Infectious plasmodium sporozoites grown in vitro
Inventors
EAPPEN, Abraham G. • Hoffman, Stephen L.
Assignees
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Publication Number
US-9878026-B2
Publication Date
2018-01-30
Expiration Date
Abstract
The application is directed to in-vitro-reared Plasmodium sporozoites of human host range wherein sporogony from gametocyte stage to sporozoite stage is external to mosquitoes, and methods of producing the same. Provided herein are in vitro-reared infectious Plasmodium sporozoites (SPZ) of human host range, particularly P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, wherein sporogony from gametocyte stage to sporozoite stage is external to mosquitoes, and methods of producing the same.
Core Innovation
The invention provides in vitro-reared infectious Plasmodium parasites of human host range that undergo sporogonic development external to mosquitoes, including development from gametocyte stage to mature sporozoite stage occurring in vitro. A culture is provided that includes in vitro-reared Plasmodium parasites of human host range at the zygote stage of development together with a matrix.
A method of culturing human host range Plasmodium parasites in vitro during sporogonic development is described. Human host range Plasmodium gametocytes are cultured to zygote stage in the absence of a matrix and in the presence of red blood cells in an exflagellation culture medium, red blood cells are agglutinated with a lectin, and the collected mixture is cultured on a substrate comprising a matrix in an ookinete culture medium in which ookinete stage parasites penetrate the matrix.
After penetration and ookinete-stage culture, the ookinete medium is exchanged with an oocyst medium and Plasmodium sporozoite stage parasites are harvested. The described in vitro production is positioned against mosquito-reared benchmarks for infectiousness and oocyst production, and functional liver-stage infectivity in human hepatocyte HC-04 cells with liver-stage development expressing PfMSP-1 is described in the provided content.
Claims Coverage
The provided independent claims cover three main aspects: a culture containing an in vitro-reared human host range Plasmodium parasite population at the zygote stage that has undergone sporogonic development in vitro together with a matrix; an in vitro culturing method that drives sporogonic development from gametocytes through sporozoite harvesting using lectin-mediated agglutination, a matrix substrate, medium exchange, and matrix-penetrating ookinete culture; and a vaccine-based immune response method using in vitro-reared infectious Plasmodium sporozoites whose sporogony from gametocyte stage to mature sporozoite stage is external to mosquitoes.
Culture with zygote stage sporogonic development and matrix
A culture comprising in vitro-reared Plasmodium parasites of human host range and a matrix, wherein said parasites are at the zygote stage of development and have undergone sporogonic development in vitro.
In vitro sporogonic culturing with lectin agglutination, matrix penetration, medium exchange, and sporozoite harvesting
A method of culturing Plasmodium parasites of human host range in vitro during sporogonic development comprising culturing human host range Plasmodium gametocytes to zygote stage in the absence of a matrix and in the presence of red blood cells in an exflagellation culture medium; agglutinating said red blood cells with a lectin; collecting a mixture comprising zygotes, gametes, gametocytes and agglutinated red blood cells; culturing said mixture on a substrate comprising a matrix and in an ookinete culture medium, wherein ookinete stage parasites penetrate said matrix; exchanging said ookinete medium with an oocyst medium; and harvesting Plasmodium sporozoite stage parasites produced thereby.
Vaccine composition using in vitro-reared infectious sporozoites with external sporogony
A method of inducing an immune response in a human subject against Plasmodium-specific antigens comprising administering to said human subject a vaccine composition comprising in vitro-reared infectious Plasmodium sporozoites of human host range wherein sporogony from gametocyte stage to mature sporozoite stage is external to mosquitoes and a pharmaceutically acceptable diluent.
Across the independent claims, the claimed subject matter combines in vitro matrix-containing sporogony, a corresponding lectin-mediated culturing workflow with matrix penetration and medium exchange, and immune-response induction by administering a vaccine composition containing in vitro-reared infectious sporozoites.
Stated Advantages
Enables sporozoite infectivity and functional liver-stage development using in vitro-reared infectious sporozoites described as at least as efficient in comparison to fresh mosquito-derived sporozoites in the 6-day hepatocyte potency assay.
Achieves increased oocyst output in vitro versus mosquito benchmarks, reported up to about 39-fold.
Eliminates attendant mosquito material by conducting sporogony external to mosquitoes.
Documented Applications
Vaccine compositions and inducing an immune response in a human subject against Plasmodium-specific antigens by administering a vaccine composition comprising in vitro-reared infectious Plasmodium sporozoites whose sporogony is external to mosquitoes.
Functional infectivity in human hepatocyte HC-04 cells with liver-stage development expressing PfMSP-1, including a 6-day hepatocyte potency assay comparison to mosquito-reared controls.
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