Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms

Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q) and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%–100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting in vivo adaptive responses have a different TH1/TH2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design

TLR Agonists as Vaccine Adjuvants Targeting Cancer and Infectious Diseases

Modern vaccines have largely shifted from using whole, killed or attenuated pathogens to being based on subunit components. Since this diminishes immunogenicity, vaccine adjuvants that enhance the immune response to purified antigens are critically needed. Further advantages of adjuvants include dose sparing, increased vaccine efficacy in immunocompromised individuals and the potential to protect against highly variable pathogens by broadening the immune response. Due to their ability to link the innate with the adaptive immune response, Toll-like receptor (TLR) agonists are highly promising as adjuvants in vaccines against life-threatening and complex diseases such as cancer, AIDS and malaria. TLRs are transmembrane receptors, which are predominantly expressed by innate immune cells. They can be classified into cell surface (TLR1, TLR2, TLR4, TLR5, TLR6) and intracellular TLRs (TLR3, TLR7, TLR8, TLR9), expressed on endosomal membranes. Besides a transmembrane domain, each TLR possesses a leucine-rich repeat (LRR) segment that mediates PAMP/DAMP recognition and a TIR domain that delivers the downstream signal transduction and initiates an inflammatory response. Thus, TLRs are excellent targets for adjuvants to provide a “danger” signal to induce an effective immune response that leads to long-lasting protection. The present review will elaborate on applications of TLR ligands as vaccine adjuvants and immunotherapeutic agents, with a focus on clinically relevant adjuvants

A kinetic response model for standardized regression analyses of inflammation-triggered hypothermic body temperature-time courses in mice

LPS is frequently used to induce experimental endotoxic shock, representing a standard model of acute inflammation in mice. The resulting inflammatory response leads to hypothermia of the experimental animals, which in turn can be used as surrogate for the severity of systemic inflammation. Although increasingly applied as a humane endpoint in murine studies, differences between obtained temperature-time curves are typically evaluated at a single time point with $\it t$-tests or ANOVA analyses. We hypothesized that analyses of the entire temperature-time curves using a kinetic response model could fit the data, which show a temperature decrease followed by a tendency to return to normal temperature, and could increase the statistical power. Using temperature-time curves obtained from LPS stimulated mice, we derived a biologically motivated kinetic response model based on a differential equation. The kinetic model includes four parameters: (i) normal body temperature $(T_{n})$, (ii) a coefficient related to the force of temperature autoregulation ($\it r$), (iii) damage strength  $(p_{0})$, and (iv) clearance rate ($\it k$). Kinetic modeling of temperature-time curves obtained from LPS stimulated mice is feasible and leads to a high goodness-of-fit. Here, modifying key enzymes of inflammatory cascades induced a dominant impact of genotypes on the damage strength and a weak impact on the clearance rate. Using a likelihood-ratio test to compare modeled curves of different experimental groups yields strongly enhanced statistical power compared to pairwise t-tests of single temperature time points. Taken together, the kinetic model presented in this study has several advantages compared to simple analysis of individual time points and therefore may be used as a standard method for assessing inflammation-triggered hypothermic response curves in mice

Non-canonical Caspase-1 signaling drives RIP2-dependent and TNF-α-mediated inflammation in vivo

Pro-inflammatory caspase-1 is a key player in innate immunity. Caspase-1 processes interleukin (IL)-1β and IL-18 to their mature forms and triggers pyroptosis. These caspase-1 functions are linked to its enzymatic activity. However, loss-of-function missense mutations in CASP1 do not prevent autoinflammation in patients, despite decreased IL-1β production. In vitro data suggest that enzymatically inactive caspase-1 drives inflammation via enhanced nuclear factor κB (NF-κB) activation, independent of IL-1β processing. Here, we report two mouse models of enzymatically inactive caspase-1-C284A, demonstrating the relevance of this pathway in vivo. In contrast to Casp1 mice, caspase-1-C284A mice show pronounced hypothermia and increased levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 when challenged with lipopolysaccharide (LPS). Caspase-1-C284A signaling is RIP2 dependent and mediated by TNF-α but independent of the NLRP3 inflammasome. LPS-stimulated whole blood from patients carrying loss-of-function missense mutations in CASP1 secretes higher amounts of TNF-α. Taken together, these results reveal non-canonical caspase-1 signaling in vivo.Reinke et al. show that enzymatically inactive caspase-1-C284A mediates non-canonical caspase-1 signaling. This pathway is RIP2 dependent and mediated by TNF-α but independent from IL-1 cytokines