Patterns of systemic and local inflammation in patients with asthma hospitalised with influenza.

BACKGROUND: Patients with asthma are at risk of hospitalisation with influenza, but the reasons for this predisposition are unknown. STUDY SETTING: A prospective observational study of adults with PCR-confirmed influenza in 11 UK hospitals, measuring nasal, nasopharyngeal and systemic immune mediators and whole-blood gene expression. RESULTS: Of 133 admissions, 40 (30%) had previous asthma; these were more often female (70% versus 38.7%, OR 3.69, 95% CI 1.67-8.18; p=0.0012), required less mechanical ventilation (15% versus 37.6%, Chi-squared 6.78; p=0.0338) and had shorter hospital stays (mean 8.3 versus 15.3 days, p=0.0333) than those without. In patients without asthma, severe outcomes were more frequent in those given corticosteroids (OR 2.63, 95% CI 1.02-6.96; p=0.0466) or presenting >4 days after disease onset (OR 5.49, 95% CI 2.28-14.03; p=0.0002). Influenza vaccination in at-risk groups (including asthma) were lower than intended by national policy and the early use of antiviral medications were less than optimal. Mucosal immune responses were equivalent between groups. Those with asthma had higher serum interferon (IFN)-α, but lower serum tumour necrosis factor, interleukin (IL)-5, IL-6, CXCL8, CXCL9, IL-10, IL-17 and CCL2 levels (all p<0.05); both groups had similar serum IL-13, total IgE, periostin and blood eosinophil gene expression levels. Asthma diagnosis was unrelated to viral load, IFN-α, IFN-γ, IL-5 or IL-13 levels. CONCLUSIONS: Asthma is common in those hospitalised with influenza, but may not represent classical type 2-driven disease. Those admitted with influenza tend to be female with mild serum inflammatory responses, increased serum IFN-α levels and good clinical outcomes.

Reproducibility of serologic assays for influenza virus A (H5N1).

Hemagglutination-inhibition (HI) and neutralization are used to evaluate vaccines against influenza virus A (H5N1); however, poor standardization leads to interlaboratory variation of results. A candidate antibody standard (07/150) was prepared from pooled plasma of persons given clade 1 A/Vietnam/1194/2004 vaccine. To test human and sheep antiserum, 15 laboratories used HI and neutralization and reassortant A/Vietnam/1194/2004, A/turkey/Turkey/1/2005 (clade 2.2), and A/Anhui/1/2005 (clade 2.3.4) viruses. Interlaboratory variation was observed for both assays, but when titers were expressed relative to 07/150, overall percentage geometric coefficient of variation for A/Vietnam/1194/2004 was reduced from 125% to 61% for HI and from 183% to 81% for neutralization. Lack of reduced variability to clade 2 antigens suggested the need for clade-specific standards. Sheep antiserum as a standard did not reliably reduce variability. The World Health Organization has established 07/150 as an international standard for antibody to clade 1 subtype H5 and has an assigned potency of 1,000 IU/ampoule.

Rapid quantitation of neuraminidase inhibitor drug resistance in influenza virus quasispecies.

BACKGROUND: Emerging resistance of influenza viruses to neuraminidase inhibitors is a concern, both in surveillance of global circulating strains and in treatment of individual patients. Current methodologies to detect resistance rely on the use of cultured virus, thus taking time to complete or lacking the sensitivity to detect mutations in viral quasispecies. Methodology for rapid detection of clinically meaningful resistance is needed to assist individual patient management and to track the transmission of resistant viruses in the community. METHODS: We have developed a pyrosequencing methodology to detect and quantitate influenza neuraminidase inhibitor resistance mutations in cultured virus and directly in clinical material. Our assays target polymorphisms associated with drug resistance in the neuraminidase genes of human influenza A H1N1 as well as human and avian H5N1 viruses. Quantitation can be achieved using viral RNA extracted directly from respiratory or tissue samples, thus eliminating the need for virus culture and allowing the assay of highly pathogenic viruses such as H5N1 without high containment laboratory facilities. RESULTS: Antiviral-resistant quasispecies are detected and quantitated accurately when present in the total virus population at levels as low as 10%. Pyrosequencing is a real-time assay; therefore, results can be obtained within a clinically relevant timeframe and provide information capable of informing individual patient or outbreak management. CONCLUSIONS: Pyrosequencing is ideally suited for early identification of emerging antiviral resistance in human and avian influenza infection and is a useful tool for laboratory surveillance and pandemic preparedness.

Safety and immunogenicity of an inactivated split-virion influenza A/Vietnam/1194/2004 (H5N1) vaccine: phase I randomised trial.

BACKGROUND: Pathogenic avian influenza A virus H5N1 has caused outbreaks in poultry and migratory birds in Asia, Africa, and Europe, and caused disease and death in people. Although person-to-person spread of current H5N1 strains is unlikely, the virus is a potential source of a future influenza pandemic. Our aim was to assess the safety and immunogenicity of a vaccine against the H5N1 strain. METHODS: We did a randomised, open-label, non-controlled phase I trial in 300 volunteers aged 18-40 years and assigned one of six inactivated split influenza A/Vietnam/1194/2004 (H5N1) influenza vaccine formulations, comprising 7.5 microg (with adjuvant n=50, without adjuvant n=49), 15 microg (n=50, n=50), or 30 microg (n=51, n=50) of haemagglutinin with or without aluminium hydroxide adjuvant. Individuals received two vaccinations (on days 0 and 21) and provided blood samples (on days 0, 21, and 42) for analysis by haemagglutination inhibition and microneutralisation. We recorded all adverse events. Analyses were descriptive. FINDINGS: All formulations were well tolerated, with no serious adverse events, few severe reactions, and no oral temperatures of more than 38 degrees C. All formulations induced an immune response, and responses were detectable in some individuals after only one dose. The adjuvanted 30 microg formulation induced the greatest response (67% haemagglutinin-inhibition seroconversion rate after two vaccinations). Adjuvant did not improve the response to the lower doses. Two vaccinations of non-adjuvanted 7.5 microg, adjuvanted 15 microg, or non-adjuvanted 15 microg seroconverted more than 40% of participants (haemagglutinin-inhibition test only). Haemagglutinin inhibition and neutralising results were comparable. INTERPRETATION: A two-dose regimen with an adjuvanted 30 microg inactivated H5N1 vaccine was safe and showed an immune response consistent with European regulatory requirements for licensure of seasonal influenza vaccine. We noted encouraging responses with lower doses of antigen that need further study to ascertain their relevance for the choice of the final pandemic vaccine.

Establishment of a UK National Influenza H5 Laboratory Network.

Avian (H5N1) influenza continues to pose a significant threat to human health, although it remains a zoonotic infection. Sensitive and robust surveillance measures are required to detect any evidence that the virus has acquired the ability to transmit between humans and emerge as the next pandemic strain. An integral part of the pandemic planning response in the UK was the creation in 2005 of the UK National H5 Laboratory Network, capable of rapidly and accurately identifying potential human H5N1 infections in all regions of the UK, and the Republic of Ireland. This review details the challenges that designing molecular detection methods for a rapidly evolving virus present, and the strategic decisions and choices required to ensure successful establishment of a functional national laboratory network, providing round the clock testing for H5N1. Laboratory partnerships have delivered improved real-time one-step multiplex PCR methodologies to ensure streamlined testing capable of not only detecting H5 but also a differential diagnosis of seasonal influenza A/B. A range of fully validated real-time PCR H5 confirmatory assays have been developed to run in parallel with a universal first-screening assay. Regular proficiency panels together with weekly surveillance runs, intermittent on-call testing for suspect cases of avian flu in returning travellers, and several outbreaks of avian influenza outbreaks in poultry that have occurred since 2005 in the UK have fully tested the network and the current diagnostic strategies for avian influenza. The network has clearly demonstrated its capability of delivering a confirmed H5N1 diagnosis within 3-4 h of receipt of a sample, an essential prerequisite for administration of the appropriate antiviral therapy, effective clinical management, disease containment and implementation of infection control measures. A functional network is an important means of enhancing laboratory capability and building diagnostic capacity for a newly emerging pandemic of influenza, and is an essential part of pandemic preparedness.

The UK's multidisciplinary response to an Ebola epidemic.

The West African Ebola virus disease (EVD) epidemic was the largest and most devastating outbreak of EVD the world has ever seen. Its impact was felt far from the shores of Guinea, Liberia and Sierra Leone, with public health systems and clinicians across the globe confronted with an international response both in the affected region and within their own borders. The UK had a prominent role in response efforts, particularly in Sierra Leone. This article highlights how UK academic, health service, military, commercial and public health professionals all played a significant role both at home and abroad.

Safety and antigenicity of whole virus and subunit influenza A/Hong Kong/1073/99 (H9N2) vaccine in healthy adults: phase I randomised trial.

BACKGROUND: In 1999, avian influenza A/Hong Kong/1073/99 (H9N2) virus emerged as a pandemic threat to human beings. We aimed to assess safety, tolerability, and antigenicity of whole virus and subunit H9N2 vaccines in healthy volunteers. METHODS: In a phase I randomised trial we randomly assigned 60 participants to whole virus or subunit H9N2 vaccine. Two doses of 7.5 microg, 15 microg, or 30 microg haemagglutinin influenza A H9N2 vaccine, were given 3 weeks apart. We measured antibody responses by haemagglutination-inhibition and microneutralisation. The primary outcome was geometric mean antibody titre 21 days after vaccination. Analysis was per protocol. FINDINGS: Both vaccines were safe and well tolerated. The antibody titres after vaccination did not differ significantly between subunit and whole virus vaccine. 24 of 60 prevaccination serum samples had unexpected reactivity to H9N2, but only in participants older than 32 years, in whom one dose of either vaccine evoked antibody responses associated with protection. In participants aged 32 years or younger, antibody responses to one dose of whole virus or subunit vaccine were poor, fulfilling none of the criteria used for yearly relicensing of interpandemic vaccines. Whole virus vaccine produced a significantly higher probability of seroconversion compared with subunit virus for this age-group. INTERPRETATION: In immunologically naive patients whole-virus vaccine produced better responses than subunit vaccine. Two doses of subunit or whole virus vaccine would leave a large proportion of the naive population (< or =32 years) unprotected against A/Hong Kong/1073/99 (H9N2). Primed patients should be protected with a single dose of either vaccine.

Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome.

BACKGROUND: The worldwide outbreak of severe acute respiratory syndrome (SARS) is associated with a newly discovered coronavirus, SARS-associated coronavirus (SARS-CoV). We did clinical and experimental studies to assess the role of this virus in the cause of SARS. METHODS: We tested clinical and postmortem samples from 436 SARS patients in six countries for infection with SARS-CoV, human metapneumovirus, and other respiratory pathogens. We infected four cynomolgus macaques (Macaca fascicularis) with SARS-CoV in an attempt to replicate SARS and did necropsies on day 6 after infection. FINDINGS: SARS-CoV infection was diagnosed in 329 (75%) of 436 patients fitting the case definition of SARS; human metapneumovirus was diagnosed in 41 (12%) of 335, and other respiratory pathogens were diagnosed only sporadically. SARS-CoV was, therefore, the most likely causal agent of SARS. The four SARS-CoV-infected macaques excreted SARS-CoV from nose, mouth, and pharynx from 2 days after infection. Three of four macaques developed diffuse alveolar damage, similar to that in SARS patients, and characterised by epithelial necrosis, serosanguineous exudate, formation of hyaline membranes, type 2 pneumocyte hyperplasia, and the presence of syncytia. SARS-CoV was detected in pneumonic areas by virus isolation and RT-PCR, and was localised to alveolar epithelial cells and syncytia by immunohistochemistry and transmission electron microscopy. INTERPRETATION: Replication in SARS-CoV-infected macaques of pneumonia similar to that in human beings with SARS, combined with the high prevalence of SARS-CoV infection in SARS patients, fulfill the criteria required to prove that SARS-CoV is the primary cause of SARS.

Confronting the avian influenza threat: vaccine development for a potential pandemic.

Sporadic human infection with avian influenza viruses has raised concern that reassortment between human and avian subtypes could generate viruses of pandemic potential. Vaccination is the principal means to combat the impact of influenza. During an influenza pandemic the immune status of the population would differ from that which exists during interpandemic periods. An emerging pandemic virus will create a surge in worldwide vaccine demand and new approaches in immunisation strategies may be needed to ensure optimum protection of unprimed individuals when vaccine antigen may be limited. The manufacture of vaccines from pathogenic avian influenza viruses by traditional methods is not feasible for safety reasons as well as technical issues. Strategies adopted to overcome these issues include the use of reverse genetic systems to generate reassortant strains, the use of baculovirus-expressed haemagglutinin or related non-pathogenic avian influenza strains, and the use of adjuvants to enhance immunogenicity. In clinical trials, conventional surface-antigen influenza virus vaccines produced from avian viruses have proved poorly immunogenic in immunologically naive populations. Adjuvanted or whole-virus preparations may improve immunogenicity and allow sparing of antigen.

Influenza-related hospitalizations among young children in Leicestershire.

BACKGROUND: Population-based data on influenza hospitalizations are unavailable in the United Kingdom, but they represent an essential component of health economic analyses that could support the use of vaccines and antiinfluenza drugs in healthy children. We collected data on hospitalizations for influenza infections among young children in Leicester, UK. METHODS: This prospective, longitudinal, noninterventional single center study was conducted at the Children's Hospital in Leicester, which provides inpatient pediatric care to a total population of approximately 1 million. We studied children <6 years of age between October 14, 2001, and June 30, 2002, who were admitted to the hospital with an acute respiratory tract illness, seizures, specified acute febrile gastrointestinal illness or any acute febrile illness or apnea or other life-threatening events in infants <12 months of age. Nasopharyngeal swabs obtained within 24 h of hospital admission were examined for influenza, respiratory syncytial virus and human metapneumovirus by PCR. RESULTS: Of 7165 clinical episodes that were assessed in the Children's Hospital between October 1 and June 30, 2441 (34.1%) were caused by acute respiratory illness. Overall 33 (5.4%) of 613 children analyzed had an influenza A or B virus infection, including 19 (5.0%) of 381 children with acute respiratory illness and 14 (6.0%) of the remaining 232 children. CONCLUSIONS: Influenza is evidently an important cause of hospitalization among young children, even during limited outbreaks of influenza. Further analyses will enable us to estimate age-related admission rates for influenza and to compare the burden from influenza with that for respiratory syncytial virus and human metapneumovirus.

Can a short period of micronutrient supplementation in older institutionalized people improve response to influenza vaccine? A randomized, controlled trial.

OBJECTIVES: To test the hypothesis that a micronutrient supplement can improve seroconversion after influenza immunization in older institutionalized people. DESIGN: : Randomized, double-blind, placebo-controlled study. SETTING: Nursing and residential homes in Liverpool, United Kingdom. PARTICIPANTS: One hundred sixty-four residents aged 60 and older from 31 homes were initially randomized; of these, 119 (72.6%) completed the study. INTERVENTION: Participants were randomized to receive a micronutrient supplement providing the reference nutrient intake for all vitamins and trace elements or identical placebo. Tablets were taken over an 8-week period during September and October 2000; influenza vaccine was administered 4 weeks after their commencement. MEASUREMENTS: The hemagglutination-inhibiting antibody response as defined by a fourfold or greater titer rise over 4 weeks and assessed separately for each of the three antigens contained in the 2000/2001 influenza vaccine (A/New Caledonia/20/99 (H1N1), A/Moscow/10/99 (H3N2), B/Beijing/184/93 (B)). RESULTS: Despite a significant increase in serum concentrations of vitamins A, C, D3, E, folate, and selenium in the supplemented group, there was no significant difference between groups (supplemented vs placebo, respectively) in the proportion of participants seroconverting to H1N1 (41% vs 49%, P=.374), H3N2 (49% vs 58%, P=.343), or B (41% vs 40%, P=.944). CONCLUSION: A micronutrient supplement providing the reference nutrient intake administered over 8 weeks had no beneficial effect on antibody response to influenza vaccine in older people living in long-term care.

Surveillance for antiviral resistance.

In the 10 years since licensure of neuraminidase inhibitor drugs, their use has steadily increased, especially during the pandemic of 2009. Experience now indicates that factors which influence the emergence of high level resistance include the nature of drug binding to target, viral subtype, the use of post exposure prophylaxis and a lack of immunity in the host as seen in children and immunocompromised individuals. These factors point towards targetted surveillance programmes for the early identification of transmissible drug resistance.

Reproducibility of serology assays for pandemic influenza H1N1: collaborative study to evaluate a candidate WHO International Standard.

Haemagglutination-inhibition (HI) and virus neutralisation (VN) assays are used to evaluate immunogenicity of pandemic H1N1 vaccines; however these bioassays are poorly standardised leading to inter-laboratory variation. A candidate International Standard (IS) for antibody to H1N1 pdm virus (09/194) was prepared from pooled sera of subjects who had either recovered from H1N1 pdm infection or who had been immunised with an adjuvanted subunit vaccine prepared from reassortant virus NYMC X-179A (derived from A/California/7/2009 virus). Ten laboratories from seven countries tested the candidate IS, 09/194 and a panel of human sera by HI and VN using the A/California/7/2009 virus (six laboratories) and/or the reassortant virus NYMC X-179A (ten laboratories). As expected, the inter-laboratory variability for HI and VN assay results was high. For results of antibody tests to NYMC X-179A, the % geometric coefficient of variation (%GCV) for 09/194 between laboratories was 83% for HI and 192% for VN. For tests of all sera, the median %GCV ranged from 95 to 345% for HI (80-fold variation) and 204 to 383% for VN (109-fold variation), but for the titres relative to 09/194 the median %GCV was much reduced (HI 34-231%; VN 44-214%). For tests of antibody to the A/California/7/2009 wild type virus there were similar reductions in %GCV when 09/194 was used. These results suggest that 09/194 will be of use to standardise assays of antibody to A/California/7/2009 vaccine and 09/194 has now been established by WHO as an IS for antibody to A/California/7/2009 with an assigned potency of 1300 IU per ml.

Safety and immunogenicity of whole-virus, alum-adjuvanted whole-virus, virosomal, and whole-virus intradermal influenza A/H9N2 vaccine formulations.

Avian influenza H9N2 viruses are considered as a pandemic threat. We assessed the safety and immunogenicity of fourteen H9N2 vaccine formulations. A randomized, phase I trial was done in 353 adults, aged 18-82 years. Subjects received two doses of A/Hong Kong/1073/99 (H9N2) whole-virus, alum-adjuvanted whole-virus, virosomal, or intradermal whole-virus vaccine at four doses (1.7, 5, 15 or 45 microg haemagglutinin). Sera were obtained before and three weeks after each vaccination (days 0, 21, and 42) for haemagglutination-inhibition (HAI) and neutralization assays. All formulations were well tolerated. Pre-vaccination sera from subjects aged below or above 40 years had baseline antibody to H9N2 in 1% and 16% of samples. Compared to intramuscular whole-virus vaccine, alum-adjuvanted vaccine was more immunogenic, intradermal vaccine was comparable, and virosomal vaccine less immunogenic. Among subjects under 40 years, two doses (45, 15, and 5 microg) of alum-adjuvanted vaccine achieved seroprotective HAI titres in 50%, 41%, and 39% respectively, and neutralization seroconversions in 83%, 82%, and 78% of recipients. Among subjects over 40 years, one dose (45, 15, and 5 microg) of alum-adjuvanted vaccine achieved seroprotective HAI titres in 50%, 25% and 0% respectively, and neutralization seroconversions in 88%, 63% and 63% of recipients. Among immunologically naive subjects under 40 years, two doses of vaccine are required and alum-adjuvanted vaccines were most immunogenic. Among immunologically primed subjects over 40 years, one dose of whole-virus or alum-adjuvanted vaccine induced immune responses; the second dose provided less additional benefit. However, no vaccine formulation satisfied all European regulatory criteria for pandemic vaccines.

Fatal cases of influenza a in childhood.

BACKGROUND: In the northern hemisphere winter of 2003-04 antigenic variant strains (A/Fujian/411/02 -like) of influenza A H3N2 emerged. Circulation of these strains in the UK was accompanied by an unusually high number of laboratory confirmed influenza associated fatalities in children. This study was carried out to better understand risk factors associated with fatal cases of influenza in children. METHODOLOGY/PRINCIPAL FINDINGS: Case histories, autopsy reports and death registration certificates for seventeen fatal cases of laboratory confirmed influenza in children were analyzed. None had a recognized pre-existing risk factor for severe influenza and none had been vaccinated. Three cases had evidence of significant bacterial co-infection. Influenza strains recovered from fatal cases were antigenically similar to those circulating in the community. A comparison of protective antibody titres in age stratified cohort sera taken before and after winter 2003-04 showed that young children had the highest attack rate during this season (21% difference, 95% confidence interval from 0.09 to 0.33, p = 0.0009). Clinical incidences of influenza-like illness (ILI) in young age groups were shown to be highest only in the years when novel antigenic drift variants emerged. CONCLUSIONS/SIGNIFICANCE: This work presents a rare insight into fatal influenza H3N2 in healthy children. It confirms that circulating seasonal influenza A H3N2 strains can cause severe disease and death in children in the apparent absence of associated bacterial infection or predisposing risk factors. This adds to the body of evidence demonstrating the burden of severe illness due to seasonal influenza A in childhood.

Safety and immunogenicity of a prototype adjuvanted inactivated split-virus influenza A (H5N1) vaccine in infants and children.

OBJECTIVE: Highly pathogenic avian influenza A virus (H5N1) is a leading candidate for the next influenza pandemic, and infants and children may play an important role in transmission in a pandemic. Our objective was to evaluate the safety and immunogenicity of a prototype inactivated, aluminium adjuvanted, split-virus, clade 1 H5N1 vaccine (A/Vietnam/1194/2004/NIBRG-14) in infants and children aged > or =6 months to < 9 years. METHODS: Healthy infants and children (N=150) received two doses of 30 microg or 45 microg H5 HA with AlPO4 adjuvant 21 days apart. Serum samples were collected for virus microneutralisation (MN) and haemagglutination inhibition (HI) assays on Days 0, 21, and 42. Six-month antibody persistence following second vaccine dose was assessed by MN, and cross-reactive HI antibodies to a clade 2 variant strain (INDO5/RG2) were evaluated at Day 42. FINDINGS: Both formulations were well-tolerated. Two doses of 30 microg or 45 microg H5 HA formulations elicited strong immune responses by both MN (98-99% > or =1:20) and HI assays (95-100% > or =1:32), with 80-87% of children having MN antibody persistence (> or =1:20) up to 6 months post-vaccination. Additionally, robust cross-clade HI antibody responses were elicited following two doses. INTERPRETATION: Two doses of prototype 30 microg or 45 microg aluminium-adjuvanted, H5N1 vaccines were highly immunogenic and well-tolerated, with considerable antibody persistence 6 months after the primary vaccination course. Additional cross-clade HI antibody responses and an acceptable safety and tolerability profile support the use of the either candidate vaccine formulations in infants and children in the event of a pandemic.

Phase I and II randomised trials of the safety and immunogenicity of a prototype adjuvanted inactivated split-virus influenza A (H5N1) vaccine in healthy adults.

OBJECTIVE: The primary objective was to evaluate the safety and immunogenicity of a prototype inactivated, split-virus H5N1 (avian influenza A) vaccine. A secondary objective was to assess the cross-reactivity of immune responses to two variant clade 2 H5N1 strains. METHODS: In two randomised, dose comparison, parallel assignment, multicentre trials conducted in Australia, healthy adult volunteers received two doses of 7.5 microg or 15 microg H5 haemagglutinin (HA) vaccine+/-AlPO4 adjuvant (phase I trial; N=400) or two doses of 30 microg or 45 microg H5 HA with AlPO4 adjuvant (phase II trial; N=400). Revaccination with a booster dose was offered 6 months after dose 2 (phase I trial only). Main outcome measures were the change in immunogenicity at each follow-up visit from baseline, measured using HA inhibition (HI) and virus microneutralisation (MN) assays, and the frequency and nature of adverse events (AEs). Computer generated tables were used to randomly allocate treatments; participants and investigators were blinded to treatment allocation. FINDINGS: All formulations were well-tolerated; no unexpected serious adverse events were reported. Two doses of 30 microg or 45 microg H5 HA adjuvanted formulations elicited the highest immune responses, with considerable MN antibody (>or=1:20) persistence up to 6 months post-vaccination. The 7.5 and 15 microg formulations (+/-adjuvant) were less immunogenic than the higher dose formulations; HI and MN antibody titres decreased to near pre-vaccination levels at 6 months but were restored to post-dose 2 levels after the booster dose. Immune responses in the phase I trial demonstrated modest levels of cross-protective MN antibodies against two currently circulating, distinct clade 2 H5N1 strains. INTERPRETATION: Two doses of prototype 30 microg or 45 microg aluminium-adjuvanted, clade 1 H5N1 vaccines were immunogenic and well-tolerated with considerable 6-month antibody persistence. The prototype H5N1 vaccine also elicited modest levels of cross-protective MN antibodies against variant clade 2 H5N1 strains [ClinicalTrials.gov identifiers: NCT00136331, NCT00320346; FUNDING: CSL Limited, Australia].