UCC iGEM

Integrated Human Practices

In the youthful stages of project design, we had researched the vast epidemiology of Leishmaniasis as a neglected tropical disease, and read into the Lutzomyia longipalpis saliva protein, LJM11, as a solid solution for immunisation against the disease. But we felt there were many questions which still needed to be answered before we could be sure that it is a worthwhile investment of both time and money;

Is our Limitless Lactis vaccination actually needed in the developing world? Is the disease as prevalent as we had read? How would it add to current available prevention/treatment and provide benefit to the population?
Is it something that the population will accept?
Would the regulatory bodies in afflicted regions approve of this therapeutic? What problems might be encountered?
Is there any way that we can improve our therapeutic design to make it more beneficial to these areas?
Outside of therapeutics, are there any other ways that we could combat the disease?

However, despite our extended efforts to find the answers to all of these questions by filtering the available online database, we couldn’t answer all of them. We were left frustrated. We soon realised that we were looking in the wrong places. These are real world questions which need real world answers. Leishmaniasis is a disease which resides primarily in the developing world, where the web is an underused resource. These answers weren’t all online, they lied in the people living on the soil of the affected areas. Thus, this realisation instigated the next step in our project - to reach out to those who live in the affected areas.

Following this bold decision, we needed to gather as much information as possible on the current global climate of the disease before choosing a location to explore. To do this, we had a Skype session with one of the key Leishmaniasis researchers worldwide, Dr. Alvaro Acosta - a parasitologist from the Liverpool School of Tropical Medicine. We set out to learn the main global problems associated with the epidemiology and management of the disease.

We learned that as Leishmaniasis is a vector-borne disease with the ability to adapt to changing environments. This is particularly worrying as Leishmaniasis is now able to spread further from the equator due to global warming. Therefore it is imperative that effective and lasting vector control systems are introduced to reduce its diffusion into new regions. The control system currently in place in many countries is indoor residual spraying (IRS). However, the usage of DDT (dichlorodiphenyltrichloroethane) spray is harmful to the environment as it has insecticidal properties. Many types of spray used are also very ineffective during the summertime when temperatures are high and there is strong radiation and accumulation of dust.

Above: Map of the Americas [New world] showing the presence or absence of Cutaneous Leishmaniasis.

Above: Map showing the absence or presence of Visceral Leishmaniasis in the Old World

The second topic we focused on was current therapies available for treatment of Leishmaniasis, which have severe problems associated with them. Such a problem is seen widely in Honduras, where pentavalent antimonial such as, Glucantime is the choice of drug for treatment. It is intravenously injected, and the course lasts for 20 days. Due to the painful treatment a culture of poor compliance among the patients has been seen at a high rate.

Next, we went onto discuss the possible issues that might arise at the distribution of vaccine if the vaccine was made. In conflict zones such as the Middle-East, the vaccine might be difficult to administer. Voluntary organisations such as MSF (Médecins Sans Frontières) who work with different developing countries in order to treat Leishmaniasis might not go if the area is seriously war ridden. A recent example of this would be Syria where the risk associated with volunteering at the capital city, Aleppo is considerably high.

The government of developing countries cannot afford expensive treatment methods, hence, there is a strong need for cost-effective therapy. Dr Acosta emphasised that we have to put ourselves in the situations of the countries we will possibly be dealing with. This helped us to divert our focus to factors that matter to a developing country. As Leishmaniasis is a tropical disease and occurs in hot climate countries, the drug needs to be stable at high temperatures. For this, a cold chain is required i.e. during the process of transferring the vaccines from one place to another, they are maintained at a temperature of approx four degrees celsius. However, as this can be very costly, our vaccine could be made as freeze dried powder and formulated in a capsule. It can then be transferred at ease and at a low cost.

With a disease such as Leishmaniasis, social stigma is bound to be prevalent especially, in poorly educated and traditional underdeveloped countries. Dr Acosta mentioned that in the Middle-East and Honduras, an unsafe treatment practice exists, known as Leishmaniasation. Many young girls proceed through this as cutaneous Leishmaniasis leaves a lifelong scar which is highly stigmatised, in their society. Military soldiers also go through this process, a mass experiment was carried out in the Islamic Republic of Iran during the war between Islamic Republic of Iran and Iraq. This was so that the recruited soldier would be immune to the disease for the duration of their service.

During the process of research, one of our team members who is originally from Nepal discovered that Leishmaniasis is another name for the disease “Kala-azar”. It translates to Black fever as characterised by darkening of the skin. This Leishmaniasis present in Nepal is of the visceral type, due to the vector species L. donovani (which was discovered by Dr Charles Donovan whose family was originally from Cork, Ireland).

In 2005, at the World Health Assembly, Nepal along with India and Bangladesh committed to a strategy to eradicate Leishmaniasis from the 3 countries by 2015. This programme was implemented in 12 districts where the most cases of Visceral Leishmaniasis were reported.

Above: Visceral Leishmaniasis trend in Nepal from years 1994 to 2010.
Source

Leishmaniasis is prevalent at the eastern and southern (central Terai region) districts of Nepal. These districts are economically and educationally under huge disadvantage. According to the annual reports of the Nepalese Department of Health Services for the fiscal years 2014/2015, out of the 12 districts in the programme, the district of Sarlahi made up 63% (449 cases) of the overall cases in 2010 (708 cases). This was mainly the result of a mass migration from nearby villages into the suburbs. The poor waste management due to lack of knowledge is one of the key reasons of transmission of Leishmaniasis into this region.

However, for the year 2014/2015, Sarlahi district only made up 8% of total 148 cases reported in the country. While, the district of Morang, on the far east of Nepal has had the highest number of reported cases in the past 5 years, it has significantly decreased over the years, from 175 cases in 2010 to 48 cases in 2015. This shows that the eradication programme has been majorly successful and this further accentuates the need for solutions to eradicate Cutaneous Leishmaniasis as well in countries in the Americas and the Middle-East.

Cases outside the 12 mainly affected and controlled districts are also increasing in numbers slightly. This highlights the importance of our discussion with Dr Acosta where along with new vector control strategies, biological vector survey needs to be carried out often to look at the indigenous transmission of the disease.

We researched the areas affected by Leishmaniasis, and debated which country would be most beneficial to visit to find these answers. Following careful deliberation, we decided to go to Honduras in Central America.

Figure 1 Figure 1

We chose Honduras due to the immense numbers of reported cases of Leishmaniasis in the country and due to the current climate of governmental support for the disease. Last year, approximately 700 cases of Leishmaniasis were reported in the Choluteca county alone (South western Honduras), and the numbers of cases seem to be increasing exponentially. Also, due to focus on the arbovirus Dengue, Chikungunya and Zika, governmental support is distracted from Leishmaniasis. Hence, the current lack of hope in Honduras in the battle against Leishmaniasis made it the perfect location to explore with our solution.

Suazo Hospital, Tegucigalpa

Our first task following arrival on Honduras soil was to visit the Alonso Suazo public hospital in the capital city, Tegucigalpa. We met with Dr. Cristian Valladares, who was working in the diagnosis laboratory for Leishmaniasis in the hospital. One of our team members, Yensi, could speak Spanish fluently, hence breaking the communication barrier with the staff. On entry, we were amazed by the differences between the laboratories that we were used to seeing at home in Ireland and the laboratory before our eyes. It was shocking how small and clustered it was, and also there was an apparent absence of modern laboratory equipment. What was more breathtaking, was the fact that this was not just the only diagnosis laboratory for Leishmaniasis in Honduras, but it was the only diagnosis laboratory for all infectious diseases in the entire country. We began to wonder whether a laboratory of such size could recognise the true prevalence of such diseases.

Photos with Dr. Cristian Valladares in Alonso Suazo hospital, Tegucigalpa

Following conversation with Dr. Valladares, we learned that several species of Leishmaniasis, from cutaneous to visceral are carried by Lutzomyia longipalpis, the sandfly which LJM11 induces immunity against. This backs the benefit of targeting our vaccine against this sandfly, as it covers a wide range of the disease subtypes. This eliminates the need for multiple vaccines.

In addition to this, we learned that diagnosis (by biopsy) is often avoided as there is a large epidemiological silence with this disease. Many people with cutaneous Leishmaniasis lesions avoid presenting to their GP for reasons such as tradition and culture, and mainly because of a lack of education about the disease. Many use “self-remedies” instead of standard treatment which can keep lesions at bay, but there is lack success in resolving of the condition as the parasite often returns. Also, the current standard treatment for Leishmaniasis in the country (Glucantime, an Antimony based drug that can cause liver and kidney damage) consists of a series of painful 15ml intramuscular injections for 20 consecutive days. Often, those infected stop in the middle of treatment, or sometimes don’t present at all due to fear of pain. This poor compliance is a major problem as without completing the full course of treatment, the parasite can continue to spread and increase in severity.

This provides the the answer to the first question.. Not only did he mention that around 2,700 cases present per year in the country, but as many people with the disease don’t present, and many others refuse treatment, the figures of those infected in reality are actually much higher than the numbers reflected by this. Also, lack of compliance to the current treatment and lack of education about the disease would suggest that a prevention may be a much better solution than a treatment. So we concluded that not only is there a need for immunisation against Leishmaniasis, but the need is actually much greater than we had ever anticipated.

Also, the fact that many attempt to treat the lesions with home-made self-remedies indicates that people do actually want to treat the condition, so a viable therapeutic should be welcomed by the population. But it appears that the inconvenience and pain involved in the injected antimony treatment is a major factor in detering people from seeking medical help for the condition. This highlights the need to ensure that the treatment is convenient and that it doesn’t instigate fear to be accepted by the population. More on this will be discussed following our visit to the Choluteca area.

A recording of part of the conversation with Dr. Valladares can be found here

National Autonomous University of Honduras, Tegucigalpa

Leading on from the visit to the hospital, we were eager to learn more about the condition in Honduras, and to deepen our understanding of Leishmaniasis on a scientific level. Thus, we visited the National Autonomous University of Honduras in Tegucigalpa, where we met Dr. Jorge Alberto Carrasco. Dr. Carrasco is head of Immunology and Parasitology in the University, and he is a leading expert on Leishmaniasis in the country. He is also part of the biosecurity council of Honduras.

Figure 3: National Autonomous University of Honduras

Here, Dr. Carrasco lectured us on both the immunology and the parasitology of Leishmaniasis. He explained the immunocellular response to the pathogen. It was noteworthy that immunity to the infection is mainly cell mediated, but humoral immunity can also play a role. This falls perfectly in line with our decision to use the LJM11 protein in our vaccine, as LJM11 is believed to induce both cell mediated and humoral immunity.

We also learned that the lack of diagnosis and treatment ensure that the life cycle of the parasite is maintained. As the parasite continues to move from sandfly to person and vice versa the untreated population act as a reservoir to ensure the infection will continue to spread rapidly. This is why there is such a high demand for immunisation against the disease. Also, as many are infected at a young age (early childhood), it is important that the vaccine can provide immunity against successive infection by different forms of the parasite leading to more severe infections, eg. mucocutaneous or visceral Leishmaniasis. Thankfully, multiple of these species are carried by the sandfly vector which we are immunising against..

A brief clip from our recording of the meeting with Dr. Carrasco can be found here:

Regulation in Honduras

Now that we understood the condition on a scientific, clinical and epidemiological level in the country, we felt the need to gain an understanding of key regulators of Leishmaniasis control in the country. Firstly, talked with Dr. Carlos Ponce, the former director of Leishmaniasis strategy control in Honduras. He approved of our Limitless lactis vaccine idea and was very impressed by it as there is a real need for such a vaccine. We also spoke with Dr. Concepcion Zuniga, the current director of Leishmaniasis strategy control who offered us further insight into the regulatory aspects of treating this disease. From these conversations we learned a great deal about the positive and negatives of introducing a vaccine such as ours into the country.

Firstly we learned that regulation in Honduras is practically non-existent in comparison to regulation in the developed world. There is no regulatory body for health products in Honduras. They do follow Pan-American regulatory guidelines, but they are far less stringent than the tight, drawn-out regulation that we are used to at home in Ireland. This may be highly beneficial for bringing the Limitless lactis vaccine to market in the country as vaccines could potentially get from initial design to the market in under a year.

This system would ensure that we would have a much better chance of avoiding failure during preformulation studies and clinical trials and reaching the market than in Ireland, where often as low as 1 in every 10,000 compounds make it to the market following drug safety and efficacy studies (1). It would also open the opportunity that the vaccine could reach the suffering population of Honduras many years earlier than in the developed world (12-15 years), in which time it could save many lives and prevent the suffering of millions. In addition to this, the reduced expenditure on clinical trials and documentation during development could contribute to a reduction in the cost of the vaccine to the government, hence having an economical benefit to the country. This is especially important as public healthcare in the country doesn’t cover everybody due to lack of funds. Taking these points into account, the potential for the Limitless lactis vaccine to be utilised by the country seems very promising.

However, even with such benefits in place, we learned that a system like this does impose some challenges. Because the regulatory system is so irregular, there is almost no concern present there among the local healthcare authority for the quality, safety and efficacy of these systems.

Regulation is particularly important in medicines as inadequate regulation may put lives at risk. Because of this, we believe that us scientists and developers in the developed world who produce vaccines such as this have an ethical responsibility to ensure the quality, safety and efficacy of the medicine before bringing it to the market in countries such as Honduras. The poor populations of the world are not lab rats, they are human beings, just like us, who require just as high a standard of healthcare as we do.

Choluteca, Honduras

Having investigated the issue of leishmaniasis with some of the main stakeholders in the country, our next focus centred on what is it really like in an epidemic area ? This question set us off to Choluteca, a province in the south west of the country, where leishmaniasis is prevalent. It was here where we encountered Fyffes who were a major employer in the area. Fyffes have demonstrated loyalty to the community by building a school and a hospital for the local populace. While in choluteca, we gathered information on the prevalence of the disease, the number of patients who seek treatment and factors that are thought to be involved in the spread of the disease.

Above: Hospital in Choluteca region.

The local doctor was very aware of the disease and provided us with information with regard to ways in which the disease could be prevented. He also highlighted issues such as the lack of education as a factor which has led to the spread of the disease in previous years. Another issue which was emphasised and which we have seen previously through interaction with Dr Alvaro Acosta and Dr Cristian Valladeres was that there is poor patient compliance with the current treatment. This series of twenty painful intramuscular injections deters patients from seeking treatment and the parasite is then allowed to spread.

Above: Yensi speaks to the Ernesto Caceres (Fyffes Medical doctor) about the prevalence of the disease.

Having informed the doctor of our aim to produce a vaccine against leishmaniasis, he was quite interested. He emphasized that the vaccine would need to be cost-effective, we therefore decided to concentrate on developing a vaccine that could be orally administered. Our strain as previously mentioned has the hylA gene integrated in it, the LLO protein is highly immunogenic and has been previously described as being capable of being an adjuvant (Sun R et Liu Y. 2013). This therefore improves the ability of our vaccine to elicit an immune-response and avoid the issue of tolerance which has been associated with some oral vaccines.

An oral vaccine is desirable from a clinical point of view as it does not require a skilled professional to administer the vaccine. This will reduce the economic cost of employing the vaccine on a large scale. The doctor also emphasised that distribution of the vaccine would be an important consideration, he spoke about the instability of certain medical products in the Honduran summer when temperatures can reach as high as forty degrees celsius. Following this recommendation, we investigated the possibility of freeze-drying our vaccine. Once freeze-dried, the requirement for a cold chain is mitigated and the stability at these higher temperatures is increased. Dr Alvaro Acosta also emphasised the need for the vaccine to be stable at high temperatures and the desirability for low cost distribution.

The doctor also highlighted that current therapies are inadequate due to poor patient compliance. Many patients were reported to stop visiting the hospital for the pentavalent antimony IM injections in the middle of the treatment because of the pain associated with the injection. One way in which they are trying to tackle this problem is through patient education, however this has only shown limited efficacy so far. When treatment is stopped, the parasite has a chance to recover as it wasn’t fully eliminated from the host and therefore can continue to spread. As previously highlighted Cristian Vallediares also focused on this and spoke about how many people stop taking the treatment. Several stakeholders have highlighted the importance of ensuring that any new medicines or vaccines must ensure patient compliance. Our L.lactis vaccine has the capabilities to be given in a capsule formulation which is easy to use and appealing for the patients.

One key issue which we identified in our discussions with stakeholders was that education is a factor in the spread of the disease. Our team tried to think of ways to tackle this problem. Having spoke to both officials in our university in Ireland and the National University of Honduras, we decided to organise the collection and donation of old, unwanted or unused equipment and consumables from our university. We organised transportation of the equipment with Fyffes.

Some of the equipment or consumables that we donated includes:

Fluoroskan Ascent plate reader
Incubator
T75 tissue culture flasks
12 well tissue culture plates
6 well tissue culture plates
96 well tissue culture plates
Gel doc imaging equipment
Gel electrophoresis powerpacks
Gel electrophoresis boxes
Gel electrophoresis trays and combs
Pipettes
Pipette tips
Biochemistry and cell biology textbooks
Research journals
Centrifuge
UV cross-linker
Flow cytometer
Analytical balance
Graduated cylinders and beakers
Sharps containers
Water purifier

Photos of us packaging equipment for shipping to Honduras

The importance of education was apparent throughout the summer. We as a team learned a tremendous amount from molecular biology techniques to designing animations. Our outreach activities proved to be the most rewarding, having interacted with school children from the ages of 11 to 17, we learned about the impact that education has from a teacher’s perspective. Whether it was witnessing a student’s reactions to painting fluorescent bacteria or learning about their ideas on GMOs, we quickly started to appreciate the effect that education can have. In this regard, we wanted to do more, we wanted to foster interest in synthetic biology in people who might otherwise never learn about it. Not only did we aim to foster scientific curiosity, we hoped that by improving the scientific education in the largest university in Honduras that it could have a positive impact on local communities and down the line mitigate the risk of disease transmission for leishmaniasis and other diseases prevalent in Honduras.

Another potential cause for the spread of the disease is due to the lifestyle of the people in the choluteca region. The region has experienced a prolonged drought in the last two years which has limited water supply and exacerbated plagues. Subsistence farmers in particular have been affected as they lack irrigation systems and have traditionally relied on rainwater. The drought has caused the loss of up to 90% of crops (Maize and beans) which has led to chronic malnutrition in 25% of children between the ages of 6 months and 30 months. Our team had very little experience in this area but decided to investigate it further. We looked at the potential of using genetically modified crops in honduras since there are very few regulations in relation to the GMOs there. Our investigations also led us to Golden Rice, we decided to use this as a tool to educate students on the benefits of producing genetically modified crops. Drought resistant crops could be extremely useful in this region if this drought persists.

Our work in Honduras highlighted the need for a vaccine for this disease. Both researchers and regulators validated that this disease will only get worse if we do not do something in the future. They will also provided us with valuable information on how it would be best to deliver this vaccine and of possible regulatory requirements that it would have to fulfill. In addition we were informed of the under-diagnosis disease and that it is actually more prevalent than any statistic would indicate. This was revealed through our interactions with staff in the Alonso Suazo Hospital and with the doctor in Choluteca. When we visited Choluteca, we observed that the cause of the disease is multifactorial. The presence of the sand-fly alone is problematic but the nature of this problem was amplified by factors such as education, hygiene and nutrition. Therefore we felt that for treatment and prevention against leishmaniasis, it should not solely be pharmacological but also should include fostering interest in education, improvement of the water supply to the region and improving the community’s diet.

Sun, Rui, and Yuqin Liu. “Listeriolysin O as a Strong Immunogenic Molecule for the Development of New Anti-Tumor Vaccines.” Human Vaccines & Immunotherapeutics 9.5 (2013): 1058–1068. PMC. Web. 18 Oct. 2016.

References

Figure 1 and 2: http://www.worldatlas.com/webimage/countrys/namerica/camerica/hn.htm Figure 3: http://www.alamy.com/stock-photo-honduras-central-district-tegucigalpa-national-autonomous-university-34439745.html Figure 4: http://rebrn.com/re/honduran-farmer-holds-up-two-ears-of-corn-one-genetically-modifi-601358/

1.Irish Pharmaceutical Healthcare Association (www.ipha.ie/GetAttachment.aspx?id=1cbb727a-abc0-455d-9ca4-f079fc456c53)