Differential Regulation of DC Function, Adaptive Immunity, and MyD88 Dependence by MF59 and AS03-like Adjuvants.

MF59 and AS03 are squalene emulsion-based vaccine adjuvants with similar compositions and droplet sizes. Despite their broad use in licensed influenza vaccines, few studies compared their adjuvant effects and action mechanisms side by side. Considering the majority of adjuvants act on dendritic cells (DCs) to achieve their adjuvant effects, this study compared MF59 and AS03-like adjuvants (AddaVax and AddaS03, respectively) to enhance antigen uptake, DC maturation, ovalbumin (OVA) and seasonal influenza vaccine-induced immune responses. Considering MF59 was reported to activate MyD88 to mediate its adjuvant effects, this study also investigated whether the above-explored adjuvant effects of AddaVax and AddaS03 depended on MyD88. We found AddaVax more potently enhanced antigen uptake at the local injection site, while AddaS03 more potently enhanced antigen uptake in the draining lymph nodes. AddaS03 but not AddaVax stimulated DC maturation. Adjuvant-enhanced antigen uptake was MyD88 independent, while AddaS03-induced DC maturation was MyD88 dependent. AddaVax and AddaS03 similarly enhanced OVA-induced IgG and subtype IgG1 antibody responses as well as influenza vaccine-induced hemagglutination inhibition antibody titers, whileAddaS03 more potently enhanced OVA-specific IgG2c antibody responses. Both adjuvants depended on MyD88 to enhance vaccine-induced antibody responses, while AddaVax depended more on MyD88 to achieve its adjuvant effects. Our study reveals similarities and differences of the two squalene emulsion-based vaccine adjuvants, contributing to our improved understanding of their action mechanisms.

Downregulation of hypocretin/orexin after H1N1 Pandemrix vaccination of adolescent mice.

Narcolepsy type 1 (NT1), characterized by the loss of hypocretin/orexin (HCRT) production in the lateral hypothalamus, has been linked to Pandemrix vaccination during the 2009 H1N1 pandemic, especially in children and adolescents. It is still unknown why this vaccination increased the risk of developing NT1. This study investigated the effects of Pandemrix vaccination during adolescence on Hcrt mRNA expression in mice. Mice received a primary vaccination (50 µL i.m.) during prepubescence and a booster vaccination during peri-adolescence. Hcrt expression was measured at three-time points after the vaccinations. Control groups included both a saline group and an undisturbed group of mice. Hcrt expression was decreased after both Pandemrix and saline injections, but 21 days after the second injection, the saline group no longer showed decreased Hcrt expression, while the Pandemrix group still exhibited a significant reduction of about 60% compared to the undisturbed control group. This finding suggests that Pandemrix vaccination during adolescence influences Hcrt expression in mice into early adulthood. The Hcrt mRNA level did not reach the low levels known to induce NT1 symptoms, instead, our finding supports the multiple-hit hypothesis of NT1 that states that several insults to the HCRT system may be needed to induce NT1 and that Pandemrix could be one such insult.

Metabolomic Signatures Differentiate Immune Responses in Avian Influenza Vaccine Recipients.

BACKGROUND: Avian influenza viruses pose significant risk to human health. Vaccines targeting the hemagglutinin of these viruses are poorly immunogenic without the use of adjuvants. METHODS: Twenty healthy men and women (18-49 years of age) were randomized to receive two doses of inactivated influenza A/H5N1 vaccine alone (IIV) or with AS03 adjuvant (IIV-AS03) one month apart. Urine and serum samples were collected on day 0 and on days 1, 3, and 7 following first vaccination and subjected to metabolomics analyses to identify metabolites, metabolic pathways, and metabolite clusters associated with immunization. RESULTS: Seventy-three differentially abundant (DA) serum and 88 urine metabolites were identified for any post-vaccination day comparison. Pathway analysis revealed enrichment of tryptophan, tyrosine and nicotinate metabolism in urine and serum among IIV-AS03 recipients. Increased urine abundance of 4-vinylphenol sulfate on Day 1 was associated with serologic response based on hemagglutination inhibition responses. In addition, 9 DA urine metabolites were identified in participants with malaise compared to those without. CONCLUSIONS: Our findings suggest that tryptophan, tyrosine, and nicotinate metabolism are upregulated among IIV-AS03 recipients compared with IIV alone. Metabolites within these pathways may serve as measures of immunogenicity and may provide mechanistic insights for adjuvanted vaccines.

Safety and immunogenicity of a variant-adapted SARS-CoV-2 recombinant protein vaccine with AS03 adjuvant as a booster in adults primed with authorized vaccines: a phase 3, parallel-group study.

Background: In a parallel-group, international, phase 3 study (ClinicalTrials.govNCT04762680), we evaluated prototype (D614) and Beta (B.1.351) variant recombinant spike protein booster vaccines with AS03-adjuvant (CoV2 preS dTM-AS03). Methods: Adults, previously primed with mRNA (BNT162b2, mRNA-1273), adenovirus-vectored (Ad26.CoV2.S, ChAdOx1nCoV-19) or protein (CoV2 preS dTM-AS03 [monovalent D614; MV(D614)]) vaccines were enrolled between 29 July 2021 and 22 February 2022. Participants were stratified by age (18-55 and ≥ 56 years) and received one of the following CoV2 preS dTM-AS03 booster formulations: MV(D614) (n = 1285), MV(B.1.351) (n = 707) or bivalent D614 + B.1.351 (BiV; n = 625). Unvaccinated adults who tested negative on a SARS-CoV-2 rapid diagnostic test (control group, n = 479) received two primary doses, 21 days apart, of MV(D614). Anti-D614G and anti-B.1.351 antibodies were evaluated using validated pseudovirus (lentivirus) neutralization (PsVN) assay 14 days post-booster (day [D]15) in 18-55-year-old BNT162b2-primed participants and compared with those pre-booster (D1) and on D36 in 18-55-year-old controls (primary immunogenicity endpoints). PsVN titers to Omicron BA.1, BA.2 and BA.4/5 subvariants were also evaluated. Safety was evaluated over a 12-month follow-up period. Planned interim analyses are presented up to 14 days post-last vaccination for immunogenicity and over a median duration of 5 months for safety. Findings: All three boosters elicited robust anti-D614G or -B.1.351 PsVN responses for mRNA, adenovirus-vectored and protein vaccine-primed groups. Among BNT162b2-primed adults (18-55 years), geometric means of the individual post-booster versus pre-booster titer ratio (95% confidence interval [CI]) were: for MV (D614), 23.37 (18.58-29.38) (anti-D614G); for MV(B.1.351), 35.41 (26.71-46.95) (anti-B.1.351); and for BiV, 14.39 (11.39-18.28) (anti-D614G) and 34.18 (25.84-45.22 (anti-B.1.351). GMT ratios (98.3% CI) versus post-primary vaccination GMTs in controls, were: for MV(D614) booster, 2.16 (1.69; 2.75) [anti-D614G]; for MV(B.1.351), 1.96 (1.54; 2.50) [anti-B.1.351]; and for BiV, 2.34 (1.84; 2.96) [anti-D614G] and 1.39 (1.09; 1.77) [anti-B.1.351]. All booster formulations elicited cross-neutralizing antibodies against Omicron BA.2 (across priming vaccine subgroups), Omicron BA.1 (BNT162b2-primed participants) and Omicron BA.4/5 (BNT162b2-primed participants and MV D614-primed participants). Similar patterns in antibody responses were observed for participants aged ≥56 years. Reactogenicity tended to be transient and mild-to-moderate severity in all booster groups. No safety concerns were identified. Interpretation: CoV2 preS dTM-AS03 boosters demonstrated acceptable safety and elicited robust neutralizing antibodies against multiple variants, regardless of priming vaccine. Funding: Sanofi and Biomedical Advanced Research and Development Authority (BARDA).

Using Dual Toll-like Receptor Agonism to Drive Th1-Biased Response in a Squalene- and α-Tocopherol-Containing Emulsion for a More Effective SARS-CoV-2 Vaccine.

A diversity of vaccines is necessary to reduce the mortality and morbidity of SARS-CoV-2. Vaccines must be efficacious, easy to manufacture, and stable within the existing cold chain to improve their availability around the world. Recombinant protein subunit vaccines adjuvanted with squalene-based emulsions such as AS03™ and MF59™ have a long and robust history of safe, efficacious use with straightforward production and distribution. Here, subunit vaccines were made with squalene-based emulsions containing novel, synthetic toll-like receptor (TLR) agonists, INI-2002 (TLR4 agonist) and INI-4001 (TLR7/8 agonist), using the recombinant receptor-binding domain (RBD) of SARS-CoV-2 S protein as an antigen. The addition of the TLR4 and TLR7/8 agonists, alone or in combination, maintained the formulation characteristics of squalene-based emulsions, including a sterile filterable droplet size (<220 nm), high homogeneity, and colloidal stability after months of storage at 4, 25, and 40 °C. Furthermore, the addition of the TLR agonists skewed the immune response from Th2 towards Th1 in immunized C57BL/6 mice, resulting in an increased production of IgG2c antibodies and a lower antigen-specific production of IL-5 with a higher production of IFNγ by lymphocytes. As such, incorporating TLR4 and TLR7/8 agonists into emulsions leveraged the desirable formulation and stability characteristics of emulsions and can induce Th1-type humoral and cell-mediated immune responses to combat the continued threat of SARS-CoV-2.

Broad neutralization against SARS-CoV-2 variants induced by ancestral and B.1.351 AS03-Adjuvanted recombinant Plant-Derived Virus-Like particle vaccines.

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulting in the coronavirus disease 2019 (COVID-19) has afflicted hundreds of millions of people in a worldwide pandemic. Several safe and effective COVID-19 vaccines are now available. However, the rapid emergence of variants and risk of viral escape from vaccine-induced immunity emphasize the need to develop broadly protective vaccines. A recombinant plant-derived virus-like particle vaccine for the ancestral COVID-19 (CoVLP) recently authorized by Canadian Health Authorities and a modified CoVLP.B1351 targeting the B.1.351 variant (both formulated with the adjuvant AS03) were assessed in homologous and heterologous prime-boost regimen in mice. Both strategies induced strong and broadly cross-reactive neutralizing antibody (NAb) responses against several Variants of Concern (VOCs), including B.1.351/Beta, B.1.1.7/Alpha, P.1/Gamma, B.1.617.2/Delta and B.1.1.529/Omicron strains. The neutralizing antibody (NAb) response was robust with both primary vaccination strategies and tended to be higher for almost all VOCs following the heterologous prime-boost regimen.

Comparative efficacy and safety of vaccines to prevent seasonal influenza: A systematic review and network meta-analysis.

Background: Influenza is one of the most common respiratory viral infections worldwide. Numerous vaccines are used to prevent influenza. Their selection should be informed by the best available evidence. We aimed to estimate the comparative efficacy and safety of seasonal influenza vaccines in children, adults and the elderly. Methods: We conducted a systematic review and network meta-analysis (NMA). We searched the Cochrane Library Central Register of Controlled Trials, MEDLINE and EMBASE databases, and websites of regulatory agencies, through December 15th, 2020. We included placebo- or no vaccination-controlled, and head-to-head randomized clinical trials (RCTs). Pairs of reviewers independently screened the studies, abstracted the data, and appraised the risk of bias in accordance to the Cochrane Handbook for Systematic Reviews of Interventions. The primary outcome was laboratory-confirmed influenza. We also synthesized data for hospitalization, mortality, influenza-like illness (ILI), pneumonia or lower respiratory-tract disease, systemic and local adverse events (AEs). We estimated summary risk ratios (RR) using pairwise and NMA with random effects. This study is registered with PROSPERO, number CRD42018091895. Findings: We identified 13,439 citations. A total of 231 RCTs were included after screening: 11 studies did not provide useful data for the analysis; 220 RCTs [100,677 children (< 18 years) and 329,127 adults (18-60 years) and elderly (≥ 61 years)] were included in the NMA. In adults and the elderly, all vaccines, except the trivalent inactivated intradermal vaccine (3-IIV ID), were more effective than placebo in reducing the risk of laboratory-confirmed influenza, with a RR between 0.33 (95% credible interval [CrI] 0.21-0.55) for trivalent inactivated high-dose (3-IIV HD) and 0.56 (95% CrI 0.41-0.74) for trivalent live-attenuated vaccine (3-LAIV). In adults and the elderly, compared with trivalent inactivated vaccine (3-IIV), no significant differences were found for any, except 3-LAIV, which was less efficacious [RR 1.41 (95% CrI 1.04-1.88)]. In children, compared with placebo, RR ranged between 0.13 (95% CrI 0.03-0.51) for trivalent inactivated vaccine adjuvanted with MF59/AS03 and 0.55 (95% CrI 0.36-0.83) for trivalent inactivated vaccine. Compared with 3-IIV, 3-LAIV and trivalent inactivated adjuvanted with MF59/AS03 were more efficacious [RR 0.52 (95% CrI 0.32-0.82) and RR 0.23 (95% CrI 0.06-0.87)] in reducing laboratory-confirmed influenza. With regard to safety, higher systemic AEs rates after vaccination with 3-IIV, 3-IIV HD, 3-IIV ID, 3-IIV MF59/AS03-adj, quadrivalent inactivated (4-IIV), quadrivalent adjuvanted (4-IIV MF59/AS03-adj), quadrivalent recombinant (4-RIV), 3-LAIV or quadrivalent live attenuated (4-LAIV) vaccines were noted in adults and the elderly [RR 1.5 (95% CrI 1.18-1.89) to 1.15 (95% CrI 1.06-1.23)] compared with placebo. In children, the systemic AEs rate after vaccination was not significantly higher than placebo. Interpretation: All vaccines cumulatively achieved major reductions in the incidence of laboratory-confirmed influenza in children, adults, and the elderly. While the live-attenuated was more efficacious than the inactivated vaccine in children, many vaccine types can be used in adults and the elderly. Funding: The directorate general of welfare, Lombardy region.

Whole-blood cytokine secretion assay as a high-throughput alternative for assessing the cell-mediated immunity profile after two doses of an adjuvanted SARS-CoV-2 recombinant protein vaccine candidate.

OBJECTIVES: We previously described the Phase I-II evaluation of SARS-CoV-2 recombinant protein candidate vaccine, CoV2-PreS-dTM, with AF03- or AS03-adjuvant systems (ClinicalTrials.gov, NCT04537208). Here, we further characterise the cellular immunogenicity profile of this vaccine candidate using a whole-blood secretion assay in parallel to intracellular cytokine staining (ICS) of cryopreserved peripheral blood mononuclear cells (PBMCs). METHODS: A randomly allocated subset of 90 healthy, SARS-CoV-2-seronegative adults aged ≥ 18 years who had received (random allocation) one or two separate injections (on study day [D]1 and D22) of saline placebo or CoV2-PreS-dTM formulated with AS03 or AF03 were included. Cytokine secretion was assessed using a TruCulture® whole-blood stimulation system in combination with multiplex bead array, and intracellular cytokine profiles were evaluated on thawed PBMCs following ex vivo stimulation with recombinant S protein at pre-vaccination (D1), post-dose 1 (D22) and post-dose 2 (D36). RESULTS: Both methods detected similar vaccine-induced responses after the first and second doses. We observed a Th1 bias (Th1/Th2 ratio > 1.0) for most treatment groups when analysed in whole blood, mainly characterised by increased IFN-γ, IL-2 and TNF-α secretion. Among participants aged ≥ 50 years, the Th1/Th2 ratio was higher for those who received vaccine candidate with AS03 versus AF03 adjuvant. ICS revealed that this higher Th1/Th2 ratio resulted from higher levels of IFN-γ expression and that the vaccine induced polyfunctional CD4+ T cells. CONCLUSIONS: The whole-blood cytokine secretion assay is a high-throughput alternative for assessing the quantity and character of vaccine-induced cellular responses.

Safety, immunogenicity, and protection provided by unadjuvanted and adjuvanted formulations of a recombinant plant-derived virus-like particle vaccine candidate for COVID-19 in nonhuman primates.

Although antivirals are important tools to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, effective vaccines are essential to control the current coronavirus disease 2019 (COVID-19) pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here, we report the immunogenicity and protection induced in rhesus macaques by intramuscular injections of a VLP bearing a SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytidine-phospho-guanosine (CpG) 1018. Although a single dose of the unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after priming) and adjuvant administration significantly improved both responses, with higher immunogenicity and protection provided by the AS03-adjuvanted CoVLP. Fifteen micrograms of CoVLP adjuvanted with AS03 induced a polyfunctional interleukin-2 (IL-2)-driven response and IL-4 expression in CD4 T cells. Animals were challenged by multiple routes (i.e., intratracheal, intranasal, and ocular) with a total viral dose of 106 plaque-forming units of SARS-CoV-2. Lower viral replication in nasal swabs and bronchoalveolar lavage fluid (BALF) as well as fewer SARS-CoV-2-infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of proinflammatory cytokines and chemotactic factors in the BALF were observed in animals immunized with the CoVLP adjuvanted with AS03. No clinical, pathologic, or virologic evidence of vaccine-associated enhanced disease was observed in vaccinated animals. The CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.

Lack of effects on female fertility or pre- and postnatal development of offspring in rats after exposure to AS03-adjuvanted recombinant plant-derived virus-like particle vaccine candidate for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulting in the coronavirus disease 2019 (COVID-19) has afflicted tens of millions of people in a worldwide pandemic. A recently developed recombinant Plant-Derived Virus-Like Particle Vaccine candidate for COVID-19 (CoVLP) formulated with AS03 has been shown to be well-tolerated and highly immunogenic in healthy adults. Since the target population for the vaccine includes women of childbearing potential, the objective of the study was to evaluate any untoward prenatal and postnatal effects of AS03-adjuvanted CoVLP administered intramuscularly to Sprague-Dawley female rats before cohabitation for mating (22 and 8 days prior) and during gestation (Gestation Days [GD] 6 and 19). The embryo-fetal development (EFD) cohort was subjected to cesarean on GD 21 and the pre/post-natal (PPN) cohort was allowed to naturally deliver. Effects of AS03-adjuvanted CoVLP was evaluated on pregnant rats, embryo-fetal development (EFD), during parturition, lactation and the development of the F1 offspring up to weaning Vaccination with AS03-adjuvanted CoVLP induced an antibody response in F0 females and anti-SARS-CoV-2 spike-specific maternal antibodies were detected in the offspring at the end of the gestation and lactation periods. Overall, there was no evidence of untoward effects of AS03-adjuvanted CoVLP on the fertility or reproductive performance of the vaccinated F0 females. There was no evidence of untoward effects on embryo-fetal development (including teratogenicity), or early (pre-weaning) development of the F1 offspring. These results support the acceptable safety profile of the AS03-adjuvanted CoVLP vaccine for administration to women of childbearing potential.

Adjuvantation of Influenza Vaccines to Induce Cross-Protective Immunity.

Influenza poses a huge threat to global public health. Influenza vaccines are the most effective and cost-effective means to control influenza. Current influenza vaccines mainly induce neutralizing antibodies against highly variable globular head of hemagglutinin and lack cross-protection. Vaccine adjuvants have been approved to enhance seasonal influenza vaccine efficacy in the elderly and spare influenza vaccine doses. Clinical studies found that MF59 and AS03-adjuvanted influenza vaccines could induce cross-protective immunity against non-vaccine viral strains. In addition to MF59 and AS03 adjuvants, experimental adjuvants, such as Toll-like receptor agonists, saponin-based adjuvants, cholera toxin and heat-labile enterotoxin-based mucosal adjuvants, and physical adjuvants, are also able to broaden influenza vaccine-induced immune responses against non-vaccine strains. This review focuses on introducing the various types of adjuvants capable of assisting current influenza vaccines to induce cross-protective immunity in preclinical and clinical studies. Mechanisms of licensed MF59 and AS03 adjuvants to induce cross-protective immunity are also introduced. Vaccine adjuvants hold a great promise to adjuvant influenza vaccines to induce cross-protective immunity.

Non-neutralizing antibody responses following A(H1N1)pdm09 influenza vaccination with or without AS03 adjuvant system.

BACKGROUND: Non-neutralizing antibodies inducing complement-dependent lysis (CDL) and antibody-dependent cell-mediated cytotoxicity (ADCC) activity may contribute to protection against influenza infection. We investigated CDL and ADCC responses in healthy adults randomized to receive either non-adjuvanted or AS03-adjuvanted monovalent A(H1N1)pdm09 vaccine (containing 15 µg/3.75 µg of hemagglutinin, respectively) on a 2-dose schedule 21 days apart. METHODS: We conducted an exploratory analysis of a subset of 106 subjects having no prior history of A(H1N1)pdm09 infection or seasonal influenza vaccination enrolled in a previously reported study (NCT00985673). Antibody responses against the homologous A/California/7/2009 (H1N1) vaccine strain and a related A/Brisbane/59/2007 (H1N1) seasonal influenza strain were analyzed up to Day 42. RESULTS: Baseline seropositivity determined with hemagglutination inhibition (HI), CDL and ADCC antibody titers against viral strains was high; A/California/7/2009 (HI [40.4-48.1%]; CDL [34.6-36.0%]; ADCC [92.1-92.3%]); A/Brisbane/59/2007 (HI [73.1-88.9%]; CDL [38.0-42.0%]; ADCC [86.8-97.0%]). CDL seropositivity increased following vaccination with both adjuvanted and non-adjuvanted formulations (A/California/7/2009 [95.9-100%]; A/Brisbane/59/2007 [75.5-79.6%]). At Day 21, increases in CDL and ADCC antibody geometric mean titers against both strains were observed for both formulations. After 2 doses of AS03-adjuvanted vaccine, vaccine responses of 95.8% (≥9-fold increase from baseline in CDL titers) and 34.3% (≥16-fold increase from baseline in ADCC titers) were seen against A/California/7/2009; and 22.4% and 42.9%, respectively, against A/Brisbane/59/2007. Vaccine responses after 2 doses of the non-adjuvanted vaccine were broadly similar. CONCLUSIONS: Broadly comparable non-neutralizing immune responses were observed following vaccination with non-adjuvanted and AS03-adjuvanted A(H1N1)pdm09 formulations; including activity against a related vaccine strain.

Single center analysis of patients with H1N1 vaccine-related narcolepsy and sporadic narcolepsy presenting over the same time period.

STUDY OBJECTIVES: We aimed to describe the clinical features of narcolepsy in patients referred to our sleep center between 2009 and 2016, and to compare these features across age groups and between sporadic vs AS03-adjuvanted H1N1 influenza vaccine-related patients. METHODS: This is a retrospective, consecutive study of adult and pediatric narcolepsy patients in the Republic of Ireland. All participants underwent structured assessments, including polysomnography and the Multiple Sleep Latency Test. Brain magnetic resonance imaging, hypocretin levels, and human leukocyte antigen typing were also carried out on the majority of patients. Patients were compared across age groups as well as etiology. RESULTS: The conditions of 40 (74%) patients were vaccine-related. The median age was 13.5 years and time from symptom onset to diagnosis was 112 weeks. Median time from vaccination to symptom onset was 26 weeks. In children, hypnogogic hallucinations and sleep paralysis were less frequent than in adults (17% vs 67%, P = .018 and 0% vs 75%, P < .0005). Sleep latency determined by the Multiple Sleep Latency Test was shorter in children than adults (median 1.75 vs 4 minutes, P = .011). Patients with vaccine-related and sporadic narcolepsies had typical clinical presentations. Vaccine-related patients had longer polysomnography latency (median 10.5 vs 5 minutes, P = .043), longer stage N2 sleep (209.6 ± 44.6 vs 182.3 ± 34.2 minutes, P = .042), and a trend toward longer total sleep times (P = .09). No differences were noted in relation to Multiple Sleep Latency Test, hypocretin, human leukocyte antigen typing, and magnetic resonance imaging. CONCLUSIONS: Results show that vaccine-related patients greatly outnumbered sporadic patients during the study period and suggest that sporadic and vaccine-related narcolepsy are clinically similar entities.

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.

No evidence of antigenic seniority in hemagglutinin specific antibody responses after adjuvanted pandemic 2009 influenza vaccination.

Influenza is a contagious respiratory illness caused by the influenza virus. The pandemic outbreak of influenza A H1N1 in 2009 (H1N1pdm09) gave us a unique opportunity to study humoral immune responses to a novel influenza vaccine strain. Here, we investigate how an individual's previous encounter with different influenza subtypes influences the humoral response after pandemic vaccination in 2009. We retrospectively chose and grouped 80 vaccinated healthcare workers (HCWs) based on their year of birth into 4 groups, reflecting which influenza subtype they were likely first exposed to during childhood. Pre- and 21 days post- vaccination sera were analyzed. We investigated antibodies to the major surface protein hemagglutinin (HA), and specifically antibodies binding to the conserved stalk domain of the HA-protein. Serological assays were used to assess the quantity and functionality of the influenza-specific antibodies, including virus neutralization and activation of natural killer (NK) cells involved in antibody-dependent cell-mediated cytotoxicity (ADCC). The AS03-adjuvanted H1N1pdm09 vaccine elicited robust antibody responses in all groups of HCWs. We found that the more antigenically experienced individuals had higher pre-vaccination antibody-levels towards the stalk domain of the HA. We also demonstrated that despite their inferior pre-vaccination antibody levels, the younger individuals reached similar antibody levels as the older birth-cohorts after pandemic vaccination. Our findings are important for understanding the effect of AS03 adjuvant on the antibody response in individuals exposed to different influenza viruses during their early childhood years, which is crucial for developing vaccine strategies against influenza.

Comparative pharmacokinetic and biodistribution study of two distinct squalene-containing oil-in-water emulsion adjuvants in H5N1 influenza vaccines.

BACKGROUND: In recent years, there has been great interest from academia, industry and government scientists for an increased understanding of the mode of action of vaccine adjuvants to characterize the safety and efficacy of vaccines. In this context, pharmacokinetic (PK) and biodistribution studies are useful for quantifying the concentration of vaccine adjuvants in mechanistically or toxicologically relevant target tissues. METHODS: In this study, we conducted a comparative analysis of the PK and biodistribution profile of radiolabeled squalene for up to 336 h (14 days) after intramuscular injection of mice with adjuvanted H5N1 influenza vaccines. The evaluated adjuvants included an experimental-grade squalene-in-water (SQ/W) emulsion (AddaVax®) and an adjuvant system (AS03®) that contained squalene and α-tocopherol in the oil phase of the emulsion. RESULTS: The half-life of the initial exponential decay from quadriceps muscle was 1.5 h for AS03 versus 12.9 h for AddaVax. At early time points (1-6 h), there was about a 10-fold higher concentration of labeled squalene in draining lymph nodes following AS03 injection compared to AddaVax. The area-under-concentration curve up to 336 h (AUC0-336hr) and peak concentration of squalene in spleen (immune organ) was about 1.7-fold higher following injection of AS03 than AddaVax. The peak systemic tissue concentration of squalene from the two adjuvants, with or without antigen, remained below 1% of injected dose for toxicologically relevant target tissues, such as spinal cord, brain, and kidney. The pharmacokinetics of AS03 was unaffected by the presence of H5N1 antigen. CONCLUSIONS: This study demonstrates a rapid decline of AS03 from the quadriceps muscles of mice as compared to conventional SQ/W emulsion adjuvant, with an increased transfer to mechanistically relevant tissues such as local lymph nodes. Systemic tissue exposure to potential toxicological target tissues was very low.

Adjuvant Systems for vaccines: 13 years of post-licensure experience in diverse populations have progressed the way adjuvanted vaccine safety is investigated and understood.

Adjuvant Systems (AS) are combinations of immune stimulants that enhance the immune response to vaccine antigens. The first vaccine containing an AS (AS04) was licensed in 2005. As of 2018, several vaccines containing AS04, AS03 or AS01 have been licensed or approved by regulatory authorities in some countries, and included in vaccination programs. These vaccines target diverse viral and parasitic diseases (hepatitis B, human papillomavirus, malaria, herpes zoster, and (pre)pandemic influenza), and were developed for widely different target populations (e.g. individuals with renal impairment, girls and young women, infants and children living in Africa, adults 50 years of age and older, and the general population). Clearly, the safety profile of one vaccine in one target population cannot be extrapolated to another vaccine or to another target population, even for vaccines containing the same adjuvant. Therefore, the assessment of adjuvant safety poses specific challenges. In this review we provide a historical perspective on how AS were developed from the angle of the challenges encountered on safety evaluation during clinical development and after licensure, and illustrate how these challenges have been met to date. Methods to evaluate safety of adjuvants have evolved based on the availability of new technologies allowing a better understanding of their mode of action, and new ways of collecting and assessing safety information. Since 2005, safety experience with AS has accumulated with their use in diverse vaccines and in markedly different populations, in national immunization programs, and in a pandemic setting. Thirteen years of experience using antigens combined with AS attest to their acceptable safety profile. Methods developed to assess the safety of vaccines containing AS have progressed the way we understand and investigate vaccine safety, and have helped set new standards that will guide and support new candidate vaccine development, particularly those using new adjuvants. FOCUS ON THE PATIENT: What is the context? Adjuvants are immunostimulants used to modulate and enhance the immune response induced by vaccination. Since the 1990s, adjuvantation has moved toward combining several immunostimulants in the form of Adjuvant System(s) (AS), rather than relying on a single immunostimulant. AS have enabled the development of new vaccines targeting diseases and/or populations with special challenges that were previously not feasible using classical vaccine technology. What is new? In the last 13 years, several AS-containing vaccines have been studied targeting different diseases and populations. Over this period, overall vaccine safety has been monitored and real-life safety profiles have been assessed following routine use in the general population in many countries. Moreover, new methods for safety assessment, such as a better determination of the mode of action, have been implemented in order to help understand the safety characteristics of AS-containing vaccines. What is the impact? New standards and safety experience accumulated over the last decade can guide and help support the safety assessment of new candidate vaccines during development.

Meeting report narcolepsy and pandemic influenza vaccination: What we know and what we need to know before the next pandemic? A report from the 2nd IABS meeting.

A group of scientific and public health experts and key stakeholders convened to discuss the state of knowledge on the relationship between adjuvanted monovalent inactivated 2009 influenza A H1N1 vaccines used during the 2009 influenza pandemic and narcolepsy. There was consensus that an increased risk of narcolepsy was consistently observed after Pandemrix (AS03-adjuvanted) vaccine, but similar associations following Arepanrix (AS03-adjuvanted) or Focetria (MF59-adjuvanted) vaccines were not observed. Whether the differences are due to vaccine composition or other factors such as the timing of large-scale vaccination programs relative to H1N1pdm09 wild-type virus circulation in different geographic regions is not clear. The limitations of retrospective observational methodologies could also be contributing to some of the differences across studies. More basic and epidemiologic research is needed to further elucidate the association between adjuvanted influenza vaccine and narcolepsy and its mechanism and to inform planning and preparation for vaccination programs in advance of the next influenza pandemic.

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.

Narcolepsy and adjuvanted pandemic influenza A (H1N1) 2009 vaccines - Multi-country assessment.

BACKGROUND: In 2010, a safety signal was detected for narcolepsy following vaccination with Pandemrix, an AS03-adjuvanted monovalent pandemic H1N1 influenza (pH1N1) vaccine. To further assess a possible association and inform policy on future use of adjuvants, we conducted a multi-country study of narcolepsy and adjuvanted pH1N1 vaccines. METHODS: We used electronic health databases to conduct a dynamic retrospective cohort study to assess narcolepsy incidence rates (IR) before and during pH1N1 virus circulation, and after pH1N1 vaccination campaigns in Canada, Denmark, Spain, Sweden, Taiwan, the Netherlands, and the United Kingdom. Using a case-control study design, we evaluated the risk of narcolepsy following AS03- and MF59-adjuvanted pH1N1 vaccines in Argentina, Canada, Spain, Switzerland, Taiwan, and the Netherlands. In the Netherlands, we also conducted a case-coverage study in children born between 2004 and 2009. RESULTS: No changes in narcolepsy IRs were observed in any periods in single study sites except Sweden and Taiwan; in Taiwan incidence increased after wild-type pH1N1 virus circulation and in Sweden (a previously identified signaling country), incidence increased after the start of pH1N1 vaccination. No association was observed for Arepanrix-AS03 or Focetria-MF59 adjuvanted pH1N1 vaccines and narcolepsy in children or adults in the case-control study nor for children born between 2004 and 2009 in the Netherlands case-coverage study for Pandemrix-AS03. CONCLUSIONS: Other than elevated narcolepsy IRs in the period after vaccination campaigns in Sweden, we did not find an association between AS03- or MF59-adjuvanted pH1N1 vaccines and narcolepsy in children or adults in the sites studied, although power to evaluate the AS03-adjuvanted Pandemrix brand vaccine was limited in our study.