“Malaria drugs fail for first time on patients in UK”

A recent news article by the BBC reported that four cases of malaria in the UK failed to respond to artemether-lumefantrine treatment, the current front-line drug combination recommended by the WHO. Drug resistance to older antimalarial drugs, like chloroquine, is already widespread in many countries with endemic malaria. It spreads faster in areas where treatments have been dispensed inappropriately, because this fosters selection for resistant strains, similarly to antibiotic resistance. WWARN (worldwide antimalarial resistance network) has an excellent series of interactive maps that you can use to explore the available data on antimalarial resistance.

Of particular concern is resistance to artemisinins – a group of drugs which are the key component in the artemisinin-combined therapies recommended by the WHO. These are well tolerated therapies with high effectiveness, assuming the full course of treatment is taken. However, in certain areas where healthcare has not been well managed, or access to drugs is available without appropriate medical advice, artemisinin resistance has spread rapidly and pervasively. This is shown well in the map produced by Ashley et al., which demonstrates the prevalence of a certain cause of resistance in South East Asia.

A map produced by Ashley et al., showing prevalence of PfKelch13-associated artemisinin resistance in S.E. Asia, and the limited spread of PfKelch13-independent resistance in Africa.

The exact mechanism of resistance to artemisinin is still contested, with several ideas proposed so far but no consensus has yet been achieved. Part of the reason for this is that we still don’t really know how artemisinin kills the parasite in the first place… What we do know however, by using Genome Wide Association Studies of parasite genomes, is that mutations in the “Kelch-propeller domain” of Plasmodium falciparum Kelch13 are strongly associated with cases of artemisinin resistance. Kelch-propeller domains are used for protein:protein interactions, and can be very specific. K13 belongs to a superfamily of proteins known to mediate ubiquitin-regulated protein degradation and oxidative stress-responses, which plays into some theories suggesting that artemisinin acts by generating reactive oxygen species, thereby causing oxidative stress.

While mutations in PfK13 are certainly common in resistant cases (again, particularly in S.E. Asia), they aren’t constant. As seen in the map above, African cases of artemisinin-resistance are mostly not found to have any mutations in PfK13. Instead, variable artemisinin efficacy appears to be linked to multi-locus genotypes involving other resistance-associated proteins such as PfMDR1 (a multi-drug resistance pump) and PfCRT (a chloroquine resistance-associated transporter protein), along with less well understood proteins such as PfUBP1 and PfAP2MU. Worryingly, the diverse causes of artemisinin resistance suggest multiple, independent evolutions of resistance in isolated Plasmodium populations.

Going back to the original point of this post, the BBC article “Malaria drugs fail for the first time on patients in the UK”, we should look at the source of this information. This is a brief report of four case histories of patients with imported malaria that were initially treated with artemether-lumefantrine but presented with recurrent parasitaemia within 6 weeks of treatment with no intervening travel to malaria-endemic areas. In English, this means patients that caught malaria outside of the UK were treated to the point where they seemed healthy, but were then readmitted at a later date with the same symptoms without an opportunity to catch it again. Two patients had been to Uganda, one to Liberia and one to Angola – all in Africa. As you might be able to guess, when profiled for resistance markers, none of the parasites these patients were infected with had any PfKelch13 mutations. Instead, they had a variety of mutations in other loci known to be associated with resistance, shown in table 1.

Ultimately all patients were successfully treated with alternative drug combinations such as atovaquone/proguanil, quinine/doxycycline or artemether/lumefantrine with doxycycline so the initial headline may be slightly exaggerated when you take the full picture into account. However, this is still an important news story as it is the first time artemisinin resistance has been seen in the UK. As resistance spreads throughout Africa these cases will only become increasingly common, so finding appropriate alternatives is a necessary task. 

The BBC article includes an interview with Dr Sutherland, the lead researcher, who stresses that while this is not yet a national health crisis it is important for doctors to be aware that drugs may not work and says drug guidelines should be reviewed. He also suggested that large scale studies of drug efficacy need to be urgently undertaken in Africa to determine the severity and scale of the problem we could be facing very soon.

Personally, I feel that while this is a very important news story, the fact that just four UK cases made headlines while thousands occur every year abroad is telling of people’s attitudes in the UK. The spread of drug resistant malaria is very real and a very serious concern to people living in malaria endemic areas, causing death on a huge scale, but is not on the radar for most people until it directly concerns them. I think awareness needs to be raised about this as an issue that faces other countries, not just ours. I’d like to know if you agree or disagree, and if you have any ideas about ways we could achieve this goal – let me know in the comments.

Malaria vaccine

By Jack Sewell

In a recent paper published in Science Translational Medicine, Kublin et al. (2017) genetically engineered a strain of the human malaria parasite Plasmodium falciparum¹. This removed its ability to infect the liver and cause illness in humans, and acted as a vaccine by increasing their resistance to future infection.

Humans become infected with malaria when they are bitten by an Anopheles mosquito that carries the malaria parasite, Plasmodium. There are many different Plasmodium species, such as P. falciparum which is the major cause of malaria in humans. The bite results in a small number of Plasmodium sporozoites entering the human blood, though this does not cause illness. Rather, the sporozoites invade the liver cells where they develop into merozoites, which eventually burst out in a process known as schizogony (Fig. 1). The merozoites then infect red blood cells (RBCs), where they undergo schizogony once more. This leads to the destruction of large numbers of red blood cells, hence causing the symptoms of malaria such as anaemia. Because of this, an effective malaria vaccine must be able to target either the sporozoite or liver-stage forms of Plasmodium, before it can infect the RBCs.

Figure 1 | Plasmodium life cycle human stages. 1. Sporozoites produced in the mosquito migrate to the salivary glands. 2. Sporozoites are injected into the host blood by the mosquito during feeding. 3. Sporozoites invade liver cells and develop into merozoites. 4. Merozoites invade and replicate inside erythrocytes. 5. Male and female gametocytes are produced inside the infected erythrocytes then taken up by a mosquito. Adapted from Hill (2011)².

Potential malaria vaccines belong to one of two categories; those which use parasite antigens and those which use whole sporozoites. The use of ‘whole sporozoites’ vaccines presents more of a risk to patients than parasite antigen vaccines, but so far is the only approach that can achieve complete, sterile protection against P. falciparum malaria. The whole-sporozoite strategy is usually achieved by one of two different approaches. The first is to produce radiation-attenuated sporozoites (RAS), which are unable to replicate in the liver due to radiation-induced DNA damage³. The second approach is chloroquine prophylaxis with sporozoites (CPS), which involves infection with infectious sporozoites, followed by administration of the antimalarial chloroquine to kill the blood-stage parasites. However, a new third approach is the creation of genetically attenuated parasites (GAP), which are genetically modified to possess a particular attenuated phenotype, and can confer long-lasting and complete sterilising immunity in mice⁴. One advantage that GAP has over RAS and CPS is that, as all the attenuated parasites have the exact same phenotype, both the efficacy and safety can be altered more easily.

In order to create the attenuated P. falciparum strain, named Pf GAP3KO, Kublin et al. removed the p52, p36 and sap1 genes. These are all expressed in the pre-erythrocytic stage and necessary for the parasite to progress from the liver-stage infection⁵. Following infection of the volunteers with Pf GAP3KO through mosquito bites, they did not suffer from any malaria symptoms, though they did experience some adverse effects from the large number of mosquito bites they received. In addition, analysis of P. falciparum 18S ribosomal RNA presence showed the absence of blood-stage parasites. This illustrated the inability of the Pf GAP3KO strain to invade erythrocytes, and hence its safety due to its inability to cause disease.

The application of the Pf GAP3KO sporozoites to the volunteers induced the development of anti-sporozoite antibodies, specifically the immunoglobulin G (IgG) that targets the circumsporozoite protein (CSP) on the sporozoite surface. Following immunisation with Pf GAP3KO, serum was taken from the volunteers and used in an in vitro assay of sporozoite traversal and liver cell invasion. This showed that the anti-CSP IgG inhibits sporozoite invasion and traversal by approximately 50%, though its activity was slightly greater when the serum was taken from the volunteer later after immunisation. Furthermore, the anti-CSP IgG was also able to inhibit liver infection in vivo, and was shown to bind to antigens on both the sporozoite and liver-stage parasites (Fig. 2). Together, these suggest that the attenuated sporozoites achieved the desired effect of a vaccine, by inducing an immune response that increases resistance to a second infection.

Figure 2 | Serum IgG binding to sporozoite and liver-stage parasites. Sporozoite and liver-stage parasites are marked with an anti-CSP or anti-Bip tag respectively (green). Day 0: Pre-immune serum does not contain IgG with sporozoite or liver-stage binding activity. Day 28: Immune serum contains IgG with both sporozoite and liver stage binding activity (red). Adapted from Kublin et al (2017)¹.

In summary, this paper showed that genetic attenuation is a viable means of producing ‘whole sporozoite’ malaria vaccines. In addition, it may be superior to previous approaches, because each sporozoite possesses the same phenotype so both safety and immunogenicity can be maximised. In addition, Pf GAP3KO was shown to be a promising candidate to be used in this approach. However, future studies on this construct still need to investigate the extent of the protection it provides against wild-type P. falciparum, as this was not investigated in this study. Finally, an obstacle to overcome is the requirement for sporozoites to be produced and maintained in mosquito salivary glands. At present, this limits the potential for widespread administration of a ‘whole sporozoite’ vaccine.

References

1. Kublin, J. G. et al. Complete attenuation of genetically engineered Plasmodium falciparum sporozoites in human subjects. Sci. Transl. Med. 9, 1-11 (2017).

2. Hill, A. V. S. Vaccines against malaria. Philos. Trans. R. Soc. 366, 2806-2814 (2011).

3. Nussenzweig, R.S., Vanderberg, J., Most, H. & Orton, C. Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. Nature 216, 160-162 (1967).

4. Bijker, E. M. et al. Novel approaches to whole sporozoite vaccination against malaria. Vaccine 33, 7462-7468 (2015).

5. Vanbuskirk, K.M. et al. Preerythrocytic, live-attenuated Plasmodium falciparum vaccine candidates by design. Proc. Natl. Acad. Sci. U.S.A. 106, 13004-13009 (2009).