by Sydney Sherman
figures by Daniel Utter

If you’ve ever received a vaccine or been prescribed a medication, then you have benefited from the contribution of animals to research. Humans have looked to animals to help combat diseases since at least 380 BC and continue to do so today. The race for COVID-19 treatments and preventatives is no exception. We usually think of animal research in terms of lab rodents. In particular, we think of mice being exposed to a contagion then treated with an experimental drug to see if it is effective. However, many other animals have proven their worth to science in a significant capacity.  This article will investigate the advances made possible by three animals you might not typically associate with a lab.

Alpaca

In May, news headlines reported that alpaca antibodies were being researched for their potential to combat COVID-19. The use of antibodies from camelids (alpacas, llamas, etc.) is not a new practice, and there’s a good reason why camelid antibodies are potentially more valuable than those from other animals.

Let’s start with a quick review on how the immune system uses antibodies to keep us healthy. Antibodies are made up of multiple proteins (called heavy-chain and light-chain proteins, named for their difference in size) linked together in a signature Y-shape formation, shown in Fig. 1. They are produced by B Cells, a type of white blood cell, and once produced continue to circulate in the bloodstream. The antigen/antibody binding process allows the immune system to mount the appropriate response to protect the body from harmful invaders. Antigens are features of pathogens that can be recognized by the immune system. Circulating antibodies detect antigens (viruses, bacteria, allergens, etc.) in the blood. They bind to the antigen, allowing the immune system to identify these pathogens, prompting an immune response and facilitating their removal from the body. When exposed to new antigens, like the novel virus that causes COVID-19 (SARS-CoV-2), the body must develop new antibodies that are capable of binding to a specific feature on the antigen. 

Camelids produce a second type of antibody called a nanobody.  Nanobodies are also Y-shaped groups of protein but are smaller than human antibodies because they lack light chain proteins. This difference in size allows nanobodies to bind to smaller features on antigens that larger antibodies are unable to, meaning they can bind to antigens that antibodies are less suited to protect against. Nanobodies are currently being tested in clinical trials as a potential therapeutic for Respiratory syncytial virus; this is just one among many pathogens that nanobodies might be more suited to protect against than antibodies. (It turns out that alpacas and their relatives aren’t alone in their ability to produce nanobodies: they are also found in sharks, but sharks are less feasible to work with for research purposes.)

Researchers have successfully demonstrated that nanobodies can bind to the spike proteins on the surface of COVID-19 and neutralize it.  Llama nanobodies are promising tools in the development of COVID-19 preventatives. Another property of nanobodies that make them more attractive for therapeutic use is their stability. In general, nanobodies can be stored for longer times and delivered to the body in a wider variety of ways. For example, they can withstand being sprayed as a mist and inhaled in a nebulizer, allowing delivery directly to the respiratory tract. Antibodies would break apart during aerosolization. 

Horseshoe Crab

Another animal hero of COVID-19 research is the horseshoe crab. Like the alpaca, the blood of the horseshoe crab has properties not found in humans and other animals. These prehistoric arthropods have a more primitive immune system than mammals. Their immunity to pathogens is derived from a type of blood cell called an amebocyte. When amebocytes encounter bacteria or toxins in circulation, they trigger a coagulation response. This response initiates the formation of a clot, or gelling of the blood, around the pathogen which prevents its spread and further harm to the body.

The clot formation caused by amebocytes is valuable for researchers because it provides an easily observable indicator of the presence of bacteria. The amebocyte protein that causes coagulation, the limulus amebocyte lysate (LAL), is extracted from harvested blood amebocytes. Concentrated LAL is then added to drug samples to ensure they are free of contamination. If a drug sample with LAL coagulates, the batch is known to be contaminated and can be discarded. Drugs being developed for COVID-19 treatments and prophylactics are no exception: their purity is guaranteed with the help of horseshoe crab blood extract. 

Although the blood extraction does not kill horseshoe crabs and they are released following the harvesting process, an estimated 50,000 still die annually as a result of biomedical blood harvesting. This is cause for serious concern: horseshoe crab populations are significantly declining, and they play an essential ecological role. 

Synthetic alternatives to both llama nanobodies and horseshoe crab LAL are being developed and utilized in drug development. The pharmaceutical company Eli Lilly is testing the use of a synthetic LAL produced by inserting horseshoe crab genes into other microorganisms. This eliminates the need for horseshoe blood harvesting. Testing has shown the synthetic alternative is equally as effective as naturally derived LAL, but further validation is needed before the alternative is widely adopted. 

Chicken

A third pandemic animal hero is the chicken. The virus causing COVID-19 is part of a genus with several coronaviruses affecting many different animals. Coronaviruses have been identified in animals for decades longer than they have in humans, which means that current researchers can look to the extensive work of veterinary virologists to gain insight into coronavirus epidemiology. One of the first coronaviruses described was isolated from a flock of chickens in the early 1930s. This avian coronavirus, called Infectious Bronchitis Virus (IBV), is highly contagious and causes tracheal lesions and respiratory symptoms in infected birds. In addition to the respiratory tract, IBV affects the kidneys and reproductive tracts. IBV leads to decreased egg production, increased mortality, and increased susceptibility to subsequent bacterial infection, and it has been isolated from chickens around the world.  

There are several lessons from the ongoing effort to combat IBV that can be applied to COVID-19. Although several vaccinations have been developed for IBV, none of them have had overwhelming success in protecting flocks from the virus. The primary struggle in preventing IBV infection is evolution, which results in variants of the virus, each with a slight difference preventing the effectiveness of a single vaccine. Furthermore, vaccine use has contributed to the increasing number of IBV strains. There are many types of vaccines, but the vaccines for IBV mainly use weakened viruses to induce protection. This type of vaccine has the potential to recombine with wild viruses, resulting in new variants. Additionally, partial immunization resulting from vaccination puts selection pressure on the virus to further adopt new mutations. 

Decades of study of animal coronaviruses has resulted in a body of research that formed a foundation for strategies currently being adopted to produce more effective preventatives and treatments. Research teams in Israel are currently developing a COVID-19 protein-based vaccine for oral delivery based on a novel IBV vaccine that they have been developing for over 4 years. As COVID-19 spread, researchers at MIGAL Research Institute, an Israeli research company, were able to switch from IBV vaccine development and apply their innovative work to COVID-19. Their methodology for vaccine development and efficacy testing was initially conducted on chickens. The vaccine is expected to begin testing in human clinical trials within the next few months. 

The use of animals and animal-derived products in research has allowed for thousands of medical advances and led to technologies used across the world daily. Countless lives have been saved as a direct result of their contribution. In Russia, the contribution of animals to research is recognized at Novosibirsk’s Institute of Cytology and Genetics in a statue of a lab mouse knitting DNA (Figure 3). Alpaca, horseshoe crabs, and chickens are far from the only creatures contributing to the mounting body of work to put an end to the COVID-19 pandemic. The world will need an army of animal statues to recognize the work made possible from their use in research.

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Sydney Sherman is a second-year Medical Engineering and Medical Physics Ph.D. student in the Harvard-MIT Health Science and Technology Program.

Daniel Utter is a 6^th year Ph.D. student in Organismic and Evolutionary Biology at Harvard.

Cover image: “Alpaca” By Diego Baravelli is licensed under CC BY-SA 4.0

For More Information:

• To learn about horseshoe crab blood and harvesting, watch this Ted-Ed video.
• For more information about regulation of animals in research, read this Speaking of Research overview
• To learn more about the advantages of using nanobodies, read this AlpaLife page.
• The Department of Health and Human Services has more information about types of vaccines