How bacteria might win the fight against malaria, dengue and Zika

Have you heard of the most prevalent parasitic microbe in the world? 

Capable of infecting up to 70% of all insects? 

With a preference for murdering males in cold blood? 

Known as “Wolbachia”, this genus of bacteria is found in many arthropods, including those responsible for the transmission of many human pathogens; such as malaria, dengue fever, and the Zika virus. It gets transmitted vertically, meaning that infected females pass it directly to their offspring. Different strains of Wolbachia infect different hosts and can have many different effects. In filiarial nematodes Wolbachia plays a mutualistic role, with elimination of the symbiotic bacteria causing host sterility and sometimes death. In mosquitos however, Wolbachia has a more sinister role - causing something known as “cytoplasmic incompatibility”. I’ll explain what exactly that means in a minute, but the end result is that infected females will always produce infected offspring, and uninfected females have half the chance of producing offspring. This is a case where it’s easier to show, than tell.

This picture, taken from the Werren lab Wolbachia biology page, shows how cytoplasmic incompatibility leads to an increase in the proportion of infected hosts.

The exact mechanism for this cytoplasmic incompatibility (CI) isn’t yet clear. What is known is that some kind of modification must take place in the infected male during spermatogenesis, as mature sperm cells do not contain Wolbachia. We also know that rescue must occur at some point in the fertilised infected egg, as the presence of Wolbachia prevents CI from occurring, but uninfected eggs don’t produce offspring. The main consequence of CI is that the male pronucleus enters mitosis later than the female pronucleus, which means the genetic information from the male does not segregate properly during the first mitosis. This leads to the production of haploid daughter cells which is embryonically lethal, preventing any offspring from being formed.

In May last year, a paper by Daniel LePage et al., was published which found that just two genes in the Wolbachia genome were needed to cause CI. The sequences for these genes were found to originate from viral DNA, integrated from the bacteriophage WO genome at some distant point in Wolbachia’s evolutionary history. These two genes, named cifA and cifB (cytoplasmic incompatibility factors A and B), were initially identified as part of a pool of 113 genes shared between CI-causing strains of Wolbachia. This pool was narrowed down by removing proteins known to be very different or absent in a non-CI-causing strain, wAu, and by removing proteins known to be expressed by infected ovarian tissue as these might be generally expressed and not play a direct role in CI. This left just two genes, WD0631 and WD0632.

In section a, a Venn diagram shows 113 genes are shared between four common CI-causing strains of Wolbachia. In section b, a Venn diagram shows how these 113 genes were narrowed down to just two candidates for CI-causing genes – WD0631 and WD0632.

To investigate if these two were indeed the genes responsible for CI, transgenic lines of Drosophila melanogaster were created with both genes under the control of a promoter that is active in germ line cells. Males of this line were crossed with infected and uninfected females and the relative hatch rate calculated. It can be seen in the graph below that having both genes caused a significant change in the embryo hatch rate when crossed with uninfected females, and that this was rescued when crossed with infected females – as would be expected if these were the culprits for CI.

A comparison of relative embryo hatch rate in D. melanogaster. White symbols indicate uninfected males and females, black symbols indicate those infected with a CI-causing strain of Wolbachia.

Further to this the team investigated whether the cytological appearance was the same between Wolbachia infected embryos and transgenic WD0631⁺/WD0632⁺ lines, further strengthening their case that these were the responsible genes.

Panels a-f show different classifications of cytological appearance in recently fertilised ovaries of D. melanogaster. a) indicates unfertilised eggs, b) shows normal nucleated cells at 1h of development, c) shows normal embryos at 2h of development, d) shows failure of nuclear division after two to three mitoses, e) shows chromatin bridging and f) shows regional mitotic failure. In section g) the relative abundance of each cytological appearance is shown in different crossing scenarios.

The paper doesn’t go on to speculate on or provide any investigation into the mechanism of action of these two genes, but sets the stage for further work into this system and tantalisingly closes with the statement 

“Finally, cifA and cifB are important for arthropod pest and vector control strategies, as they could be an alternative or adjunct to current Wolbachia-based efforts aimed at controlling agricultural pests or curbing arthropod-borne transmission of infectious diseases”

This brings me on to the conclusion of this post and refers all the way back to the title – how can bacteria be used to fight insect-vector based disease?

There are already programs in place that are attempting to use Wolbachia-based methods of eradicating the insect vector of the dengue fever virus, the Aedes aegypti mosquito. By introducing large numbers of infected males into an uninfected population, the population can be reduced without the use of pesticides. The work done by LePage could provide the means to supplement Wolbachia in insect systems or possibly induce CI in other systems. An alternative to herbicides, or maybe maintenance-free pest control – can you think of any other applications? Let me know in the comments.

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).

Is RTS,S the malaria vaccine we need?

The World Health Organisation has announced their intention to run three pilot projects within Sub-Saharan Africa for the first malaria vaccine. Due to begin in 2017, the pilots will will answer remaining questioning regarding RTS,S/AS01, which is the result of an enormous 32 years of research from partnerships between GlaxoSmithKline, the PATH Malaria Vaccine Initiative (MVI) and Walter Reed Army Institute of Research. Composed of a viral Hepatitis B surface antigen-like particle, an adjuvant to improve immunogenicity and a section of Plasmodium falciparum circumsporozoite protein, RTS,S represents the only candidate to have completed Phase II trials. 

Despite this success, vaccine efficacy has been found to fall far short of the 75% benchmark. With protection levels reported to be controversially low, what can we really expect from RTS,S?

Development timeline of the RTS,S vaccine shows the path from 1987 to 2014. Since creation of this figure, the vaccine has received a positive opinion from the European Medicines Agency and African countries are applying to national regulatory authorities for vaccine approval. 

The WHO seeks to answer these questions, with the pilot programmes aiming to "evaluate the feasibility of delivering the required 4 doses of RTS,S; the impact of RTS,S on lives saved; and the safety of the vaccine in the context of routine use". The replicability of protection levels has also been queried, with researchers advising four doses to provide maximum efficacy. This in itself could be problematic without comprehensive healthcare infrastructure. Summaries of the available Phase III trail data by Gosling and von Seidlein (2016) found the addition of the fourth booster justified, as in 6 - 12 week infants vaccine efficency rose from 18% to 25%, and in children 5 - 17 months old this rose from 28.3% to 36.3%. 

If RTS,S isn't the answer, where are the other candidates for malaria vaccines? Jack has previously posted regarding the types of malaria vaccine, and the WHO estimates the 20 candidates currently at various stages of the pipeline are all 5 - 10 years behind RTS,S. One of these candidates - PfSPZ, Sanaria - is composed of radiation-attentuated, whole sporozoites and has recently been reported to deliver full protection up to 25 weeks after innoculation. A recent review by Cowman et al (2016) highlights that this candidate lacks the stability of RTS,S, requiring liquid nitrogen to maintain viability which will undoubtedly be a logistical issue. It will be interesting to follow PfSPZ through more extensive trials to see if those statistics hold, but it should also be noted that this vaccine was also using a 4 dose schedule. 

Summarising the benefits and drawbacks of RTS,S, what can we expect from this vaccine? Whilst RTS,S could be integrated into current malaria control schemes, it is easy to be disappointed by the low levels of protection granted. At the beginning of this post I underlined the sheer amount of effort and time funneled into vaccine development, and for it still to fall short is hugely discouraging. Whilst the more cynical among us might argue that RTS,S is no solution in our fight against malaria, I find it important to consider the severity of the disease in sub-saharan Africa. Within this context any new tools are welcome, especially should we wish to continue reducing malarial burden. The WHO's decision to further test RTS,S is prudent but should they not seek to implement vaccination programs after pilot programmes have finalised it would be interesting to see if RTS,S would survive. If nothing else, the public and academic support for malaria vaccines generated by RTS,S should ease the passage of future vaccine candidates.

References

1. Gosling, R., and von Seidlein, L. (2016). The Future of the RTS,S/AS01 Malaria Vaccine: An Alternative Development Plan. PLoS Medicine 13(4) pp. 312-315
   
2. Ishizuka, A.S., Lyke, K.E., DeZure, A., Berry, A.A., Richie, T.L., Mendoza, F.H., Enama, M.E., Gordon, I.J., Chang, L.-J., Sarwar, U.N., et al. (2016). Protection against malaria at 1 year and immune correlates following PfSPZ vaccination. Nature Medicine 6, pp. 614–623.
   
3. Cowman, A.F., Healer, J., Marapana, D., and Marsh, K. (2016). Malaria: Biology and Disease. Cell 167, pp.610–624.