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Objective: CD4+ T cells are critical mediators of immunity to Plasmodium spp. infection, but their characteristics during malarial episodes and immunopathology in naturally infected adults are poorly defined. Flow cytometric analysis of PBMCs from patients with either P. falciparum or P. knowlesi malaria revealed a pronounced population of CD4+ T cells co‐expressing very high levels of CD4 and CD38 we have termed CD4hiCD38hi T cells. We set out to gain insight into the function of these novel cells. Methods: CD4+ T cells from 18 patients with P. falciparum or P. knowlesi malaria were assessed by flow cytometry and sorted into populations of CD4hiCD38hi or CD4norm T cells. Gene expression in the sorted populations was assessed by qPCR and NanoString. Results: CD4hiCD38hi T cells expressed high levels of CD4 mRNA and canonical type 1 regulatory T‐cell (TR1) genes including IL10, IFNG, LAG3 and HAVCR2 (TIM3), and other genes with relevance to cell migration and immunomodulation. These cells increased in proportion to malaria disease severity and were absent after parasite clearance with antimalarials. Conclusion: In naturally infected adults with acute malaria, a prominent population of type 1 regulatory T cells arises that can be defined by high co‐expression of CD4 and CD38 (CD4hiCD38hi) and that correlates with disease severity in patients with falciparum malaria. This study provides fundamental insights into T‐cell biology, including the first evidence that CD4 expression is modulated at the mRNA level. These findings have important implications for understanding the balance between immunity and immunopathology during malaria. [ABSTRACT FROM AUTHOR]

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 Plasmodium yoelii 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

An effective vaccine against the Plasmodium 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 Plasmodium yoelii 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. [ABSTRACT FROM AUTHOR]

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. Trial Registration: ClinicalTrials.gov clinical trial numbers , and [ABSTRACT FROM AUTHOR]

CD103+ dermal dendritic cells (d DCs) are a recently described DC subset of the skin shown to be the principal migratory DCs capable of efficiently cross-presenting antigens and activating CD8+ T cells. Harnessing their activity would promote vaccine efficacy, but it has been unclear how this can be achieved. We tested a panel of adjuvants for their ability to affect d DCs. In comparison to the other adjuvants tested, the capacity of cholera toxin ( CT) to induce the migration of d DCs was unique. Within 24 h of CT injection, large numbers of highly activated d DCs (including CD103+ d DCs) migrated to the draining lymph nodes and cross-presented coinjected antigens, potently activating naïve CD8+ T cells. Peptide vaccines adjuvanted with CT induced T-cell responses uniquely characterized by dynamic cytokine responses including the production of IL-2, and such vaccines were protective in situations reliant on CD8+ T-cell responses, including liver-stage Plasmodium challenge, or tumor challenge. This study is the first to examine the effects of adjuvants on CD103+ d DCs and identifies CT as a prototypical adjuvant for the activation of CD103+ d DCs, opening the way to development of vaccines and adjuvants that specifically target d DCs and generate effective CD8+ T-cell responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The pre-erythrocytic stages of Plasmodiumspp. 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. [ABSTRACT FROM AUTHOR]

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 Plasmodium yoelii 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., (Copyright © 2020 Pattinson, Apte, Wibowo, Rivera-Hernandez, Groves, Middelberg and Doolan.)

An effective vaccine against the Plasmodium 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 Plasmodium yoelii 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.

Adjuvant development and understanding the physicochemical properties of particles and interpreting the subsequent immunological responses is a challenge faced by many researchers in the vaccine field. We synthesized and investigated the physicochemical properties and immunogenicity of a library of multiple epitope self-adjuvant lipopeptides in a novel asymmetric arrangement. Vaccine candidates were synthesized using a combination of solid-phase peptide synthesis and copper-mediated click chemistry. In vivo studies showed that vaccine constructs containing a single OVA CD8 + T-cell epitope and two N-terminally located C16 lipid moieties were more effective at generating robust cellular immune responses compared to the same molecule containing multiple copies of the OVA CD8 + T-cell epitope with or without the C16 moieties. Furthermore, attachment of the two C16 lipids to the N-terminus provoked formation of long β-sheet fibrils and was shown to induce a higher CD8 + donor T-cell frequency and IFN-γ secretion, compared to vaccine constructs with an internal lipid placement. A regression analysis indicated that particle secondary structure had a significant impact on CD8 + donor T-cell frequency and cytolytic activity. In addition, IFN-γ production was influenced significantly by particle shape. The findings of this research will impact the future design of a vaccine intended to elicit cellular immune responses., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pathogen-specific polyfunctional T cell responses have been associated with favorable clinical outcomes, but it is not known whether molecular differences exist between polyfunctional and monofunctional cytokine-producing T cells. Here, we report that polyfunctional CD4 + T cells induced during Plasmodium falciparum ( P . falciparum ) blood-stage infection in humans have a unique transcriptomic profile compared with IFN-γ monofunctional CD4 + T cells and, thus, are molecularly distinct. The 14-gene signature revealed in P . falciparum -reactive polyfunctional T cells is associated with cytokine signaling and lymphocyte chemotaxis, and systems biology analysis identified IL-27 as an upstream regulator of the polyfunctional gene signature. Importantly, the polyfunctional gene signature is largely conserved in Influenza -reactive polyfunctional CD4 + T cells, suggesting that polyfunctional T cells have core characteristics independent of pathogen specificity. This study provides the first evidence to our knowledge that consistent molecular differences exist between polyfunctional and monofunctional CD4 + T cells., Competing Interests: The authors have declared that no conflict of interest exists.

CD103⁺ dermal dendritic cells (dDCs) are a recently described DC subset of the skin shown to be the principal migratory DCs capable of efficiently cross-presenting antigens and activating CD8⁺ T cells. Harnessing their activity would promote vaccine efficacy, but it has been unclear how this can be achieved. We tested a panel of adjuvants for their ability to affect dDCs. In comparison to the other adjuvants tested, the capacity of cholera toxin (CT) to induce the migration of dDCs was unique. Within 24 h of CT injection, large numbers of highly activated dDCs (including CD103⁺ dDCs) migrated to the draining lymph nodes and cross-presented coinjected antigens, potently activating naïve CD8⁺ T cells. Peptide vaccines adjuvanted with CT induced T-cell responses uniquely characterized by dynamic cytokine responses including the production of IL-2, and such vaccines were protective in situations reliant on CD8⁺ T-cell responses, including liver-stage Plasmodium challenge, or tumor challenge. This study is the first to examine the effects of adjuvants on CD103⁺ dDCs and identifies CT as a prototypical adjuvant for the activation of CD103⁺ dDCs, opening the way to development of vaccines and adjuvants that specifically target dDCs and generate effective CD8⁺ T-cell responses., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)