Sequential disruption of PM4 and MSP7 in Plasmodium berghei induces a progressive virulence-attenuated phenotype.

Malaria remains one of the most significant infectious diseases affecting human populations in developing countries. We investigated the occurrence of experimental cerebral malaria (ECM) and the development of parasitemia, in different mouse strains, infected with P. berghei parasites showing a mild in vivo growth impairment phenotype as a consequence of targeted disruption of either the plasmepsin 4 (pm4) or the merozoite surface protein 7 (msp7) gene. Both PM4 and MSP7 single knockout parasites failed to induce ECM in susceptible mouse strains though most animals died with later as a consequence of high parasitemia. Notably, parasites lacking both pm4 and msp7 genes showed a dramatic virulence-attenuated phenotype characterized by a self-resolving infection with lower levels of parasitemia. Furthermore, convalescent mice were completely protected against subsequent challenge with the lethal P. berghei or P.yoelii parasites for several months. We showed that the cellular and humoral immune response are both crucial for immune protection. This work demonstrates that the sequential disruption of parasite genes can generate progressively induces virulence-attenuated blood stage parasites that are capable of stimulate a long lasting protective immunity. These observations provide a proof-of-concept step for the development of human malaria vaccines based on genetically attenuated blood stage parasites.