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Dengue in the crosshairs

A biosensor able to diagnose dengue in 20 minutes is one of several new weapons to combat the disease

ilus_Dengue_fmauricio pierroVaccines, genetically modified insects and traps that act as data collectors, plus a rapid diagnostic test: these are some of the strategies already being used or studied to combat dengue in Brazil and worldwide. According to the Centers for Disease Control and Prevention of the United States (CDC), today about 2.5 billion people, or 40% of the world’s population, live in areas where there is risk of dengue transmission. Estimates indicate that the disease affects between 50 million and 100 million people each year, including 500,000 cases of hemorrhagic dengue and 22,000 deaths, mostly among children.

Brazil is one of the 30 countries where the disease is most endemic, from among more than 100 countries reporting the disease. As of April 2014, according to the Ministry of Health, there were 215,000 reported cases of dengue fever, a reduction of 70% compared with the first four months of 2013. “Like all vector-borne diseases, the incidence of dengue is spatially localized, so that some areas experience an increase while others just the reverse. Everything depends on where you look,” says Dr. Marcelo Burattini, an infectious diseases specialist and professor of medical informatics at the University of São Paulo School of Medicine and professor of infectious diseases at the Federal University of São Paulo (Unifesp). The vectors are mosquitoes of the genus Aedes. In the Americas, the Aedes aegypti species is responsible for transmission of dengue. Circulation of the four dengue virus serotypes (1, 2, 3 and 4) transmitted by mosquitoes is in constant flux around the world.

Because the symptoms of the infection by the virus are very similar to other diseases, early detection makes all the difference in the world to the patient. With that in mind, researchers at the São Carlos Institute of Physics of the University of São Paulo (IFSC/USP), coordinated by Professor Francisco Eduardo Gontijo Guimarães, have developed a diagnostic test that takes only 20 minutes to get the results, which can be accessed in real time by mobile phone or other electronic devices. In an effort to reduce the price of the final product and compete with an imported test, used only in cases requiring confirmation, such as severe or complicated dengue and for epidemiological surveillance, the researchers resorted to the antigen-antibody interaction to detect the dengue virus. The virus secretes a structural protein, known as NS1, into the bloodstream during the early days of the infection. Then the human body produces specific antibodies to fight the NS1 protein after the fifth day of infection. “The good news for the project was the high concentration of antibodies produced in chicken eggs,” says Guimarães. These antibodies are then immobilized on a metal membrane (biosensor) which, when they come in contact with infected blood, react to the NS1 protein and produce an electrical signal. “Although the body has millions of proteins, only the NS1 generated by the dengue virus binds to the antibody.”

058-063_Dengue_220-01Currently the test most commonly used by health clinics to detect the disease can only be performed as of the sixth day, which means that dengue may be confused with other infections and may not always be treated properly. “The conventional test can not be used in the early days because it measures the antibody concentration as of the sixth day,” he says. But the biosensor test evaluates a small concentration of the NS1 protein, which is produced by the virus as soon as it enters the bloodstream of an infected person. USP filed a patent for the innovation in September of 2013.

The project was developed under the National Institute for Organic Electronics (Ineo), part of the National Institutes of Science and Technology (INCT) based at the São Carlos Institute of Physics (USP), funded by FAPESP and the National Council for Scientific and Technological Development (CNPq ). “We had already worked with biosensors when, three years ago, we got the idea to develop a portable and low-cost device to detect dengue,” says Guimarães. The idea is that every health clinic, even in remote locations, can do the test without having to transport the blood to a major health center for testing. The project is in the final stages of implementation.

Another line of research involves an innovative tactic; the goal is to block the dengue virus in Aedes aegypti by using Wolbachia bacteria. These symbiotic bacteria are present in the cells of about 70% of all insect species, including fruit flies, mosquitoes and butterflies. The project, initiated in 2006 and coordinated by Professor Scott O’Neill of Monash University in Melbourne, Australia, also involves researchers from six other countries, including Brazil. In Brazil, the studies are being conducted at the Oswaldo Cruz Foundation (Fiocruz), based in Rio de Janeiro, and are being coordinated by Luciano Moreira with a staff of 15 researchers.

058-063_Dengue_220-02The bacteria are withdrawn from fruit flies and inserted into the Aedes eggs. After colonies of mosquitoes are established in the laboratory, they are released into nature. In addition to blocking the transmission of the mosquito-borne dengue virus, the bacteria upset their reproduction. A study published in the August 25, 2011, issue of the journal Nature described the results of an experimental field test carried out in two locations in the city of Cairns, Queensland, Australia for 10 weeks. Three hundred thousand Aedes adults with Wolbachia were released, and after five weeks, 100% of the wild mosquitoes were carrying the bacteria in one of the locations, and 90% of them in the other location; this meant they were unable to transmit the virus. Field tests are being done in Vietnam and Indonesia, and they are scheduled to begin this year in Brazil, after approval by the regulatory authorities.

Along similar lines, genetically modified mosquitoes represent a different approach. They were developed in 2002 by the British company Oxford Insect Technologies (Oxitec) and were released for commercial use in April 2014 by the Brazilian National Biosecurity Commission (CTNBio). However, their production still needs commercial registration with the Ministry of Health, subject to a technical analysis by the Brazilian Health Surveillance Agency (ANVISA). The mosquitoes carry a gene that is lethal to their offspring, which die before reaching adulthood. The method consists of releasing a large number of genetically modified males in infested areas; males do not sting, and therefore do not transmit dengue. When they copulate with females in the wild, their descendants die in the early stages of development (see Pesquisa FAPESP Issue No. 180). In Brazil, Oxitec has partnered with the USP Biomedical Sciences Institute, which is responsible for laboratory tests using the transgenic strain, and the social organization Moscamed, which carries out field testing and has a bio-factory that produces mosquitoes in Juazeiro, Bahia State. “We have been doing tests with these strains since 2002, including in Brazil,” says Glen Slade, Oxitec’s global business development director.

Local governments are the major customers for transgenic mosquitoes. “We have not yet settled on costs, but we have studies for an area of 50,000 people,” says Slade. The first year, which is considered an intensive release in order to reduce the number of adult Aedes in the wild, will cost between R$2 million and R$5 million. The second year, referred to as maintenance, will cost around R$1 million. “There is also the option of partnering with cities to train people to carry out the maintenance program, which would reduce its cost,” he says. During the four-to-six-month intensive phase, a release of between 100 and 200 mosquitoes per week per person is necessary. Oxitec is starting to produce the first mosquitoes in cages in its own factory in Campinas, which has a production capacity of two million transgenic mosquitoes per week. The production capacity is small, able to handle an area of 10,000 people. The plan is to set up other nearby factories for future projects.

Vaccines offer another very promising front to combat the disease, but there is as yet none on the market. The most advanced of all those in development is that of the Sanofi Pasteur laboratory, which is in phase 3 clinical development, with ongoing large-scale trials to assess its effectiveness. Preliminary results released by the laboratory in April of this year showed a 56% reduction of dengue cases in a study done between 2011 and 2013 with more than 10,000 volunteers in Asia. Children ranging in age from 2 to 14 in dengue endemic areas in Indonesia, Malaysia, the Philippines, Thailand and Vietnam participated. Participants received three injections of the vaccine or a placebo every six months. “The children were chosen because they are the ones who suffer the most severely from the disease,” says Dr. Sheila Homsani, manager of the medical department of Sanofi Pasteur.

058-063_Dengue_220-03Yet studies have yet to show whether immunization is effective against all four serotypes of the virus. “Preliminary results from Asia will be complemented in the second half of 2014 by data from a second study being conducted in Latin America; it has more than 20,000 volunteers between the ages of 9 and 16, from Brazil, Colombia, Honduras, Mexico and Puerto Rico,” says Dr. Homsani. The efficacy analysis will be done by serotype. Regarding criticisms by immunologists and infectious disease specialists concerning the study done in Thailand in 2012, which reported that the vaccine did not protect against serotype 2, Dr. Homsani says that it was still in an early stage of development. “Only 4,000 people were evaluated and only in a single region,” she says. “The recently released study was done in different epidemiological environments of Asia, as well as in Latin America.”

The reported 56% efficacy rate of the vaccine was also questioned by some experts. The hypothesis put forth by critics is that unless the vaccine offers total protection against all serotypes, then if a vaccinated person becomes re-infected, the risk of contracting dengue hemorrhagic fever increases. “This risk is theoretical and has not been observed in the tests done so far,” says Dr. Homsani. “We have been vaccinating patients since the beginning of the study, and the good safety profile of the vaccine has remained consistent with previous studies.”

The vaccine is made ​​with attenuated and recombinant viruses. “Part of the envelope of the four serotypes of dengue virus is attenuated and the inside is filled with yellow fever virus vaccine, which is stable in terms of efficacy and safety,” says Dr. Homsani. The research that resulted in the vaccine began 20 years ago. It is scheduled to be available in Brazil by the end of 2015.

Apart from Sanofi, other Brazilian research groups such as the Butantan Institute in São Paulo, and the Immunobiologics Technology Institute of the Oswaldo Cruz Foundation (Bio-Manguinhos/Fiocruz) based in Rio de Janeiro, are working on developing tetravalent vaccines for dengue. The Butantan Institute, whose research began in 2005, is partnering with the National Institutes of Health (NIH) in the United States. The technology uses the live attenuated virus through genetic modification of the virus in the wild. The infectious agent is still capable of stimulating the production of the body’s defense response, but does not cause the disease. Several phase 1 studies in humans have been carried out in the United States to select the four attenuated viruses comprising the vaccine and to prove the safety and immune response generated by the four-virus combination. Phase 2 of the study, the first clinical trials in humans in Brazil aimed at evaluating the safety and immunogenicity (ability to induce immune response) of the tetravalent vaccine, began in October 2013. By the beginning of June 50 volunteers who had never had dengue and were between the ages of 18 and 59 had been vaccinated. The vaccine was shown to be safe in this group. In the second stage 250 more volunteers with or without a previous dengue infection will be recruited. The results of this study should be known by mid-2015 and, if they are favorable, a phase 2 study with thousands of participants will evaluate the effectiveness of the vaccine.

The Bio-Manguinhos vaccine has been made in collaboration with the British laboratory GlaxoSmithKline (GSK) since 2009, when they signed a research and development agreement to obtain a tetravalent inactivated vaccine for dengue. After tests in mice, in 2013 tests were done in monkeys, as part of the pre-clinical studies. The next step will be the start of human clinical trials, scheduled for 2015. Epidemiological studies to determine locations for the evaluation of the vaccine have been done ​​in Fortaleza, Rio de Janeiro, Salvador and Manaus. In parallel, another partnership between Fiocruz and GSK is already testing a dengue vaccine in adults in the Caribbean and the United States; this also involves the U.S.-based Walter Reed Army Institute of Research.

058-063_Dengue_220-04Dr. Burattini believes vaccines will be the most effective way to combat dengue epidemics in the future. However, he notes that none of the vaccines developed so far are 100% effective. “Since the vaccines require a regimen of two to three doses, which greatly limits their actual effectiveness, and there is a long interval between doses, it is unclear whether they will work in practice,” says Dr. Burattini, who coordinates a survey commissioned by the Ministry of Health that is scheduled to be finished by the end of the year. It aims to establish the best vaccination strategy for dengue.

In his opinion it is important to study and differentiate between the efficacy of the vaccine and how effective it will be under public health conditions. “Effectiveness depends on its ability to elicit a protective and durable immune response against all four serotypes of the virus, since actual effectiveness also depends on a number of factors related to the operating conditions and the characteristics of the populations where the vaccines are used,” he says. Another aspect to be addressed, in his opinion, is that the ideal vaccine should be applied in a single dose or should be administered with the shortest possible interval of time between doses. And the third is that the vaccine should have no long-term adverse effects. “The vaccine has to demonstrate in clinical trials that the antigens used are not related to adverse effects, such as a predisposition to severe disease if re-infected with another serotype, due to an exaggerated inflammatory response.”

One of the ways to control adult insects currently in use by 10 Brazilian cities, including Santos, Vitória, Porto Alegre and Uberaba, all with populations of more than 300,000 people, is an information system called intelligent dengue monitoring (MI-Dengue), developed by a partnership between the Federal University of Minas Gerais (UFMG) and the company Ecovec, based in Belo Horizonte in the state of Minas Gerais. “It’s a platform that uses traps to capture adult Aedes as data collectors, which has the tremendous advantage of providing weekly photographs of the vector population for each city block,” explains Gustavo Mamão, director of the company.

Invented by Professor Álvaro Eiras of UFMG, the Mosquitrap simulates a breeding site for Aedes adult females. When the females enter the black plastic cylinders to deposit their eggs, attracted by a chemical compound, they end up stuck to an adhesive card (see Pesquisa FAPESP Issue No. 142). The collected data are sent by the local field agent from a mobile phone to the MI-Dengue central platform, which includes among its functions the ability to generate maps of the locality with color-coded spots to indicate vector infestation. Santos, on the coast of São Paulo State, has been using MI-Dengue since 2012. “461 traps checked by six agents and a supervisor are scattered throughout the city,” says Mamão. Starting in July 2014, the project will be implemented experimentally in a county of Florida in the United States for six months. Since the system was created, 50 Brazilian cities have tested the platform, in addition to countries such as Australia, Singapore and Colombia.