In September 2015, the 193 countries of the United Nations adopted 17 Sustainable Development Goals (SDGs) to end poverty, protect the planet and ensure prosperity for all. As outlined in the 2030 Agenda for Sustainable Development, specific targets are set for each goal over the next 15 years. The fight against malaria is one of the targets of goal 3 (ensuring healthy lives and promote well-being for all at all ages). The research carried out by Prof. Dr. Frischknecht and Mirko Singer from the Centre for Infectious Diseases at Heidelberg University Hospital is one of several steps towards eradicating malaria.
Malaria is the most common tropical disease and is caused by parasites that are transmitted by female mosquitoes of the genus Anopheles. There are around 300 million new cases of malaria and nearly half a million deaths from malaria every year. Researchers around the world have been working on finding an effective malaria vaccine for many decades. The latest of these, RTS,S, gives reason for some hope despite its limited efficacy. New research from the US suggests that releasing genetically modified, Plasmodia-immune mosquitoes could be a way to block the transmission of the malaria parasite. Resistance against Plasmodia was achieved by introducing resistance genes using the powerful but controversial CRISPR gene-editing technology. The idea is that the mosquitoes carrying the genes that block the transmission of the malaria parasite will propagate and eventually eradicate the parasites. The problem is that over 60 malaria-transmitting parasite species would need to be modified, and releasing genetically modified parasites could have unpredictable effects on the ecosystem.
Prof. Dr. Frischknecht from the Centre for Infectious Diseases at Heidelberg University Hospital pursues a different route involving the genetic modification of Plasmodia. He has recently published his results in the journal Genome Biology1. Plasmodia are mobile, unicellular, eukaryotic organisms that have evolved a deceptive multiplication strategy: they enter the bloodstream of human individuals through the bite of an infected female Anopheles mosquito and invade the liver. They grow and multiply in the liver cells and subsequently in the red blood cells. The parasites are safely enclosed in the erythrocytes where the human immune system is unable to detect and destroy them. The parasites inside the red blood cells multiply, the infected cells burst and the parasites continue to infect other erythrocytes, generating cyclic symptoms. The infected mosquito also carries the disease from one human being to another. Infected individuals become very weak: typical malaria symptoms include recurrent fever and anaemia. Some species cause severe malaria and death; it is known that both Alexander the Great and Tutankhamun died from malaria.
“The evolution of eukaryotic parasites is a perfect example of a development that is driven by sexual reproduction and recombination,” says Mirko Singer, lead author of the Genome Biology paper. His institute is equipped with several different imaging and biophysical technologies as well as standard equipment used for molecular and genetic research. The researchers use all these instruments to study model organisms to find out how parasites invade the human body and how they can be prevented from causing malaria.
As part of their research, Frischknecht and Singer use genetically attenuated parasites that lack the genes essential for the liver stage of the parasite and therefore only develop to a limited extent. They can be recognised and eliminated by the human immune system. An effect similar to that achieved by polio vaccines, which actively ‘train’ the immune system to fight off potential polio virus infections, can be achieved with genetically attenuated parasites.
The researchers have so far specifically focused on deleting between one and three parasite genes to stop the parasite developing in the liver and make it easy prey for the immune system. However, such parasites, along with parasites that have been attenuated with γ radiation, can still cause breakthrough infections in rodents during immunisation and result in pathological blood-stage infections. This is one of the major reasons why transferring the method to humans is difficult and risky. The Genome Biology paper describes an alternative method for producing genetically attenuated parasites (GAP). The goal is to generate parasites that could potentially be used as malaria vaccines. “We wanted to develop a method that can stop parasite development at a specific stage by precisely timing the point of developmental arrest,” says Singer. In order to achieve parasites that would not lead to breakthrough infections, Frischknecht introduced zinc-finger nuclease (ZFN) genes into one of the parasite’s 14 chromosomes. ZFNs are restriction enzymes that create double-strand breaks in DNA at user-specified locations. When activated, ZFNs specifically recognise a chosen target site within a genome and cleave the DNA at user-specified locations.
Nevertheless, some parasites survived and led to an infection in the rodents. Singer was also able to identify the parasites’ DNA repair mechanism, which is called rudimentary microhomology-mediated end joining, that enabled the parasites to survive, thus causing breakthrough infections. “Little is yet known about this repair mechanism,” Singer said. It is also possible that vaccination with genetically attenuated parasites might cause the zing-finger nucleases to mutate. “So we need to use several, mutually supportive safety mechanisms,” says Singer alluding to what could happen with immunisations involving insufficiently attenuated parasites.
Plasmodia are highly adaptive single-celled organisms that have evolved resistance to almost all standard malaria drugs. Numerous attempts to eradicate the disease have therefore failed. “I do not believe that malaria will be eradicated within the coming decades,” says Singer. Although the WHO Malaria Report found that increased prevention and control measures have led to reduced malaria mortality, Frischknecht is not so sure that it can be eradicated as quickly as people are hoping. “In order to further reduce the number of malaria mortalities, and work towards the eradication of the disease, the WHO is planning to provide three times more annual funding than it actually has available for achieving this goal.”
Frischknecht and Singer have shown that in principle it is possible to attenuate the parasites despite the fact that some of them mutate and become pathogenic again. A potential vaccine would present the entire repertoire of surface antigens to the human immune cells. Whether the researchers’ attenuated parasites could become an effective anti-malaria vaccine is not yet known. “There might be a completely different solution to eradicating malaria, or none at all,” concludes Singer.
1Original publication: Singer et al.: Zinc finger nuclease-based double-strand breaks attenuate malaria parasites and reveal rare microhomology-mediated end joining, Genome Biology (2015) 16:249, DOI 10.1186/s13059-015-0811-1