Further Improvements of the P. falciparum Humanized Mouse Model Ludovic Arnold.¤a, Rajeev Kumar Tyagi., Pedro Meija¤b, Claire Swetman, James Gleeson, Jean-Louis Pe´ rignon, Pierre Druilhe* Malaria Vaccine Development Laboratory, Institut Pasteur, Paris, France Abstract Background: It has been shown previously that it is possible to obtain growth of Plasmodium falciparum in human erythrocytes grafted in mice lacking adaptive immune responses by controlling, to a certain extent, innate defences with liposomes containing clodronate (clo-lip). However, the reproducibility of those models is limited, with only a proportion of animals supporting longstanding parasitemia, due to strong inflammation induced by P. falciparum. Optimisation of the model is much needed for the study of new anti-malarial drugs, drug combinations, and candidate vaccines. Materials/Methods: We investigated the possibility of improving previous models by employing the intravenous route (IV) for delivery of both human erythrocytes (huRBC) and P. falciparum, instead of the intraperitoneal route (IP), by testing various immunosuppressive drugs that might help to control innate mouse defences, and by exploring the potential benefits of using immunodeficient mice with additional genetic defects, such as those with IL-2Rc deficiency (NSG mice). Results: We demonstrate here the role of aging, of inosine and of the IL-2 receptor c mutation in controlling P. falciparum induced inflammation. IV delivery of huRBC and P. falciparum in clo-lip treated NSG mice led to successful infection in 100% of inoculated mice, rapid rise of parasitemia to high levels (up to 40%), long-lasting parasitemia, and consistent results from mouse-to-mouse. Characteristics were closer to human infection than in previous models, with evidence of synchronisation, partial sequestration, and receptivity to various P. falciparum strains without preliminary adaptation. However, results show that a major IL-12p70 inflammatory response remains prevalent. Conclusion: The combination of the NSG mouse, clodronate loaded liposomes, and IV delivery of huRBC has produced a reliable and more relevant model that better meets the needs of Malaria research. Citation: Arnold L, Tyagi RK, Meija P, Swetman C, Gleeson J, et al. (2011) Further Improvements of the P. falciparum Humanized Mouse Model. PLoS ONE 6(3): e18045. doi:10.1371/journal.pone.0018045 Editor: Lisa Ng Fong Poh, Agency for Science, Technology and Research - Singapore Immunology Network, Singapore Received December 20, 2010; Accepted February 18, 2011; Published March 31, 2011 Copyright: 2011 Arnold et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded, in part, by a grant from the French Ministry of Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: druilhe@pasteur.fr . These authors contributed equally to this work. ¤a Current address: Immunite´ et Infection, UMR-S 945 INSERM Universite´ Pierre et Marie Curie, Faculte´ de Me´decine, Paris, France ¤b Current address: Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America Introduction The development of a small laboratory model capable of tolerating and sustaining human malaria infection has almost unlimited applications in areas such as parasite biology, novel drug development and vaccine discovery. Currently, the majority of in vivo investigations into malaria biology are performed using rodent malaria species such as P. berghei, and P. yoelii that are much easier to handle however, their relevance to human malaria has been questioned [1,2,3]. A convenient model capable of sustaining P. falciparum would undoubtedly be beneficial. For example, such a model could serve to harmonise in vitro studies that use P. falciparum in culture, with the in vivo models that presently use rodent species. While these approaches are complementary, the current mismatch of Plasmodium species is a very serious limitation. With the development of resistance against all existing anti-malarial drugs, the need for better tools to discover and develop novel classes and combinations of anti-malarials is imperative [4,5]. The advent of several new mouse strains with genetic immune deficiencies has greatly benefited the development of a small laboratory malaria model, and results have shown that such a model is indeed both achievable and useful [6,7,8,9,10]. Experiments performed so far have used mouse strains such as SCID, NIH III (Beige Xid Nude), and NOD/SCID, together with pharmacological agents to control their remaining innate defences, or mouse adapted parasites. It was shown that by grafting them with either human erythrocytes [6,11] or human hepatocytes [12] these animals can support, respectively, the asexual blood cycle, or the intra-hepatic cycle of the human parasite P. falciparum. However, on a day-to-day basis they are quite cumbersome to manage [6,11], and this onus, combined with their currently poor reproducibility of successful infection, limits their usefulness, and is likely to have contributed to the persistent use of rodent Plasmodium PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e18045