Zika outbreak gives rise to global fears

Zika may not be a new virus but its newly confirmed ability to cause foetal brain abnormalities makes the search for a treatment or a vaccine a high priority. Dr Sarah Houlton looks at the latest initiatives

Aedes aegypti mosquito

Given the number of headlines generated in recent months by the Zika virus, one might be forgiven for thinking it’s something new. In fact, it was first isolated in 1947 during research on yellow fever in Uganda’s Zika Forest.

The first documented human case outside the occasional outbreak of mild disease in Africa was in the Federated States of Micronesia in 2007. The mosquito-borne virus spread east across the Pacific and is now pandemic in some parts of Latin America, with the Pan American Health Organization reporting the first confirmed Zika infection in Brazil in May 2015. Since then, it has spread across the continent and confirmed cases have been reported in returning travellers in North America. The World Health Organization (WHO) expects it may spread to all countries in the Americas with the exception of Canada and Chile, thanks to the endemic nature of Aedes mosquitoes that spread it.

The WHO is, rightly, concerned about the virus’s potential impact. At a briefing session at the WHO’s executive board meeting in January, Director General Margaret Chan cited the reasons for concern as the possible association of infection with birth malformations and neurological syndromes; the potential for further international spread, given the wide geographical distribution of the mosquito vector; the lack of population immunity in newly affected areas; and the absence of any vaccines, specific treatments or rapid diagnostic tests.

The disease it causes in humans has only mild symptoms, and there it would end were it not for the apparent association with microcephaly in babies born to women who have been infected by the virus. There may also be an association with Guillain-Barré syndrome. Brazil has been particularly badly affected – according to figures from the Brazilian Health Ministry in early March, there had been 745 confirmed cases of microcephaly since reporting became mandatory on 22 October 2015. In contrast, there were 147 cases reported in the whole of 2014.

Whether the virus is the cause of microcephaly has been the subject of much debate

Whether the virus is the cause of microcephaly has been the subject of much debate. In mid-April, scientists from the US Center for Disease Control published an evaluation of the available data using criteria proposed for the assessment of potential teratogens in the New England Journal of Medicine. They concluded that there is a causal relationship between prenatal infection and brain anomalies including microcephaly. Factors that support their conclusions include infection having happened at points in gestation that would be consistent with the birth defects. They also cited a specific and rare phenotype involving brain anomalies in foetuses and infants with congenital Zika infection, and the identification of the virus in their brain tissue supported biological plausibility.

‘Many questions that are critical to our prevention efforts remain, including the spectrum of defects caused by prenatal Zika virus infection, the degree of relative and absolute risks of adverse outcomes among foetuses whose mothers were infected at different times during pregnancy, and factors that might affect a woman’s risk of adverse pregnancy or birth outcomes,’ they concluded.

The nature of the virus

The Zika virus is a flavivirus, the same family as the yellow fever, dengue, West Nile and Japanese encephalitis viruses. At the end of March, scientists at Purdue University in West Lafayette, Indiana published the first structural determination of the Zika virus in Science. Importantly, they managed to identify regions within the viral structure where it differs from other flaviviruses.

The structure of the virus provides a map that shows potential regions that could be targeted by a therapeutic treatment, used to create an effective vaccine or to diagnose and distinguish Zika infection from related viruses

‘The structure of the virus provides a map that shows potential regions that could be targeted by a therapeutic treatment, used to create an effective vaccine or to improve our ability to diagnose and distinguish Zika infection from that of other related viruses,’ says Richard Kuhn, Director of the Purdue Institute for Inflammation, Immunology and Infectious Diseases, where the research was carried out.

The team used cryo-electron microscopy, which is becoming increasingly important in pharmaceutical research for the determination of target structures, as it provides a level of resolution previously only attainable using X-ray crystallography. ‘[Crystallography] requires a relatively large amount of virus, which isn’t always available; it can be very difficult to do, especially for viruses like Zika that have a lipid membrane and don’t organise accurately in a crystal; and it takes a long time,’ says Michael Rossman, who co-led the team. ‘Now, we can do it through electron microscopy and view the virus in a more native state.’

The virus was isolated from a patient infected during the French Polynesia epidemic. They determined it was structurally very similar to other flaviviruses, comprising an RNA genome surrounded by a lipid membrane, inside an icosahedral protein shell. It bears a glycosylation site protruding from the surface of the virus that binds to receptors on the surface of human cells. The amino acid sequence surrounding this glycosylation site differs between flaviviruses, which may have an impact on the human cells it is able to infect. The Purdue team speculates that this could prove a successful target for an antiviral drug.

The Zika virus is structurally very similar to other flaviviruses, comprising an RNA genome surrounded by a lipid membrane, inside an icosahedral protein shell

The search for a vaccine

The speed at which the virus has become a problem has led to various governments announcing funding and research programmes to combat Zika. Brazil, for example, is providing additional funds to the Butantan Institute in São Paulo, which is Latin America’s largest immunobiology lab and produces about 90% of the vaccines in Brazil. The strategy is to produce antibodies to the virus in animals, in an analogous fashion to their work on Ebola. In the longer term, they plan to adapt their investigational dengue vaccine by inserting antigens from Zika.

And with cases now appearing in the US, the National Institutes of Health (NIH) is prioritising research in the area. It is already funding, via the National Institute of Allergy and Infectious Diseases (NIAID), Zika science such as the structural determination carried out at Purdue. And a bipartisan bill has been passed by the US Senate’s health committee that would add Zika to the Food and Drug Administration’s (FDA) priority review voucher programme, which promotes the development of treatments for neglected tropical diseases.

Sanofi Pasteur is the first of the big pharma companies to announce a strategic development programme for a Zika vaccine. Others, including GlaxoSmithKline, Pfizer, Johnson & Johnson, Merck & Co and Takeda, have indicated that they are assessing their options.

Zika is an obvious target for Sanofi Pasteur, as it already has licensed vaccines against the flaviviruses yellow fever, Japanese encephalitis and dengue. Its Dengvaxia product was approved at the end of 2015, and the company believes it will be able to leverage the technology and infrastructure used for Dengvaxia to help understand the spread of Zika and, potentially, speed up the identification of a vaccine candidate for further clinical development.

Our invaluable collaborations with scientific and public health experts, both globally and in the regions affected by the outbreaks of Zika virus, will expedite efforts to research and develop a vaccine for this disease

‘Our invaluable collaborations with scientific and public health experts, both globally and in the regions affected by the outbreaks of Zika virus, together with the mobilisation of our best experts, will expedite efforts to research and develop a vaccine for this disease,’ says the company’s Global Head of R&D, John Shiver.

Outside the world of big pharma, numerous biotechs and academic groups are focusing on the challenge of developing a Zika vaccine, particularly those who already have expertise with related viruses. For example, US biotech Inovio and South Korea-based GeneOne Life Science are collaborating on the development of a DNA-based vaccine for preventing and treating Zika infection. They are working alongside academic groups, including the University of Pennsylvania and the National Microbiology Laboratory in Winnipeg, Canada. Inovio has already had positive immunogenicity and challenge protection results for its vaccine candidates against the flaviviruses dengue and West Nile.

Positive preclinical results on a Zika vaccine, made using Inovio’s SynCon synthetic vaccine technology, were released in February. In the study, DNA vaccine constructs targeting multiple Zika virus antigens were generated synthetically and then administered using its Cellectra electroporation delivery technology. The vaccine resulted in seroconversion – the development of detectable specific antibodies in the blood – in all of the mice that had been vaccinated. The company says vaccination also generated robust, broad T-cell responses, as analysed by the standardised T-cell Elispot assay. They claim these findings are vital, given the potential importance of neutralising antibodies in preventing infection and the role T-cells play in clearing infection by killing those cells that harbour the virus.

Zika is a flavivirus – the same family as the yellow fever, dengue, West Nile and Japanese encephalitis

GeoVax Labs is applying its modified vaccinia virus Ankara – virus-like particle (MVA-VLP) vaccine platform to Zika, a combination technology that is already being applied to an HIV vaccine candidate. It uses the recombinant virus to produce self-assembling non-infectious virus-like particles in cells of the person being vaccinated, and in trials on its HIV vaccines this strategy has been shown to be safe while eliciting strong and durable humoral and cellular immune responses.

It is working with scientists at the University of Georgia to accelerate the vaccine’s development. The academic scientists are to develop vaccine antigens that elicit broadly reactive immunity against Zika viruses from different lineages, and test them in preclinical models.

GeoVax also announced in March that it has entered into a research collaboration agreement with the US Centers for Disease Control and Prevention (CDC) to evaluate the candidate Zika vaccine’s immunogenicity and protective efficacy. The company believes its platform is particularly suitable for Zika virus and has already been shown to be safe in more than 500 people in HIV clinical trials.

Scientists at the Mayo Clinic’s vaccine research group in Minnesota are working with a team at Brazil’s Butantan Institute. Mayo scientists designed a technique to identify the parts of a virus to which immune cells develop immunity that has already been successfully applied to measles and smallpox. They plan to use mass spectrometry to study human cells infected by the virus and identify the component parts of the virus as they are broken down by the cells. This information will then be used to reverse engineer a vaccine.

The advantages of a peptide vaccine would be a long shelf life and the absence of a requirement for cold storage

The result would be a peptide vaccine, and as humans do not normally get good responses from this type of vaccine, it will be adjuvanted to enhance the response – either using packaging with nanoparticles developed alongside by a team at the Karolinska Institute in Sweden or, if that is not successful, a chemical adjuvant. The advantages of a peptide vaccine would be a long shelf life and the absence of a requirement for cold storage. South San Francisco-based Vaxart has also announced that it has started preclinical testing, this time of an oral Zika virus vaccine. The company’s vaccine platform allows recombinant vaccines to be delivered in a tablet that is stable at room temperature. It has already carried out a Phase I study in an H1 influenza vaccine delivered in this way, which generated a robust and broad immune response.

The technology combines a suitable antigen with an adjuvant and a non-replicating adenovirus type 5 vector as the delivery vehicle that delivers both antigen and adjuvant to the mucosal cells in the gut. Delivery to the gut avoids the problem of pre-existing immunity to adenoviruses that can block viral uptake on vector administration, as it does not induce the same type of anti-vector immunity. The adjuvant is an important component in gaining efficacy in the gut, and Vaxart’s adjuvant works through a toll-like receptor. The platform offers a modular approach, with the same vector being used with different antigens for different diseases.

NewLink Genetics has also entered the race to develop treatment options, alongside its main focus on immuno-oncology product development. Although it has not detailed its approach, it has said that it hopes its scientists’ long experience in developing vaccines against other flaviviruses, and its experience of collaboration during the Ebola crisis, will assist its search. ‘Given the deep expertise and experience of our infectious disease team, we believe we are well positioned to develop a vaccine candidate for the Zika virus,’ claims the company’s Chief Executive and Chief Scientific Officer, Charles Link.

Canadian biotech Immunovaccine, based in Halifax, Nova Scotia, is starting a programme using its DepoVax platform to create a Zika vaccine. It has already developed candidate vaccines for Ebola, anthrax and respiratory syncytial virus (RSV) using this technology, as well as two in-house projects in the immuno-oncology field that are in the clinic. A candidate antigen has already been identified, and the company is looking to establish collaborations with manufacturers to produce the antigen for formulation into the vaccine.

Single doses may be effective, which would be important for a pandemic vaccine

To create the vaccine, the antigen and an adjuvant are mixed and encapsulated in a liposome; these are then suspended in oil. The formulation gives controlled and prolonged exposure of both antigen and adjuvant to the immune system, resulting in a strong, specific, sustained immune response. Single doses may be effective, which would be important for a pandemic vaccine. Also important is the absence of water from the vaccines, which should extend shelf life at room temperature.

Research is under way in Australia too, with a collaboration between Melbourne-based biotech Sementis and scientists at the University of South Australia’s experimental therapeutics laboratory. The two groups have developed a protective vaccine for mosquito-borne Chikungunya virus that is now in the clinical manufacturing process, and they believe it should be possible to adapt this for the Zika virus. Although Chikungunya is an alphavirus rather than a flavivirus, it is spread by the same type of mosquito, and the Australian scientists are now embarking on clinical studies on using the vaccine platform for Zika.

The platform is a bit like a cassette system that we can plug and play, so we plug the platform with different antigens to target a specific condition

‘The platform is a bit like a cassette system that we can plug and play, so we plug the platform with different antigens to target a specific condition,’ says the university lab head John Hayball. It uses genetic engineering to insert genes for antigens for different diseases.

India’s Bharat Biotech has filed for global patents for its Zikavac candidate vaccines. It has two potential Zika vaccines in development, one of which is an inactivated vaccine that has reached preclinical animal testing, and the company is focusing on scale-up and characterisation of the vaccine product. At the same time, it is working on a recombinant vaccine using the surface antigens of the virus. The company has already been working on the project for more than a year – since before the microcephaly issue came to light in Brazil – citing as a driving force the fact that the clinical features at an early stage of infection are indistinguishable from dengue and Chikungunya.

Another US biotech company, Novavax, has also indicated that it is in the early stages of Zika vaccine research. No further details have been released, but it has a pipeline of vaccine candidates based on recombinant protein nanoparticles and virus-like particles, with products for RSV and influenza in the clinic. Similarly, California-based PaxVax has said it plans to bring a candidate into preclinical trials in the first half of this year. Preclinical studies are already under way at Hawaii Biotech, which is already working on vaccines to counteract Chikungunya, tick-borne encephalitis and West Nile virus.

The speed at which the virus has spread across Latin America has highlighted the threat it poses to human health and the desperate need for a preventative vaccine. As the WHO’s Chan says, ‘Conditions associated with this year’s El Niño weather pattern are expected to increase mosquito populations greatly in many areas. The level of concern is high, as is the level of uncertainty. Questions abound. We need to get some answers quickly.’

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