PD-1 Co-inhibitory and OX40 Co-stimulatory Crosstalk Regulates Helper T Cell Differentiation and Anti-Plasmodium Humoral Immunity

SummaryThe differentiation and protective capacity of Plasmodium-specific T cells are regulated by both positive and negative signals during malaria, but the molecular and cellular details remain poorly defined. Here we show that malaria patients and Plasmodium-infected rodents exhibit atypical expression of the co-stimulatory receptor OX40 on CD4 T cells and that therapeutic enhancement of OX40 signaling enhances helper CD4 T cell activity, humoral immunity, and parasite clearance in rodents. However, these beneficial effects of OX40 signaling are abrogated following coordinate blockade of PD-1 co-inhibitory pathways, which are also upregulated during malaria and associated with elevated parasitemia. Co-administration of biologics blocking PD-1 and promoting OX40 signaling induces excessive interferon-gamma that directly limits helper T cell-mediated support of humoral immunity and decreases parasite control. Our results show that targeting OX40 can enhance Plasmodium control and that crosstalk between co-inhibitory and co-stimulatory pathways in pathogen-specific CD4 T cells can impact pathogen clearance

Protein-Specific Features Associated with Variability in Human Antibody Responses to Plasmodium falciparum Malaria Antigens

The magnitude of antibody responses varies across the individual proteins that constitute any given microorganism, both in the context of natural infection and vaccination with attenuated or inactivated pathogens. The protein-specific factors underlying this variability are poorly understood. In 267 individuals exposed to intense seasonal malaria, we examined the relationship between immunoglobulin G (IgG) responses to 861 Plasmodium falciparum proteins and specific features of these proteins, including their subcellular location, relative abundance, degree of polymorphism, and whether they are predicted to have human orthologs. We found that IgG reactivity was significantly higher to extracellular and plasma membrane proteins and also correlated positively with both protein abundance and degree of protein polymorphism. Conversely, IgG reactivity was significantly lower to proteins predicted to have human orthologs. These findings provide insight into protein-specific factors that are associated with variability in the magnitude of antibody responses to natural P. falciparum infection-data that could inform vaccine strategies to optimize antibody-mediated immunity as well as the selection of antigens for sero-diagnostic purposes

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

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). 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. 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. 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

A Positive Correlation between Atypical Memory B Cells and Plasmodium falciparum Transmission Intensity in Cross-Sectional Studies in Peru and Mali

transmission intensity correlates with atypical memory B cell expansion.. In Peru, the mean level of atypical memory B cells, as a percent of total B cells, was higher than U.S. adults (Peru mean: 5.4% [95% CI: 3.61–7.28]; U.S. mean: 1.4% [95% CI: 0.92–1.81]; p<0.0001) but lower than Malian adults (Mali mean 13.1% [95% CI: 10.68–15.57]; p = 0.0001). In Peru, individuals self-reporting ≥1 prior malaria episodes had a higher percentage of atypical memory B cells compared to those reporting no prior episodes (≥1 prior episodes mean: 6.6% [95% CI: 4.09–9.11]; no prior episodes mean: 3.1% [95% CI: 1.52–4.73]; p = 0.028). infection could contribute to our understanding of naturally-acquired malaria immunity

Extent and Dynamics of Polymorphism in the Malaria Vaccine Candidate Plasmodium falciparum Reticulocyte-Binding Protein Homologue-5 in Kalifabougou, Mali

Reticulocyte-binding homologues (RH) are a ligand family that mediates merozoite invasion of erythrocytes in Plasmodium falciparum. Among the five members of this family identified so far, only P. falciparum reticulocyte-binding homologue-5 (PfRH5) has been found to be essential for parasite survival across strains that differ in virulence and route of host-cell invasion. Based on its essential role in invasion and early evidence of sequence conservation, PfRH5 has been prioritized for development as a vaccine candidate. However, little is known about the extent of genetic variability of RH5 in the field and the potential impact of such diversity on clinical outcomes or on vaccine evasion. Samples collected during a prospective cohort study of malaria incidence conducted in Kalifabougou, in southwestern Mali, were used to estimate genetic diversity, measure haplotype prevalence, and assess the within-host dynamics of PfRH5 variants over time and in relation to clinical malaria. A total of 10 nonsynonymous polymorphic sites were identified in the Pfrh5 gene, resulting in 13 haplotypes encoding unique protein variants. Four of these variants have not been previously observed. Plasmodium falciparum reticulocyte-binding homologue-5 had low amino acid haplotype (h = 0.58) and nucleotide (π = 0.00061) diversity. By contrast to other leading blood-stage malaria vaccine candidate antigens, amino acid differences were not associated with changes in the risk of febrile malaria in consecutive infections. Conserved B- and T-cell epitopes were identified. These results support the prioritization of PfRH5 for possible inclusion in a broadly cross-protective vaccine

The Plasmodium falciparum-Specific Human Memory B Cell Compartment Expands Gradually with Repeated Malaria Infections

Immunity to Plasmodium falciparum (Pf) malaria is only acquired after years of repeated infections and wanes rapidly without ongoing parasite exposure. Antibodies are central to malaria immunity, yet little is known about the B-cell biology that underlies the inefficient acquisition of Pf-specific humoral immunity. This year-long prospective study in Mali of 185 individuals aged 2 to 25 years shows that Pf-specific memory B-cells and antibodies are acquired gradually in a stepwise fashion over years of repeated Pf exposure. Both Pf-specific memory B cells and antibody titers increased after acute malaria and then, after six months of decreased Pf exposure, contracted to a point slightly higher than pre-infection levels. This inefficient, stepwise expansion of both the Pf-specific memory B-cell and long-lived antibody compartments depends on Pf exposure rather than age, based on the comparator response to tetanus vaccination that was efficient and stable. These observations lend new insights into the cellular basis of the delayed acquisition of malaria immunity

Transcriptomic evidence for modulation of host inflammatory responses during febrile Plasmodium falciparum malaria

Identifying molecular predictors and mechanisms of malaria disease is important for understanding how Plasmodium falciparum malaria is controlled. Transcriptomic studies in humans have so far been limited to retrospective analysis of blood samples from clinical cases. In this prospective, proof-of-principle study, we compared whole-blood RNA-seq profiles at pre-and post-infection time points from Malian adults who were either asymptomatic (n = 5) or febrile (n = 3) during their first seasonal PCR-positive P. falciparum infection with those from malaria-naïve Dutch adults after a single controlled human malaria infection (n = 5). Our data show a graded activation of pathways downstream of pro-inflammatory cytokines, with the highest activation in malaria-naïve Dutch individuals and significantly reduced activation in malaria-experienced Malians. Newly febrile and asymptomatic infections in Malians were statistically indistinguishable except for genes activated by pro-inflammatory cytokines. The combined data provide a molecular basis for the development of a pyrogenic threshold as individuals acquire immunity to clinical malaria

Plasmodium falciparum transcription in different clinical presentations of malaria associates with circulation time of infected erythrocytes

Following Plasmodium falciparum infection, individuals can remain asymptomatic, present with mild fever in uncomplicated malaria cases, or show one or more severe malaria symptoms. Several studies have investigated associations between parasite transcription and clinical severity, but no broad conclusions have yet been drawn. Here, we apply a series of bioinformatic approaches based on P. falciparum’s tightly regulated transcriptional pattern during its ~48-hour intraerythrocytic developmental cycle (IDC) to publicly available transcriptomes of parasites obtained from malaria cases of differing clinical severity across multiple studies. Our analysis shows that within each IDC, the circulation time of infected erythrocytes without sequestering to endothelial cells decreases with increasing parasitaemia or disease severity. Accordingly, we find that the size of circulating infected erythrocytes is inversely related to parasite density and disease severity. We propose that enhanced dhesiveness of infected erythrocytes leads to a rapid increase in parasite burden, promoting higher parasitaemia and increased disease severity

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence

Evidence for exposure dependent carriage of malaria parasites across the dry season: modelling analysis of longitudinal data

Abstract Background In malaria endemic regions, transmission of Plasmodium falciparum parasites is often seasonal with very low transmission during the dry season and high transmission in the wet season. Parasites survive the dry season within some individuals who experience prolonged carriage of parasites and are thought to ‘seed’ infection in the next transmission season. Methods Dry season carriers and their role in the subsequent transmission season are characterized using a combination of mathematical simulations and data analysis of previously described data from a longitudinal study in Mali of individuals aged 3 months–12 years (n = 579). Results Simulating the life-history of individuals experiencing repeated exposure to infection predicts that dry season carriage is more likely in the oldest, most exposed and most immune individuals. This hypothesis is supported by the data from Mali, which shows that carriers are significantly older, experience a higher biting rate at the beginning of the transmission season and develop clinical malaria later than non-carriers. Further, since the most exposed individuals in a community are most likely to be dry season carriers, this is predicted to enable a more than twofold faster spread of parasites into the mosquito population at the start of the subsequent wet season. Conclusions Carriage of malaria parasites over the months-long dry season in Mali is most likely in the older, more exposed and more immune children. These children may act as super-spreaders facilitating the fast spread of parasites at the beginning of the next transmission season