Development and evaluation of a new Plasmodium falciparum 3D7 blood stage malaria cell bank for use in malaria volunteer infection studies

Abstract Background New anti-malarial therapeutics are required to counter the threat of increasing drug resistance. Malaria volunteer infection studies (VIS), particularly the induced blood stage malaria (IBSM) model, play a key role in accelerating anti-malarial drug development. Supply of the reference 3D7-V2 Plasmodium falciparum malaria cell bank (MCB) is limited. This study aimed to develop a new MCB, and compare the safety and infectivity of this MCB with the existing 3D7-V2 MCB, in a VIS. A second bank (3D7-V1) developed in 1995 was also evaluated. Methods The 3D7-V2 MCB was expanded in vitro using a bioreactor to produce a new MCB designated 3D7-MBE-008. This bank and 3D7-V1 were then evaluated using the IBSM model, where healthy participants were intravenously inoculated with blood-stage parasites. Participants were treated with artemether-lumefantrine when parasitaemia or clinical thresholds were reached. Safety, infectivity and parasite growth and clearance were evaluated. Results The in vitro expansion of 3D7-V2 produced 200 vials of the 3D7-MBE-008 MCB, with a parasitaemia of 4.3%. This compares to 0.1% in the existing 3D7-V2 MCB, and < 0.01% in the 3D7-V1 MCB. All four participants (two per MCB) developed detectable P. falciparum infection after inoculation with approximately 2800 parasites. For the 3D7-MBE-008 MCB, the parasite multiplication rate of 48 h (PMR48) using non-linear mixed effects modelling was 34.6 (95% CI 18.5–64.6), similar to the parental 3D7-V2 line; parasitaemia in both participants exceeded 10,000/mL by day 8. Growth of the 3D7-V1 was slower (PMR48 of 11.5 [95% CI 8.5–15.6]), with parasitaemia exceeding 10,000 parasites/mL on days 10 and 8.5. Rapid parasite clearance followed artemether-lumefantrine treatment in all four participants, with clearance half-lives of 4.01 and 4.06 (weighted mean 4.04 [95% CI 3.61–4.57]) hours for 3D7-MBE-008 and 4.11 and 4.52 (weighted mean 4.31 [95% CI 4.16–4.47]) hours for 3D7-V1. A total of 59 adverse events occurred; most were of mild severity with three being severe in the 3D7-MBE-008 study. Conclusion The safety, growth and clearance profiles of the expanded 3D7-MBE-008 MCB closely resemble that of its parent, indicating its suitability for future studies. Trial Registration: Australian New Zealand Clinical Trials registry numbers: P3487 (3D7-V1): ACTRN12619001085167. P3491 (3D7-MBE-008): ACTRN12619001079134

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Development and evaluation of a new Plasmodium falciparum 3D7 blood stage malaria cell bank for use in malaria volunteer infection studies

10.21203/rs.3.rs-90708/v3 ◽

2021 ◽

Author(s):

Stephen Derek Woolley ◽

Melissa Fernandez ◽

Maria Rebelo ◽

Stacey Llewellyn ◽

Louise Marquart ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Parasite Clearance ◽

Blood Stage ◽

Multiplication Rate ◽

Weighted Mean ◽

Cell Bank ◽

Parasite Multiplication ◽

Clinical Trials Registry ◽

Blood Stage Malaria

Abstract Background New anti-malarial therapeutics are required to counter the threat of increasing drug resistance. Malaria volunteer infection studies (VIS), particularly the induced blood stage malaria (IBSM) model, play a key role in accelerating anti-malarial drug development. Supply of the reference 3D7-V2 Plasmodium falciparum malaria cell bank (MCB) is limited. This study aimed to develop a new MCB, and compare the safety and infectivity of this MCB with the existing 3D7-V2 MCB, in a VIS. A second bank (3D7-V1) developed in 1995 was also evaluated.Methods The 3D7-V2 MCB was expanded in vitro using a bioreactor to produce a new MCB designated 3D7-MBE-008. This bank and 3D7-V1 were then evaluated using the IBSM model, where healthy participants were intravenously inoculated with blood-stage parasites. Participants were treated with artemether-lumefantrine when parasitaemia or clinical thresholds were reached. Safety, infectivity and parasite growth and clearance were evaluated. Results The in vitro expansion of 3D7-V2 produced 200 vials of the 3D7-MBE-008 MCB, with a parasitaemia of 4.3%. This compares to 0.1% in the existing 3D7-V2 MCB, and <0.01% in the 3D7-V1 MCB. All four participants (two per MCB) developed detectable P. falciparum infection after inoculation with approximately 2800 parasites. For the 3D7-MBE-008 MCB, the parasite multiplication rate of 48 hours (PMR48) using non-linear mixed effects modelling was 34.6 (95% CI: 18.5 – 64.6), similar to the parental 3D7-V2 line; parasitaemia in both participants exceeded 10,000/mL by day 8. Growth of the 3D7-V1 was slower (PMR48 of 11.5 [95% CI: 8.5 – 15.6]), with parasitaemia exceeding 10,000 parasites/mL on days 10 and 8.5. Rapid parasite clearance followed artemether-lumefantrine treatment in all four participants, with clearance half-lives of 4.01 and 4.06 (weighted mean 4.04 [95% CI: 3.61 – 4.57]) hours for 3D7-MBE-008 and 4.11 and 4.52 (weighted mean 4.31 [95% CI: 4.16 – 4.47]) hours for 3D7-V1. A total of 59 adverse events occurred; most were of mild severity with three being severe in the 3D7-MBE-008 study. Conclusion The safety, growth and clearance profiles of the expanded 3D7-MBE-008 MCB closely resemble that of its parent, indicating its suitability for future studies. Trial Registration Australian New Zealand Clinical Trials registry numbers:P3487 (3D7-V1): ACTRN12619001085167P3491 (3D7-MBE-008): ACTRN12619001079134

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Development and evaluation of a new P. falciparum 3D7 blood stage malaria cell bank for use in malaria volunteer infection studies

10.21203/rs.3.rs-90708/v2 ◽

2021 ◽

Author(s):

Stephen Derek Woolley ◽

Melissa Fernandez ◽

Maria Rebelo ◽

Stacey Llewellyn ◽

Louise Marquart ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Parasite Clearance ◽

Blood Stage ◽

Multiplication Rate ◽

Weighted Mean ◽

Cell Bank ◽

Parasite Multiplication ◽

Clinical Trials Registry ◽

Blood Stage Malaria

Abstract Background New antimalarial therapeutics are required to counter the threat of increasing drug resistance. Malaria volunteer infection studies (VIS), particularly the induced blood stage malaria (IBSM) model, play a key role in accelerating antimalarial drug development. Supply of the reference 3D7-V2 Plasmodium falciparum malaria cell bank (MCB) is limited. We aimed to develop a new MCB, and compare the safety and infectivity of this MCB with the existing 3D7-V2 MCB, in a VIS. A second bank (3D7-V1) developed in 1995 was also evaluated.Methods We expanded the 3D7-V2 MCB in vitro using a bioreactor to produce a new MCB designated 3D7-MBE-008. This bank and 3D7-V1 were then evaluated using the IBSM model, where healthy participants were intravenously inoculated with blood-stage parasites. Participants were treated with artemether-lumefantrine when parasitaemia or clinical thresholds were reached. Safety, infectivity and parasite growth and clearance were evaluated. Results The in vitro expansion of 3D7-V2 produced 200 vials of the 3D7-MBE-008 MCB, with a parasitaemia of 4.3%. This compares to 0.1% in the existing 3D7-V2 MCB, and <0.01% in the 3D7-V1 MCB. All four participants (two per MCB) developed detectable Plasmodium falciparum infection after inoculation with approximately 2800 parasites. For the 3D7-MBE-008 MCB, the parasite multiplication rate of 48 hours (PMR48) using non-linear mixed effects modelling was 34.6 (95% CI: 18.5 – 64.6), similar to the parental 3D7-V2 line; parasitaemia in both participants exceeded 10,000/mL by day 8. Growth of the 3D7-V1 was slower (PMR48 of 11.5 [95% CI: 8.5 – 15.6]), with parasitaemia exceeding 10,000 parasites/mL on days 10 and 8.5. Rapid parasite clearance followed artemether-lumefantrine treatment in all four participants, with clearance half-lives of 4.01 and 4.06 (weighted mean 4.04 [95% CI: 3.61 – 4.57]) hours for 3D7-MBE-008 and 4.11 and 4.52 (weighted mean 4.31 [95% CI: 4.16 – 4.47]) hours for 3D7-V1. A total of 59 adverse events occurred; most were of mild severity with three being severe in the 3D7-MBE-008 study. Conclusion The safety, growth and clearance profiles of the expanded 3D7-MBE-008 MCB closely resemble that of its parent, indicating its suitability for future studies.Trial Registration Australian New Zealand Clinical Trials registry numbers:P3487 (3D7-V1): ACTRN12619001085167P3491 (3D7-MBE-008): ACTRN12619001079134

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Development and Evaluation of a New P. Falciparum 3D7 Blood Stage Malaria Cell Bank for Use in Malaria Volunteer Infection Studies

10.21203/rs.3.rs-90708/v1 ◽

2020 ◽

Author(s):

Stephen Derek Woolley ◽

Melissa Fernandez ◽

Maria Rebelo ◽

Stacey Llewellyn ◽

Louise Marquart ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Parasite Clearance ◽

Blood Stage ◽

Cell Bank ◽

Future Studies ◽

Parasite Multiplication ◽

Clinical Trials Registry ◽

Healthy Participants ◽

Blood Stage Malaria

Abstract BackgroundNew antimalarial therapeutics are required to counter the threat of increasing drug resistance. Malaria volunteer infection studies (VIS), particularly the induced blood stage malaria (IBSM) model, play a key role in accelerating antimalarial drug development. Supply of the reference 3D7-V2 Plasmodium falciparum malaria cell bank (MCB) is limited. We aimed to develop a new MCB, and compare the safety and infectivity of this MCB with the existing 3D7-V2 MCB, in a VIS. A second bank (3D7-V1) developed in 1995 was also evaluated.MethodsWe expanded the 3D7-V2 MCB in vitro using a bioreactor to produce a new MCB designated 3D7-MBE-008. This bank and 3D7-V1 were then evaluated using the IBSM model, where healthy participants were intravenously inoculated with blood-stage parasites. Participants were treated with artemether-lumefantrine when parasitaemia or clinical thresholds were reached. Safety, infectivity and parasite growth and clearance were evaluated. ResultsThe in vitro expansion of 3D7-V2 produced 200 vials of the 3D7-MBE-008 MCB, with a parasitaemia of 4.3%. This compares to 0.1% in the existing 3D7-V2 MCB, and <0.01% in the 3D7-V1 MCB. All four participants, (two per MCB) developed detectable Plasmodium falciparum infection after inoculation with approximately 2800 parasites. The parasite multiplication rates of 48 hours (PMR48) for the two participants inoculated with 3D7-MBE-008 MCB were 26 and 61, similar to the parental (3D7-V-2) line, with both parasitaemia in both participants exceeding 10,000/mL by day 8. Growth of the 3D7-V1 was slower (PMR48 of 8 and 18), with parasitaemia exceeded 10,000 parasites/mL on days 10 and 8.5 respectively. Rapid parasite clearance followed artemether-lumefantrine treatment in all four participants (clearance half-lives of 4.01 and 4.06 hours for 3D7-MBE-008 and 4.11 and 4.52 hours for 3D7-V1). A total of 59 adverse events occurred, most were of mild severity with three being severe in the 3D7-MBE-008 study. ConclusionThe safety, growth and clearance profiles of the expanded 3D7-MBE-008 MCB closely resemble that of its parent, indicating its suitability for future studies.Trial RegistrationAustralian New Zealand Clinical Trials registry numbers:P3487 (3D7-V1): ACTRN12619001085167P3491 (3D7-MBE-008): ACTRN12619001079134

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A bioreactor system for the manufacture of a genetically modified Plasmodium falciparum blood stage malaria cell bank for use in a clinical trial

Malaria Journal ◽

10.1186/s12936-018-2435-x ◽

2018 ◽

Vol 17 (1) ◽

Cited By ~ 6

Author(s):

Rebecca Pawliw ◽

Rebecca Farrow ◽

Silvana Sekuloski ◽

Helen Jennings ◽

Julie Healer ◽

...

Keyword(s):

Clinical Trial ◽

Plasmodium Falciparum ◽

Genetically Modified ◽

Blood Stage ◽

Cell Bank ◽

Bioreactor System ◽

Blood Stage Malaria

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Plasmodium falciparum-specific IgM B cells dominate in children, expand with malaria and produce parasite inhibitory IgM

10.1101/2020.04.12.030049 ◽

2020 ◽

Author(s):

Christine S. Hopp ◽

Ababacar Diouf ◽

Kazutoyo Miura ◽

Kristin Boswell ◽

Padmapriya Sekar ◽

...

Keyword(s):

Plasmodium Falciparum ◽

B Cells ◽

Cell Receptor ◽

Blood Stage ◽

Igg Antibodies ◽

Influenza Hemagglutinin ◽

Specific Memory ◽

Specific Igg ◽

Blood Stage Malaria

AbstractIgG antibodies are known to play a central role in naturally acquired immunity to blood-stage malaria in humans, but little is known about the IgM response to blood-stage malaria, the mechanisms by which IgM may protect, or the underlying biology of Plasmodium falciparum (Pf)-specific IgM B cells. In a Mali cohort spanning infants to adults we conducted a longitudinal analysis of B cells specific for the Pf blood-stage antigens AMA1 and MSP1, as well as the comparator antigen influenza hemagglutinin (HA). At the uninfected baseline, before the malaria season, Pf-specific memory B cells (MBCs) in children are disproportionally IgM+ and only gradually shift to IgG+ with age, in contrast to HA-specific MBCs that are predominantly IgG+ from infancy to adulthood. In response to acute febrile malaria, Pf-specific IgM B cells increase in frequency and upregulate activation and co-stimulatory markers. B cell receptor (BCR) analysis showed that Pf-specific IgM B cells are somatically hypermutated at levels comparable to HA-specific IgG B cells. Finally, IgM antibodies from the plasma of malaria-exposed individuals were comparable to IgG in inhibiting Pf blood-stage growth in vitro, and significantly better at enhancing phagocytosis of Pf merozoites, suggesting that IgM may protect through both direct neutralization and opsonization. Thus, somatically hypermutated Pf-specific IgM MBCs dominate in early life, are activated and expand during acute malaria and are associated with plasma IgM that inhibits parasite growth in vitro.

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Antibody Combinations Targeting the Essential Antigens CyRPA, RH5, and MSP-119 Potently Neutralize Plasmodium falciparum Clinical Isolates From India and Africa

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa608 ◽

2020 ◽

Author(s):

Hina Singh ◽

Syed Yusuf Mian ◽

Alok K Pandey ◽

Sri Krishna ◽

Gaurav Anand ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Neutralizing Antibodies ◽

Central India ◽

Blood Stage ◽

Clinical Isolates ◽

In Vitro Cultivation ◽

Invasion Pathways ◽

Blood Stage Malaria

Abstract Background Targeting multiple key antigens that mediate distinct Plasmodium falciparum erythrocyte invasion pathways is an attractive approach for the development of blood-stage malaria vaccines. However, the challenge is to identify antigen cocktails that elicit potent strain-transcending parasite-neutralizing antibodies efficacious at low immunoglobulin G concentrations feasible to achieve through vaccination. Previous reports have screened inhibitory antibodies primarily against well adapted laboratory parasite clones. However, validation of the parasite-neutralizing efficacy against clinical isolates with minimal in vitro cultivation is equally significant to better ascertain their prospective in vivo potency. Methods We evaluated the parasite-neutralizing activity of different antibodies individually and in combinations against laboratory adapted clones and clinical isolates. Clinical isolates were collected from Central India and Mozambique, Africa, and characterized for their invasion properties and genetic diversity of invasion ligands. Results In our portfolio, we evaluated 25 triple antibody combinations and identified the MSP-Fu+CyRPA+RH5 antibody combination to elicit maximal parasite neutralization against P. falciparum clinical isolates with variable properties that underwent minimal in vitro cultivation. Conclusions The MSP-Fu+CyRPA+RH5 combination exhibited highly robust parasite neutralization against P. falciparum clones and clinical isolates, thus substantiating them as promising candidate antigens and establishing a proof of principle for the development of a combinatorial P. falciparum blood-stage malaria vaccine.

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The Structure of the Cysteine-Rich Domain of Plasmodium falciparum P113 Identifies the Location of the RH5 Binding Site

mBio ◽

10.1128/mbio.01566-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Ivan Campeotto ◽

Francis Galaway ◽

Shahid Mehmood ◽

Lea K. Barfod ◽

Doris Quinkert ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Binding Site ◽

Structural Information ◽

Blood Stage ◽

Site Directed Mutagenesis ◽

Effective Vaccine ◽

Binding Region ◽

Fab Fragment ◽

Content Type ◽

Blood Stage Malaria

ABSTRACT Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5. IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum. It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it—and the other parasite proteins it interacts with—promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

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