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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.

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., SCOPUS: ar.j, info:eu-repo/semantics/published

Adjuvants are present in all vaccines, either as part of the killed or live attenuated pathogens, or added to the vaccine. An efficient immune response cannot exist without the antigen and the adjuvant activating together the antigen presenting cells. There are more than 10 adjuvants that are part of licensed vaccines. They can be tailored to the pathogen, type of immune response expected and targeted population. They will continue to be part of vaccines, even for mRNA vaccines as they are already present as part of the mRNA sequence.

RÉSUMÉ L’explosion des vaccins pendant le XXe siècle a permis le contrôle de nombreux fléaux infectieux mais de multiples défis s’opposent à la préservation et l’extension de ces succès. L’hésitation des sociétés modernes face aux vaccinations nécessite, outre une meilleure compréhension par des recherches en sciences humaines et sociales, l’amélioration de l’acceptabilité et de la tolérance des vaccins et adjuvants. Le vieillissement des populations et l’augmentation des sujets à risque nécessitent d’améliorer l’immunogénicité et l’efficacité des vaccins existants. L’émergence constante de nouvelles épidémies, le développement de l’antibio-résistance imposent la création de nouveaux vaccins mais les difficultés du développement de vaccins contre le paludisme, la tuberculose ou le sida, illustrent la nécessité de dépasser les approches classiques pour élaborer de nouveaux vecteurs et adjuvants vaccinaux, mieux comprendre l’immunité vaccinale et des populations, définir des corrélats de protection et développer des voies nouvelles d’immunisation. Des recherches multidisciplinaires utilisant les progrès les plus récents de la biologie structurale et cellulaire, de la microbiologie, de l’immunologie et du génie biomoléculaire s’imposent pour répondre à ces défis.

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.

The exponential evolution of scientific knowledge during the first half of the twentieth century led to the emergence of new and improved ways of producing vaccines. Vaccines were produced from cultivating the pathogens, but this has not always been possible in sufficient quantities. The rise of molecular biology and a better understanding of the key components of immune protection have allowed the development and production of what is known as recombinant antigens. Most, if not all, purified and recombinant antigens, require to be effective, the addition of what is known today as adjuvants. They are an important part of the development of improved or new vaccines against infectious diseases, alongside DNA or vector-based vaccines.

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 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.

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.

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

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 14 days 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 30 days 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, SCOPUS: ar.j, info:eu-repo/semantics/published

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.

Combination of tumor antigens with immunostimulants is a promising approach in cancer immunotherapy. We assessed animal model toxicity of AS15 combined with various tumor antigens: WT1 (rabbits), or p501, dHER2 and recPRAME (cynomolgus monkeys), administered in seven or 20 dose regimens versus a saline control. Clinical and ophthalmological examinations, followed by extensive post‐mortem pathological examinations, were performed on all animals. Blood hematology and biochemistry parameters were also assessed. Antigen‐specific antibody titers were determined by enzyme‐linked immunosorbent assay. Additional assessments in monkeys included electrocardiography and immunohistochemical evaluations of the p501 expression pattern. Transient increases in body temperature were observed 4 h or 24 h after injections of recPRAME + AS15 and dHER2 + AS15. Edema and erythema were observed up to 1 week after most injections of recPRAME + AS15 and all injections of dHER2 + AS15. No treatment‐related effects were observed for electrocardiography parameters. Mean fibrinogen levels were significantly higher in all treated groups compared to controls, but no differences could be observed at the end of the treatment‐free period. Transient but significant differences in biochemistry parameters were observed post‐injection: lower albumin/globulin ratios (p501 + AS15), and higher bilirubin, urea and creatinine (dHER2 + AS15). Pathology examinations revealed significant increases in axillary lymph node mean weights (recPRAME + AS15) compared to controls. A 100% seroconversion rate was observed in all treated groups, but not in controls. p501 protein expression was observed in prostates of all monkeys from studies assessing p501 + AS15. These results suggest a favorable safety profile of the AS15‐containing candidate vaccines, supporting the use of AS15 for clinical development of potential anticancer vaccines. Copyright © 2015 The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd., The aim of the current paper was to assess the safety profile of vaccine candidates containing the AS15 immunostimulant combined with different antigens in two animal models. Several antigens were tested for this purpose: WT1 (rabbits), p501, dHER2 and recPRAME (cynomolgus monkeys). Only transient differences in hematology and biochemical parameters could be observed, while pathology testing revealed no safety concerns. Our findings support the use of AS15 for clinical development of potential immunotherapeutic cancer vaccines.

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. Copyright © 2015 John Wiley & Sons, Ltd.

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. Copyright © 2015 John Wiley & Sons, Ltd.

Background Recombinant hepatitis B surface antigen (HBsAg) was used as a model antigen to evaluate persistence of cellular and humoral immune responses when formulated with three different Adjuvant Systems containing 3- O -desacyl-4′-monophosphoryl lipid A (MPL) and QS-21, in an oil-in-water emulsion (AS02 B and AS02 V ), or with liposomes (AS01 B ). Methods This is an open, 4-year follow-up of a previous randomised, double-blind study. Healthy subjects aged 18–40 years received three vaccine doses on a month 0, 1, 10 schedule and were initially followed for 18 months. A total of 93 subjects (AS02 B : n = 30; AS02 V : n = 28; AS01 B : n = 35) were enrolled in this follow-up and had an additional blood sample taken at Year 4 (NCT02153320). The primary endpoint was the frequency of HBsAg-specific CD4 + and CD8 + T-cells expressing cytokines upon short-term in vitro stimulation of peripheral blood mononuclear cells with HBsAg-derived peptides. Secondary endpoints were anti-HBs antibody titres and frequency of HBsAg-specific memory B-cells. Results A strong and persistent specific CD4 + T-cell response was observed at Year 4 in all groups. HBsAg-specific CD4 + T-cells expressed mainly CD40L and IL-2, and to a lesser extent TNF-α and IFN-γ. HBsAg-specific CD8 + T-cells were not detected in any group. A high, persistent HBsAg-specific humoral immune response was observed in all groups, with all subjects seroprotected (antibody titre ≥10 mIU/mL) at Year 4. The geometric mean antibody titre at Year 4 was above 100,000 mIU/mL in all groups. A strong memory B-cell response was observed post-dose 2, which tended to increase post-dose 3 and persisted at Year 4 in all groups. Conclusion The MPL/QS-21/HBsAg vaccine formulations induced persistent immune responses up to 4 years after first vaccination. These Adjuvant Systems offer potential for combination with recombinant, synthetic or highly purified subunit vaccines, particularly for vaccination against challenging diseases, or in specific populations, although additional studies are needed.

We assessed potential toxic effects of the MAGE-A3 Cancer Immunotherapeutic on female fertility and embryo-fetal, pre- and post-natal development in rats and on male fertility in rats and monkeys. Three groups of 48 female (Study 1) or 22 male (Study 2) CD rats received 5 or 3 injections of 100 μL of saline, AS15 immunostimulant, or MAGE-A3 Cancer Immunotherapeutic (MAGE-A3 recombinant protein combined with AS15) at various timepoints pre- or post-mating. Male Cynomolgus monkeys (Study 3) received 8 injections of 500 μL of saline ( n = 2) or the MAGE-A3 Cancer Immunotherapeutic ( n = 6) every 2 weeks. Rats were sacrificed on gestation day 20 or lactation day 25 (Study 1) or 9 weeks after first injection (Study 2) and monkeys, 3 days or 8 weeks after last injection. Injections were well tolerated. Female rat mating performance or fertility, pre- and post-natal survival, offspring development up to 25 days of age, and male mating performance (rats) or fertility parameters (rats and monkeys) were unaffected.

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.

Adjuvant system AS01 is used in two candidate vaccines that have recently demonstrated efficacy in phase III trials: the malaria vaccine, RTS,S and the herpes zoster vaccine, HZ/su. AS01 is also in efficient in other candidate vaccines targeted against tuberculosis and human immunodeficiency virus/AIDS, two diseases in addition to malaria that represent global health priorities. AS01 contains liposomes and two immunostimulants, 3-O-desacyl-4ʹ-monophosphoryl lipid A and the purified saponin, QS-21. Both immunostimulants in AS01 contribute to the local and transient induction of innate immunity immediately after the vaccine's injection, and this induction appears to be critical for the promotion of antigen-specific cell-mediated and antibody-mediated immunity. The promotion of adaptive immunity by AS01 can occur in diverse populations ranging from infants to the elderly, and in the immunocompromised. Future challenges include further elucidation of the relationship between AS01's mode of action and vaccine correlates of protection and vaccine reactogenicity profiles, to shape strategies for improving existing AS01-containing vaccines and for aiding the design of new vaccines.

Combining immunostimulants in adjuvants can improve the quality of the immune response to vaccines. Here, we report a unique mechanism of molecular and cellular synergy between a TLR4 ligand, 3-O-desacyl-4’-monophosphoryl lipid A (MPL), and a saponin, QS-21, the constituents of the Adjuvant System AS01. AS01 is part of the malaria and herpes zoster vaccine candidates that have demonstrated efficacy in phase III studies. Hours after injection of AS01-adjuvanted vaccine, resident cells, such as NK cells and CD8+ T cells, release IFNγ in the lymph node draining the injection site. This effect results from MPL and QS-21 synergy and is controlled by macrophages, IL-12 and IL-18. Depletion strategies showed that this early IFNγ production was essential for the activation of dendritic cells and the development of Th1 immunity by AS01-adjuvanted vaccine. A similar activation was observed in the lymph node of AS01-injected macaques as well as in the blood of individuals receiving the malaria RTS,S vaccine. This mechanism, previously described for infections, illustrates how adjuvants trigger naturally occurring pathways to improve the efficacy of vaccines.

During the past two decades, GSK Vaccines has been developing new Adjuvant Systems (ASs) intended to promote a faster, stronger, and longer protection through the induction of high and persistent antibody titer and induction of cell-mediated immunity (CMI). One such proprietary AS, AS04, has been developed for prophylactic vaccines. AS04 is composed of two adjuvants: aluminum salt and 3-O-desacyl-4′- monophosphoryl lipid A (MPL).

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. Copyright © 2014 GlaxoSmithKline Vaccines. Journal of Applied Toxicology published by John Wiley & Sons, Ltd.

A novel human papillomavirus (HPV) vaccine has been formulated with virus-like particles of the L1 protein of HPV-16 and HPV-18, and the Adjuvant System 04 (AS04). AS04 is a combination of the toll-like receptor 4 agonist monophosphoryl lipid A (MPL) and aluminum hydroxide. The AS04-adjuvanted HPV vaccine induces a high and sustained immune response against HPV, including high levels of neutralizing antibodies at the cervical mucosa in women aged 15-55 years. Recently, the mechanism of action of AS04 has been evaluated in vitro in human cells and in vivo in mice and the data provide evidence for the molecular and cellular basis of the observed immunogenicity, efficacy, and safety profile of this formulation. In this review, we discuss how the results of GlaxoSmithKline's clinical studies on immunogenicity, protection, and reactogenicity with the AS04-adjuvanted HPV vaccine are supported by the observed mechanism of action for the adjuvant. The adjuvant activity of AS04, as measured by enhanced antibody response to HPV antigens, was found to be strictly dependent on AS04 and the HPV antigens being injected at the same intramuscular site within 24 hours of each other. The addition of MPL to aluminum salt enhances humoral and cell-mediated response by rapidly triggering a local and transient cytokine response that leads to an increased activation of antigen-presenting cells and results in an improved presentation of antigen to CD4+ T cells. The added value of MPL in AS04 for an HPV vaccine was demonstrated in clinical studies by high vaccine-elicited antibody responses and the induction of high levels of memory B cells. The vaccine elicits cross protection against some other oncogenic HPV types (specifically HPV-31, -33, and -45) not contained in the vaccine. The localized and transient nature of the innate immune response supports the acceptable safety profile observed in clinical studies.

Novel adjuvants that contain immunoenhancer molecules are now present in human vaccines either registered or in clinical trials. These adjuvants have the potential to provide clear benefits in improving the magnitude and duration of various aspects of the adaptive immune response. However, the use of immunoenhancers in vaccine formulations may be perceived as introducing theoretical safety risks that need to be addressed during the course of vaccine development. In addition to classical clinical safety evaluation, the licensing authorities recommend that novel adjuvants should be evaluated in non-clinical toxicology studies, both as separate entities and as part of the final vaccine formulation. We present here our approach for the safety evaluation of adjuvanted vaccines using AS04-adjuvanted vaccines as example. This evaluation consists of three tiers: non-clinical toxicology, adjuvant mode-of-action investigations and clinical safety assessment in controlled clinical trials and post-marketing surveillance. We also discuss how the knowledge of adjuvant mode of action can support the current practice of safety evaluation.

The immunostimulants 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and the saponin QS-21 are part of licensed or candidate vaccines. MPL and QS-21 directly affect the innate immune response to orchestrate the quality and intensity of the adaptive immune response to the vaccine antigens. The combination of immunostimulants in different adjuvant formulations forms the basis of Adjuvant Systems (AS) as a way to promote appropriate protective immune responses following vaccination. MPL and aluminum salts are present in AS04, and both MPL and QS-21 are present in AS01 and AS02, which are liposome- and emulsion-based formulations, respectively. The recent clinical performance of AS01-, AS02- and AS04-adjuvanted vaccines will be discussed in the context of the diseases being targeted. The licensing of two AS04-adjuvanted vaccines and the initiation of Phase III trials with an AS01-adjuvanted vaccine demonstrate the potential to develop new or improved human vaccines that contain MPL or MPL and QS-21.

Combining immunostimulants in adjuvants can improve the quality of the immune response to vaccines. Here, we report a unique mechanism of molecular and cellular synergy between a TLR4 ligand, 3-O-desacyl-4’-monophosphoryl lipid A (MPL), and a saponin, QS-21, the constituents of the Adjuvant System AS01. AS01 is part of the malaria and herpes zoster vaccine candidates that have demonstrated efficacy in phase III studies. Hours after injection of AS01-adjuvanted vaccine, resident cells, such as NK cells and CD8+ T cells, release IFNγ in the lymph node draining the injection site. This effect results from MPL and QS-21 synergy and is controlled by macrophages, IL-12 and IL-18. Depletion strategies showed that this early IFNγ production was essential for the activation of dendritic cells and the development of Th1 immunity by AS01-adjuvanted vaccine. A similar activation was observed in the lymph node of AS01-injected macaques as well as in the blood of individuals receiving the malaria RTS,S vaccine. This mechanism, previously described for infections, illustrates how adjuvants trigger naturally occurring pathways to improve the efficacy of vaccines., Adjuvants: Vaccine components working in synergy to improve beneficial effects of vaccination A mechanism is revealed by which vaccine components co-operate to stimulate the immune system and improve vaccine efficacy. Some vaccines are formulated with adjuvants—compounds that induce a greater immune response to the vaccine and help to elicit greater protection against future infections. Arnaud Didierlaurent and his team of researchers at GSK Vaccines, Belgium, demonstrate that the two immunostimulants in the adjuvant AS01, used in several recently developed vaccines, works in tandem to trigger the activation of important immune system moderators. The synergistic effect of the immunostimulants modulate specific immune cells at the site of the vaccination to better prepare the body against future infection. Studies such as this allow us to better understand how vaccines work and lay the foundation for more informed research into future vaccine development.

Protection against Plasmodium falciparum sporozoite infection can be achieved by vaccination with the recombinant circumsporozoite protein-based vaccine RTS,S formulated with the AS02A Adjuvant System. Since this protection is only partial and wanes over time, we have developed a new RTS,S-based vaccine adjuvanted with AS01B. RTS,S/AS01B-induced high specific antibody titers and increased the frequency of mouse CD4(+) and CD8(+) T cells expressing IFN-gamma, and of monkey CD4(+) T cells expressing IL-2 and/or IFN-gamma and/or TNF-alpha upon stimulation with vaccine antigens. Our data provides clear evidence that combining RTS,S antigen with a potent adjuvant induces strong humoral and cellular responses in vivo.

The need for potentiating immune responses to recombinant or subunit antigens has prompted GlaxoSmithKline (GSK) Biologicals to develop various Adjuvant Systems for the design of prophylactic and therapeutic vaccines. Adjuvant Systems are formulations of classical adjuvants mixed with immunomodulators, specifically adapted to the antigen and the target population. They can activate the appropriate innate immune system and subsequently impact on adaptive immune responses. AS04 is an Adjuvant System that has demonstrated significant achievements in several vaccines against viral diseases. AS02, another Adjuvant System, is being evaluated in various contexts, where a strong T-cell response is needed to afford protection. Likewise, AS01 has been developed for vaccines where the induction of a yet stronger T-cell-mediated immune response is required. Altogether, the promising clinical results strongly support the concept of Adjuvant Systems and allow for further development of new vaccines, best adapted to the target population and the immune mechanisms of protection.

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.

Background Studies are underway to identify more immunogenic formulations of the existing anti-falciparum malaria vaccine RTS,S/AS02A. To supplement in vitro immunogenicity assays, cutaneous delayed-type hypersensitivity (DTH) may be a useful indicator of functional, cell-mediated immunogenicity. Methods Adult rhesus monkeys were immunized with saline or one of four RTS,S/adjuvant formulations: RTS,S/AS01B, RTS,S/AS02A-standard (current formulation), RTS,S/AS05 or RTS,S/AS06 at 0, 4, and 12 weeks. An additional cohort received RTS,S/AS02A-accelerated, at 0, 1, and 4 weeks. Six months after completing immunizations, five vaccine-relevant antigens (high and low doses) and two controls were administered intradermally. DTH reactivity (induration) was measured at 48 and 72 h, and selected sites were biopsied for histological confirmation. Results In comparison with RTS,S/AS02A-standard, RTS,S/AS01B and RTS,S/AS05 each had larger mean reactions (induration) at 5 of 10 (p

Recombinant subunit protein vaccines generally elicit good humoral immune responses, weak helper T cell responses and no cytotoxic T cell responses. Certain adjuvants are known to enhance humoral and cellular immune responses. This study evaluated the humoral, CD4+ T helper and CTL responses induced by the recombinant SL* protein adjuvanted with AS02A in comparison with non-adjuvanted SL* in PBS in two groups of 15 healthy adult volunteers. The AS02A adjuvant contains monophosphoryl lipid A (MPL), QS21 and an oil in water emulsion. The adjuvanted vaccine induced fast and vigorous humoral and helper T cell responses of the Th1 type. Using a pool of overlapping 20mer peptides a cytotoxic response was detected in 6 out of 14 HLA-A2-positive (+) and HLA-A2-negative (-) recipients of the adjuvanted vaccine. All HLA-A2-positive subjects in the adjuvanted group and up to 30% of the subjects in the SL* PBS group displayed a CTL response against selected HLA-A2-restricted CD8+ T cell epitopes. The non-adjuvanted vaccine induced a very weak antibody response and no helper T cell responses. Local and general reactions were more frequently reported by AS02A recipients than in the non-adjuvanted group but the safety profile was considered acceptable. AS02A can be considered as a useful adjuvant that strongly enhances the cellular and humoral responses of subunit protein vaccines.

During the past decade, tremendous progress has been made in process development allowing for the production of large quantities of recombinant antigens, as well as in the understanding of the immune mechanisms underlying protection. Parallel to this, various and numerous adjuvant systems have been developed and tested in animal models and in clinical trials but have rarely induced protection. This review will discuss the development of a new adjuvant system (AS02) in combination with a malaria vaccine antigen candidate. To date, this vaccine is the only one to demonstrate protection in man in artificial challenge as well as in natural field trials. It has been established that this adjuvant system is capable of eliciting high antibody titers along with strong cell-mediated immunity which both contribute to the efficacy of the vaccine.

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.

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.

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 MHCIIhigh dendritic cells were higher after the injection of the AS01-adjuvanted vaccine than after Ag alone. However, only MHCIIhigh 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.

Several formulations of a recombinant chimeric respiratory syncytial virus (RSV) vaccine consisting of the extramembrane domains of the F and G glycoproteins (FG) were tested in cotton rats to evaluate efficacy and safety. The FG vaccine was highly immunogenic, providing nearly complete resistance to pulmonary infection at doses as low as 25 ng in spite of inducing relatively low levels of serum neutralizing antibody at low vaccine doses. Upon RSV challenge animals primed with FG vaccine showed quite mild alveolitis and interstitial pneumonitis, which were eliminated by the addition of monophosphoryl lipid A to the formulation.

Background The candidate vaccines against malaria are poorly immunogenic and thus have been ineffective in preventing infection. We developed a vaccine based on the circumsporozoite protein of Plasmodium falciparum that incorporates adjuvants selected to enhance the immune response. Methods The antigen consists of a hybrid in which the circumsporozoite protein fused to hepatitis B surface antigen (HBsAg) is expressed together with unfused HBsAg. We evaluated three formulations of this antigen in an unblinded trial in 46 subjects who had never been exposed to malaria. Results Two of the vaccine formulations were highly immunogenic. Four subjects had adverse systemic reactions that may have resulted from the intensity of the immune response after the second dose, which led us to reduce the third dose. Twenty-two vaccinated subjects and six unimmunized controls underwent a challenge consisting of bites from mosquitoes infected with P. falciparum. Malaria developed in all six control subjects, seven of eight ...

When liposomes are used to present haptens and T-independent antigens to the immune system the response is primarily of the IgM type. This response can be switched to a potent IgG type by the incorporation within the liposomal structure of either exogenous T-epitopes, immunostimulants, or both.The capability of liposomes to co-present separate B-and T-epitopes and immunostimulants, without the necessity for covalent conjugation, thus makes them ideal candidates as carriers for vaccines where the immune response is limited to recognition of T-epitopes within the antigen.

Introduction: Inflammatory reactions are one of the potential safety concerns that are evaluated in the framework of vaccine safety testing. In nonclinical studies, the assessment of the inflammation relies notably on the measurement of biomarkers. C-reactive protein (CRP) is an acute-phase plasma protein of hepatic origin that could be used for that purpose in toxicity studies with rabbits. Methods: To evaluate the utility of CRP as an additional inflammatory biomarker in adjuvant or vaccine toxicity studies, rabbits were injected on Day 0 with saline, aluminium phosphate, aluminium hydroxide, Adjuvant System (AS)01, AS03, AS15, or diphtheria-tetanus-whole cell pertussis-hepatitis B vaccine (DTPw-HB). Body weights, haematology parameters, CRP and fibrinogen levels were measured daily up to Day 7. Macroscopic changes at the injection site were also evaluated up to Day 7. At Day 7, a histopathological examination of the injection site was performed. Results: Like fibrinogen, CRP levels rapidly increased after the injection of Adjuvant Systems or DTPw-HB, peaking at Day 1, and returning to baseline in less than a week. The magnitude of the CRP increase was consistently higher than that of fibrinogen with a larger fold increase from background, providing a more sensitive evaluation. The number of circulating heterophils was also increased on Day 1 after the injection of Adjuvant Systems or DTPw-HB. The highest increases in CRP levels were observed after the injection of DTPw-HB or AS03, and were also associated with the persistence of mixed inflammatory cell infiltrates (including heterophils) at the injection sites on Day 7. No increases in CRP levels and in circulating heterophils were observed after injection of the aluminium salt adjuvants. Discussion: Our study supports the use of CRP as an accurate biomarker of acute inflammation in rabbits for vaccine toxicity studies and highlights an association between increased CRP levels and the recruitment of heterophils. © 2013 Elsevier Inc.

AS03 is an Adjuvant System composed of α-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion. In various nonclinical and clinical studies, high levels of antigen-specific antibodies were obtained after administration of an AS03-adjuvanted vaccine, permitting antigen-sparing strategies. AS03 has been shown to enhance the vaccine antigen-specific adaptive response by activating the innate immune system locally and by increasing antigen uptake and presentation in draining lymph nodes, a process that is modulated by the presence of α-tocopherol in AS03. In nonclinical models of the AS03-adjuvanted prepandemic H5N1 influenza vaccine, increased levels of anti-influenza antibody afforded protection against disease and against virus replication of influenza strains homologous and heterologous to the vaccine strain. By incorporating AS03 in the pandemic H1N1/2009 vaccine, vaccine immunogenicity was increased compared with nonadjuvanted H1N1 vaccines. High H1N1/2009/AS03 vaccine effectiveness was demonstrated in several assessments in multiple populations. Altogether, the nonclinical and clinical data illustrate the ability of AS03 to induce superior adaptive responses against the vaccine antigen, principally in terms of antibody levels and immune memory. In general, these results support the concept of Adjuvant Systems as a plausible approach to develop new effective vaccines.