Fluorescence and Failure

By Jack Sewell

Project aims

This post is about the malaria lab project that we've been working on full-time this semester. To avoid giving away too much information about what the lab is investigating, I won't mention the specific proteins under study. Essentially, the project has involved each of us studying the expression and localisation of a separate protein during the life cycle of Plasmodium, the malaria parasite. In addition, we aimed to study the morphology of the parasite when the protein was absent using a PTD construct. This removed expression of the protein from every stage in the life cycle, except for the blood stage where it is known to be essential. The point of studying the proteins was to identify a potential drug target for malaria treatment. The first thing we did was to transfect Plasmodium with a fusion gene, which coded for our protein of interest followed by the green fluorescence protein (GFP) tag. This would later allow us to see where our protein was in the cell. However, first we had to battle through the process of producing a Western blot, in order to show that the protein and GFP had been successfully fused. As Carrie has written about previously, this resulted in lots of failed attempts, with each one inevitably falling foul of a mistake that would leave us scratching our heads.

It's in there somewhere.

Life cycle imaging

Eventually though, we all had a band that we could confidently call our protein, and could then focus on the fluorescence imaging of the various Plasmodium life cycle stages. Because each attempt to transfect our PTDs into Plasmodium failed, this meant we could only study the protein localisation and not the phenotypes of the knockout. What I would consider to be the major difficulty in this part of the project was the time taken to culture the different life cycle stages. The process of growing Plasmodium in mouse blood takes around 3-4 days, after which time the mouse must be euthanised to prevent unnecessary suffering. The blood can then be used to image the parasite blood-stages (rings and trophozoites), or cultured for a further 24 hours to produce either schizonts or a mix of gametocytes, zygotes and ookinetes.

Plasmodium stages that can be imaged from mouse blood

Furthermore, studying the mosquito stages of the life cycle takes a total of 21 days following the collection of mouse blood. The mice must first be treated to eliminate all parasites except the gametocyte stages, then the mosquitoes are permitted to feed on them. At this point, the gametocytes in the mosquito gut become activated and leave the red blood cells. You then need to wait 10 days before some of the mosquitoes can be checked for oocysts lining the midgut, and mature oocysts can be imaged on day 14. After another week, on day 21, it should be possible to image sporozoites in the mosquito salivary glands. This completes the stages of the life cycle that can be studied, because the host's liver-stage parasite infections cannot be studied without complex dissection that would also render the mouse useless for study of the other stages. Unfortunately, the day 10 check of the mosquito infection for my protein showed no oocysts, meaning that it may not be possible to complete the oocyst and sporozoite imaging. This has therefore been a potential waste of 10 days, and would need the use of more mice and mosquitoes if a second attempt is to be made.

The Fluorescence Microscope

The actual process of taking parasite fluorescent images starts by mixing infected blood with DAPI, a DNA-specific stain that highlights the nucleus blue. In addition, the activated female, zygote and ookinete stages can also be tagged in red. This works by using a red fluorophore conjugated to an antibody the specifically binds to a protein expressed on the surface of these cell types. Once the fluorophores have been added, the blood can be searched for the desired stages under the fluorescence microscope. Any cells of interest must then be measured for their blue and red fluorescence, as well as the green fluorescence emitted by the GFP-tagged protein.

Activated female gametocyte with its membrane, nucleus and protein highlighted.

Getting a good image was always more difficult than you'd think, given that we had to make do with a single microscope shared between 4+ people, who often all had parasites to image at the exact same time. As well as this, we had to get the cell concentration exactly right, or they would be too sparse to find anything of interest or too dense to get the cell on its own. After many mice though, we were each able to create a complete collage to show the localisation of our protein at each of the ring-to-ookinete stages. All that remains now is to finish the study of the mosquito stages, and the project will be completed.

All in all there's been a lot of frustration and disappointment, but with some perseverance we got good results in the end. We also gained a lot from being able to adapt and learn from the things that went wrong, which in the end is a very important skill for a lab scientist to have. 

Ectogenesis

By Jack Sewell

The concept of ectogenesis, or in vitro pregnancy, is an interesting one, with huge ethical and social implications. Essentially it involves the use of an “artificial womb” machine that would facilitate the development of a foetus, by replicating the processes that normally happen during a natural pregnancy. This includes the provision of maternal blood or a suitable substitute, which could supply oxygen and nutrients to the foetus, as well as remove waste materials. To achieve this, an artificial placenta would have to mediate the transfer of these substances to and from the foetal circulation. Research in this field began in the 1980s, when in 1989 a human embryo was implanted in ex vivo uterus for the first time¹, though this line of study was quickly halted due to ethical concerns. Further advancements have been made since then, but currently there is no technology which is capable of supporting foetal development from conception through to birth.

Taken from motherboard.vice.com.

There are two potential uses for ectogenesis technology. The first would be as an improvement on current technology for the incubation of premature babies, which would likely give a much greater survival rate by more closely imitating the final stage of gestation. At present, incubators are only effective for babies born after at least 24 weeks of gestation¹. The second use is as an alternative to natural pregnancy, in which the majority or entirety of the gestation could be completed within the machine. An advantage that this could have over a natural pregnancy is the ability to monitor the developing foetus more closely throughout the process, perhaps as though it were on life support. This may be useful if the unborn baby is known or suspected to have a condition that would benefit from close monitoring. It could also make prenatal diagnosis tests safer and more viable, as the current tests (amniocentesis and chorionic villus tests) pose a small risk to the foetus.

Unsurprisingly, this technology raises significant ethical issues relating to the role of the mother and the reliance on technology as a substitute for a natural human process. As with many other scientific controversies, just because science can do something doesn’t necessarily mean that it should. Therefore, we must ask who - if and when this technology becomes available - should be allowed to use it to facilitate an entirely ex vivo pregnancy. For instance, should it only be available to those who cannot carry a child naturally, such as women with damaged uteri or gay men? In these cases, it would remove the need to find a willing surrogate mother, which is currently their only option if they wish to have a biological child. On the other hand, if the technology were made available to everyone, one could imagine that a pregnant woman may be offered the choice to transfer the embryo into an artificial womb for the remainder of the gestation. One ethical issue that arises here is whether this could mean taking away the control that women have over their own body and their own pregnancy. It may even lead to a world where artificial gestation is medically advised or legally enforced, due to reasons such as the poor health of the unborn child or the unfitness of the mother to take care of it during the pregnancy. Therefore, as this technology develops, there are serious decisions that lawmakers must prepare to make concerning the ethics of its use. For now though, there is plenty of time as it is estimated that the necessary technology for a full ex vivo pregnancy will not exist for several decades.

References

1. Bulletti, C., Palagiano, A., Pace, C., Cerni, A., Borini, A. and de Ziegler, D. The artificial womb. Ann. NY Acad. Sci. 1221, 124-128 (2011).

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).