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Production and Immunogenicity of a Tag-Free Recombinant Chimera Based on PfMSP-1 and PfMSP-3 Using Alhydrogel and Dipeptide-Based Hydrogels.

ABSTRACT: A fusion chimeric vaccine comprising multiple protective domains of different blood-stage Plasmodium falciparum antigens is perhaps necessary for widening the protective immune responses and reducing the morbidity caused by the disease. Here we continue to build upon the prior work of developing a recombinant fusion chimera protein, His-tagged PfMSP-Fu24, by producing it as a tag-free recombinant protein. In this study, tag-free recombinant PfMSPFu24 (rFu24) was expressed in Escherichia coli, and the soluble protein was purified using a three-step purification involving ammonium sulphate precipitation followed by 2-step ion exchange chromatography procedures and shown that it was highly immunogenic with the human-compatible adjuvant Alhydrogel. We further investigated two dipeptides, phenylalanine-α, β-dehydrophenylalanine (FΔF) and Leucine-α, β-dehydrophenylalanine (LΔF) based hydrogels as effective delivery platforms for rFu24. These dipeptides self-assembled spontaneously to form a highly stable hydrogel under physiological conditions. rFu24 was efficiently entrapped in both the F∆F and L∆F hydrogels, and the three-dimensional (3D) mesh-like structures of the hydrogels remained intact after the entrapment of the antigen. The two hydrogels significantly stimulated rFu24-specific antibody titers, and the sera from the immunized mice showed an invasion inhibitory activity comparable to that of Alhydrogel. Easily synthesized dipeptide hydrogels can be used as an effective antigen delivery platform to induce immune responses.

SUBMITTER: Anand G

PROVIDER: S-EPMC8310097 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Defective interfering particles (DIPs) of influenza A virus (IAV) are suggested for use as broad-spectrum antivirals. We discovered a new type of IAV DIP named "OP7" that carries point mutations in its genome segment (Seg) 7 instead of a deletion as in conventional DIPs (cDIPs). Recently, using genetic engineering tools, we generated "OP7 chimera DIPs" that carry point mutations in Seg 7 plus a deletion in Seg 1. Together with cDIPs, OP7 chimera DIPs were produced in shake flasks in the absence of infectious standard virus (STV), rendering UV inactivation unnecessary. However, only part of the virions harvested were OP7 chimera DIPs (78.7%) and total virus titers were relatively low. Here, we describe the establishment of an OP7 chimera DIP production process applicable for large-scale production. To increase total virus titers, we reduced temperature from 37 to 32 °C during virus replication. Production of almost pure OP7 chimera DIP preparations (99.7%) was achieved with a high titer of 3.24 log10(HAU/100 µL). This corresponded to an 11-fold increase relative to the initial process. Next, this process was transferred to a stirred tank bioreactor resulting in comparable yields. Moreover, DIP harvests purified and concentrated by steric exclusion chromatography displayed an increased interfering efficacy in vitro. Finally, a perfusion process with perfusion rate control was established, resulting in a 79-fold increase in total virus yields compared to the original batch process in shake flasks. Again, a very high purity of OP7 chimera DIPs was obtained. This process could thus be an excellent starting point for good manufacturing practice production of DIPs for use as antivirals. KEY POINTS: • Scalable cell culture-based process for highly effective antiviral OP7 chimera DIPs • Production of almost pure OP7 chimera DIPs in the absence of infectious virus • Perfusion mode production and purification train results in very high titers.

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Production and Immunogenicity of a Tag-Free Recombinant Chimera Based on PfMSP-1 and PfMSP-3 Using Alhydrogel and Dipeptide-Based Hydrogels.

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