Malaria, caused by Plasmodium parasite infection, continues to be one of the leading causes of worldwide morbidity and mortality. Vaccine is crucial for prevention of infectious diseases and in the case of malaria, whole parasite immunization strategies employing genetically attenuated parasites (GAP), which arrest during liver-stage development has been shown to be effective in rodents and recently also in human. Here we describe a new approach for the development of liver stage arrested GAP parasites that while inducing high level of protection upon sporozoites challenge overcomes the problem of residual infectivity often encountered with attenuated parasites carrying a disrupted liver stage gene. We have engineered Plasmodium berghei parasite to express at the liver stage destabilized variants of I-PpoI gene, a homing nuclease that selectively recognize a highly conserved ribosomal sequences present in the parasite ribosome genes. Immunization experiments showed that a regimen of three doses of 10,000 transgenic sporozoites injected intravenously induced complete sterile protection upon challenge with wild type parasites. Notably, we never observed blood stage infected mice even after injection of 1x106 transgenic sporozoites per mice. The recent observation that CRISPR/Cas9 endonuclease can be engineered to selectively attack ribosome specific sequence opens the possibility of developing Plasmodium parasites expressing toxic molecules able to target specifically only parasites ribosomal sequence essential during liver stage development, providing the basis for a new class of GAP strategy.
Nuclease mediated disruption of Plasmodium berghei ribosomes in the liver arrested parasite development and induced complete protective immunity
Roberta Spaccapelo
2017
Abstract
Malaria, caused by Plasmodium parasite infection, continues to be one of the leading causes of worldwide morbidity and mortality. Vaccine is crucial for prevention of infectious diseases and in the case of malaria, whole parasite immunization strategies employing genetically attenuated parasites (GAP), which arrest during liver-stage development has been shown to be effective in rodents and recently also in human. Here we describe a new approach for the development of liver stage arrested GAP parasites that while inducing high level of protection upon sporozoites challenge overcomes the problem of residual infectivity often encountered with attenuated parasites carrying a disrupted liver stage gene. We have engineered Plasmodium berghei parasite to express at the liver stage destabilized variants of I-PpoI gene, a homing nuclease that selectively recognize a highly conserved ribosomal sequences present in the parasite ribosome genes. Immunization experiments showed that a regimen of three doses of 10,000 transgenic sporozoites injected intravenously induced complete sterile protection upon challenge with wild type parasites. Notably, we never observed blood stage infected mice even after injection of 1x106 transgenic sporozoites per mice. The recent observation that CRISPR/Cas9 endonuclease can be engineered to selectively attack ribosome specific sequence opens the possibility of developing Plasmodium parasites expressing toxic molecules able to target specifically only parasites ribosomal sequence essential during liver stage development, providing the basis for a new class of GAP strategy.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
