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Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4+ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4+ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4+ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38+ CD4+ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38+ CD4+ T cells could be generated in vitro from CD38- CD4+ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38+ CD4+ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38+ CD4+ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense. ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752.

Despite extensive research, the development of an effective malaria vaccine remains elusive. The induction of robust and sustained T cell and antibody response by vaccination is an urgent unmet need. Chimeric virus-like particles (VLPs) are a promising vaccine platform. VLPs are composed of multiple subunit capsomeres which can be rapidly produced in a cost-effective manner, but the ability of capsomeres to induce antigen-specific cellular immune responses has not been thoroughly investigated. Accordingly, we have compared chimeric VLPs and their sub-unit capsomeres for capacity to induce CD8 and CD4 T cell and antibody responses. We produced chimeric murine polyomavirus VLPs and capsomeres each incorporating defined CD8 T cell, CD4 T cell or B cell repeat epitopes derived from CSP. VLPs and capsomeres were evaluated using both homologous or heterologous DNA prime/boost immunization regimens for T cell and antibody immunogenicity. Chimeric VLP and capsomere vaccine platforms induced robust CD8 T cell responses at similar levels which was enhanced by a heterologous DNA prime. The capsomere platform was, however, more efficient at inducing CD4 T cell responses and less efficient at inducing antigen-specific antibody responses. Our data suggest that capsomeres, which have significant manufacturing advantages over VLPs, should be considered for diseases where a T cell response is the desired outcome.

The pre-erythrocytic stages of Plasmodium spp. are increasingly recognised as ideal targets for prophylactic vaccines and drug treatments. Intense research efforts in the last decade have been focused on in vitro culture and in vivo detection and quantification of liver stage parasites to assess the effects of candidate vaccines or drugs. Typically, the onset of blood stage parasitaemia is used as a surrogate endpoint to estimate the efficacy of vaccines and drugs targeting pre-erythrocytic parasite stages in animal models. However, this provides no information on the parasite burden in the liver after vaccination or treatment and therefore does not detect partial efficacy of any vaccine or drug candidates. Herein, we describe a quantitative RT-PCR method adapted to detect and quantitate Plasmodium yoelii liver stages in mice with increased sensitivity even after challenge with as few as 50 cryopreserved sporozoites (corresponding to approximately 5-10 freshly isolated sporozoites). We have validated our quantitative RT-PCR assay according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and established high reproducibility and accuracy. Our assay provides a rapid and reproducible assessment of liver stage parasite burden in rodent malaria models, thereby facilitating the evaluation of the efficacy of anti-malarial drugs or prophylactic vaccines with high precision and efficacy.

An effective vaccine against the parasite is likely to require the induction of robust antibody and T cell responses. Chimeric virus-like particles are an effective vaccine platform for induction of antibody responses, but their capacity to induce robust cellular responses and cell-mediated protection against pathogen challenge has not been established. To evaluate this, we produced chimeric constructs using the murine polyomavirus structural protein with surface-exposed CD8 or CD4 T cell or B cell repeat epitopes derived from the circumsporozoite protein, and assessed immunogenicity and protective capacity in a murine model. Robust CD8 T cell responses were induced by immunization with the chimeric CD8 T cell epitope virus-like particles, however CD4 T cell responses were very low. The B cell chimeric construct induced robust antibody responses but there was no apparent synergy when T cell and B cell constructs were administered as a pool. A heterologous prime/boost regimen using plasmid DNA priming followed by a VLP boost was more effective than homologous VLP immunization for cellular immunity and protection. These data show that chimeric murine polyomavirus virus-like particles are a good platform for induction of CD8 T cell responses as well as antibody responses.

Vaccines against many pathogens for which conventional approaches have failed remain an unmet public health priority. Synthetic peptide-based vaccines offer an attractive alternative to whole protein and whole organism vaccines, particularly for complex pathogens that cause chronic infection. Previously, we have reported a promising lipid core peptide (LCP) vaccine delivery system that incorporates the antigen, carrier, and adjuvant in a single molecular entity. LCP vaccines have been used to deliver several peptide subunit-based vaccine candidates and induced high titre functional antibodies and protected against Group A streptococcus in mice. Herein, we have evaluated whether LCP constructs incorporating defined CD4(+) and/or CD8(+) T cell epitopes could induce epitope-specific T cell responses and protect against pathogen challenge in a rodent malaria model. We show that LCP vaccines failed to induce an expansion of antigen-specific CD8(+) T cells following primary immunization or by boosting. We further demonstrated that the LCP vaccines induced a non-specific type 2 polarized cytokine response, rather than an epitope-specific canonical CD8(+) T cell type 1 response. Cytotoxic responses of unknown specificity were also induced. These non-specific responses were able to protect against parasite challenge. These data demonstrate that vaccination with lipid core peptides fails to induce canonical epitope-specific T cell responses, at least in our rodent model, but can nonetheless confer non-specific protective immunity against Plasmodium parasite challenge.

Plant-derived compounds that modulate the immune responses are emerging as frontline treatment agents for cancer, infectious diseases and autoimmunity. Herein we have isolated 40 phytochemicals from five Bhutanese Sowa Rigpa medicinal plants—Aconitum laciniatum, Ajania nubegina, Corydalis crispa, Corydalis dubia and Pleurospermum amabile—and tested 14 purified compounds for their immunomodulatory properties using a murine dendritic cell (DC) line, and cytotoxicity against a human cholangiocyte cell line using xCELLigence real time cell monitoring. These compounds were: pseudaconitine, 14-veratryolpseudaconitine, 14-O-acetylneoline, linalool oxide acetate, (E)-spiroether, luteolin, luteolin-7-O-β-d-glucopyranoside, protopine, ochrobirine, scoulerine, capnoidine, isomyristicin, bergapten, and isoimperatorin. Of the 14 compounds tested here, scoulerine had adjuvant-like properties and strongly upregulated MHC-I gene and protein expression whereas bergapten displayed immunosuppressive properties and strongly down-regulated gene and protein expression of MHC-I and other co-stimulatory molecules. Both scoulerine and bergapten showed low cytotoxicity against normal healthy cells that were consistent with their immunoregulatory properties. These findings highlight the breadth of immunomodulatory properties of defined compounds from Bhutanese medicinal plants and show that some of these compounds exert their mechanisms of action by modulating DC activity.

Alveolar macrophages (AM) must perform three seemingly opposing roles including homeostasis, driving inflammation, and facilitating tissue repair. Whilst there is now consensus (supported by a large body of human single cell RNA sequencing (scRNA-seq) data) that the cell subsets that perform these tasks can readily be found based on their transcriptome, their ontogeny has remained unclear. Moreover, there is agreement that in all types of pulmonary fibrosis (PF) there is an expanded population of profibrotic AM that may aberrantly drive PF. From a therapeutic viewpoint, there is great appeal in the notion that the transcriptional program in different AM subsets is not fixed but remains plastic and amenable to pharmacological reprogramming. Accordingly, this study addresses this question by performing scRNA-seq on human AM following treatment with drugs or perturbagens including pioglitazone, trametinib, nintedanib, lipopolysaccharide and the natural compound endiandrin A. Each treatment induced a unique global transcriptional change, driving the cells towards distinct subsets, further supported by trajectory analysis, confirming a high level of plasticity. Confirmatory experiments using qPCR demonstrated that single exposure to a compound induced a relatively stable transcriptome, whereas serial exposure to a different compound allowed the cells to be reprogrammed yet again to a different phenotype. These findings add new insight into the biology of AM and support the development of novel therapies to treat PF.

Idiopathic pulmonary fibrosis (IPF) is the most common and lethal form of the interstitial pneumonias. The cause of the disease is unknown, and new therapies that stop or reverse disease progression are desperately needed. Recent advances in next-generation sequencing have led to an abundance of freely available, clinically relevant, organ-and-disease-specific, single-cell transcriptomic data, including studies from patients with IPF. We mined data from published IPF data sets and identified gene signatures delineating pro-fibrotic or antifibrotic macrophages and then used the Enrichr platform to identify compounds with the potential to drive the macrophages toward the antifibrotic transcriptotype. We then began testing these compounds in a novel in vitro phenotypic drug screening assay utilising human lung macrophages recovered from whole-lung lavage of patients with silicosis. As predicted by the Enrichr tool, glitazones potently modulated macrophage gene expression towards the antifibrotic phenotype. Next, we assayed a subset of the NatureBank pure compound library and identified the cyclobutane lignan, endiandrin A, which was isolated from the roots of the endemic Australian rainforest plant, Endiandra anthropophagorum, with a similar antifibrotic potential to the glitazones. These methods open new avenues of exploration to find treatments for lung fibrosis.

Abstract Background The effect of timing of exposure to first Plasmodium falciparum infections during early childhood on the induction of innate and adaptive cytokine responses and their contribution to the development of clinical malaria immunity is not well established. Methods As part of a double-blind, randomized, placebo-controlled trial in Mozambique using monthly chemoprophylaxis with sulfadoxine-pyrimethamine plus artesunate to selectively control timing of malaria exposure during infancy, peripheral blood mononuclear cells collected from participants at age 2.5, 5.5, 10.5, 15, and 24 months were stimulated ex vivo with parasite schizont and erythrocyte lysates. Cytokine messenger RNA expressed in cell pellets and proteins secreted in supernatants were quantified by reverse-transcription quantitative polymerase chain reaction and multiplex flow cytometry, respectively. Children were followed up for clinical malaria from birth until 4 years of age. Results Higher proinflammatory (interleukin [IL] 1, IL-6, tumor necrosis factor) and regulatory (IL-10) cytokine concentrations during the second year of life were associated with reduced incidence of clinical malaria up to 4 years of age, adjusting by chemoprophylaxis and prior malaria exposure. Significantly lower concentrations of antigen-specific T-helper 1 (IL-2, IL-12, interferon-γ) and T-helper 2 (IL-4, IL-5) cytokines by 2 years of age were measured in children undergoing chemoprophylaxis compared to children receiving placebo (P < .03). Conclusions Selective chemoprophylaxis altering early natural exposure to malaria blood stage antigens during infancy had a significant effect on T-helper lymphocyte cytokine production >1 year later. Importantly, a balanced proinflammatory and anti-inflammatory cytokine signature, probably by innate cells, around age 2 years was associated with protective clinical immunity during childhood. Clinical Trials Registration NCT00231452. Prevention of exposure to Plasmodium falciparum infection during infancy significantly impacted antigen-specific T-helper 1 and 2 cytokine responses at age 2 years, and protection from clinical malaria was associated with balanced proinflammatory and regulatory cytokine/chemokine signatures characteristic of innate immune cells.

Systematically evaluate lipid core peptide vaccine delivery platforms to identify core features promoting strong CD8(+) T-cell responses. Three different self-adjuvanting lipid core peptide nanovaccines each comprising four copies of the dominant ovalbumin CD8(+) T-cell epitope and varying in the utilization of a polylysine or glucose core with 2-amino-hexadecanoic acid (C16) or 2-amino-dodecanoic acid (C12) lipids were synthesized. Vaccines were tested for ability to induce CD8(+) T-cell responses and inhibit tumor growth in vivo. The construct utilizing C12 lipids and polylysine core induced very robust effector T cells shown to have in vivo effector capability as demonstrated by in vivo cytotoxicity and ability to inhibit tumor growth as well as modulation of dendritic cell activation. The C12 polylysine platform was an effective configuration for induction of potent CD8(+) T-cell responses.