Toll-like receptor 4 signaling in hematopoietic-lineage cells contributes to the enhanced activity of the human vaccine adjuvant AS01.

The 3-O-desacyl-4'-monophosphoryl lipid A (MPL) activates immunity through Toll-like receptor 4 (TLR4) signaling. The Adjuvant System AS01 contains MPL and is used in the candidate malaria vaccine and the licensed zoster vaccine. Recent studies reported that AS01 adjuvant activity depends on a transient inflammation at the site of vaccination, but the role of stromal or structural cells in the adjuvant effect is unknown. We investigated this question in mouse models by assessing the role of TLR4 on hematopoietic versus resident structural cells during immunization with AS01-adjuvanted vaccines. We first established that TLR4-deficient animals had a reduced immune response to an AS01-adjuvanted vaccine. Using bone marrow chimera, we consistently found that Tlr4 expression in radio-sensitive cells, i.e., hematopoietic cells, was required for an optimal adjuvant effect on antibody and T-cell responses. At day 1 after injection, the pro-inflammatory reaction at the site of injection was strongly dependent on TLR4 signaling in hematopoietic cells. Similarly, activation of dendritic cells in muscle-draining lymph nodes was strictly associated with the radio-sensitive cells expressing Tlr4. Altogether, these data suggest that MPL-mediated TLR4-signaling in hematopoietic cells is critical in the mode of action of AS01.

The Lymphatic Immune Response Induced by the Adjuvant AS01: A Comparison of Intramuscular and Subcutaneous Immunization Routes.

The liposome-based adjuvant AS01 incorporates two immune stimulants, 3-O-desacyl-4'-monophosphoryl lipid A and the saponin QS-21. AS01 is under investigation for use in several vaccines in clinical development. i.m. injection of AS01 enhances immune cell activation and dendritic cell (DC) Ag presentation in the local muscle-draining lymph node. However, cellular and Ag trafficking in the lymphatic vessels that connect an i.m. injection site with the local lymph node has not been investigated. The objectives of this study were: 1) to quantify the in vivo cellular immune response induced by AS01 in an outbred ovine model, 2) to develop a lymphatic cannulation model that directly collects lymphatic fluid draining the muscle, and 3) to investigate the function of immune cells entering and exiting the lymphatic compartments after s.c. or i.m. vaccination with AS01 administered with hepatitis B surface Ag (HBsAg). We show that HBsAg-AS01 induces a distinct immunogenic cellular signature within the blood and draining lymphatics following both immunization routes. We reveal that MHCII(high) migratory DCs, neutrophils, and monocytes can acquire Ag within muscle and s.c. afferent lymph, and that HBsAg-AS01 uniquely induces the selective migration of Ag-positive neutrophils, monocytes, and an MHCII(high) DC-like cell type out of the lymph node via the efferent lymphatics that may enhance Ag-specific immunity. We report the characterization of the immune response in the lymphatic network after i.m. and s.c. injection of a clinically relevant vaccine, all in real time using a dose and volume comparable with that administered in humans.

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.

Risk of Monkeypox virus (MPXV) transmission through the handling and consumption of food.

Monkeypox (MPX) is a zoonotic infectious disease caused by Monkeypox virus (MPXV), an enveloped DNA virus belonging to the Poxviridae family and the Orthopoxvirus genus. Since early May 2022, a growing number of human cases of Monkeypox have been reported in non-endemic countries, with no history of contact with animals imported from endemic and enzootic areas, or travel to an area where the virus usually circulated before May 2022. This qualitative risk assessment aimed to investigate the probability that MPXV transmission occurs through food during its handling and consumption. The risk assessment used "top-down" (based on epidemiological data) and "bottom-up" (following the agent through the food chain to assess the risk of foodborne transmission to human) approaches, which were combined. The "top-down" approach first concluded that bushmeat was the only food suspected as a source of contamination in recorded cases of MPXV, by contact or ingestion. The "bottom-up" approach then evaluated the chain of events required for a human to become ill after handling or consuming food. This approach involves several conditions: (i) the food must be contaminated with MPXV (naturally, by an infected handler or after contact with a contaminated surface); (ii) the food must contain viable virus when it reaches the handler or consumer; (iii) the person must be exposed to the virus and; (iv) the person must be infected after exposure. Throughout the risk assessment, some data gaps were identified and highlighted. The conclusions of the top-down and bottom-up approaches are consistent and suggest that the risk of transmission of MPXV through food is hypothetical and that such an occurrence was never reported. In case of contamination, cooking (e.g., 12 min at 70°C) could be considered effective in inactivating Poxviridae in foods. Recommendations for risk management are proposed. To our knowledge, this is the first risk assessment performed on foodborne transmission of MPXV.

Innate Immune Responses to Chimpanzee Adenovirus Vector 155 Vaccination in Mice and Monkeys.

Replication-deficient chimpanzee adenovirus (ChAd) vectors represent an attractive vaccine platform and are thus employed as vaccine candidates against several infectious diseases. Since inducing effective immunity depends on the interplay between innate and adaptive immunity, a deeper understanding of innate immune responses elicited by intramuscularly injected ChAd vectors in tissues can advance the platform's development. Using different candidate vaccines based on the Group C ChAd type 155 (ChAd155) vector, we characterized early immune responses in injected muscles and draining lymph nodes (dLNs) from mice, and complemented these analyses by evaluating cytokine responses and gene expression patterns in peripheral blood from ChAd155-injected macaques. In mice, vector DNA levels gradually decreased post-immunization, but local transgene mRNA expression exhibited two transient peaks [at 6 h and Day (D)5], which were most obvious in dLNs. This dynamic pattern was mirrored by the innate responses in tissues, which developed as early as 1-3 h (cytokines/chemokines) or D1 (immune cells) post-vaccination. They were characterized by a CCL2- and CXCL9/10-dominated chemokine profile, peaking at 6 h (with CXCL10/CCL2 signals also detectable in serum) and D7, and clear immune-cell infiltration peaks at D1/D2 and D6/D7. Experiments with a green fluorescent protein-expressing ChAd155 vector revealed infiltrating hematopoietic cell subsets at the injection site. Cell infiltrates comprised mostly monocytes in muscles, and NK cells, T cells, dendritic cells, monocytes, and B cells in dLNs. Similar bimodal dynamics were observed in whole-blood gene signatures in macaques: most of the 17 enriched immune/innate signaling pathways were significantly upregulated at D1 and D7 and downregulated at D3, and clustering analysis revealed stronger similarities between D1 and D7 signatures versus the D3 signature. Serum cytokine responses (CXCL10, IL1Ra, and low-level IFN-α) in macaques were predominantly observed at D1. Altogether, the early immune responses exhibited bimodal kinetics with transient peaks at D1/D2 and D6/D7, mostly with an IFN-associated signature, and these features were remarkably consistent across most analyzed parameters in murine tissues and macaque blood. These compelling observations reveal a novel aspect of the dynamics of innate immunity induced by ChAd155-vectored vaccines, and contribute to ongoing research to better understand how adenovectors can promote vaccine-induced immunity.

CCR2- and Flt3-Dependent Inflammatory Conventional Type 2 Dendritic Cells Are Necessary for the Induction of Adaptive Immunity by the Human Vaccine Adjuvant System AS01.

The Adjuvant System AS01 contains monophosphoryl lipid A (MPL) and the saponin QS-21 in a liposomal formulation. AS01 is included in recently developed vaccines against malaria and varicella zoster virus. Like for many other adjuvants, induction of adaptive immunity by AS01 is highly dependent on the ability to recruit and activate dendritic cells (DCs) that migrate to the draining lymph node for T and B cell stimulation. The objective of this study was to more precisely address the contribution of the different conventional (cDC) and monocyte-derived DC (MC) subsets in the orchestration of the adaptive immune response after immunization with AS01 adjuvanted vaccine. The combination of MPL and QS-21 in AS01 induced strong recruitment of CD26+XCR1+ cDC1s, CD26+CD172+ cDC2s and a recently defined CCR2-dependent CD64-expressing inflammatory cDC2 (inf-cDC2) subset to the draining lymph node compared to antigen alone, while CD26-CD64+CD88+ MCs were barely detectable. At 24 h post-vaccination, cDC2s and inf-cDC2s were superior amongst the different subsets in priming antigen-specific CD4+ T cells, while simultaneously presenting antigen to CD8+ T cells. Diphtheria toxin (DT) mediated depletion of all DCs prior to vaccination completely abolished adaptive immune responses, while depletion 24 h after vaccination mainly affected CD8+ T cell responses. Vaccinated mice lacking Flt3 or the chemokine receptor CCR2 showed a marked deficit in inf-cDC2 recruitment and failed to raise proper antibody and T cell responses. Thus, the adjuvant activity of AS01 is associated with the potent activation of subsets of cDC2s, including the newly described inf-cDC2s.

Application of Modeling Approaches to Explore Vaccine Adjuvant Mode-of-Action.

Novel adjuvant technologies have a key role in the development of next-generation vaccines, due to their capacity to modulate the duration, strength and quality of the immune response. The AS01 adjuvant is used in the malaria vaccine RTS,S/AS01 and in the licensed herpes-zoster vaccine (Shingrix) where the vaccine has proven its ability to generate protective responses with both robust humoral and T-cell responses. For many years, animal models have provided insights into adjuvant mode-of-action (MoA), generally through investigating individual genes or proteins. Furthermore, modeling and simulation techniques can be utilized to integrate a variety of different data types; ranging from serum biomarkers to large scale "omics" datasets. In this perspective we present a framework to create a holistic integration of pre-clinical datasets and immunological literature in order to develop an evidence-based hypothesis of AS01 adjuvant MoA, creating a unified view of multiple experiments. Furthermore, we highlight how holistic systems-knowledge can serve as a basis for the construction of models and simulations supporting exploration of key questions surrounding adjuvant MoA. Using the Systems-Biology-Graphical-Notation, a tool for graphical representation of biological processes, we have captured high-level cellular behaviors and interactions, and cytokine dynamics during the early immune response, which are substantiated by a series of diagrams detailing cellular dynamics. Through explicitly describing AS01 MoA we have built a consensus of understanding across multiple experiments, and so we present a framework to integrate modeling approaches into exploring adjuvant MoA, in order to guide experimental design, interpret results and inform rational design of vaccines.

Activation of the endoplasmic reticulum stress sensor IRE1α by the vaccine adjuvant AS03 contributes to its immunostimulatory properties.

The oil-in-water emulsion Adjuvant System 03 (AS03) is one of the few adjuvants used in licensed vaccines. Previous work indicates that AS03 induces a local and transient inflammatory response that contributes to its adjuvant effect. However, the molecular mechanisms involved in its immunostimulatory properties are ill-defined. Upon intramuscular injection in mice, AS03 elicited a rapid and transient downregulation of lipid metabolism-related genes in the draining lymph node. In vitro, these modifications were associated with profound changes in lipid composition, alteration of endoplasmic reticulum (ER) morphology and activation of the unfolded protein response pathway. In vivo, treatment with a chemical chaperone or deletion of the ER stress sensor kinase IRE1α in myeloid cells decreased AS03-induced cytokine production and its capacity to elicit high affinity antigen-specific antibodies. In summary, our results indicate that IRE1α is a sensor for the metabolic changes induced by AS03 in monocytic cells and may constitute a canonical pathway that could be exploited for the design of novel vaccine adjuvants.

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.].

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.

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

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.

Central Role of CD169+ Lymph Node Resident Macrophages in the Adjuvanticity of the QS-21 Component of AS01.

Saponins represent a promising class of vaccine adjuvant. Together with the TLR4-ligand MPL, QS-21 is part of the Adjuvant System AS01, a key component of the malaria and zoster candidate vaccines that display demonstrated clinical efficacy. However, the mechanism of action of QS-21 in this liposomal formulation is poorly understood. Upon intra-muscular immunisation, we observed that QS-21 rapidly accumulated in CD169+ resident macrophages of the draining lymph node where it elicited a local innate immune response. Depletion of these cells abrogated QS-21-mediated innate cell recruitment to the lymph node, dendritic cell (DC) phenotypic maturation as well as the adjuvant effect on T-cell and antibody responses to co-administered antigens. DCs rather than lymph node-resident macrophages were directly involved in T-cell priming by QS-21, as revealed by the decrease in antigen-specific T-cell response in Batf3-/- mice. Further analysis showed that the adjuvant effect of QS-21 depended on the integration of Caspase-1 and MyD88 pathways, at least in part through the local release of HMGB1. Taken together, this work unravels the key role of lymph node sentinel macrophage in controlling the adjuvant effect of a molecule proven to improve vaccine response in humans.