Evaluating the stability of host-reference gene expression and simultaneously quantifying parasite burden and host immune responses in murine malaria.

The efficacy of pre-erythrocytic stage malaria antigens or vaccine platforms is routinely assessed in murine models challenged with Plasmodium sporozoites. Relative liver-stage parasite burden is quantified using reverse transcription quantitative PCR (RTqPCR), which relies on constitutively expressed endogenous control reference genes. However, the stability of host-reference gene expression for RTqPCR analysis following Plasmodium challenge and immunization has not been systematically evaluated. Herein, we evaluated the stability of expression of twelve common RTqPCR reference genes in a murine model of Plasmodium yoelii sporozoite challenge and DNA-adenovirus IV 'Prime-Target' immunization. Significant changes in expression for six of twelve reference genes were shown by one-way ANOVA, when comparing gene expression levels among challenge, immunized, and naïve mice groups. These changes were attributed to parasite challenge or immunization when comparing group means using post-hoc Bonferroni corrected multiple comparison testing. Succinate dehydrogenase (SDHA) and TATA-binding protein (TBP) were identified as stable host-reference genes suitable for relative RTqPCR data normalisation, using the RefFinder package. We defined a robust threshold of 'partial-protection' with these genes and developed a strategy to simultaneously quantify matched host parasite burden and cytokine responses following immunisation or challenge. This is the first report systematically identifying reliable host reference genes for RTqPCR analysis following Plasmodium sporozoite challenge. A robust RTqPCR protocol incorporating reliable reference genes which enables simultaneous analysis of host whole-liver cytokine responses and parasite burden will significantly standardise and enhance results between international malaria vaccine efficacy studies.

Small extracellular vesicles but not microvesicles from Opisthorchis viverrini promote cell proliferation in human cholangiocytes.

Chronic infection with O. viverrini has been linked to the development of cholangiocarcinoma (CCA), which is a major public health burden in the Lower Mekong River Basin countries, including Thailand, Lao PDR, Vietnam and Cambodia. Despite its importance, the exact mechanisms by which O. viverrini promotes CCA are largely unknown. In this study, we characterized different extracellular vesicle populations released by O. viverrini (OvEVs) using proteomic and transcriptomic analyses and investigated their potential role in host-parasite interactions. While 120k OvEVs promoted cell proliferation in H69 cells at different concentrations, 15k OvEVs did not produce any effect compared to controls. The proteomic analysis of both populations showed differences in their composition that could contribute to this differential effect. Furthermore, the miRNAs present in 120k EVs were analysed and their potential interactions with human host genes was explored by computational target prediction. Different pathways involved in inflammation, immune response and apoptosis were identified as potentially targeted by the miRNAs present in this population of EVs. This is the first study showing specific roles for different EV populations in the pathogenesis of a parasitic helminth, and more importantly, an important advance towards deciphering the mechanisms used in establishment of opisthorchiasis and liver fluke infection-associated malignancy.

Comparative Analysis of the Humoral Immune Response to the EBV Proteome across EBV-Related Malignancies.

BACKGROUND: Epstein-Barr virus (EBV) is linked to multiple cancers, including classical Hodgkin lymphoma (cHL), endemic Burkitt lymphoma (eBL), nasopharyngeal carcinoma (NPC), and extranodal natural killer/T-cell lymphoma (NKTCL). METHODS: Anti-EBV IgG and IgA antibody responses targeting 202 sequences from 86 EBV proteins were measured using the same EBV whole proteome array across four case-control studies investigating EBV-positive cHL, eBL, NPC, and NKTCL (407 cases/620 controls). We grouped EBV-targeted antibodies into pathways by immunoglobulin type (IgA and IgG) and life-cycle stage (latent, immediate early lytic, early lytic, late lytic, and glycoprotein) and evaluated their association with each cancer type. In an additional analysis, we focused on the subset of 46 individual antibodies representing the top candidates for each cancer and compared their associations across the four cancer types using multivariable linear regression models. RESULTS: IgA antibody responses targeting all EBV life-cycle stages were associated with NPC but limited to anti-early lytic stage for cHL. NPC and eBL were associated with IgG antibodies across the viral life cycle; cHL with antibodies in the early lytic, late lytic and glycoprotein stages; and NKTCL with antibodies in the latent, immediate early lytic and early lytic phases. EBNA3A, BBLF1, BDLF4, and BLRF2 IgG antibodies were associated with all cancer types. CONCLUSIONS: Our observed similarities and differences across four EBV-associated cancers may inform EBV-related oncogenesis. IMPACT: Understanding the comparative humoral immune response across EBV-related cancers may aid in identifying shared etiologic roles of EBV proteins and inform unique pathogenic processes for each cancer.

Recommendations for Epstein-Barr virus-based screening for nasopharyngeal cancer in high- and intermediate-risk regions.

A meeting of experts was held in November 2021 to review and discuss available data on performance of Epstein-Barr virus (EBV)-based approaches to screen for early stage nasopharyngeal carcinoma (NPC) and methods for the investigation and management of screen-positive individuals. Serum EBV antibody and plasma EBV DNA testing methods were considered. Both approaches were found to have favorable performance characteristics and to be cost-effective in high-risk populations. In addition to endoscopy, use of magnetic resonance imaging (MRI) to investigate screen-positive individuals was found to increase the sensitivity of NPC detection with minimal impact on cost-effectiveness of the screening program.

Characterizing and correcting immune dysfunction in non-tuberculous mycobacterial disease.

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic, progressive, and growing worldwide health burden associated with mounting morbidity, mortality, and economic costs. Improvements in NTM-PD management are urgently needed, which requires a better understanding of fundamental immunopathology. Here, we examine temporal dynamics of the immune compartment during NTM-PD caused by Mycobacterium avium complex (MAC) and Mycobactereoides abscessus complex (MABS). We show that active MAC infection is characterized by elevated T cell immunoglobulin and mucin-domain containing-3 expression across multiple T cell subsets. In contrast, active MABS infection was characterized by increased expression of cytotoxic T-lymphocyte-associated protein 4. Patients who failed therapy closely mirrored the healthy individual immune phenotype, with circulating immune network appearing to 'ignore' infection in the lung. Interestingly, immune biosignatures were identified that could inform disease stage and infecting species with high accuracy. Additionally, programmed cell death protein 1 blockade rescued antigen-specific IFN-γ secretion in all disease stages except persistent infection, suggesting the potential to redeploy checkpoint blockade inhibitors for NTM-PD. Collectively, our results provide new insight into species-specific 'immune chatter' occurring during NTM-PD and provide new targets, processes and pathways for diagnostics, prognostics, and treatments needed for this emerging and difficult to treat disease.

A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs.

Immunoassays that quantitate cytokines and other surrogate markers of immunity from peripheral blood mononuclear cells (PBMCs), such as flow cytometry or Enzyme-Linked Immunosorbent Spot (ELIspot), allow highly sensitive measurements of immune effector function. However, those assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). To address this issue, we developed a sensitive and specific reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay, specifically designed to quantify surrogate markers of immunity from very low numbers of PBMCs. We systematically evaluated the volumes and concentrations of critical reagents within the RT-qPCR protocol, miniaturizing the assay and ultimately reducing the cost by almost 90% compared to current standard practice. We assessed the suitability of this cost-optimized RT-qPCR protocol as an HTS tool and determined the assay exceeds HTS uniformity and signal variance testing standards. Furthermore, we demonstrate this technique can effectively delineate a hierarchy of responses from as little as 50,000 PBMCs stimulated with CD4+ or CD8+ T cell peptide epitopes. Finally, we establish that this HTS-optimized protocol has single-cell analytical sensitivity and a diagnostic sensitivity equivalent to detecting 1:10,000 responding cells (i.e., 100 Spot Forming Cells/106 PBMCs by ELIspot) with over 90% accuracy. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration.

The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples.

Whole-blood-derived transcriptional profiling is widely used in biomarker discovery, immunological research, and therapeutic development. Traditional molecular and high-throughput transcriptomic platforms, including molecular assays with quantitative PCR (qPCR) and RNA-sequencing (RNA-seq), are dependent upon high-quality and intact RNA. However, collecting high-quality RNA from field studies in remote tropical locations can be challenging due to resource restrictions and logistics of post-collection processing. The current study tested the relative performance of the two most widely used whole-blood RNA collection systems, PAXgene® and Tempus™, in optimal laboratory conditions as well as suboptimal conditions in tropical field sites, including the effects of extended storage times and high storage temperatures. We found that Tempus™ tubes maintained a slightly higher RNA quantity and integrity relative to PAXgene® tubes at suboptimal tropical conditions. Both PAXgene® and Tempus™ tubes gave similar RNA purity (A260/A280). Additionally, Tempus™ tubes preferentially maintained the stability of mRNA transcripts for two reference genes tested, Succinate dehydrogenase complex, subunit A (SDHA) and TATA-box-binding protein (TBP), even when RNA quality decreased with storage length and temperature. Both tube types preserved the rRNA transcript 18S ribosomal RNA (18S) equally. Our results suggest that Tempus™ blood RNA collection tubes are preferable to PAXgene® for whole-blood collection in suboptimal tropical conditions for RNA-based studies in resource-limited settings.

Novel antiinflammatory biologics shaped by parasite-host coevolution.

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.

CD161 expression defines new human γδ T cell subsets.

γδ T cells are a highly versatile immune lineage involved in host defense and homeostasis, but questions remain around their heterogeneity, precise function and role during health and disease. We used multi-parametric flow cytometry, dimensionality reduction, unsupervised clustering, and self-organizing maps (SOM) to identify novel γδ T cell naïve/memory subsets chiefly defined by CD161 expression levels, a surface membrane receptor that can be activating or suppressive. We used middle-to-old age individuals given immune blockade is commonly used in this population. Whilst most Vδ1+subset cells exhibited a terminal differentiation phenotype, Vδ1- subset cells showed an early memory phenotype. Dimensionality reduction revealed eight γδ T cell clusters chiefly diverging through CD161 expression with CD4 and CD8 expression limited to specific subpopulations. Comparison of matched healthy elderly individuals to bronchiectasis patients revealed elevated Vδ1+ terminally differentiated effector memory cells in patients potentially linking this population with chronic proinflammatory disease.

Identifying Epstein-Barr virus peptide sequences associated with differential IgG antibody response.

BACKGROUND: Epstein-Barr virus (EBV) infection contributes to cancers in a fraction of seropositive individuals, but much remains to be learned about variation in EBV-directed humoral immunity in cancer-free adults. METHODS: A protein microarray was used to probe serum from 175 Taiwanese and 141 Northern European adults for immunoglobulin G (IgG) antibody responses to 115 different peptide sequences, representing protein segments or protein variants, from 45 EBV proteins. It was posited that this antibody-based approach could identify EBV peptide sequences representing immunodominant regions relevant for B-cell immunity. RESULTS: Analyses of 45 EBV proteins with multiple protein segments or variants printed on the array identified eight EBV peptide sequences that appear to play a role in immunogenicity. This included: (1) three proteins with segments/regions associated with IgG reactivity (BALF5, LMP1, LMP2A); and (2) five proteins with sequence variants/amino acid changes associated with IgG reactivity (BDLF4, EBNA3A, EBNA3B, EBNA-LP, LF1). CONCLUSION: This examination of IgG antibody responses against 115 EBV peptide sequences in 316 cancer-free adults represents an important step toward identifying specific EBV protein sequences that play a role in generating B-cell immunity in humans.

Estimating the global burden of Epstein-Barr virus-related cancers.

BACKGROUND: More than 90% of the adult population globally is chronically infected by the Epstein-Barr virus (EBV). It is well established that EBV is associated with a number of malignancies, and advances in knowledge of EBV-related malignancies are being made every year. Several studies have analysed the global epidemiology and geographic distribution of EBV-related cancers. However, most have only described a single cancer type or subtype in isolation or limited their study to the three or four most common EBV-related cancers. This review will present an overview on the spectrum of cancers linked to EBV based on observations of associations and proportions in the published literature while also using these observations to estimate the incidence and mortality burden of some of these cancers. METHOD: We have reviewed the literature on defining features, distribution and outcomes across six cancers with a relatively large EBV-related case burden: Nasopharyngeal carcinoma (NPC), Gastric carcinoma (GC), Hodgkin lymphoma (HL), Burkitt lymphoma (BL), Diffuse large B-cell lymphoma (DLBCL) and Extranodal NK/T-cell lymphoma, Nasal type (ENKTL-NT). We retrieved published region-specific EBV-related case proportions for NPC, GC, HL and BL and performed meta-analyses on pooled region-specific studies of EBV-related case proportions for DLBCL and ENKTL-NT. We match these pooled proportions with their respective regional incidence and mortality numbers retrieved from a publicly available cancer database. Additionally, we also reviewed the literature on several other less common EBV-related cancers to summarize their key characteristics herein. CONCLUSION: We estimated that EBV-related cases from these six cancers accounted for 239,700-357,900 new cases and 137,900-208,700 deaths in 2020. This review highlights the significant global impact of EBV-related cancers and extends the spectrum of disease that could benefit from an EBV-specific therapeutic.

Proteomic identification of the contents of small extracellular vesicles from in vivo Plasmodium yoelii infection.

Small extracellular vesicles, including exosomes, are formed by the endocytic pathway and contain genetic and protein material which reflect the contents of their cells of origin. These contents have a role in vesicle-mediated information transfer, as well as physiological and pathological functions. Thus, these vesicles are of great interest as therapeutic targets, or as vehicles for immunomodulatory control. In Plasmodium spp. infections, vesicles derived from the parasite or parasite-infected cells have been shown to induce the expression of pro-inflammatory elements, which have been correlated with manifestations of clinical disease. Herein, we characterised the protein cargo of naturally occurring sEVs in the plasma of P. yoelii-infected mice. After in vivo infections, extracellular vesicles in the size range of exosomes were collected by sequential centrifugation/ultracentrifugation followed by isopycnic gradient separation. Analysis of the vesicles was performed by transmission electron microscopy, dynamic light scattering, SDS-PAGE and flow cytometry. LC-MS analysis followed by bioinformatics analysis predicted parasite protein cargo associated with exosomes. Within these small extracellular vesicles, we identified proteins of interest as vaccine candidates, uncharacterized proteins which may be targets of T cell immunoreactivity, and proteins involved in metabolic processes, regulation, homeostasis and immunity. Importantly, the small extracellular vesicles studied in our work were obtained from in vivo infection rather than from the supernatant of in vitro cultures. These findings add to the growing interest in parasite small extracellular vesicles, further our understanding of the interactions between host and parasite, and identify novel proteins which may represent potential targets for vaccination against malaria.

A point-of-care lateral flow assay for neutralising antibodies against SARS-CoV-2.

BACKGROUND: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing. METHODS: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques. FINDINGS: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test. INTERPRETATION: Taken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care. FUNDING: Support was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individual researchers were supported by an NHMRC Emerging Leadership Level 1 Investigator Grants (1194036), NHMRC APPRISE Research Fellowship (1116530), NHMRC Leadership Investigator Grant (1173871), NHMRC Principal Research Fellowship (1137285), NHMRC Investigator Grants (1177174 and 1174555) and NHMRC Senior Principal Research Fellowships (1117766 and 1136322). Grateful support was also received from the A2 Milk Company and the Jack Ma Foundation.

Characterization of the humoral immune response to the EBV proteome in extranodal NK/T-cell lymphoma.

Extranodal natural killer/T-cell lymphoma (NKTCL) is an aggressive malignancy that has been etiologically linked to Epstein-Barr virus (EBV) infection, with EBV gene transcripts identified in almost all cases. However, the humoral immune response to EBV in NKTCL patients has not been well characterized. We examined the antibody response to EBV in plasma samples from 51 NKTCL cases and 154 controls from Hong Kong and Taiwan who were part of the multi-center, hospital-based AsiaLymph case-control study. The EBV-directed serological response was characterized using a protein microarray that measured IgG and IgA antibodies against 202 protein sequences representing the entire EBV proteome. We analyzed 157 IgG antibodies and 127 IgA antibodies that fulfilled quality control requirements. Associations between EBV serology and NKTCL status were disproportionately observed for IgG rather than IgA antibodies. Nine anti-EBV IgG responses were significantly elevated in NKTCL cases compared with controls and had ORshighest vs. lowest tertile > 6.0 (Bonferroni-corrected P-values < 0.05). Among these nine elevated IgG responses in NKTCL patients, three IgG antibodies (all targeting EBNA3A) are novel and have not been observed for other EBV-associated tumors of B-cell or epithelial origin. IgG antibodies against EBNA1, which have consistently been elevated in other EBV-associated tumors, were not elevated in NKTCL cases. We characterize the antibody response against EBV for patients with NKTCL and identify IgG antibody responses against six distinct EBV proteins. Our findings suggest distinct serologic patterns of this NK/T-cell lymphoma compared with other EBV-associated tumors of B-cell or epithelial origin.

Malaria research in Australia: looking through the lens of the past towards the future.

Malaria remains a global health priority, with substantial resources devoted to control and intervention since the causative parasite was first identified in 1880. Major advances have been made in discovery and translational research activities aimed at prevention, treatment and control. Laboratory-based, clinical, and field-based studies have complemented public health approaches. Australian scientists have played important roles, developing and applying innovative approaches, novel research tools and cutting-edge technologies in animal and human models of disease, as well as in disease-endemic settings. This article will provide an insight into 50 years of Australian efforts to discover mechanisms and targets of immunity and pathogenesis; develop new diagnostics, drugs, vaccines, and therapeutics; and assess new public health interventions and control measures in malaria-endemic settings.

Identification of the Glycan Binding Profile of Human and Rodent Plasmodium Sporozoites.

The transmission of Plasmodium spp. sporozoites to the mammalian host is the first step in the initiation of the mosquito-borne disease known as malaria. The exact route of transmission from the bloodstream to the liver is still not clearly elucidated, and identification of the host glycan structures bound by the sporozoites may inform as to which host cells are involved. Here, we provide a comprehensive analysis of the glycan structures that sporozoites from the human pathogen, P. falciparum, and the rodent pathogen, P. yoelii, recognize and bind. Glycan array analysis was used to profile the glycans bound by the sporozoites, and the binding affinities of these sporozoite-glycan interactions were then determined by surface plasmon resonance. Data showed that the different Plasmodium spp. bind different classes of glycans. P. falciparum was observed to bind to glycans with terminal N-acetylgalactosamine (GalNAc) or Galactose (Gal) linked to a GalNAc, and the highest-affinity observed was with the GalNAc monosaccharide (12.5 nM). P. yoelii bound glycosaminoglycans, mannosyl glycans, Gal linked to N-acetylglucosamine structures, and the αGal epitope. The highest-affinity interaction for P. yoelii was with the αGal epitope (31.4 nM). This is the first study to identify the key host glycan structures recognized by human and rodent Plasmodium spp. sporozoites. An understanding of how Plasmodium sporozoites interact with the specific glycan structures identified here may provide further insight into this infectious disease that could help direct the design of an effective therapeutic.

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.

Systems serology detects functionally distinct coronavirus antibody features in children and elderly.

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics.

Memory CD8+ T cell compartment associated with delayed onset of Plasmodium falciparum infection and better parasite control in sickle-cell trait children.

OBJECTIVES: Study of individuals with protection from Plasmodium falciparum (Pf) infection and clinical malaria, including individuals affected by the sickle-cell trait (HbAS), offers the potential to identify cellular targets that could be translated for therapeutic development. We previously reported the first involvement of cellular immunity in HbAS-associated relative protection and identified a novel subset of memory-activated NK cells that was enriched in HbAS children and associated with parasite control. We hypothesised that other memory cell subsets might distinguish the baseline profile of HbAS children and children with normal haemoglobin (HbAA). METHODS: Subsets of memory T cells and NK cells were analysed by flow cytometry in paired samples collected from HbAS and HbAA children, at baseline and during the first malaria episode of the ensuing transmission season. Correlations between cell frequencies and features of HbAS-mediated protection from malaria were determined. RESULTS: HbAS children displayed significantly higher frequency of memory CD8+ T cells at baseline than HbAA children. Baseline frequency of memory CD8+ T cells correlated with features of HbAS-mediated protection from malaria. Exploration of memory CD8+ T cell subsets revealed that central memory CD8+ T cell frequency was higher in HbAS children than in HbAA children. CONCLUSION: This study shows that HbAS children develop a larger memory CD8+ T cell compartment than HbAA children, and associates this compartment with better control of subsequent onset of infection and parasite density. Our data suggest that central memory CD8+ T cells may play an important role in the relative protection against malaria experienced by HbAS individuals, and further work to investigate this is warranted.