Maíra Menezes (IOC/Fiocruz)
After five years of research, a project led by Fiocruz has reached encouraging results in the search for a therapy to prevent congenital zika syndrome. In an article published on Antiviral Research, the authors show an intense protective effect of sofosbuvir, the antiviral drug currently indicated to treat hepatitis C. In trials with Rhesus monkeys, considered to be the experimental model closest to humans, the drug prevented zika virus transmission from mother to offspring during pregnancy and avoided congenital malformation in 100% of the tests.
Tests confirmed protective action and did not indicate toxicity to the pups. The next stage will compare the placentas of monkeys and humans (Photo: Josué Damacena)
According to Marcelo Alves Pinto, researcher of the Laboratory of Technical Development in Virology of the Oswaldo Cruz Institute (IOC/Fiocruz) and coordinator of the trial, the findings in monkeys are a crucial phase to take therapy to patients. “This work began with in vitro studies [in cell cultures] led by Fiocruz, showing that sofosbuvir was active against the zika virus. But to make sure the therapy had potential, we need to test it on the model that is the most similar to humans, and those are Rhesus monkeys. The amount of results, with healthy offspring born out of treated mothers, indicates that sofosbuvir can be an effective option to prevent congenital zika syndrome in pregnant women that have been in contact with the virus”, says the researcher.
Congenital zika syndrome was detected in Brazil in October 2015, when the country faced an outbreak of microcephaly. Between 2015 and 2016, when the disease was considered a public health emergency of international significance, almost three thousand pregnancies were affected by the virus in the country, resulting in microcephaly and other malformations, in addition to neurological issues and deaths of fetuses and newborns. These numbers fell progressively in the following years, but scientists warn that the problem has not gone away.
“Infectious diseases have cycles. When a massive epidemic takes place, such as the zika, a large part of the population is infected and acquires immunity. It takes some time for there to be a contingent of people susceptible to it again. Congenital zika virus is a very serious problem. Localized cases continue to occur and a new outbreak may take place in the future”, states the head of the Laboratory of Technological Development in Virology of the IOC and author of the study, Jaqueline Mendes de Oliveira.
When it employed a drug approved to treat hepatitis C, the trial used a strategy known as drug repositioning (or repurposing), which allows for the quicker development of therapies. In addition to previous studies with culture cells and mice, showing an action against the zika virus, scientists also take into account the drug’s safety profile. “Sofosbuvir targets a region of the viral genome that is also present in several other viruses, and it is used with high effectiveness against hepatitis C. It has not been cleared for pregnant women yet, but there are clinical trials currently evaluating therapy in this group. It is therefore a viable alternative. As for the possibility of harmful effects of the drug on the fetus, there are data in the literature that show the drug is safe for use by pregnant women. We did not observe toxicity in our work”, highlights the lead author of the study, Noemi Gardinali, a biotechnician at the Laboratory of Viral Technology at Bio-Manguinhos.
Action on the placenta
The research studies eight female monkeys and their offspring. The Macaca mulatta monkeys, popularly known as Rhesus monkeys, were inoculated in their early pregnancy with a lineage of the zika virus isolated in the state of Espírito Santo in 2015, associated with cases of the congenital syndrome. The researchers ran tests on saliva, blood, urine, and vaginal secretions, and also carried out ultrasound exams in the monkeys during their pregnancy. Once the monkeys had given birth, samples were collected from their placentas, and blood and tissue samples were collected from their offspring.
The first stage of the work had the goal of confirming that the zika virus caused, in animals, the same issues observed in humans. The first two case studies confirmed the validity of the model. The virus was detected in blood samples drawn from the mothers, which also presented symptoms of the disease, such as fever and spots on their bodies. One of the babies developed a severe case of the congenital syndrome and died right after birth. The other was born with no apparent alterations. In spite of the different outcomes, the zika virus was found in samples of blood or tissues of both babies.
“The model was a reproduction of what we observe in human infections. Some babies are severely affected, while others present no lesions at all. There are also cases in which issues appear later, as the child develops”, comments Oliveira.
In the following phase, researchers intended to assess the impact of sofosbuvir therapy. Over two years, six pregnant monkeys were included in the study, for a total of eight. Five were treated right after the first symptoms of infection and one was not medicated so that the number of animals in the non-treated group was a total of three. According to the scientists, the higher number of individuals in the group submitted to therapy took into account the ethics of research on animals, in addition to the complexity and costs of the project. “In studies with experimental models, we must always attempt to reduce the number of animals to the minimum necessary”, says Oliveira.
Another severe case was observed in the absence of treatment, with fetal death occurring during pregnancy and with the identification of high viral load in the fetus. Conversely, no malformations were observed in the treated group. Three animals were born healthy and one fetus, victim of a miscarriage following an accident, showed no alterations. The zika virus was not detected in any of these four babies. The fifth case of the related group was considered inconclusive: a monkey with endometriosis and signs of preeclampsia (a kind of hypertension that is specific to pregnant women) suffered placental abruption with hemorrhage, resulting in a miscarriage. The zika virus was found in the fetus, but contamination may have occurred due to maternal bleeding.
“The set of data of the study, including serology, viral load, and histopathological exams, is very significant. The lesions observed in the placenta and in the central nervous system of the offspring in the non-treated cases strongly suggest inflammation caused by the zika virus”, states Renato Marchevsky, researcher of the Laboratory of Neurovirulence of the Institute of Technology in Immunobiology (Bio-Manguinhos/Fiocruz).
The study also mentioned that sofosbuvir does not eliminate the virus in the mothers, but its action on the placenta may explain the success of the therapy. The placenta is responsible for providing the fetus with nutrients and oxygen during the pregnancy, and it also works as a barrier against infectious agents. Previous studies had indicated that the zika virus is capable of breaking this protection when it infects cells of the immune system of the placenta itself. “When we look at the placenta, we observe that in treated mothers the rate of infection in these cells is close to zero, while it reaches 42% in non-treated monkeys. The placental barrier seems to be more effective in the mothers treated with sofosbuvir”, says Pinto.
In the next phase of the research, scientists intend to compare monkey placentas with human cases, hoping to acquire new knowledge on the dynamics of this infection. Possible perspectives for possible trials with patients are currently being discussed. “Our research has not shown toxicity or teratogenic effects, and there are data showing the sofosbuvir can be well tolerated during pregnancy. But we need to discuss with clinical researchers the possibility of establishing a protocol to evaluate treatment in pregnant women exposed to the zika virus, considering that the drug can reduce the risk of malformations”, states Pinto.
The authors highlight that cooperation between different Fiocruz research groups and external collaborators was crucial to carry out the study. Overall, thirty-five scientists took part in the study. Led by the IOC, the work was a partnership with five Fiocruz units: Bio-Manguinhos; Institute of Science and Technology in Biomodels (ICTB); Service of Equivalence and Pharmacokinetics of the Vice-Presidency of Production and Innovation in Health (Sefar/VPPIS); and Center for Technological Development in Health (CDTS). At the IOC, in addition to the Laboratory of Technological Development in Virology, the Pathology Lab and the Biotechnology and Physiology of Viral Infections Lab were also involved.
The research also counted on the collaboration of the Veterinarian Diagnostic Center (Cevet) of Niterói (state of Rio de Janeiro), the Federal University of ABC (UFABC), National Institute of Science and Technology of Innovation Management for Neglected Diseases (INCT/IDN), Dr. Julio Moran Laboratories (Switzerland), and Bernhard Nocth Institute of Tropical Medicine (Germany). The project was financed by funds provided by the Carlos Chagas Filho Foundation for Research of the State of Rio de Janeiro (Faperj), the National Council for Scientific and Technological Development (CNPq), the Department of Science and Technology of the Ministry of Health (Decit/MS), and the International Development Research Center (Canada).