A maximum bias of 20% compared to the nontreated QC2 was set as the acceptance criterion

A maximum bias of 20% compared to the nontreated QC2 was set as the acceptance criterion. infectious disease, killing nearly 1 million people each year. The development of a protective vaccine against malaria is LY 345899 considered a high priority in order to prevent the disease (WHO,http://www.who.int/mediacenter/news/notes/2006/np35/en/print.html; Malaria Vaccine Initiative,http://www.malariavaccineroadmap.net/pdfs/vision_document.pdf). Although the feasibility of a malaria vaccine using irradiated sporozoites was exhibited more than 30 years ago (3), an effective malaria vaccine has not been generated as yet. The protective effect of irradiated sporozoites is likely due to a protective antibody response. Immunological studies over the following decades, however, have indicated that both antibodies and T cells are required for protection against contamination with malaria parasites. Adenoviral vectors have been proven highly efficient in inducing both cellular and humoral immune responses in various disease models (1,16,20). Therefore, recombinant adenoviral vectors are considered to be a potential vaccine to prevent malaria disease. We have developed such a candidate malaria vaccine based on adenovirus serotype 35 (Ad35) expressing aPlasmodium falciparumcircumsporozoite (CS) transgene (Ad35.CS) which is produced on PER.C6 cells. This vaccine induced strong immune responses in mice (17) and rhesus macaques (14,18). Although heterologous prime-boost vaccine strategies are likely required to preventP. falciparuminfection (12), Ad35 is proposed to be an important component of such a combination. Following the successful preclinical investigations on Ad35.CS, a clinical phase I trial has started in LY 345899 the United States. The primary objective of this phase I study is to show security and tolerability, and the secondary objective is to explore the immunogenicity of the vaccine by measuring T cell responses and antibody responses. In this statement we describe the development and validation of an antibody enzyme-linked immunosorbent assay (ELISA) that determines the antibody titer in human serum against LY 345899 CS induced by the vaccine, in order to LY 345899 support the secondary objective. Although the correlates of protection against malaria are not determined yet, the antibody assay explained here is required to provide an accurate indication of the humoral immunogenicity of the vaccine. Therefore, we selected the immunodominant epitope in the CS antigen as the target for the antibody assay in order to detect the majority of human responses induced by the vaccine. Since the repeat region of CS is the immunodominant region for B cell epitopes (2224) and antibodies against the NANP3epitope in the repeat region reflect exposure to naturalP. falciparuminfection (25), it is assumed that this antibodies directed to this region correlate with the total amount of anti-CS antibodies. The (NANP)6C peptide, as explained by Stoute et al. (19), is the optimal representation of the repeat region and was selected to be capture antigen in our assay. In general, an antibody ELISA would be based on the total antigen of interest. The CS protein, however, is usually not readily available to the scientific community, LY 345899 and recombinant CS proteins from different sources may hamper the comparability of results between different laboratories. Using a synthetic peptide as target Mouse monoclonal to SRA antigen could facilitate comparability between laboratories, since the antigen quality is much easier to assurance for any peptide than for any recombinant protein. Here we describe the development of the CS antibody ELISA to determine the humoral immunogenicity of Ad35. CS in human subjects and its validation as a strong and reproducible assay to support clinical vaccine development. == MATERIALS AND METHODS == == CS antibody ELISA protocol. == The peptide (NANP)6C, comprising 6 repeats of NANP, mimics a part of the repeat sequence ofP. falciparumCS (4,19), and this peptide was used as capture antigen to bind CS-specific antibodies. The peptide was obtained from Pepscan (Lelystad, Netherlands) at a purity of 90%. The peptide was coated at a concentration of 2 g/ml in 0.05 M carbonate buffer at 2 to 8C overnight or for a maximum of 3 days. Reference serum, serum samples, and.