Poison and Antidote - antivenom VHH production

By Alwin Schönberger - profil Nachrichtenmagazin

More than five million people are bitten by venomous snakes every year, and at least 100,000 die. Researchers in Vienna have developed a new method to save lives. How the technology works, and why it could also combat other diseases.

“There are seven steps until death,” says a proverb in East Africa. According to legend, after being bitten by a black mamba, a person can take exactly seven steps before collapsing lifeless to the ground. This is greatly exaggerated, but in fact the snake is one of the most dangerous in the world: a single bite releases enough venom to kill 40 people.

Hundreds of other snake species can inject venom with their fangs or spit it at their victims. They paralyze the nervous system or muscles, attack the cardiovascular system, or destroy cellular tissue. Roughly every five minutes, somewhere on the planet, a person dies from a venomous snakebite. At least 100,000 people die each year from snakebites, and more than four times as many suffer amputations of limbs. More than five million people per year are attacked by snakes.

Only in April, a 57-year-old German man died in Egypt after being bitten during the performance of a snake charmer. The snake, presumably a cobra, had previously crawled into the tourist’s trousers.

The situation is especially critical, however, for local populations in regions of the world where venomous snakes are common, medical care is poor, and money is scarce: in sub-Saharan Africa. Particularly with regard to these regions, the World Health Organization (WHO) has classified snakebites among the most neglected tropical diseases.

“We are working on a passive immunization that neutralizes snake venoms.”— Jürgen Mairhofer, CEO and biotechnologist

“People often do not even know which snake bit them because it disappears into the bushes,” says Jürgen Mairhofer, biotechnologist and CEO of the internationally active company enGenes Biotech GmbH, which develops technologies for the microbial production of biological active substances. Together with Danish partners, Mairhofer is researching a completely new generation of antivenoms, immune sera against snake venoms, commonly referred to as antidotes. The researchers recently described their technology in the renowned scientific journal Trends in Biotechnology.

Their most important equipment stands on laboratory benches at enGenes in a wing of the University of Natural Resources and Life Sciences (BOKU) in Vienna’s 19th district, where university spin-offs are based: fermenters, bioreactors, chromatographs. The laboratory technology serves a single purpose: inside bacteria, protein fragments mature into antibodies against snake venoms. “It is a passive immunization that neutralizes snake venoms,” says Mairhofer. The central objective is to develop a standardized production of antibodies with homogeneous quality and a broad spectrum of activity, clean, fast, efficient, and comparatively inexpensive.

Milking Snakes

Traditionally, antidotes are produced in a surprisingly antiquated way, hardly any different from more than a century ago, when the principle was discovered. In 1894, the French physician Albert Calmette devised a method for treating snakebite victims. It was based on the idea that cobras are immune to their own venom. Therefore, Calmette reasoned, one should attempt to strengthen the human immune system against toxins.

In a modified form, Calmette’s method is still used today: small doses of venom are injected into large mammals such as horses or sheep. The venom is obtained by “milking” snakes, forcing them to empty their venom glands into a container. After the venom is administered, horses develop antibodies against the snake toxin: proteins that bind to venom molecules and neutralize their effects, for example on nerve cells. The antibodies matured in the blood serum of horses or sheep are harvested and, when needed, injected into the bloodstream of bite victims.

The method basically works, but it has considerable disadvantages. First, the composition of the antivenom can vary greatly depending on the venom cocktail of the particular snake and the organism of the horses whose serum was used. Second, the side effects can be substantial. After all, the therapy essentially amounts to a blood transfusion from horse to human. Furthermore, such methods can usually only produce a remedy against one specific type of venom or even the toxin of a single snake. Yet cobras alone possess completely different venoms: that of the Cape cobra targets the brain and nervous system, while the biochemical weapons of the spitting cobra are cytotoxic and attack cellular tissue.

And finally, people in the regions most affected, especially in sub-Saharan Africa, must first be able to afford the treatment, which can cost around 600 dollars per dose, assuming it is available at all. Barely 50 laboratories worldwide specialize in the production of antivenoms.

Genetic Engineering Instead of Horses

What is currently being developed in the Viennese research facilities follows a completely different approach: modern molecular biology instead of extraction from animal organisms.

Why are scientists in Central Europe focusing on novel antivenoms? The reason is that the team around Jürgen Mairhofer possesses a patented technology for recombinantly cultivating proteins , producing them specifically through genetic engineering methods, and that a Danish pioneer in the field of innovative antivenoms considers the Viennese method best suited for producing his antibody variants.

Andreas Hougaard Laustsen-Kiel is a professor of molecular biology at the Technical University of Denmark (DTU) and knows the cruel consequences of snakebites firsthand. Fifteen years ago in Tanzania, he saw children whose arms or legs had to be amputated at the elbow or knee in order to save their lives.

As a scientist, Laustsen-Kiel focused on researching modern antidotes: molecular-biologically produced fragments of antibodies known as nanobodies. These small protein fragments are more stable than conventional antibodies and bind precisely to various toxins, allowing them to be neutralized. The professor’s research group developed such nanobodies against 18 medically relevant venomous snakes in Africa and then faced the question of how these could be produced economically while maintaining consistently high quality.

At this point, the Viennese researchers entered the picture with their method. “Andreas approached us, and it was an easy decision,” says Mairhofer, who participated without compensation or funding. “Scientifically, it is a perfect application for our platform, and at the same time it is about saving human lives.”

The basic principle of the Viennese technology: bacteria serve as incubators, breeding grounds for the nanobodies researched in Denmark. Specifically, the bacteria belong to the species Escherichia coli, into which the researchers introduce the genetic blueprint of the nanobody proteins. The genetic sequence is first transcribed into messenger ribonucleic acid, mRNA, whose function became widely known during the Covid vaccinations. mRNA is a molecular message that instructs body cells to produce certain proteins, in this case antibody fragments for immunization against snake venoms.

Bacteria as Unwilling Helpers

Inside the E. coli bacteria, the blueprint is translated into proteins during growth. At a certain point, the cell division of the bacteria must be stopped. For this purpose, Mairhofer’s researchers use bacteriophages, special viruses that attack bacteria and are also considered an alternative to classical antibiotics. “The phages take control of the bacteria and make them, so to speak, devoid of will,” says Mairhofer. This allows the gene sequence of the nanobodies inserted using mRNA technology to mature into the desired antibody proteins.

Mairhofer points to a glass vessel from which metal rods, tubes, and hoses protrude. This is the bioreactor in which the entire process takes place. It contains mainly water and sugar — the most important fuel for bacteria. Within 48 hours, the entire process is completed. From one liter of bacterial culture, ten to twenty grams of purified antibodies can then be extracted using a method that exploits the affinity of proteins to metal ions.

The clever part of the procedure is its efficiency: six different antibodies can be cultivated simultaneously in the bioreactor. “We produce six lines quasi in one pot,” says Mairhofer. These six antibodies cover the most important venom groups of African and partly Asian snakes, including those of mamba and cobra species, explains Anne Ljungars, biotechnologist at the Technical University of Denmark. “It is not about individual snake species, but about broad neutralization of entire toxin families that occur in many snake species,” says Ljungars. Specifically, the six antibody lines target four toxin families that primarily include neurotoxins and cytotoxins.

The rapid “one-pot” technology also raises hopes that the novel treatments can be offered relatively cheaply. With sufficiently large production volumes, a dose could cost less than 50 dollars, barely one tenth of some current preparations. The principle of stable nanobodies offers yet another advantage: they are likely to have a long shelf life, which is particularly important in hot regions of the world where transportation routes for medical goods are often lengthy.

A New Principle in Medicine

However, there are not yet any finished medicines, only the results of experimental studies involving the new antidotes, according to Ljungars. These results are promising, as detailed in the Trends in Biotechnology article. After initial successful tests in mice, the next step is to demonstrate the effectiveness of the molecular-biologically designed antibodies in larger mammals, and then in humans. Ultimately, says Mairhofer, the goal is to show that “the production can be scaled up”: that active substances can also grow in bioreactors with capacities of 100 liters while maintaining consistent quality. Only then could the necessary quantities of antidote be produced at acceptable prices.

If everything works as hoped, the researchers’ principle could also be transferred to other fields of medicine: recombinant proteins matured in bacteria could be suitable for many immunotherapies, for combating cancer, infectious diseases, or allergies. It would be yet another example of how genetic engineering can bring about a paradigm shift in medicine.