A Full-Length Plasmodium falciparum Recombinant Circumsporozoite Protein Expressed by Pseudomonas fluorescens Platform as a Malaria Vaccine Candidate

Abstract

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.

Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria

“…Sixteen weeks after HSC engraftment, when CD4+ T cells and B cells were fully reconstituted in AAV9-transduced NSG mice, the HIS-CD4/B mice were immunized with a recombinant circumsporozoite (CS) protein of Plasmodium falciparum, PfCS protein (Noe et al, 2014).…”

“…The indirect immunofluorescence assay (IFA) was carried out using transgenic P. berghei sporozoites expressing PfCS protein repeats or wild-type P. berghei sporozoites, as an antigen, as we previously performed (Noe et al, 2014). Briefly, after air-dried sporozoites on multi-well glass slides were incubated with diluted sera collected from immunized HIS-CD4/B mice, for 1 hour, the slides were washed and incubated with Alexa Fluor 488-labeled goat anti-human IgG antibody (Invitrogen) for 1 hour.…”

“…After 24-hr incubation, the plate was washed and incubated with biotinylated anti-human IFN-γ or anti-human IL-2 antibody, developed by adding AEC substrate (BD Bioscience), and the numbers of spot-forming cells counted as described (Noe et al, 2014).…”

“…HepG2 cells that express human CD81 were first seeded onto a 96 well plate at 2 x 10 5 cells/well, and incubated for overnight at 37°C 5% CO 2 (Noe et al, 2014;Shiratsuchi et al, 2010).…”

“…After two-hour incubation, uninfected sporozoites were washed out, and then the cells were cultured for 42 hours. Total RNA was prepared and the relative amount of parasite 18S ribosomal RNA to human GAPDH was determined by a real-time qRT-PCR, using 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) (Noe et al, 2014;Shiratsuchi et al, 2010).…”