mRNA vaccine against malaria tailored for liver-resident memory T cells.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.

A high-throughput screen for ligand binding reveals the specificities of three amino acid chemoreceptors from Pseudomonas syringae pv. actinidiae.

Chemoreceptors play a central role in chemotaxis, allowing bacteria to detect chemical gradients and bias their swimming behavior in order to navigate toward favorable environments. The genome of the kiwifruit pathogen, Pseudomonas syringae pv. actinidiae (Psa) strain NZ-V13 encodes 43 predicted chemoreceptors, none of which has been characterized. We developed a high-throughput fluorescence-based thermal shift assay for identifying the signal molecules that are recognized by a given chemoreceptor ligand binding domain (LBD). Using this assay, we characterized the ligand binding profiles of three Psa homologs of the P. aeruginosa PAO1 amino acid chemoreceptors PctA, PctB and PctC. Each recombinant LBD was screened against 95 potential ligands. The three Psa homologs, named pscA, pscB and pscC (Psa chemoreceptors A, B and C) bound 3, 10 and 3 amino acids respectively. In each case, their binding profiles were distinct from their P. aeruginosa PAO1 homologs. Notably, Psa PscA-LBD only bound the acidic amino acids l-aspartate, d-aspartate and l-glutamate, whereas P. aeruginosa PctA-LBD binds all of the l-proteinogenic amino acids except for l-aspartate and l-glutamate. A combination of homology modeling, site-directed mutagenesis and functional screening identified a single amino acid residue in the Psa PscA-LBD (Ala146) that is critically important for determining its narrow specificity.

Using Full-Spectrum Flow Cytometry to Phenotype Memory T and NKT Cell Subsets with Optimized Tissue-Specific Preparation Protocols.

Full-spectrum flow cytometry is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With capacity to use larger and more complex staining panels, standardized protocols are required for optimal panel design and analysis. Importantly, for ex vivo analysis, tissue preparation methods also need to be optimized to ensure samples are truly representative of tissues in situ. This is particularly relevant given the recent interest in adaptive immune cells that form residency in specific organs. Here we provide optimized protocols for tissue processing and phenotyping of memory T cells and natural killer T (NKT) cell subsets from liver, lung, spleen, and lymph node using full-spectrum flow cytometry. We provide a 21-color antibody panel for identification of different memory subsets, including tissue-resident memory T (TRM ) cells, which are increasingly regarded as important effectors in adaptive immunity. We show that processing procedures can affect outcomes, with liver TRM cells particularly sensitive to heat, such that accurate evaluation requires fast processing at defined temperatures. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Processing mouse liver for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 2: Processing mouse spleen for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 3: Processing mouse lungs for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 4: Processing mouse lymph nodes for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 5: Staining and flow cytometric analysis of samples for memory T and NKT cell subsets Support Protocol: Obtaining cell counts from flow cytometry data.

Cryo-EM structure of the deltaretroviral intasome in complex with the PP2A regulatory subunit B56γ.

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.

Inhibition of HTLV-1 Infection by HIV-1 First- and Second-Generation Integrase Strand Transfer Inhibitors.

More than 10 million people worldwide are infected with the retrovirus human T-cell lymphotropic virus type 1 (HTLV-1). Infection phenotypes can range from asymptomatic to severe adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy. HTLV-1, like human immunodeficiency virus type 1 (HIV-1), is a blood-borne pathogen and viral infection happens in a similar fashion, with the major mode of transmission through breastfeeding. There is a strong correlation between time of infection and disease development, with a higher incidence of ATLL in patients infected during childhood. There is no successful therapeutic or preventative regimen for HTLV-1. It is therefore essential to develop therapies to inhibit transmission or block the onset/development of HTLV-1 associated diseases. Recently, we have seen the overwhelming success of integrase strand transfer inhibitors (INSTIs) in the treatment of HIV-1. Previously, raltegravir was shown to inhibit HTLV-1 infection. Here, we tested FDA-approved and two Phase II HIV-1 INSTIs in vitro and in a cell-to-cell infection model and show that they are highly active in blocking HTLV-1 infection, with bictegravir (EC50 = 0.30 ± 0.17 nM) performing best overall. INSTIs, in particular bictegravir, are more potent in blocking HTLV-1 transmission than tenofovir disproxil fumarate (TDF), an RT inhibitor. Our data suggest that HIV-1 INSTIs could present a good clinical strategy in HTLV-1 management and justifies the inclusion of INSTIs in clinical trials.