Mary Lopez-Perez

Background: Antibody Fc regions have important roles in clearance of Plasmodium falciparum-infected erythrocytes. One such role is engagement with Fcγ receptors on host leukocytes. In the case of FcγRIIIa and b, this interaction is greatly enhanced when the IgG glycan is afucosylated. Methods: In this study, we used a fucose-sensitive enzyme-linked immunosorbent assay (FEASI) to assess afucosylation in IgG specific for placental malaria protein VAR2CSA from n = 139 malaria-exposed pregnant Malawian women, correlating these data with mass spectrometry-based analysis. Furthermore, we measured the effect of afucosylation on Fc-mediated leukocyte functions, using both plasma and a monoclonal antibody, PAM2.8, with varying levels of afucosylation. Results: There were significantly higher levels of VAR2CSA-specific IgG afucosylation in women with no placental malaria measured by FEASI (P < .0001), which correlated strongly with mass spectrometry analysis (R = 0.8, P < .0001). In addition, highly afucosylated IgG mediated significantly greater neutrophil phagocytosis of antigen-coated beads and induction of NK cell degranulation by infected erythrocytes. Conclusions: Afucosylated IgG to VAR2CSA, measured by FEASI or mass spectrometry, was a correlate of protection from placental malaria, and afucosylated IgG activated NK cells and neutrophils. Naturally acquired or therapeutic afucosylated IgG antibody could have a role in protection from malaria infection.

Soft X-ray tomography (SXT) is an emerging modality for whole-cell 3D imaging in near-native states. However, the effective spatial resolution is limited by optical artifacts characterized by the point spread function (PSF). To achieve optimal resolution via PSF inversion, we propose a measurement-supervised deep learning framework. Bypassing purely data-driven neural networks that are prone to hallucinations, we employ a measurement-supervised, instance-specific optimization strategy strictly constrained by a differentiable SXT formation forward model. The structural fidelity was validated using split-tilt Fourier ring correlation (FRC), ensuring the recovered high-frequency features reflect genuine specimen features rather than random artifacts. Our results demonstrate that this optimization consistently increases FRC resolution and enhances visual ultrastructural details across diverse biological structures. Furthermore, by recovering high-frequency features from sparse-angular projections, we show that spatial resolution can be maintained using only half the radiation exposure. This approach effectively compensates for the degradations caused by angular sparsity, providing a hardware-free computational solution to minimize radiation damage, maximize imaging speed, and overcome the optical and dosimetric limits of SXT.

Background: IgG-mediated effector functions are modulated by the antibody Fc-region glycan composition, and the absence of core fucose is a strong modifier of NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Most plasma IgG is fully Fc-fucosylated, but antigens displayed on host-derived membranes can selectively induce Fc-afucosylated IgG. These antigens include alloantigens, enveloped virus surface proteins, and PfEMP1, a malaria parasite protein expressed on the surface of Plasmodium falciparum-infected erythrocytes (IEs). Methods: We determined levels and Fc-fucosylation of IgG specific for several P. falciparum antigens in plasma from children naturally exposed to P. falciparum and from vaccinated adults. Results: IgG specific for PfEMP1 (known to be exposed on the IE surface) and PfGARP, a putative IE surface antigen, were markedly Fc-afucosylated, whereas IgG specific for antigens on parasite membranes (merozoites, gametocytes/gametes, and sporozoites) were fully Fc-fucosylated in most of the children naturally exposed to P. falciparum studied here. Immunization of adults with recombinant proteins (MSP1, Pfs230, and CSP) also consistently induced fully Fc-fucosylated IgG. Children with Fc-afucosylated PfGARP-specific IgG had reduced risk of carrying P. falciparum parasites, independent of levels of PfGARP-specific IgG. Conclusions: These results reinforce the hypothesis that Fc-afucosylation of antigen-specific IgG requires exposure of the antigen on a host membrane (context) and is a function of antigen exposure. Host-specific factors appear to play a minor role in this context.

Early diagnosis of upper respiratory infections is essential to control infectious disease transmission within the community and to initiate relevant antiviral treatments. Nonetheless, variable sensitivities of sampling sites are often overlooked. We conducted a clinical trial at a COVID-19 outpatient test center, where healthcare workers collected nasopharyngeal, throat, nasal swabs, and saliva specimens. Each specimen was tested by RT-PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A and B, and respiratory syncytial virus (RSV). Clinical information was collected at enrollment and again 13 months later using an online questionnaire. From 4 March to 31 March 2023, 253 individuals were enrolled. Data from 250 participants were included in the analysis. SARS-CoV-2 was the most frequent viral infection (48%), followed by influenza A or B (5%), and a combination of SARS-CoV-2 and influenza (9%). RSV was not detected in any specimen. Several participants carried two or three viruses simultaneously. Throat swabs were significantly more sensitive for detecting SARS- CoV-2 (79%) and influenza (64%) than samples from other sites. In contrast, saliva had the lowest sensitivity for SARS- CoV-2 (43 % ) and was unsuitable for detecting influenza. The sensitivity for SARS- CoV-2 (88 % ) and influenza (100 % ) improved when results from throat and nasal swabs were combined. Throat swabs were more sensitive than nasopharyngeal swabs and saliva for molecular detection of SARS-CoV-2 and influenza. A combined throat and nasal swab is highly recommended to increase test sensitivity in patients presenting with upper respiratory infection symptoms. IMPORTANCE: Upper respiratory infections are the most common condition in primary care. Therefore, their early diagnosis is essential to control infectious disease transmission within the community. Here, we show that throat swabs were more sensitive than nasopharyngeal swabs and saliva for detecting severe acute respiratory syndrome coronavirus 2. Furthermore, throat and nasal swabs were more effective in detecting influenza compared to nasopharyngeal swabs. CLINICAL TRIALS: Registered at ClinicalTrials.gov NCT05765838 .

Soft X-Ray tomography (SXT) is a non-invasive bio-imaging technique that enables 3D imaging of cellular structures in large volume, with a unique resolution range that bridges the gap between fluorescence and transmission electron microscopy. However, a fundamental limitation, the missing wedge artefact caused by incomplete tilt-series acquisition, introduces systematic structural elongation in the reconstructed tomograms. This artifact compromises accurate quantitative biological analysis by overestimating cellular and organelle volumes. To overcome this persistent issue, we introduce a novel, self-supervised missing wedge correction model that learns key sine-wave patterns from existing SXT sinograms of the tilt series stacks. This model can be applied to recover the missing-angle region of the sinogram, reducing distortions and elongations in the reconstructed tomograms. We demonstrate a significant quantitative improvement in artifact removal, achieving faithful recovery of the spherical morphology of lipid droplets. We further applied this method to Plasmodium falciparum hemozoin crystals, a biomarker in antimalarial drug efficacy studies. Our model successfully reduced volume overestimation, achieving up to a 16% decrease in distorted volume. This level of precision is paramount for correctly interpreting the mode of action of antimalarial drugs.

Placental malaria (PM) causes mortality and severe morbidity in areas with stable Plasmodium falciparum transmission. The selective placental accumulation of P. falciparum -infected erythrocytes (IEs) is mediated by VAR2CSA, a PfEMP1-type parasite ligand that binds exclusively to placenta-restricted CSA. VAR2CSA-specific IgG is therefore generally restricted to women exposed to P. falciparum infection during pregnancy. However, widespread acquisition of VAR2CSA-reactive IgG outside pregnancy among Colombian and Brazilian individuals has been reported, supposedly due to cross-reactivity between VAR2CSA and the P. vivax -specific antigen PvDBP. Here, we measured levels and Fc-afucosylation of VAR2CSA-reactive IgG in plasma from pregnant Brazilian women at delivery, using full-length VAR2CSA (FV2) expressed in baculovirus-transfected insect cells (FV2 _BIC ) or Chinese hamster ovary cells (FV2 _CHO ) as well as the corresponding native antigen (IT4VAR04) on the IE surface. We also measured levels of IgG specific for GLURP ( P. falciparum- specific) and PvDBP ( P. vivax -specific). FV2 _CHO -specific IgG levels were lower than FV2 _BIC -reactive IgG levels. Furthermore, only FV2 _CHO -specific IgG was restricted to women exposed to P. falciparum during pregnancy. Levels of PvDBP-specific IgG were significantly higher among P. vivax -exposed pregnant women but did not correlate with FV2-specific IgG levels. Finally, FV2 _CHO -specific IgG was markedly Fc-afucosylated in contrast to FV2 _BIC -reactive IgG. Our findings caution against using levels of IgG reacting with recombinant proteins expressed in insect cells as a measure of exposure to VAR2CSA during pregnancy, at least in South America. Furthermore, our data do not support the hypothesis that exposure to PvDBP induces IgG that cross-reacts with VAR2CSA and contributes to protection against PM.

Protective immunity to malaria depends on acquisition of parasite-specific antibodies, with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) being one of the most important target antigens. The effector functions of PfEMP1-specific IgG include inhibition of infected erythrocyte (IE) sequestration and opsonization of IEs for cell-mediated destruction. IgG glycosylation modulates antibody functionality, with increased affinity to FcγRIIIa for IgG lacking fucose in the Fc region (Fc-afucosylation). We report here that selective Fc-afucosylation of PfEMP1-specific IgG1 increases with age in P. falciparum-exposed children and is associated with reduced risk of anemia, independent of the IgG levels. A similar association was found for children having PfEMP1-specific IgG1 inducing multiple effector functions against IEs, particularly those associated with antibody-dependent cellular cytotoxicity (ADCC) by NK cells. Our findings provide new insights regarding protective immunity to P. falciparum malaria and highlight the importance of cell-mediated destruction of IgG-opsonized IEs.

Background: Acquired immunity to Plasmodium falciparum malaria is mainly mediated by immunoglobulin G (IgG) targeting erythrocyte membrane protein 1 (PfEMP1). These adhesins mediate infected erythrocyte (IE) sequestration, protecting IEs from splenic destruction. PfEMP1-specific IgG is therefore thought to protect mainly by inhibiting IE sequestration. VAR2CSA-type PfEMP1 mediates placental IE sequestration, putting pregnant women exposed to P falciparum parasites at risk of placental malaria (PM). Methods: Levels and Fc-afucosylation of VAR2CSA-specific plasma IgG were measured by a modified enzyme-linked immunosorbent assay (FEASI). We also measured the ability of the IgG to inhibit IE adhesion and to induce natural killer (NK) cell degranulation. The results were related to parity and clinical pregnancy outcomes. Results: Parity was positively correlated with levels and Fc-afucosylation of VAR2CSA-specific IgG, and with birth weight and plasma IgG inhibition of IE adhesion in vitro. Fc-afucosylation of VAR2CSA-specific IgG increased NK–cell degranulation. Women with Fc-afucosylated VAR2CSA–specific IgG had a reduced risk of delivering a low birth weight (LBW) baby, but not of PM or anemia. Conclusions: Fc-afucosylated VAR2CSA-specific IgG effectively induced NK–cell degranulation and was associated with protection against LBW, independent of IgG levels. Our study has implications for the development of VAR2CSA-based subunit vaccines, which exclusively induce Fc-fucosylated IgG.

Background: Glucose-6-Phosphate Dehydrogenase deficiency (G6PDd) is a common genetic enzymopathy that can induce haemolysis triggered by various factors, including some anti-malarial drugs. Although many Point-of-Care (PoC) tests, such as Standard G6PD™ are available to detect G6PDd, its pooled diagnostic test accuracy (DTA) remains unknown. Methods: To estimate the DTA of StandG6PD-BS at various thresholds of G6PDd, a systematic review with a DTA meta-analysis were conducted, searching EMBASE, MEDLINE, and SciELO databases up to April 4, 2024.The included studies were those that measured G6PD activity using StandG6PD-BS (reference test) and spectrophotometry (gold standard) in patients suspected of having G6PDd. The risk of bias (RoB) of the studies was assessed using the QUADAS-2 tool and the certainty of evidence (CoE) with the GRADE approach. For the estimation of within-study DTA, a random-effect bivariate meta-analysis was performed to determine the pooled sensitivity and specificity for 30%, 70%, and 80% enzyme levels’ thresholds, and a graphical analysis of the heterogeneity using crosshair and Confidence Regions on receiver operating characteristic (ROC) space plots. Results: After screening 2496 reports, four studies were included with 7864 participants covering all thresholds. Two studies had high RoB in QUADAS-2 domains 2 and 3, and the others had low RoB, with low, moderate, and high heterogeneity at the 30%, 70%, and 80% thresholds, respectively. The pooled sensitivity was 99.1%, 95.7%, and 90% for 30%, 70%, and 80% thresholds, respectively. The pooled specificity was 97.4%; 92.9%; and 89.0% for 30%, 70%, and 80% thresholds, respectively. Conclusion: StandG6PD-BS is a PoC test with high sensitivity and specificity to detect G6PDd at different thresholds.