Potent Malaria Transmission-Blocking Antibody Responses Elicited by Plasmodium falciparum Pfs25 Expressed in Escherichia coli after Successful Protein Refolding

Abstract

Production of Pfs25, a Plasmodium falciparum transmission-blocking vaccine target antigen, in functional conformation with the potential to elicit effective immunogenicity still remains a major challenge. In the current study, codon-harmonized recombinant Pfs25 (CHrPfs25) was expressed in Escherichia coli, and purified protein after simple oxidative refolding steps retained reduction-sensitive conformational epitopes of transmission-blocking monoclonal antibodies. CHrPfs25 formulated in several adjuvants elicited strong immunogenicity in preclinical studies in mice. Antibodies elicited after immunization recognized native Pfs25 on the surface of live gametes of P. falciparum and demonstrated complete malaria transmission-blocking activity. The transmission-blocking efficacy was 100% even after a 1:128 dilution of sera from immunized mice in the complete Freund's adjuvant and Montanide ISA51 groups and after a 1:16 dilution of sera from mice in the alum group. The blocking was mediated by antibodies; purified IgG at concentrations as low as 31.25 g/ml exhibited 100% transmission blocking in membrane feeding assays employing two different species of mosquitoes, Anopheles gambiae and Anopheles stephensi. This study provides the first evidence for successful expression of biologically functional rPfs25 in E. coli. The extremely potent malaria transmission-blocking activity of antibodies elicited by immunization with purified protein provides strong support for further evaluation of E. coli-derived CHrPfs25 as a malaria transmission-blocking vaccine in human clinical trials. N early half the world's population lives in regions where malaria is endemic, and this accounts for approximately 219 million clinical cases with up to 660,000 deaths, mostly of children under five years of age (1, 2). The parasite has displayed a remarkable ability to develop resistance to almost any antimalaria drug used, and there has been a recent report on artemisinin-resistant Plasmodium falciparum in Cambodia (3). While efforts to develop vaccines targeting various life cycle stages of the parasite are under way, in both the human host and the mosquito vector, none is available currently. Vaccines targeting the transmission stages of the parasites, the sexual stages, are considered essential to achieve the goal of gradual elimination of malaria. Malaria transmission reduction can be achieved either by blocking the development of gametocytes, the sexual stages of the parasite, or by reducing further development of these transmission stages in the mosquito vector (4-6). Recent emphasis on global elimination and eradication of malaria has outlined a critical role for a malaria transmission-blocking vaccine (TBV) as an effective tool for reducing malaria transmission.The long-term success of a TBV depends upon induction of high functional antibody titers in order to effectively block the parasite transmission cycle (7). In P. falciparum, Pfs230, Pfs48/45, and Pfs25 have been identified as target antigens, and antibodies directed against any o...

Evaluation of the Impact of Codon Optimization and N-Linked Glycosylation on Functional Immunogenicity of Pfs25 DNA Vaccines Delivered by In Vivo Electroporation in Preclinical Studies in Mice

“…Recombinant Pfs25 (rPfs25) (25) was run on a 12.5% polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked with 5% milk-PBST (PBS ϩ 0.1% Tween 20), and individual strips were incubated for 1 h with pooled immune sera at 1:4,000 dilution and processed using enhanced chemiluminescence (ECL) (Amersham Biosciences, NJ) (26).…”

Alga-Produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated with a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium falciparum Infection of Mosquitoes

“…The many different systems, i.e., E. coli (10), tobacco (38,39), Saccharomyces cerevisiae (11,37,40,41), Pichia pastoris (12,42), DNA vaccines (43,44), wheat germ (15), insect cells via baculovirus (13,16), protein-peptide conjugates (45), Pseudomonas aeruginosa ExoProtein A (rEPA) (46), cholera toxin adjuvant (12), and virally vectored vaccines (47,48), by which recombinant Pfs25 has been produced have uniformly found that recombinant Pfs25 administered in animal models in the absence of adjuvant is poorly immunogenic, with relatively low IgG titers. The importance of a safe and effective adjuvant for Pfs25-regardless of the heterologous expression system-was highlighted in a phase I clinical trial in which Montanide IS151 combined with Pichia-produced Pfs25 was associated with leukemoid reactions and erythema nodosum in humans (21).…”

“…Parasite-produced Pfs25 contains 4 epidermal growth factor (EGF)-like domains and is not glycosylated, which makes it biotechnologically challenging to produce the properly folded, conformationally correct recombinant protein(s) required for the induction of effective transmission-blocking antibodies (7)(8)(9). Heterologous expression systems used to produce Pfs25 as a recombinant subunit immunogen include Escherichia coli (10), Saccharomyces cerevisiae (11), Pichia pastoris (7,12), baculovirus (13), Nicotiana benthamiana (14), Triticum vulgare (wheat germ) extract (6,15,16), and most recently, the chloroplast of the microalga Chlamydomonas reinhardtii (17,18).…”

Salmonella flagellin acted as an effective fusion partner for expression of Plasmodium falciparum surface protein 25 in Escherichia coli

“…However, studies showed that Pfs25 was not only lowly expressed but also aggregated in the form of inclusion body in E. coli. 2,3 The ability of insoluble Pfs25 to induce functional antibodies was greatly reduced. 2 Co-expression with a fusion partner is one of the common strategies to improve the expression of some hard-to-express and hard-to-solubilize proteins in E. coli.…”

“…1B, Lane 2). Compared to the single expression of Pfs25 in E. coli, which presented only a faint protein band on SDS-PAGE gel, 3 the fusion expression with FliC remarkably enhanced the expression level of Pfs25 in E. coli. The solubility of FliC-Pfs25 was evidenced by the fact that a large amount of the fusion protein existed in the sonicated cell supernatant of E. coli after IPTG induction (Fig.…”