RESUMEN
BACKGROUND: The development of vaccine candidates for COVID-19, and the administration of booster vaccines, has meant a significant reduction in COVID-19 related deaths world-wide and the easing of global restrictions. However, new variants of SARS-CoV-2 have emerged with less susceptibility to vaccine induced immunity leading to breakthrough infections among vaccinated people. It is generally acknowledged that immunoglobulins play the major role in immune-protection, primarily through binding to the SARS-COV-2 receptor binding domain (RBD) and thereby inhibiting viral binding to the ACE2 receptor. However, there are limited investigations of anti-RBD isotypes (IgM, IgG, IgA) and IgG subclasses (IgG1-4) over the course of vaccination and breakthrough infection. METHOD: In this study, SARS-CoV-2 humoral immunity is examined in a single subject with unique longitudinal sampling. Over a two year period, the subject received three doses of vaccine, had two active breakthrough infections and 22 blood samples collected. Serological testing included anti-nucleocapsid total antibodies, anti-RBD total antibodies, IgG, IgA, IgM and IgG subclasses, neutralization and ACE2 inhibition against the wildtype (WT), Delta and Omicron variants. RESULTS: Vaccination and breakthrough infections induced IgG, specifically IgG1 and IgG4 as well as IgM and IgA. IgG1 and IgG4 responses were cross reactive and associated with broad inhibition. CONCLUSION: The findings here provide novel insights into humoral immune response characteristics associated with SARS-CoV-2 breakthrough infections.
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COVID-19 , SARS-CoV-2 , Humanos , Inmunidad Humoral , Enzima Convertidora de Angiotensina 2 , Inmunoglobulina G , Inmunoglobulina A , Inmunoglobulina MRESUMEN
BACKGROUND: Psoriatic Arthritis (PsA) is an immune-mediated disease with heterogenous symptoms indicating differences in the underlying immunopathogenesis. The primary objective of the study explored the dynamic mechanisms and interplay between immune cell subtypes constituting the immune response driving PsA to evaluate possible differences in immune cellular phenotypes, and secondary examined associations between emerging immune cellular phenotypes and disease outcomes. METHODS: Peripheral blood was collected from 70 PsA patients. Frequencies of nine immune cell subtypes were determined by multicolor flow cytometry. The interplay between immune cells were examined with principal component analysis (PCA) to establish immune cellular phenotypes. Disease characteristics, Disease Activity in Psoriatic Arthritis (DAPSA) and Psoriasis Area Severity Index (PASI) were retrieved to examine associations to individual cellular phenotypes. RESULTS: Four components were identified using PCA resembling four immune cellular phenotypes. Component 1, explaining 25.6% of the variance with contribution from T-helper 17 cells (Th17), memory T regulatory cells (mTregs), dendritic cells and monocytes, was associated with longer disease duration and higher DAPSA. Component 2, driven by Th1, naïve Tregs and mTregs, was associated with shorter disease duration. Component 3 was driven by both Th1, Th17 and CD8+ T cells, while component 4 was characterized by a reverse correlation between CD8+ T cells and natural killer cells. CONCLUSION: Four immune cellular phenotypes of PsA were suggested at baseline demonstrating complex immune cellular mechanisms in PsA implying the possibility of improving PsA patient stratification based on both clinical and immune cellular phenotypes.
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Artritis Psoriásica , Psoriasis , Humanos , Inmunofenotipificación , Monocitos , FenotipoRESUMEN
BACKGROUND: Health-care workers are thought to be highly exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We aimed to investigate the prevalence of antibodies against SARS-CoV-2 in health-care workers and the proportion of seroconverted health-care workers with previous symptoms of COVID-19. METHODS: In this observational cohort study, screening was offered to health-care workers in the Capital Region of Denmark, including medical, nursing, and other students who were associated with hospitals in the region. Screening included point-of-care tests for IgM and IgG antibodies against SARS-CoV-2. Test results and participant characteristics were recorded. Results were compared with findings in blood donors in the Capital Region in the study period. FINDINGS: Between April 15 and April 23, 2020, we screened 29â295 health-care workers, of whom 28â792 (98·28%) provided their test results. We identified 1163 (4·04% [95% CI 3·82-4·27]) seropositive health-care workers. Seroprevalence was higher in health-care workers than in blood donors (142 [3·04%] of 4672; risk ratio [RR] 1·33 [95% CI 1·12-1·58]; p<0·001). Seroprevalence was higher in male health-care workers (331 [5·45%] of 6077) than in female health-care workers (832 [3·66%] of 22â715; RR 1·49 [1·31-1·68]; p<0·001). Frontline health-care workers working in hospitals had a significantly higher seroprevalence (779 [4·55%] of 16â356) than health-care workers in other settings (384 [3·29%] of 11â657; RR 1·38 [1·22-1·56]; p<0·001). Health-care workers working on dedicated COVID-19 wards (95 [7·19%] of 1321) had a significantly higher seroprevalence than other frontline health-care workers working in hospitals (696 [4·35%] of 15â983; RR 1·65 [1·34-2·03]; p<0·001). 622 [53·5%] of 1163 seropositive participants reported symptoms attributable to SARS-CoV-2. Loss of taste or smell was the symptom that was most strongly associated with seropositivity (377 [32·39%] of 1164 participants with this symptom were seropositive vs 786 [2·84%] of 27â628 without this symptom; RR 11·38 [10·22-12·68]). The study is registered at ClinicalTrials.gov, NCT04346186. INTERPRETATION: The prevalence of health-care workers with antibodies against SARS-CoV-2 was low but higher than in blood donors. The risk of SARS-CoV-2 infection in health-care workers was related to exposure to infected patients. More than half of seropositive health-care workers reported symptoms attributable to COVID-19. FUNDING: Lundbeck Foundation.
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COVID-19/epidemiología , Personal de Salud/estadística & datos numéricos , Salud Laboral/estadística & datos numéricos , Adulto , Anticuerpos Antivirales/sangre , Donantes de Sangre/estadística & datos numéricos , COVID-19/diagnóstico , COVID-19/inmunología , COVID-19/patología , Estudios de Cohortes , Dinamarca/epidemiología , Femenino , Personal de Salud/clasificación , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Pruebas en el Punto de Atención , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Seroconversión , Estudios SeroepidemiológicosRESUMEN
BACKGROUND: Malaria in pregnancy has major impacts on mother and child health. To complement existing interventions, such as intermittent preventive treatment and use of impregnated bed nets, we developed a malaria vaccine candidate with the aim of reducing sequestration of asexual "blood-stage" parasites in the placenta, the major virulence mechanism. METHODS: The vaccine candidate PAMVAC is based on a recombinant fragment of VAR2CSA, the Plasmodium falciparum protein responsible for binding to the placenta via chondroitin sulfate A (CSA). Healthy, adult malaria-naive volunteers were immunized with 3 intramuscular injections of 20 µg (n = 9) or 50 µg (n = 27) PAMVAC, adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) or in a liposomal formulation with QS21 (GLA-LSQ). Allocation was random and double blind. The vaccine was given every 4 weeks. Volunteers were observed for 6 months following last immunization. RESULTS: All PAMVAC formulations were safe and well tolerated. A total of 262 adverse events (AEs) occurred, 94 (10 grade 2 and 2 grade 3) at least possibly related to the vaccine. No serious AEs occurred. Distribution and severity of AEs were similar in all arms. PAMVAC was immunogenic in all participants. PAMVAC-specific antibody levels were highest with PAMVAC-GLA-SE. The antibodies inhibited binding of VAR2CSA expressing P. falciparum-infected erythrocytes to CSA in a standardized functional assay. CONCLUSIONS: PAMVAC formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area. CLINICAL TRIALS REGISTRATION: EudraCT 2015-001827-21; ClinicalTrials.gov NCT02647489.
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Vacunas contra la Malaria/uso terapéutico , Adulto , Hidróxido de Aluminio/química , Sulfatos de Condroitina/metabolismo , Método Doble Ciego , Femenino , Humanos , Inyecciones Intramusculares , Liposomas/química , Vacunas contra la Malaria/administración & dosificación , Plasmodium falciparum/inmunología , Plasmodium falciparum/patogenicidad , Embarazo , Adulto JovenRESUMEN
Isolation of metastatic circulating tumor cells (CTCs) from cancer patients is of high value for disease monitoring and molecular characterization. Despite the development of many new CTC isolation platforms in the last decade, their isolation and detection has remained a challenge due to the lack of specific and sensitive markers. In this feasibility study, we present a method for CTC isolation based on the specific binding of the malaria rVAR2 protein to oncofetal chondroitin sulfate (ofCS). We show that rVAR2 efficiently captures CTCs from hepatic, lung, pancreatic, and prostate carcinoma patients with minimal contamination of peripheral blood mononuclear cells. Expression of ofCS is present on epithelial and mesenchymal cancer cells and is equally preserved during epithelial-mesenchymal transition of cancer cells. In 25 stage I-IV prostate cancer patient samples, CTC enumeration significantly correlates with disease stage. Lastly, rVAR2 targets a larger and more diverse population of CTCs compared to anti-EpCAM strategies.
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Antígenos de Protozoos/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Células Neoplásicas Circulantes/metabolismo , Adaptación Fisiológica , Línea Celular Tumoral , Separación Celular , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Humanos , Leucocitos Mononucleares/metabolismo , Magnetismo , Masculino , Mesodermo/metabolismo , Microesferas , Estadificación de Neoplasias , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Recombinantes/metabolismoRESUMEN
Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP) based vaccines (e.g., the licensed human papillomavirus vaccines) have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTagTM) can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA)-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1st and 2nd immunization; however, this difference was not statistically significant after 3rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of parasites to CSA. This study demonstrates that the described Avi-L1 VLP-platform may serve as a versatile system for facilitating optimal VLP-display of large and complex vaccine antigens.
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Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Placenta/parasitología , Virión/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Biotinilación , Western Blotting , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Malaria Falciparum/sangre , Malaria Falciparum/inmunología , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Parásitos/inmunología , Embarazo , Reproducibilidad de los Resultados , Ultracentrifugación , VacunaciónRESUMEN
The disease caused by Plasmodium falciparum (Pf) involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM) is one such manifestation in which Pf infected erythrocytes (IE) bind to chondroitin sulphate A (CSA) through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2) expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a broadly strain-transcending antibody response.
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Formación de Anticuerpos/inmunología , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/prevención & control , Placenta/parasitología , Animales , Reacciones Cruzadas/inmunología , Drosophila melanogaster/metabolismo , Femenino , Humanos , Malaria Falciparum/inmunología , Placenta/inmunología , Plasmodium falciparum/inmunología , Embarazo , Ingeniería de Proteínas/métodos , Proteínas RecombinantesRESUMEN
Placental malaria is a major health problem for both pregnant women and their fetuses in malaria endemic regions. It is triggered by the accumulation of Plasmodium falciparum-infected erythrocytes (IE) in the intervillous spaces of the placenta and is associated with foetal growth restriction and maternal anemia. IE accumulation is supported by the binding of the parasite-expressed protein VAR2CSA to placental chondroitin sulfate A (CSA). Defining specific CSA-binding epitopes of VAR2CSA, against which to target the immune response, is essential for the development of a vaccine aimed at blocking IE adhesion. However, the development of a VAR2CSA adhesion-blocking vaccine remains challenging due to (i) the large size of VAR2CSA and (ii) the extensive immune selection for polymorphisms and thereby non-neutralizing B-cell epitopes. Camelid heavy-chain-only antibodies (HcAbs) are known to target epitopes that are less immunogenic to classical IgG and, due to their small size and protruding antigen-binding loop, able to reach and recognize cryptic, conformational epitopes which are inaccessible to conventional antibodies. The variable heavy chain (VHH) domain is the antigen-binding site of camelid HcAbs, the so called Nanobody, which represents the smallest known (15 kDa) intact, native antigen-binding fragment. In this study, we have used the Nanobody technology, an approach new to malaria research, to generate small and functional antibody fragments recognizing unique epitopes broadly distributed on VAR2CSA.
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Antígenos de Protozoos/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Plasmodium falciparum/inmunología , Anticuerpos de Dominio Único/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Camélidos del Nuevo Mundo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Epítopos/química , Epítopos/inmunología , Eritrocitos/parasitología , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/química , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Datos de Secuencia Molecular , Biblioteca de Péptidos , Plasmodium falciparum/química , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/químicaRESUMEN
Malaria during pregnancy in Plasmodium falciparum endemic regions is a major cause of mortality and severe morbidity. VAR2CSA is the parasite ligand responsible for sequestration of Plasmodium falciparum infected erythrocytes to the receptor chondroitin sulfate A (CSA) in the placenta and is the leading candidate for a placental malaria vaccine. Antibodies induced in rats against the recombinant DBL4ε domain of VAR2CSA inhibit the binding of a number of laboratory and field parasite isolates to CSA. In this study, we used a DBL4ε peptide-array to identify epitopes targeted by DBL4ε-specific antibodies that inhibit CSA-binding of infected erythrocytes. We identified three regions of overlapping peptides which were highly antigenic. One peptide region distinguished itself particularly by showing a clear difference in the binding profile of highly parasite blocking IgG compared to the IgG with low capacity to inhibit parasite adhesion to CSA. This region was further characterized and together these results suggest that even though antibodies against the synthetic peptides which cover this region did not recognize native protein, the results using the mutant domain suggest that this linear epitope might be involved in the induction of inhibitory antibodies induced by the recombinant DBL4ε domain.
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Antígenos de Protozoos/química , Epítopos de Linfocito B/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/química , Anticuerpos Antiprotozoarios/inmunología , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos de Protozoos/inmunología , Adhesión Celular , Mapeo Epitopo , Epítopos de Linfocito B/química , Eritrocitos/parasitología , Femenino , Humanos , Sueros Inmunes/inmunología , Modelos Lineales , Modelos Moleculares , Datos de Secuencia Molecular , Análisis Multivariante , Proteínas Mutantes/química , Proteínas Mutantes/inmunología , Parásitos/inmunología , Péptidos/química , Péptidos/inmunología , Plasmodium falciparum/citología , Plasmodium falciparum/inmunología , Embarazo , Estructura Terciaria de Proteína , Ratas , Alineación de SecuenciaRESUMEN
Malaria is a major global health problem. Pregnant women are susceptible to infection regardless of previously acquired immunity. Placental malaria is caused by parasites capable of sequestering in the placenta. This is mediated by VAR2CSA, a parasite antigen that interacts with chondroitin sulfate A (CSA). One vaccine strategy is to block this interaction with VAR2CSA-specific antibodies. It is a priority to define a small VAR2CSA fragment that can be used in an adhesion blocking vaccine. In this, the obvious approach is to define regions of VAR2CSA involved in receptor binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with nanomolar affinity, and that the CSA-binding site lies in the N-terminal part of the protein. In this study we define the minimal binding region by truncating VAR2CSA and analyzing CSA binding using biosensor technology. We show that the core CSA-binding site lies within the DBL2X domain and parts of the flanking interdomain regions. This is in contrast to the idea that single domains do not possess the structural requirements for specific CSA binding. Small-angle x-ray scattering measurements enabled modeling of VAR2CSA and showed that the CSA-binding DBL2X domain is situated in the center of the structure. Mutating classic sulfate-binding sites in VAR2CSA, along with testing dependence of ionic interactions, suggest that the CSA binding is not solely dependent on the sulfated CSA structure. Based on these novel PfEMP1 structure-function studies, we have constructed a small VAR2CSA antigen that has the capacity to induce highly adhesion-blocking antibodies.
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Antígenos de Protozoos/inmunología , Sulfatos de Condroitina/inmunología , Malaria Falciparum/inmunología , Placenta/inmunología , Plasmodium falciparum/inmunología , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Sitios de Unión/genética , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Femenino , Interacciones Huésped-Parásitos , Humanos , Sueros Inmunes/inmunología , Sueros Inmunes/metabolismo , Inmunización , Cinética , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Modelos Moleculares , Mutación , Placenta/metabolismo , Placenta/parasitología , Plasmodium falciparum/fisiología , Embarazo , Complicaciones Parasitarias del Embarazo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
BACKGROUND: In Plasmodium falciparum malaria endemic areas placental malaria (PM) is an important complication of malaria. The recurrence of malaria in primigravidae women irrespective of acquired protection during childhood is caused by the interaction between the parasite-expressed VAR2CSA antigen and chondroitin sulfate A (CSA) in the placental intervillous space and lack of protective antibodies. PM impairs fetal development mainly by excessive inflammation processes. After infections during pregnancy women acquire immunity to PM conferred by antibodies against VAR2CSA. Ideally, a vaccine against PM will induce antibody-mediated immune responses that block the adhesion of infected erythrocytes (IE) in the placenta. PRINCIPAL FINDINGS: We have previously shown that antibodies raised in rat against individual domains of VAR2CSA can block IE binding to CSA. In this study we have immunized mice, rats and rabbits with each individual domain and the full-length protein corresponding to the FCR3 VAR2CSA variant. We found there is an inherently higher immunogenicity of C-terminal domains compared to N-terminally located domains. This was irrespective of whether antibodies were induced against single domains or the full-length protein. Species-specific antibody responses were also found, these were mainly directed against single domains and not the full-length VAR2CSA protein. CONCLUSIONS/SIGNIFICANCE: Binding inhibitory antibodies appeared to be against conformational B-cell epitopes. Non-binding inhibitory antibodies reacted highly against the C-terminal end of the VAR2CSA molecule especially the highly polymorphic DBL6ε domain. Differential species-specific induction of antibody responses may allow for more direct analysis of functional versus non-functional B-cell epitopes.
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Antígenos de Protozoos/inmunología , Inmunoglobulina G/inmunología , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Placenta/inmunología , Placenta/parasitología , Plasmodium falciparum/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Formación de Anticuerpos/inmunología , Antígenos de Protozoos/química , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Eritrocitos/parasitología , Femenino , Humanos , Malaria Falciparum/parasitología , Ratones , Embarazo , Análisis por Matrices de Proteínas , Estructura Terciaria de Proteína , Conejos , Ratas , Proteínas Recombinantes/inmunología , Especificidad de la EspecieRESUMEN
Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDR(PAM) and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments.
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Sulfatos de Condroitina/química , Mapeo Peptídico , Plasmodium falciparum/química , Animales , Antígenos de Protozoos , Técnicas Biosensibles/métodos , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/inmunología , Sulfatos de Condroitina/metabolismo , Eritrocitos/inmunología , Eritrocitos/metabolismo , Eritrocitos/parasitología , Femenino , Humanos , Vacunas contra la Malaria/química , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/metabolismo , Malaria Falciparum/epidemiología , Malaria Falciparum/genética , Malaria Falciparum/inmunología , Malaria Falciparum/metabolismo , Malaria Falciparum/prevención & control , Placenta/inmunología , Placenta/metabolismo , Placenta/parasitología , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Plasmodium falciparum/metabolismo , Embarazo , Complicaciones Parasitarias del Embarazo/epidemiología , Complicaciones Parasitarias del Embarazo/genética , Complicaciones Parasitarias del Embarazo/inmunología , Complicaciones Parasitarias del Embarazo/metabolismo , Complicaciones Parasitarias del Embarazo/prevención & control , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
GMZ2 adjuvanted by aluminum hydroxide is a candidate malaria vaccine that has successfully passed phase 1 clinical testing in adult German and Gabonese volunteers and Gabonese children under five. Here we report a preclinical study screening a series of adjuvant vehicles and Toll-like receptor (TLR) agonists in CB6F1 mice to identify an improved formulation of GMZ2 suitable for further human clinical studies. GMZ2 formulated in an oil-in-water emulsion plus the synthetic TLR4 agonist GLA elicits the highest (a) vaccine-specific IgG2a and total IgG titers, (b) parasite-specific IFA titers, (c) levels of Type 1 cytokine responses (IFN-γ), and (d) number of long-lived-plasma cells (LLPC) secreting antibodies against both the GMZ2 fusion and its two components. Thus, GLA helps to elicit a vaccine-specific Type 1 antibody profile together with high levels of LLPC, both of which are thought to be essential for the development of long-term protective immunity against clinical malaria.
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Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Antiprotozoarios/sangre , Lípido A/administración & dosificación , Vacunas contra la Malaria/inmunología , Células TH1/inmunología , Receptor Toll-Like 4/agonistas , Animales , Femenino , Inmunoglobulina G/sangre , Vacunas contra la Malaria/administración & dosificación , Ratones , Ratones Endogámicos C57BLRESUMEN
Malaria during pregnancy is a major cause of intra-uterine growth-retardation and infant death in sub-Saharan Africa. Ideally, this could be prevented by a vaccine delivered before the first pregnancy. Antibodies against domain DBL4É from VAR2CSA has been shown to inhibit adhesion of laboratory isolates to the placental receptor chondroitin sulfate A. In this study, the binding inhibitory efficacy of IgG elicited by two different DBL4É recombinant proteins was tested on a panel of fresh clinical isolates from pregnant women living in Benin and Tanzania. The most promising recombinant protein elicited antibodies with similar efficacy as pooled plasma from immune multi-gravid African women.
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Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Adhesión Celular , Enzimas Reparadoras del ADN/metabolismo , Eritrocitos/parasitología , Malaria Falciparum/inmunología , Complicaciones Infecciosas del Embarazo/inmunología , Factores de Transcripción/metabolismo , Benin , Femenino , Humanos , Inmunoglobulina G/inmunología , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Embarazo , Proteínas Recombinantes/inmunología , TanzaníaRESUMEN
IgM is an ancestral Ab class found in all jawed vertebrates, from sharks to mammals. This ancient ancestry is shared by malaria parasites (genus Plasmodium) that infect all classes of terrestrial vertebrates with whom they coevolved. IgM, the least studied and most enigmatic of the vertebrate Igs, was recently shown to form an intimate relationship with the malaria parasite Plasmodium falciparum. In this article, we discuss how this association might have come about, building on the recently determined structure of the human IgM pentamer, and how this interaction could affect parasite survival, particularly in light of the just-discovered Fc mu R localized to B and T cell surfaces. Because this parasite may exploit an interaction with IgM to limit immune detection, as well as to manipulate the immune response when detected, a better understanding of this association may prove critical for the development of improved vaccines or vaccination strategies.
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Interacciones Huésped-Parásitos/inmunología , Evasión Inmune/inmunología , Inmunoglobulina M/metabolismo , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Plasmodium falciparum/inmunología , Receptores Fc/metabolismo , Animales , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Femenino , Variación Genética/inmunología , Interacciones Huésped-Parásitos/genética , Humanos , Evasión Inmune/genética , Inmunoglobulina M/sangre , Inmunoglobulina M/deficiencia , Malaria Falciparum/prevención & control , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Unión Proteica/genética , Unión Proteica/inmunología , Receptores Fc/sangre , Receptores Fc/fisiología , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
BACKGROUND: Malaria caused by Plasmodium falciparum can result in several different syndromes with severe clinical consequences for the about 200 million individuals infected each year. During pregnancy, women living in endemic areas become susceptible to malaria due to lack of antibodies against a unique P. falciparum membrane protein, named VAR2CSA. This antigen is not expressed in childhood infections, since it binds chondroitin sulphate A (CSA) expressed on the intervillous space in the placenta. A vaccine appears possible because women acquire protective antibodies hindering sequestration in the placenta as a function of parity. A challenge for vaccine development is to design small constructs of this large antigen, which can induce broadly protective antibodies. It has previously been shown that one domain of VAR2CSA, DBL4-FCR3, induces parasite adhesion-blocking antibodies. In this study, it is demonstrated that other domains of VAR2CSA also can induce antibodies with inhibitory activity. METHODS: All VAR2CSA domains from the 3D7 and HB3 parasites were produced in Baculovirus-transfected insect cells. Groups of three rats per protein were immunized and anti-sera were tested for surface reactivity against infected erythrocytes expressing FCR3 VAR2CSA and for the ability to inhibit FCR3CSA parasite adhesion to CSA. The fine specificity of the immune sera was analysed by VAR2CSA peptide arrays. RESULTS: Inhibitory antibodies were induced by immunization with DBL3-HB3 T1 and DBL1-3D7. However, unlike the previously characterised DBL4-FCR3 response the inhibitory response against DBL1-3D7 and DBL3-HB3 T1 was poorly reproduced in the second rounds of immunizations. CONCLUSION: It is possible to induce parasite adhesion-blocking antibodies when immunizing with a number of different VAR2CSA domains. This indicates that the CSA binding site in VAR2CSA is comprised of epitopes from different domains.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Adhesión Celular/inmunología , Vacunas contra la Malaria/inmunología , Animales , Antígenos de Protozoos/genética , Baculoviridae/genética , Femenino , Vectores Genéticos , Humanos , Insectos , Vacunas contra la Malaria/genética , Embarazo , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Plasmodium falciparum malaria remains one of the world's leading causes of human suffering and poverty. Each year, the disease takes 1-3 million lives, mainly in sub-Saharan Africa. The adhesion of infected erythrocytes (IEs) to vascular endothelium or placenta is the key event in the pathogenesis of severe P. falciparum infection. In pregnant women, the parasites express a single and unique member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family named VAR2CSA, which is associated with the ability of the IEs to adhere specifically to chondroitin sulphate A (CSA) in the placenta. Several Duffy-binding-like domains from VAR2CSA molecules have been shown in vitro to bind to CSA, but it has also been demonstrated that Duffy-binding-like domains from PfEMP1 proteins other than VAR2CSA can bind CSA. In addition, the specificity of the binding of VAR2CSA domains to glycosaminoglycans does not match that of VAR2CSA-expressing IEs. This has led to speculation that the domains of native VAR2CSA need to come together to form a specific binding site or that VAR2CSA might bind to CSA through a bridging molecule. Here, we describe the expression and purification of the complete extracellular region of VAR2CSA secreted at high yields from insect cells. Using surface plasmon resonance, we demonstrate that VAR2CSA alone binds with nanomolar affinity to human chondroitin sulphate proteoglycan and with significantly weaker affinity to other glycosaminoglycans, showing a specificity similar to that observed for IEs. Antibodies raised against full-length VAR2CSA completely inhibit recombinant VAR2CSA binding, as well as parasite binding to chondroitin sulphate proteoglycan. This is the first study to describe the successful production and functionality of a full-length PfEMP1. The specificity of the binding and anti-adhesion potency of induced IgG, together with high-yield production, encourages the use of full-length PfEMP1 in vaccine development strategies.
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Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Antiprotozoarios/metabolismo , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Sulfatos de Condroitina/metabolismo , Eritrocitos/metabolismo , Animales , Antígenos de Protozoos/genética , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/parasitología , Citometría de Flujo , Glicosaminoglicanos/metabolismo , Malaria Falciparum/genética , Malaria Falciparum/inmunología , Malaria Falciparum/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Individuals living in areas with high Plasmodium falciparum transmission acquire immunity to malaria over time and adults have a markedly reduced risk of contracting severe disease. However, pregnant women constitute an important exception. Pregnancy-associated malaria is a major cause of mother and offspring morbidity, such as severe maternal anaemia and low birth-weight, and is characterised by selective accumulation of parasite-infected erythrocytes (IE) in the placenta. A P. falciparum protein named VAR2CSA, which belongs to the large P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family, enables the IE to bind chondroitin sulphate A (CSA) in the placenta. Knock-out studies have demonstrated the exclusive capacity of VAR2CSA to mediate IE binding to CSA, and it has been shown that four of the six Duffy-binding-like (DBL) domains of VAR2CSA have the ability to bind CSA in vitro. In this study, we confirm the CSA-binding of these DBL domains, however, the analysis of a number of DBL domains of a non-VAR2CSA origin shows that CSA-binding is not exclusively restricted to VAR2CSA DBL domains. Furthermore, we show that the VAR2CSA DBL domains as well as other DBL domains also bind heparan sulphate. These data explain a number of publications describing CSA-binding domains derived from PfEMP1 antigens not involved in placental adhesion. The data suggest that the ability of single domains to bind CSA does not predict the functional capacity of the whole PfEMP1 and raises doubt whether the CSA-binding domains of native VAR2CSA have been correctly identified.