PMC

Fabrication of protein microarrays for antibody target identification. Protein microarrays are fabricated using a method involving high throughput amplification of each predicted open reading frame from the genomic sequence using gene-specific primers with adapter sequences, followed by homologous recombination into a T7 expression vector that has an N-terminus HIS tag and a C-terminus HA tag. The plasmids are transformed into Escherichia coli cells, minipreped, and proteins expressed using in an in vitro cell-free transcription/translation system supplemented with T7 RNA polymerase. The proteins are then printed directly onto microarray chips without further purification. Microarrays are probed with anti-HIS and anti-HA antibodies to confirm expression of full-length proteins in quality control (QC) assays, and with sera from experimentally immunized or naturally exposed subjects. From http://www.antigendiscovery.com/technology.php, with permission.

Epitope-based T cell screening strategy for T cell target identification. Complete amino acid sequences translated from the genes of interest are scanned using motif identification software for the presence of HLA motif-containing sequences. For each antigen (Ag), peptides representing the top 10 epitopes predicted to bind with highest affinity to each of five common HLA supertypes are identified and synthesized. Peptides are pooled and screened for capacity to induce recall IFN-g immune responses using peripheral blood mononuclear cells from volunteers immunized with irradiated Plasmodium falciparum sporozoites or naturally exposed to malaria. Antigens can be prioritized on the basis of immune reactivity, and the peptide pools deconvoluted to identify the minimal peptide epitopes and their HLA restriction elements.

Genomes to vaccines schematic illustration. Methodologies for utilizing genomic sequence data to identify new candidate antigens for use in malaria vaccines require the complex interplay of bioinformatics (denoted by dotted arrows) and molecular manipulations (denoted by solid arrows), many of which involve novel technologies that are amenable to high-throughput processing (see Section 3 for details). Open reading frames (ORF) are denoted by open boxes, and their expressed protein product by solid boxes. There are multiple alternative pathways for many of the steps, only some of which are illustrated. For example, either transcriptionally active polymerase chain reaction (TAP) fragments (linear DNA) or the proteins they encode as expressed in cell-free systems, or circular plasmid DNA generated by recombinatorial cloning, can be used to transfect or transduce antigen presenting cells (APCs) such as dendritic cells (DC); similarly, protein arrays can be used to screen either sera or cells from protected volunteers. This figure is modified from with permission. MHC, major histocompatibility complex; HLA, human leukocyte antigen; PBMC, peripheral blood mononuclear cell; ISI, inhibition of sporozoite invasion assay; ILSDA, inhibition of liver stage development assay; GIA, growth inhibition assay (blood stage); IFAT, immunofluorescent antibody test; Pf, P. falciparum.