Various approaches to the modification of mosquitoes are emerging as a tool to prevent the diffusion of several types of viral vectors

There are many different diseases that spread in the environment and infect men and animals through the diffusion of viral vectors transmitted by insects. Mosquitoes play a relevant role in the diffusion of the viruses responsible for diseases such as malaria, Zika, Chikungunya, Dengue, yellow fever, encephalitis and West Nile fever.

Many of these pathologies still lack effective remedies, being typically diffused mainly within the African and Asian emerging countries where the health facilities and the wide access to medicines are not so easily available as in the Western economies. According to the “World Malaria Report 2015” published by the World Health Organisation (WHO), 214 mln new cases of malaria were diagnosed just in 2015 (88% in the African region), and 438 thousands were the number of deaths.

New Zika cases are becoming more frequent even in Europe and in the United States. A Dengue’s epidemic caused a health alarm in the Hawaii Islands last February, with more than 250 confirmed cases of the disease since the last quarter 2015. Italy was concerned in 2007 by a Chikungunya’s epidemic, with 217 confirmed cases.

GMO-mosquitoes emerging from the labs

Andrea Crisanti, Imperial College London
Andrea Crisanti, Imperial College London

«Mosquitoes are the main health issue at the global level, since they are responsible of approx. 500-600 thousands deaths for malaria each year. Insecticidal agents are a useful tool just on the short time. They were introduced in the 1950s; since then, many advancements have been made to control the diffusion of the vectors. Nevertheless, it has not been possible to eradicate the diseases: insecticides are not the proper solution in certain areas of the globe, and there are other reasons ranging from the will to the lack of logistics and to the resistance developed by many types of mosquitoes», tells Andrea Crisanti.

The Italian researcher of the Imperial College London is involved in the identification of novel strategies to obtain genetically modified (GMO) mosquitoes in order to prevent the diffusion of malaria as well as of other diseases. He used the so-called gene drive technique to engineering Anopheles gambiae (the main vector for malaria) using CRISPR-Cas 9 technologies. The team inserted three genes able to sterilise the female mosquitoes with recessive phenotype1. «Our hypothesis is that the genetically modified mosquitoes cannot transmit the disease; if their fertility or reproductive capacity is reduced they might undergo extinction. The genes we have inserted are selectively activated in the females. Once released, these genetic modifications are able to propagate», explains the researcher.

This technology is just one of the many examples which are under scrutiny in many different laboratories world-wide. There are more than 10 thousands different species of mosquitoes, and just approx. 30-35 of them are responsible for the transmission of viral vectors to men and animals, tells the expert to Pharma World. «We have demonstrated that our method works. We are now working at the second and third generation prototypes, and we plan to be on-field in approx. two years», tells Crisanti.

Anopheles mosquitoes are typical of the African ecological niche and are not able to adapt to the temperate climate typical of the Mediterranean region. Aedes aegypti is widely diffused in Africa, Asia, South and Central America and is the responsible of the transmission of viruses such as Dengue or Zika. Aedes albopictus, the so-called “tiger mosquito”, in an highly invasive species which is able to adapt to the different climates, from tropical to temperate ones. «Albopictus has colonised the south of Europe during the last 25 years. It is able to transmit the virus of several different diseases, among which Chinkungunya and Zika», tells the expert.

Other emerging technologies

The self-limiting gene technique is the core technology of U.K.based Oxitec, used to modify the genetic material of the male mosquitoes through the insertion of the tTAV (tetracycline repressible activator variant) suicidal gene. Once released in the environment, the mutation is transmitted by the males to their progeny, which undergo death before reaching the adult age. The technique limits the spread of mosquitoes, possibly up to the complete extinction of the original pool carrying the diseases’ vectors. According to Oxitec, the tTAV gene does not produce toxic proteins, thus the GMO technology would be safe and should not have any consequence for animals feeding with mosquitoes. The approach has been subject to on-field trials in Brasil, Panama and Cayman Islands; according to the company, the success in the reduction of the native mosquitoes populations has been of at least 90% in all tests. An issue affecting the Oxitec’s technology could be represented by the inactivation of the tTAV gene in livestock treated with tetracycline to prevent diseases. «Even in such an instance, the quantity is low and the residual fertility is approx. of 5%. This is not a negative effect, because it causes an increase of the effect in the following generations. The genetic modification auto-extinguishes, there is a decrease of its frequency generation after generation», says Crisanti. In order to better trace the fate of the modified mosquitoes once released in the environment, the OX513A strain is characterised by a fluorescent marker which helps in the identification of the progeny.

Not-GMO mosquitoes

MosquitoMate is a start up company of the Kentucky University focusing on a different approach to the problem, without genetic modification of the mosquitoes’ genome. The company core technology is based on the insertion in the mosquito’s organism of the Wolbachia bacterium, typical of many species of flies. The National Institutes of Health issued a $ 1.3 mln grant in favour of MosquitoMate in order to develop new bio-pesticides approaches to fight the diffusion of the tiger mosquitoes. The on-field trials begun in June 2015 in an 5,7 acres area of South El Monte, California, under the coordination of the Greater Los Angeles County Vector Control Agency. The Wolbachia bacterium sterilise the male mosquitoes; the fertilesed eggs cannot develop more than up to the very early phases. According to the Greater Los Angeles County Vector Control Agency, the area has been characterised by a great increase in the presence of the West Nile virus (42% in October 2015 vs. 16% of 2014).

«The use of the Wolbachia bacterium does not require a genetic modification: it stimulates the immune system of the insect, which become able to destroy the virus. This type of mosquitoes are semi-immune of the Dengue virus, it is still not clear if also for Zika», tells Crisanti.

The bacterium Wolbachia pipientis is studied also by the researcher of the Monash University, Australia, who received a grant by the Bill & Melinda Gates Foundation. According to the MIT Technology Review, the approach could be tested on-field in Colombia as a part of a project aimed to protect the city of Medellin (3 mln inhabs.) from the Dengue virus. The costs for the intervention using the Wolbachia technology would be lower (1$ per protected person) with respect to the GMO-mosquitoes (7$ per protected person), writes the MIT magazine.

The regulatory impact

«We are in an unexplored field from the regulatory point of view, there are no legislative frameworks available for these technologies – tells Andrea Crisanti. – The situation is rapidly evolving, work is on going to better understand the limits and risks of these techniques, and the important issues to be evaluated. For Zika, for example, there are several levels of risk. It is very low for the Oxitec’s technology, because the genetic modification does not propagates, or it does so at a very low level. From the ecological and food chain point of view, there are so many mosquitoes that it is probable that the extinction of a species in the food chain would be replaced by another species. Technologies such as the one we are developing are more problematic, as they imply the propagation of a genetically modified element. It is necessary to evaluate the rate and type of mutation, the phenotype it confers, the possibility to limit mutations and the occurrence of adverse effects. Several discussion tables are active on these issues, including also the authorities of the countries involved in the on-field trials. We need also to consider the technology transfer and educational processes: an informed evaluation would be the optimal for the adoption of a technology by a country, they should be able to directly judge it». The WHO and the Food and Drug Administration have issued statements to clarify their position on these emerging technologies, with a particular focus on the Oxitec’s one. We wait the results of the many on-field trials to better understand the sustainability for the human and animal health and for the environment of this new type of emerging technology.

The position of the authorities

According to the WHO note dated 17 February 2016, the presumed association of Zika virus circulation with an increased detection of Guillain-Barré syndrome (a pluricausal autoimmune disorder) has been reported in eight countries, including French Polynesia, Brazil, El Salvador, Martinique, Colombia, Suriname, Venezuela and Honduras. In some of these countries, Zika is the only circulating flavivirus, an observation supporting the association.

The WHO highlights that Zika virus has frequently co-circulated with Dengue and Chikungunya viruses. “These viruses cross-react in diagnostic tests, making test results unreliable and putting better tests at the top of the list of most-needed new medical tools”, writes the UN’s Organisation on its web-site. WHO estimated at least 18 months before Zika vaccines could be tested in large-scale trials and encouraged the adoption of newer tools for mosquito control in addiction to traditional methods, in order to achieve an integrated approach that tackle all life stages of the mosquito and fully engage local communities.

The WHO Vector Control Advisory Group (VCAG) has evaluated genetically modified prototype mosquito and has recommended further field trials and risk assessment to evaluate the impact of this new tool on disease transmission. The VCAG also issued in March 2016 a positive recommendation in support of Oxitec’s self-limiting mosquito (OX513A). The Advisory Group noted the way forward for control of Aedes mosquitoes has to change from a reactive approach to sustained, proactive control intervention and remarked on the need to improve “the quality and extent of implementation of vector control interventions to ensure optimal impact – both within the context of the immediate response to Zika virus disease and, more broadly, against all Aedes-borne diseases.

The U.S. Food and Drug Administration released a preliminary Finding of No Significant Impact (FONSI) on Oxitec’s solution for an investigational trial in the Florida Keys. The finding concluded a field trial of the Company’s OX513A mosquitoes in Key Haven, Florida, would not result in a significant impact on the environment.