Candidate Optimization and Potency in Mice We previously reported that several LbL-MP vaccine candidates containing epitopes of the PfCS protein elicited parasite-neutralizing antibodies and protected mice from in vivo difficulties with live [14]. rhesus macaque. Base layer-crosslinked LbL-MP loaded with T1BT* peptide with or without covalently linked Pam3Cys elicited T1B-specific antibody responses and T1BT*-specific T-cell responses dominated by IFN secretion with lower levels of IL-5 secretion. The Pam3Cys-modified construct was more potent, generating antibody responses that neutralized wild-type in an in vitro hepatocyte invasion assay. IgG purified from individual macaques immunized with Pam3Cys.T1BT* LbL-MP protected na?ve mice from difficulties with transgenic sporozoites that expressed the full-length PfCS protein, with 50C88% IEM 1754 Dihydrobromide of passively immunized mice parasite-free for 15 days. Substitution of serine for an unpaired cysteine in the T* region of the T1BT* subunit did not adversely impact immune potency in the mouse while simplifying the manufacture of the antigenic peptide. In a Good Laboratory Practices compliant rabbit toxicology study, the base layer-crosslinked, Pam3Cys-modified, serine-substituted candidate was shown to be safe and immunogenic, eliciting parasite-neutralizing antibody responses and establishing the dose/route/regimen for any clinical evaluation of this novel synthetic microparticle pre-erythrocytic malaria vaccine candidate. Keywords: malaria vaccine, microparticle, non-human primate, peptide, sporozoite 1. Introduction The latest estimates provided by the World Health Business show an increase in malaria IEM 1754 Dihydrobromide cases worldwide, from 230 million in 2015 to 247 million in 2021, while the quantity of malaria deaths worldwide rose from 577,000 to 619,000 over the same time period [1]. Africa accounts for 90% of all cases and deaths worldwide, and is the causative agent of nearly all malaria cases in sub-Saharan Africa. Current methods of malaria control are limited to vector control and post-infection therapeutics, but these have limited utility due to sporadic use, environmental concerns, and the emergence of resistance in both the vector and parasite. The development of widely useful vaccine methods has been hampered by the complex life cycle of strains, and the T* epitope that is recognized by many HLA Class Rabbit Polyclonal to RUFY1 II molecules. Those studies showed that LbL-MP loaded with T1BT* designed peptides could elicit specific antibody and T-cell IEM 1754 Dihydrobromide responses and safeguard the murine host from difficulties with PfPb, a transgenic expressing the CS subunits of difficulties in the mouse model. The most promising candidates IEM 1754 Dihydrobromide incorporated base layer crosslinking with or without the inclusion of the TLR2 ligand and were tested in a non-human primate (NHP) model. The results showed that this TLR2 ligand-modified candidate elicited the most potent antibody responses that conferred protection against parasite difficulties upon passive transfer to na?ve mice. The final modification of cysteine serine substitution yielded a vaccine candidate that was easier to manufacture, retained immunogenicity and efficacy in mice, demonstrated a large therapeutic index, and elicited parasite-neutralizing antibodies in a Good Laboratory Practices compliant toxicology study in rabbits. The current results set the stage for any clinical evaluation of the security and immunogenicity of this novel LbL-MP vaccine in healthy malaria-na?ve adults. 2. Materials and Methods 2.1. LbL Particle Fabrication CaCO3 microparticle cores were prepared by quick mixing of equivalent volumes of 0.33 M Na2CO3 (containing 1.2 g/L poly-l-glutamic acid [PGA]) and 0.33 M CaCl2 in a circulation reaction vessel. The producing spherical porous CaCO3 microparticles made up of a surface layer of PGA were collected in fractions throughout the circulation process, and the particle IEM 1754 Dihydrobromide sizes of individual fractions were measured by dynamic light scattering (DLS) using a Malvern Mastersizer 3000 particle analyzer (Malvern Panalytical, Boston MA). Fractions made up of microparticles with a mean particle diameter (by volume) <5.0 m and 90% of the particles <10 m were pooled to yield a particle suspension containing approximately 1.6% solids in 0.33 M NaCl and excess PGA. LbL-MP were fabricated as previously reported [19] by alternately layering poly-l-lysine (PLL, positive charge) and PGA (unfavorable charge) around the CaCO3 cores to build up a 7-layer base film and capping with an outermost layer of the designed peptide (DP). Where indicated (bXL), the PGA:PLL base film was chemically crosslinked by treatment with 200 mM EDC and 50 mM sulfo-NHS (Sigma-Aldrich, St. Louis MO, USA) in 0.2 M phosphate buffer, pH 6.5, for 30 min at room temperature prior to layering the DP. DPs were synthesized and analyzed by standard techniques [19]. The wild-type T1BT* DP sequence derived from CS protein is DPNANPNVDPNANPNV(NANP)3epitope is usually italicized, and K20 is usually a poly-lysine.