Hope in the face of Zika

In initial tests, immunizing formulations and drugs against other diseases protected cells and mice from the virus

An invasion in progress: copies of the Zika virus (pink) interact with the receptors (green) of a cell

David Goodwill / Wikipedia An invasion in progress: copies of the Zika virus (pink) interact with the receptors (green) of a cellDavid Goodwill / Wikipedia

After researchers in the northeastern Brazilian state of Bahia confirmed the presence of the Zika virus in that area one year ago, the virus has spread throughout almost all of the country. Caught by surprise, researchers and health authorities had to mobilize and direct their efforts toward understanding the virus and finding ways to stop it. Their first results are beginning to appear and are promising.

Throughout June 2016, the results of important studies were made public. These studies demonstrated an understanding of the reactions that the virus awakens in the immune system, a situation of particular interest to the Brazilian population: cases in which the Zika virus infects people who have already been in contact with any of the four varieties of the dengue virus (in certain areas of the Northeast, 90% of the population has had dengue fever). Published in the journal Nature Immunology by researchers from England, France, Polynesia and Thailand, this work confirms that the antibodies that protect against dengue also act against Zika, but are unable to neutralize it completely. Immunologists call this a cross reaction. What has been found to be true for Zika in relation to antibodies against dengue fever may also occur with dengue in relation to anti-Zika antibodies.

This partial immunization, researchers suggest, could occur because antibodies produced against a virus are insufficient in number or not effective enough against another virus. To complicate the situation even more, according to a hypothesis called antibody dependent enhancement (ADE), incomplete immunization appears to facilitate viral entry into cells in which the virus replicates, thereby increasing the number of copies in the body and the severity of the infection. This data, corroborated days later by another study presented in the journal Proceedings of the National Academy of Sciences (PNAS), may help to explain the severity of Zika cases in Brazil and influence vaccine development, since vaccinating a population against only one disease could lead to more serious cases of another.

There was also some more encouraging news. Later in June 2016, signs emerged that it is possible to contain the virus with drugs that have been in use for a long time or even with a vaccine, which would be more desirable from a public health standpoint, because of its preventive nature. Neurologist Arnold Kriegstein and his team at the University of California at San Francisco investigated how Zika penetrates placental cells and brain tissue and found that azithromycin, an antibiotic produced in the early 1980s and widely used against a variety of infections, including in pregnant women, stopped the spread of the virus and prevented cell damage in laboratory tests. This data adds to the work coordinated by virologist Amílcar Tanuri at the Federal University of Rio de Janeiro (UFRJ). Tanuri and his team have observed that one of the compounds most commonly used to fight malaria, chloroquine, discovered in 1934, was also able to contain the Zika virus in experiments done with cells.

In late June 2016, researchers from Brazil and the United States took the first effective step toward demonstrating that it is possible to produce a vaccine against Zika, although its development will take some time. At Harvard Medical School’s Center for Virology and Vaccine Research (CVVR) in the United States, two candidate vaccine formulations successfully passed their first tests with laboratory animals. Each candidate vaccine, applied in a single dose, protected the mice from the Zika infection, the researchers reported in an article published on June 28, 2016 in the journal Nature. In experiments with mice, the two vaccines were effective against both the variety of the virus circulating in Brazil and against the strain found in Puerto Rico in the Caribbean.

“We showed that it is possible to produce an anti-Zika vaccine,” says Brazilian immunologist Rafael Larocca, the lead author, along with his colleague Peter Abbink, of the study done at CVVR. “The results are strong and compelling, but we have to be cautious and wait for more tests with animals and trials with humans,” he adds. “As far as we know, this is the first demonstration in an animal model of protection against Zika with a vaccine,” says the U.S. physician Dan Barouch, who coordinates the laboratory where Larocca works and heads CVVR.

The researchers used two classes of vaccine for the experiments. One was a formulation containing chemically inactivated copies of the virus. It was developed by the U.S. Defense Department’s Walter Reed Army Institute of Research, which used the Zika virus circulating in Puerto Rico. Larocca and Abbink adopted another strategy at CVVR. They analyzed the genome of the virus and produced a synthetic copy of the segment containing the protein complex sequence covering Zika’s exterior: the pre-membrane protein (prM) and envelope protein (E), by which immune cells identify the virus. At the body’s first contact with the virus, special types of immune cells—the antigen-presenting cells—detect these proteins, process them and display portions of them for the B lymphocytes, which produce antibodies to neutralize the virus when the body is again exposed to it.

The researchers transferred this synthetic gene to bacteria and allowed them to act as copying machines, producing a large number of replicas used later to immunize mice—these copies are what researchers call a DNA vaccine. “We created six vaccine formulations from DNA, but only one, the one expressing the complete protein complex, worked,” says Larocca, who has both a master’s and doctoral degree from the University of São Paulo (USP) supported by FAPESP. He was supervised, respectively, by immunologists Luis Vicente Rizzo and Niels Olsen Saraiva Câmara and since 2012 has worked at CVVR.

On the left, copies of the Zika virus (red) isolated from an infant in Ceará

National Institute of Allergy and Infectious Diseases, National Institutes of Health On the left, copies of the Zika virus (red) isolated from an infant in CearáNational Institute of Allergy and Infectious Diseases, National Institutes of Health

Larocca and his collaborators used the two formulations separately to immunize mice and, days later, injected the animals with the Brazilian Zika virus or the Puerto Rican Zika virus to see whether the vaccine offered protection. None of the immunized mice developed signs of infection or showed detectable quantities of the virus in their blood, while Zika proliferated in abundance in unvaccinated mice.

Mechanism of action
A subsequent battery of tests helped identify what appears to be the main form of protection against Zika. Larocca and Abbink collected blood samples from vaccinated animals, extracted specific antibodies against the virus, and transferred those antibodies only to unimmunized mice. By injecting the Brazilian Zika or the Puerto Rican Zika into these animals, the researchers determined that the mice were not infected.

“This experiment shows that the antibodies produced against the virus are sufficient to offer protection from the infection,” notes USP neuro-immunologist Jean Pierre Peron, who along with virologist Paolo Zanotto, is co-author of the June Nature study and a member of the Zika Network, a consortium of São Paulo researchers investigating the virus with support from FAPESP. “This does not eliminate the possibility that a formulation capable of producing immunity also works in another way, stimulating immune cells, called T cells, to produce compounds that prevent the virus from replicating.” Experiments performed by other teams have suggested that the production of a signaling molecule called interferon prevents the Zika virus from multiplying.

“We had some indication that a vaccine could produce immunity against Zika,” says biologist Paulo Lee Ho, director of the Technological Development and Production Division of the Butantan Institute, which works, with funding from the U.S. government, on the development of an immunizing candidate against Zika using the inactivated virus. At USP in Ribeirão Preto, immunologist Benedito Fonseca and his team had already performed a simpler version of the experiment. They extracted blood serum from the mice immunized with the inactivated virus and added cells cultured in the laboratory. The test results have not yet been published, but they do indicate that the virus failed to infect the cells treated with serum. “The Nature study is important because it shows that a very complex immunological response is not necessary to protect against the virus, and provides clues as to how to overcome some barriers that we were finding to inactivate it,” says Ho.

Culture medium in which copies of the virus multiply in monkey cells

Léo Ramos Culture medium in which copies of the virus multiply in monkey cellsLéo Ramos

“This work is relevant and demonstrates that developing a Zika vaccine is technically feasible,” says veterinarian Marcos da Silva Freire, deputy director of Technological Development at the Institute of Technology in Immunobiology of the Oswaldo Cruz Foundation (Fiocruz) in Rio de Janeiro. “But, as the authors also note, it is difficult to extend the test results on mice to a potential clinical efficacy in humans.”

Freire is also working to develop a vaccine and is focusing on two approaches: one with an inactivated virus and the other with a recombinant live virus that uses the yellow fever virus to express Zika proteins. Both approaches will begin animal testing in September 2016. He notes that there are still many questions to be answered before arriving at a safe and effective vaccine, and it is not helpful to create a false sense of protection, especially among women of childbearing age. “We do not know if the formulations tested in the Nature study can safely be given to children and adults, especially to  women of childbearing age or to pregnant women, or how long the protection lasts or whether booster doses are needed,” says Freire. “These questions can only be answered by clinical studies.”

Another open question, intensified by the cross-reaction between antibodies against dengue and antibodies against Zika, is whether developing a vaccine against just one of the diseases would aggravate another. “Does a Zika vaccine increase the number of dengue cases and their severity and vice versa?” asks Ho. Perhaps, he says, the solution is a pentavalent vaccine that protects against all four serotypes of the dengue virus and against Zika, such as the one the Butantan Institute is trying to develop in collaboration with the U.S. National Institutes of Health.

The search for a vaccine against Zika became a global health priority after evidence emerged that the virus infects the fetus of pregnant women and causes microcephaly. Between October 2015 and June 18, 2016, the Ministry of Health in Brazil recorded 8,039 suspected cases of microcephaly, of which 1,616 were confirmed (233 tested positive for infection by the Zika virus). While Brazilian groups work to find different formulations and develop, on an industrial scale, strategies for the production of safe vaccines for use in humans, the experiments continue at Harvard.

Larocca and his colleagues have used the two formulations to immunize monkeys and are awaiting the results due in the coming months. In partnership with the group led by USP’s Jean Pierre Peron, they will soon begin immunization tests on pregnant female mice, in order to determine whether the formulations actually protect against microcephaly. “Our results have made us optimistic that the development of a safe and effective vaccine for humans against the Zika virus is likely to be successful,” says CVVR’s Dan Barouch. “Clinical trials should start as soon as possible.”

1. The role of the tryptophan-kinureninas axis in regulating the immune response through NMDA glutamate receptors in experimental autoimmune encephalomyelitis and cerebral ischemia and reperfusion (nº 2011/18703-2); Grant Mechanism Young Investigators Award; Principal Investigator Jean Pierre Schatzmann Peron (ICB-USP); Investment R$1,077,384.82.
2. A systematic approach to study permissivity on the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (nº 2014/17766-9); Grant Mechanism Regular Research Grant; Principal Investigator Paolo Marinho Zanotto (ICB-USP); Investment R$500,009.45.

Scientific articles
LAROCCA, R. A. et alVaccine protection against Zika virus from BrazilNature. June 28, 2016.
DEJNIRATTISAI, W. et al. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with Zika virus. Nature Immunology. June 23, 2016.