPX seroprevalence was recorded 4.18% in the Punjab province. The BPX was recorded 5.48% in Nili Ravi breed during winter (7.42%), aged 5-7 years (7.46%) under loose housing (5.51%) in the Faisalabad region (8.03%). Further, BPX was 5.37% in pregnant, 6.86% pregnant milking buffaloes during the 3rd lactation period (7.28%) in dairy herds (5.20%). The BPX was 5.22% in non-vaccinated buffaloes where multiple animals were reared together (4.99%) in the herds having 21-30 total number of animals. A total of 49 scab samples were found positive for the BPX virus via PCR with C18L gene amplification. Grossly, inflammatory lesions with pits in the center and wart-like nodules were seen on teats and udder of buffaloes. Increased leukocytes, especially neutrophils and lymphocytes, were seen in the blood of the infected animals. These results provide a broader window to understand the effect of associated risk factors, strengthen the diagnostic aid, and to contain the current spread of BPX in Pakistan to safeguard large ruminant-based livelihood.

Effect of Repeated Freeze-Thaw Cycles on Influenza Virus Antibodies.

Background: Vaccine effectiveness relies on various serological tests, whose aim is the measurement of antibody titer in serum samples collected during clinical trials before and after vaccination. Among the serological assays required by the regulatory authorities to grant influenza vaccine release there are: Hemagglutination inhibition (HAI), microneutralization (MN), and Single Radial Hemolysis (SRH). Although antibodies are regarded to be relatively stable, limited evidences on the effect of multiple freeze–thaw cycles on the stability of antibodies in frozen serum samples are available so far. In view of this, the present paper aimed to evaluate the impact of multiple freeze–thaw cycles on influenza antibody stability, performing HAI, MN and SRH assays. Methods: Ten serum samples were divided into 14 aliquots each, stored at −20 °C and taken through a total of 14 freeze–thaw cycles to assess influenza antibody stability. Each assay measurement was carried out following internal procedures based on World Health Organization (WHO) guidelines. Results: No statistically significant effect of 14 freeze–thaw cycles on antibody stability, measured through three different assays, was observed. Conclusions: Collectively, these data demonstrated that specific influenza antibody present in serum samples are stable up to 14 freeze–thaw cycles.

Immunogenicity of Calvenza-03 EIV/EHV® Vaccine in Horses: Comparative In Vivo Study.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines that is caused mainly by the H3N8 subtype of influenza A virus. Vaccinating horses against EI is the most effective strategy to prevent the infection. The current study aimed to compare the kinetics of EI-specific humoral- and cell-mediated immunity (CMI) in horses receiving either identical or mixed vaccinations. Two groups of horses were previously (six months prior) vaccinated with either Calvenza 03 EIV EHV® (G1) or Fluvac Innovator® (G2) vaccine. Subsequently, both groups received a booster single dose of Calvenza 03 EIV EHV®. Immune responses were assessed after 10 weeks using single radial hemolysis (SRH), virus neutralization (VN), and EliSpot assays. Our results revealed that Calvenza-03 EIV/EHV®-immunized horses had significantly higher protective EI-specific SRH antibodies and VN antibodies. Booster immunization with Calvenza-03 EIV/EHV® vaccine significantly stimulated cell-mediated immune response as evidenced by significant increase in interferon-γ-secreting peripheral blood mononuclear cells. In conclusion, Calvenza-03 EIV/EHV® vaccine can be safely and effectively used for booster immunization to elicit optimal long persisting humoral and CMI responses even if the horses were previously immunized with a heterogeneous vaccine.

An Evaluation of Three Different Primary Equine Influenza Vaccination Intervals in Foals

In order to evaluate the effect of three different primary vaccination intervals on EI vaccine response, 21 unvaccinated thoroughbred foals were randomly divided into three groups of 7 and vaccinated with three different intervals of primary immunization (i.e., with 1, 2 or 3 months intervals between V1 and V2, respectively). The antibody response was measured for up to 1 year after the third immunization V3 (administered 6 months after V2) by single radial hemolysis (SRH) assay. All weanlings had seroconverted and exceeded the clinical protection threshold 2 weeks after V2 and 1 month after V3 until the end of the study. Significant differences were measured at the peak of immunity after V2 and for the duration of the immunity gap between V2 and V3. The group with one month primary vaccination interval had a lower immunity peak after V2 (158.05 ± 6.63 mm2) and a wider immunity gap between V2 and V3 (18 weeks) when compared with other groups (i.e., 174.72 ± 6.86 mm2 and 16 weeks for a two months interval, 221.45 ± 14.48 mm2 and 12 weeks for a 3-month interval). The advantage observed in the group with 1 month primary vaccination interval, which induces an earlier protective immunity, is counterbalance with a lower peak of immunity and a wider immunity gap after V2, when compared with foals vaccinated with 2- and 3-month intervals.

Correlation of Influenza B Haemagglutination Inhibiton, Single-Radial Haemolysis and Pseudotype-Based Microneutralisation Assays for Immunogenicity Testing of Seasonal Vaccines.

Influenza B is responsible for a significant proportion of the global morbidity, mortality and economic loss caused by influenza-related disease. Two antigenically distinct lineages co-circulate worldwide, often resulting in mismatches in vaccine coverage when vaccine predictions fail. There are currently operational issues with gold standard serological assays for influenza B, such as lack of sensitivity and requirement for specific antigen treatment. This study encompasses the gold standard assays with the more recent Pseudotype-based Microneutralisation assay in order to study comparative serological outcomes. Haemagglutination Inhibition, Single Radial Haemolysis and Pseudotype-based Microneutralisation correlated strongly for strains in the Yamagata lineage; however, it correlated with neither gold standard assays for the Victoria lineage.

Retrospective serological survey for influenza in horses from Brazil.

Equine influenza (EI) virus is one of the most economically important pathogens of respiratory diseases of horses worldwide. Despite availability of vaccines for control of EI, the highly contagious nature and variability properties of the virus mean global outbreaks occur. Thus, continuous surveillance programs, including seroprevalence studies of disease in different countries, may contribute to better control of the disease. In this study, the seroprevalence of equine influenza in 850 horses from Brazil was investigated. The serodiagnosis was based on the single radial hemolysis (SRH) assay using influenza A/equine/Richmond/1/2007 (H3N8) antigen. Antibodies against A/equine/Richmond/1/07 (H3N8) were detected in 44.7% (380/850, 95% CI: 41.4–48.1%) of horses. Seroprevalence was significantly lower (p = 0.001) in younger animals (< 5 years, 38.6%) than in “adult” animals (5–14 years, 52.1%). There was also a significant relationship between the year of sampling and seroprevalence (p < 0.0005). The mean SRH antibody value was 42.0 mm2 (range 4–238.9 mm2), with the majority of horses (95.3%) having an SRH value ≤ 150 mm2, which is considered an insufficient level for protection of equine hosts against influenza infections and potential virus shedding. These findings indicate the need to reinforce preventive/control measures against equine influenza in Brazil.

Serologically-Based Evaluation of Cross-Protection Antibody Responses among Different A(H1N1) Influenza Strains.

After the influenza H1N1 pandemic of 2009, the seasonal A/Brisbane/59/2007 strain was replaced by the A/California/07/2009 strain for the influenza virus vaccine composition. After several seasons with no indications on the occurrence of antigenic drift, A/Michigan/45/2015 was chosen as the H1N1 vaccine strain for the 2017/2018 season. Since the immune response to influenza is shaped by the history of exposure to antigenically similar strains, the potential cross-protection between seasonal human influenza vaccine strains and the emerging pandemic strains was investigated. Human serum samples were tested by hemagglutination inhibition and single radial hemolysis assays against A/Brisbane/59/2007, A/California/07/2009, and A/Michigan/45/2015 strains. Strong cross-reactions between A/California/07/2009 and A/Michigan/45/2015 strains were observed in 2009/2010, most likely induced by the start of the 2009 pandemic, and the subsequent post-pandemic seasons from 2010/2011 onward when A/California/07/2009 became the predominant strain. In the 2014/2015 season, population immunity against A/California/07/2009 and A/Michigan/45/2015 strains increased again, associated with strong cross-reactions. Whereas hemagglutination inhibition assay has a higher sensitivity for detection of new seasonal drift, the single radial hemolysis assay is an excellent tool for determining the presence of pre-existing immunity, allowing a potential prediction on the booster potential of influenza vaccines against newly emerging drifted strains.

Evaluation of a Pseudotyped Virus Neutralisation Test for the Measurement of Equine Influenza Virus-Neutralising Antibody Responses Induced by Vaccination and Infection.

Equine influenza is a major respiratory disease of horses that is largely controlled by vaccination in some equine populations. Virus-neutralising antibodies, the mainstay of the protective immune response, are problematic in assaying for equine influenza virus, as most strains do not replicate efficiently in cell culture. Surrogate measures of protective antibody responses include the haemagglutination inhibition (HI) test and single radial haemolysis (SRH) assay. For this study, a pseudotyped virus, bearing an envelope containing the haemagglutinin (HA) from the Florida clade 2 equine influenza virus strain A/equine/Richmond/1/07 (H3N8), was generated to measure HA-specific neutralising antibodies in serum samples (n = 134) from vaccinated or experimentally-infected ponies using a pseudotyped virus neutralization test (PVNT). Overall, the results of PVNT were in good agreement with results from the SRH assay (100% sensitivity, 68.53% specificity) and HI test (99.2% sensitivity, 49.03% specificity). The PVNT was apparently more sensitive than either the SRH assay or the HI test, which could be advantageous for studying the antibody kinetics, particularly when antibody levels are low. Nevertheless, further studies are required to determine whether a protective antibody level can be defined for the SRH assay and to ascertain the inter-laboratory reproducibility. In conclusion, the PVNT efficiently measures neutralising antibodies after immunization and/or experimental infection in the natural host, and may complement existing antibody assays.

Serosurvey of pandemic H1N1 influenza A virus in dogs in Andalusia (southern Spain).

In April 2009, a new influenza A virus (IAV) subtype (A(H1N1)pdm09) spread worldwide and triggered the first human influenza pandemic of the 21st century. Since then, exposure to the pandemic H1N1 IAV has been confirmed in different animal species. Serological evidence and clinical infection with A(H1N1)pdm09 have been reported in canines, but the information available about the role of dogs in the epidemiology of this IAV subtype is still very limited in Europe. A cross-sectional study was carried out to determine the seroprevalence of A(H1N1)pdm09 in dogs in southern Spain, a region with endemic seasonal circulation in human. Sera from 750 companion dogs were collected during the period 2013-2016. Antibodies against pandemic H1N1 IAV were analysed using the haemagglutination inhibition test. Positive samples were also tested by single radial haemolysis assay. Seropositivity was only confirmed by both methods in one (0.13%; 95% CI: 0.00-0.38) adult animal sampled in 2013. To the best of the authors' knowledge, this is the first report of A(H1N1)pdm09 exposure in dogs in Spain. The low seroprevalence obtained indicates a limited exposure history to A(H1N1)pdm09 IAV in dogs in this country and suggests a low risk of transmission of this zoonotic IAV subtype between humans and dogs.

Response of Sport Horses to Different Formulations of Equine Influenza Vaccine.

The international governing body of equestrian sports requires that horses be vaccinated against equine influenza within 6 months and 21 days of competing. The aim of this study was to compare the antibody response of young sport horses to six-monthly booster vaccination with equine influenza vaccines of different formulations. An inactivated vaccine was allocated to 35 horses and subunit and recombinant vaccines were allocated to 34 horses each. After vaccination, all horses were monitored for evidence of adverse reactions. Whole blood samples were collected at the time of vaccination and on nine occasions up to six months and 21 days post vaccination. Antibodies against equine influenza were measured by single radial haemolysis. Transient fever and injection site reactions were observed in several horses vaccinated with each vaccine. Only two horses failed to seroconvert post booster vaccination but there was a delayed response to the recombinant vaccine. The antibody response to the recombinant vaccine was lower than that induced by the whole-inactivated and subunit vaccines up to three months post vaccination. Thereafter, there was no significant difference. By six months post vaccination, the majority of horses in all three groups were clinically but not virologically protected. There was minimal decline in antibody titres within the 21-day grace period.

Immunogenicity Measures of Influenza Vaccines: A Study of 1164 Registered Clinical Trials.

Influenza carries an enormous burden each year. Annual influenza vaccination is the best means of reducing this burden. To be clinically effective, influenza vaccines must be immunogenic, and several immunological assays to test their immunogenicity have been developed. This study aimed to describe the patterns of use of the various immunological assays available to measure the influenza vaccine-induced adaptive immune response and to determine its correlates of protection. A total of 76.5% of the studies included in our analysis measured only the humoral immune response. Among these, the hemagglutination-inhibition assay was by far the most widely used. Other, less common, humoral immune response assays were: virus neutralization (21.7%), enzyme-linked immunosorbent (10.1%), single radial hemolysis (4.6%), and assays able to quantify anti-neuraminidase antibodies (1.7%). By contrast, cell-mediated immunity was quantified in only 23.5% of studies. Several variables were significantly associated with the use of single assays. Specifically, some influenza vaccine types (e.g., adjuvanted, live attenuated and cell culture-derived or recombinant), study phase and study sponsorship pattern were usually found to be statistically significant predictors. We discuss the principal findings and make some suggestions from the point of view of the various stakeholders.

Evaluation of Current Equine Influenza Vaccination Protocols Prior to Shipment, Guided by OIE Standards.

To facilitate the temporary importation of horses for competition and racing purposes, with a minimum risk of transmitting equine influenza, the World Organisation for Animal Health (Office International des Epizooties, or OIE), formally engaged in a public–private partnership with the Federation Equestre Internationale (FEI) and the International Federation for Horseracing Authorities (IFHA) to establish, within the context of existing OIE standards, a science-based rationale to identify the ideal time period for equine influenza vaccination prior to shipment. Field trials using vaccines based on different technologies were carried out on three continents. The antibody response post-booster vaccination at intervals aligned with the different rules/recommendations of the OIE, FEI, and IFHA, was monitored by single radial haemolysis. It was determined that 14 days was the optimum period necessary to allow horses adequate time to respond to booster vaccination and for horses that have previously received four or more doses of vaccine and are older than four years, it is adequate to allow vaccination within 180 days of shipment. In contrast, the results indicate that there is a potential benefit to younger (four years old or younger) horses in requiring booster vaccination within 90 days of shipment, consistent with the current OIE standard.

Epidemiological and virological findings during an outbreak of equine influenza in Uruguay in 2018.

Equine influenza is one of the major respiratory infectious diseases in horses. In 2018, equine influenza virus (EIV) was confirmed as the cause of outbreaks of respiratory disease in horses in Chile and Argentina. In the same year, for the first time in Uruguay, EIV infection was confirmed by isolation and molecular analysis to be the cause of respiratory disease among hundreds of clinically affected thoroughbred horses in training and racing facilities. The virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A realtime reverse transcription polymerase chain reaction (rRT-PCR). The partial nucleotide sequence of the haemagglutinin 1 (HA1 ) gene (994 base pairs) was determined and analysed phylogenetically using MEGA X software. Amino acid sequence alignments were constructed, and serum samples were tested by haemagglutination inhibition and single radial haemolysis. The diagnosis of EIV was confirmed by rRT-PCR, virus isolation and serological testing. The phylogenetic analysis of the partial HA1 gene sequence of the isolated virus indicated that it belongs to clade 1 of the Florida sub-lineage of the American lineage and is closely related to viruses isolated in the recent past. Study of the HA1 region (331 amino acids) of the virus identified in horses in racing facilities in Uruguay displayed the highest amino acid sequence identity with viruses detected in Argentina, Chile and the United Kingdom in 2018. The surveillance data reported illustrate the international spread of EIVs and support the recommendation of the World Organisation for Animal Health (OIE) Expert Surveillance Panel to include viruses of the Florida sub-lineage in vaccines.

Influenza Anti-Stalk Antibodies: Development of a New Method for the Evaluation of the Immune Responses to Universal Vaccine.

Growing interest in universal influenza vaccines and novel administration routes has led to the development of alternative serological assays that are able to detect antibodies against conserved epitopes. We present a competitive ELISA method that is able to accurately determine the ratio of serum immunoglobulin G directed against the different domains of the hemagglutinin, the head and the stalk. Human serum samples were treated with two variants of the hemagglutinin protein from the A/California/7/2009 influenza virus. The signals detected were assigned to different groups of antibodies and presented as a ratio between head and stalk domains. A subset of selected sera was also tested by hemagglutination inhibition, single radial hemolysis, microneutralization, and enzyme-linked lectin assays. Pre-vaccination samples from adults showed a quite high presence of anti-stalk antibodies, and the results were substantially in line with those of the classical serological assays. By contrast, pre-vaccination samples from children did not present anti-stalk antibodies, and the majority of the anti-hemagglutinin antibodies that were detected after vaccination were directed against the head domain. The presented approach, when supported by further assays, can be used to assess the presence of specific anti-stalk antibodies and the potential boost of broadly protective antibodies, especially in the case of novel universal influenza vaccine approaches.

A Phase 2/3 double blinded, randomized, placebo-controlled study in healthy adult participants in Vietnam to examine the safety and immunogenicity of an inactivated whole virion, alum adjuvanted, A(H5N1) influenza vaccine (IVACFLU-A/H5N1)

BackgroundA global shortfall of vaccines for avian influenza A(H5N1) would occur, especially in low- and-middle income countries, if a pandemic were to occur. To address this issue, development of a pre-pandemic influenza vaccine was initiated in 2012, leveraging a recently established influenza vaccine manufacturing capacity in Vietnam. MethodsThis was a Phase 2/3, double-blinded, randomized, placebo-controlled study to test the safety and immunogenicity of IVACFLU-A/H5N1 vaccine in healthy adults. Phase 2 was a dose selection study, in which 300 participants were randomized to one of the three groups (15 mcg, 30 mcg, or placebo). Safety and immunogenicity were assessed in all participants. In Phase 3, 630 participants were randomized to receive the IVACFLU-A/H5N1 vaccine dose selected in Phase 2 (15 mcg, n = 525) or placebo (n = 105). Safety was assessed in all Phase 3 participants and immunogenicity was measured in a subset of participants. ResultsThe vaccine was well tolerated and most of the adverse events were mild and of short duration. Mild pain at the injection site was the most common adverse event seen in 60 percent of participants in the vaccine group in Phase 3. In Phase 2, both 15 mcg and 30 mcg doses were immunogenic, so the lower dose was selected for further testing in Phase 3. In Phase 3 overall seroconversion rates were 68 percent for hemagglutination inhibition (HI), 51 percent for microneutralization (MN) and 56 percent for single radial hemolysis (SRH). The seroprotection rates were 44 percent for HI, 41 percent for MN and 55 percent for SRH. The GMT ratio was 5.31 and 3.7 for HI and MN respectively; GMA was 4.75 for the SRH. ConclusionThe IVACFLU A/H5N1 was safe and immunogenic. Development of this pandemic avian influenza vaccine is a welcome addition to the limited global pool of these vaccines.ClinicalTrials.gov register NCT02612909.