Intestinal stroma guides monocyte differentiation to macrophages through GM-CSF.

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142-/low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142-/low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Survival of P. falciparum infected red blood cell aggregates in elongational shear flow.

Rosetting, the formation of red blood cell aggregates, is a life-threatening condition in malaria tropica and not yet fully understood. We study rosette stability using a set of microfluidic stenotic channels, with varied narrowing angle and erythrocytes of blood groups O and A. We find reduced ability of a rosette to pass a stenosis without disruption, the longer the tapered part of the constriction and the narrower the stenosis is. In general, this ability increases with rosette size and is 5-15% higher in blood group A. The experimental results are substantiated by equivalent experiments using lectin-induced red blood cell aggregates and a simulation of the underlying protein binding kinetics.

Antibiotics against Pseudomonas aeruginosa on Human Skin Cell Lines: Determination of the Highest Non-Cytotoxic Concentrations with Antibiofilm Capacity for Wound Healing Strategies.

Pseudomonas aeruginosa is one of the most common microorganisms causing infections of severe skin wounds. Antibiotic or antiseptic treatments are crucial to prevent and curb these infections. Antiseptics have been reported to be cytotoxic to skin cells and few studies evaluate the impact of commonly used antibiotics. This study evaluates how clinical antibiotics affect skin cells' viability, proliferation, migration, and cytokine secretion and defines the highest non-cytotoxic concentrations that maintain antibacterial activity. Cell proliferation, viability, and migration were evaluated on cell monolayers. Cytokines related to the wound healing process were determined. The minimum inhibitory concentrations and the impact on bacterial biofilm were assessed. Results showed that 0.02 mg/mL ciprofloxacin and 1 mg/mL meropenem are the highest non-cytotoxic concentrations for fibroblasts and keratinocytes while 1.25 mg/mL amikacin and 0.034 mg/mL colistin do not affect fibroblasts' viability and cytokine secretion but have an impact on keratinocytes. These concentrations are above the minimum inhibitory concentration but only amikacin could eradicate the biofilm. For the other antibiotics, cytotoxic concentrations are needed to eradicate the biofilm. Combinations with colistin at non-cytotoxic concentrations effectively eliminate the biofilm. These results provide information about the concentrations required when administering topical antibiotic treatments on skin lesions, and how these antibiotics affect wound management therapies. This study set the basis for the development of novel antibacterial wound healing strategies such as antibiotic artificial skin substitutes.

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase. METHODS: Bacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry. RESULTS: Here, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation. CONCLUSIONS: Our findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

Targeted plasma proteomics reveals signatures discriminating COVID-19 from sepsis with pneumonia.

BACKGROUND: COVID-19 remains a major public health challenge, requiring the development of tools to improve diagnosis and inform therapeutic decisions. As dysregulated inflammation and coagulation responses have been implicated in the pathophysiology of COVID-19 and sepsis, we studied their plasma proteome profiles to delineate similarities from specific features. METHODS: We measured 276 plasma proteins involved in Inflammation, organ damage, immune response and coagulation in healthy controls, COVID-19 patients during acute and convalescence phase, and sepsis patients; the latter included (i) community-acquired pneumonia (CAP) caused by Influenza, (ii) bacterial CAP, (iii) non-pneumonia sepsis, and (iv) septic shock patients. RESULTS: We identified a core response to infection consisting of 42 proteins altered in both COVID-19 and sepsis, although higher levels of cytokine storm-associated proteins were evident in sepsis. Furthermore, microbiologic etiology and clinical endotypes were linked to unique signatures. Finally, through machine learning, we identified biomarkers, such as TRIM21, PTN and CASP8, that accurately differentiated COVID-19 from CAP-sepsis with higher accuracy than standard clinical markers. CONCLUSIONS: This study extends the understanding of host responses underlying sepsis and COVID-19, indicating varying disease mechanisms with unique signatures. These diagnostic and severity signatures are candidates for the development of personalized management of COVID-19 and sepsis.

Red blood cell blood group A antigen level affects the ability of heparin and PfEMP1 antibodies to disrupt Plasmodium falciparum rosettes.

BACKGROUND: The histo-blood group ABO system has been associated with adverse outcomes in COVID-19, thromboembolic diseases and Plasmodium falciparum malaria. An integral part of the severe malaria pathogenesis is rosetting, the adherence of parasite infected red blood cells (RBCs) to uninfected RBCs. Rosetting is influenced by the host's ABO blood group (Bg) and rosettes formed in BgA have previously been shown to be more resilient to disruption by heparin and shield the parasite derived surface antigens from antibodies. However, data on rosetting in weak BgA subgroups is scarce and based on investigations of relatively few donors. METHODS: An improved high-throughput flow cytometric assay was employed to investigate rosetting characteristics in an extensive panel of RBC donor samples of all four major ABO Bgs, as well as low BgA expressing samples. RESULTS: All non-O Bgs shield the parasite surface antigens from strain-specific antibodies towards P. falciparum erythrocyte membrane protein 1 (PfEMP1). A positive correlation between A-antigen levels on RBCs and rosette tightness was observed, protecting the rosettes from heparin- and antibody-mediated disruption. CONCLUSIONS: These results provide new insights into how the ABO Bg system affects the disease outcome and cautions against interpreting the results from the heterogeneous BgA phenotype as a single group in epidemiological and experimental studies.

High-dimensional profiling reveals phenotypic heterogeneity and disease-specific alterations of granulocytes in COVID-19.

Since the outset of the COVID-19 pandemic, increasing evidence suggests that the innate immune responses play an important role in the disease development. A dysregulated inflammatory state has been proposed as a key driver of clinical complications in COVID-19, with a potential detrimental role of granulocytes. However, a comprehensive phenotypic description of circulating granulocytes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients is lacking. In this study, we used high-dimensional flow cytometry for granulocyte immunophenotyping in peripheral blood collected from COVID-19 patients during acute and convalescent phases. Severe COVID-19 was associated with increased levels of both mature and immature neutrophils, and decreased counts of eosinophils and basophils. Distinct immunotypes were evident in COVID-19 patients, with altered expression of several receptors involved in activation, adhesion, and migration of granulocytes (e.g., CD62L, CD11a/b, CD69, CD63, CXCR4). Paired sampling revealed recovery and phenotypic restoration of the granulocytic signature in the convalescent phase. The identified granulocyte immunotypes correlated with distinct sets of soluble inflammatory markers, supporting pathophysiologic relevance. Furthermore, clinical features, including multiorgan dysfunction and respiratory function, could be predicted using combined laboratory measurements and immunophenotyping. This study provides a comprehensive granulocyte characterization in COVID-19 and reveals specific immunotypes with potential predictive value for key clinical features associated with COVID-19.

Enhanced virulence of Plasmodium falciparum in blood of diabetic patients.

Rising prevalence of diabetes in sub-Saharan Africa, coupled with continued malaria transmission, has resulted more patients dealing with both communicable and non-communicable diseases. We previously reported that travelers with type 2 diabetes mellitus (T2DM) infected with Plasmodium falciparum were three times more likely to develop severe malaria than non-diabetics. Here we explore the biological basis for this by testing blood from uninfected subjects with type 1 and type 2 diabetes, ex vivo, for their effects on parasite growth and rosetting (binding of infected erythrocytes to uninfected erythrocytes). Rosetting was associated with type 2 diabetes, blood glucose and erythrocyte sedimentation rate (ESR), while parasite growth was positively associated with blood glucose, glycated hemoglobin (HbA1c), body mass index (BMI), fibrinogen and triglycerides. This study establishes a link between diabetes and malaria virulence assays, potentially explaining the protective effect of good glycemic control against severe malaria in subjects with diabetes.

Blood group and size dependent stability of P. falciparum infected red blood cell aggregates in capillaries.

For Plasmodium falciparum related malaria (B50), one of the outstanding host factors for the development of severe disease is the ABO blood group of malaria patients, where blood group O reduces the probability of severe disease as compared to individuals of groups A, B, or AB. In this report, we investigate the stability of rosette aggregates in malaria caused by Plasmodium falciparum in microflows. These flows are created in microfluidic channels with stenosis-like constrictions of different widths down to ones narrower as the rosette's diameter. High speed videos were recorded and analyzed by a MATLAB© based tracking software (SURF: SUrvival of Rosettes in Flow). We find a correlation of rosette size, channel diameter, and blood group regarding the mobility of the rosettes. Following the concept of a thermodynamic model, we find a critical width of the stenosis for rosette rupture during their passage. Our data reveal that under physiologically relevant conditions, rosettes in blood group A have a higher rosette frequency and stability as compared to blood group O (BG O), which constitutes a crucial factor promoting the observed protection in BG O individuals against severe malaria in non-O individuals.

SURGE complex of Plasmodium falciparum in the rhoptry-neck (SURFIN4.2-RON4-GLURP) contributes to merozoite invasion.

Plasmodium falciparum invasion into red blood cells (RBCs) is a complex process engaging proteins on the merozoite surface and those contained and sequentially released from the apical organelles (micronemes and rhoptries). Fundamental to invasion is the formation of a moving junction (MJ), a region of close apposition of the merozoite and the RBC plasma membranes, through which the merozoite draws itself before settling into a newly formed parasitophorous vacuole (PV). SURFIN4.2 was identified at the surface of the parasitized RBCs (pRBCs) but was also found apically associated with the merozoite. Using antibodies against the N-terminus of the protein we show the presence of SURFIN4.2 in the neck of the rhoptries, its secretion into the PV and shedding into the culture supernatant upon schizont rupture. Using immunoprecipitation followed by mass spectrometry we describe here a novel protein complex we have named SURGE where SURFIN4.2 forms interacts with the rhoptry neck protein 4 (RON4) and the Glutamate Rich Protein (GLURP). The N-terminal cysteine-rich-domain (CRD) of SURFIN4.2 mediates binding to the RBC membrane and its interaction with RON4 suggests its involvement in the contact between the merozoite apex and the RBC at the MJ. Supporting this suggestion, we also found that polyclonal antibodies to the extracellular domain (including the CRD) of SURFIN4.2 partially inhibit merozoite invasion. We propose that the formation of the SURGE complex participates in the establishment of parasite infection within the PV and the RBCs.

ABO Blood Group Antigen Decorated Giant Unilamellar Vesicles Exhibit Distinct Interactions with Plasmodium falciparum Infected Red Blood Cells.

Severe malaria is considered to be the deadliest disease of this century, primarily among children in sub-Saharan Africa. It stems from infection by the virulent parasite Plasmodium falciparum. The pathogenesis of the disease is based on the rosetting phenomenon, which occurs during the life cycle of the parasite in red blood cells (RBCs) and promotes the binding of parasitized RBCs to healthy ones. The role of the ABO blood group antigens in relation to the phenomenon has previously only been investigated in clinical isolates obtained from malaria patients. Here, we aim to clarify their role using synthetic ABO-decorated giant unilamellar vesicles (GUVs), which serve as simple biomimetic models of RBC-size cell membranes. Our results suggest clearly and for the first time that the blood group A and O antigens have a direct impact on receptor-specific rosetting phenomena when compared to the B antigen, which only participates in rosetting to an insignificant degree. Thus, glycodecorated GUVs represent a practical tool for studying cell-surface interactions.

Antibodies in children with malaria to PfEMP1, RIFIN and SURFIN expressed at the Plasmodium falciparum parasitized red blood cell surface.

Naturally acquired antibodies to proteins expressed on the Plasmodium falciparum parasitized red blood cell (pRBC) surface steer the course of a malaria infection by reducing sequestration and stimulating phagocytosis of pRBC. Here we have studied a selection of proteins representing three different parasite gene families employing a well-characterized parasite with a severe malaria phenotype (FCR3S1.2). The presence of naturally acquired antibodies, impact on rosetting rate, surface reactivity and opsonization for phagocytosis in relation to different blood groups of the ABO system were assessed in a set of sera from children with mild or complicated malaria from an endemic area. We show that the naturally acquired immune responses, developed during malaria natural infection, have limited access to the pRBCs inside a blood group A rosette. The data also indicate that SURFIN4.2 may have a function at the pRBC surface, particularly during rosette formation, this role however needs to be further validated. Our results also indicate epitopes differentially recognized by rosette-disrupting antibodies on a peptide array. Antibodies towards parasite-derived proteins such as PfEMP1, RIFIN and SURFIN in combination with host factors, essentially the ABO blood group of a malaria patient, are suggested to determine the outcome of a malaria infection.

Inhibition of merozoite invasion and transient de-sequestration by sevuparin in humans with Plasmodium falciparum malaria.

SEVERE MALARIA: Even with the best available treatment, the mortality from severe Plasmodium falciparum malaria remains high. Typical features at death are high parasite loads and obstructed micro- vasculature. Infected erythrocytes (IE) containing mature parasites bind to the host receptor heparan sulfate, which is also an important receptor for merozoite invasion. To block merozoite invasion has not previously been proposed as an adjunctive therapeutic approach but it may preclude the early expansion of an infection that else leads to exacerbated sequestration and death. SEVUPARIN IN PHASE I STUDY: The drug sevuparin was developed from heparin because heparan sulfate and heparin are nearly identical, so the rationale was that sevuparin would act as a decoy receptor during malaria infection. A phase I study was performed in healthy male volunteers and sevuparin was found safe and well tolerated. SEVUPARIN IN PHASE I/II CLINICAL STUDY: A phase I/II clinical study was performed in which sevuparin was administered via short intravenous infusions to malaria patients with uncomplicated malaria who were also receiving atovaquone/proguanil treatment. This was a Phase I/II, randomized, open label, active control, parallel assignment study. Sevuparin was safe and well tolerated in the malaria patients. The mean relative numbers of ring-stage IEs decreased after a single sevuparin infusion and mature parasite IEs appeared transiently in the circulation. The effects observed on numbers of merozoites and throphozoites in the circulation, were detected already one hour after the first sevuparin injection. Here we report the development of a candidate drug named sevuparin that both blocks merozoite invasion and transiently de-sequesters IE in humans with P. falciparum malaria. TRIAL REGISTRATION: ClinicalTrials.gov NCT01442168.

Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing.

Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.

Development of drug-loaded immunoliposomes for the selective targeting and elimination of rosetting Plasmodium falciparum-infected red blood cells.

Parasite proteins exported to the surface of Plasmodium falciparum-parasitized red blood cells (pRBCs) have a major role in severe malaria clinical manifestation, where pRBC cytoadhesion and rosetting processes have been strongly linked with microvascular sequestration while avoiding both spleen filtration and immune surveillance. The parasite-derived and pRBC surface-exposed PfEMP1 protein has been identified as one of the responsible elements for rosetting and, therefore, considered as a promising vaccine candidate for the generation of rosette-disrupting antibodies against severe malaria. However, the potential role of anti-rosetting antibodies as targeting molecules for the functionalization of antimalarial drug-loaded nanovectors has never been studied. Our manuscript presents a proof-of-concept study where the activity of an immunoliposomal vehicle with a dual performance capable of specifically recognizing and disrupting rosettes while simultaneously eliminating those pRBCs forming them has been assayed in vitro. A polyclonal antibody against the NTS-DBL1α N-terminal domain of a rosetting PfEMP1 variant has been selected as targeting molecule and lumefantrine as the antimalarial payload. After 30min incubation with 2µM encapsulated drug, a 70% growth inhibition for all parasitic forms in culture (IC50: 414nM) and a reduction in ca. 60% of those pRBCs with a rosetting phenotype (IC50: 747nM) were achieved. This immunoliposomal approach represents an innovative combination therapy for the improvement of severe malaria therapeutics having a broader spectrum of activity than either anti-rosetting antibodies or free drugs on their own.

Phagocytosis-inducing antibodies to Plasmodium falciparum upon immunization with a recombinant PfEMP1 NTS-DBL1α domain.

BACKGROUND: Individuals living in endemic areas gradually acquire natural immunity to clinical malaria, largely dependent on antibodies against parasite antigens. There are many studies indicating that the variant antigen PfEMP1 at the surface of the parasitized red blood cell (pRBC) is one of the major targets of the immune response. It is believed that antibodies against PfEMP1 confer protection by blocking sequestration (rosetting and cytoadherence), inducing antibody-dependent cellular-inhibitory effect and opsonizing pRBCs for phagocytosis. METHODS: A recombinant NTS-DBL1α domain from a rosette-mediating PfEMP1 was expressed in Escherichia coli. The resulting protein was purified and used for immunization to generate polyclonal (goat) and monoclonal (mouse) antibodies. The antibodies' ability to opsonize and induce phagocytosis in vitro was tested and contrasted with the presence of opsonizing antibodies naturally acquired during Plasmodium falciparum infection. RESULTS: All antibodies recognized the recombinant antigen and the surface of live pRBCs, however, their capacity to opsonize the pRBCs for phagocytosis varied. The monoclonal antibodies isotyped as IgG2b did not induce phagocytosis, while those isotyped as IgG2a were in general very effective, inducing phagocytosis with similar levels as those naturally acquired during P. falciparum infection. These monoclonal antibodies displayed different patterns, some of them showing a concentration-dependent activity while others showed a prozone-like effect. The goat polyclonal antibodies were not able to induce phagocytosis. CONCLUSION: Immunization with an NTS-DBL1-α domain of PfEMP1 generates antibodies that not only have a biological role in rosette disruption but also effectively induce opsonization for phagocytosis of pRBCs with similar activity to naturally acquired antibodies from immune individuals living in a malaria endemic area. Some of the antibodies with high opsonizing activity were not able to disrupt rosettes, indicating that epitopes of the NTS-DBL1-α other than those involved in rosetting are exposed on the pRBC surface and are able to induce functional antibodies. The ability to induce phagocytosis largely depended on the antibody isotype and on the ability to recognize the surface of the pRBC regardless of the rosette-disrupting capacity.

Rosette-Disrupting Effect of an Anti-Plasmodial Compound for the Potential Treatment of Plasmodium falciparum Malaria Complications.

The spread of artemisinin-resistant parasites could lead to higher incidence of patients with malaria complications. However, there are no current treatments that directly dislodge sequestered parasites from the microvasculature. We show that four common antiplasmodial drugs do not disperse rosettes (erythrocyte clusters formed by malaria parasites) and therefore develop a cell-based high-throughput assay to identify potential rosette-disrupting compounds. A pilot screen of 2693 compounds identified Malaria Box compound MMV006764 as a potential candidate. Although it reduced rosetting by a modest 20%, MMV006764 was validated to be similarly effective against both blood group O and A rosettes of three laboratory parasite lines. Coupled with its antiplasmodial activity and drug-likeness, MMV006764 represents the first small-molecule compound that disrupts rosetting and could potentially be used in a resource-limited setting to treat patients deteriorating rapidly from malaria complications. Such dual-action drugs that simultaneously restore microcirculation and reduce parasite load could significantly reduce malaria morbidity and mortality.

ARAM: an automated image analysis software to determine rosetting parameters and parasitaemia in Plasmodium samples.

BACKGROUND: Rosetting is associated with severe malaria and a primary cause of death in Plasmodium falciparum infections. Detailed understanding of this adhesive phenomenon may enable the development of new therapies interfering with rosette formation. For this, it is crucial to determine parameters such as rosetting and parasitaemia of laboratory strains or patient isolates, a bottleneck in malaria research due to the time consuming and error prone manual analysis of specimens. Here, the automated, free, stand-alone analysis software automated rosetting analyzer for micrographs (ARAM) to determine rosetting rate, rosette size distribution as well as parasitaemia with a convenient graphical user interface is presented. METHODS: Automated rosetting analyzer for micrographs is an executable with two operation modes for automated identification of objects on images. The default mode detects red blood cells and fluorescently labelled parasitized red blood cells by combining an intensity-gradient with a threshold filter. The second mode determines object location and size distribution from a single contrast method. The obtained results are compared with standardized manual analysis. Automated rosetting analyzer for micrographs calculates statistical confidence probabilities for rosetting rate and parasitaemia. RESULTS: Automated rosetting analyzer for micrographs analyses 25 cell objects per second reliably delivering identical results compared to manual analysis. For the first time rosette size distribution is determined in a precise and quantitative manner employing ARAM in combination with established inhibition tests. Additionally ARAM measures the essential observables parasitaemia, rosetting rate and size as well as location of all detected objects and provides confidence intervals for the determined observables. No other existing software solution offers this range of function. The second, non-malaria specific, analysis mode of ARAM offers the functionality to detect arbitrary objects. CONCLUSIONS: Automated rosetting analyzer for micrographs has the capability to push malaria research to a more quantitative and statistically significant level with increased reliability due to operator independence. As an installation file for Windows © 7, 8.1 and 10 is available for free, ARAM offers a novel open and easy-to-use platform for the malaria community to elucidate resetting.

Evasion of Immunity to Plasmodium falciparum: Rosettes of Blood Group A Impair Recognition of PfEMP1.

The ABO blood group antigens are expressed on erythrocytes but also on endothelial cells, platelets and serum proteins. Notably, the ABO blood group of a malaria patient determines the development of the disease given that blood group O reduces the probability to succumb in severe malaria, compared to individuals of groups A, B or AB. P. falciparum rosetting and sequestration are mediated by PfEMP1, RIFIN and STEVOR, expressed at the surface of the parasitized red blood cell (pRBC). Antibodies to these antigens consequently modify the course of a malaria infection by preventing sequestration and promoting phagocytosis of pRBC. Here we have studied rosetting P. falciparum and present evidence of an immune evasion mechanism not previously recognized. We find the accessibility of antibodies to PfEMP1 at the surface of the pRBC to be reduced when P. falciparum forms rosettes in blood group A RBC, as compared to group O RBC. The pRBC surrounds itself with tightly bound normal RBC that makes PfEMP1 inaccessible to antibodies and clearance by the immune system. Accordingly, pRBC of in vitro cloned P. falciparum devoid of ABO blood group dependent rosetting were equally well detected by anti-PfEMP1 antibodies, independent of the blood group utilized for their propagation. The pathogenic mechanisms underlying the severe forms of malaria may in patients of blood group A depend on the ability of the parasite to mask PfEMP1 from antibody recognition, in so doing evading immune clearance.