Vaccine adjuvants: Tailoring innate recognition to send the right message.

Adjuvants play pivotal roles in vaccine development, enhancing immunization efficacy through prolonged retention and sustained release of antigen, lymph node targeting, and regulation of dendritic cell activation. Adjuvant-induced activation of innate immunity is achieved via diverse mechanisms: for example, adjuvants can serve as direct ligands for pathogen recognition receptors or as inducers of cell stress and death, leading to the release of immunostimulatory-damage-associated molecular patterns. Adjuvant systems increasingly stimulate multiple innate pathways to induce greater potency. Increased understanding of the principles dictating adjuvant-induced innate immunity will subsequently lead to programming specific types of adaptive immune responses. This tailored optimization is fundamental to next-generation vaccines capable of inducing robust and sustained adaptive immune memory across different cohorts.

Non-canonical inflammasome activation mediates the adjuvanticity of nanoparticles.

The non-canonical inflammasome sensor caspase-11 and gasdermin D (GSDMD) drive inflammation and pyroptosis, a type of immunogenic cell death that favors cell-mediated immunity (CMI) in cancer, infection, and autoimmunity. Here we show that caspase-11 and GSDMD are required for CD8+ and Th1 responses induced by nanoparticulate vaccine adjuvants. We demonstrate that nanoparticle-induced reactive oxygen species (ROS) are size dependent and essential for CMI, and we identify 50- to 60-nm nanoparticles as optimal inducers of ROS, GSDMD activation, and Th1 and CD8+ responses. We reveal a division of labor for IL-1 and IL-18, where IL-1 supports Th1 and IL-18 promotes CD8+ responses. Exploiting size as a key attribute, we demonstrate that biodegradable poly-lactic co-glycolic acid nanoparticles are potent CMI-inducing adjuvants. Our work implicates ROS and the non-canonical inflammasome in the mode of action of polymeric nanoparticulate adjuvants and establishes adjuvant size as a key design principle for vaccines against cancer and intracellular pathogens.

Group 2 Innate Lymphoid Cells Are Detrimental to the Control of Infection with Francisella tularensis.

Innate lymphoid cells (ILCs) are capable of rapid response to a wide variety of immune challenges, including various respiratory pathogens. Despite this, their role in the immune response against the lethal intracellular bacterium Francisella tularensis is not yet known. In this study, we demonstrate that infection of the airways with F. tularensis results in a significant reduction in lung type 2 ILCs (ILC2s) in mice. Conversely, the expansion of ILC2s via treatment with the cytokine IL-33, or by adoptive transfer of ILC2s, resulted in significantly enhanced bacterial burdens in the lung, liver, and spleen, suggesting that ILC2s may favor severe infection. Indeed, specific reduction of ILC2s in a transgenic mouse model results in a reduction in lung bacterial burden. Using an in vitro culture system, we show that IFN-γ from the live vaccine strain-infected lung reduces ILC2 numbers, suggesting that this cytokine in the lung environment is mechanistically important in reducing ILC2 numbers during infection. Finally, we show Ab-mediated blockade of IL-5, of which ILC2s are a major innate source, reduces bacterial burden postinfection, suggesting that IL-5 production by ILC2s may play a role in limiting protective immunity. Thus, overall, we highlight a negative role for ILC2s in the control of infection with F. tularensis. Our work therefore highlights the role of ILC2s in determining the severity of potentially fatal airway infections and raises the possibility of interventions targeting innate immunity during infection with F. tularensis to benefit the host.

Type II NKT Cell Agonist, Sulfatide, Is an Effective Adjuvant for Oral Heat-Killed Cholera Vaccines.

Oral vaccination has the potential to offer a safer and more efficacious approach for protection against enteric pathogens than injection-based approaches, especially in developing countries. One key advantage is the potential to induce intestinal immune responses in addition to systemic immunity. In general, antigen delivery via the oral route triggers weak immune responses or immunological tolerance. The effectiveness of oral vaccination can be improved by co-administering adjuvants. However, a major challenge is the absence of potent and safe oral adjuvants for clinical application. Here, the Type II NKT cell activator sulfatide is shown for the first time to be an effective oral adjuvant for Vibrio cholerae vaccine antigens in a mouse model. Specifically, administration of sulfatide with the oral cholera vaccine Dukoral® resulted in enhancement of intestinal antigen-specific IgA in addition to Th1 and Th17 immune responses. In summary, sulfatide is a promising adjuvant for inclusion in an oral cholera vaccine and our data further support the potential of adjuvants targeting NKT cells in new vaccine strategies.

Chitin-derived polymer deacetylation regulates mitochondrial reactive oxygen species dependent cGAS-STING and NLRP3 inflammasome activation.

Chitosan is a cationic polysaccharide that has been evaluated as an adjuvant due to its biocompatible and biodegradable nature. The polysaccharide can enhance antibody responses and cell-mediated immunity following vaccination by injection or mucosal routes. However, the optimal polymer characteristics for activation of dendritic cells (DCs) and induction of antigen-specific cellular immune responses have not been resolved. Here, we demonstrate that only chitin-derived polymers with a high degree of deacetylation (DDA) enhance generation of mitochondrial reactive oxygen species (mtROS), leading to cGAS-STING mediated induction of type I IFN. Additionally, the capacity of the polymers to activate the NLRP3 inflammasome was strictly dependent on the degree and pattern of deacetylation and mtROS generation. Polymers with a DDA below 80% are poor adjuvants while a fully deacetylated polyglucosamine polymer is most effective as a vaccine adjuvant. Furthermore, this polyglucosamine polymer enhanced antigen-specific Th1 responses in a NLRP3 and STING-type I IFN-dependent manner. Overall these results indicate that the degree of chitin deacetylation, the acetylation pattern and its regulation of mitochondrial ROS are the key determinants of its immune enhancing effects.

Regulation of barrier immunity and homeostasis by integrin-mediated transforming growth factor ß activation.

Transforming growth factor ß (TGF-ß) is a multifunctional cytokine that regulates cell growth, differentiation, adhesion, migration and death dependent on cell type, developmental stage, or tissue conditions. Various cell types secrete TGF-ß, but always as an inactive complex. Hence, for TGF-ß to function, this latent complex must somehow be activated. Work in recent years has highlighted a critical role for members of the αv integrin family, including αv ß1 , αv ß3 , αv ß5 , αv ß6 and αv ß8 that are involved in TGF-ß activation in various contexts, particularly at barrier sites such as the gut, lung and skin. The integrins facilitating this context- and location-specific regulation can be dysregulated in certain diseases, so are potential therapeutic targets in a number of disorders. In this review, we discuss the role of TGF-ß at these barrier sites with a focus on how integrin-mediated TGF-ß activation regulates tissue and immune homeostasis, and how this is altered in disease.

Type I IFN signalling is required for cationic adjuvant formulation (CAF)01-induced cellular immunity and mucosal priming.

Despite being in the midst of a global pandemic of infections caused by the pathogen Chlamydia trachomatis, a vaccine capable of inducing protective immunity remains elusive. Given the C. trachomatis mucosal port of entry, a formulation compatible with mucosal administration and capable of eliciting potent genital tract immunity is highly desirable. While subunit vaccines are considered safer and better tolerated, these are typically poorly immunogenic and require co-formulation with immune-potentiating adjuvants. However, of the adjuvants licensed for use in humans, very few drive robust cellular responses, a pre-requisite for protection against C. trachomatis infection. Recently, the cationic adjuvant formulations (CAF) have been shown to induce robust humoral and cellular immunity in pre-clinical models of chlamydia, malaria and tuberculosis (TB). Here, we demonstrate that CAF01 induces potent immune responses when combined with the major outer membrane protein (MOMP) of C. trachomatis following parenteral immunisation and also as part of a heterologous prime/boost regime. We show that a subcutaneous prime with CAF01-adjuvanted recombinant MOMP licenses antigen-specific immunity at distant mucosal sites which can be activated following oral antigen re-encounter in the absence of concomitant adjuvant stimulation. Finally, we shed light on the mechanism(s) through which CAF01 elicits robust antigen-specific immunity to co-formulated MOMP via type I interferon (IFN) signalling.

An oral alpha-galactosylceramide adjuvanted Helicobacter pylori vaccine induces protective IL-1R- and IL-17R-dependent Th1 responses.

Helicobacter pylori causes chronic gastric infection that can lead to peptic ulcers and is an identified risk factor for gastric cancer development. Although much effort has been put into the development of a Helicobacter pylori vaccine over the last three decades, none has yet reached clinical application. Specific challenges pertaining to effective H. pylori vaccine development include the lack of proven vaccine-effective antigens and safe mucosal adjuvants to enhance local immune responses as well as the lack of accepted correlates of protection. Herein, we demonstrate that prophylactic intragastric immunisation with a whole-cell killed H. pylori antigen administered together with the non-toxic oral adjuvant α-galactosylceramide (α-GalCer) induced effective immune protection against H. pylori infection in mice, which was of similar magnitude as when using the "gold standard" cholera toxin as adjuvant. We further describe that this α-GalCer-adjuvanted vaccine formulation elicited strong intestinal and systemic Th1 responses as well as significant antigen-specific mucosal and systemic antibody responses. Finally, we report that the protective intestinal Th1 responses induced by α-GalCer are dependent on CD1d, IL-1R as well as IL-17R signalling. In summary, our results show that α-GalCer is a promising adjuvant for inclusion in an oral vaccine against H. pylori infection.

Migratory DCs activate TGF-ß to precondition naïve CD8+ T cells for tissue-resident memory fate.

Epithelial resident memory T (eTRM) cells serve as sentinels in barrier tissues to guard against previously encountered pathogens. How eTRM cells are generated has important implications for efforts to elicit their formation through vaccination or prevent it in autoimmune disease. Here, we show that during immune homeostasis, the cytokine transforming growth factor ß (TGF-ß) epigenetically conditions resting naïve CD8+ T cells and prepares them for the formation of eTRM cells in a mouse model of skin vaccination. Naïve T cell conditioning occurs in lymph nodes (LNs), but not in the spleen, through major histocompatibility complex class I-dependent interactions with peripheral tissue-derived migratory dendritic cells (DCs) and depends on DC expression of TGF-ß-activating αV integrins. Thus, the preimmune T cell repertoire is actively conditioned for a specialized memory differentiation fate through signals restricted to LNs.

Divergent Roles for the IL-1 Family in Gastrointestinal Homeostasis and Inflammation.

Inflammatory disorders of the gastro-intestinal tract are a major cause of morbidity and significant burden from a health and economic perspective in industrialized countries. While the incidence of such conditions has a strong environmental component, in particular dietary composition, epidemiological studies have identified specific hereditary mutations which result in disequilibrium between pro- and anti-inflammatory factors. The IL-1 super-family of cytokines and receptors is highly pleiotropic and plays a fundamental role in the pathogenesis of several auto-inflammatory conditions including rheumatoid arthritis, multiple sclerosis and psoriasis. However, the role of this super-family in the etiology of inflammatory bowel diseases remains incompletely resolved despite extensive research. Herein, we highlight the currently accepted paradigms as they pertain to specific IL-1 family members and focus on some recently described non-classical roles for these pathways in the gastrointestinal tract. Finally, we address some of the shortcomings and sources of variance in the field which to date have yielded several conflicting results from similar studies and discuss the potential effect of these factors on data interpretation.

Alpha-galactosylceramide enhances mucosal immunity to oral whole-cell cholera vaccines.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3-4 million cases globally with 100,000-150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses.

Human monocytes and macrophages regulate immune tolerance via integrin αvß8-mediated TGFß activation.

Monocytes are crucial immune cells involved in regulation of inflammation either directly or via differentiation into macrophages in tissues. However, many aspects of how their function is controlled in health and disease are not understood. Here we show that human blood monocytes activate high levels of the cytokine TGFß, a pathway that is not evident in mouse monocytes. Human CD14+, but not CD16+, monocytes activate TGFß via expression of the integrin αvß8 and matrix metalloproteinase 14, which dampens their production of TNFα in response to LPS. Additionally, when monocytes differentiate into macrophages, integrin expression and TGFß-activating ability are maintained in anti-inflammatory macrophages but down-regulated in pro-inflammatory macrophages. In the healthy human intestine, integrin αvß8 is highly expressed on mature tissue macrophages, with these cells and their integrin expression being significantly reduced in active inflammatory bowel disease. Thus, our data suggest that integrin αvß8-mediated TGFß activation plays a key role in regulation of monocyte inflammatory responses and intestinal macrophage homeostasis.

A novel adjuvanted capsule based strategy for oral vaccination against infectious diarrhoeal pathogens.

Diarrhoeal infections are a major cause of morbidity and mortality with enterotoxigenic Escherichia coli (ETEC) and cholera imposing a significant global burden. There is currently no licensed vaccine for ETEC. Development of new nonliving oral vaccines has proven difficult due to the physicochemical and immunological challenges associated with the oral route. This demands innovative delivery solutions to protect antigens, control their release and build in immune-stimulatory activity. We describe the Single Multiple Pill® (SmPill®) vaccine formulation which combines the benefits of enteric polymer coating to protect against low gastric pH, a dispersed phase to control release and aid the solubility of non-polar components and an optimized combination of adjuvant and antigen to promote mucosal immunity. We demonstrate the effectiveness of this system with whole cell killed E. coli overexpressing colonization factor antigen I (CFA/I), JT-49. Alpha-galactosylceramide was identified as a potent adjuvant within SmPill® that enhanced the immunogenicity of JT-49. The bacteria associated with the dispersed phase were retained within the capsules at gastric pH but released at intestinal pH. Vaccination with an optimized SmPill® formulation promoted CFA/I-specific immunoglobulin A (IgA) responses in the intestinal mucosa in addition to serum IgG and a solubilized adjuvant was indispensable for efficacy.

The Vaccine Adjuvant Chitosan Promotes Cellular Immunity via DNA Sensor cGAS-STING-Dependent Induction of Type I Interferons.

The cationic polysaccharide chitosan is an attractive candidate adjuvant capable of driving potent cell-mediated immunity, but the mechanism by which it acts is not clear. We show that chitosan promotes dendritic cell maturation by inducing type I interferons (IFNs) and enhances antigen-specific T helper 1 (Th1) responses in a type I IFN receptor-dependent manner. The induction of type I IFNs, IFN-stimulated genes and dendritic cell maturation by chitosan required the cytoplasmic DNA sensor cGAS and STING, implicating this pathway in dendritic cell activation. Additionally, this process was dependent on mitochondrial reactive oxygen species and the presence of cytoplasmic DNA. Chitosan-mediated enhancement of antigen specific Th1 and immunoglobulin G2c responses following vaccination was dependent on both cGAS and STING. These findings demonstrate that a cationic polymer can engage the STING-cGAS pathway to trigger innate and adaptive immune responses.