Construct design, production, and characterization of Plasmodium falciparum 48/45 R0.6C subunit protein produced in Lactococcus lactis as candidate vaccine

Singh, Susheel K.; Roeffen, Will; Mistarz, Ulrik H.; Chourasia, Bishwanath Kumar; Yang, Fen; Rand, Kasper D.; Sauerwein, Robert W.; Theisen, Michael

doi:10.1186/s12934-017-0710-0

Cited by 38 publications

(58 citation statements)

References 33 publications

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“…Surprisingly, SpyCatcher fusion enhanced soluble expression in E. coli of the disulfide-rich Pfs25 from P. falciparum ( 75 ). Fusions to Catcher and Tag sequences have been expressed in various systems, including Gram-positive and Gram-negative bacteria ( 73 , 89 , 90 ), mammalian cells ( 73 ), insect cells ( 75 ), plant cells ( 91 ), and yeast ( 92 ).…”

Section: Adoption Of Catcher/tag Technology For Vaccine Assemblymentioning

confidence: 99%

New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue

2018

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Vaccines based on virus-like particles (VLPs) can induce potent B cell responses. Some non-chimeric VLP-based vaccines are highly successful licensed products (e.g., hepatitis B surface antigen VLPs as a hepatitis B virus vaccine). Chimeric VLPs are designed to take advantage of the VLP framework by decorating the VLP with a different antigen. Despite decades of effort, there have been few licensed chimeric VLP vaccines. Classic approaches to create chimeric VLPs are either genetic fusion or chemical conjugation, using cross-linkers from lysine on the VLP to cysteine on the antigen. We describe the principles that make these classic approaches challenging, in particular for complex, full-length antigens bearing multiple post-translational modifications. We then review recent advances in conjugation approaches for protein-based non-enveloped VLPs or nanoparticles, to overcome such challenges. This includes the use of strong non-covalent assembly methods (stick), unnatural amino acids for bio-orthogonal chemistry (click), and spontaneous isopeptide bond formation by SpyTag/SpyCatcher (glue). Existing applications of these methods are outlined and we critically consider the key practical issues, with particular insight on Tag/Catcher plug-and-display decoration. Finally, we highlight the potential for modular particle decoration to accelerate vaccine generation and prepare for pandemic threats in human and veterinary realms.

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Section: Adoption Of Catcher/tag Technology For Vaccine Assemblymentioning

confidence: 99%

New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue

2018

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“…The C-terminal 6-Cys domain of Pfs 48/45 contains the conformational epitope I, which is targeted by the most potent TB monoclonal antibody described to date, mAb45.1 (18). We have recently used the Lactococcus lactis expression system for the production of the C-terminal 6-Cys domain of Pf s48/45 (6C) as a fusion protein (R0.6C) with the N-terminal GLURP-R0 region (19, 20). The resulting fusion protein can be produced in high yields of properly folded monomeric protein which elicited high levels of TB antibodies in small rodents (19, 20).…”

Section: Introductionmentioning

confidence: 99%

“…We have recently used the Lactococcus lactis expression system for the production of the C-terminal 6-Cys domain of Pf s48/45 (6C) as a fusion protein (R0.6C) with the N-terminal GLURP-R0 region (19, 20). The resulting fusion protein can be produced in high yields of properly folded monomeric protein which elicited high levels of TB antibodies in small rodents (19, 20). In the case of Pf s230, the C fragment spanning the N-terminal pro-domain and first three 6-Cys domains has been shown to elicit the most potent TB antibodies (21).…”

Section: Introductionmentioning

confidence: 99%

Pfs230 and Pfs48/45 Fusion Proteins Elicit Strong Transmission-Blocking Antibody Responses Against Plasmodium falciparum

et al. 2019

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The Plasmodium falciparum Pf s230 and Pf s48/45 proteins are expressed during transmission from man to mosquito and are leading candidates for a malaria transmission blocking vaccine. Individually they generate transmission blocking (TB) antibodies in rodent models. Whether the single protein vaccines are suitable to use in field settings will primarily depend on their potency to elicit functional antibodies. We hypothesized that a combination of both proteins will be more potent than each protein individually. Therefore we designed chimeric proteins composed of fragments of both Pf s230 and Pf s48/45 as well as single protein fragments, and expressed these in Lactoccus lactis . Both the individual Pf s230 and Pf s48/45 fragments and chimeras elicited high levels of functional antibodies in mice. Importantly, one of the chimeric proteins elicited over threefold higher transmission blocking antibody responses than the single antigens alone. Furthermore the immunogenicity of one of the chimeras could be enhanced through coupling to a virus-like particle (VLP). Altogether these data support further clinical development of these novel constructs.

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“…Culture supernatants were clarified by centrifugation at 9000 g for 20 min and protein expression levels were assessed in the culture supernatants by ELISA using HRP-conjugated anti-His antibody (MACS, Miltenyi biotech, Germany) and by SDS-PAGE. L. lactis MG1363 harboring expression constructs was grown in a 1 l stirred bioreactors for 15 h at 30 °C [ 23 ]. Cells were removed by centrifugation at 9000 rpm for 30 min and the raw culture supernatant was clarified by filtration.…”

Section: Methodsmentioning

confidence: 99%

Lactococcus lactis provides an efficient platform for production of disulfide-rich recombinant proteins from Plasmodium falciparum

et al. 2018

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BackgroundThe production of recombinant proteins with proper conformation, appropriate post-translational modifications in an easily scalable and cost-effective system is challenging. Lactococcus lactis has recently been identified as an efficient Gram positive cell factory for the production of recombinant protein. We and others have used this expression host for the production of selected malaria vaccine candidates. The safety of this production system has been confirmed in multiple clinical trials. Here we have explored L. lactis cell factories for the production of 31 representative Plasmodium falciparum antigens with varying sizes (ranging from 9 to 90 kDa) and varying degree of predicted structural complexities including eleven antigens with multiple predicted structural disulfide bonds, those which are considered difficult-to-produce proteins.ResultsOf the 31 recombinant constructs attempted in the L. lactis expression system, the initial expression efficiency was 55% with 17 out of 31 recombinant gene constructs producing high levels of secreted recombinant protein. The majority of the constructs which failed to produce a recombinant protein were found to consist of multiple intra-molecular disulfide-bonds. We found that these disulfide-rich constructs could be produced in high yields when genetically fused to an intrinsically disorder protein domain (GLURP-R0). By exploiting the distinct biophysical and structural properties of the intrinsically disordered protein region we developed a simple heat-based strategy for fast purification of the disulfide-rich protein domains in yields ranging from 1 to 40 mg/l.ConclusionsA novel procedure for the production and purification of disulfide-rich recombinant proteins in L. lactis is described.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0902-2) contains supplementary material, which is available to authorized users.

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Construct design, production, and characterization of Plasmodium falciparum 48/45 R0.6C subunit protein produced in Lactococcus lactis as candidate vaccine

Cited by 38 publications

References 33 publications

New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue

New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue

Pfs230 and Pfs48/45 Fusion Proteins Elicit Strong Transmission-Blocking Antibody Responses Against Plasmodium falciparum

Lactococcus lactis provides an efficient platform for production of disulfide-rich recombinant proteins from Plasmodium falciparum

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