The invisible enemy: the unknown about ZIKA virus.

Have you ever wondered what are you afraid of? Lots of people are so scared about big nature phenomena such as earthquakes, tsunamis, tornados or volcanos. Other are scared about spiders or snakes. But all in all I think it is quite possible to avoid all the above threats or at least to escape without any injuries. What I think is super scary though is what I call THE INVISIBLE ENEMY. Can you imagine that? Something that could harm you or your family, without any warning…. An enemy which you cannot see, smell or listen, that it’s out there ready to attack. Thing is, invisible enemies exist in real life and they are called VIRUSES.

Although we deal with viruses everyday and that we have developed a quite efficient immune response against them, they keep coming back. Sometimes there are new kinds of viruses, sometimes “pimped” viruses which have acquired mutations that can make them more infectious or even old, once-thought- erradicated viruses can return on stage.

Some of this enemies can be transmitted by air, others by sexual intercourse or blood transfusions, others can even be transmited by vectors such as mosquitoes. Either way, we have had some serious health emergencies in the 2000s due to viruses:

2002: An outbreak of Severe Acute Respiratory Syndrome (SARS) caused by the SARS coronavirus began in China. Three months after the first diagnosed infection, a 64-year-old Chinese doctor who had treated cases in China arrived in Hong Kong to attend a wedding. He checked into the Metropole Hotel and died 2 weeks later. About 80% of the Hong Kong cases have been traced back to this doctor. Thus this was a highly contagious virus which could be transmitted by respiratory droplets (coughs or sneezes) or even by touching a contaminated surface. In an 8 month-window, the SARS outbreak in southern China caused an eventual 8,096 cases and 774 deaths reported in multiple countries with the majority of cases in Hong Kong (9.6% fatality rate). Within weeks, SARS spread to infect individuals in 37 countries in early 2003. It then was eradicated by January the following year.

2009: A new flu virus (H1N1) spread quickly across the United States and the world. A total of 74 countries were affected by the pandemic. Luckily, an H1N1 vaccine was developed and a total of 80 million people were vaccinated. It was estimated that 43 million to 89 million people had H1N1 between April 2009 and April 2010 and deaths were estimated between 8,870 and 18,300. In 2010, the WHO declared an end to the global H1N1 flu pandemic.

2013: The most widespread epidemic of Ebola virus disease in history began in Guinea and continued with significant loss of life for over two years. It has caused significant mortality, with reported case fatality rates of up to 70%. As of January 2016, although the epidemic is no longer out of control, the WHO has not yet declared the epidemic over due to the continuation of flare-ups.

2015: A new viral outbreak have come to invade us again and the responsable is the Zika virus (ZIKAV). It quietly started by the end of 2015 and just now, the WHO has declared a global health emergency due to the high number of Zika infections and its connection to microcephaly in newborns and other neurological disorders. Zika virus moved outside of Africa and Asia in 2007 and 2013 with outbreaks in Yap Island and French Polynesia, respectively. The first cases in the Americas were detected in Brazil in May 2015. The virus circulating in Brazil is an Asian genotype, possibly imported during the World Cup of 2014. But is Zika a new virus? But what do we know about this invisible enemy?

70 years since ZIKAV discovery. Monkeys got it before humans

In 1947, a Yellow Fever Virus (YFV) study was setup in the Zika forest in Uganda as part of Rockefeller Foundation’s program for research on jungle YFV. The location was selected because it was known to harbour a lot of previously YFV infected, antibody-positive monkeys. In the first experiment, six platforms were set up in the forest canopy and upon each, a caged Rhesus monkey was placed. The monkey, Rhesus 766, was a sentinel animal in the Rockefeller Foundation’s program for research on jungle yellow fever (sentinel animals are used to detect risks to humans by providing advance warning of a danger).

On 18th April, the daily temperature recording for “Rhesus 766” had increased to 39.7°C and rising to 40°C the next day. It was taken to the Foundation’s lab at Entebbe where a blood sample was collected and its serum was injected into mice intraperitoneally (showing no signs of illness) or intracerebrally (becoming ill from day 10 post-injection). But why injecting the serum into mice? Well, because at that time, cell lines were not available for studying viruses, so serum from the febrile monkey was inoculated intracerebrally into mice. But it was also injected underneath the skin of Rhesus 771 (which did not develop any signs of illness). By the way, the only sign or symptom of illness in 766 was the fever.

Well, it happened that a filterable transmisible agent, later named Zika virus (strain 766), was isolated from the mouse brains and also from the serum of 766. The virus’s growth could be hampered by antibodies which developed in the serum of 766 a month later, identifying that the Zika virus was capable of triggering a specific immune response, despite a mild illness.

ZIKAV second appearence: Jesus! It’s in mosquitoes!!!!

In early 1948, during a different study also set up for YFV, a second virus (later called strain E/1) was acquired from a ground up, unfiltered preparation of Aedes africanus mosquitoes, which had been caught January 1948 in the same forest.

The preparation was injected intracerebrally into 6 mice and subcutaneously into Rhesus 758 via 9 inoculations across 2 weeks. Primate 758 showed no signs of illnes, but the mice had at the 7th day. At days 8, 9 and 10 after 758’s injection, blood was collected from it and serum injected intracerebrally into groups of 6 new mice:

-       From the first primate sample (say 8), 2 injected mice died and virus could be passed on to additional mice from filtered preparations of their infected brain tissue.

-       From the second sample (day 9), 1 mouse died (and its brain tisue filtrate could also produce new infections in mice)

-       No mice died from the 3rd inoculation of serum

-       The serum from 758 was shown to block infection of animals by Zika virus after it was first preincubated with preparations of the agent isolate from 758 serum after inoculation with the A. Africanus preparation or the E/1 or 766 Zika virus strains.

The authors concluded that Zika virus was a new virus and that it triggered a specific antibody response which did not cross-react with YFV, dengue virus or the TMEV.

It was notable that the disease was mild or went unnoticed in primates. Primates are usually considered to be close animals substitutes for humans in studies of disease progression. Also, no studies on pregnant primates were conducted which does raise the question of  doing it on every virus capable of replicating in us.

ZIKAV reached humans

In 1952, serologic studies indicated that humans could also be infected. Transmission of ZIKV by artificially fed Ae. aegypti mosquitoes to mice and a monkey in a laboratory was reported in 1956. But it wasn’t until 1968 that Zika was isolated from humans, specifically from Nigeria.

Who are you ZIKAV???

Zika virus is a member of the flavivirus family, which also includes yellow fever virus, dengue virus, Japanese encephalitis virus, and West Nile virus. The genome is a ~10.8 kilobase, positive strand RNA enclosed in a capsid and surrounded by a membrane. The envelope (E) glycoprotein, embedded in the membrane, allows attachment of the virus particle to the host cell receptor to initiate infection. As for other flaviviruses, antibodies against the E glycoprotein are likely important for protection against infection.

Zika virus is transmitted among humans by mosquito bites. The virus has been found in various mosquitoes of the Aedes genus, including Aedes africanus, Aedes apicoargenteus, Aedes leuteocephalus, Aedes aegypti, Aedes vitattus, and Aedes furcifer.Aedes albopictus was identified as the primary vector for Zika virus transmission in the Gabon outbreak of 2007. Whether there are non-human reservoirs for Zika virus has not been established yet.

Signs, symptoms, control, the known blablabla in four lines.

Mild or no symptoms. Could be rash, fever, joint pain, red eyes 2-10 days after infection. The frightening stuff: There is association with Guillain-Barré syndrome and Microcephaly. But I emphazise in “association”, it has not been demostrated yet. No current drugs or vaccine available. Mosquito control is THE option for restricting Zika virus infection for now.

How can ZIKAV be detected?

Diagnostic tests for ZIKV infection include:

1.    PCR tests on acute-phase serum samples, which detect viral RNA. This test can be conducted on samples obtained less than 10 days after illness onset.

2.    ELISA to detect specific antibody (IgM) against ZIKV in serum. IgM is detectable as early as 3 to 5 days after onset of illness. The problema is that IgM antibodies against Zika virus, dengue viruses, and other flaviviruses (e.g., yellow fever and West Nile virus) have strong crossreactivity possibly generating false positive results in serological tests. Therefore an IgM positive result in a dengue or Zika IgM ELISA test should be considered indicative of a recent flavivirus infection. Plaque-reduction neutralization tests (PRNT) can be performed to measure virus-specific neutralizing antibodies and may be able to discriminate between cross-reacting antibodies in primary flavivirus infections. For primary flavivirus infections, a fourfold or greater increase in virus-specific neutralizing antibodies between acute- and convalescent-phase serum specimens collected 2 to 3 weeks apart may be used to confirm recent infection. In patients who have been immunized against (e.g., received yellow fever or Japanese encephalitis vaccination) or infected with another flavivirus (e.g., West Nile or St. Louis encephalitis virus) in the past, cross-reactive antibodies in both the IgM and neutralizing antibody assays may make it difficult to identify which flavivirus is causing the patient’s current illness.

The thing EVERYBODY wants RIGHT NOW: a ZIKA vaccine

I would love it. I happen to live in Brazil and in a fertile age. But, is it posible to get a quick vaccine? Brazil says it could take from 3 to 5 years to develop a vaccine, a “fairly well” timing for me. Translating lab research into vaccines is costly (up to $1 billion per bug, according to some estimates) and time consuming (up to 15 years). But in an outbreak, R&D needs to be able to attack pathogens and timelines. From my point of view, developing a vaccine is a thing about MONEY (which involves TIMING) and PREDICTIONS (not that I agree with how things are done at all).

The capacity to generate vaccines and therapies within months, rather than years, could have spared thousands in the Ebola outbreak. It could save millions in a severe flu pandemic. To deal with the threat of unpredictable health emergencies (bioterrorism or global epidemics) the US has set aside a small collection of previously developed drugs and vaccines. But stockpiling drugs against a few known pathogens gives protection against only some of the full spectrum of biological threats. Moreover, predictive stockpiling didn’t work for Ebola. An Ebola vaccine actually made the shortlist for the US program in 2003, largely because officials worried about this virus in the hands of a terrorist. But making the list and making medicine are different. Thanks to a decade of federal assistance, several Ebola vaccine candidates were in the pipeline in 2014. Even so, the dollars directed toward Ebola were inadequate to develop vaccines that were outbreak ready. Few had made the leap from the lab into human trials. As a consequence, West Africa lost precious months last winter to preliminary safety tests. 

The most interesting questions I’ve read about this whole issue are:

-       Can we build generic vaccine scaffolds that require minor modifications for different pathogens?

-       Can we do this in a way that accelerates development times, lowers costs, and facilitates regulatory review?

In answer, one group of scientists at Massachusetts General Hospital is responding with VaxCelerate, a consortium of biotechnology companies and academic research labs. VaxCelerate generates vaccines on demand, using a two-part vaccine platform: a standard delivery vehicle and a specialized “payload” that can be tailored to an emerging disease. Delivery vehicles, or vectors, often take the form of a harmless bacterium or virus. These can be prepared, tested, and stockpiled in advance. Computer algorithms identify peptides most likely to produce a safe and effective human immune response to the disease of concern. These peptides are loaded into the delivery vehicle to quickly identify pre-clinical candidates.

VaxCelerate’s first vaccine targeted Lassa fever, another African virus that is virtually unknown in the West. Lassa fever infects thousands each year and kills ten percent of those infected. The consortium’s first test produced a pre-clinical vaccine for $1 million in just four months—fifteen times faster and ten times cheaper a typical vaccine project at this stage.

This generic vaccine platform shows that it’s possible to address a range of pathogenic threats while shaving years (and millions of dollars) off of the pre-clinical research phase. And using a standardized platform doesn’t just speed research; it can also simplify industry’s manufacturing process and the FDA’s evaluation requirements.

A handful of other companies are investigating vaccine and drug development platforms that promise similar capabilities. Last fall, Novavax was able to generate an Ebola vaccine candidate for human trials in three months using a nanoparticle vaccine platform. When H7N9 broke out in China in 2013, Novartis was able to generate pre-clinical vaccine candidates in 13 days using a lipid delivery system for mRNA.

Anyway, researchers and regulators must improve technologies and operational efficiency in each phase: detection, diagnostics, discovery, development, manufacturing, clinical trials, and delivery. The greatest challenge and the greatest benefit will come from integrating new technologies as they mature into a streamlined emergency medical countermeasure development system. 

The example of Ebola is what happened with a disease that was on a short list. What will happen if the next disease is not? In public health emergencies, scientists don’t have the luxury of time. And yet they work within research traditions that assume that they do.

For the existing Zika emergency, let the development timeline be driven wholly by legitimate scientific challenges and not by operational inefficiency. Lets get this invisible enemy!

Great things start small: an insight into fragment based drug discovery

Have you ever noticed that great things almost always start small? Take for example Richard Branson, he started the Virgin brand with a student magazine!.

We want ‘big’ things, we have ‘big’ aspirations, we want to jump ‘big’ too soon without thinking that maybe starting from small is the best way to go.

Nowadays, drug development is adopting this thought as well: start small. And this can be achieved by screening for small chemical fragments that bind only weakly to a biological target and only then growing them or combining them to produce a compound with higher affinity. This method is known as fragment-based drug discovery (FBDD) and the main topic of this post.

Think small, think FBDD

Developing a new drug is a complex process. Once the target has been chosen, the process of lead discovery begins. A lead is a compound from a series of related compounds that has some of a desired biological activity. A variety of methods to identify hit molecules exist, but the most commonly used is high-throughput screening (HTS), in which libraries with up to millions of compounds with molecular weights of around 500 daltons are screened and nM binding affinities to target are sought. But could we think ‘small’? Smaller molecules, smaller affinities? Definitely YES.

In contrast with HTS, in FBDD typically about 1,000 fragments are screened using biophysical techniques. The fragments are between 150-250 daltons and mM affinities can be considered useful. After biophysical analysis, candidate fragments are grown to form new interactions using structure-based drug design. Although initial fragment hits have low potency due to their small size, they form high-quality interactions and can be readily optimized into potent lead molecules.

Does size matters then? YES, because the size, complexity and physical properties of small molecules can be more easily controlled than when starting from higher affinity HTS hit.

How can fragment binding be screened?

Biophysical methods to screen fragment binding to its target include NMR spectroscopy, X-ray diffraction, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and mass spectrometry. Which one to use? Each method has advantages and disadvantages in terms of sample consumption, degree of automation and assay complexity, but the general rule is that whatever method you choose, it has to be FAST, EFICIENT, PRECISE and to REDUCE THE NUMBER OF FALSE POSITIVES AND FALSE NEGATIVES. Remember that the number of fragments that are frequently analyzed can go from hundreds to thousands, so it is very important to choose well.

A new effective method for fragment screening

An emerging technique has appeared very recently for fragment screening¹. Microscale thermophoresis (MST) has been broadly applied to investigate biomolecular interaction of a variety of drug targets, thus it seemed perfect for trying it in FBDD… and it is.

In a recent fragment screening made by Sanofi, MST was used in an automated manner to screen a library of fragments targeted against the kinase MEK1 from the MAPK signalling cascade, thus with looks into developing anticancer therapies. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods.

But what I found great about this technology and what marks a difference is related to an important aspect of fragment screening campaigns: The EXCLUSION OF FALSE POSITIVES AND FALSE NEGATIVES. Binding of fragments and small molecules can either stabilize or destabilize target proteins. Destabilization or denaturation of proteins is often accompanied by protein aggregation and MST provides a direct feedback on ligand-induced aggregation and other secondary effects. Thus the information provided by MST prevents false-positive hits from entering later stages of hit expansion, as well as rescuing fragments classified as false negatives.

All in all, this speed technology, used in conjunction with other biophysical techniques has the potential to significantly improve FBDD workflows.

In the early days of FBDD there was some scepticism that it would ever work, for example, “How can such low-affinity compounds be identified?” or “Drug discovery is hard enough starting with a high affinity hit so why go to smaller and lower affinity compounds?”. Today, the consensus has changed. At least nine FBLD projects have led to compounds in phase II or III studies, including one approved drug. The fragment approach has been adopted throughout academia and large pharma and biotech companies, and there are a number of commercially available fragment libraries.

So think about it……… maybe it’s time for you to start small.

“Start small, think big. Don’t worry about too many things at once. Take a handful of simple things to begin with, and then progress to more complex ones. Think about not just tomorrow, but the future. Put a ding in the universe”. Steve Jobs.

1. Linke, P., Amaning, K., Maschberger, M., Vallee, F., Steier, V., Baaske, P., Duhr, S., Breitsprecher, D., Rak, A. An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery. 2015. Journal of Biomolecular Screening. doi:10.1177/1087057115618347

Biosimilars are the next money makers of pharma industry

It has happened to everyone: got sick, went to the doctor, got diagnosed and received that desired white piece of paper named “drug prescription”. We run to the drugstore and just ask straight ahead for what has been prescribed. Until recently not many options regarding price of the drug or effect were available, but now some alternatives are there for us.

GENERIC DRUGS are one alternative. They are copies of brand-name drugs, and by ‘copies’ I mean in dosage, safety, strength, route of administration, quality, performance, characteristics and intended use. They have the exact same ‘active component’ of the brand name drug, which is the part that has the therapeutic function, while the ‘inactive components” which in general do not have any pharmacological effect can be different, and they can include dyes, preservatives, flavouring agents, etc.

Generic drugs are important options that allow greater access to health care… why?? BECAUSE THEY ARE WAY CHEAPER THAN BRAND NAME DRUGS. The latter are developed under a patent, which protect a drug company’s investment in developing the drug. This gives the company EXCLUSIVE RIGHTS to sell the drug while the patent is in effect for up to 17 years. After the patent expires, other drug companies can produce the drug. But generic drugs are able to be sold for lower prices because they are not required to repeat the costly clinical trials of new drugs and generally do not pay for costly advertising and marketing. In addition, multiple generic companies are often approved to market a single product; this creates competition in the market place often resulting in lower prices. Good for us consumers. (Story not told, is that 70 to 80% of all generic drugs are produced by the same companies who make brand name drugs… obviously).

BIOSIMILARS, the new kid on the block, are another alternative. They are biological products (not chemicals such as brand name and generic drugs) highly similar to brand name drugs with no clinically meaningful differences in terms of safety and effectiveness from the reference product. Only minor differences in clinically inactive components are allowable in biosimilar products. They also differ from ‘generics’ in that THEY ARE NOT EXACT COPIES of brand name drugs.

Producing generic small-molecule drugs is relatively simple–it’s like following a recipe with standard ingredients. Biosimilars are much more challenging because living cells (where they are produced) are highly sensitive to their environments, and manufacturers have to create their own, unique process to make these cells to produce an identical outcome to an existing treatment.

Biosimilars have made drug approvals challenging. Generics are approved based on matching chemical structure, but that doesn’t work for biosimilars. Each new biosimilar has to run clinical trials to prove the outcome matches that of the biologic it is imitating, even though it looks structurally different, according to recently announced guidelines from the Food and Drug Administration.

Let’s talk about money

When talking about Pharma business there is a lot of money involved and the opportunities for biosimilars are huge for both manufacturers and consumers. Many leading biologic medicines worth more than $81 billion global annual sales will lose their patent protections by 2020… only 4 more years, that means the war between pharma companies has begun.

Much like generics, biosimilars can help cut drug costs, though the savings are smaller because of their complexity as well as regulatory challenges of getting FDA approvals. Biosimilars cost about $75 million to $250 million to reach the approval stage, versus around $2 million to $3 million for a generic small-molecule medicine.

So far, an inflexion point in the pharmaceutical industry has been seen with the approval of two biosimilars: the Hospira’s biosimilar version of infliximab (a monoclonal antibody used to treat autoimmune diseases) in Europe and the subsequent approval in the U.S. of its first biosimilar in history, Novartis’ Zarxio (which targets Amgen’s Neupogen).

In September 2015, Pfizer  shelled out big bucks–$17 billion to be exact–to buy the much smaller drugmaker Hospira. A big reason for the rather expensive acquisition is to gain access to the company’s biosimilar portfolio, an insight on how important these new drugs are becoming for pharma industry.

What can be expected then? Well, that some of the big firms — including Abbvie, Roche and Pfizer — will be highly impacted by how this biosimilar market shapes up in near future. There are around 11 biosimilars under development to compete with Abbvie’s Humira (infliximab competitor) alone, which loses its patent exclusivity in the U.S. in 2016.

Biosimilars are extending to the rest of the world as well. Last month, Brazil registered the first latin-american biosimilar (filgrastim), one of the 20 existing biosimilars to date.

What’s great about these drugs is that many people will definitely benefit as biosimilars take off. Significant savings for medical programs will be seen, since the worldwide trends is that governments are pushing theirselves for granting marketing authorizations for biosimilars…. I hope less money spend in health means more money spent for good purposes for us all tax-payers.