Induction of protective mucosal T cell memory remains a formidable challenge to vaccinologists. Using a combination adjuvant strategy that elicits potent CD8 and CD4 T cell responses, we define the tenets of vaccine-induced pulmonary T cell immunity. An acrylic-acid-based adjuvant (ADJ), in combination with Toll-like receptor (TLR) agonists glucopyranosyl lipid adjuvant (GLA) or CpG, promotes mucosal imprinting but engages distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of T H 1/T C 1/T H 17/T C 17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampens T cell receptor (TCR) signaling, mitigates terminal differentiation of effectors, and enhances the development of CD4 and CD8 T RM cells that protect against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells play a vital role in optimal programming of CD8 T RM and viral control. Taken together, these findings provide further insights into vaccine-induced multifaceted mucosal T cell immunity with implications in the development of vaccines against respiratorypathogens, including influenza virus and SARS-CoV-2.

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.

Eliciting durable and protective T cell-mediated immunity in the respiratory mucosa remains a significant challenge. Polylactic-co-glycolic acid (PLGA)-based cationic pathogen-like particles (PLPs) loaded with TLR agonists mimic biophysical properties of microbes and hence, simulate pathogen-pattern recognition receptor interactions to safely and effectively stimulate innate immune responses. We generated micro particle PLPs loaded with TLR4 (glucopyranosyl lipid adjuvant, GLA) or TLR9 (CpG) agonists, and formulated them with and without a mucosal delivery enhancing carbomer-based nanoemulsion adjuvant (ADJ). These adjuvants delivered intranasally to mice elicited high numbers of influenza nucleoprotein (NP)-specific CD8+ and CD4+ effector and tissue-resident memory T cells (TRMs) in lungs and airways. PLPs delivering TLR4 versus TLR9 agonists drove phenotypically and functionally distinct populations of effector and memory T cells. While PLPs loaded with CpG or GLA provided immunity, combining the adjuvanticity of PLP-GLA and ADJ markedly enhanced the development of airway and lung TRMs and CD4 and CD8 T cell-dependent immunity to influenza virus. Further, balanced CD8 (Tc1/Tc17) and CD4 (Th1/Th17) recall responses were linked to effective influenza virus control. These studies provide mechanistic insights into vaccine-induced pulmonary T cell immunity and pave the way for the development of a universal influenza and SARS-CoV-2 vaccines.

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Development of T-cell-based subunit protein vaccines against diseases, such as tuberculosis and malaria, remains a challenge for immunologists. Here, we have identified a nano-emulsion adjuvant Adjuplex (ADJ), which enhanced dendritic cell (DC) cross-presentation and elicited effective memory T cell-based immunity to Listeria monocytogenes (LM). We further evaluated whether cross-presentation induced by ADJ, can be combined with the immunomodulatory effects of TLR agonists (CpG or glucopyranosyl lipid adjuvant [GLA]) to evoke systemic CD8 T cell-based immunity to LM. Mechanistically, vaccination with ADJ, alone or in combination with CpG or GLA augmented activation and antigen uptake by CD103+ migratory and CD8α+ resident DCs and up-regulated CD69 expression on B and T lymphocytes in vaccine-draining lymph nodes. By engaging basic leucine zipper ATF-like transcription factor 3-dependent cross-presenting DCs, ADJ potently elicited effector CD8 T cells that differentiated into granzyme B-expressing CD27LO effector-like memory CD8 T cells, which provided effective immunity to LM in spleen and liver. CpG or GLA alone did not elicit effector-like memory CD8 T cells and induced moderate protection in spleen, but not in the liver. Surprisingly, combining CpG or GLA with ADJ reduced the number of ADJ-induced memory CD8 T cells and compromised protective immunity to LM, especially in the liver. Taken together, data presented in this manuscript provides a glimpse of protective CD8 T cell memory differentiation induced by a nano-emulsion adjuvant and demonstrates the unexpected negative effects of TLR signaling on the magnitude of CD8 T cell memory and protective immunity to LM, a model intracellular pathogen.

We describe the isolation, molecular characterization, and drug sensitivity of Mycobacterium tuberculosis recovered from lung tissues of four rescued captive sloth bears (Melursus ursinus) at Bannerghatta Biological Park (BBP), Bangalore, India. These bears had lived most of their life with humans in circus companies. They were rescued and housed in the Bear Rescue Center (BRC) of BBP. Upon rescue, they showed signs of unthriftiness, chronic debility, and failed to respond to symptomatic treatments. Over the period of the next 12–14 months, the four sloth bears died and the post-mortem examination revealed nodular lesions in the lungs that showed the presence of acid-fast bacilli. Polymerase chain reaction (PCR), culture, and nucleotide sequencing confirmed the bacilli as Mycobacterium tuberculosis. Histopathology of the lungs revealed characteristic granulomatous reaction with caseation. We determined the sensitivity of these isolates to rifampicin and isoniazid drugs by a WHO approved test, Line Probe Assay (LPA) using Genotype MTBDRplus VER 2.0. We discuss the role of unnatural habitat with the human environment in predisposing captive sloth bears for tuberculosis (TB). In the absence of any other reliable ante-mortem diagnostic test, this study recommends the use of LPA for early detection of TB in captive wild animals, which will help in taking necessary steps to prevent its further spread to animal caretakers and other susceptible animals in captivity.

Induction of protective mucosal T-cell memory remains a formidable challenge to vaccinologists. Using a novel adjuvant strategy that elicits unusually potent CD8 and CD4 T-cell responses, we have defined the tenets of vaccine-induced pulmonary T-cell immunity. An acrylic acid-based adjuvant (ADJ), in combination with TLR agonists glucopyranosyl lipid adjuvant (GLA) or CpG promoted mucosal imprinting but engaged distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of TH1/TC1/TH17/TC17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampened TCR signaling, mitigated terminal differentiation of effectors and enhanced the development of CD4 and CD8 TRM that protected against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells played a vital role in optimal programming of CD8 TRM and anti-viral immunity. Taken together, these findings provide new insights into vaccine-induced multi-faceted mucosal T-cell immunity with significant implications in the development of vaccines against respiratory pathogens.

ADJ induces inflammasome activation in DCs, but deficiency for NLRP3, ASC or caspase 1 did not affect ADJ-mediated cross-presentationNext, we examined the effects of ADJ on the expression profiles of cytokines, chemokines and canonical cell surface markers of DC activation. Compared to untreated DCs, ADJ-treated DCs showed statistically significant, yet modest increases in expression of CD40, CD80, and CD86; no significant differences in expression were observed for MHC-I, MHC-II, or CCR7 (Fig. 1C).ADJ-treated DCs produced higher levels of IL-12 (p70), TNF-, IL-1, CCL3, CCL4, CXCL1, and RANTES, as compared to untreated DCs; no significant differences in expression were observed for IL-6, IL-10, and IFN- (Fig. 1D). Particularly, ADJ-stimulated DCs also produced significantly elevated levels of IL-1 and IL-18 (Fig. 1D), which is suggestive of inflammasome activation. Since inflammasome activation has been implicated in modulation of antigen presentation by DCs (Sokolovska et al., 2013, Li et al., 2019, we performed B3Z assays using DCs deficient in NLRP3, ASC, or caspase 1 to interrogate whether inflammasome activation is required for ADJ-induced cross-presentation in BMDCs. Surprisingly, loss of NLRP3, ASC or caspase 1 activity did not affect ADJ-induced cross-presentation by DCs, in vitro (Fig. 1E). To validate whether caspase 1 is required for cross-presentation in vivo, we immunized cohorts of wild type (WT) and caspase 1-deficient mice with ADJ+OVA, and quantified OVA SIINFEKLspecific CD8 T cells in spleens using MHC I tetramers at day 8 after immunization. Consistent with our results from B3Z assays, caspase 1 deficiency did not significantly affect the activation of SIINFEKL-specific CD8 T cells in spleens (Fig. 1F), suggesting that caspase 1 is not essential for ADJ-driven cross-presentation to CD8 T cells in vivo.