Direct contact between Plasmodium falciparum and human B-cells in a novel co-culture increases parasite growth and affects B-cell growth.

BACKGROUND: Plasmodium falciparum parasites cause malaria and co-exist in humans together with B-cells for long periods of time. Immunity is only achieved after repeated exposure. There has been a lack of methods to mimic the in vivo co-occurrence, where cells and parasites can be grown together for many days, and it has been difficult with long time in vitro studies. METHODS AND RESULTS: A new method for growing P. falciparum in 5% CO2 with a specially formulated culture medium is described. This knowledge was used to establish the co-culture of live P. falciparum together with human B-cells in vitro for 10 days. The presence of B-cells clearly enhanced parasite growth, but less so when Transwell inserts were used (not allowing passage of cells or merozoites), showing that direct contact is advantageous. B-cells also proliferated more in presence of parasites. Symbiotic parasitic growth was verified using CESS cell-line and it showed similar results, indicating that B-cells are indeed the cells responsible for the effect. In malaria endemic areas, people often have increased levels of atypical memory B-cells in the blood, and in this assay it was demonstrated that when parasites were present there was an increase in the proportion of CD19 + CD20 + CD27 - FCRL4 + B-cells, and a contraction of classical memory B-cells. This effect was most clearly seen when direct contact between B-cells and parasites was allowed. CONCLUSIONS: These results demonstrate that P. falciparum and B-cells undoubtedly can affect each other when allowed to multiply together, which is valuable information for future vaccine studies.

Factors influencing the induction of high affinity antibodies to Plasmodium falciparum merozoite antigens and how affinity changes over time.

Understanding the functional characteristics of naturally acquired antibodies against P. falciparum merozoite antigens is crucial for determining the protective functions of antibodies. Affinity (measured as kd) of naturally acquired antibodies against two key targets of acquired immunity, EBA175 and PfRh2, was determined using Surface Plasmon Resonance (SPR) in a longitudinal survey in Nigeria. A majority of the participants, 79% and 67%, maintained stable antibody affinities to EBA175 and PfRh2, respectively, over time. In about 10% of the individuals, there was a reciprocal interaction with a reduction over time in antibody affinity for PfRh2 and an increase for EBA175. In general, PfRh2 elicited antibodies with higher affinity compared to EBA175. Individuals with higher exposure to malaria produced antibodies with higher affinity to both antigens. Younger individuals (5-15 years) produced comparable or higher affinity antibodies than adults (>15 years) against EBA175, but not for PfRh2. Correlation between total IgG (ELISA) and affinity varied between individuals, but PfRh2 elicited antibodies with a higher correlation in a majority of the participants. There was also a correlation between antibody inhibition of erythrocyte invasion by merozoites and PfRh2 affinity. This work gives new insights into the generation and maintenance of antibody affinity over time.

Development of Plasmodium falciparum specific naïve, atypical, memory and plasma B cells during infancy and in adults in an endemic area.

BACKGROUND: B-cells are essential in immunity against malaria, but which sub-sets of B-cells specifically recognize Plasmodium falciparum and when they appear is still largely unknown. RESULTS: Using the flow cytometry technique for detection of P. falciparum specific (Pf+) B-cells, this study for the first time measured the development of Pf+ B cell (CD19+) phenotypes in Ugandan babies from birth up to nine months, and in their mothers. The babies showed increases in Pf+ IgG memory B-cells (MBCs), atypical MBCs, and plasma cells/blasts over time, but the proportion of these cells were still lower than in the mothers who displayed stable levels (5, 18, and 3%, respectively). Pf+ non-IgG+ MBCs and naïve B-cells binding to P. falciparum antigens were higher in the babies compared to the mothers (12 and 50%). In ELISA there was an increase in IgG and IgM antibodies over time in babies, and stable levels in mothers. At baby delivery, multigravidae mothers had a higher proportion of Pf+ IgG MBCs and less Pf+ naïve B-cells than primigravidae mothers. CONCLUSIONS: In newborns, naïve B-cells are a major player in recognizing P. falciparum. In adults, the high proportion of Pf+ atypical MBCs suggests a major role for these cells. Both in infants and adults, non-IgG+ MBCs were higher than IgG MBCs, indicating that these cells deserve more focus in future.

Novel flow cytometry technique for detection of Plasmodium falciparum specific B-cells in humans: increased levels of specific B-cells in ongoing infection.

BACKGROUND: Malaria caused by Plasmodium falciparum is still a major health threat in endemic areas especially for children below 5 years of age. While it is recognized that antibody immunity plays an important role in controlling the disease, knowledge of the mechanisms of sustenance and natural boosting of immunity is very limited. Before, it has not been possible to investigate malaria specific B-cells directly in flow cytometry, making it difficult to know how much of a B cell response is due to malaria, or how much is due to other immunological stimulators. METHODS: This study developed a technique using quantum dots and schizont extract made from ghosts of infected erythrocytes, to be able to investigate P. falciparum specific B-cells, something that has never been done before. RESULTS: Major differences in P. falciparum specific B-cells were found between samples from immune (22.3 %) and non-immune (1.7 %) individuals. Samples from parasite positive individuals had the highest proportions of specific B-cells (27.9 %). CONCLUSION: The study showed increased levels of P. falciparum-specific B-cells in immune individuals, with the highest levels in active malaria infections, using a new technique that opens up new possibilities to study how these cells are sustained in vivo after natural infections. It will also be useful in vaccine studies.

Differences in affinity of monoclonal and naturally acquired polyclonal antibodies against Plasmodium falciparum merozoite antigens.

BACKGROUND: Malaria is a major global cause of deaths and a vaccine is urgently needed. RESULTS: We have employed the P. falciparum merozoite antigens MSP2-3D7/FC27 and AMA1, used them in ELISA, and coupled them in different ways using surface plasmon resonance (SPR) and estimated affinity (measured as kd) of monoclonal as well as naturally-acquired polyclonal antibodies in human plasma. There were major differences in kd depending on how the antigens were immobilized and where the His-tag was placed. For AMA1 we could see correlations with invasion inhibition. Using different immobilizations of proteins in SPR, we could see only moderate correlations with levels of antibodies in ELISA, indicating that in ELISA the proteins were not uniformly bound and that antibodies with many specificities exist in natural immunisation. The correlations between ELISA and SPR were enhanced when only parasite positive samples were included, which may indicate that high affinity antibodies are difficult to maintain over long periods of time. We found higher kd values for MSP2 (indicating lower affinity) compared to AMA1, which might be partly explained by MSP2 being an intrinsically disordered protein, while AMA1 is globular. CONCLUSIONS: For future vaccine studies and for understanding immunity, it is important to consider how to present proteins to the immune system to achieve highest antibody affinities.

High affinity antibodies to Plasmodium falciparum merozoite antigens are associated with protection from malaria.

BACKGROUND: Malaria kills almost 1 million people every year, but the mechanisms behind protective immunity against the disease are still largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, surface plasmon resonance technology was used to evaluate the affinity (measured as k(d)) of naturally acquired antibodies to the Plasmodium falciparum antigens MSP2 and AMA1. Antibodies in serum samples from residents in endemic areas bound with higher affinities to AMA1 than to MSP2, and with higher affinities to the 3D7 allele of MSP2-3D7 than to the FC27 allele. The affinities against AMA1 and MSP2-3D7 increased with age, and were usually within similar range as the affinities for the monoclonal antibodies also examined in this study. The finding of MSP2-3D7 type parasites in the blood was associated with a tendency for higher affinity antibodies to both forms of MSP2 and AMA1, but this was significant only when analyzing antibodies against MSP2-FC27, and individuals infected with both allelic forms of MSP2 at the same time showed the highest affinities. Individuals with the highest antibody affinities for MSP2-3D7 at baseline had a prolonged time to clinical malaria during 40 weeks of follow-up, and among individuals who were parasite positive at baseline higher antibody affinities to all antigens were seen in the individuals that did not experience febrile malaria during follow up. CONCLUSIONS/SIGNIFICANCE: This study contributes important information for understanding how immunity against malaria arises. The findings suggest that antibody affinity plays an important role in protection against disease, and differs between antigens. In light of this information, antibody affinity measurements would be a key assessment in future evaluation of malaria vaccine formulations.

Acoustic wave immunosensing of a meningococcal antigen using gold nanoparticle-enhanced mass sensitivity.

Bacterial meningitis is an infection of the thin membranes covering the brain and spinal cord by a number of microorganisms including Neisseria meningitidis, which can lead to permanent neurological damage in the event of late diagnosis. Given the quick onset and severity of the disease, there is a clear need for a rapid, sensitive and specific diagnostic technique. Here, we describe the development and evaluation of an acoustic wave sensor, the quartz crystal microbalance (QCM), as a rapid immunosensor employing antibodies against the cell surface outer membrane protein 85 (OMP85) of N. meningitidis as an immobilized selective layer. These antibodies were directionally orientated as receptors by thin film deposition of structured polyvinylidene fluoride and Protein A. The sensitivity of this QCM immunosensor was further increased by conjugation of the OMP85 antigen to 50 nm gold nanoparticles providing reproducible detection of the target down to 300 ng/mL. Subsequent treatment of the QCM surface with an acidic glycine solution regenerated the immunosensor allowing each crystal to be used several times.