Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens

Abstract

Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens.

Prime-and-Trap Malaria Vaccination To Generate Protective CD8+ Liver-Resident Memory T Cells

“…With respect to such Ag discovery, our study additionally demonstrated a novel method for rapidly evaluating candidate Ags and for making Go/No Go decisions about their protective potential. Immunogenicity testing is fraught with misleading outcomes because many Ags are immunogenic in immunized animals but are ultimately nonprotective (30,36,91). In the DNA-only acute challenge model described in this article (Fig.…”

“…For all vaccinations, a plasmid encoding the Escherichia coli heat-labile lymphotoxin (LT) (29) was used as an adjuvant in a 1:10 ratio with the CSP-minigene vector. DNA was purified using an endotoxin-free purification kit (Qiagen), loaded onto gold beads (1-2-mm diameter; InBio Gold), and coated on tubing as cartridges for helium gas-propelled bombardment via gene gun on trimmed abdominal skin as described previously (30). Mice were vaccinated using a PowderJect-style gene gun by cluster priming for the first DNA vaccination using two cartridges (0.5 mg DNA per cartridge), each 2 d apart as shown in the figures (hereafter called "cluster primed" and denoted in figures with a " + "), and were boosted a single day later as indicated using two cartridges.…”

Gene gun DNA immunization of cattle induces humoral and CD4 T-cell-mediated immune responses against the Theileria parva polymorphic immunodominant molecule

“…Cartridges were loaded using traditional methods [28] . Cartridge batches were quality controlled by elution of single cartridges with 50 µL of water and quantified for DNA concentration using a NanoDrop (Thermo Scientific, Waltham, MA).…”

“…GG DNA immunization has been successfully utilized in vaccination trials for viral and neoplastic diseases in humans, primates, pigs and mice, and was recently used in mice to discover new Plasmodium yoelii sporozoite candidate antigens [22] , [25] , [27] , [28] . To our knowledge, GG DNA immunization using complex protozoal antigens has never been tested in a large, outbred species such as cattle.…”

Update on treatment and preventive interventions against COVID-19: an overview of potential pharmacological agents and vaccines

“…DC and CTL cells play a key role in viral clearance during the immune process, so it is important to induce vaccines that produce strong, long-lasting, cross-T cell responses [319][320][321][322]. This minigene can express a segment of amino acid residue peptide through viral infection or the synthesis of a minigene.…”