What Are AB5 Toxins?
Many bacteria produce toxins-harmful substances that can disrupt normal cellular functions and lead to disease symptoms. Bacteria have developed arsenals of all kinds of toxins over centuries. One common example, named for their structure, is the class of AB5 toxins. Cholera toxin, which causes the severe watery diarrhea associated with cholera, may be the most famous AB5 toxin. Another famous one, pertussis toxin, is produced by the bacterium that causes pertussis, or whooping cough. These toxins help create an environment in the host that facilitates bacterial survival and spread. But how do they work?
It's all about structure. AB5 toxins are made of an enzymatically active (A) subunit, and a binding (B5) subunit. The B5 subunit is made up of 5 proteins-monomers-arranged into a donut-shaped pentamer. The A subunit has a linking section that sits right in the "donut hole" and loosely attaches it to the "donut." At the other end, the A subunit has an active domain, which acts as an enzyme once it's inside a host cell. When all of the pieces are fully assembled, it's referred to as an AB5 holotoxin.
The B5 subunit is the key to the whole system. It binds to receptors on epithelial and mucosal cells, like the ones that line our respiratory and intestinal tracts. The cell then pulls the toxin into itself through endocytosis. Once the toxin is inside, the A subunit detaches and goes on to have some enzymatic effect on the host cell.
So, with our example of cholera toxin, its B5 subunit lets it "in the door" of intestinal cells, then its A subunit goes on to cause cholera's grossest symptom- watery diarrhea-by opening up cellular ion channels; as ions flood out, water follows. Pertussis toxin works the same way to get "in the door," but it acts in the lungs and on a different cellular pathway, ultimately leading to the whooping cough pertussis is known for.
How Can AB5 Toxins Be Used in Vaccines?
Some scientists are working on repurposing the B5 subunit of AB5 bacterial toxins as a delivery platform to taxi or ferry vaccine antigens into the body. Using genetic engineering techniques, scientists can remove the toxic A subunit and replace it with some other bacterial or viral antigens, with the goal of generating an immune response that will protect the host upon re-exposure to that particular antigen.
In theory, the replacement antigen could be any antigen at all-a SARS-CoV-2 spike protein or an influenza hemagglutinin protein, for example. In practice, however, swapping in a new antigen for the A subunit is a bit more difficult. The biochemistry has to be just right for that newly introduced protein subunit (antigen) to fold properly and sit happily in the "donut hole" of the B subunit, though some progress has been made.
In the end, AB5 toxin-based vaccines are just proteins like many other vaccines. But, they offer a new and exciting option for the delivery route. Regardless of the antigen, the idea is to harness the action of the B5 subunit and use it to deliver the antigen of choice directly to skin or mucosal membranes, where many immune cells await stimulation. That means no needles! A vaccine could be applied as a skin patch, as a smear across a nasal mucus membrane or stabilized in a sugar capsule to be swallowed. Scientists think AB5 toxin-based vaccines could stimulate a robust immune response by targeting the immune system at skin or mucosal barriers (e.g., the linings of our gastrointestinal and respiratory tracts), which protects against pathogens that invade the body through these barriers (think influenza virus), as well as the systemic immune system, which is key for attacking pathogens once they bypass those barriers.
Indeed, both barrier and systemic immune responses start at the skin or mucosal membrane. Immune cells called antigen presenting cells, or APCs, live in these barrier regions of our bodies. An APC's job is to recognize a foreign invader or a vaccine. The APC takes up the vaccine antigen and physically transports it to the lymph node, where it introduces the antigen to naive T and B cells. These cells are then activated and exit the lymph node, surveilling for pathogens that look like the vaccine. Some cells return to the site of vaccination (i.e., the mucosal membrane or skin), and some travel the body systemically, searching for the invader they've been trained to recognize.
Our lab is trying to harness the AB5 toxin-based vaccine system in our own research. We've engineered a vaccine intended to protect dairy cows from mastitis, a severe inflammation of the udder that is quite painful for cows and ruins their milk, costing farmers millions in economic losses every year. Because the vaccine is based on cholera toxin, we can administer it without needles by swiping it into nasal passages or applying it directly into the udders through the teat. In recent and ongoing studies, cows that we've vaccinated have shown a positive immune response to the vaccine, and so far, it has an excellent safety profile. Of course, as always with science, we need to do a lot more work to make sure the vaccine is safe and works the way we want it to.
Where Are AB5 Toxin-Based Vaccines Today?
While there are currently no vaccines approved by the U.S. Food and Drug Administration (FDA) that use the strategy of replacing the A subunit of AB5 toxins with an "antigen of choice," there are existing vaccines that harness these toxins in other ways.
For example, because the B5 subunit of cholera toxin binds receptors in the gut, it can be administered orally. CTB-the B5 subunit of cholera toxin without the A subunit-is itself an ingredient in the oral cholera vaccine Dukoral, which is approved by the World Health Organization (WHO) and administered around the world, although it is not available in the U.S. One research group has gone so far as to put CTB in rice for a more accessible and transportable vaccine. Unfortunately, research has revealed that cooking the rice ruins the vaccine, and raw rice is not very appetizing or consumable. In ongoing human trials, researchers grind up the rice and mix it with corn starch and water, then volunteer subjects drink the rice vaccine slurry. While this delivery method is not ideal, and there's more work to be done, some people might still find it more appealing than getting an injection.
Detoxified versions of pertussis toxin and diphtheria toxin are ingredients in FDA-approved pediatric DTaP vaccines (Daptacel, Pentacel, Quadracel, Infanrix, Kinrix, Pediarix, Vaxelis) and Tdap vaccines (Adacel, Boostrix). The toxins used in these vaccines are treated with chemicals to inactivate the A subunits.
In 2016, Thailand licensed 2 pertussis vaccines (Pertagen, Boostagen) that use a genetically detoxified version of pertussis toxin. Its amino acids are modified so that the A subunit is non-toxic.
Different versions of the AB5 toxin called heat-labile enterotoxin, or LT, are also being tested in vaccines against Enterotoxigenic Escherichia coli, which produces LT and is a leading cause of travelers' diarrhea.
What Else Can the AB5 Taxi Do?
Vaccine scientists aren't the only ones repurposing bacterial toxins. In fact, CTB in particular is being used or investigated for a variety of medical applications.
Some labs have employed the CTB taxi service to, for example, reduce allergic responses to peanuts in mice. If an allergen is conjugated to CTB, the CTB taxi service can quickly deliver the allergen to the immune system. This way, the body can be trained to see those allergens as friends, not foes, which leads to a reduction in allergic response. These are the same basic principles as traditional allergy shots. The difference is that the use of CTB directly targets APCs, which are instrumental in triggering an immune response. Because of this cellular specificity, only a small dose of allergen is needed-it reaches the correct cells right away.
In contrast, traditional allergy shots require a large dose of allergen to ensure a small amount reaches APCs and stimulates an immune response. This difference is one reason why researchers hypothesize the CTB taxi service could be a safer route of administration. Scientists have also used the CTB taxi service to intentionally cause allergic responses in mice in order to understand mechanisms underlying development of allergies, including those to milk or egg whites.
In addition to allergies, researchers have used the CTB taxi to treat autoimmune disorders. In hemophilia, for example, a patient's immune system attacks their own blood clotting factors. One group harnessed the CTB taxi service to induce tolerance to coagulation factors in rat and canine models and is hoping to translate this work to humans soon. Notably, the taxi is actually produced and delivered in the cells of lettuce. When the lettuce is eaten, CTB is transferred to the gut, where it goes through the natural CTB process: CTB binds mucosal receptors on intestinal cells, then those cells pull the taxi and its "passenger" (in this case, a coagulation factor) in through endocytosis. Inside the gut cells, the coagulation factors are cleaved and enzymatically separated from CTB. The coagulation factors then make their way into the circulatory system. The end result is delivery the coagulation factors to the patient and treatment of their hemophilia. What a motivation to eat your greens!
Still others are investigating CTB's potential to help the immune system target cancer cells. If the CTB taxi service is carrying a prostate cancer marker, for example, then the immune system can be trained to recognize prostate cancer cells. The idea is that the immune system would clear out the cancer, reducing the need for chemotherapy or radiation.
Beyond acting as a taxi service, CTB's powers can be used to cross nearly impenetrable barriers, like the blood-brain barrier. CTB binds to GM1, a surface receptor on many cell types, including neurons. The GM1 receptors allow the CTB taxi to carry its cargo right into the central nervous system like a molecular express lane pass. Scientists have used CTB attached to various fluorophores as a neuronal tracer to help study complex neuronal connections since the 1970s. More modern work is looking at attaching better tracers, such as carbon dots. Some scientists worry about CTB crossing the blood-brain barrier. Delivering neuronal tracers might be okay, but what if a vaccine makes its way up there? Is it safe? So far, the answer appears to be "yes," but researchers are actively looking into this.
Plenty of Potential
While cholera toxin is probably the most well-researched, it's clear that AB5 toxins could have multiple therapeutic uses. Harnessing their extensive immunomodulatory properties could change the vaccine game. Someday, getting vaccinated could be as easy as eating a salad-or even a piece of candy.
For more insights into how bacterial toxins are being used to advance health and prevent disease, check out this next article.
