Research interests and areas of expertise

• Generation and analysis of genetically attenuated parasites for vaccine development
• Genetic modification of malaria parasites and application of post-genomic technologies to identify and validate new drug and vaccine targets
• Parasite-host interactions involved in pathology and disease

The Leiden malaria group developed Plasmodium genetic modification in 1995 and continues to innovate and lead research into Plasmodium transgenesis.

Gene deletion mutants of rodent malaria parasites have been used to analyze a wide range of parasite gene functions. In 2005 they were one of the first groups to create genetically attenuated parasites and demonstrated in rodent malaria models that these could serve as a live-parasite vaccine against malaria.

 Leiden has created a number of (reference) transgenic parasite lines expressing heterologous proteins such as fluorescent, bioluminescent or OVA reporter proteins, which have proven to be valuable tools in dissecting parasite-host interactions and these have been shared with many different labs world-wide. In addition, chimeric rodent malaria parasites have been created that express antigens of human malaria parasites that have been used to validate vaccine target antigens. They have created over 160 transgenic/chimeric mutants and have distributed these mutants to more than 50 groups across the world.

Genetic modification has also been applied to the human parasite P. falciparum and novel methods to efficiently generate stable genetically modified P. falciparum parasites have been developed that are free of heterologous drug selectable. These methods are key in creating genetically attenuated vaccines. CRISPR-Cas9 technologies for genetic modification of P. falciparum are continually refined.

Leiden Malaria facilities

In vitro cultivation of the blood stages of malaria parasite

Technologies are available for short- and long-term in vitro culture of blood stages of the human malaria parasite P. falciparum  and rodent malaria parasites. These are used for a wide range of applications in drug screening, phenotype analysis, production of parasites for genetic modification and isolation of the different parasite forms for molecular analysis.

Infections of mosquitoes by malaria parasites

Facilities are available for breeding of Anopheles stephensi mosquitoes and for mosquito transmission of the human malaria parasite P. falciparum and rodent malaria parasites.

In vitro cultivation of mosquito stages of malaria parasites

Standardized techniques are applied for in vitro gamete production, fertilization and zygote development of P. berghei.

In vitro cultivation of liver stages of malaria parasites

Facilities and technologies are available for in vitro cultivation of liver stages of rodent malaria parasites and for in vitro cultivation of liver stages of the human parasite P. falciparum in human primary hepatocytes. Technologies are available for analyzing liver stages of rodent malaria parasites in vivo in mice by imaging technologies.

Molecular biology techniques and genomics and post-genomic analyses

All standard technologies for analysis of DNA and RNA are applied. These activities have generated resources that can be requested. In collaboration with other groups detailed genomic and high-throughput post-genomic expression analyses have been performed using rodent malaria parasites.

Genetic modification of malaria parasites

Technologies for genetic modification the human malaria parasite P. falciparum and rodent malaria parasites have been developed and are applied. A large number of transgenic/chimeric parasites have been generated expressing reporter proteins providing a broad range of genetic tools for the study of malaria parasite biology. New tools (including CRISPR/Cas9 genome editing) are continuously under development and a number of well-defined gene knockout, transgenic and chimeric mutant parasite lines are available.

Technologies to study phenotype and cellular aspects of malaria parasite development

The combination of in vitro cultivation of human and rodent parasites and in vivo infections of rodent parasites permit the detailed analysis of the phenotype of the parasites. For example, gamete fertilization, zygote development, synchronous blood stage development, gametocyte formation. In addition: FACS analysis of growth characteristics of blood stages; drug screening assays; light- and fluorescence microscopy; visualization of live, fluorescent/luminescent  parasites.

(In vivo) imaging of parasite-host interactions

Interactions are studied between malaria parasites and their host in live animals using rodent models of malaria.  For this research the latest (real-time, in vivo) imaging technologies are used and an array of transgenic (fluorescent and/or bio-luminescent) malaria parasites.

Preclinical and clinical evaluation of vaccine candidates

We evaluate novel vaccine candidates in preclinical studies using mouse models of malaria and in clinical studies in human volunteers using Controlled Human Malaria Infections (CHMI)

Genetically attenuated malaria vaccines

Leiden is at the forefront of international research on malaria vaccine development using live-parasites for immunization. They were one of the first groups to develop the concept of immunization with genetically attenuated parasites (GAP) and showed that GAP based immunity can effectively protect mice against malaria (van Dijk et al., 2005). They have translated these findings from murine malaria into the generation of human malaria, P. falciparum, GAPs. Collectively the LUMC are experts in Plasmodium genetic modification, immunization and protection studies in both rodents and human malaria and have developed robust pre-clinical and clinical screening protocols to evaluate their suitability for vaccination. 

The development of GAP vaccines in Leiden is the culmination of studies that were first initiated here in the mid-90s: 

• The Leiden Malaria group was the first group to develop genetic modification in malaria parasites (2 papers published in Science in 1995 and 1996)
• Through analysis of gene-deletion mutants in rodent models of malaria, we discovered that it was possible to create attenuated parasites, which were able to invade the liver but were unable to proceed into the pathogenic blood stage infection. Importantly mice infected (immunized) with these live-attenuated parasites developed protective immunity against an infection with wild type parasites (published in PNAS in 2005).
• In 2008 we started to work on translating these findings into a human vaccine against malaria. This was performed in collaboration with the RadboudUMC (Nijmegen) and the US company Sanaria, and was supported by a grant provided by TI-Pharma. This involved testing and refining many live genetically-attenuated vaccines to demonstrate that all the necessary pre-clinical safety and protective efficacy standards were met.
• In 2017 the first-in-human safety trials with a genetically modified malaria vaccine (i.e. PfSPZ-GA1) were performed at the LUMC. The GAP vaccine, PfSPZ-GA1, was manufactured by Sanaria and administered to volunteers intravenously by needle and syringe; the clinical trial was led by Dr. Meta Roestenberg.  
• In a joint clinical study, 67 volunteers in Leiden and Nijmegen received injections of the PfSPZ GA1 vaccine - a first in the world for an injectable, genetically weakened malaria vaccine. A high and a low dose were administered. In May 2020 the results of this clinical trial were published in Science Translational Medicine, showing that the vaccine is safe. It does not lead to infection of the blood and therefore does not cause malaria symptoms. Vaccinated volunteers developed an immune defense against a malaria infection, although this protection was not complete. The measured immune responses and demonstrated safety are strong incentives to further develop a vaccine based on GAP.
• 2018-2020: Studies started to create a so-called ‘second generation GAP’, a GAP with increased immunogenicity compared to the first generation GAP (PfSPZ-GA1; GA1 vaccine). 
  One of the likely ways to improve the immunogenicity of GAP vaccines is to broaden the array of antigens displayed to the immune system and by extending the duration of parasite exposure to the immune system. In mouse models of malaria, a GAP that arrest late during development in the liver has indeed proven to induce significantly higher protective immune responses as compared to both radiation-attenuated sporozoites (RAS) and early-arresting GAP (such as the first generation GAP), most likely resulting from increased antigen breadth and biomass of a LA-GAP. The current studies are therefore focused on generating a late-arresting GAP for the human malaria parasite P. falciparum 
• 2021-2022: A second generation GAP (GA2), created by the LUMC, has passed regulatory approval for use in human studies by the Gene Therapy Office of the Dutch Ministry of Infrastructure and the Environment in the Netherlands, licensed to the Leiden University Medical Centre (GGO IM-MV 20-018_000) and subsequent approval of the Central Committee on Research Involving Human Subjects (CCMO; NL75577.000.21). Initial safety and preliminary efficacy studies in healthy volunteers are currently ongoing.