Infectious plasmodium sporozoites grown in vitro
Inventors
EAPPEN, Abraham G. • Hoffman, Stephen L.
Assignees
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Publication Number
US-11207395-B2
Publication Date
2021-12-28
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 mature infectious Plasmodium sporozoites of human host range in which sporogony from the gametocyte stage to the mature sporozoite stage occurs external to mosquitoes. The in vitro-reared mature infectious Plasmodium sporozoites of human host range are genetically attenuated.
The document describes an in vitro sporogonic development concept in which parasites progress from gametocyte stage to mature sporozoite stage without mosquito-associated development, including an option for formulations where attendant mosquito material is absent. It further defines and uses in vitro sporogonic culture systems including exflagellation culture medium, lectin-mediated red blood cell agglutination, an ookinete culture medium, and an oocyst culture medium.
The document also describes in vitro production systems intended to increase oocyst/sporozoite outputs relative to mosquito production, including culture on a substrate comprising a matrix and an ookinete culture medium in which ookinetes penetrate the matrix and differentiate to oocysts, followed by replacement of the ookinete medium with an oocyst culture medium. The harvested in vitro-produced parasites are obtained at a mature sporozoite stage.
Claims Coverage
The document includes three independent claims. Claim 1 covers genetically attenuated in vitro-reared mature infectious Plasmodium sporozoites of human host range with sporogony external to mosquitoes. Claim 11 covers a stepwise in vitro method to culture genetically attenuated Plasmodium parasites through sporogonic development to mature sporozoites. Claim 12 covers an in vitro method to increase production of genetically attenuated Plasmodium oocysts relative to mosquito production from an equivalent number of gametocytes.
Sporogony external to mosquitoes for in vitro-reared infectious sporozoites
In vitro-reared mature infectious Plasmodium sporozoites of human host range, wherein sporogony from gametocyte stage to mature sporozoite stage is external to mosquitoes.
Genetically attenuated in vitro-reared mature infectious sporozoites for human host range
The in vitro-reared mature infectious Plasmodium sporozoites of human host range are genetically attenuated.
Gametocyte-to-mature-sporozoite in vitro sporogonic culturing workflow
A method of culturing genetically attenuated Plasmodium parasites of human host range in vitro during sporogonic development, comprising culturing gametocyte-stage parasites to zygote stage in an exflagellation culture medium in the presence of red blood cells; agglutinating red blood cells with a lectin; collecting a mixture comprising zygotes, gametes, gametocytes and agglutinated red blood cells; culturing the mixture on a substrate comprising a matrix in an ookinete culture medium to differentiate to ookinetes and allow ookinetes to penetrate the matrix; exchanging the ookinete medium with an oocyst medium; culturing in the oocyst medium to differentiate to mature sporozoites; and harvesting at a mature sporozoite stage.
Matrix-based ookinete-to-oocyst differentiation with medium substitution to increase oocyst production
A method for increasing the production of genetically attenuated Plasmodium oocysts of human host range relative to genetically attenuated Plasmodium oocyst production from the same species and an equivalent number of human Plasmodium gametocytes in a mosquito, comprising culturing gametocyte-stage parasites to zygote stage in an exflagellation culture medium; agglutinating red blood cells with a lectin; collecting a mixture comprising zygotes, gametes, gametocytes and agglutinated red blood cells; culturing the mixture on a substrate comprising a matrix in an ookinete medium where parasites differentiate to ookinetes and enter the matrix and differentiate to oocysts; replacing the ookinete medium with an oocyst culture medium; and quantitating the oocysts, wherein more oocysts develop in vitro than in mosquitoes from the equivalent number of gametocytes.
Across the independent claims, the central coverage is genetically attenuated, in vitro-reared mature infectious Plasmodium sporozoites of human host range generated via sporogony external to mosquitoes, and in vitro culturing workflows that proceed through gametocyte-to-zygotic stages using lectin-mediated red blood cell agglutination, matrix-based ookinete penetration and oocyst development, medium exchange to oocyst culture medium, and harvesting at a mature sporozoite stage, including an explicit increase in oocyst production compared to mosquito development from equivalent gametocytes.
Stated Advantages
In vitro-produced genetically attenuated Plasmodium oocysts are produced in greater quantity than genetically attenuated oocysts developed in mosquitoes from an equivalent number of human host range gametocytes.
Documented Applications
Vaccine compositions comprising in vitro-reared sporozoites for use in immune response induction in a subject against Plasmodium-specific antigens.
In vitro use of in vitro-reared sporozoites to support testing of drugs or vaccines (sporozoites for drug/vaccine testing).
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