High Prevalence Of Antibody Response Against Plasmodium Falciparum (Pf) Antigens In A Holoendemic Area Of Benin (1994-1995)

The present study aimed at measuring the capacity of naturally occurring antibodies to bind Pf83/AMA-1 and MSP-1/19 antigens, two malaria vaccine candidates, in an immunoassay. According to the fact that antibody prevalence reflects endemicity of malaria, we further aimed at using the results obtained here as baseline data set to follow up and evaluate the expected decline in endemicity in 2016, 8 years after the change in drug policy in Benin. Therefore, individuals, 2 – 19 and above 20 years old, living in Awansori, a malaria holoendemic area in the suburb of Cotonou, Benin were bled during the dry and raining seasons of the years 1994/1995. Antibody responses were measured using direct, indirect and competition ELISA. We found a very high prevalence of antibody responses (89 to 96%) in the studied population. The results indicate for Pf83/AMA-1, that naturally occurring antibodies bind to protective epitopes in a competition ELISA with a parasite inhibiting monoclonal antibody. The data and samples analysed here were collected during the rainy season 1994 and the following dry season 1994/1995.

Pharmacokinetic analysis of orally administered puerarin in human saliva using an indirect competition ELISA

The pharmacokinetics of puerarin in human saliva following oral administration of pueraria capsules were successfully studied by an icELISA method.

The Diversity of Anti-Factor VIII B-Cell Epitopes in Hemophilia a Patients with Inhibitors

The most significant complication in the management of patients with hemophilia A continues to be the development of anti-factor VIII antibodies in response to infusions of the fVIII protein. Patients with hemophilia A typically develop a polyclonal B-cell response to fVIII with antibodies to the A2 and C2 domains being the most prevalent. Within the A2 and C2 domains, we have identified non-overlapping epitopes with unique characteristics. There are 5 non-overlapping epitopes in the A2 domain and 3 non-overlapping epitopes in the C2 domain. Within the C2 domain, antibody epitope was more important than inhibitory titer in predicting response to fVIII treatment in a murine in vivo bleeding model. Similar findings were seen within the A2 domain. In previous studies we developed a competition ELISA that assessed the ability of patient plasma to compete with biotinylated monoclonal antibodies (MAbs) with known epitopes for binding to fVIII. Competition with at least 1 of 3 non-overlapping anti-fVIII C2 domain MAbs was seen in 19 of 26 (73%) patient plasmas. The high volume of plasma needed for each monoclonal antibody assessed restricts the use of this assay to map a limited number of epitopes from a single plasma sample. Given the number of non-overlapping epitopes identified on the fVIII protein, the goal of this study was to modify the previous competition ELISA to better define the diversity of the immune response to fVIII using a clinically feasible volume of plasma. Plasma samples and limited clinical data from hemophilia A patients with inhibitors who were HIV negative were obtained from the Biologic Specimen and Data Repository Information Center (BioLINCC) of the NHLBI. A single sample was available from 27 patients while 2 samples from different time periods were available from an additional 23 patients. Epitope mapping was performed using a modified competition ELISA utilizing a single biotinylated MAb concentration with patient plasma (~50 µl total for all experiments) diluted 1:40. The rate of MAb binding in the presence of patient plasma was compared to the rate of MAb binding in the presence of control severe hemophilia A plasma without inhibitors. Competition was considered present when the binding was reduced more than 2 standard deviations below control. The anti-fVIII MAbs in this study were 4 anti-A2 antibodies with non-overlapping epitopes (A2-A, A2-B, A2-D, A2-E) and 3 anti-C2 antibodies with non-overlapping epitopes(C2-A, C2-B, C2-C) as well as one inhibitory antibody from both the A3 and C1 domains. A modified Bethesda assay was performed on each sample which allowed us to group the plasmas into 3 inhibitory titer ranges: < 6 BU/ml, 6-16 BU/ml, and >16 BU/ml. Of the 73 plasma samples 20 showed competition with at least 1 MAb as shown in Table 1. Six of 36 plasmas (17%) with titers of <6 BU/ml showed competition with a single MAb. Three of 20 plasmas (15%) with titers of 6-16 BU/ml showed competition with either 1 or 2 MAbs. Eleven of 17 plasmas with titers of >16 BU/ml showed competition with between 1 and 9 MAbs. No competition was detected in the 6 patients with moderate or mild hemophilia A. In 12 of the 23 patients with 2 samples available, epitopes were detected in at least 1 sample but none of these patients had identical epitope spectra in both samples. Despite the lower overall detection rate compared to the previous assay, our results highlight the diversity of epitope spectra present in patient plasmas with high inhibitory titers. This diversity underscores the need to better understand the makeup of the immune response to fVIII in patients with hemophilia A at the individual epitope level. Given the success of detecting each of the 9 MAb epitopes in at least one patient plasma, work is ongoing to improve the sensitivity of this assay to further define differences in response between patients. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.