An innovative pipeline to establish the complete Plasmodium vivax life-cycle in the laboratory – VICTOR

The VICTOR project relied on four complementary components to attempt the reconstruction of the complete Plasmodium vivax life cycle in the laboratory.

The first focused on the collection and cryopreservation of clinical isolates in Madagascar and Ethiopia, resulting in a unique biobank of 139 African samples. This resource represents a valuable foundation for experimental work and for the broader scientific community.

The second component targeted the production of sporogonic stages through experimental mosquito infections (An. stephensi, An. arabiensis). Although the limited post-cryopreservation viability of gametocytes reduced infection success, these experiments identified critical parameters and helped refine protocols for future mosquito challenges.

The third component addressed liver stages. Primary human hepatocyte cultures were optimized, with viability extended up to 80 days through co-culture with fibroblasts and circadian training. The use of an innovative peptide significantly increased infection efficiency by sporozoites, opening new perspectives for investigating the longevity, activation, and reactivation of hypnozoites.

The fourth component dealt with the production of permissive blood cells. Reticulocytes generated from CD34? cells, cord blood, and immortalized erythroblast lines such as BeLA were tested for their capacity to sustain early blood-stage development. Although short-lived, these assays demonstrated the feasibility of such systems to study asexual replication and, in some cases, gametocyte production.

All experimental activities were supported by advanced tools. qPCR enabled sensitive monitoring of parasite stages, microscopy and immunofluorescence provided direct visualization, and genomic analyses documented the diversity of the isolates. Innovative devices, such as the PetakaG3 bioreactor, were also tested to create controlled physio-mimetic environments favorable to parasite survival and development.

By combining these four components—biobank, mosquito infections, liver cultures, and blood-stage systems—VICTOR implemented an integrated and multidisciplinary strategy. Although a complete life cycle reconstruction has not yet been achieved, the project identified major bottlenecks, explored innovative solutions, and laid the foundations for a partial but functional experimental model of P. vivax.

he VICTOR project generated several major scientific achievements. The establishment of a biobank of 139 cryopreserved isolates from Madagascar and Ethiopia represents a unique resource for P. vivax research. These samples now provide a solid foundation for exploring the genetic diversity of African parasites and for better understanding their adaptive potential. The project also highlighted the limited post-cryopreservation viability of gametocytes, a critical bottleneck for experimental transmission, pointing to new avenues for optimizing preservation and thawing conditions.

Work on blood stages produced, for the first time, short-term cultures using human reticulocytes derived from CD34? cells, cord blood, or immortalized BeLA lines. Although limited in duration, these assays confirmed the feasibility of a reproducible system to study asexual multiplication and gametocytogenesis.

Research on liver stages represented another important advance. Primary human hepatocyte cultures were extended in longevity from 21 to nearly 80 days through co-culture with fibroblasts and circadian training. The addition of an innovative peptide significantly improved sporozoite infection efficiency, opening the way to study hypnozoites and their reactivation.

Scientific output included two international publications: one on the presence of cryptic reservoirs in Duffy-negative individuals in Ethiopia (PLOS NTDs, 2023), and another on the genomic diversity of African isolates (Communications Biology, 2025). A further manuscript on hepatocyte infection by P. falciparum is in preparation. Two oral communications complemented this output: one presented at the 9th International Conference on P. vivax Research in India (2025), the other at the Parasites and Insect Vectors Department meeting at Institut Pasteur.

Finally, three young researchers received extensive training in the techniques developed during the project, including cell culture, microscopy, qPCR, and management of cryopreserved isolates. This investment contributes to strengthening local scientific capacity and to consolidating a research network on African P. vivax.

Although a complete life cycle reconstruction has not yet been achieved, VICTOR has successfully addressed several critical steps and laid the foundations for a partial but functional experimental model, paving the way for future scientific and translational advances.

The perspectives opened by the VICTOR project are numerous and structuring. The first priority is to produce infective sporozoites directly from mosquitoes infected on site in Africa. Project results showed that cryopreservation severely reduced gametocyte viability, limiting experimental transmission. The next step will therefore be to organize, in collaboration with field sites, mosquito infection cycles to obtain fresh, infective sporozoites, an essential condition for advancing studies on liver stages.

This work will be closely linked to strengthening hypnozoite cultures and studying their reactivation. The extension of primary hepatocyte viability achieved during VICTOR has already opened new opportunities to investigate the dynamics of dormant forms. Future research will aim to identify the signals that trigger hypnozoite reactivation and to test new molecules active against these latent forms, which remain poorly targeted by current treatments.

Exploitation of the African isolate biobank established during the project represents another major perspective. This unique resource will enable comparative genomic analyses, improve understanding of parasite adaptation mechanisms, and document regional differences. It can also be used for immunological studies and drug screening, providing access to parasites representative of African diversity.

Beyond P. vivax, the methods developed in VICTOR can be adapted to other neglected parasites such as P. ovale or P. cynomolgi. These species, still poorly studied, will directly benefit from the innovations introduced, particularly extended hepatocyte culture, the generation of permissive reticulocytes, and the use of physio-mimetic culture systems.

Finally, VICTOR has consolidated a transnational network linking European and African partners. This network now provides a foundation for future collaborative projects integrating entomology, parasitology, cell biology, genomics, and immunology, while ensuring capacity building and technology transfer in endemic countries.

The VICTOR project generated high-level scientific output, reflecting the richness of the results obtained and their dissemination to the international community. Two open-access international publications were produced. The first, published in 2023 in PLOS Neglected Tropical Diseases (Abebe et al.), demonstrated the presence of Plasmodium vivax in Duffy-negative individuals in Ethiopia, previously considered naturally resistant. This discovery reshaped the understanding of P. vivax epidemiology in Africa and highlighted the need to adapt surveillance strategies.

The second publication, released in 2025 in Communications Biology (Bouyssou et al.), described the genomic diversity of African isolates collected within the project. Using high-throughput sequencing and bioinformatic analysis, it revealed the richness and originality of these populations, providing a reference framework for studying parasite resistance, immunity, and adaptation.

A third manuscript is in preparation (Real et al., Priming Plasmodium falciparum sporozoites for liver infection). Although it does not focus directly on P. vivax, it sheds light on hepatocyte infection mechanisms that will inform cross-species comparisons.

Two oral communications further disseminated project findings. Liliana Mancio-Silva presented advances on hypnozoite biology at the 9th International Conference on P. vivax Research (Pondicherry, February 2025). Catherine Bourgouin reported at Institut Pasteur (June 2025) on experimental mosquito infections using cryopreserved patient blood, discussing both the limitations and perspectives for improvement.

No patents were filed, but several technical innovations provide lasting value. The protocols developed, the use of PetakaG3 bioreactors that create favorable physio-mimetic environments, and BeLA erythroblast lines capable of continuously producing permissive reticulocytes represent transferable tools for future projects. Their potential extends beyond P. vivax and can be applied to other neglected malaria parasites.

In summary, the scientific production of VICTOR combined impactful publications, international communications, and technical innovations. Together, these outputs demonstrate the consortium’s capacity to advance knowledge on P. vivax while providing the scientific community with a strong technological foundation for future research.

Research on P. vivax is greatly lagging behind P. falciparum mainly due to the absence of an efficient laboratory cultivation system on one hand. On the other hand most of the current investigations are compartmentalized, focusing specifically on either individual P. vivax stage (blood stage, liver stage, mosquito stage). As a major breakthrough we propose in the VICTOR project to join forces and expertise for implementing an innovative pipeline establishing the complete Plasmodium vivax life-cycle in the laboratory, independent of transmission seasons and heavy logistic constraints for establishing connections with collection sites located in malaria endemic settings. This platform will provide the today major missing piece of the puzzle to enlarge our general knowledge on P. vivax biology and pave the way for boosting research on P. vivax and promoting the development of potential innovative novel interventions such as new therapeutics and vaccines.