Summary: Inflammasomes are multi‐protein complexes that assemble within the cytoplasm of mammalian cells in response to pathogen‐associated molecular patterns (PAMPs) or damage‐associated molecular patterns (DAMPs), driving the secretion of the pro‐inflammatory cytokines IL‐1β and IL‐18, and pyroptosis. The best‐characterized inflammasome complexes are the NLRP3, NAIP‐NLRC4, NLRP1, AIM2, and Pyrin canonical caspase‐1‐containing inflammasomes, and the caspase‐11 non‐canonical inflammasome. Newer inflammasome sensor proteins have been identified, including NLRP6, NLRP7, NLRP9, NLRP10, NLRP11, NLRP12, CARD8, and MxA. These inflammasome sensors can sense PAMPs from bacteria, viruses and protozoa, or DAMPs in the form of mitochondrial damage, ROS, stress and heme. The mechanisms of action, physiological relevance, consequences in human diseases, and avenues for therapeutic intervention for these novel inflammasomes are beginning to be realized. Here, we discuss these emerging inflammasome complexes and their putative activation mechanisms, molecular and signaling pathways, and physiological roles in health and disease.

This study demonstrates that the fate of a vaccine is influenced by the cytokines produced by the innate lymphoid cells (ILC) recruited to the vaccination site, and it is vaccine route and adjuvant dependent. Intranasal virus vaccination induced ST2/IL-33R⁺ ILC2 in lung, while intramuscular vaccination induced exclusively IL-25R⁺ ILC2 in muscle. Interestingly, a larger proportion of IL-13⁺ ILC2s were detected in muscle following i.m. viral vector vaccination compared to lung post i.n. delivery. These observations revealed that ILC2 were the main source of IL-13 at the vaccination site (24 h post vaccination) responsible for inducing T cells of varying avidities. Moreover, recombinant fowlpox viral vector-based vaccines expressing adjuvants that transiently block IL-13 signalling at the vaccination site using different mechanisms (IL-4R antagonist or IL-13Rα2 adjuvants), revealed that the level of IL-13 present in the milieu also significantly influenced IFN-γ, IL-22 or IL-17A expression by ILC1/ILC3. Specifically, an early IL-13 and IFN-γ co-dependency at the ILC level may also be associated with shaping the downstream antibody responses, supporting the notion that differentially regulating IL-13 signalling via STAT6 or IL-13Rα2 pathways can modify ILC function and the resulting adaptive T- and B-cell immune outcomes reported previously. Moreover, unlike chronic inflammatory or experimentally induced conditions, viral vector vaccination induced uniquely different ILC profiles (i.e., expression of CD127 only on ILC2 not ILC1/ILC3; expression of IFN-γ in both NKP46⁺ and NKp46⁻ ILCs). Collectively, our data highlight that tailoring a vaccine vector/adjuvant to modulate the ILC cytokine profile according to the target pathogen, may help design more efficacious vaccines in the future.

Subunit vaccines exhibit favorable safety and immunogenicity profiles and can be designed to mimic native antigen structures. However, pairing with an appropriate adjuvant is imperative in order to elicit effective humoral and cellular immune responses. In this study, we aimed to determine an optimal adjuvant pairing with the prefusion form of influenza haemagglutinin (HA) or respiratory syncytial virus (RSV) fusion (F) subunit vaccines in BALB/c mice in order to inform future subunit vaccine adjuvant selection. We tested a panel of adjuvants, including aluminum hydroxide (alhydrogel), QS21, Addavax, Addavax with QS21 (AdQS21), and Army Liposome Formulation 55 with monophosphoryl lipid A and QS21 (ALF55). We found that all adjuvants elicited robust humoral responses in comparison to placebo, with the induction of potent neutralizing antibodies observed in all adjuvanted groups against influenza and in AdQS21, alhydrogel, and ALF55 against RSV. Upon HA vaccination, we observed that none of the adjuvants were able to significantly increase the frequency of CD4⁺ and CD8⁺ IFN-γ⁺ cells when compared to unadjuvanted antigen. The varying responses to antigens with each adjuvant highlights that those adjuvants most suited for pairing purposes can vary depending on the antigen used and/or the desired immune response. We therefore suggest that an adjuvant trial for different subunit vaccines in development would likely be necessary in preclinical studies.

This study demonstrates that route and viral vector can significantly influence the innate lymphoid cells (ILC) and dendritic cells (DC) recruited to the vaccination site, 24 h post delivery. Intranasal (i.n.) vaccination induced ST2/IL-33R⁺ ILC2, whilst intramuscular (i.m.) induced IL-25R⁺ and TSLPR⁺ (Thymic stromal lymphopoietin protein receptor) ILC2 subsets. However, in muscle a novel ILC subset devoid of the known ILC2 markers (IL-25R⁻ IL-33R⁻ TSLPR⁻) were found to express IL-13, unlike in lung. Different viral vectors also influenced the ILC-derived cytokines and the DC profiles at the respective vaccination sites. Both i.n. and i.m. recombinant fowlpox virus (rFPV) priming, which has been associated with induction of high avidity T cells and effective antibody differentiation exhibited low ILC2-derived IL-13, high NKp46⁺ ILC1/ILC3 derived IFN-γ and low IL-17A, together with enhanced CD11b⁺ CD103⁻ conventional DCs (cDC). In contrast, recombinant Modified Vaccinia Ankara (rMVA) and Influenza A vector priming, which has been linked to low avidity T cells, induced opposing ILC derived-cytokine profiles and enhanced cross-presenting DCs. These observations suggested that the former ILC/DC profiles could be a predictor of a balanced cellular and humoral immune outcome. In addition, following i.n. delivery Rhinovirus (RV) and Adenovius type 5 (Ad5) vectors that induced elevated ILC2-derived IL-13, NKp46⁺ ILC1/ILC3-derived-IFN-γ and no IL-17A, predominantly recruited CD11b⁻ B220⁺ plasmacytoid DCs (pDC). Knowing that pDC are involved in antibody differentiation, we postulate that i.n. priming with these vectors may favour induction of effective humoral immunity. Our data also revealed that vector-specific replication status and/or presence or absence of immune evasive genes can significantly alter the ILC and DC activity. Collectively, our findings suggest that understanding the route- and vector-specific ILC and DC profiles at the vaccination site may help tailor/design more efficacious viral vector-based vaccines, according to the pathogen of interest.

A HIV vaccine that provides mucosal immunity is urgently needed. We evaluated an intranasal recombinant Fowlpox virus (rFPV) priming vaccine followed by intramuscular Modified Vaccinia Ankara (rMVA) booster vaccine, both expressing SIV antigens. The vaccination generated mucosal and systemic SIV-specific CD4⁺ T cell mediated immunity and was associated with partial protection against high-dose intrarectal SIV_mac251 challenge in outbred pigtail macaques. Three of 12 vaccinees were completely protected and these animals elicited sustained Gag-specific poly-functional, cytotoxic mucosal CD4⁺ T cells, complemented by systemic poly-functional CD4⁺ and CD8⁺ T cell immunity. Humoral immune responses, albeit absent in completely protected macaques, were associated with partial control of viremia in animals with relatively weaker mucosal/systemic T cell responses. Co-expression of an IL-4R antagonist by the rFPV vaccine further enhanced the breadth and cytotoxicity/poly-functionality of mucosal vaccine-specific CD4⁺ T cells. Moreover, a single FPV-gag/pol/env prime was able to induce rapid anamnestic gp140 antibody response upon SIV encounter. Collectively, our data indicated that nasal vaccination was effective at inducing robust cervico-vaginal and rectal immunity, although cytotoxic CD4⁺ T cell mediated mucosal and systemic immunity correlated strongly with ‘complete protection’, the different degrees of protection observed was multi-factorial.

This study demonstrates that modulation of IL-25 and IL-33 cytokines responsible for innate lymphoid cell 2 (ILC2) activation/function can differentially regulate ILC profiles at the vaccination site, in a vaccine route-dependent manner. Specifically, recombinant fowlpox (rFPV) vector-based vaccine co-expressing an adjuvant that transiently sequestered IL-25 (FPV-HIV-IL-25 binding protein), delivered intramuscularly (i.m.) was able to induce significantly lower IL-25R+ ILC2-deived IL-13 and ILC1/ILC3-derived IFN-γ expression with significantly elevated IL-17A in muscle. In contrast, intranasal (i.n.) delivery was able to induce all three known ILC2 subsets (ST2/IL-33R+, IL-25R+, and TSLPR+ ILC2) to express varying amounts of IL-13 in lung, and also the TSLPR+ ILC2 to express IL-4, unlike the unadjuvanted control, which only showed ST2/IL-33R+ ILC2 to express IL-13. Interestingly, the sequestration of IL-25 in lung also induced a unique lineage- ST2/IL-33R- IL-25R- TSLPR- ILC2 population to express elevated IL-13 and IL-4. Moreover, both i.m. and, i.n. FPV-HIV-IL-25BP vaccination induced significantly elevated ILC1/ILC3-derived IL-17A in lung, indicating that ILC2 could directly impact ILC1/ILC3 activity. To our surprise, transient sequestration of IL-33 at the lung mucosae did not alter the lung ILC2 profiles or activity. These inhibitor studies showed that in the context of i.n. viral vector vaccination, IL-25 plays a predominant role in early ILC development/regulation than IL-33, and likely acts as a master regulator of ILC. Our previous findings have indicated that level of IL-4/IL-13 at the vaccination site impacts the quality/avidity of T cell immunity. Taken together data suggest that IL-25 binding protein could be used as an effective i.m. not an i.n. adjuvant to enhance quality of vaccine-specific T cell immunity. These findings evoke the notion that route-dependent manipulation of ILCs according to the target pathogen could be exploited to design more effective vaccines against chronic pathogens in the future.