Immunogenicity of Self-Associated Aggregates and Chemically Cross-Linked Conjugates of the 42 kDa Plasmodium falciparum Merozoite Surface Protein-1

Self-associated protein aggregates or cross-linked protein conjugates are, in general, more immunogenic than oligomeric or monomeric forms. In particular, the immunogenicity in mice of a recombinant malaria transmission blocking vaccine candidate, the ookinete specific Plasmodium falciparum 25 kDa protein (Pfs25), was increased more than 1000-fold when evaluated as a chemical cross-linked protein-protein conjugate as compared to a formulated monomer. Whether alternative approaches using protein complexes improve the immunogenicity of other recombinant malaria vaccine candidates is worth assessing. In this work, the immunogenicity of the recombinant 42 kDa processed form of the P. falciparum merozoite surface protein 1 (MSP142) was evaluated as a self-associated, non-covalent aggregate and as a chemical cross-linked protein-protein conjugate to ExoProtein A, which is a recombinant detoxified form of Pseudomonas aeruginosa exotoxin A. MSP142 conjugates were prepared and characterized biochemically and biophysically to determine their molar mass in solution and stoichiometry, when relevant. The immunogenicity of the MSP142 self-associated aggregates, cross-linked chemical conjugates and monomers were compared in BALB/c mice after adsorption to aluminum hydroxide adjuvant, and in one instance in association with the TLR9 agonist CPG7909 with an aluminum hydroxide formulation. Antibody titers were assessed by ELISA. Unlike observations made for Pfs25, no significant enhancement in MSP142 specific antibody titers was observed for any conjugate as compared to the formulated monomer or dimer, except for the addition of the TLR9 agonist CPG7909. Clearly, enhancing the immunogenicity of a recombinant protein vaccine candidate by the formation of protein complexes must be established on an empirical basis

Changeover behavior under pairs of fixed-ratio and variable-ratio schedules of reinforcement

Three pigeons were studied under a pair of equal fixed-ratio schedules and a pair of equal variable-ratio schedules. Each pair was arranged as independent, concurrent schedules and also in a non-independent relation where each peck in a schedule counted toward the response requirement of both schedules. The non-independent pair of variable-ratio schedules maintained much higher changeover rates than any of the other three arrangements. Thus, two factors seemed necessary for generating high changeover rates. Responding on a schedule had to count toward the response requirement of both schedules, and the component schedules had to be variable. These data are consistent with the hypothesis that changeovers are at least partly controlled by the probability of reinforcement following a changeover

Forms and volatilities of trace minor elements in coal

pt. 1. A comparison of three pyrolysis units / Richard A. Cahill, Richard H. Shiley, Neil F. Shimp -- pt. 2. Feasibility of a graphite trapping system / Richard H. Shiley ... [et al.]

Choice between fixed-interval schedules: Graded versus step-like choice functions

Pigeons chose between two fixed-interval schedules of food reinforcement. A single peck on one of two lighted keys started the fixed-interval schedule correlated with that key. The schedule had to be completed before the next choice opportunity. The durations of the fixed intervals were varied over conditions from 15 s to 40 s. To maximize the rate of reinforcement, the pigeons had to choose exclusively the shorter of the two schedules. Nevertheless, choice was not all-or-none. Instead, relative choice, and the rates of producing the fixed intervals, varied in a graded fashion with the disparity between the two schedules. Choice ratios under this procedure (single response to choose) were highly sensitive to the ratios of the fixed-interval schedules

Forms and volatilities of trace minor elements in coal

pt. 1. A comparison of three pyrolysis units / Richard A. Cahill, Richard H. Shiley, Neil F. Shimp -- pt. 2. Feasibility of a graphite trapping system / Richard H. Shiley ... [et al.]

Identification and Characterization of the Plasmodium yoelii PyP140/RON4 Protein, an Orthologue of Toxoplasma gondii RON4, Whose Cysteine-Rich Domain Does Not Protect against Lethal Parasite Challenge Infection▿

Previously, we identified a Plasmodium yoelii YM 140-kDa merozoite protein, designated PyP140, which formed a complex with apical membrane antigen 1 (AMA1). Furthermore, we produced a nonprotective monoclonal antibody (MAb), 48F8, that immunoprecipitated metabolically labeled PyP140 and localized the protein to the merozoite's apical end and, less frequently, to the merozoite surface, as observed by immunofluorescence assay (IFA). Here, using MAb 48F8, we have identified the pyp140 gene by screening a P. yoelii λ-Zap cDNA expression library. The pyp140 cDNA covers approximately 90% of the putative open reading frame (ORF) of PY02159 from the P. yoelii NL genome sequencing project. Analysis of the complete gene identified the presence of two introns. The ORF encodes a 102,407-Da protein with an amino-terminal signal sequence, a series of three unique types of repeats, and a cysteine-rich region. The binding site of MAb 48F8 was also identified. A BLAST search with the deduced amino acid sequence shows significant similarity with the Toxoplasma gondii RON4 protein and the Plasmodium falciparum RON4 protein, and the sequence is highly conserved in other Plasmodium species. We produced the cysteine-rich domain of PyP140/RON4 by using the Pichia pastoris expression system and characterized the recombinant protein biochemically and biophysically. BALB/c mice immunized with the protein formulated in oil-in-water adjuvants produced antibodies that recognize parasitized erythrocytes by IFA and native PyP140/RON4 by immunoblotting but failed to protect against a lethal P. yoelii YM infection. Our results show that PyP140/RON4 is located within the rhoptries or micronemes. It may associate in part with AMA1, but the conserved cysteine-rich domain does not appear to elicit inhibitory antibodies, a finding that is supported by the marked sequence conservation in this protein within Plasmodium spp., suggesting that it is not under immune pressure