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Resistance to clinical malaria takes years to develop even in hyperendemic regions and sterilizing immunity has rarely been observed. To evaluate the maturation of the host response against controlled repeat exposures to P. falciparum (Pf) NF54 strain-infected mosquitoes, we systematically monitored malaria-naïve participants through an initial exposure to uninfected mosquitoes and 4 subsequent homologous exposures to Pf-infected mosquitoes over 21 months (n = 8 males) (ClinicalTrials.gov# NCT03014258). The primary outcome was to determine whether protective immunity against parasite infection develops following repeat CHMI and the secondary outcomes were to track the clinical signs and symptoms of malaria and anti-Pf antibody development following repeat CHMI. After two exposures, time to blood stage patency increases significantly and the number of reported symptoms decreases indicating the development of clinical tolerance. The time to patency correlates positively with both anti-Pf circumsporozoite protein (CSP) IgG and CD8 + CD69+ effector memory T cell levels consistent with partial pre-erythrocytic immunity. IFNγ levels decrease significantly during the participants' second exposure to high blood stage parasitemia and could contribute to the decrease in symptoms. In contrast, CD4-CD8 + T cells expressing CXCR5 and the inhibitory receptor, PD-1, increase significantly after subsequent Pf exposures, possibly dampening the memory response and interfering with the generation of robust sterilizing immunity.
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Malaria Falciparum , Plasmodium falciparum , Proteínas Protozoarias , Humanos , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Malaria Falciparum/sangre , Plasmodium falciparum/inmunología , Masculino , Proteínas Protozoarias/inmunología , Animales , Adulto , Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Antiprotozoarios/sangre , Interferón gamma/metabolismo , Interferón gamma/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Adulto Joven , Linfocitos T CD8-positivos/inmunología , Mosquitos Vectores/parasitología , Mosquitos Vectores/inmunología , Anopheles/parasitologíaRESUMEN
The accurate and efficient measurement of white blood cell (WBC) counts is vital for monitoring general patient health and can aid in the diagnosis of a range of possible infections or diseases. Even with their importance universally acknowledged, access to WBC counts is largely limited to those with access to phlebotomists and centralized clinical laboratories, which house the instruments that perform the tests. As a result, large populations of people (e.g., those that are home-bound or live in remote locations) lack facile access to testing. Dried blood spot (DBS) cards are often used to bridge these gaps in access to testing by offering the ability to collect blood at home for ambient shipping to laboratories. However, it is well understood that these cards, which are prepared from cellulose cardstocks without further modification, suffer from variabilities in accuracy and precision due to uncontrolled sample spreading and hematocrit effects, which have hindered their use to determine WBC counts. In this paper, we present a method to obtain an accurate WBC count using a patterned dried blood spot (pDBS) card, which comprises collection zones that meter volumes of dried blood. Using an input volume of 75 µL of whole blood, we demonstrate that, unlike the gold standard DBS card (Whatman 903), our pDBS design allows for the collection of replicate zones containing a reproducible, average volume of dried blood (12.1 µL, 7.8% CV) over the range of hematocrits from 25 to 55%. We then used qPCR to quantify the 18S rRNA gene to determine WBC counts from the volumes of blood that are metered in pDBS zones. We observe that WBC counts generated from our method are comparable to those measured with a HemoCue point-of-care WBC analyzer. Our approach to using pDBS cards as a blood collection device has the potential to support at-home sampling and other patient populations that need WBC counts but lack access to clinical facilities.
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Recolección de Muestras de Sangre , Pruebas con Sangre Seca , Humanos , Hematócrito , Pruebas con Sangre Seca/métodos , Recuento de Leucocitos , CelulosaRESUMEN
Recent work demonstrating that asymptomatic carriers of P. falciparum parasites make up a large part of the infectious reservoir highlights the need for an effective malaria vaccine. Given the historical challenges of vaccine development, multiple parasite stages have been targeted, including the sexual stages required for transmission. Using flow cytometry to efficiently screen for P. falciparum gamete/zygote surface reactivity, we identified 82 antibodies that bound live P. falciparum gametes/zygotes. Ten antibodies had significant transmission-reducing activity (TRA) in a standard membrane feeding assay and were subcloned along with 9 nonTRA antibodies as comparators. After subcloning, only eight of the monoclonals obtained have significant TRA. These eight TRA mAbs do not recognize epitopes present in any of the current recombinant transmission-blocking vaccine candidates, Pfs230D1M, Pfs48/45.6C, Pf47 D2 and rPfs25. One TRA mAb immunoprecipitates two surface antigens, Pfs47 and Pfs230, that are expressed by both gametocytes and gametes/zygotes. These two proteins have not previously been reported to associate and the recognition of both by a single TRA mAb suggests the Pfs47/Pfs230 complex is a new vaccine target. In total, Pfs230 was the dominant target antigen, with five of the eight TRA mAbs and 8 of 11 nonTRA gamete/zygote surface reactive mAbs interacting with Pfs230. Of the three remaining TRA mAbs, two recognized non-reduced, parasite-produced Pfs25 and one bound non-reduced, parasite-produced Pfs48/45. None of the TRA mAbs bound protein on an immunoblot of reduced gamete/zygote extract and two TRA mAbs were immunoblot negative, indicating none of the new TRA epitopes are linear. The identification of eight new TRA mAbs that bind epitopes not included in any of the constructs currently under advancement as transmission-blocking vaccine candidates may provide new targets worthy of further study.
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Vacunas contra la Malaria , Malaria Falciparum , Humanos , Plasmodium falciparum , Anticuerpos Bloqueadores , Epítopos , Anticuerpos Antiprotozoarios , Anticuerpos Monoclonales , Proteínas Protozoarias , Antígenos de ProtozoosRESUMEN
γδ T cells are generally considered innate-like lymphocytes, however, an "adaptive-like" γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αß T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct "innate-effector" transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.
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Receptores de Antígenos de Linfocitos T gamma-delta , Subgrupos de Linfocitos T , Adulto , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Preescolar , Granzimas/metabolismo , Humanos , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
Blocking malaria transmission is critical to malaria control programs but remains a major challenge especially in endemic regions with high levels of asymptomatic infections. New strategies targeting the transmissible sexual stages of the parasite, called gametocytes, are needed. This review focuses on P. falciparum gametocytogenesis in vivo and in vitro. Highlighting advances made elucidating genes required for gametocyte production and identifying key questions that remain unanswered such as the factors and regulatory mechanisms that contribute to gametocyte induction, and the mechanism of sequestration. Tools available to begin to address these issues are also described to facilitate advances in our understanding of this important stage of the life cycle.
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Malaria Falciparum , Malaria , Animales , Infecciones Asintomáticas , Estadios del Ciclo de Vida , Plasmodium falciparum/genéticaRESUMEN
Repeated Plasmodium falciparum infections drive the development of clinical immunity to malaria in humans; however, the immunological mechanisms that underpin this response are only partially understood. We investigated the impact of repeated P. falciparum infections on human γδ T cells in the context of natural infection in Malian children and adults, as well as serial controlled human malaria infection (CHMI) of U.S. adults, some of whom became clinically immune to malaria. In contrast to the predominant Vδ2+ T cell population in malaria-naïve Australian individuals, clonally expanded cytotoxic Vδ1effector T cells were enriched in the γδ T cell compartment of Malian subjects. Malaria-naïve U.S. adults exposed to four sequential CHMIs defined the precise impact of P. falciparum on the γδ T cell repertoire. Specifically, innate-like Vδ2+ T cells exhibited an initial robust polyclonal response to P. falciparum infection that was not sustained with repeated infections, whereas Vδ1+ T cells increased in frequency with repeated infections. Moreover, repeated P. falciparum infection drove waves of clonal selection in the Vδ1+ T cell receptor repertoire that coincided with the differentiation of Vδ1naïve T cells into cytotoxic Vδ1effector T cells. Vδ1+ T cells of malaria-exposed Malian and U.S. individuals were licensed for reactivity to P. falciparum parasites in vitro. Together, our study indicates that repeated P. falciparum infection drives the clonal expansion of an adaptive γδ T cell repertoire and establishes a role for Vδ1+ T cells in the human immune response to malaria.
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Malaria Falciparum , Plasmodium falciparum , Adulto , Australia , Niño , Humanos , Malaria Falciparum/parasitología , Receptores de Antígenos de Linfocitos T gamma-delta , Linfocitos TRESUMEN
Subclinical infections that serve as reservoir populations to drive transmission remain a hurdle to malaria control. Data on infection dynamics in a geographical area is required to strategically design and implement malaria interventions. In a longitudinal cohort, we monitored Plasmodium falciparum infection prevalence and persistence, and anti-parasite immunity to gametocyte and asexual antigens for 10 weeks. Of the 100 participants, only 11 were never infected, whilst 16 had persistent infections detected by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and one participant had microscopic parasites at all visits. Over 70% of the participants were infected three or more times, and submicroscopic gametocyte prevalence was high, ≥ 48% of the parasite carriers. Naturally induced responses against recombinant Pfs48/45.6C, Pfs230proC, and EBA175RIII-V antigens were not associated with either infection status or gametocyte carriage, but the antigen-specific IgG titers inversely correlated with parasite and gametocyte densities consistent with partial immunity. Longitudinal analysis of gametocyte diversity indicated at least four distinct clones circulated throughout the study period. The high prevalence of children infected with distinct gametocyte clones coupled with marked variation in infection status at the individual level suggests ongoing transmission and should be targeted in malaria control programs.
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Inmunidad , Malaria Falciparum/inmunología , Infección Persistente/inmunología , Plasmodium falciparum/inmunología , Niño , Femenino , Ghana/epidemiología , Humanos , Estudios Longitudinales , Malaria Falciparum/epidemiología , Masculino , Infección Persistente/epidemiología , PrevalenciaRESUMEN
Antibody sequence repertoire analysis of plasma cells (PC) isolated before and 1 week after a vaccine provides time-specific snapshots of the antibody response. Comparison of the immunoglobulin (Ig) sequences pre- and post-vaccination allows analysis of maturation over time and identification of antigen specific Ig. Here we compare the Ig heavy chain (Ig-H) repertoire of circulating PCs isolated from 109 peripheral blood mononuclear cells (PBMC) collected by apheresis 1 week after a tetanus toxoid vaccine booster with the Ig-H repertoire of PCs collected 2 and 11 weeks prior to the booster. A total of 21,060 unique Ig nucleotide sequences encoding 14,307 unique heavy chain complementarity determining region 3 (CDR-H3) amino acid sequences, also called clonotypes, were identified. Only 466 clonotypes (3.3%) were present at all 3 time points. In contrast, 90% of the 30 highest frequency CDR-H3 regions at +1w were also identified at another time point and 50% were present at all time points, suggesting the rapid expansion of a memory B cell population. The tetanus toxoid specificity of the CDR-H3 region with the 7th highest frequency at +1w was confirmed using immunoprecipitation and mass spectroscopy, and two public tetanus toxoid-specific CDR-H3 regions were also overrepresented at +1w. In summary, we have used the tetanus vaccine model system to demonstrate that bulk PC Ig repertoire analysis can identify PC populations that expand and mature following antigen exposure. The application of this approach before and after clinical infections should advance our understanding of clinical protection and facilitate vaccine design.
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Regiones Determinantes de Complementariedad/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Leucocitos Mononucleares/inmunología , Células Plasmáticas/inmunología , Toxoide Tetánico/inmunología , Vacunación/métodos , Regiones Determinantes de Complementariedad/inmunología , Voluntarios Sanos , Humanos , Leucocitos Mononucleares/metabolismo , Toxoide Tetánico/administración & dosificaciónRESUMEN
Malaria is spread by the transmission of sexual stage parasites, called gametocytes. However, with Plasmodium falciparum, gametocytes can only be detected in peripheral blood when they are mature and transmissible to a mosquito, which complicates control efforts. Here, we identify the set of genes overexpressed in patient blood samples with high levels of gametocyte-committed ring stage parasites. Expression of all 18 genes is regulated by transcription factor AP2-G, which is required for gametocytogenesis. We select three genes, not expressed in mature gametocytes, to develop as biomarkers. All three biomarkers we validate in vitro using 6 different parasite lines and develop an algorithm that predicts gametocyte production in ex vivo samples and volunteer infection studies. The biomarkers are also sensitive enough to monitor gametocyte production in asymptomatic P. falciparum carriers allowing early detection and treatment of infectious reservoirs, as well as the in vivo analysis of factors that modulate sexual conversion.
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Estadios del Ciclo de Vida/genética , Malaria Falciparum/transmisión , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Factor de Transcripción AP-2/genética , Transcriptoma , Animales , Biomarcadores/sangre , Portador Sano , Eritrocitos/parasitología , Gametogénesis/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Humanos , Malaria Falciparum/diagnóstico , Malaria Falciparum/parasitología , Anotación de Secuencia Molecular , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Factor de Transcripción AP-2/metabolismoRESUMEN
Drug resistance is a constant threat to malaria control efforts making it important to maintain a good pipeline of new drug candidates. Of particular need are compounds that also block transmission by targeting sexual stage parasites. Mature sexual stages are relatively resistant to all currently used antimalarials except the 8-aminoquinolines that are not commonly used due to potential side effects. Here, we synthesized a new Torin 2 derivative, NCATS-SM3710 with increased aqueous solubility and specificity for Plasmodium and demonstrate potent in vivo activity against all P. berghei life cycle stages. NCATS-SM3710 also has low nanomolar EC50s against in vitro cultured asexual P. falciparum parasites (0.38 ± 0.04 nM) and late stage gametocytes (5.77 ± 1 nM). Two independent NCATS-SM3710/Torin 2 resistant P. falciparum parasite lines produced by growth in sublethal Torin 2 concentrations both had genetic changes in PF3D7_0509800, annotated as a phosphatidylinositol 4 kinase (Pfâ¯PI4KIIIß). One line had a point mutation in the putative active site (V1357G), and the other line had a duplication of a locus containing Pfâ¯PI4KIIIß. Both lines were also resistant to other Pfâ¯PI4K inhibitors. In addition NCATS-SM3710 inhibited purified Pfâ¯PI4KIIIß with an IC50 of 2.0 ± 0.30 nM. Together the results demonstrate that Pfâ¯PI4KIIIß is the target of Torin 2 and NCATS-SM3710 and provide new options for potent multistage drug development.
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BACKGROUND: Red blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development. METHODS: Approximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy. RESULTS: Merozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p > 0.05). CONCLUSIONS: Parasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.
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Eritrocitos/parasitología , Malaria Falciparum/sangre , Plasmodium falciparum/crecimiento & desarrollo , Adolescente , Adulto , Antígenos de Grupos Sanguíneos/clasificación , Niño , Estudios Transversales , Femenino , Ghana , Hemoglobinas/clasificación , Humanos , Malaria Falciparum/parasitología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Individuals growing up in malaria endemic areas gradually develop protection against clinical malaria and passive transfer experiments in humans have demonstrated that this protection is mediated in part by protective antibodies. However, neither the target antigens, specific effector mechanisms, nor the role of continual parasite exposure have been elucidated, which complicates vaccine development. Progress has been made in defining the innate signaling pathways activated by parasite components, including DNA, RNA, hemozoin, and phospholipids, which initiate the immune response and will be the focus of this review. The challenge that remains within the field is to understand the role of these early responses in the development of protective adaptive responses that clear iRBC and block merozoite invasion so that optimal vaccines and therapeutics may be produced.
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Eritrocitos/parasitología , Inmunidad/inmunología , Estadios del Ciclo de Vida/inmunología , Merozoítos/inmunología , Parásitos/inmunología , Inmunidad Adaptativa/inmunología , Animales , Antígenos de Protozoos/inmunología , Antimaláricos/inmunología , Células Dendríticas , Hemoproteínas , Humanos , Células Asesinas Naturales , Malaria/inmunología , Malaria/prevención & control , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Fosfolípidos/inmunología , Pigmentos Biológicos/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Plasmodium sexual differentiation is required for malaria transmission, yet much remains unknown about its regulation. Here, we quantify early gametocyte-committed ring (gc-ring) stage, P. falciparum parasites in 260 uncomplicated malaria patient blood samples 10 days before maturation to transmissible stage V gametocytes using a gametocyte conversion assay (GCA). Seventy six percent of the samples have gc-rings, but the ratio of gametocyte to asexual-committed rings (GCR) varies widely (0-78%). GCR correlates positively with parasitemia and is negatively influenced by fever, not hematocrit, age or leukocyte counts. Higher expression levels of GDV1-dependent genes, ap2-g, msrp1 and gexp5, as well as a gdv1 allele encoding H217 are associated with high GCR, while high plasma lysophosphatidylcholine levels are associated with low GCR in the second study year. The results provide a view of sexual differentiation in the field and suggest key regulatory roles for clinical factors and gdv1 in gametocytogenesis in vivo.
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Interacciones Huésped-Parásitos/fisiología , Malaria Falciparum/parasitología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/fisiología , Diferenciación Sexual/fisiología , Factores de Edad , Niño , Preescolar , Femenino , Gametogénesis/fisiología , Genes Protozoarios/fisiología , Ghana , Humanos , Lisofosfatidilcolinas/sangre , Malaria Falciparum/sangre , Masculino , Parasitemia/parasitología , Plasmodium falciparum/aislamiento & purificaciónRESUMEN
In recent work, Paton et al. (2019) report that mosquitoes exposed to atovaquone, a known antimalarial, are resistant to infection by the malaria parasite Plasmodium. Their results may be the foundation for the discovery of a new arsenal of antimalarials.
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Anopheles , Antimaláricos , Malaria , Parásitos , Plasmodium , AnimalesRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMEN
In the progression of the life cycle of Plasmodium falciparum, a small proportion of asexual parasites differentiate into male or female sexual forms called gametocytes. Just like their asexual counterparts, gametocytes are contained within the infected host's erythrocytes (RBCs). However, unlike their asexual partners, they do not exit the RBC until they are taken up in a blood meal by a mosquito. In the mosquito midgut, they are stimulated to emerge from the RBC, undergo fertilization, and ultimately produce tens of thousands of sporozoites that are infectious to humans. This transmission cycle can be blocked by antibodies targeting proteins exposed on the parasite surface in the mosquito midgut, a process that has led to the development of candidate transmission-blocking vaccines (TBV), including some that are in clinical trials. Here we review the leading TBV antigens and highlight the ongoing search for additional gametocyte/gamete surface antigens, as well as antigens on the surfaces of gametocyte-infected erythrocytes, which can potentially become a new group of TBV candidates.
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Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Animales , Humanos , Estadios del Ciclo de Vida , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (>98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate.