Deep metabolic profiling of immune cells by spectral flow cytometry-A comprehensive validation approach.

The advancing field of immunometabolism requires tools that link single-cell metabolism with immune function. Metabolic flow cytometry provides this capability, but its broad adoption has been limited by costly custom reagents and a lack of standardized methods for validating metabolic targets. Here, we present a standardized and user-friendly spectral flow cytometry panel that profiles eight key metabolic pathways at single-cell resolution using only commercially available antibodies, enabling simultaneous analysis of immune phenotype and metabolic activity .

An adjuvanted chimeric spike antigen boosts lung-resident memory T-cells and induces pan-sarbecovirus protective immunity.

Next-generation vaccines are essential to address the evolving nature of SARS-CoV-2 and to protect against emerging pandemic threats from other coronaviruses. These vaccines should elicit broad protection, provide long-lasting immunity and ensure equitable access for all populations. In this study, we developed a panel of chimeric, full-length spike antigens incorporating mutations from previous, circulating and predicted SARS-CoV-2 variants.

An LNP-mRNA vaccine modulates innate cell trafficking and promotes polyfunctional Th1 CD4 T cell responses to enhance BCG-induced protective immunity against Mycobacterium tuberculosis.

Background: Mycobacterium tuberculosis remains the largest infectious cause of mortality worldwide, even with over a century of widespread administration of the only licenced tuberculosis (TB) vaccine, Bacillus Calmette-Guérin (BCG). mRNA technology remains an underexplored approach for combating chronic bacterial infections such as TB.

Methods: We have developed a lipid nanoparticle (LNP)-mRNA vaccine, termed mRNA, encoding for the M.

Effect of Bacille Calmette-Guérin vaccination on immune responses to SARS-CoV-2 and COVID-19 vaccination.

Objectives: Bacille Calmette-Guérin (BCG) vaccination has off-target effects on disease risk for unrelated infections and immune responses to vaccines. This study aimed to determine the immunomodulatory effects of BCG vaccination on immune responses to vaccines against SARS-CoV-2.

Methods: Blood samples, from a subset of 275 SARS-CoV-2-naïve healthcare workers randomised to BCG vaccination (BCG group) or no BCG vaccination (Control group) in the BRACE trial, were collected before and 28 days after the primary course (two doses) of ChAdOx1-S (Oxford-AstraZeneca) or BNT162b2 (Pfizer-BioNTech) vaccination.

Optimisation of a multiplexed, high throughput assay to measure neutralising antibodies against SARS-CoV-2 variants.

A multiplexed, lentivirus-based pseudovirus neutralisation assay (pVNT) was developed for high-throughput measurement of neutralising antibodies (nAbs) against three distinct SARS-CoV-2 spike variants. Intra-assay variability was minimised by optimising the plate layout and determining an optimal percentage transduction for the pseudovirus inoculum. Comparison of EC titres between single and multiplexed pVNT assays showed no significant differences, indicating reliability of the multiplexed assay.

Immunogenicity and Protective Efficacy of a Multi-Antigen Subunit Vaccine in Mice.

There is an urgent need for an effective TB vaccine capable of controlling both acute and chronic infection in populations with diverse genetic backgrounds. In this study, we characterised the immunogenicity and protective efficacy of a novel protein-in-adjuvant subunit vaccine. The protein component is a fusion protein of three different antigens, which we termed CysVac5: CysD, a major component of the sulfate activation pathway that is highly expressed during the chronic stage of infection, is fused with two major secreted mycobacterial antigens, Ag85B and MPT83.

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a cellular receptor for delta inulin adjuvant.

Delta inulin, or Advax, is a polysaccharide vaccine adjuvant that significantly enhances vaccine-mediated immune responses against multiple pathogens and was recently licensed for use in the coronavirus disease 2019 (COVID-19) vaccine SpikoGen. Although Advax has proven effective as an immune adjuvant, its specific binding targets have not been characterized. In this report, we identify a cellular receptor for Advax recognition.

Colony morphotype governs innate and adaptive pulmonary immune responses to Mycobacterium abscessus infection in C3HeB/FeJ mice.

Mycobacterium abscessus is an emerging pathogen that causes chronic pulmonary infection. Treatment is challenging owing in part to our incomplete understanding of M. abscessus virulence mechanisms that enable pathogen persistence, such as the differing pathogenicity of M.

Lung IL-17A-Producing CD4 T Cells Correlate with Protection after Intrapulmonary Vaccination with Differentially Adjuvanted Tuberculosis Vaccines.

Tuberculosis (TB), caused by , results in approximately 1.6 million deaths annually. BCG is the only TB vaccine currently in use and offers only variable protection; however, the development of more effective vaccines is hindered by a lack of defined correlates of protection (CoP) against .

A unique cytotoxic CD4 T cell-signature defines critical COVID-19.

Objectives: SARS-CoV-2 infection causes a spectrum of clinical disease presentation, ranging from asymptomatic to fatal. While neutralising antibody (NAb) responses correlate with protection against symptomatic and severe infection, the contribution of the T-cell response to disease resolution or progression is still unclear. As newly emerging variants of concern have the capacity to partially escape NAb responses, defining the contribution of individual T-cell subsets to disease outcome is imperative to inform the development of next-generation COVID-19 vaccines.

Clinical and Experimental Determination of Protection Afforded by BCG Vaccination against Infection with Non-Tuberculous Mycobacteria: A Role in Cystic Fibrosis?

is a nontuberculous mycobacterium (NTM) of particular concern in individuals with obstructive lung diseases such as cystic fibrosis (CF). Treatment requires multiple drugs and is characterised by high rates of relapse; thus, new strategies to limit infection are urgently required. This study sought to determine how Bacille Calmette-Guérin (BCG) vaccination may impact NTM infection, using a murine model of infection and observational data from a non-BCG vaccinated CF cohort in Sydney, Australia and a BCG-vaccinated CF cohort in Cape Town, South Africa.

L-selectin-dependent and -independent homing of naïve lymphocytes through the lung draining lymph node support T cell response to pulmonary Mycobacterium tuberculosis infection.

Recruiting large numbers of naïve lymphocytes to lymph nodes is critical for mounting an effective adaptive immune response. While most naïve lymphocytes utilize homing molecule L-selectin to enter lymph nodes, some circulating cells can traffic to the lung-draining mediastinal lymph node (mLN) through lymphatics via the intermediate organ, lung. However, whether this alternative trafficking mechanism operates in infection and contributes to T cell priming are unknown.

Neutrophil Conversion to a Tumor-Killing Phenotype Underpins Effective Microbial Therapy.

Unlabelled: The inflammatory microenvironment of solid tumors creates a protumorigenic milieu that resembles chronic inflammation akin to a subverted wound healing response. Here, we investigated the effect of converting the tumor microenvironment from a chronically inflamed state to one of acute microbial inflammation by injecting microbial bioparticles directly into tumors. Intratumoral microbial bioparticle injection led to rapid and dramatic changes in the tumor immune composition, the most striking of which was a substantial increase in the presence of activated neutrophils.

Mucosal immunization with a delta-inulin adjuvanted recombinant spike vaccine elicits lung-resident immune memory and protects mice against SARS-CoV-2.

Multiple SARS-CoV-2 vaccine candidates have been approved for use and have had a major impact on the COVID-19 pandemic. There remains, however, a significant need for vaccines that are safe, easily transportable and protective against infection, as well as disease. Mucosal vaccination is favored for its ability to induce immune memory at the site of infection, making it appealing for SARS-CoV-2 vaccine strategies.

Mucosal TLR2-activating protein-based vaccination induces potent pulmonary immunity and protection against SARS-CoV-2 in mice.

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (PamCys), delivered to mice parenterally or mucosally.

Stromal structure remodeling by B lymphocytes limits T cell activation in lymph nodes of Mycobacterium tuberculosis-infected mice.

An effective adaptive immune response depends on the organized architecture of secondary lymphoid organs, including the lymph nodes (LNs). While the cellular composition and microanatomy of LNs under steady state are well defined, the impact of chronic tissue inflammation on the structure and function of draining LNs is incompletely understood. Here we showed that Mycobacterium tuberculosis infection remodeled LN architecture by increasing the number and paracortical translocation of B cells.

Calpains Released from Necrotic Tumor Cells Enhance Antigen Cross-Presentation to Activate CD8 T Cells In Vitro.

The initiation of CD8 T cell responses against dead cell-associated Ags is tightly regulated, facilitating adaptive immunity against pathogens and tumors while preventing autoimmunity. It is now well established that dying cells actively regulate the generation of CD8 T cell responses via the release or exposure of damage-associated molecular patterns. However, it is unclear whether nonproteasomal proteases (activated in stressed and dying cells) can influence the availability of Ags for cross-presentation.

Characterization of the Protective Immune Responses Conferred by Recombinant BCG Overexpressing Components of Sec Protein Export System.

Bacillus Calmette-Guérin (BCG) is the only approved vaccine against tuberculosis (TB). However, its efficacy in preventing pulmonary TB in adults is limited. Despite its variable efficacy, BCG offers a number of unique and beneficial characteristics, which make it suitable as a vaccine vehicle to express recombinant molecules.

Virulence Mechanisms of : Current Knowledge and Implications for Vaccine Design.

is a member of the non-tuberculous mycobacteria (NTM) group, responsible for chronic infections in individuals with cystic fibrosis (CF) or those otherwise immunocompromised. While viewed traditionally as an opportunistic pathogen, increasing research into in recent years has highlighted its continued evolution into a true pathogen. This is demonstrated through an extensive collection of virulence factors (VFs) possessed by this organism which facilitate survival within the host, particularly in the harsh environment of the CF lung.

High-Titer Neutralizing Antibodies against the SARS-CoV-2 Delta Variant Induced by Alhydroxyquim-II-Adjuvanted Trimeric Spike Antigens.

Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained, and cross-variant SARS-CoV-2 neutralizing antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a Toll-Like Receptor (TLR) 7/8 small-molecule agonist chemisorbed on aluminum hydroxide (Alhydrogel).

A single dose, BCG-adjuvanted COVID-19 vaccine provides sterilising immunity against SARS-CoV-2 infection.

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum.

TCR Affinity Controls the Dynamics but Not the Functional Specification of the Antimycobacterial CD4 T Cell Response.

The quality of T cell responses depends on the lymphocytes' ability to undergo clonal expansion, acquire effector functions, and traffic to the site of infection. Although TCR signal strength is thought to dominantly shape the T cell response, by using TCR transgenic CD4 T cells with different peptide:MHC binding affinity, we reveal that TCR affinity does not control Th1 effector function acquisition or the functional output of individual effectors following mycobacterial infection in mice. Rather, TCR affinity calibrates the rate of cell division to synchronize the distinct processes of T cell proliferation, differentiation, and trafficking.

Advax adjuvant formulations promote protective immunity against aerosol Mycobacterium tuberculosis in the absence of deleterious inflammation and reactogenicity.

The development of safe and effective adjuvants is a critical goal of vaccine development programs. In this report, we defined the immunostimulatory profile and protective effect against aerosol Mycobacterium tuberculosis infection of vaccine formulations incorporating the semi-crystalline adjuvant δ-inulin (Advax). Advax formulated with CpG oligonucleotide and the QS-21 saponin (Advax) was the most effective combination, demonstrated by the capacity of CysVac2/Advax to significantly reduce the bacterial burden in the lungs of M.

Intrapulmonary vaccination with delta-inulin adjuvant stimulates non-polarised chemotactic signalling and diverse cellular interaction.

There is an urgent need for novel vaccination strategies to combat respiratory pathogens. Mucosal vaccine delivery is an attractive option as it directly targets the site of infection; however, preclinical development has been hindered by a lack of suitable mucosal adjuvants and a limited understanding of their immune effects in the lung environment. Herein, we define the early immune events following the intrapulmonary delivery of a vaccine incorporating the adjuvant delta-inulin.

Mucosal delivery of a multistage subunit vaccine promotes development of lung-resident memory T cells and affords interleukin-17-dependent protection against pulmonary tuberculosis.

The development of effective vaccines against bacterial lung infections requires the induction of protective, pathogen-specific immune responses without deleterious inflammation within the pulmonary environment. Here, we made use of a polysaccharide-adjuvanted vaccine approach to elicit resident pulmonary T cells to protect against aerosol Mycobacterium tuberculosis infection. Intratracheal administration of the multistage fusion protein CysVac2 and the delta-inulin adjuvant Advax™ (formulated with a TLR9 agonist) provided superior protection against aerosol M.