Expression of malaria transmission-blocking vaccine antigen Pfs25 in Pichia pastoris for use in human clinical trials

Zou, Lanling; Miles, Aaron P.; Wang, Jin; Stowers, Anthony W.

doi:10.1016/s0264-410x(02)00701-6

Cited by 69 publications

(56 citation statements)

References 18 publications

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“…The truncated versions of the PfCSP and Pfs25-3D7 genes were amplified by PCR from the genomic DNA of the P. falciparum 3D7 strain and subcloned into pEU3 (a vector carrying the Cterminal His 6 tag) (29) at the EcoRV site. The gene encoding Pfs25-TBV was a generous gift from Anthony W. Stowers (NIAID, NIH, Rockville, MD) (35). Pfs25-TBV, a synthetic version of the Pfs25 gene, was codon optimized for expression in the yeast Saccharomyces cerevisiae, and the replacement of Asn with Gln at three N-glycosylation sites was performed (20).…”

Section: Methodsmentioning

confidence: 99%

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates

Takeo²,

et al. 2008

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One of the major bottlenecks in malaria research has been the difficulty in recombinant protein expression. Here, we report the application of the wheat germ cell-free system for the successful production of malaria proteins. For proof of principle, the Pfs25, PfCSP, and PfAMA1 proteins were chosen. These genes contain very high A/T sequences and are also difficult to express as recombinant proteins. In our wheat germ cell-free system, native and codon-optimized versions of the Pfs25 genes produced equal amounts of proteins. PfCSP and PfAMA1 genes without any codon optimization were also expressed. The products were soluble, with yields between 50 and 200 g/ml of the translation mixture, indicating that the cell-free system can be used to produce malaria proteins without any prior optimization of their biased codon usage. Biochemical and immunocytochemical analyses of antibodies raised in mice against each protein revealed that every antibody retained its high specificity to the parasite protein in question. The development of parasites in mosquitoes fed patient blood carrying Plasmodium falciparum gametocytes and supplemented with our mouse anti-Pfs25 sera was strongly inhibited, indicating that both Pfs25-3D7/WG and Pfs25-TBV/WG retained their immunogenicity. Lastly, we carried out a parallel expression assay of proteins of blood-stage P. falciparum. The PCR products of 124 P. falciparum genes chosen from the available database were used directly in a small-scale format of transcription and translation reactions. Autoradiogram testing revealed the production of 93 proteins. The application of this new cell-free system-based protocol for the discovery of malaria vaccine candidates will be discussed.Plasmodium falciparum is the protozoan responsible for the widespread return of malaria to tropical countries, particularly in Africa. This reemergence is generally credited to two causes: the development of multidrug-resistant parasites and the development of insecticide-resistant mosquitoes (10). Through decades of work, scientists have learned that vaccination could be a potent curative, but efforts to develop a successful vaccine have not yet succeeded (25). One of the bottlenecks in vaccine development is at the malaria protein production step and is mainly due to the lack of a methodology to enable preparation of quality proteins in an efficient manner. P. falciparum genes have a very high A/T content (average, 76% per gene), and a number of them encode repeated stretches of amino acid sequences (8); these features have been proposed as the major factors limiting P. falciparum protein expression in cell-based systems. Moreover, the presence of glycosylation machinery in eukaryotic cell-based systems can produce inappropriately glycosylated recombinant malaria proteins, resulting in incorrect immune responses (9,21,26). In fact, the three pioneering genome-wide studies on the production of P. falciparum proteins in cell-based systems faced serious problems. For instance, Aguiar et al. (1) were able to obtain exp...

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Section: Methodsmentioning

confidence: 99%

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates

Takeo²,

et al. 2008

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“…It was purified from the culture supernatant by microfiltration and ultrafiltration, affinity chromatography on nickeltrilotriacetic acid Superflow, hydrophobic interaction chromatography on a Phenyl Sepharose column, and size-exclusion chromatography with Superdex 75 (17). OMPC (nonproduct lots) was purified from a detergent extract of N. meningitidis strain B11 being isolated by ultracentrifugation, diafiltration, and sterile filtration (18).…”

Section: Methodsmentioning

confidence: 99%

Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex

Przysiecki²,

Flanagan³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

118

113

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The development of protein subunit vaccines to combat some of the world's deadliest pathogens such as a malaria parasite, Plasmodium falciparum, is stalled, due in part to the inability to induce and sustain high-titer antibody responses. Here, we show the induction of persistent, high-titer antibody responses to recombinant Pfs25H, a human malarial transmission-blocking protein vaccine candidate, after chemical conjugation to the outer-membrane protein complex (OMPC) of Neisseria meningitidis serogroup B and adsorption to aluminum hydroxyphosphate. In mice, the Pfs25H-OMPC conjugate vaccine was >1,000 times more potent in generating anti-Pfs25H ELISA reactivity than a similar 0.5-g dose of Pfs25H alone in Montanide ISA720, a water-in-oil adjuvant. The immune enhancement requires covalent conjugation between Pfs25H and the OMPC, given that physically mixed Pfs25H and OMPC on aluminum hydroxyphosphate failed to induce greater activity than the nonconjugated Pfs25H on aluminum hydroxyphosphate. The conjugate vaccine Pfs25H-OMPC also was highly immunogenic in rabbits and rhesus monkeys. In rhesus monkeys, the antibody responses were sustained over 18 months, at which time another vaccination with nonconjugated Pfs25H induced strong anamnestic responses. The vaccine-induced anti-Pfs25-specific antibodies in all animal species blocked the transmission of parasites to mosquitoes. Protein antigen conjugation to OMPC or other protein carrier may have general application to a spectrum of protein subunit vaccines to increase immunogenicity without the need for potentially reactogenic adjuvants. malaria ͉ Pfs25 ͉ transmission-blocking vaccine A s reported by the World Health Organization in 2004, the worldwide incidence of malaria is Ϸ300 million clinical cases and 1.3 million deaths annually (1). Of the four species of malaria parasites that infect humans, Plasmodium falciparum is responsible for the majority of these deaths, and Plasmodium vivax accounts for Ͼ50% of all malarial infections outside Africa and 10% of those in Africa. Mounting drug resistance by the malaria parasite makes chemotherapy increasingly difficult. Three types of malarial vaccines are under research and development: (i) vaccines targeting the liver-stage parasite development for sterile immunity; (ii) vaccines targeting the blood-stage parasite to reduce disease burden; and (iii) vaccines targeting the parasite development in the mosquito stage to block transmission, called transmission-blocking vaccines (TBVs). For all three types of vaccines, antibody is important. For TBVs, antibody is the only mechanism for providing immune protection. TBVelicited functional antibodies, ingested with the sexual stages of the parasite in a blood meal by a mosquito, will inhibit or block parasite development in the mosquito.Pxs25 proteins encoded by orthologous genes and expressed on the surface of zygotes and ookinetes during the development of the malaria parasite P. falciparum (Pfs25) and P. vivax (Pvs25) are leading candidates for mosquito-stage transmission...

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“…Clinical grade Pvs25 [13] and Pfs25 [14] were prepared as described previously. In brief, the parasite gene encoding Pvs25 was cloned into the plasmid YEpRPEU-3, transformed into Saccharomyces cerevisiae VK1 cells.…”

Section: Antigen Preparationmentioning

confidence: 99%

Development and characterization of a standardized ELISA including a reference serum on each plate to detect antibodies induced by experimental malaria vaccines

Miura

Orcutt

Muratova

et al. 2008

Vaccine

203

188

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Enzyme linked immunosorbent assay (ELISA) has been widely used to measure antibody titers for evaluating the immunogenicity of a vaccine. However, there is as yet no generally accepted way of expressing the ELISA results in the case of experimental vaccines, since there is usually no uniform standard. Both end point and single dilution methods have significant disadvantages. In this paper, we obtained reproducible data with fewer dilutions of samples by addition of serially diluted standard serum to each ELISA plate. Since this ELISA method gives reliable antibody titer with less labor than other methods, it can strongly support vaccine development.

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Expression of malaria transmission-blocking vaccine antigen Pfs25 in Pichia pastoris for use in human clinical trials

Cited by 69 publications

References 18 publications

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates

Wheat Germ Cell-Free System-Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates

Sustained high-titer antibody responses induced by conjugating a malarial vaccine candidate to outer-membrane protein complex

Development and characterization of a standardized ELISA including a reference serum on each plate to detect antibodies induced by experimental malaria vaccines

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