Characterization of AMA1-RON2L complex with native gel electrophoresis and capillary isoelectric focusing.

Rhoptry neck protein 2 (RON2) binds to the hydrophobic groove of apical membrane antigen 1 (AMA1), an interaction essential for invasion of red blood cells (RBCs) by Plasmodium falciparum (Pf) parasites. Vaccination with AMA1 alone has been shown to be immunogenic, but unprotective even against homologous challenge in human trials. However, the AMA1-RON2L (L is referred to as the loop region of RON2 peptide) complex is a promising candidate, as preclinical studies with Freund's adjuvant have indicated complete protection against lethal challenge in mice and superior protection against virulent infection in Aotus monkeys. To prepare for clinical trials of the AMA1-RON2L complex, identity and integrity of the candidate vaccine must be assessed, and characterization methods must be carefully designed to not dissociate the delicate complex during evaluation. In this study, we developed a native Tris-glycine gel method to separate and identify the AMA1-RON2L complex, which was further identified and confirmed by Western blotting using anti-AMA1 monoclonal antibodies (mAbs 4G2 and 2C2) and anti-RON2L polyclonal Ab coupled with mass spectrometry. The formation of complex was also confirmed by Capillary Isoelectric Focusing (cIEF). A short-term (48 h and 72 h at 4°C) stability study of AMA1-RON2L complex was also performed. The results indicate that the complex was stable for 72 h at 4°C. Our research demonstrates that the native Tris-glycine gel separation/Western blotting coupled with mass spectrometry and cIEF can fully characterize the identity and integrity of the AMA1-RON2L complex and provide useful quality control data for the subsequent clinical trials.

Long term stability of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with Montanide(®) ISA 720 and stabilized with glycine.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is an asexual blood-stage vaccine candidate against the malaria parasite. AMA1-C1/ISA 720 refers to a mixture of recombinant AMA1 proteins representing the FVO and 3D7 alleles in 1:1 mass ratio, formulated with Montanide(®) ISA 720 as a water-in oil emulsion. In order to develop the AMA1-C1/ISA 720 vaccine for human use, it was important to determine the shelf life of this formulation. Previously it was found 267 mM glycine stabilized the proteins in Montanide(®) ISA 720 formulations for a short period of time at 2-8°C [25]. We now test the long term stability of AMA1-C1 at 10 and 40 µg/mL formulated with Montanide(®) ISA 720 with 50mM glycine as a stabilizer. Stability of AMA1-C1/ISA 720 at different time points following formulation (0, 5, 12 or 18 months) was evaluated by determining the mean particle size (diameter of the mean droplet volume), total protein content by a Modified Lowry assay, identity and integrity using western blot and SDS-PAGE. Our results showed that the mean particle size of these emulsions increased over time, whereas protein content, as determined by an ELISA method using a monoclonal antibody against penta-his, decreased over time. For the 10 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was 6.5±2.2 µg/mL, and for the 40 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was only 8.2±2.3 µg/mL after 18 months of storage at 2-8°C. These results suggest that the integrity of the protein was affected by long-term storage. The results of the present study indicate that the AMA1-C1/ISA 720 emulsion was unstable after 12 months of storage, after which AMA1-C1 proteins were partially degraded.

Role of brain-derived neurotrophic factor and NF-kappaB in neuronal plasticity and survival: From genes to phenotype.

PURPOSE: Brain-derived neurotrophic factor (BDNF) is a member of the family of neurotrophins and promotes diverse effects in neurons including development, maintenance of function, synaptic plasticity, and survival in different animal models. We present advances in our understanding of the genomics of the BDNF gene (bdnf) and its regulation by calcium-activated transcription factors, including cAMP response element binding protein (CREB) and more recently, nuclear factor kappaB (NF-kappaB) and discuss these findings in the context of neuronal plasticity and survival. METHODS: We used amplified bdnf complementary DNAs (cDNAs) and genomic DNA templates for direct sequencing and sequence variant discovery, information mining of public databases, and conventional molecular and cellular biology approaches to screen bdnf for novel regulatory elements, alternatively spliced exons, and functional sequence variants. RESULTS: We discovered a candidate NF-kappaB site in promoter 3 of bdnf and showned that activation of N-methyl-D-aspartate (NMDA) inotropic glutamate receptors increased bdnf expression through an NF-kappaB-dependent pathway and extended the finding to show that NF-kappaB was required for NMDA neuroprotection in vitro. In addition, sequence analysis of bdnf cDNAs from different brain regions predicted at least three pre-pro-BDNF protein isoforms, two of which were previously unknown. Each isoform differs at the amino terminus and may have functional importance. CONCLUSIONS: Given the central role that BDNF plays in the developing and adult nervous system, understanding how BDNF is regulated and how it functions will enhance our knowledge of its diverse effects, which may lead to more effective treatments for neurodegenerative disorders and reveal the role of BDNF in complex phenotypes related to behavior.