Antigen and checkpoint receptor engagement recalibrates T cell receptor signal strength.

How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength-and its manipulation-can provide powerful metrics for monitoring outcomes to immunotherapy.

Nanoparticle-Fusion Protein Complexes Protect against Mycobacterium tuberculosis Infection.

Tuberculosis (TB) is the leading cause of death from infectious disease, and the current vaccine, Bacillus Calmette-Guerin (BCG), is inadequate. Nanoparticles (NPs) are an emerging vaccine technology, with recent successes in oncology and infectious diseases. NPs have been exploited as antigen delivery systems and also for their adjuvantic properties. However, the mechanisms underlying their immunological activity remain obscure. Here, we developed a novel mucosal TB vaccine (Nano-FP1) based upon yellow carnauba wax NPs (YC-NPs), coated with a fusion protein consisting of three Mycobacterium tuberculosis (Mtb) antigens: Acr, Ag85B, and HBHA. Mucosal immunization of BCG-primed mice with Nano-FP1 significantly enhanced protection in animals challenged with low-dose, aerosolized Mtb. Bacterial control by Nano-FP1 was associated with dramatically enhanced cellular immunity compared to BCG, including superior CD4+ and CD8+ T cell proliferation, tissue-resident memory T cell (Trm) seeding in the lungs, and cytokine polyfunctionality. Alongside these effects, we also observed potent humoral responses, such as the generation of Ag85B-specific serum IgG and respiratory IgA. Finally, we found that YC-NPs were able to activate antigen-presenting cells via an unconventional IRF-3-associated activation signature, without the production of potentially harmful inflammatory mediators, providing a mechanistic framework for vaccine efficacy and future development.

A polymeric immunoglobulin-antigen fusion protein strategy for enhancing vaccine immunogenicity.

In this study, a strategy based on polymeric immunoglobulin G scaffolds (PIGS) was used to produce a vaccine candidate for Mycobacterium tuberculosis. A genetic fusion construct comprising genes encoding the mycobacterial Ag85B antigen, an immunoglobulin γ-chain fragment and the tailpiece from immunoglobulin µ chain was engineered. Expression was attempted in Chinese Hamster Ovary (CHO) cells and in Nicotiana benthamiana. The recombinant protein assembled into polymeric structures (TB-PIGS) in N. benthamiana, similar in size to polymeric IgM. These complexes were subsequently shown to bind to the complement protein C1q and FcγRs with increased affinity. Modification of the N-glycans linked to TB-PIGS by removal of xylose and fucose residues that are normally found in plant glycosylated proteins also resulted in increased affinity for low-affinity FcγRs. Immunization studies in mice indicated that TB-PIGS are highly immunogenic with and without adjuvant. However, they did not improve protective efficacy in mice against challenge with M. tuberculosis compared to conventional vaccination with BCG, suggesting that additional or alternative antigens may be needed to protect against this disease. Nevertheless, these results establish a novel platform for producing polymeric antigen-IgG γ-chain molecules with inherent functional characteristics that are desirable in vaccines.

Plant-expressed Fc-fusion protein tetravalent dengue vaccine with inherent adjuvant properties.

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly-immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor-targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell-mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen-specific CD4+ and CD8+ T-cell proliferation, IFN-γ and antibody production. The purified polymeric fraction of dengue PIGS (D-PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low-affinity Fcγ receptors on antigen-presenting cells. These results show that the plant-expressed D-PIGS have the potential for translation towards a safe and easily scalable single antigen-based tetravalent dengue vaccine.

Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

In order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen-presenting cells. These self-adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII-PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII-PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII-PIGS also induced a significant cellular immune response, IFN-γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof-of-principle study shows that the potent antibody Fc-mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad-ranging immune response.

Murine IL-4Δ2 splice variant down-regulates IL-4 activities independently of IL-4Rα binding and STAT-6 phosphorylation.

IL-4 is a pleiotropic cytokine that is highly Th2 polarizing. The ratio of IL-4 and its splice variant IL-4Δ2 observed in human health and disease suggests a role for both isoforms. In the present study, the biological function of murine IL-4Δ2 and the potential mechanism of action were studied. We report for the first time the generation of a functional, recombinant murine IL-4Δ2 form which is suggestive of its possible biological role in this species. Recombinant murine IL-4Δ2 inhibited IL-4 mediated cellular processes in macrophages and lymphocytes. Specifically, (i) it reversed IL-4 mediated inhibition of IFN-γ induced nitric oxide release by macrophages, (ii) inhibited IL-4 mediated induction of T cell proliferation, and (iii) prevented IL-4 stimulation of IgE synthesis by B cells. However, IL-4Δ2 did not compete with IL-4 for IL-4Rα binding and did not interfere with the downstream STAT-6 phosphorylation in T cells, suggesting an alternative mechanism for its antagonism of specific IL4-driven effects. These findings suggest that the mouse is a suitable experimental model for studies of the biology of IL-4 and its alternative splice variant.

LOS oligosaccharide modification enhances dendritic cell responses to meningococcal native outer membrane vesicles expressing a non-toxic lipid A.

Outer membrane vesicles (OMV) are released by many bacteria, and contain immunogenic antigens in addition to harmful inflammatory factors, like lipopolysaccharides. Chemically detoxified OMV have been used in vaccines against Neisseria meningitidis (Nm); however, little is known about their interaction with antigen presenting cells. In this study, we investigated the interaction of Nm OMV with human dendritic cells (DC) to gain further understanding of their biological activity. We engineered a novel serogroup B Nm that is unencapsulated (siaD), expresses pentacylated lipid A (lpxL1), hence conferring reduced toxicity, and expresses an lgtB oligosaccharide structure designed to target OMV to DC via DC-SIGN. We show that the lgtB moiety is critical for internalization of NOMV by DC. Furthermore, the lgtB moiety significantly enhances DC maturation, IL-10 and IL-23 production in the presence of a pentacylated lipid A. While different DC phenotypes were observed for each NOMV, this had little effect on Th1 and Th2 cell differentiation; however, lgtBsignificantly increased Th17 cell expansion in the presence of pentacylated lipid A. We believe that lpxL1/lgtB NOMV should be considered further as a vaccine vector, particularly considering the importance of lgtB in antigen uptake and further human studies on antigen-specific responses should be considered.

Pseudaminic acid on Campylobacter jejuni flagella modulates dendritic cell IL-10 expression via Siglec-10 receptor: a novel flagellin-host interaction.

INTRODUCTION: Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. At present the identity of host-pathogen interactions that promote successful bacterial colonisation remain ill defined. Herein, we aimed to investigate C. jejuni-mediated effects on dendritic cell (DC) immunity. RESULTS: We found C. jejuni to be a potent inducer of human and murine DC interleukin 10 (IL-10) in vitro, a cellular event that was MyD88- and p38 MAPK-signalling dependent. Utilizing a series of C. jejuni isogenic mutants we found the major flagellin protein, FlaA, modulated IL-10 expression, an intriguing observation as C. jejuni FlaA is not a TLR5 agonist. Further analysis revealed pseudaminic acid residues on the flagella contributed to DC IL-10 expression. We identified the ability of both viable C. jejuni and purified flagellum to bind to Siglec-10, an immune-modulatory receptor. In vitro infection of Siglec-10 overexpressing cells resulted in increased IL-10 expression in a p38-dependent manner. Detection of Siglec-10 on intestinal CD11c(+) CD103(+) DCs added further credence to the notion that this novel interaction may contribute to immune outcome during human infection. CONCLUSIONS: We propose that unlike the Salmonella Typhimurium flagella-TLR5 driven pro-inflammatory axis, C. jejuni flagella instead promote an anti-inflammatory axis via glycan-Siglec-10 engagement.

Exploring the sequence determinants of amyloid structure using position-specific scoring matrices.

Protein aggregation results in beta-sheet-like assemblies that adopt either a variety of amorphous morphologies or ordered amyloid-like structures. These differences in structure also reflect biological differences; amyloid and amorphous beta-sheet aggregates have different chaperone affinities, accumulate in different cellular locations and are degraded by different mechanisms. Further, amyloid function depends entirely on a high intrinsic degree of order. Here we experimentally explored the sequence space of amyloid hexapeptides and used the derived data to build Waltz, a web-based tool that uses a position-specific scoring matrix to determine amyloid-forming sequences. Waltz allows users to identify and better distinguish between amyloid sequences and amorphous beta-sheet aggregates and allowed us to identify amyloid-forming regions in functional amyloids.

T cell biomarkers come to the fore in cancer immunotherapy.

A comprehensive study by van der Sluis et al.1 demonstrates immunotherapeutic targeting of OX40 and PD-L1 results in enhanced tumor clearance, which is linked to the dynamic emergence of distinct subsets of CD8+ T cells.

Spore-FP1 tuberculosis mucosal vaccine candidate is highly protective in guinea pigs but fails to improve on BCG-conferred protection in non-human primates.

Tuberculosis remains a major health threat globally and a more effective vaccine than the current Bacillus Calmette Guerin (BCG) is required, either to replace or boost it. The Spore-FP1 mucosal vaccine candidate is based on the fusion protein of Ag85B-Acr-HBHA/heparin-binding domain, adsorbed on the surface of inactivated Bacillus subtilis spores. The candidate conferred significant protection against Mycobacterium. tuberculosis challenge in naïve guinea pigs and markedly improved protection in the lungs and spleens of animals primed with BCG. We then immunized rhesus macaques with BCG intradermally, and subsequently boosted with one intradermal and one aerosol dose of Spore-FP1, prior to challenge with low dose aerosolized M. tuberculosis Erdman strain. Following vaccination, animals did not show any adverse reactions and displayed higher antigen specific cellular and antibody immune responses compared to BCG alone but this did not translate into significant improvement in disease pathology or bacterial burden in the organs.

Bacterial cancer therapy in autochthonous colorectal cancer affects tumor growth and metabolic landscape.

Bacterial cancer therapy (BCT) shows great promise for treatment of solid tumors, yet basic mechanisms of bacterial-induced tumor suppression remain undefined. Attenuated strains of Salmonella enterica serovar Typhimurium (STm) have commonly been used in mouse models of BCT in xenograft and orthotopic transplant cancer models. We aimed to better understand the tumor epithelium-targeted mechanisms of BCT by using autochthonous mouse models of intestinal cancer and tumor organoid cultures to assess the effectiveness and consequences of oral treatment with aromatase A-deficient STm (STmΔaroA). STmΔaroA delivered by oral gavage significantly reduced tumor burden and tumor load in both a colitis-associated colorectal cancer (CAC) model and in a spontaneous Apcmin/+ intestinal cancer model. STmΔaroA colonization of tumors caused alterations in transcription of mRNAs associated with tumor stemness, epithelial-mesenchymal transition, and cell cycle. Metabolomic analysis of tumors demonstrated alteration in the metabolic environment of STmΔaroA-treated tumors, suggesting that STmΔaroA imposes metabolic competition on the tumor. Use of tumor organoid cultures in vitro recapitulated effects seen on tumor stemness, mesenchymal markers, and altered metabolome. Furthermore, live STmΔaroA was required, demonstrating active mechanisms including metabolite usage. We have demonstrated that oral BCT is efficacious in autochthonous intestinal cancer models, that BCT imposes metabolic competition, and that BCT has direct effects on the tumor epithelium affecting tumor stem cells.

Mucosal Therapy of Multi-Drug Resistant Tuberculosis With IgA and Interferon-γ.

New evidence has been emerging that antibodies can be protective in various experimental models of tuberculosis. Here, we report on protection against multidrug-resistant Mycobacterium tuberculosis (MDR-TB) infection using a combination of the human monoclonal IgA 2E9 antibody against the alpha-crystallin (Acr, HspX) antigen and mouse interferon-gamma in mice transgenic for the human IgA receptor, CD89. The effect of the combined mucosal IgA and IFN-γ; treatment was strongest (50-fold reduction) when therapy was applied at the time of infection, but a statistically significant reduction of lung bacterial load was observed even when the therapy was initiated once the infection had already been established. The protection involving enhanced phagocytosis and then neutrophil mediated killing of infected cells was IgA isotype mediated, because treatment with an IgG version of 2E9 antibody was not effective in human IgG receptor CD64 transgenic mice. The Acr antigen specificity of IgA antibodies for protection in humans has been indicated by their elevated serum levels in latent tuberculosis unlike the lack of IgA antibodies against the virulence-associated MPT64 antigen. Our results represent the first evidence for potential translation of mucosal immunotherapy for the management of MDR-TB.

Cross-beta spine architecture of fibrils formed by the amyloidogenic segment NFGSVQFV of medin from solid-state NMR and X-ray fiber diffraction measurements.

Over 30 polypeptides are known to assemble into highly ordered fibrils associated with pathological disorders known collectively as amyloidoses. Structural studies of short model peptides are beginning to reveal trends in the types of molecular interactions that drive aggregation and stabilize the packing of beta-sheet layers within fibrillar assemblies. This work investigates the molecular architecture of fibrils formed by the peptide AMed42-49 representing residues 42-49 of the 50 amino acid polypeptide medin associated with aortic medial amyloid, the most common form of senile localized amyloid. The peptide aggregates within 2 days to form bundles of microcrystalline-like needles displaying a high degree of order. Fibrils were prepared from peptides containing up to 23 13C labels, and the solid-state nuclear magnetic resonance (SSNMR) method rotational resonance (RR) was used to determine constraints on the distances between selective atomic sites within fibrils. The constraints are consistent with unbroken beta-strands hydrogen bonded in a parallel in-register arrangement within beta-sheets. Further RR measurements identify close (>6.5 A) contacts between residues F43 and V46 and between S45 and V46, which can only occur between beta-sheet layers and which are consistent with two principal models of beta-sheet arrangements. X-ray fiber diffraction from partially aligned fibrils revealed the classical amyloid diffraction pattern, and comparison of patterns calculated from model coordinates with experimental data allowed determination of a consistent molecular model.

Inactivated Lactobacillus plantarum Carrying a Surface-Displayed Ag85B-ESAT-6 Fusion Antigen as a Booster Vaccine Against Mycobacterium tuberculosis Infection.

Vaccination is considered the most effective strategy for controlling tuberculosis (TB). The existing vaccine, the Bacille Calmette-Guérin (BCG), although partially protective, has a number of limitations. Therefore, there is a need for developing new TB vaccines and several strategies are currently exploited including the use of viral and bacterial delivery vectors. We have previously shown that Lactobacillus plantarum (Lp) producing Ag85B and ESAT-6 antigens fused to a dendritic cell-targeting peptide (referred to as Lp_DC) induced specific immune responses in mice. Here, we analyzed the ability of two Lp-based vaccines, Lp_DC and Lp_HBD (in which the DC-binding peptide was replaced by an HBD-domain directing the antigen to non-phagocytic cells) to activate antigen-presenting cells, induce specific immunity and protect mice from Mycobacterium tuberculosis infection. We tested two strategies: (i) Lp as BCG boosting vaccine (a heterologous regimen comprising parenteral BCG immunization followed by intranasal Lp boost), and (ii) Lp as primary vaccine (a homologous regimen including subcutaneous priming followed by intranasal boost). The results showed that both Lp constructs applied as a BCG boost induced specific cellular immunity, manifested in T cell proliferation, antigen-specific IFN-γ responses and multifunctional T cells phenotypes. More importantly, intranasal boost with Lp_DC or Lp_HBD enhanced protection offered by BCG, as shown by reduced M. tuberculosis counts in lungs. These findings suggest that Lp constructs could be developed as a potential mucosal vaccine platform against mycobacterial infections.

Bacillus Calmette-Guérin Induces PD-L1 Expression on Antigen-Presenting Cells via Autocrine and Paracrine Interleukin-STAT3 Circuits.

Bacillus Calmette-Guérin (BCG) is the only licensed vaccine for tuberculosis (TB), and is also used as an immunotherapy for bladder cancer and other malignancies due to its immunostimulatory properties. Mycobacteria spp., however, are well known for their numerous immune evasion mechanisms that limit the true potential of their therapeutic use. One such major mechanism is the induction of programmed death ligand-1 (PD-L1), which mitigates adaptive immune responses. Here, we sought to unravel the molecular pathways behind PD-L1 up-regulation on antigen-presenting cells (APCs) by BCG. We found that infection of APCs with BCG induced PD-L1 up-regulation, but that this did not depend on direct infection, suggesting a soluble mediator for this effect. BCG induced potent quantities of IL-6 and IL-10, and the downstream transcription factor STAT3 was hyper-phosphorylated. Intracellular analyses revealed that levels of PD-L1 molecules were associated with the STAT3 phosphorylation state, suggesting a causal link. Neutralisation of the IL-6 or IL-10 cytokine receptors dampened STAT3 phosphorylation and BCG-mediated up-regulation of PD-L1 on APCs. Pharmacological inhibition of STAT3 achieved the same effect, confirming an autocrine-paracrine cytokine loop as a mechanism for BCG-mediated up-regulation of PD-L1. Finally, an in vivo immunisation model showed that BCG vaccination under PD-L1 blockade could enhance antigen-specific memory CD4 T-cell responses. These novel findings could lead to refinement of BCG as both a vaccine for infectious disease and as a cancer immunotherapy.

Immunization With Mycobacterium tuberculosis Antigens Encapsulated in Phosphatidylserine Liposomes Improves Protection Afforded by BCG.

Liposomes have been long considered as a vaccine delivery system but this technology remains to be fully utilized. Here, we describe a novel liposome-based subunit vaccine formulation for tuberculosis (TB) based on phosphatidylserine encapsulating two prominent TB antigens, Ag85B, and ESAT-6. We show that the resulting liposomes (Lipo-AE) are stable upon storage and can be readily taken up by antigen presenting cells and that their antigenic cargo is delivered and processed within endosomal cell compartments. The Lipo-AE vaccine formulation combined with the PolyIC adjuvant induced a mixed Th1/Th17-Th2 immune response to Ag85B but only a weak response to ESAT-6. An immunization regimen based on systemic delivery followed by mucosal boost with Lipo-AE resulted in the accumulation of resident memory T cells in the lungs. Most importantly though, when Lipo-AE vaccine candidate was administered to BCG-immunized mice subsequently challenged with low dose aerosol Mycobacterium tuberculosis, we observed a significant reduction of the bacterial load in the lungs and spleen compared to BCG alone. We therefore conclude that the immunization with mycobacterial antigens delivered by phosphatidylserine based liposomes in combination with Poly:IC adjuvant may represent a novel BCG boosting vaccination strategy.

Foxp3 Molecular Dynamics in Treg in Juvenile Idiopathic Arthritis.

Since the identification of the regulatory T-cell (Treg)-associated transcription factor Foxp3, there have been intensive research efforts to understand its biology and roles in maintaining immune homeostasis. It is well established that thymic selection of a repertoire of self-reactive Foxp3+ T-cells provides an essential mechanism to minimize reactions to self-antigens in the periphery, and thus aid in the prevention of autoimmunity. It is clear from both genetic and immunological analyses of juvenile idiopathic arthritis (JIA) patients that T-cells have a strong role to play in both the initiation and propagation of disease. The current paradigm is to view autoimmunity as a consequence of an imbalance between inflammatory and immunoregulatory mechanisms. This view has led to the assigning of cells and inflammatory mediators to different classes based on their assumed pro- or anti-inflammatory roles. This is typically reported as ratios of effector T-cells to Treg cells. Problematically, many analyses are based on static "snapshots-in-time," even though both mouse models and human patient studies have highlighted the dynamic nature of Foxp3+ T-cells in vivo, which can exhibit plasticity and time-dependent functional states. In this review, we discuss the role of Foxp3 dynamics in the control of T-cell responses in childhood arthritis, by reviewing evidence in humans and relevant mouse models of inflammatory disease. Whilst the cellular dynamics of Treg have been well evaluated-leading to standard data outputs such as frequency, quantity and quality (often assessed by in vitro suppressive capacity)-we discuss how recent insights into the molecular dynamics of Foxp3 transcription and its post-translational control may open up tantalizing new avenues for immunotherapies to treat autoimmune arthritis.

Mucosal Delivery of Fusion Proteins with Bacillus subtilis Spores Enhances Protection against Tuberculosis by Bacillus Calmette-Guérin.

Tuberculosis (TB) is the most deadly infectious disease in existence, and the only available vaccine, Bacillus Calmette-Guérin (BCG), is almost a century old and poorly protective. The immunological complexity of TB, coupled with rising resistance to antimicrobial therapies, necessitates a pipeline of diverse novel vaccines. Here, we show that Bacillus subtilis spores can be coated with a fusion protein 1 ("FP1") consisting of Mycobacterium tuberculosis (Mtb) antigens Ag85B, ACR, and HBHA. The resultant vaccine, Spore-FP1, was tested in a murine low-dose Mtb aerosol challenge model. Mice were primed with subcutaneous BCG, followed by mucosal booster immunizations with Spore-FP1. We show that Spore-FP1 enhanced pulmonary control of Mtb, as evidenced by reduced bacterial burdens in the lungs. This was associated with elevated antigen-specific IgG and IgA titers in the serum and lung mucosal surface, respectively. Spore-FP1 immunization generated superior antigen-specific memory T-cell proliferation in both CD4+ and CD8+ compartments, alongside bolstered Th1-, Th17-, and Treg-type cytokine production, compared to BCG immunization alone. CD69+CD103+ tissue resident memory T-cells (Trm) were found within the lung parenchyma after mucosal immunization with Spore-FP1, confirming the advantages of mucosal delivery. Our data show that Spore-FP1 is a promising new TB vaccine that can successfully augment protection and immunogenicity in BCG-primed animals.