Microbial Solutions
Norman Wainwright

COVID, Horseshoe Crabs and Serendipitous Science

The blue blood of Limulus polyphemus has been invaluable during the pandemic 

It may be a bit ironic that the horseshoe crab, Limulus polyphemus , whose blood is the source for making the LAL test for bacterial endotoxin detection, cannot benefit from the very COVID-19 vaccines and drugs that rely on the LAL test to make sure those vaccines are safe for patients. In fact, the LAL test has been keeping all injectable pharmaceuticals and medical devices safe from microbial contamination for decades, saving countless lives that bacterial endotoxin might otherwise have caused. The reason for the irony is that horseshoes do not have antibodies as part of their primitive immune systems. In fact, all invertebrates – animals without backbones – share this point. So, while the vaccines are designed to elicit a specific antibody production in the human recipient to attack the COVID-19 virus, they would do the horseshoe crab no good at all.

It is widely known that horseshoe crabs are a primitive species, surviving millions of years relatively unchanged in appearance. Indeed, fossils over 400 million years old look almost identical to modern horseshoe crabs. The very primitive immune systems that are utilized in the LAL test may well have played a part in their longevity. While they do not have antibodies to combat microbial infections, they do have a very rapid and non-specific protective response whenBlue blood of horseshoe crab w/lab worker bacteria enter their blood system—for example, if their shell was injured, allowing bacteria in seawater to enter. Horseshoe crabs have a single type of white blood cell that is activated when it comes into contact with the cell wall of gram-negative bacteria. Endotoxin molecules reside in the cell wall, and when endotoxin binds to some very unique enzymes, they become activated and cause the blood to clot, sealing any wounds and entrapping the bacteria in the clot. What is so special about these enzymes that become activated is that they act in concert to magnify the response to the bacterial trigger. In other words, the first enzyme acts on the second, and the second acts on the third enzyme. Each enzyme magnifies the response about 10-fold, much like a chain letter magnifies its numbers as the chain grows. The result is that within minutes of contact with the bacterial cell wall—and its endotoxin molecules—the clot has formed, limiting infection and presumably improving the horseshoe crab’s survival.

The discovery of this clotting mechanism was one of those moments of serendipity in science. Dr. Frederick Bang, a medical researcher from Johns Hopkins University, working at his summer lab at the Marine Biological Laboratory in Woods Hole, Massachusetts was studying bacterial infection models using marine animals. When horseshoe crabs were injected with a bacterial suspension, he noticed their blood clotted very quickly, making their use for his research a problem. Rather than ignoring the result and moving on using other animals, Dr. Bang became curious about the cause of this clotting, and that led to discovering that endotoxin was the culprit. Soon, another medical researcher, Dr. Jack Levin, formulated a preparation of the blood cell enzymes as a very sensitive test for the presence of bacterial endotoxin, and in humans, endotoxin was one of the major triggers causing fever, and in high enough quantities, death by septic shock. This was the birth of the LAL test: L for Limulus, the scientific name of the crab, A for amoebocytes, the source of the enzymes; and L for lysate, a process to extract the enzymes from the cells. Eventually, the FDA approved the use of the LAL test to assure that pharmaceuticals and medical devices coming into contact with human blood were free of dangerous levels of endotoxin, and today LAL is recognized as the gold standard for measuring endotoxin.

Horseshoe crab population concern

Back to COVID-19. The realization that Operation Warp Speed put multiple candidate vaccines and drugs into the development pipeline at multiple pharmaceutical companies, and that potentially billions of doses would be needed worldwide, began to raise questions, mostly from conservationists, activists, and journalists, over whether this increase in demand would put pressure on the horseshoe crab populations, jeopardizing both the crabs and humans relying on Gathering horseshoe crabs in an ocean areathe new vaccines. LAL manufacturers jointly put those concerns to rest, knowing that billions of doses of drugs don’t require billions of LAL tests; rather, large batches of products, each containing perhaps millions of doses, are qualified by a relatively small number of LAL tests during quality control. This is not unlike the many thousands of other pharmaceutical products that routinely get similarly tested.

However, that is not to say the concern for horseshoe crab populations is unwarranted. In the 19th and 20th centuries, horseshoe crab populations have been horribly overharvested for use as fertilizer or bait. Modern population growth along key waterways, particularly in Delaware Bay and New England, have encroached and destroyed on their habitats.

Collecting the crabs for blood donation for LAL production does not kill the crabs. They are returned to their natural habitat within the same a day, even within hours. The Atlantic Marine Fisheries routinely do population counts along the east coast and have deemed the biomedical harvesting for LAL production to not be a significant factor.

Alternative recombinant sources for endotoxin tests

The cost / benefit controversy about horseshoe crab collection will likely go on for some time. Public awareness that there may be a threat to this beloved species is enough to bring up the question of whether we can find an alternative synthetic source of the horseshoe crab enzymes employed in LAL. The introduction of recombinant DNA technology in the 1980’s brought new hope that cloning the genes for these enzymes could produce a replacement for the natural LAL. Indeed, scientists in Singapore were among the first to clone the first enzyme in the LAL cascade, named Factor C. Several versions of this enzyme have been formulated into tests by different research groups to quantify endotoxin and have been offered to research and pharmaceutical markets. There is controversy around this point, though. Could a single enzyme perform equally to the enzyme cascade that amplifies the endotoxin trigger in natural LAL? LAL has been validated and tested with many different bacterial species of endotoxin over the last 50 years, with an unrivaled safety record of protecting the drug supply. While some are ready to make the switch, others are being more cautious, and want to make sure that there would never be a case where the synthetic enzyme(s) might miss a critical source of bacterial contamination.

As gene cloning technology has improved, it is now possible to clone all three enzymes in the LAL cascade, and several LAL manufacturers are pursuing this path to create an LAL that matches, or exceeds, the performance of the original natural LAL. The day may be here soon when validation of this new technology is complete, and we can rely on it as we have natural LAL for all these years. Hopefully, the FDA will certify and regulate this, assuring the public that the synthetic LAL is safe. Where will that leave the horseshoe crab?

Regardless of the industry’s efforts to ease the burden on the horseshoe crab populations, thereOcean marsh at sunset is good reason to support conservation efforts. While many states have reduced or stopped the taking of horseshoe crabs for bait, habitat loss is an issue that will likely not improve soon, especially in densely populated New England. While we have been most concerned about our native American horseshoe crab, Asian species have continued suffering losses to their populations with little to no regulation until very recently. So, the need for innovative ideas to address this is very real indeed.

In 2012, Rutgers University began the horseshoe crab enhancement project to collect fertilized eggs from the shores of Delaware Bay, bringing them into the aquaculture lab, allowing them to hatch and grow to size where they would better survive in the wild, then releasing them back into local waters. In 2018, Charles River Laboratories joined to support that effort with the intention of duplicating the technology up and down the east coast as needed. Mystic Aquarium became the next partner to join, adding a state-of-the-art aquaculture lab to their campus, and lots of attention of local Citizen Scientists to get involved. While this endeavor is still in its beginning stages, it has the potential to grow into a positive impact on threatened populations, both in North America as well as Asia.

The future: Aquaria, other marine species, serendipity, and discovery

Horseshoe crabs are so loved by many that it is unlikely the conservation efforts will be in vain. Whether natural or synthetic, the crabs have contributed to our ability to combat COVID-19, as well as future challenges to our health, as they have done for many decades assuring the new drugs, we develop are safe for injection into humans. When you get your COVID-19 vaccine injection, give a little thanks to the horseshoe crab.

Our thoughts for the future should also include thousands of other species just waiting for future scientists to discover interesting applications that could have equally impressive benefits. Public aquaria, like Mystic and South Carolina, while providing entertainment, also serve as safe havens or repositories of marine species that are or could become threatened. Many of the marine mammals, for instance, are rescued as injured or sick, and would have a difficult time competing and surviving back in the wild. It also brings the public in contact with species they may have never come upon personally. Being exposed to the fantastic diversity of both vertebrate and invertebrate species as a child, shrieking with delight at seeing a horseshoe crab crawling towards their toes on the beach, could return benefits many years later, influencing how they think about nature, or careers in the arts, science, engineering, or teaching.