A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.

Reliable protein production in a Pseudomonas fluorescens expression system.

A bottleneck to product development can be reliable expression of active target protein. A wide array of recombinant proteins in development, including an ever growing number of non-natural proteins, is being expressed in a variety of expression systems. A Pseudomonas fluorescens expression platform has been developed specifically for recombinant protein production. The development of an integrated molecular toolbox of expression elements and host strains, along with automation of strain screening is described. Examples of strain screening and scale-up experiments show rapid development of expression strains producing a wide variety of proteins in a soluble active form.

Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens.

Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens. The human g-csf gene was optimized for expression in P. fluorescens and cloned into a set of periplasmic expression vectors. These plasmids were transformed into a variety of P. fluorescens host strains each having a unique phenotype, to evaluate soluble expression in a 96-well growth and protein expression format. To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfenex Expression Technology™ toolbox were screened in parallel using biolayer interferometry (BLI) to quantify active G-CSF binding to its receptor. A subset of these strains was screened by LC-MS analysis to assess the quality of the expressed G-CSF protein. A single strain with an antibiotic resistance marker insertion in the pfaI gene was identified that produced>99% Met-GCSF. A host with a complete deletion of the autotransporter-coding gene pfaI from the genome was constructed, and expression of soluble, active Met-GSCF in this strain was observed to be 350mg/L at the 1 liter fermentation scale.

Transport of heterologous proteins to the periplasmic space of Pseudomonas fluorescens using a variety of native signal sequences.

Bacterial expression of recombinant proteins containing disulfide bonds is facilitated by transport of the proteins to the periplasmic space. Several Pseudomonas fluorescens signal sequences have been identified that efficiently direct proteins to the periplasm and provide solubility and yield advantages over the production of proteins fused to the PelB signal sequence in E. coli. For a single chain antibody fragment, the final yield varied from about 1 g/l to 10 g/l when expression in P. fluorescens involved fusion to various P. fluorescens signal sequences.

Characterization of the SOS response of Pseudomonas fluorescens strain DC206 using whole-genome transcript analysis.

DNA microarray technology was used to survey changes in gene expression in Pseudomonas fluorescens after mitomycin C treatment. As expected, genes associated with the SOS response were upregulated, such as those encoding the recombination protein RecA, DNA repair protein RecN, excinuclease ABC subunit A UvrA, and the LexA repressor protein. Interestingly, expression of 33 clustered bacteriophage-like genes was upregulated, suggesting that mitomycin C (MMC) may induce a prophage resident in the P. fluorescens genome. However, no phage particles were detected in P. fluorescens strain DC206 that had been treated with MMC using transmission electron microscopy. The same preparation failed to produce phage plaques on lawns of any of 10 different Pseudomonas strains tested, indicating that the 33 bacteriophage-like gene cluster represents a defective prophage.

Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions.

BACKGROUND: In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD). RESULTS: The antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR). CONCLUSION: We have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales.