Squalene emulsion-based vaccine adjuvants stimulate CD8 T cell, but not antibody responses, through a RIPK3-dependent pathway

Abstract

The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remain unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hr, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8+ DCs. Consistent with this, RIPK3 deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene emulsion-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.

Emerging concepts in the science of vaccine adjuvants

“…Given the similarity of AS03 and MF59 both being squalene-based adjuvants, it is likely that they engage common pathways of innate immune activation. As described, both adjuvants stimulate innate cytokines and chemokines, but the extent to which they both depend on the IRE1α and RIPK3-dependent pathways 76 remains to be determined.…”

“…Interestingly, and consistent with the study by Seubert et al, the response was intact in Nlrp3 -knockout and Casp1 -knockout mice 75 , which led the authors to conclude that MF59-adjuvanted vaccines induce antibody responses via an ASC-dependent, but inflammasome-independent, pathway. Finally, our recent work demonstrates that subcutaneous immunization of mice with MF59 plus antigen stimulates antigen-specific CD8 + T cell responses (which are stimulated at very low magnitude in the draining lymph nodes, but can be detected at much higher frequencies in tissues such as the lung and liver), via a mechanism dependent on receptor-interacting serine/threonine protein kinase 3 (RIPK3), which is a key mediator of necroptosis 76 . Thus, immunization of mice with antigen mixed with MF59 or its mimetic Addavax, but not with alum, induced rapid RIPK3-dependent necroptosis of lymph node macrophages.…”

“…RIPK3-deficient mice were impaired in their capacity to mount antigen-specific CD8 + T cell responses in the lung and liver, in response to subcutaneous immunization with antigen plus Addavax. However, surprisingly, such responses were normal in mice deficient in mixed-lineage kinase domain-like protein (MLKL), a downstream mediator of necroptosis, suggesting that the impaired CD8 + T cell responses observed in RIPK3-deficient mice occurred through a mechanism independent of necroptosis 76 . In contrast to the effects observed on CD8 + T cells, antibody responses were not affected in RIPK3-deficient mice or in caspase 1-deficient mice.…”

“…In particular, recent work has highlighted that receptor-interacting serine/threonine protein kinase 3 (RIPK3)-mediated necroptosis can synergize with NF-κB-dependent inflammation to promote CD8 + T cell responses. Furthermore, recent work shows that the adjuvant effects of MF59 in promoting CD8 + T cell response are largely mediated by the key mediator of necroptosis RIPK3 but via a necroptosis-independent pathway 76 . Epigenetic adjuvants: recent work suggests that innate cells including monocytes and macrophages undergo epigenetic modifications and acquire memory-like characteristics following stimulation with pathogen-associated molecular patterns (PAMPs) 161 – 166 .…”

Squalene-Based Influenza Vaccine Adjuvants and Their Impact on the Hemagglutinin-Specific B Cell Response

“… Immunocompetent environment induced by squalene-based emulsion (SE) adjuvants at the injection site. SE adjuvants cause local muscle cell damage (e.g., cell lysis by detergent-like molecules in the formulation), inducing the transient release of damage-associated molecular pattern (DAMP) signals such as ATP and double-stranded DNA, and the change in expression of as many as 891 genes in mice, compared to 312 genes by Alum at the injection site [ 35 , 42 , 43 , 44 ]. These DAMP signals bind to receptors on antigen-presenting cells (APCs) cells, trigger cytokine and chemokine secretion cascades as early as 3–6 h post-injection in mice [ 45 ].…”