Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation

Faber, Bart W.; Hellwig, Stephan; Houard, Sophie; Havelange, Nicolas; Drossard, Jürgen; Mertens, Hubert; Croon, Alexander; Kastilan, Robin; Byrne, Richard; Werff, Nicole van der; Eijk, Marjolein van der; Thomas, Alan W.; Kocken, Clemens H. M.; Remarque, Edmond J.

doi:10.1371/journal.pone.0164053

Cited by 9 publications

(5 citation statements)

References 45 publications

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“…Although AMA-1 is the major target in naturally-acquired invasion inhibitory antibodies, this protein has a high degree of allelic diversity 56 , 57 ; several studies analyzing AMA-1 sequences from different geographical regions have shown that the ama-1 gene is under balancing selection, thereby posing a challenge when designing a vaccine based on this antigen 58 – 61 . Even though different authors have suggested including multiple alleles in a vaccine to induce antibodies having wide-scale reactivity and thus covering the parasite population’s global genetic diversity 62 , 63 , it is also important to ascertain which AMA-1 regions are involved in this protein’s vital functions to guide any immune response towards these regions thereby leading to developing control methods covering Plasmodium ’s broad allele spectrum.…”

Section: Introductionmentioning

confidence: 99%

Plasmodium vivax ligand-receptor interaction: PvAMA-1 domain I contains the minimal regions for specific interaction with CD71+ reticulocytes

Arévalo-Pinzón

Bermúdez

Hernández

et al. 2017

Sci Rep

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The malarial parasite’s invasion is complex, active and coordinated, involving many low and high affinity interactions with receptors on target cell membrane. Proteomics analysis has described around 40 proteins in P. vivax which could be involved in reticulocyte invasion; few have been studied with the aim of elucidating how many of them establish specific interactions with their respective host cells. Given the importance of knowing which of the parasite’s protein regions are functionally important for invasion, minimum regions mediating specific interaction between Plasmodium vivax apical membrane antigen 1 (PvAMA-1) and its host cell were here elucidated. The region covering PvAMA-1 domains I and II (PvAMA-DI-II) specifically bound to the CD71+ red blood cell subpopulation. A 20 residue-long region (81EVENAKYRIPAGRCPVFGKG100) located in domain I was capable of inhibiting PvAMA-DI-II recombinant protein binding to young reticulocytes (CD71+CD45−) and rosette formation. This conserved peptide specifically interacted with high affinity with reticulocytes (CD71+) through a neuraminidase- and chymotrypsin-treatment sensitive receptor. Such results showed that, despite AMA-1 having universal functions during late Plasmodium invasion stages, PvAMA-1 had reticulocyte-preferring binding regions, suggesting that P. vivax target cell selection is not just restricted to initial interactions but maintained throughout the erythrocyte invasion cycle, having important implications for designing a specific anti-P. vivax vaccine.

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Section: Introductionmentioning

confidence: 99%

Plasmodium vivax ligand-receptor interaction: PvAMA-1 domain I contains the minimal regions for specific interaction with CD71+ reticulocytes

Arévalo-Pinzón

Bermúdez

Hernández

et al. 2017

Sci Rep

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“…The field of malaria vaccines has historically seen many candidate antigens from all lifecycle stages produced for clinical trial as recombinant proteins in bacterial- and yeast-based expression platforms including E. coli , 41 – 47 Lactococcus lactis , 48 Saccharomyces cerevisiae 49 , 50 and Pichia pastoris . 51 – 54 However, generation of full-length PfRH5 protein proved particularly problematic in these heterologous expression platforms. Consequently, viral-vectored immunization led to the first promising results in animal models, 15 whereby antigen is expressed in situ from virally infected muscle cells.…”

Section: Discussionmentioning

confidence: 99%

Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells

et al. 2018

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Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is a leading asexual blood-stage vaccine candidate for malaria. In preparation for clinical trials, a full-length PfRH5 protein vaccine called “RH5.1” was produced as a soluble product under cGMP using the ExpreS2 platform (based on a Drosophila melanogaster S2 stable cell line system). Following development of a high-producing monoclonal S2 cell line, a master cell bank was produced prior to the cGMP campaign. Culture supernatants were processed using C-tag affinity chromatography followed by size exclusion chromatography and virus-reduction filtration. The overall process yielded >400 mg highly pure RH5.1 protein. QC testing showed the MCB and the RH5.1 product met all specified acceptance criteria including those for sterility, purity, and identity. The RH5.1 vaccine product was stored at −80 °C and is stable for over 18 months. Characterization of the protein following formulation in the adjuvant system AS01B showed that RH5.1 is stable in the timeframe needed for clinical vaccine administration, and that there was no discernible impact on the liposomal formulation of AS01B following addition of RH5.1. Subsequent immunization of mice confirmed the RH5.1/AS01B vaccine was immunogenic and could induce functional growth inhibitory antibodies against blood-stage P. falciparum in vitro. The RH5.1/AS01B was judged suitable for use in humans and has since progressed to phase I/IIa clinical trial. Our data support the future use of the Drosophila S2 cell and C-tag platform technologies to enable cGMP-compliant biomanufacture of other novel and “difficult-to-express” recombinant protein-based vaccines.

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“…The levels of antibodies against recombinant PfAMA1 and PfCSP, as well as those against two conserved synthetic PfCSP peptides, were measured by in-house optimized ELISAs. The recombinant PfAMA1 antigen (amino acids 25 – 545) was based on the 3D7 parasite clone antigen (NCBI Reference Sequence: NC_037282), expressed in Pichia pastoris and purified by a methodology that has been previously described ( Faber et al., 2008 ; Faber et al., 2016 ). The recombinant PfCSP antigen (amino acids 26 – 383) contained 4 NVDP and 38 NANP repeats (NCBI Reference Sequence: XM_001351086.1) from the 3D7 clone and was expressed in Lactococcus lactis as previously described ( Singh et al., 2018 ; Singh et al., 2020 ).…”

Section: Methodsmentioning

confidence: 99%

Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana

Kusi,

Amoah,

Acquah

et al. 2024

Front. Cell. Infect. Microbiol.

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IntroductionDiversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity.MethodsThe study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA. ResultsOf the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons. DiscussionThese data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene.

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Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation

Cited by 9 publications

References 45 publications

Plasmodium vivax ligand-receptor interaction: PvAMA-1 domain I contains the minimal regions for specific interaction with CD71+ reticulocytes

Plasmodium vivax ligand-receptor interaction: PvAMA-1 domain I contains the minimal regions for specific interaction with CD71+ reticulocytes

Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells

Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana

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