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The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria transmission-blocking vaccine (TBV). Previously, we showed that a Pfs230-Pfs48/45 fusion protein elicits higher levels of functional antibodies than the individual antigens, but low yields hampered progression to clinical evaluation. Here we identified a modified construct (ProC6C) with a circumsporozoite protein (CSP) repeat-linker sequence that enhances expression. A scalable and reproducible process in the Lactococcus lactis expression system was developed and ProC6C was successfully transferred for manufacturing under current Good Manufacturing Practices (cGMP). In addition, a panel of analytical assays for release and stability were developed. Intact mass spectrometry analysis and multiangle light scattering showed that the protein contained correct disulfide bonds and was monomeric. Immunogenicity studies in mice showed that the ProC6C adsorbed to Alhydrogel®, with or without Matrix-MTM, elicited functional antibodies that reduced transmission to mosquitoes and sporozoite invasion of human hepatocytes. Altogether, our data support manufacture and clinical evaluation of ProC6C as a multistage malaria-vaccine candidate.

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel®) elicited functional antibodies in small rodents and that adding Matrix-M™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine.

Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL)-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95%) and expression of surface markers FcεRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cell-specific transcripts, i.e. β-tryptase, serglycin and FcεRI α-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP), charcot leyden crystal (CLC), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and basophils ar