Team:UCC Ireland/Application Scenario
Application Scenario
Introduction
Leishmaniasis is a tropical disease that currently affects over 12 million people worldwide, in 98 different countries. These are mostly in developing and newly industrialised countries in Latin America, Africa and the Middle East. There are two main forms of the disease, visceral and cutaneous Leishmaniasis (affecting the internal organs and the skin, respectively) and a third rarer form, mucocutaneous Leishmaniasis (affecting the mucous membranes).There is currently no vaccine against the disease, and the current treatment is quite aggressive.(Oliveira et al., 2015) In light of this, the aim of our project is to develop an L.lactis platform for the production of an immunogenic protein, LJM11, found in the saliva of the main vector for the Leishmania parasite in Latin America, Lutzomyia longipalpis.
While our wet lab work consisted mostly of working out the finer details of engineering the bacteria, we also did extensive research into the practical applications of our design, its current need and the long road to producing a functional vaccine. Our work constitutes the first sketches in the blueprint of a product which could potentially ameliorate and even save many lives in disadvantaged communities.
Design CriteriaAs with any other newly developed medicinal product, the basic design of our protein producing platform had to meet certain criteria that would render it viable and safe for usage in a healthcare setting. The first measure we took was choosing the right chassis for our purposes. L.lactis is a GRAS bacterium which is used in the production of milk and so is highly safe for human consumption. It has also been shown to be efficient at producing recombinant proteins and so is an ideal organism for our purposes. This is especially true as the gene encoding the immunogenic protein is under the control of the efficacious p44 promoter on the pNZ44 plasmid. (d’Souaza R, Pandeya DR, Hong ST, 2012) Biocontainment is also ensured in our design as our strain of L.lactis has the PyrG gene knocked, ensuring that it cannot replicate in the wild.(Hanin A, Culligan EP et al. 2014) Thus, at the molecular and cellular level, our vaccine is specially designed to be safe and fitting for use in a healthcare setting.Our design also took into account the natural presence of extracellular proteases, we therefore choose a strain that has a Htr A knockout which is one of the main extracellular proteases of L.lactis. Our protein could therefore be secreted from L.lactis without being degraded.
Another important design criterion is that the vaccine must have few side effects. Our vaccine will be used in areas with very few healthcare resources, so it is imperative that additional care be kept to a minimum. As mentioned before, with regards to the chassis, there is no issue-L.lactis is widely consumed due to its role in the production of dairy products such as milk. The one small risk that exists is that of cross reactivity with self antigen. The antibody against our recombinant protein, LJM11, has been reported to cross react with desmoglein 1, a structural protein involved in cell adhesion. This causes a loss of adhesion between epidermal keratinocytes, resulting in superficial skin blisters.(Qian, Y., et al. 2016) However, mutations in the hypervariable HLA-DBR1 gene determine this cross reactivity, and this is not present in all individuals. So, an important part of our vaccine strategy could be screening individuals for this genetic profile, but of course this could prove to be very expensive-especially in the impoverished areas where Leishmaniasis is endemic. Thus, some additional measures would be required in order for our product to be functional.
Another essential design criteria for our vaccine is that it must be easily administered and not impinge significantly on patients’ quality of life. Our plan satisfies this criterion-the vaccine is designed to be administered orally. This is a painless and simple method of delivery, with no difficult or cumbersome instructions to follow. Ease of delivery is especially important in the case of Leishmaniasis, as the member of our team who investigated the treatment options for this disease learned from Honduran healthcare professionals that the remedies currently cause great discomfort and pain for patients. The options are a series of highly painful intramuscular injections over the course of a few weeks, or purposefully acquiring sandfly bites on concealed areas of the body in order to gain immunity against the disease. These measures are so harsh that many people chose to live with the disease rather than receive treatment, which of course causes it to spread uncontrollably. Thus, a pain free and non invasive vaccine like ours could constitute a big step forward in the fight against Leishmaniasis. This was a key aspect of the design of our bacterial strain.
Target Manufacturers/Users
Finding manufacturers for neglected tropical diseases is a difficult process, due to the economic uncertainty of such a venture and the difficulties of adapting a therapeutic product for a target market that is so distinct from those of Westernised countries. However, some options do exist. For example, the Developing Countries Vaccine Manufacturers Network (DCVMN) is a body which incorporates over fifty manufacturers, with seven of these located in Latin America. Another company which could take on this venture would be Fio Cruz in Brazil. With a heavy involvement in research and development of drugs and a strong workforce of 7,500 employees, such a manufacturer would be very interested in our design, and would be ideally located to carry it on to more advanced stages of development. Companies with such similar goals to ours and in such locations would potentially be willing to take on our idea and manufacture the vaccine in the quantities required.
In addition, companies located in developed countries could have an interest in working on our protein producing platform. While there would be inherent difficulties for them in doing so, there would also be advantages. Working on treatments for Neglected Tropical Diseases (NTDs) improves the ethical profile of pharmaceutical companies, which is a particularly important factor in an age where there is a great degree of public scepticism towards the morality of of the major players in the industry. Thus, companies involved in the production of vaccines such as Pfizer, GlaxoSmith Kline, Eli Lilly and Johnson and Johnson could be candidates for these reasons. Furthermore, some companies in the developed world have already entered into collaborations with humanitarian organisations to branch into this field. In particular, Gilead Sciences are currently working with the WHO in the surveillance and control of Leishmaniasis. Such collaborative efforts could certainly be enhanced by the undertaking of a project such as ours. Clearly, the public image benefits and facilitation from large charitable organisations would be a major reason for manufacturers to produce our vaccine.
The users of our vaccine, should it be successfully produced, would most definitely be numerous. As a member of our team learned from first hand experience, the people of Honduras would benefit greatly from this vaccine. Over the course of discussions with epidemiologists in the country, she learned that the incidence of Leishmaniasis is rising, due to public health efforts being directed primarily towards arbovirus. The situation is no doubt similar in other Latin American countries such as Brazil where public health campaigns are largely being focused on Zika virus. Furthermore, she found that Leishmaniasis cases are not actively sought, thus affirming that a vaccine is the best means of fighting this disease in a feasible way. A prophylactic measure such as this would prevent the terrible complications of this disease, as well as the tribulations associated with the current treatments.
Populations in the Middle East would also benefit greatly from our bacterial delivery system. Leishmaniasis was formerly contained to endemic areas here, such as Damascus and Aleppo in Syria. However, with the spread of Syrian refugees into countries such as Lebanon and Turkey, Leishmaniasis has been reported in these countries too (between the years 2000 and 2012 there were only six reported cases of Leishmaniasis in Lebanon, while in 2013 alone there were 1,033 new cases).(Alawieh A et al., 2014) A case of Leishmaniasis was even reported in a refugee in Holland, suggesting that the disease could begin to migrate even further afield.(de Wild et al 2016) Thus, it is clear that a vaccine against this dolorous and often stigmatising disease would be very much welcomed in these parts of the world. It could prevent potentially serious outbreaks of Leishmaniasis in countries located near endemic parts of Syria, and even European countries.
Asian populations could also avail of our proposed product. A member of our team with contacts in Nepal decided to investigate this and gained access to the annual reports of the Nepalese Department of Health Services for the fiscal years 2013/2014 and 2014/2015. According to these reports, Leishmaniasis was the most common vector borne disease among Nepalese inpatients outside of miscellaneous viral fevers in the 2013/2014 fiscal year and the fourth most common vector borne disease outside of unspecified viral encephalitis, dengue fever and unspecified malaria in the 2014/2015 fiscal year. A vaccine to a disease which is so common in such a country would take a great weight off the shoulders of the public health system here. Again, this is evidence that our design could have very far reaching effects.
Needs and Costs Involved
So, from what has been outlined so far, it is clear that our protein producing platform satisfies the design criteria for a pragmatic vaccine, and that there are producers and consumers to whom our project would be of great relevance and benefit. However, it is necessary to consider the needs and costs involved in allowing this strategy to come to fruition.
Needs
First of all, the vaccine must undergo clinical trials. While our design is sound at a molecular and biochemical level, it would have to be tested for safety and efficacy before being used in a real healthcare setting. Phase Ia and IIa trials could be carried out in Ireland or some other developed country where the vaccine is being developed, but the Phase Ib and IIb would have to be carried out in subjects whose ethnic profile is the same as that of the people receiving the vaccine. This would mean that there would have to be sufficient technological infrastructure in place for data collection, and also healthcare professionals trained in carrying out clinical trials.
The vaccine must also be readily available to the local people of the countries involved. Lengthy bureaucratic processes and having to travel long distances would act as deterrents to these people, who would likely have many other concurrent difficulties in their lives. The solution lies in having sponsored vaccination programs by companies such as Fyffes. Fyffes employ many people in Honduras, and so sponsoring a vaccination program is in their interests as it ensures the wellbeing of their workforce. Humanitarian organisations such as PATH would also potentially be interested in following through in making our vaccine available to vulnerable people.
On the other side of things, it is imperative that people in places such as Honduras and Syria understand the need for a vaccine, and know about the benefits of herd immunity. It is only through insight into the true downstream effects of prophylactic treatment that people will feel motivated to participate in the vaccination program. Again, companies such as Fyffes have a huge role to play here. Fyffes currently run educational workshops for women, so perhaps the need for vaccination could be incorporated into these workshops, should our product be licensed. Educating women is especially important, as they will most likely pass on what they learn to their children. This will cause knowledge about vaccinations to be propagated through generations.Having witnessed the good work of Fyffes we aimed to tackle education as well, having observed the good work of Fyffes we decided to help promote education in this region. Our team collected some unused lab equipment from the different labs around our university and in collaboration with Fyffes sent this to one of the universities in Honduras. We had also contacted students and investigators who were interested in iGEM but are unable to take part due to the lack of financial resources, it is the hope that the equipment that we have sent will be of use for them in future years and promote education in the area.
Another essential need for our project to come to fruition is the improvement of transport networks in places such as Honduras. The member of our team who went to Honduras found these to be very inefficient and cumbersome. Large lorries had to travel along winding roads that were often poorly surfaced. Due to the fact that our vaccine would have to be transported from the site of manufacture to several different vaccine clinics, transport networks would have to be ameliorated in order to ensure that delivery can be swift and cheap. Not only do long and convoluted roads make delivery times longer, but they also waste money in that more fuel has to be used unnecessarily. Thus, infrastructural changes would also need to be put in place in order to make our product viable in the real world.
The limitless vaccine can also be freeze-dried and therefore stability issues during transport would be mitigated by the lack of needing a cold chain. We were informed by stakeholders Dr Jorge Carassco and Dr Alvaro Acosta in both the middle east and Honduras of the need of a cost-effective distribution system. Freeze dried products also have the advantage over liquid formulations in that the product can be shipped and the likelihood of damage to the product would be less likely. Both of these factors would lower the cost of our vaccine and therefore make it more applicable for developing countries.
Also, in areas such as the middle east, where Leishmaniasis is spreading to adjacent populations due to migration of refugees from endemic areas, there would be a need to monitor movement of refugees so that areas of greatest risk can be anticipated and vaccination campaigns set up. This would be essential to ensure that the disease does not infiltrate into new populations. Again, this would require trained public health workers in these areas who could work together to analyse the movement of infected people. Such an effort could be funded by the WHO or other charitable organisations.
Costs
One of the main costs involved in the development of our project is, of course, the cost of production of the vaccine. From start to finish, it is estimated that a given vaccine costs between $200 million and $500 million dollars (Serdobova I and Kieny M-P, 2006) to develop fully and produce. Raising such funds is, of course no mean feat-especially for a vaccine for use in the developing world where profit will be virtually nonexistent. There is a lack of innate “pull” factors in such a project, which may deter many manufacturers. However, strategies do exist for procuring funds for such non lucrative but philanthropic projects. For example, in similar situations, companies have charged at a surplus for vaccines in the developed world, and used this profit margin for development of vaccines for use in the developing world. Should a manufacturer who is also developing vaccines in the first world take on our project, this would most certainly be a viable option.
Another way to finance our project which has been used with success in the past is to have public funds put aside in developed countries to accumulate money for vaccine development in the third world. This strategy would also be effective as not only do the receiving countries benefit, but also the donor countries. Countries such as Honduras have important primary industries, such as the growing of coffee and bananas, which will be enhanced should an effective vaccine for Leishmaniasis be produced. Furthermore, European countries would benefit from the introduction of the vaccine to Syria and neighbouring countries as it will ensure that refugees will not bring the vaccine into this continent. As a result, important healthcare resources could be saved. So, it is clear that the use of public funds from developed countries would benefit both the donors and the recipients.
Legal issues of patenting
Genetically modified bacteria, even though are living organisms, are capable of being patented. In our case, the patent would be specific for L.lactis that has the genomic modifications such as the Htr knockout, pyrG knockout and insertion of the LLO gene and that expresses the protein LJM11 from a plasmid.
Regulatory requirements
In order for a L.lactis LJM11 vaccine to be licensed by regulators, it would need to be shown to possess quality, safety and efficacy in phase I, II and III clinical trials. In Europe, the process to regulate a L.lactis vaccine would have to go through the European Medical Agency’s (EMA) Committee for Advanced Therapies (CAT). Once approval is granted by the committee for advanced therapies, health services in the 28 EU countries would be able to supply the therapy to their citizens. The success rate of advanced drug therapies is quite low currently. Regulators are cautious to approve these therapies because they have no standards to apply in the regulation process. Unlike small molecules, it is not possible to possess information regarding the physicochemical properties of the medicine which is often the first piece of information that is requested by the regulatory bodies.
Very few advanced therapies have been licensed by the EMA and even less so from the FDA. Only 4 out of 250 advanced therapies have made it through clinical trials and have received a license from the EMA. However new licensing measures have been introduced in the last couple of years such as adaptive licensing which aims to speed up the process of licensing. One of the main reasons why some of the medicines were not licensed is because of sub-standard GMP practices. Many of the therapies are being developed solely by clinicians, researchers and small research companies who do not have the facilities like the larger pharmaceutical companies. Our vaccine would hopefully be developed commercially by larger pharmaceutical companies as outlined in our manufacturers section.
With a combination of stakeholders working on the development of a vaccine for leishmaniasis, it would likely come to market a lot sooner than would otherwise be possible. In Honduras, the guidelines set by the Pan American Network on Drug Regulatory Harmonization apply for the licensing of drugs. The Honduran Ministry of Health would however have the ability to implement any new therapies such as our vaccine, if the clinical trials and quality, safety and efficacy data of the vaccine were deemed satisfactory.
Potential Safety concerns
Safety concerns may arise because of the fact that the administration of this vaccine is via live bacteria. However L.lactis are a GRAS organism and are naturally present in the body as part of the microbiome. There have been no known reports of the bacteria possessing any pathogenicity. One of the major reasons for choosing L.lactis as the protein delivery bacteria was the safety of the bacteria. However, the vaccine in question may not be completely safe. The LJM11 antigen has been found to cross-react with a self-antigen in the body: desmoglein 1 which is a desmosomal core glycoprotein that is cell adhesion molecule (Qian.Y et al 2016) Cross-reaction of the saliva protein naturally introduced via the sandfly can cause an autoimmune disease Fogo Selvagem which causes blistering of the skin (Li N et al 2003). The cross-reactivity only occurs in specific individuals with specific genetic traits which are more common in certain ethnic groups. The mutations responsible for susceptibility to this autoimmune disease are rare but there are some ethnic groups like the Terano Indians in brazil where it is more common. Approx 2.6% of the tribe have been diagnosed with Fogo Selvagem due to genetic predisposition (Moraes et al 1997).
The cause of the susceptibility is a mutation in the HLA-DRB1 gene on an epitope in one of the hypervariable regions. This leads to the production of antibodies that recognise both antigenic determinant sites in LJM11 and desmoglein 1 (Moraes et al 1997). IgGG4 antibodies are produced which are anti-epidermal antibodies and trigger the detachment of epidermal cells and intra-epidermal blister formation.
This therefore looks like it is a possible safety concern in the development of the vaccine, however it can be avoided through genetic screening of the individuals who may be of an ethnic group where the recessive allele has been observed. It is noteworthy that Caucasian and African ethnic groups have a much lower chance of developing this auto-immune reaction due to the fact that this particular allele is rare in these ethnicities.
It may the case that this would not be observed in phase III clinical trials and therefore clinicians would need to be aware of this particular concern even though it may occur rarely. Another potential safety concern is due to the LJM11 gene being present in a plasmid. Horizontal transfer may occur and other bacteria in the GI tract may utilise this plasmid. The concern here would be the antibiotic resistance gene. The concern over the spread of antibiotic resistance may be alleviated by gnomically inserting the LJM11 gene into the bacterial chromosome.
Social issues
Stigmatization of the disease is currently a problem. We discussed the social impact of leishmaniasis with Alvaro Acosta from the institute of neglected tropical diseases in Liverpool. We were informed about the stigma that surrounds cutaneous leishmaniasis in countries such as Honduras. The social stigma is so pronounced that people are willing to go to great and dangerous lengths to avoid the lesions of cutaneous leishmaniasis in exposed areas of the body. This is done through leishmaniazation. A person is purposefully bitten by an infected sandfly and develops cutaneous leishmaniasis, but in a concealed part of the body. As cutaneous leishmaniasis can often be self-limiting, the disease may abate after some time. The person then has immunity against leishmaniasis, and only has the scars from the lesions in a concealed area. However, problems exist with this approach, such as the possible development of a secondary bacterial infection at the site of the lesion.
Indeed these stigmatizing lesions are often the only reason people seek treatment for leishmaniasis in the first place, but at this stage it is often too late to get rid of them completely.
Thus, if a vaccine for leishmaniasis were developed and made available to the people of Honduras, visible lesions could be prevented in a safe and effective way, and this social stigma prevented. This realisation which resulted from our human practices reinforced our understanding of the need for a vaccination against leishmaniasis.
Ethical issues
The ethics of vaccines has long been debated both in the scientific community and the popular media. The issue of herd immunity and the protection of vulnerable individuals it provides has long been used as the argument in favour of vaccination. However, because vaccination involves interfering with healthy individuals, it can be argued that vaccines should be of very low risk; i.e., the benefits should greatly outweigh the risks. The fact that our proposed vaccine is: bacterially based and a genetically modified organism, and intended for used in a developing country, raises some important ethical issues that will be discussed here.
The obvious issue that first comes to mind when considering a genetically modified bacterium intended to be orally ingested is safety. Our chassis, Lactococcus lactis subsp. cremoris, is a GRAS (Generally Recognised As Safe) organism. It contains a pyrG knockout as previously discussed which acts as a biological containment strategy. However, the safety of our vaccination strategy would have to be rigorously tested in the regulatory process. But would this process be as rigorous in Honduras as through regulatory bodies such as the European Medicines Agency (EMA) and the Food and Drug Administration (FDA)
As mentioned previously one possible safety issue would be the cross reactivity of anti-desmoglein 1 antibodies with LJM11. (Qian.Y et al 2016) It has been suggested that LJM11 may be an environmental factor which contributes to the development of an auto-immune disease called endemic pemphigus foliaceus, known as Fogo Selvagem in Brazil. This is a disease which is caused both by genetic and environmental factors.
Another relevant question concerns the fact that our vaccine is intended for use in a developing country. Would those who need it get the vaccine even if it was approved? Would the government be in a financial position to implement a nationwide vaccination programme, or would the vaccine only be available to those who can afford it? The Honduran public health officials would have to carefully consider the pharmacoeconomic factors inherent in all their decisions, and decide whether or not to provide the vaccination on a wide scale. While the current treatment for leishmaniasis in Choluteca in Honduras is afforded at no cost to the patient, it is unclear whether or not this would be the case for a vaccination. It is possible, of course, that a multinational company such as Fyffes which employs many people in Honduras would possibly provide the vaccination to the people of Honduras.
In our techno-moral scenario, we explored the consequences the possible spread of leishmaniasis to Europe due to the Syrian refugee crisis. This scenario is set in a future where a lactococcal-based vaccine against leishmaniasis is available, in European countries through their respective health systems as well as in countries such as Syria through organisations such as Médecins Sans Frontières (MSF). But due to a new outbreak of conflict in the country, MSF have to pull out of Syria. In this scenario, the people of Europe are still availing of this vaccine while those in Syria have to do without, leaving them vulnerable to the disease. Thus the ethical dilemma concerning the denial of this vaccine to the people for whom it is designed is raised. It is clear that if a vaccine against leishmaniasis were made available, it should be made available to as many people who need it as possible.
It is clear the development of this vaccine is an ethically complex issue; many considerations would have to be taken into account if our potential vaccine were to be developed.
Available or conceivable alternatives
Besides the unsafe practice of leishmaniasation, no alternative vaccination strategy for leishmaniasis currently exists. Many efforts are being made to develop such a vaccine, (Cutaneous Leishmaniasis Vaccine: A Matter of Quality) but there are none currently in use clinically. As one of our team members found out on her trip to Honduras, the treatment of choice is Glucantime, an Antimony based drug that causes liver and kidney damage. This drug is administered through an intramuscular injection that is extremely painful. The usual doses of the drug is 15 ml of the solution (1.5 g) per day, for 20 days.
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