CRIg on liver macrophages clears pathobionts and protects against alcoholic liver disease.

Complement receptor of immunoglobulin superfamily (CRIg) is expressed on liver macrophages and directly binds complement component C3b or Gram-positive bacteria to mediate phagocytosis. CRIg plays important roles in several immune-mediated diseases, but it is not clear how its pathogen recognition and phagocytic functions maintain homeostasis and prevent disease. We previously associated cytolysin-positive Enterococcus faecalis with severity of alcohol-related liver disease. Here, we demonstrate that CRIg is reduced in liver tissues from patients with alcohol-related liver disease. CRIg-deficient mice developed more severe ethanol-induced liver disease than wild-type mice; disease severity was reduced with loss of toll-like receptor 2. CRIg-deficient mice were less efficient than wild-type mice at clearing Gram-positive bacteria such as Enterococcus faecalis that had translocated from gut to liver. Administration of the soluble extracellular domain CRIg-Ig protein protected mice from ethanol-induced steatohepatitis. Our findings indicate that ethanol impairs hepatic clearance of translocated pathobionts, via decreased hepatic CRIg, which facilitates progression of liver disease.

Increasing the efficacy and safety of a human complement inhibitor for treating post-transplant cardiac ischemia reperfusion injury by targeting to a graft-specific neoepitope.

BACKGROUND: Post-transplant ischemia reperfusion injury (IRI) is a recognized risk factor for subsequent organ dysfunction, alloresponsiveness, and rejection. The complement system is known to play a role in IRI and represents a therapeutic target. Complement is activated in transplanted grafts when circulating IgM antibodies bind to exposed ischemia-induced neoepitopes upon reperfusion, and we investigated the targeting of a human complement inhibitor, CR1, to a post-transplant ischemia-induced neoepitope. METHODS: A fragment of human CR1 was linked to a single chain antibody construct (C2 scFv) recognizing an injury-specific neoepitope to yield C2-CR1. This construct, along with a soluble untargeted counterpart, was characterized in a cardiac allograft transplantation model of IRI in terms of efficacy and safety. RESULTS: CR1 was similarly effective against mouse and human complement. C2-CR1 provided effective protection against cardiac IRI at a lower dose than untargeted CR1. The increased efficacy of C2-CR1 relative to CR1 correlated with decreased C3 deposition, and C2-CR1, but not CR1, targeted to cardiac allografts. At a dose necessary to reduce IRI, C2-CR1 had minimal impact on serum complement activity, in contrast to CR1 which resulted in a high level of systemic inhibition. The circulatory half-life of CR1 was markedly longer than that of C2-CR1, and whereas a minimum therapeutic dose of CR1 severely impaired host susceptibility to infection, C2-CR1 had no impact. CONCLUSION: We show the translational potential of a human complement inhibitor targeted to a universal ischemia-induced graft-specific epitope, and demonstrate advantages compared to an untargeted counterpart in terms of efficacy and safety.

A novel soluble complement receptor 1 fragment with enhanced therapeutic potential.

Human complement receptor 1 (HuCR1) is a pivotal regulator of complement activity, acting on all three complement pathways as a membrane-bound receptor of C3b/C4b, C3/C5 convertase decay accelerator, and cofactor for factor I-mediated cleavage of C3b and C4b. In this study, we sought to identify a minimal soluble fragment of HuCR1, which retains the complement regulatory activity of the wildtype protein. To this end, we generated recombinant, soluble, and truncated versions of HuCR1 and compared their ability to inhibit complement activation in vitro using multiple assays. A soluble form of HuCR1, truncated at amino acid 1392 and designated CSL040, was found to be a more potent inhibitor than all other truncation variants tested. CSL040 retained its affinity to both C3b and C4b as well as its cleavage and decay acceleration activity and was found to be stable under a range of buffer conditions. Pharmacokinetic studies in mice demonstrated that the level of sialylation is a major determinant of CSL040 clearance in vivo. CSL040 also showed an improved pharmacokinetic profile compared with the full extracellular domain of HuCR1. The in vivo effects of CSL040 on acute complement-mediated kidney damage were tested in an attenuated passive antiglomerular basement membrane antibody-induced glomerulonephritis model. In this model, CSL040 at 20 and 60 mg/kg significantly attenuated kidney damage at 24 h, with significant reductions in cellular infiltrates and urine albumin, consistent with protection from kidney damage. CSL040 thus represents a potential therapeutic candidate for the treatment of complement-mediated disorders.

Two novel antithetical KN blood group antigens may contribute to more than a quarter of all KN antisera in Europe.

BACKGROUND: All antigens described in the KN blood group system are located in the long homologous repeat D (LHR-D) of complement receptor 1 (CR1). While there have been reports that some sera react only with the long homologous repeat C (LHR-C), the antigens in LHR-C are unknown. STUDY DESIGN AND METHODS: Recombinant LHR-C and LHR-D were used to identify antibodies directed against LHR-C of CR1, into which a point mutation was introduced to characterize the underlying blood group antigens. In addition, database studies to define haplotypes of CR1 were performed. RESULTS: Several antisera were identified that were specific against CR1 p.1208His and against CR1 p.1208Arg, located in LHR-C. Fifteen KN haplotypes were found in the Ensembl genome browser. It was shown that due to a linkage disequilibrium anti-CR1 p.1208His may be mistaken for anti-KCAM. CONCLUSION: A novel antithetical KN blood group antigen pair was found at position p.1208 of CR1, for which the names DACY and YCAD are proposed. Antibodies against these two novel antigens seem to contribute to more than a quarter of all KN sera in Europe.

Mechanistic Understanding of Cell Recognition and Immune Reaction via CR1/CR3 by HAP- and SiO2-NPs.

Nanodrug carrier will eventually enter the blood when intravenously injected or in other ways. Meanwhile, a series of toxic effects were caused to the body with the formation of nanoparticle protein corona. In our studies, we try to reveal the recognition mechanism of nanoparticle protein corona by monocyte and the damage effect on immune cells by activated complement of hydroxyapatite nanoparticles (HAP-NPs) and silicon dioxide nanoparticles (SiO2-NPs). So expressions of TLR4/CR1/CR were analyzed by flow cytometry (FCM) in order to illuminate the recognition mechanism of nanoparticle protein corona by monocyte. And the expression of ROS, cytokines, adhesion molecules, and arachidonic acid was measured when THP-1 and HUVECs were stimulated by NP-activated complement. The results showed that HAP-NPs can be recognized by the opsonin receptor (iC3b/CR3) model, while plasma protein, opsonin receptor, and Toll-like receptors are all likely launch cell recognition of SiO2-NPs. And it was considerate that NP-activated complement can damage THP-1 and HUVECs, including oxidative stress, inflammation, and increased vascular permeability. So the surface of nanodrug carrier can be modified to avoid being clear and reduce the efficacy according to the three receptors (TLR4/CR1/CR3).

Complement Receptor 1 (CR1/CD35)-expressing retinal pigment epithelial cells as a potential therapy for age-related macular degeneration.

The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential "enhanced" cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.

CD35 and CD64 of Neutrophils Can Differentiate Between Bacterial and Viral Infections in Children by Simultaneous Quantitative Analysis.

BACKGROUND The aim of this study was to determine the relationship between the expression of CD35 and CD64 from white blood cells (neutrophil, monocytes, and lymphocytes) and acute infectious diseases in children. MATERIAL AND METHODS The blood samples were collected from 104 children with infections (42 viral infections and 62 bacterial infections). Blood samples were stained with CD45-PC5, CD35-FITC, and CD64-PE, and the fluorescence intensities were measured by flow cytometer, and then the ratio of CD35 to CD64 was calculated. RESULTS The ratio of CD64/CD35 on neutrophils (NCD35/NCD64) was significantly different between the bacterial group, the virus group, and the healthy control group. According to receiver operating characteristic (ROC) analysis, a cutoff value of 7.256 (sensitivity: 90.0%, specificity: 93.7%) was determined for the NCD35/NCD64 ratio. CONCLUSIONS This study shows that NCD35/NCD64 is helpful in the differential diagnosis of acute viral infection and bacterial infection in children.

Altered Expression of Complement Regulatory Proteins CD35, CD46, CD55, and CD59 on Leukocyte Subsets in Individuals Suffering From Coronary Artery Disease.

Studies conducted in animal models have suggested that membrane complement regulatory proteins play an important role in the pathophysiology of coronary artery disease (CAD). In this study, a total of 100 individuals, with stable CAD and 100 healthy controls, both groups predominantly male, were recruited. We evaluated the plasma levels of complement regulatory proteins (Cregs) CD35, CD46, CD55, and CD59 and their surface expression on granulocytes, lymphocytes, and monocytes by flow cytometry. The mRNA expression of these Cregs in total leukocytes was determined by quantitative PCR. The soluble forms of Cregs, C3c, Mannose binding protein-associated serine protease 2 (MASP-2), Platelet activating factor-acetyl hydrolase (PAF-AH), and inflammatory cytokines were quantified by ELISA. High plasma levels of C3c, indicative of complement activation, in addition to significantly low levels of Cregs, were observed in CAD patients. A significantly lower expression of CD46 and CD55 on the surface of lymphocytes, monocytes, and granulocytes and higher surface expression of CD35 and CD59 on granulocytes (p < 0.0001) was seen in CAD patients as compared to healthy donors. The high expression of CD59 on granulocytes positively correlated with the severity of disease and may serve as a potential marker of disease progression in CAD.

Key role of the number of complement receptor 1 on erythrocytes for binding of Escherichia coli to erythrocytes and for leukocyte phagocytosis and oxidative burst in human whole blood.

AIM: To study the role of complement receptor 1 (CR1) for binding of Escherichia coli (E. coli) to erythrocytes, for leukocyte phagocytosis, oxidative burst and complement activation in human whole blood from a CR1 deficient (CR1D) patient and healthy controls with low, medium and high CR1 numbers. METHODS: Alexa-labelled bacteria were used to quantify erythrocyte-bound bacteria, free bacteria in plasma and phagocytosis using flow cytometry. Complement activation in plasma was measured by enzyme-linked immunosorbent assay. The CR1 numbers as well as C3bc and C4bc deposition on erythrocytes were measured by flow cytometry. Cytokines were measured using multiplex technology, and bacterial growth was measured by colony forming units. CR1 was blocked using the anti-CR1 blocking mAb 3D9. RESULTS: Approximately 85% of E. coli bound to erythrocytes after 15 min incubation in donor blood with high and medium CR1 numbers, 50% in the person with low CR1 numbers and virtually no detectable binding in the CR1D (r2 = 0.87, P < 0.0007). The number of free bacteria in plasma was inversely related to erythrocyte CR1 numbers (r2 = 0.98, P < 0.0001). E. coli-induced phagocytosis and oxidative burst were significantly enhanced by the anti-CR1 mAb 3D9 and in the CR1D and the donor with low CR1 numbers. E. coli-induced complement activation in plasma, C3bc and C4bc deposition on erythrocytes, and bacterial growth were similar in all four cases. CONCLUSIONS: CR1D and low CR1 numbers prevented E. coli binding to erythrocytes, increased free bacteria in plasma, phagocytosis and oxidative burst, but did not affect plasma or surface complement activation and bacterial growth.

Protein-engineered molecules carrying GAD65 epitopes and targeting CD35 selectively down-modulate disease-associated human B lymphocytes.

Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti-CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti-GAD65 specific B cells by the co-cross-linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65-specific B lymphocytes and the production of anti-GAD65 IgG autoantibodies by co-cross-linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells.

Complement serves as a switch between CD4+ T cell-independent and -dependent RBC antibody responses.

RBC alloimmunization represents a significant immunological challenge for patients requiring lifelong transfusion support. The majority of clinically relevant non-ABO(H) blood group antigens have been thought to drive antibody formation through T cell-dependent immune pathways. Thus, we initially sought to define the role of CD4+ T cells in formation of alloantibodies to KEL, one of the leading causes of hemolytic transfusion reactions. Unexpectedly, our findings demonstrated that KEL RBCs actually possess the ability to induce antibody formation independent of CD4+ T cells or complement component 3 (C3), two common regulators of antibody formation. However, despite the ability of KEL RBCs to induce anti-KEL antibodies in the absence of complement, removal of C3 or complement receptors 1 and 2 (CR1/2) rendered recipients completely reliant on CD4+ T cells for IgG anti-KEL antibody formation. Together, these findings suggest that C3 may serve as a novel molecular switch that regulates the type of immunological pathway engaged following RBC transfusion.

C1q and Mannose-Binding Lectin Interact with CR1 in the Same Region on CCP24-25 Modules.

Complement receptor type 1 (CR1) is a multi modular membrane receptor composed of 30 homologous complement control protein modules (CCP) organized in four different functional regions called long homologous repeats (LHR A, B, C, and D). CR1 is a receptor for complement-opsonins C3b and C4b and specifically interacts through pairs of CCP modules located in LHR A, B, and C. Defense collagens such as mannose-binding lectin (MBL), ficolin-2, and C1q also act as opsonins and are involved in immune clearance through binding to the LHR-D region of CR1. Our previous results using deletion variants of CR1 mapped the interaction site for MBL and ficolin-2 on CCP24-25. The present work aimed at deciphering the interaction of C1q with CR1 using new CR1 variants concentrated around CCP24-25. CR1 bimodular fragment CCP24-25 and CR1 CCP22-30 deleted from CCP24-25 produced in eukaryotic cells enabled to highlight that the interaction site for both MBL and C1q is located on the same pair of modules CCP24-25. C1q binding to CR1 shares with MBL a main common interaction site on the collagen stalks but also subsidiary sites most probably located on C1q globular heads, contrarily to MBL.

Follicular dendritic cells in normal and infected human appendix

Follicular dendritic cells (FDCs) that reside within the lymphoid follicles play a central role in humoral immunity. They bind immune complexes and present antigen to follicular B cells and in the generation of B cell memory. This study aims to demonstrate the distribution of CD35 positive FDCs in normal and infected appendix by immunohistochemistry. Four normal and 5 infected appendix specimens were used for the study. Tissues collected were processed for immunohistochemistry, stained with mouse antihuman CD35 monoclonal antibody using the Polymer - HRP Detection System. Double immunostaining was done with mouse antihuman CD20 monoclonal antibody to find out the association CD35 positive Langerhans cells with B lymphocytes. Cells were viewed under the light microscope (Olympus DP21). In the normal appendix, CD35 positive FDCs were present in a reticular pattern in the germinal centre of the follicle. In acute appendicitis, the lymphatic follicles were not intact and FDCs were scattered in the mucosa of the appendix. Few discrete CD35 positive cells were seen surrounding the intestinal glands. CD20 positive B lymphocytes were noted in the lymphatic follicle, interfollicular areas, around the crypts and in the lamina propria. Apposition of CD35 and CD20 cells was noted. The dendrites of FDCs demonstrated in the follicles of appendix displayed an antigen-retaining reticulum which aids in trapping of immune complexes. Their association with CD20 positive B cells confirm the role of appendix in humoral immunity

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Complement Receptor Type 1 Suppresses Human B Cell Functions in SLE Patients.

Complement receptors (CRs) play an integral role in innate immunity and also function to initiate and shape the adaptive immune response. Our earlier results showed that complement receptor type 1 (CR1, CD35) is a potent inhibitor of the B cell receptor- (BCR-) induced functions of human B lymphocytes. Here we show that this inhibition occurs already at the initial steps of B cell activation since ligation of CR1 reduces the BCR-induced phosphorylation of key signaling molecules such as Syk and mitogen activated protein kinases (MAPKs). Furthermore, our data give evidence that although B lymphocytes of active systemic lupus erythematosus (SLE) patients express lower level of CR1, the inhibitory capacity of this complement receptor is still maintained and its ligand-induced clustering results in significant inhibition of the main B cell functions, similar to that found in the case of healthy individuals. Since we have found that reduced CR1 expression of SLE patients does not affect the inhibitory capacity of the receptor, our results further support the therapeutical potential of CD35 targeting the decrease of B cell activation and autoantibody production in autoimmune patients.

Complement receptor immunoglobulin: a control point in infection and immunity, inflammation and cancer.

The B7 family-related protein, V-set and Ig domain (VSIG4) / Z39Ig / complement receptor immunoglobulin (CRIg), is a new player in the regulation of immunity to infection and inflammation. The unique features of this receptor as compared with classical complement receptors, CR3 and CR4, have heralded the emergence of new concepts in the regulation of innate and adaptive immunity. Its selective expression in tissue macrophages and dendritic cells has been considered of importance in host defence and in maintaining tolerance against self-antigens. Although a major receptor for phagocytosis of complement opsonised bacteria, its array of emerging functions which incorporates the immune suppressive and anti-inflammatory action of the receptor have now been realised. Accumulating evidence from mouse experimental models indicates a potential role for CRIg in protection against bacterial infection and inflammatory diseases, such as rheumatoid arthritis, type 1 diabetes and systemic lupus erythematosus, and also in promotion of tumour growth. CRIg expression can be considered as a control point in these diseases, through which inflammatory mediators, including cytokines, act. The ability of CRIg to suppress cytotoxic T cell proliferation and function may underlie its promotion of cancer growth. Thus, the unique properties of this receptor open up new avenues for understanding of the pathways that regulate inflammation during infection, autoimmunity and cancer with the potential for new drug targets to be identified. While some complement receptors may be differently expressed in mice and humans, as well as displaying different properties, mouse CRIg has a structure and function similar to the human receptor, suggesting that extrapolation to human diseases is appropriate. Furthermore, there is emerging evidence in human conditions that CRIg may be a valuable biomarker in infection and immunity, inflammatory conditions and cancer prognosis.

Control of Methicillin-Resistant Staphylococcus aureus Pneumonia Utilizing TLR2 Agonist Pam3CSK4.

The spread of methicillin-resistant Staphylococcus aureus (MRSA) is a critical health issue that has drawn greater attention to the potential use of immunotherapy. Toll-like receptor 2 (TLR2), a pattern recognition receptor, is an essential component in host innate defense system against S. aureus infection. However, little is known about the innate immune response, specifically TLR2 activation, against MRSA infection. Here, we evaluate the protective effect and the mechanism of MRSA murine pneumonia after pretreatment with Pam3CSK4, a TLR2 agonist. We found that the MRSA-pneumonia mouse model, pretreated with Pam3CSK4, had reduced bacteria and mortality in comparison to control mice. As well, lower protein and mRNA levels of TNF-α, IL-1ß and IL-6 were observed in lungs and bronchus of the Pam3CSK4 pretreatment group. Conversely, expression of anti-inflammatory cytokine IL-10, but not TGF-ß, increased in Pam3CSK4-pretreated mice. Our additional studies showed that CXCL-2 and CXCL1, which are necessary for neutrophil recruitment, were less evident in the Pam3CSK4-pretreated group compared to control group, whereas the expression of Fcγ receptors (FcγⅠ/Ⅲ) and complement receptors (CR1/3) increased in murine lungs. Furthermore, we found that increased survival and improved bacterial clearance were not a result of higher levels of neutrophil infiltration, but rather a result of enhanced phagocytosis and bactericidal activity of neutrophils in vitro and in vivo as well as increased robust oxidative activity and release of lactoferrin. Our cumulative findings suggest that Pam3CSK4 could be a novel immunotherapeutic candidate against MRSA pneumonia.

Further studies of the down-regulation by Factor I of the C3b feedback cycle using endotoxin as a soluble activator and red cells as a source of CR1 on sera of different complotype.

In this paper we have extended our earlier studies of the action of increasing Factor I concentration on complement activation by using a soluble activator, lipopolysaccharide (LPS) endotoxin, and using human erythrocytes as a source of CR1 - the co-factor needed for the final clip of iC3b to C3dg by Factor I. Using this more physiological system, the results show that we can predict that a quite modest increase in Factor I concentration - 22 µg/ml of extra Factor I - will convert the activity of the highest risk sera to those of the lowest risk. Preliminary experiments have been performed with erythrocytes allotyped for CR1 number. While we have not been able to perform an adequate study of their co-factor activities in our assays, preliminary experiments suggest that when Factor I levels are increased the difference produced by different allotypes of red cells is largely overcome. This suggests that in patients with paroxysmal nocturnal haemoglobinuria (PNH) treated with eculizumab, additional treatment with Factor I may be very useful in reducing the need for blood transfusion. We have also explored the age-related allele frequency for the two polymorphisms of Factor H and the polymorphism of C3. In our population, unlike the 1975 study, we found no age variation in the allele frequency in these polymorphisms. This may, however, reflect that the Cambridge BioResource volunteers do not include many very young or very elderly patients, and in general comprise a population not greatly at risk of death from infectious disease.

The immune adherence receptor CR1-like existed on porcine erythrocytes membrane.

In the present study, we obtain a mouse anti-porcine complement receptor type 1 (CR1)-like monoclonal antibody (McAb) and use this McAb to verify the existence of CR1-like protein on porcine erythrocytes. Our results confirm that CR1-like protein is localized on the surface of porcine erythrocytes. Mouse immunoglobulin G inhibited the binding of serum-opsonized green fluorescent protein-expressing Escherichia coli to porcine erythrocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicates that CR1-like McAb reacts with biochemically-purified porcine erythrocyte membrane fractions, with a clear band at 135 kDa to 140 kDa. We postulate that the 135 kDa to 140 kDa membrane protein is the equivalent of the porcine erythrocyte CR1-like protein.

TNF signals are dispensable for the generation of CD23+ CD21/35-high CD1d-high B cells in inflamed lymph nodes.

Tumor necrosis factor (TNF) is a key cytokine in rheumatoid arthritis (RA) pathogenesis, as underscored by the clinical effectiveness of TNF antagonists. While several of TNF's key targets in RA are well understood, its many pleiotropic effects remain to be elucidated. TNF-transgenic mice develop inflammatory-erosive arthritis associated with disruption of draining lymph node histology and function, and accumulation of B cells with unique phenotypic and functional features consistent with contribution to pathogenesis (B cells in inflamed nodes, Bin). Bin cell induction depends on the inflamed microenvironment, but the specific signals are unknown. Using anti-TNF treatment and TNF-receptor-deficient mice, here we show that Bin cells are induced and maintained independently of B cell-intrinsic TNF signals.

Analysis of Erythrocyte Invasion Mechanisms of Plasmodium falciparum Clinical Isolates Across 3 Malaria-Endemic Areas in Ghana.

BACKGROUND: Plasmodium falciparum invades human erythrocytes by using an array of ligands that interact with several receptors, including sialic acid (SA), complement receptor 1 (CR1), and basigin. We hypothesized that in malaria-endemic areas, parasites vary invasion pathways under immune pressure. Therefore, invasion mechanisms of clinical isolates collected from 3 zones of Ghana with different levels of endemicity (from lowest to highest, Accra, Navrongo, and Kintampo) were compared using standardized methods. METHODS: Blood samples were collected from children aged 2-14 years in whom malaria was diagnosed, and erythrocyte invasion phenotypes were determined using the enzymes neuraminidase, chymotrypsin, and trypsin, which differentially cleave receptors from the erythrocyte surface. In addition, antibodies against CR1 and basigin were used to determine the contributions of these receptors to invasion. Gene expression levels of P. falciparum invasion ligands were also examined. RESULTS: The parasites generally expressed SA-independent invasion phenotypes across the malaria-endemic areas, with parasites from Kintampo showing the highest invasion rates in neuraminidase-treated erythrocytes. CR1 was a major mediator of SA-independent invasion, while basigin was essential for both SA-dependent and SA-independent invasion mechanisms. Furthermore, expression of the basigin ligand PfRh5 was the best predictor of donor parasitemia. CONCLUSIONS: Erythrocyte invasion phenotypes expressed by P. falciparum are influenced by endemicity levels, and the PfRh5-basigin pathway is a potential vaccine target.