Non-clinical safety assessment of single and repeated administration of gE/AS01 zoster vaccine in rabbits.

HZ/su is an investigational recombinant subunit vaccine for the prevention of shingles, a disease resulting from the reactivation of varicella zoster virus. The vaccine is composed of recombinant varicella zoster virus glycoprotein E (gE), and liposome-based Adjuvant System AS01. To evaluate the potential local and systemic effects of this vaccine, three studies were performed in rabbits. In the first two studies, rabbits received a single intramuscular (IM; study 1) or subcutaneous (SC; study 2) dose of gE/AS01, AS01 alone (in study 2 only) or saline, and the local tolerance was evaluated up to 3 days after administration. Under these conditions, only local inflammatory reactions at the injection sites were detected by microscopic evaluation. In the third study, gE/AS01, AS01 alone or saline, were injected SC or IM on four occasions at 2 week intervals. General health status, local tolerance, ophthalmology, haematology and blood chemistry parameters were monitored. Macroscopic and microscopic evaluations were performed after termination of the study. The only treatment-related changes included a transient increase in neutrophils, C-reactive protein and fibrinogen levels and microscopic signs of inflammation at the injection sites, which are expected observations related to the elicited inflammatory reaction. The SC and IM routes of administration produced similar systemic effects. However, microscopic findings at the injection sites differed. One month after the last injection, recovery was complete in all groups. In conclusion, the single and repeated SC and IM administration of the gE/AS01 vaccine were locally and systemically well-tolerated in rabbits and support the clinical development of the vaccine. Copyright © 2016 John Wiley & Sons, Ltd.

Séance bi-académique de l'Académie nationale de médecine et de l'Académie des Sciences: « Confiance et défiance vis-à-vis des vaccins ¼.

SUMMARYThe explosion of vaccines during the 20th century allowed the control of numerous infectious plagues but multiple challenges oppose conservation and extension of these successes. The hesitation of modern societies in front of vaccinations requires researches in life, human and social sciences in order to reach a better understanding of vaccines mechanism of action and to improve the tolerance and acceptability of vaccines and additives. The ageing of the populations and the increase of subjects at risk also require to improve the immunogenicity and the efficiency of existing vaccines. The constant emergence of new epidemics or the development of the antibio-resistance imposes innovation and development of new vaccines. The recent difficulties faced by the development of vaccines against malaria, tuberculosis or AIDS illustrate the necessity of moving beyond classical recipes and of elaborating new vectors and new adjuvants, of better understanding the heterogeneity of vaccine immunity and of developing alternative routes of immunization. Multidisciplinary researches using the most recent advances in molecular, structural and cellular biology, in microbiology, immunology and of genetic engineering to answer these worldwide challenges.

Impact of adjuvants on CD4(+) T cell and B cell responses to a protein antigen vaccine: Results from a phase II, randomized, multicenter trial.

Immunogenicity and safety of different adjuvants combined with a model antigen (HBsAg) were compared. Healthy HBV-naïve adults were randomized to receive HBs adjuvanted with alum or Adjuvant Systems AS01B, AS01E, AS03A or AS04 at Days 0 and 30. Different frequencies of HBs-specific CD4+ T cells 14days post dose 2 but similar polyfunctionality profiles were induced by the different adjuvants with frequencies significantly higher in the AS01B and AS01E groups than in the other groups. Antibody concentrations 30days post-dose 2 were significantly higher in AS01B, AS01E and AS03A than in other groups. Limited correlations were observed between HBs-specific CD4+ T cell and antibody responses. Injection site pain was the most common solicited local symptom and was more frequent in AS groups than in alum group. Different adjuvants formulated with the same antigen induced different adaptive immune responses and reactogenicity patterns in healthy naïve adults. The results summary for this study (GSK study number 112115 - NCT# NCT00805389) is available on the GSK Clinical Study Register and can be accessed at www.gsk-clinicalstudyregister.com.

Enhancement of adaptive immunity by the human vaccine adjuvant AS01 depends on activated dendritic cells.

Adjuvant System AS01 is a liposome-based vaccine adjuvant containing 3-O-desacyl-4'-monophosphoryl lipid A and the saponin QS-21. AS01 has been selected for the clinical development of several candidate vaccines including the RTS,S malaria vaccine and the subunit glycoprotein E varicella zoster vaccine (both currently in phase III). Given the known immunostimulatory properties of MPL and QS-21, the objective of this study was to describe the early immune response parameters after immunization with an AS01-adjuvanted vaccine and to identify relationships with the vaccine-specific adaptive immune response. Cytokine production and innate immune cell recruitment occurred rapidly and transiently at the muscle injection site and draining lymph node postinjection, consistent with the rapid drainage of the vaccine components to the draining lymph node. The induction of Ag-specific Ab and T cell responses was dependent on the Ag being injected at the same time or within 24 h after AS01, suggesting that the early events occurring postinjection were required for these elevated adaptive responses. In the draining lymph node, after 24 h, the numbers of activated and Ag-loaded monocytes and MHCII(high) dendritic cells were higher after the injection of the AS01-adjuvanted vaccine than after Ag alone. However, only MHCII(high) dendritic cells appeared efficient at and necessary for direct Ag presentation to T cells. These data suggest that the ability of AS01 to improve adaptive immune responses, as has been demonstrated in clinical trials, is linked to a transient stimulation of the innate immune system leading to the generation of high number of efficient Ag-presenting dendritic cells.

Effects of Adjuvant Systems on the cardiovascular and respiratory functions in telemetered conscious dogs and anaesthetised rats.

Adjuvants Systems (AS) containing immunostimulant combinations are used in human vaccines. Safety pharmacology studies evaluated the cardiorespiratory effects of AS in conscious telemetered dogs and in anaesthetised rats. Sixteen telemetered beagle dogs (4/group) received intramuscular injections of saline at Day 0, and one clinical dose of AS01, AS03, AS04 or AS15 at Day 7 (7× the equivalent human dose on a bodyweight basis). Bodyweights were measured through Day 14 and cardiorespiratory parameters and body temperature through 72 h post-treatment. Anaesthetised rats (4/group) received one intravenous injection of AS01, AS03 or AS15 at 1 mL/kg bodyweight (140× the equivalent human dosages), or saline. Cardiorespiratory parameters were measured for 120 min post-dose. In dogs, food consumption and mean bodyweight decreased with AS03, and mean body temperature slightly increased with AS01, AS03 and AS15, but were not considered to be adverse. Cardiovascular effects (a slight, reversible increase in mean heart rate and shortened mean RR/PR/QT-intervals) were observed with AS15. No relevant clinical effects or effects on QRS-complex/QTc-interval durations, arterial pressure or respiratory parameters were observed. In rats, there were no consistent treatment-related effects. Collectively, this suggests that AS01, AS03, AS04 and AS15 are not associated with potentially deleterious effects on ventricular repolarisation, atrio/intra-ventricular conductivities or respiratory functions.

Local tolerance and systemic toxicity of single and repeated intramuscular administrations of two different formulations of the RTS,S malaria candidate vaccine in rabbits.

RTS,S malaria antigen is weakly immunogenic as such and needs to be formulated with an adjuvant to improve the magnitude and duration of the immune responses to RTS,S. Two Adjuvant Systems, AS01 and AS02 were evaluated during the development of the RTS,S vaccine. The evaluation included non-clinical studies in rabbits to evaluate the local intramuscular tolerance following administration on a single occasion, and the local and systemic effects following repeated administrations of RTS,S/AS01 or RTS,S/AS02 formulations. In the first study, rabbits were injected on one occasion with RTS,S/AS01, RTS,S/AS02 or controls, and the local intramuscular tolerance was evaluated up to 3 days after injection. In the second study, the different formulations were injected on Days 0, 14, 28 and 42. General health status, haematology and blood chemistry parameters were monitored on a regular basis. Macroscopic and microscopic evaluations were made after termination of the study. No sign of toxicity was detected following single or repeated administrations of the adjuvanted RTS,S formulations. Changes in haematology or clinical chemistry parameters were indicative of a developing immune response in the groups receiving either RTS,S formulation. All examined parameters returned to normal within 28 days after the last injection. The absence of toxicological effects following the injection of RTS,S/AS01 or RTS,S/AS02 in rabbits was supportive of further clinical evaluation of these two formulations.

Non-clinical safety assessment of single and repeated intramuscular administration of a human papillomavirus-16/18 vaccine in rabbits and rats.

The human papillomavirus (HPV)-16/18 vaccine (Cervarix®) is a prophylactic vaccine for the prevention of cervical cancer. The vaccine contains recombinant virus-like particles assembled from the L1 major capsid proteins of the cervical cancer-causing viral types HPV-16 and HPV-18, and Adjuvant System 04 (AS04), which contains the immunostimulant MPL and aluminium salt. To evaluate potential local and systemic toxic effects of the HPV-16/18 vaccine or AS04 alone, three repeated-dose studies were performed in rabbits and rats. One rabbit study also included a single-dose evaluation. In rabbits (~2.5 kg), the full human dose (HD) of the vaccine was evaluated (0.5 ml per injection site), and in rats (~250 g), 1/5 HD of vaccine was evaluated, corresponding to ≥ 12 times the dosage in humans relative to body weight. In both animal models, the treatment-related changes included a slight transient increase in the number of circulating neutrophils as well as a local inflammatory reaction at the injection site. These treatment-related changes were less pronounced after four doses of AS04 alone than after four doses of the HPV-16/18 vaccine. Additional treatment-related changes in the rat included lower albumin/globulin ratios and microscopic signs of inflammation in the popliteal lymph nodes. In both animal models, 13 weeks after the fourth dose, recovery was nearly complete, although at the injection site in some animals there were signs of discoloration, muscle-fibre regeneration and focal points of macrophage infiltration. Therefore, in these non-clinical models, the single and repeated dose administrations of the HPV-16/18 vaccine or AS04 alone were safe and well tolerated.

Non-clinical safety evaluation of single and repeated intramuscular administrations of MAGE-A3 Cancer Immunotherapeutic in rabbits and cynomolgus monkeys.

The MAGE-A3 recombinant protein combined with AS15 immunostimulant (MAGE-A3 Cancer Immunotherapeutic) is under development by GlaxoSmithKline for the treatment of lung cancer and melanoma. We performed non-clinical safety studies evaluating potential local and systemic toxic effects induced by MAGE-A3 Cancer Immunotherapeutic in rabbits (study 1) and cynomolgus monkeys (study 2). Animals were allocated to two groups to receive a single (rabbits) or 25 repeated (every 2 weeks) injections (monkeys) of MAGE-A3 Cancer Immunotherapeutic (treatment groups) or saline (control groups). All rabbits were sacrificed 3 days post-injection and monkeys 3 days following last injection (3/5 per gender per group) or after a 3-month treatment-free period (2/5 per gender per group). Local and systemic reactions and MAGE-A3-specific immune responses (monkeys) were assessed. Macroscopic and microscopic (for rabbits, injection site only) post-mortem examinations were performed on all animals. No systemic toxicity or unscheduled mortalities were recorded. Single (rabbits) and repeated (monkeys; up to four times at the same site) injections were well tolerated. Following five to seven repeated injections, limb circumferences increased up to 26% (5 h post-injection), but returned to normal after 1-8 days. Three days after the last injection, enlargements of iliac, popliteal, axillary and inguinal lymph nodes, and increased incidence or severity of mononuclear inflammatory cell infiltrates was observed in injected muscles of treated monkeys. No treatment-related macroscopic findings were recorded after the treatment-free period. MAGE-A3-specific antibody and T-cell responses were raised in all treated monkeys, confirming test item exposure. Single or repeated intramuscular injections of MAGE-A3 Cancer Immunotherapeutic were well tolerated in rabbits and monkeys.

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.

Predictive markers of safety and immunogenicity of adjuvanted vaccines.

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.

AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity.

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-kappaB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4(+) T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

Antibody avidity, persistence, and response to antigen recall: comparison of vaccine adjuvants.

Differences in innate immune 'imprinting' between vaccine adjuvants may mediate dissimilar effects on the quantity/quality of persisting adaptive responses. We compared antibody avidity maturation, antibody/memory B cell/CD4+ T cell response durability, and recall responses to non-adjuvanted fractional-dose antigen administered 1-year post-immunization (Day [D]360), between hepatitis B vaccines containing Adjuvant System (AS)01B, AS01E, AS03, AS04, or Alum (NCT00805389). Both the antibody and B cell levels ranked similarly (AS01B/E/AS03 > AS04 > Alum) at peak response, at D360, and following their increases post-antigen recall (D390). Proportions of high-avidity antibodies increased post-dose 2 across all groups and persisted at D360, but avidity maturation appeared to be more strongly promoted by AS vs. Alum. Post-antigen recall, frequencies of subjects with high-avidity antibodies increased only markedly in the AS groups. Among the AS, total antibody responses were lowest for AS04. However, proportions of high-avidity antibodies were similar between groups, suggesting that MPL in AS04 contributes to avidity maturation. Specific combinations of immunoenhancers in the AS, regardless of their individual nature, increase antibody persistence and avidity maturation.

Safety biomarkers for development of vaccines and biologics: Report from the safety biomarkers symposium held on November 28-29, 2017, Marcy l'Etoile, France.

Vaccines prevent infectious diseases, but vaccination is not without risk and adverse events are reported although they are more commonly reported for biologicals than for vaccines. Vaccines and biologicals must undergo vigorous assessment before and after licensure to minimise safety concerns. Potential safety concerns should be identified as early as possible during the development for vaccines and biologicals to minimize investment risk. State-of-the art tools and methods to identify safety concerns and biomarkers that are predictive of clinical outcomes are indispensable. For vaccines and adjuvant formulations, systems biology approaches, supported by single-cell microfluidics applied to translational studies between preclinical and clinical studies, could improve reactogenicity and safety predictions. Next-generation animal models for clinical assessment of injection-site reactions with greater relevance for target human population and criteria to define the level of acceptability of local reactogenicity at vaccine injection sites in pre-clinical animal species should be assessed. Advanced in silico machine-learning-based analytics, species-specific cell or tissue expression, receptor occupancy and kinetics and cell-based assays for functional activity are needed to improve pre-clinical safety assessment of biologicals. The in vitro MIMIC® system could be used to compliment preclinical and clinical studies for assessing immune-toxicity, immunogenicity, immuno-inflammatory and mode of action of biologicals and vaccines. Sanofi Pasteur brought together leading experts in this field to review the state-of-the-art at a unique 'Safety Biomarkers Symposium' on 28-29 November 2017. Here we summarise the proceedings of this symposium. This unique scientific meeting confirmed the importance for institutions and industrial organizations to collaborate to develop tools and methods needed for predicting reactogenicity and immune-inflammatory reactions to vaccines and biologicals, and to develop more accuracy, reliability safety biomarkers, to inform decisions on the attrition or advancement of vaccines and biologicals.

The science of vaccine safety: Summary of meeting at Wellcome Trust.

Vaccines are everywhere hugely successful but are also under attack. The reason for the latter is the perception by some people that vaccines are unsafe. However that may be, vaccine safety, life any other scientific subject, must be constantly studied. It was from this point of view that a meeting was organized at the Wellcome Trust in London in May 2019 to assess some aspects of vaccine safety as subjects for scientific study. The objective of the meeting was to assess what is known beyond reasonable doubt and conversely what areas need additional studies. Although the meeting could not cover all aspects of vaccine safety science, many of the most important issues were addressed by a group of about 30 experts to determine what is already known and what additional studies are merited to assess the safety of the vaccines currently in use. The meeting began with reviews of the current situation in different parts of the world, followed by reviews of specific controversial areas, including the incidence of certain conditions after vaccination and the safety of certain vaccine components. Lastly, information about the human papillomavirus vaccine was considered because its safety has been particularly challenged by vaccine opponents. The following is a summary of the meeting findings. In addition to this summary, the meeting organizers will explore opportunities to perform studies that would enlarge knowledge of vaccine safety.

Safety of AS03-adjuvanted influenza vaccines: A review of the evidence.

Clinical and post-licensure data have demonstrated that AS03-adjuvanted inactivated split virion vaccines, many with reduced antigen content, are effective against influenza infection. The objective of this review is to provide a comprehensive assessment of the safety of trivalent seasonal, monovalent pre-pandemic and pandemic AS03-adjuvanted influenza vaccines, based on non-clinical, clinical and post-licensure data in various populations. Non-clinical studies on local tolerance, toxicology and safety pharmacology did not raise any safety concerns with AS03 administered alone or combined with various influenza antigens. Data from clinical trials with over 55,000 vaccinated subjects showed that AS03-adjuvanted influenza vaccines were generally well tolerated and displayed an acceptable safety profile, although the power to detect rare events was limited. Approximately 90 million doses of A/H1N1pdm09 pandemic influenza vaccines (Pandemrix and Arepanrix H1N1) were administered worldwide, which contributed post-licensure data to the collective safety data for AS03-adjuvanted influenza vaccines. An association between Pandemrix and narcolepsy was observed during the A/H1N1pdm09 pandemic, for which a role of a CD4 T cell mimicry sequence in the haemagglutinin protein of A/H1N1pdm09 cannot be excluded. Provided that future AS03-adjuvanted influenza vaccines do not contain this putative mimicry sequence, this extensive safety experience supports the further development and use of AS03-adjuvanted inactivated split virion candidate vaccines against seasonal and pandemic influenza infections.

Signal management in pharmacovigilance and human risk assessment of CpG 7909, integrating embryo-fetal and post-natal developmental toxicity studies in rats and rabbits.

The potential reproductive and developmental toxicity of the synthetic oligodeoxynucleotide (ODN) CpG 7909, a component of GSK's AS15 immunostimulant, was examined in rat and rabbit studies following intermittent intramuscular injections. Previous studies using subcutaneous and intraperitoneal injections in mice, rats and rabbits revealed that CpG ODNs induced developmental effects. To analyze the safety signal, GSK conducted additional animal studies using the intended clinical route of administration. CpG 7909 injections were administered intramuscularly to rats or rabbits 28 and 14days before pairing, on 4 or 5 occasions during gestation, and on lactation day 7. The No Observed Adverse Effect Level for female fertility, embryo-fetal and pre- and post-natal development was 4.2mg/kg in both species, approximately 500-fold higher than the anticipated human dose. In conclusion, the anticipated risk to humans is considered low for sporadic intramuscular exposure to CpG 7909.

Erratum: Author Correction: Cellular and molecular synergy in AS01-adjuvanted vaccines results in an early IFNγ response promoting vaccine immunogenicity.

[This corrects the article DOI: 10.1038/s41541-017-0027-3.].

From discovery to licensure, the Adjuvant System story.

Adjuvants are substances added to vaccines to improve their immunogenicity. Used for more than 80 years, aluminum, the first adjuvant in human vaccines, proved insufficient to develop vaccines that could protect against new challenging pathogens such as HIV and malaria. New adjuvants and new combinations of adjuvants (Adjuvant Systems) have opened the door to the delivery of improved and new vaccines against re-emerging and difficult pathogens. Adjuvant Systems concept started through serendipity. The access to new developments in technology, microbiology and immunology have been instrumental for the dicephering of what they do and how they do it. This knowledge opens the door to more rational vaccine design with implications for developing new and better vaccines.

Intramuscularly administered herpes zoster subunit vaccine has no effects on fertility, pre- and post-natal development in Sprague-Dawley rats.

The herpes zoster subunit vaccine (HZ/su) is an investigational vaccine for the prevention of shingles, a disease caused by the varicella zoster virus (VZV). It is composed of recombinant VZV glycoprotein E (gE) and AS01. We assessed the potential toxic effects of gE/AS01 and AS01 alone on female and male fertility, and on embryo-fetal, pre- and post-natal development in Sprague-Dawley rats. Females were immunized before pairing and during gestation. Half of the pregnant rats were used for embryo-fetal investigations. The ones that gave birth were immunized during lactation and offspring were analysed. In a male fertility study, rats were immunized before pairing. After mating, the untreated females were sacrificed and the fetuses examined. In addition, male fertility parameters were evaluated. Results indicated that female mating performance and fertility, pre- and post-natal survival and offspring development, male mating performance and fertility were unaffected by intramuscular administration of the zoster candidate vaccine gE/AS01.

Adjuvant system AS01: helping to overcome the challenges of modern vaccines.

INTRODUCTION: Adjuvants are used to improve vaccine immunogenicity and efficacy by enhancing antigen presentation to antigen-specific immune cells with the aim to confer long-term protection against targeted pathogens. Adjuvants have been used in vaccines for more than 90 years. Combinations of immunostimulatory molecules, such as in the Adjuvant System AS01, have opened the way to the development of new or improved vaccines. Areas covered: AS01 is a liposome-based vaccine adjuvant system containing two immunostimulants: 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and the saponin QS-21. Here we describe studies investigating the mode of action of AS01, and consider the role of AS01 in enhancing specific immune responses to the antigen for selected candidate vaccines targeting malaria and herpes zoster. The effects of AS01 are rapid and transient, being localized to the injected muscle and draining lymph node. AS01 is efficient at promoting CD4+ T cell-mediated immune responses and is an appropriate candidate adjuvant for inclusion in vaccines targeting viruses or intracellular pathogens. Expert commentary: AS01 activity to enhance adaptive responses depends on synergistic activities of QS-21 and MPL. AS01 adjuvantation shows good prospects for use in new vaccines targeted to populations with challenging immune statuses and against diseases caused by complex pathogens.