Clinical phase II and III studies of an AS03-adjuvanted H5N1 influenza vaccine produced in an EB66® cell culture platform.

BACKGROUND: We have developed an AS03-adjuvanted H5N1 influenza vaccine produced in an EB66® cell culture platform (KD-295). OBJECTIVES: In accordance with Japanese guidelines for development of pandemic prototype vaccines, the phase II study was conducted in a double-blind, randomized, parallel-group comparison study and the phase III study was conducted in an open-label, non-randomized, uncontrolled study. METHODS: Healthy adult volunteers aged 20 - 64 years enrolled in the phase II and III studies (N = 248 and N = 369) received KD-295 intramuscularly twice with a 21-day interval. After administration, immune response and adverse events were evaluated. In the phase II study, four different vaccine formulations were compared: MA (3.75 µg hemagglutinin [HA] antigen + AS03 adjuvant system), MB (3.75 µg HA + 1/2AS03), HA (7.5 µg HA + AS03), and HB (7.5 µg HA + 1/2AS03). In the phase III study, the MA formulation was further evaluated. RESULTS: In the phase II study, all four vaccine formulations were well-tolerated and no SAE related to vaccination were observed. The MA formulation was slightly more immunogenic and less reactogenic among the vaccine formulations. Therefore, the MA formulation was selected for the phase III study, and it was well-tolerated and no serious adverse drug reactions were observed. The vaccine fulfilled the three immunogenicity criteria described in the Japanese guidelines. CONCLUSIONS: These data indicate that the MA formulation of KD-295 was well-tolerated and highly immunogenic and it can be considered a useful pandemic and pre-pandemic influenza vaccine.

Formulation Design, Optimization and In Vivo Evaluations of an α-Tocopherol-Containing Self-Emulsified Adjuvant System using Inactivated Influenza Vaccine.

α-Tocopherol has been used as an immune supplement in humans, as an emulsion adjuvant component in several veterinary vaccines as well as an immunomodulatory component of AS03, an emulsion adjuvant that was used in an H1N1 pandemic vaccine (Pandemrix). AS03 is manufactured using microfluidization and high-pressure homogenization. Such high energy and complex manufacturing processes make it difficult and expensive to produce emulsion adjuvants on a large scale, especially in developing countries. In this study we have explored simpler, comparatively inexpensive methods, to formulate emulsion adjuvants containing α-tocopherol, that have the potential to be made in any well-established scale-up facility. This might facilitate producing and stock-piling adjuvant doses and therefore aide in pandemic preparedness. We used design of experiment as a tool to explore incorporating α-tocopherol into self-emulsified systems containing squalene oil and polysorbate 80. We created novel self-emulsified adjuvant systems (SE-AS) and evaluated their potency in vivo in BALB/c mice with inactivated quadrivalent influenza vaccine (QIV) and tested the cellular and humoral immune responses against the four vaccine strains.

Vaccine adjuvants: Understanding the structure and mechanism of adjuvanticity.

In conjugate, inactivated, recombinant, and toxoid vaccines, adjuvants are extensively and essentially used for enhanced and long-lasting protective immune responses. Depending on the type of diseases and immune responses required, adjuvants with different design strategies are developed. With aluminum salt-based adjuvants as the most used ones in commercial vaccines, other limited adjuvants, e.g., AS01, AS03, AS04, CpG ODN, and MF59, are used in FDA-approved vaccines for human use. In this paper, we review the uses of different adjuvants in vaccines including the ones used in FDA-approved vaccines and vaccines under clinical investigations. We discuss how adjuvants with different formulations could affect the magnitude and quality of adaptive immune response for optimized protection against specific pathogens. We emphasize the molecular mechanisms of various adjuvants, with the aim to establish structure-activity relationships (SARs) for designing more effective and safer adjuvants for both preventative and therapeutic vaccines.

AS03-Adjuvanted H5N1 Avian Influenza Vaccine Modulates Early Innate Immune Signatures in Human Peripheral Blood Mononuclear Cells.

BACKGROUND: Adjuvant System 03 (AS03) markedly enhances responses to influenza A/H5N1 vaccines, but the mechanisms of this enhancement are incompletely understood. METHODS: Using ribonucleic acid sequencing on peripheral blood mononuclear cells (PBMCs) from AS03-adjuvanted and unadjuvanted inactivated H5N1 vaccine recipients, we identified differentially expressed genes, enriched pathways, and genes that correlated with serologic responses. We compared bulk PBMC findings with our previously published assessments of flow-sorted immune cell types. RESULTS: AS03-adjuvanted vaccine induced the strongest differential signals on day 1 postvaccination, activating multiple innate immune pathways including interferon and JAK-STAT signaling, Fcγ receptor (FcγR)-mediated phagocytosis, and antigen processing and presentation. Changes in signal transduction and immunoglobulin genes predicted peak hemagglutinin inhibition (HAI) titers. Compared with individual immune cell types, activated PBMC genes and pathways were most similar to innate immune cells. However, several pathways were unique to PBMCs, and several pathways identified in individual cell types were absent in PBMCs. CONCLUSIONS: Transcriptomic analysis of PBMCs after AS03-adjuvanted H5N1 vaccination revealed early activation of innate immune signaling, including a 5- to 8-fold upregulation of FcγR1A/1B/1C genes. Several early gene responses were correlated with HAI titer, indicating links with the adaptive immune response. Although PBMCs and cell-specific results shared key innate immune signals, unique signals were identified by both approaches.

Evaluation of a primary course of H9N2 vaccine with or without AS03 adjuvant in adults: A phase I/II randomized trial.

BACKGROUND: Avian influenza A H9N2 strains have pandemic potential. METHODS: In this randomized, observer-blind study (ClinicalTrials.gov: NCT01659086), 420 healthy adults, 18-64years of age, received 1 of 10 H9N2 inactivated split-virus vaccination regimens (30 participants per group), or saline placebo (120 participants). H9N2 groups received 2 doses (days 0, 21) of 15µg hemagglutinin (HA) without adjuvant, or 1.9µgHA+AS03A, 1.9µgHA+AS03B, 3.75µgHA+AS03A, or 3.75µgHA+AS03B; followed by the same H9N2 formulation or placebo (day 182). AS03 is an adjuvant system containing α-tocopherol (AS03A: 11.86mg; AS03B: 5.93mg) and squalene in an oil-in-water emulsion. Immunogenicity (hemagglutination inhibition [HI] and microneutralization assays) and safety were assessed up to day 546. RESULTS: All adjuvanted formulations exceeded regulatory immunogenicity criteria at days 21 and 42 (HI assay), with seroprotection and seroconversion rates of ≥94.9% and ≥89.8% at day 21, and 100% and ≥98.1% at day 42. Immunogenicity criteria were also met for unadjuvanted vaccine, with lower geometric mean titers. In groups administered a third vaccine dose (day 182), an anamnestic immune response was elicited with robust increases in HI and microneutralization titers. Injection site pain was reported more frequently with adjuvanted vaccines. No vaccine-related serious adverse events were observed. CONCLUSIONS: All H9N2 vaccine formulations were immunogenic with a clinically acceptable safety profile; adjuvanted formulations were 4-8 times dose-sparing (3.75-1.9vs 15µgHA). TRIAL REGISTRATION: Registered on ClinicalTrials.gov: NCT01659086.

Increased humoral immunity by DNA vaccination using an α-tocopherol-based adjuvant.

DNA vaccines induce broad immunity, which involves both humoral and strong cellular immunity, and can be rapidly designed for novel or evolving pathogens such as influenza. However, the humoral immunogenicity in humans and higher animals has been suboptimal compared with that of traditional vaccine approaches. We tested whether the emulsion-based and α-tocopherol containing adjuvant Diluvac Forte® has the ability to enhance the immunogenicity of a naked DNA vaccine (i.e., plasmid DNA). As a model vaccine, we used plasmids encoding both a surface-exposed viral glycoprotein (hemagglutinin) and an internal non-glycosylated nucleoprotein in the Th1/Th2 balanced CB6F1 mouse model. The naked DNA (50 µg) was premixed at a 1:1 volume/volume ratio with Diluvac Forte®, an emulsion containing different concentrations of α-tocopherol, the emulsion alone or endotoxin-free phosphate-buffered saline (PBS). The animals received 2 intracutaneous immunizations spaced 3 weeks apart. When combined with Diluvac Forte® or the emulsion containing α-tocopherol, the DNA vaccine induced a more potent and balanced immunoglobulin G (IgG)1 and IgG2c response, and both IgG subclass responses were significantly enhanced by the adjuvant. The DNA vaccine also induced CD4+ and CD8+ vaccine-specific T cells; however, the adjuvant did not exert a significant impact. We concluded that the emulsion-based adjuvant Diluvac Forte® enhanced the immunogenicity of a naked DNA vaccine encoding influenza proteins and that the adjuvant constituent α-tocopherol plays an important role in this immunogenicity. This induction of a potent and balanced humoral response without impairment of cellular immunity constitutes an important advancement toward effective DNA vaccines.

Adjuvanted (AS03) A/H1N1 2009 Pandemic Influenza Vaccines and Solid Organ Transplant Rejection: Systematic Signal Evaluation and Lessons Learnt.

INTRODUCTION: We investigated a signal of solid organ transplant (SOT) rejection after immunisation with (AS03) A/H1N1 2009 pandemic influenza vaccines. METHODS: Potential immunological mechanisms were reviewed and quantitative analyses were conducted. The feasibility of pharmacoepidemiological studies was explored. RESULTS: Overall results, including data from a pharmacoepidemiological study, support the safety of adjuvanted (AS03) pandemic influenza vaccination in SOT recipients. The regulatory commitment to evaluate the signal through a stepwise investigation was closed in 2014. CONCLUSION: Lessons learned highlight the importance of investigating plausible biological mechanisms between vaccines and potentially associated adverse outcomes, and the importance of selecting appropriate study settings and designs for safety signal investigations.

Persistence of Antibody to Influenza A/H5N1 Vaccine Virus: Impact of AS03 Adjuvant.

The adjuvant AS03 is stockpiled for future formulations with new and existing vaccines for the control of pandemic influenza virus. We previously reported the immunogenicity of an A/H5N1 vaccine extemporaneously mixed with the AS03 adjuvant for 42 days following vaccination. This report extends those findings to 1 year after vaccination.

A clinical phase I study of an EB66 cell-derived H5N1 pandemic vaccine adjuvanted with AS03.

BACKGROUND: We conducted a phase I clinical trial of a cell culture-derived AS03-adjuvanted influenza vaccine containing HA antigen (A/Indonesia/05/2005(H5N1)/PR8-IBCDC-RG2) derived from EB66 cells (KD-295). METHODS: Healthy male adult volunteers (20-40 years old, N=60) enrolled in the study were divided into 3 groups, the MA group (3.8 µg of HA+AS03), HA group (7.5 µg of HA+AS03), and 1/2 MA group (half the volume of the MA group), and received KD-295 intramuscularly twice with a 21-day interval. After administration of KD-295, adverse events, clinical laboratory parameters, and immune response to the vaccine strain and heterologous virus strains were evaluated. RESULTS: No severe adverse events leading to discontinuation of vaccine administration occurred. The vaccine was well-tolerated. There was no dose dependency in the rate, timing, or duration of the adverse events. Immunogenicity of the vaccines was evaluated by HI (hemagglutination inhibition) assay, which confirmed that the antibody response to the vaccine strain and heterologous strain in all groups met the three criteria for immunogenicity described in the Japanese guidelines for development of a pandemic prototype vaccine. We also measured the neutralizing antibody titers against several virus strains, and confirmed a significant rise in antibody levels to both the vaccine strain and heterologous strains. CONCLUSION: The EB66-derived H5N1 influenza vaccine adjuvanted with AS03 elicited a broad cross-reactive antibody response among H5N1 strains with acceptable reactogenicity. Therefore, KD-295 can be considered a useful pandemic and pre-pandemic influenza vaccine candidate.

Single dose vaccination of the ASO3-adjuvanted A(H1N1)pdm09 monovalent vaccine in health care workers elicits homologous and cross-reactive cellular and humoral responses to H1N1 strains.

Healthcare workers (HCW) were prioritized for vaccination during the 2009 influenza A(H1N1)pdm09 pandemic. We conducted a clinical trial in October 2009 where 237 HCWs were immunized with a AS03-adjuvanted A(H1N1)pdm09 monovalent vaccine. In the current study, we analyzed the homologous and cross-reactive H1N1 humoral responses using prototype vaccine strains dating back to 1977 by the haemagglutinin inhibition (HI), single radial hemolysis SRH), antibody secreting cell (ASC) and memory B cell (MBC) assays. The cellular responses were assessed by interferon-γ (IFN-γ) ELISPOT and by intracellular staining (ICS) for the Th1 cytokines IFN-γ, interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). All assays were performed using blood samples obtained prior to (day 0) and 7, 14 and 21 d post-pandemic vaccination, except for ASC (day 7) and ICS (days 0 and 21). Vaccination elicited rapid HI, SRH and ASC responses against A(H1N1)pdm09 which cross reacted with seasonal H1N1 strains. MBC responses were detected against the homologous and seasonal H1N1 strains before vaccination and were boosted 2 weeks post-vaccination. An increase in cellular responses as determined by IFN-γ ELISPOT and ICS were observed 1-3 weeks after vaccination. Collectively, our data show that the AS03-adjuvanted A(H1N1)pdm09 vaccine induced rapid cellular and humoral responses against the vaccine strain and the response cross-reacted against prototype H1N1 strains dating back to 1977.

Immunogenicity and safety of an AS03-adjuvanted H5N1 pandemic influenza vaccine in Korean adults: a phase IV, randomized, open-label, controlled study.

BACKGROUND: AS03-adjuvanted H5N1 pandemic influenza vaccines have been assessed in an extensive clinical development program conducted in North America, Europe, and Asia including children from 6 months of age, adults, and elderly adults. We evaluated AS03-H5N1 in Korean adults 18 through 60 years of age. METHODS: This Phase IV, randomized, study was conducted to assess the immunogenicity, reactogenicity, and safety of two doses (3.75µg of hemagglutinin antigen) of A/Indonesia/5/2005 (H5N1) adjuvanted with AS03 given 21 days apart in Korean adults. Antibody responses were assessed using hemagglutination-inhibition (HI) assays against the vaccine strain and a vaccine-heterologous strain (A/Vietnam/1194/2004) 21 days after the second dose. A control group (safety) received a licensed seasonal inactivated trivalent influenza vaccine (TIV). Reactogenicity was assessed for 7 days after each vaccination, and unsolicited adverse events were assessed for 182 days following vaccination in both study groups (NCT01730378). RESULTS: AS03-H5N1 was immunogenic and elicited robust HI antibody responses with seroconversion rates of 100% for the vaccine strain and 69.1% for the heterologous strain (N=81). HI antibody responses fulfilled the European licensure criteria for immunogenicity (primary endpoint). The incidence of local and systemic solicited adverse events (reactogenicity) was higher with AS03-H5N1 than TIV. There was no apparent difference in the rate of unsolicited adverse events in the AS03-H5N1 and TIV groups. CONCLUSION: The results indicate that AS03-H5N1 vaccine is immunogenic with reactogenicity and safety findings that are consistent with the established profile of AS03-H5N1 vaccine.

Non-clinical safety and biodistribution of AS03-adjuvanted inactivated pandemic influenza vaccines.

Pandemic-influenza vaccines containing split-inactivated-virus antigen have been formulated with the immunostimulatory Adjuvant System AS03 to enhance the antigen immunogenicity and reduce antigen content per dose. AS03 is an oil-in-water emulsion containing α-tocopherol, squalene and polysorbate 80. To support the clinical development of AS03-adjuvanted pandemic-influenza vaccines, the local and systemic toxicity of test articles containing split-influenza A(H5N1) and/or AS03 were evaluated after 3-4 intramuscular (i.m.) injections in rabbits. Treatment-related effects were restricted to mild inflammatory responses and were induced primarily by the test articles containing AS03. The injection-site inflammation was mild at 3 days, and minimal at 4 weeks after the last injection; and was reflected by signs of activation in the draining lymph nodes and by systemic effects in the blood including a transient increase of neutrophils. In addition, a study in mice explored the biodistribution of A(H5N1) vaccines or AS03 through radiolabelling the antigen or constituents of AS03 prior to injection. In this evaluation, 57-73% of AS03's principal constituents had cleared from the injection site 3 days after injection, and their different clearance kinetics were suggestive of AS03's dissociation. All these AS03 constituents entered into the draining lymph nodes within 30 min after injection. In conclusion, the administration of repeated doses of the H5N1/AS03 vaccine was well tolerated in the rabbit, and was primarily associated with transient mild inflammation at the injection site and draining lymph nodes. The biodistribution kinetics of AS03 constituents in the mouse were consistent with AS03 inducing this pattern of inflammation.

Phase II, randomized, open, controlled study of AS03-adjuvanted H5N1 pre-pandemic influenza vaccine in children aged 3 to 9 years: follow-up of safety and immunogenicity persistence at 24 months post-vaccination.

BACKGROUND: An AS03-adjuvanted H5N1 influenza vaccine elicited broad and persistent immune responses with an acceptable safety profile up to 6 months following the first vaccination in children aged 3-9 years. METHODS: In this follow-up of the Phase II study, we report immunogenicity persistence and safety at 24 months post-vaccination in children aged 3-9 years. The randomized, open-label study assessed two doses of H5N1 A/Vietnam/1194/2004 influenza vaccine (1·9 µg or 3·75 µg hemagglutinin antigen) formulated with AS03A or AS03B (11·89 mg or 5·93 mg tocopherol, respectively). Control groups received seasonal trivalent influenza vaccine. Safety was assessed prospectively and included potential immune-mediated diseases (pIMDs). Immunogenicity was assessed by hemagglutination-inhibition assay 12 and 24 months after vaccination; cross-reactivity and cell-mediated responses were also assessed. (NCT00502593). RESULTS: The safety population included 405 children. Over 24 months, five events fulfilled the criteria for pIMDs, of which four occurred in H5N1 vaccine recipients, including uveitis (n = 1) and autoimmune hepatitis (n = 1), which were considered to be vaccine-related. Overall, safety profiles of the vaccines were clinically acceptable. Humoral immune responses at 12 and 24 months were reduced versus those observed after the second dose of vaccine, although still within the range of those observed after the first dose. Persistence of cell-mediated immunity was strong, and CD4(+) T cells with a TH 1 profile were observed. CONCLUSIONS: Two doses of an AS03-adjuvanted H5N1 influenza vaccine in children showed low but persistent humoral immune responses and a strong persistence of cell-mediated immunity, with clinically acceptable safety profiles up to 24 months following first vaccination.

Long-term booster schedules with AS03A-adjuvanted heterologous H5N1 vaccines induces rapid and broad immune responses in Asian adults.

BACKGROUND: The pandemic potential of avian influenza A/H5N1 should not be overlooked, and the continued development of vaccines against these highly pathogenic viruses is a public health priority. METHODS: This open-label extension booster study followed a Phase III study of 1206 adults who had received two 3.75 µg doses of primary AS03A-adjuvanted or non-adjuvanted H5N1 split-virus vaccine (A/Vietnam/1194/2004; clade 1) (NCT00449670). The aim of the extension study was to evaluate different timings for heterologous AS03A-adjuvanted booster vaccination (A/Indonesia/5/2005; clade 2.1) given at Month 6, 12, or 36 post-primary vaccination. Immunogenicity was assessed 21 days after each booster vaccination and the persistence of immune responses against the primary vaccine strain (A/Vietnam) and the booster strain (A/Indonesia) was evaluated up to Month 48 post-primary vaccination. Reactogenicity and safety were also assessed. RESULTS: After booster vaccination given at Month 6, HI antibody responses to primary vaccine, and booster vaccine strains were markedly higher with one dose of AS03A-H5N1 booster vaccine in the AS03A-adjuvanted primary vaccine group compared with two doses of booster vaccine in the non-adjuvanted primary vaccine group. HI antibody responses were robust against the primary and booster vaccine strains 21 days after boosting at Month 12 or 36. At Month 48, in subjects boosted at Month 6, 12, or 36, HI antibody titers of ≥1:40 against the booster strain persisted in 39.2%, 61.2%, and 95.6% of subjects, respectively. Neutralizing antibody responses and cell-mediated immune responses also showed that AS03A-H5N1 heterologous booster vaccination elicited robust immune responses within 21 days of boosting at Month 6, 12, or 36 post-primary vaccination. The booster vaccine was well tolerated, and no safety concerns were raised. CONCLUSIONS: In Asian adults primed with two doses of AS03A-adjuvanted H5N1 pandemic influenza vaccine, strong cross-clade anamnestic antibody responses were observed after one dose of AS03A-H5N1 heterologous booster vaccine given at Month 6, 12, or 36 after priming, suggesting that AS03A-adjuvanted H5N1 vaccines may provide highly flexible prime-boost schedules. Although immunogenicity decreased with time, vaccinated populations could potentially be protected for up to three years after vaccination, which is likely to far exceed the peak of the a pandemic.

Narcolepsy, 2009 A(H1N1) pandemic influenza, and pandemic influenza vaccinations: what is known and unknown about the neurological disorder, the role for autoimmunity, and vaccine adjuvants.

The vaccine safety surveillance system effectively detected a very rare adverse event, narcolepsy, in subjects receiving AS03-adjuvanted A(H1N1) pandemic vaccine made using the European inactivation/purification protocol. The reports of increased cases of narcolepsy in non-vaccinated subjects infected with wild A(H1N1) pandemic influenza virus suggest a role for the viral antigen(s) in disease development. However, additional investigations are needed to better understand what factor(s) in wild influenza infection trigger(s) narcolepsy in susceptible hosts. An estimated 31 million doses of European AS03-adjuvanted A(H1N1) pandemic vaccine were used in more than 47 countries. The Canadian AS03-adjuvanted A(H1N1) pandemic vaccine was used with high coverage in Canada where an estimated 12 million doses were administered. As no similar narcolepsy association has been reported to date with the AS03-adjuvanted A(H1N1) pandemic vaccine made using the Canadian inactivation/purification protocol, this suggests that the AS03 adjuvant alone may not be responsible for the narcolepsy association. To date, no narcolepsy association has been reported with the MF59®-adjuvanted A(H1N1) pandemic vaccine. This review article provides a brief background on narcolepsy, outlines the different types of vaccine preparations including the ones for influenza, reviews the accumulated evidence for the safety of adjuvants, and explores the association between autoimmune diseases and natural infections. It concludes by assimilating the historical observations and recent clinical studies to formulate a feasible hypothesis on why vaccine-associated narcolepsy may not be solely linked to the AS03 adjuvant but more likely be linked to how the specific influenza antigen component of the European AS03-adjuvanted pandemic vaccine was prepared. Careful and long-term epidemiological studies of subjects who developed narcolepsy in association with AS03-adjuvanted A(H1N1) pandemic vaccine prepared with the European inactivation/purification protocol are needed.

Vitamin E, vitamin A, and zinc status are not related to serologic response to influenza vaccine in older adults: an observational prospective cohort study.

It has been hypothesized that micronutrient levels play a role in the immune response to vaccination; however, population-level research on the association between micronutrient levels and immune response to influenza vaccination is needed. In this study, we hypothesized that decreasing levels of nutrients would be associated with decreased hemagglutination inhibition (HAI) responses to influenza vaccination. Therefore, the purpose of this study was to determine whether serum vitamin A, vitamin E, or zinc levels are associated with influenza vaccine response determined by HAI titer in adults 65 years or older. Participants in this study included 205 community-dwelling adults 65 years or older who resided in Marshfield, WI, USA, from fall 2008 through spring 2009. Participants received trivalent influenza vaccine and donated blood samples before and 21 to 28 days after vaccination. Prevaccination levels of serum retinol, α-tocopherol, and zinc as well as prevaccination and postvaccination HAI titer levels were measured. No participants were vitamin A or vitamin E deficient; 20% had low serum zinc levels (<70 µg/dL). Continuous variables and categorical quartiles coding for vitamin A, vitamin E, and zinc levels were not related to prevaccination or postvaccination seroprotection or seroconversion for any of the vaccine components (influenza A [H1N1], A [H3N2], or B), after adjusting for age, sex, body mass index, and prevaccination HAI geometric mean titer. In conclusion, our study population showed no association between variations in levels of serum vitamin A, vitamin E, or zinc and influenza vaccine response as measured by HAI in adults older than 65 years. Thus, associations between micronutrients and other measures of vaccine response, such as cell-mediated immune parameters, should also be explored.

Long-term immunogenicity of an AS03-adjuvanted influenza A(H1N1)pdm09 vaccine in young and elderly adults: an observer-blind, randomized trial.

BACKGROUND: This study (NCT00979602) evaluated the immunogenicity and relative protective efficacy of one dose of influenza A(H1N1)pdm09 vaccine with or without AS03 (an α-tocopherol oil-in-water emulsion based Adjuvant System). METHODS: Four thousands and forty-eight healthy adults aged ≥ 18 years were randomized (1:1) to receive one dose of either the adjuvanted split virion (3.75 µg hemagglutinin antigen [HA]/AS03) or non-adjuvanted (15 µg HA) vaccine. Hemagglutination inhibition [HI] antibody response was evaluated before vaccination and at Days 21, 42 and 182 (Month 6). Safety of the study vaccines was evaluated during the entire study duration. RESULTS: At Day 21, both study vaccines induced HI immune responses meeting the US regulatory criteria in subjects 18-64 years (seroprotection rate [SPR]: 98.0% [97.1-98.6]; seroconversion rate [SCR]: 89.7% [88.0-91.2] in the AS03-adjuvanted group; SPR: 91.4% [89.9-92.8]; SCR: 74.6% [72.3-76.9] in the non-adjuvanted group) and >64 years of age (SPR: 86.0% [82.5-89.0]; SCR: 75.3% [71.1-79.2] in the AS03-adjuvanted group; SPR: 69.1% [64.6-73.3]; SCR: 56.7% [52.0-61.3] in the non-adjuvanted group). The AS03-adjuvanted vaccine induced higher HI geometric mean titers than the non-adjuvanted vaccine at all time points. At Month 6, only subjects 18-64 years of age from both vaccine groups still met the US regulatory criteria (SPR: 82.1% [80.0-84.1]; SCR: 62.3% [59.6-64.8] in the AS03-adjuvanted group; SPR: 75.3% [72.9-77.5]; SCR: 53.7% [51.0-56.4] in the non-adjuvanted group). Protective efficacy was not evaluated due to low number of RT-qPCR-confirmed A(H1N1)pdm09 influenza cases. Through Month 12, 216 serious adverse events (in 157 subjects: 84 in the AS03-adjuvanted and 73 in the non-adjuvanted group) and 12 potentially immune mediated diseases (5 in the AS03-adjuvanted and 7 in the non-adjuvanted group) were reported. CONCLUSION: A single dose of either adjuvanted or non-adjuvanted influenza A(H1N1)pdm09 vaccine induced protective HI antibody levels against the A/California/7/2009 strain that persisted through Month 6 in the 18-64 years population.

Long-term persistence of humoral and cellular immune responses induced by an AS03A-adjuvanted H1N1 2009 influenza vaccine: an open-label, randomized study in adults aged 18-60 years and older.

This manuscript presents data on the persistence of Hemagglutination Inhibition (HI) immune response against the A/California/7/2009 strain, six and 12 mo after adults received one dose (n = 138) or two doses (n = 102; 21 d apart) of a 3.75 µg Hemagglutinin antigen AS03-adjuvanted H1N1 2009 vaccine (NCT00968526). Two hundred forty subjects (18-60 y: 120;>60 y: 120) were vaccinated. Immunogenicity end points were based on the European licensure criteria for pandemic influenza vaccines. Exploratory analyses assessed the cell-mediated immune response (CMI) up to Month 12 and the influence of previous influenza vaccination on persistence of immune response. At Month 6, the CHMP criteria were met in subjects aged 18-60 y who received one or two vaccine doses and in subjects aged>60 y who received two vaccine doses. At Month 12, the CHMP criteria were met only in subjects aged 18-60 y who received two vaccine doses. Persistence of HI immune response against the vaccine strain was higher in subjects without prior influenza vaccination. Exploratory analyses showed that two doses of the H1N1 2009 vaccine induced persistence of H1N1-specific CD4+ T cells up to Month 6 and memory B cells up to Month 12. In conclusion, HI immune responses persisted up to 12 mo after vaccination with one-dose and two-dose regimens of the AS03-adjuvanted 3.75 µg HA H1N1 2009 pandemic influenza vaccine, although not all three CHMP guidance criteria for both groups were met at Month 6 and Month 12. The CD4+ T cell and B cell responses also persisted up to Month 12.

AS03-adjuvanted versus non-adjuvanted inactivated trivalent influenza vaccine against seasonal influenza in elderly people: a phase 3 randomised trial.

BACKGROUND: We aimed to compare AS03-adjuvanted inactivated trivalent influenza vaccine (TIV) with non-adjuvanted TIV for seasonal influenza prevention in elderly people. METHODS: We did a randomised trial in 15 countries worldwide during the 2008-09 (year 1) and 2009-10 (year 2) influenza seasons. Eligible participants aged at least 65 years who were not in hospital or bedridden and were without acute illness were randomly assigned (1:1) to receive either AS03-adjuvanted TIV or non-adjuvanted TIV. Randomisation was done in an internet-based system, with a blocking scheme and stratification by age (65-74 years and 75 years or older). Participants were scheduled to receive one vaccine in each year, and remained in the same group in years 1 and 2. Unmasked personnel prepared and gave the vaccines, but participants and individuals assessing any study endpoint were masked. The coprimary objectives were to assess the relative efficacy of the vaccines and lot-to-lot consistency of the AS03-adjuvanted TIV (to be reported elsewhere). For the first objective, the primary endpoint was relative efficacy of the vaccines for prevention of influenza A (excluding A H1N1 pdm09) or B, or both, that was confirmed by PCR analysis in year 1 (lower limit of two-sided 95% CI had to be greater than zero to establish superiority). From Nov 15, to April 30, in both years, participants were monitored by telephone or site contact and home visits every week or 2 weeks to identify cases of influenza-like illness. After onset of suspected cases, we obtained nasal and throat swabs to identify influenza RNA with real-time PCR. Efficacy analyses were done per protocol. This trial is registered with ClinicalTrials.gov, number NCT00753272. FINDINGS: We enrolled 43 802 participants, of whom 21 893 were assigned to and received the AS03-adjuvanted TIV and 21 802 the non-adjuvanted TIV in year 1. In the year 1 efficacy cohort, fewer participants given AS03-adjuvanted than non-adjuvanted TIV were infected with influenza A or B, or both (274 [1·27%, 95% CI 1·12-1·43] of 21 573 vs 310 [1·44%, 1·29-1·61] of 21 482; relative efficacy 12·11%, 95% CI -3·40 to 25·29; superiority not established). Fewer participants in the year 1 efficacy cohort given AS03-adjuvanted TIV than non-adjuvanted TIV were infected with influenza A (224 [1·04%, 95% CI 0·91-1·18] vs 270 [1·26, 1·11-1·41]; relative efficacy 17·53%, 95% CI 1·55-30·92) and influenza A H3N2 (170 [0·79, 0·67-0·92] vs 205 [0·95, 0·83-1·09]; post-hoc analysis relative efficacy 22·0%, 95% CI 5·68-35·49). INTERPRETATION: AS03-adjuvanted TIV has a higher efficacy for prevention of some subtypes of influenza than does a non-adjuvanted TIV. Future influenza vaccine studies in elderly people should be based on subtype or lineage-specific endpoints. FUNDING: GlaxoSmithKline Biologicals SA.

Multi-centre observational study of transplacental transmission of influenza antibodies following vaccination with AS03(A)-adjuvanted H1N1 2009 vaccine.

INTRODUCTION: Illness and death from influenza increase during pregnancy. In the United Kingdom pregnant women were targeted in a national programme for vaccination during the H1N1 2009-10 pandemic. METHODS: In this study, pregnant women were recruited in labour from November 9, 2009 to March 10, 2010. Pandemic vaccination status was determined. Venous cord blood collected at delivery was evaluated for transplacental transfer of antibodies by measurement of haemagglutination inhibition and microneutralization titres. RESULTS: Samples were collected from 77 vaccinated and 27 unvaccinated women. Seroprotection (HI titre ≥1:40) was detected in 58 (75.3%, 95% CI 64.2-84.4) cord blood samples from vaccinated women and 5 (18.5%, 95% CI 6.3-38.1) from unvaccinated women (P<0.0001). There was evidence of transplacental seroprotection 8 days after maternal immunization (77.9%, 95 CI 66.2-87.1), maintained in most cases for at least 16 weeks. DISCUSSION: Immunization of pregnant women with AS03(A)-adjuvanted vaccine is followed by transplacental transfer of passive immunity at titres consistent with clinical protection in three-quarters of new-born infants. The findings support national and international pandemic H1N1 2009 recommendations for immunization during pregnancy.