At the Sharp End
High tech is coming to the fore in the struggle against viruses and bacteria. Modern vaccines transport genetic information from pathogens into target human cells in order to trigger an immune reaction there. The transportation of these "blueprints" requires tiny lipid nanoparticles of the kind that Evonik is producing in Canada
TEXTINGA OLFEN
When the country doctor Edward Jenner took a knife to his gardener's son in the English county of Gloucestershire on May 14, 1796, he could not have suspected that he was starting a medical revolution. After making an incision in the healthy eight-year-old boy's skin, Jenner rubbed into it some pus taken from a milkmaid who was sick with cowpox. One week later, the child had fever and a headache, as well as small blisters at the places that Jenner had infected. A few days later, these symptoms disappeared.
Six weeks later, the doctor repeated this procedure, this time with secretions from a patient suffering from smallpox, an illness that was killing 400,000 people a year in Europe during the 18th century. The boy developed no symptoms of illness whatsoever: Vaccination had been invented. Jenner called his process vaccination, a word derived from vacca, the Latin word for cow. The scientific term "vaccine" is also derived from this Latin word.
Today, a good two centuries after the invention of vaccine, scientists are researching completely new types of vaccine technology. This time, they are not working behind the doors of a country doctor's small office; instead, they are doing their work in full view of the international public. They are driven by a pandemic caused by a virus that can be deadly: the coronavirus SARS-CoV-2. They are working under tremendous time pressure. "Until just a few years ago, people estimated that it would take around 15 to 20 years to go from the analysis of a virus to the approval of a vaccine," explains Stefan Randl. As the head of research, development, and innovation at Evonik's Health Care business line, he's familiar with the challenges along the way toward new or improved medicines and vaccine serums. "Empirical data and very new technologies, such as gene-based vaccines, can significantly accelerate the process," he adds.
Evonik, as a contract development and manufacturing organization for the pharmaceutical industry, is also part of this effort. As a specialty chemicals company, it has the expertise and the technology that are needed for the development and manufacture of complex and highly specialized injectable drug products. These products can require drug delivery technologies such as lipid nanoparticles (LNPs), which are a hundred times smaller than a human blood cell and play a very important role when it's necessary to encapsulate unstable active ingredients and release them at exactly the right place in the human body. "Today, when we're discussing topics such as gene-based vaccines that have much shorter development times yet are more effective than conventional serums, lipid nanoparticles play a central role," Randl says.
"The Covid-19 pandemic is a catalyst for the development of gene-based vaccines"
STEFAN RANDL, HEAD OF RESEARCH, DEVELOPMENT, AND INNOVATION, EVONIK HEALTH CARE BUSINESS LINE
By producing lipid nanoparticles, the chemical industry is making an important contribution to the development of the vaccines of the future, says Professor Hartmut Hengel. "The substances we call transfection reagents play a crucial role in the effectiveness of vaccines," says Hengel, who is the medical director of the Institute of Virology at the Freiburg University Medical Center and the deputy chairperson of the Scientific Advisory Board of the Paul-Ehrlich-Institut (see here the complete interview starting). "These reagents determine which cells the vaccine penetrates and how effective and stable it is."
The researchers have not yet determined which form of vaccination will have the best outcomes in the coronavirus pandemic. Scientific institutes, startups, and companies all over the world are working on a range of technologies. It is assumed that many different serums will be used. However, one thing is already obvious today: A vaccine is absolutely essential in order to block the virus.
FROM SMALLPOX VIA MEASLES TO THE CORONAVIRUS
Ever since Edward Jenner's pioneering work, some of the worst threats to human health have been mitigated or even eliminated through vaccination. That includes devastating diseases such as rabies, plague, diphtheria, and tuberculosis. For example, thanks to worldwide vaccination programs, smallpox has been considered eradicated since 1980. In 2002 the World Health Organization (WHO) declared that Europe was "polio-free," and a few weeks ago it extended that declaration to include the continent of Africa.
The WHO had also sought to eradicate the measles virus by 2020. Measles can lead to serious complications, including blindness or fatal meningitis, especially in children younger than five. About 2.6 million people died of measles worldwide every year before the first vaccination became available in 1964. After that, the figures rapidly decreased—until 2016. Unfortunately, since then the illness has once again been gaining ground. In 2019 the WHO sounded the alarm: The number of registered cases of measles had increased by 700 percent in Africa and by about 300 percent in Europe in a single year. Many people who live in poor regions have no access to vaccines, but the spread of measles in rich countries is primarily due to growing skepticism about vaccination. In reaction to these figures, mandatory measles vaccination was introduced in Germany in March 2020.
In the struggle against the novel coronavirus, there are no plans for mandatory vaccination, even though fears about such a mandate are being voiced by vaccination skeptics and believers in conspiracy theories. At the moment the focus is on the search for a suitable vaccine and on the associated hopes for ending the pandemic as soon as possible. "There's a lot of optimism," said Professor Klaus Cichutek, the president of the Paul-Ehrlich-Institut (PEI—the Federal Institute for Vaccines and Biomedicines), which is responsible for approving vaccines in Germany, back in August. The initial results of ongoing studies have shown "that some vaccines can actually induce a specific immune reaction in human beings against SARS-CoV-2," he said.
The new vaccine technologies are based on the classic processes that make use of the memory of our immune system. When it is invaded by a virus or a bacterium, the immune system reacts by forming antibodies. The information about these antibodies remains stored in special white blood cells. If there is a new infection by the same pathogen, these white blood cells can produce these antibodies very quickly and render the intruders harmless.
As a rule, a vaccination actively introduces pathogens into the human body in order to stimulate the immune system to form antibodies. This is usually done by means of an injection into a muscle or subcutaneously. We distinguish between various classes of vaccine. Live vaccines contain viruses or bacteria that have been attenuated to such an extent that they can still reproduce themselves but can no longer cause the disease. The protection afforded by a live vaccine lasts for many years. Examples include the live vaccines against measles, mumps, rubella, and chickenpox.
In inactivated or killed vaccines, the pathogen is first killed, so that it is unable to reproduce itself and cause the disease. In this case the protection gradually decreases and must therefore be regularly renewed with a booster shot. Vaccines of this type protect the recipients from polio, tick-borne encephalitis (TBE), and hepatitis B. Other vaccines, such as those against tetanus, diphtheria, whooping cough, and the flu, only contain components of the pathogen, such as proteins or sugars that are recognized by our immune system. These vaccines also protect recipients for only a limited period of time.
GENE-BASED VACCINES OFFER HOPE
The time to develop traditional vaccines can typically take several years as a general rule. For one thing, large amounts of virus material are required. For another, the large-scale production of a vaccine requires a lot of time and effort. "For example, in the case of inactivated vaccines the pathogens have to be precisely specified under strict safety conditions," explains PEI President Cichutek. "Next, the strain is produced, cultured in large amounts, and only then inactivated."
This is why the researchers who are searching for effective protection against the coronavirus as well as diseases such as AIDS and certain types of cancer have been focusing in recent years on completely new candidates: gene-based vaccines that contain not the virus itself but only a blueprint that the human body can use in order to produce exactly that part of the virus that triggers the immune response. For example, in the case of the coronavirus this part could be the "spike protein" on the virus envelope. Serums of this kind can be produced in large amounts relatively quickly, and if the pathogens should mutate the serums can be adapted as necessary.
Vector vaccines are a variant of these innovative vaccines. In vector vaccines, genetic material of the pathogen is inserted into harmless carrier viruses (such as the virus used for measles vaccine or attenuated adenoviruses). Initial vaccines against dengue fever and Ebola have already been approved. And Russia already dashed forward in August and produced a vector serum against Covid-19.
Scientists believe that additional opportunities are offered by vaccines based on nucleic acids, which are the carriers of genetic information inside cells in the form of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Because the production of DNA or RNA vaccines requires not the entire virus but only its genetic material, it is much easier and faster than the production of other types of vaccine. Dozens of studies are now being conducted in this area. However, by mid-October no vaccines against Covid-19 had been approved for use on human beings.
In the case of DNA vaccines, the sequence of the desired antigen is inserted into the genetic information of a bacterium. After the bacterium has entered the target cell, this information is read in the cell's nucleus and the antigen is produced directly inside the cell. Researchers have already been working on these vaccines for many years. Pharmaceutical companies are currently working on DNA vaccines against about 20 diseases including rabies, leukemia, and AIDS. So far, the possibility of foreign DNA being inserted into human genetic information, which in the worst case could lead to increased tumor formation, has not been documented in any studies. "We have spent many long decades investigating a theoretical risk that might be harbored by DNA vaccines, but in fact this risk has never materialized in animal testing or in clinical trials," said PEI President Cichutek at a press briefing in April 2020 in order to calm any fears.
In order to completely exclude this risk, there is the option to use not the entire DNA of a protein but only its mRNA, or messenger RNA. A protein's mRNA is basically a copy of its blueprint, which is read out from the DNA. The mRNA transports this blueprint directly to those places in the cell where the desired protein is produced. In other words, it is not incorporated into the cell's nucleus, and thus it cannot be inserted into the DNA there.
THE EPICENTER FOR LIPID FORMULATIONS
However, in order to unfold its effect the mRNA must first reach the right place inside the human body. "For a long time this was a big problem for scientists, because it is a very unstable construct," explains Stefan Randl. This is where the lipid nanoparticles from Evonik—ultrafine particles of fats and waxes—once again come into play. "If I were to inject mRNA without having previously formulated it—in other words, without having packaged it inside a protective layer—it would disintegrate in the bloodstream within seconds."
Evonik produces lipid nanoparticles and complete mRNA serums at a facility in the town of Burnaby near Vancouver, Canada. In 2016 Evonik expanded its portfolio for advanced drug delivery to include the development and production of liposomal formulation technologies by acquiring the local company Transferra Nanosciences. "Vancouver is an epicenter for the development and the production of LNPs," says Randl. Research on lipid nanoparticles has been conducted there for almost 30 years. "The researchers have already deistered via injection—against diseases such as cancer or amyloidosis, which is triggered by protein deposits in the body and can lead to organ dysfunction. This was the first application of an RNA-based therapy. "Certain combinations of active ingredients, as well as personalized medications, would also be unthinkable without LNPs," Randl adds. In the future, serums based on lipid nanoparticles could play an important role in the market for vaccines and many therapeutic drug products.
By means of its highly specialized and complex production processes for LNP-based medicines, Evonik develops formulations for pharmaceutical companies in Vancouver from start to finish. "For example, the customer sends us the mRNA, and we then conduct research in order to find out the proportions in which lipids must be mixed with other ingredients," says Jay Natarajan, the head of research in Burnaby. The tiny lipid particles have to protect the nucleic acids from destructive enzymes and thus enable them to pass through the cell membrane.
LAYERED LIKE AN ONION
"To make sure the mRNA safely reaches its target, the LNPs themselves have to consist of many different lipid and buffer components, so there's a long list of ingredients," explains Natarajan. The lipid ingredients are first dissolved using ethanol and combined with the mRNA, which has been dissolved in a buffer solution. This is done using a very rapid micromixing process that creates lipid nanoparticles encapsulating the mRNA just like the layers of an onion. These lipid nanoparticles are then subjected to a downstream purification process to form a final drug product that is ready for clinical trials on humans.
"The lipid nanoparticles have to be put together out of a large number of different components"
JAY NATARAJAN, HEAD OF LIPID RESEARCH AT EVONIK IN VANCOUVER, CANADA
When the particles reach the target cells, they fuse with the cell membrane and release the mRNA into the cell exactly where it is needed. There the information that is required to manufacture the desired protein is read out, and the production of the antigens begins.
As soon as the right formulation for the customer's mRNA has been determined in Burnaby, serums can be produced in amounts sufficient for reaching Phase I /II of clinical testing. Looking ahead, Evonik is planning future operations that go beyond this stage. The laboratories at the company's location in Birmingham, Alabama have the capability to produce larger batches. The company has already developed and produced drugs based on bioresorbable polymer microparticles.
Incidentally, the story of the little boy in England had a good ending. Not only did the vaccination make him immune to smallpox—to show his gratitude, the country doctor Edward Jenner later on gave him a cottage to live in with his family. Eventually, this house became the first Jenner Museum.
Inga Olfen is a science journalist based in Hamburg. She has a degree in biology and previously worked for eight years as an editor in the science department of Stern magazine. She founded the communications agency Kontenta in 2017.
Photos: Ollanski, Stefan Wildhirt/Evonik, Mohammed Munawar
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