Hongfan Jin

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.

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 sporozoites 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.

Comparison of Methods for Chemical Conjugation of an Influenza Peptide to Wild-Type and Cysteine-Mutant Virus-Like Particles Expressed in Pseudomonas fluorescens

Chemical conjugation of the influenza peptide antigen M2E to different variants of virus-like particles (VLPs) was investigated. Wild-type cowpea chlorotic mottle virus (CCMV) and two novel cysteine mutants of CCMV, all expressed in Pseudomonas fluorescens, were utilized in this study. Two different conjugation schemes, primary amine-directed and cysteine-directed, were tested and compared. Both strategies were successfully used to attach M2E peptides to the surface of these VLPs. Ultimately, the cysteine-directed conjugation strategy using the CCMV cysteine mutant particles displayed key advantages over the primary amine-directed strategy.