Vaccines’ Little Helpers

The news was so exciting that New York City’s mayor decided to spill the details on his radio show in late 1944. A research team led by Jules Freund, then an immunologist at the city’s Public Health Research Institute, had developed the first malaria vaccine that worked in lab animals. The achievement thrilled the mayor because millions of Allied soldiers were stationed in parts of the world where malaria was prevalent—more than 60 percent of U.S. soldiers and sailors in the Pacific Theater came down with the illness. The hope was that “the discovery may lead to a vaccine which will lick World War II’s No. 1 disease,” as Time magazine put it.

The mayor’s enthusiasm was premature. Freund’s animal findings did not lead to a malaria vaccine that licked the disease. The results were significant in another way, however, because a key ingredient in his vaccine made a huge impact on science over the next several decades. That component, now known as Freund’s adjuvant, is a mixture of mineral oil and water spiked with dead tuberculosis (TB) bacteria. Adjuvants are immune system boosters, and Freund’s is one of the most potent ever developed.

Adjuvants are often added to vaccines to increase their effectiveness, but Freund’s formulation proved more important for research. The mixture “has been an incredibly valuable tool to study the immune system,” says microbiologist Marcel Behr of McGill University. Scientists used the adjuvant to discover mechanisms that protect us from pathogens, including a new branch of the immune system. Freund’s innovation also allowed researchers to create animal models of multiple sclerosis (MS) and arthritis that were vital for deciphering what goes wrong in those illnesses and for developing and testing treatments. Even today, “it’s the ‘gold standard’ adjuvant” for work on those diseases, says immunologist Jay Reddy of the University of Nebraska–Lincoln.

The adjuvant played such a large role in immunology that it earned Freund the 1959 Albert Lasker Basic Medical Research Award. Despite having never appeared in a commercial human vaccine because of its side effects, his original mixture has served as a model for scientists hoping to create safer but still powerful alternatives. That research has yielded five new adjuvants in the United States in the last couple of decades, and scientists are developing a variety of others. Freund’s adjuvant remains the benchmark “for what you can achieve in terms of an immune response,” says immunologist Harm HogenEsch of Purdue University, who is testing a new adjuvant for veterinary vaccines. “It’s what we are aiming for.”

Search for the Magic Ingredient
Freund was born in 1890 in Budapest, which was then in the Austro-Hungarian Empire. He studied medicine at the Royal Hungarian University and later served as a commissioner for hygiene for the Austro-Hungarian army during World War I. After the war, Freund taught preventive medicine at his alma mater and in 1922 moved to the United States, where he established himself as a leading immunologist and vaccinologist. His work stood out, according to his obituary, because it “was deliberate and methodical, with exploration in depth set above elaboration in breadth.”

In the 1930s, Freund began to explore in depth how to make a better TB vaccine. The only option then—and still the only TB vaccine available—was the Bacillus Calmette–Guérin (BCG) vaccine, which contains living but weakened cattle bacteria closely related to those that cause TB in humans. Worried about the safety of injecting live microorganisms into patients, researchers had been testing whether they could substitute dead TB bacteria for the cattle bacteria. The results had been disappointing. In experiments with rabbits, however, Freund and a colleague discovered that heat-killed TB bacteria worked almost as well as the BCG vaccine.

Hoping to increase the protective power of the approach, Freund turned to adjuvants, which French immunologist Gaston Ramon had discovered in the 1920s. While studying antibody production in horses, Ramon had noticed that substances that caused local inflammation increased the ensuing immune system reaction to immunization. The catch was that “at the time, people didn’t know how to activate immune responses,” says immunologist Ed Lavelle of Trinity College Dublin. So Ramon tried a seemingly random assortment of candidates, including bread crumbs, milk, pus, rubber, and petroleum jelly. Tapioca was particularly effective, he found.

But for his adjuvant, Freund chose a type of oil. His decision drew on the work of other researchers, including German bacteriologist Lydia Rabinowitsch-Kempner, who had shown that butter could amplify immune responses to certain microbes. In the vaccine field, “the idea of an oil for immune stimulation was there,” Lavelle says. Freund and colleagues mingled heat-killed TB bacteria with mineral oil and injected the combination into guinea pigs and rabbits. The scientists reported in 1937 that the animals produced more antibodies than did their counterparts receiving the BCG vaccine, a sign that the mixture provoked a stronger response from the immune system. Freund and his team continued to refine the adjuvant and found that it worked better if they added a wax used in skin cream or other chemicals to stabilize the mixture. By the 1940s, they were performing animal tests and hoping to move on to human trials.

Second Life of Freund’s Adjuvant
Freund’s adjuvant proved too powerful for human vaccines, however. It can cause pain, severe inflammation, abscesses, tissue destruction, and other problems. Take the case of a lab technician who accidentally injected her hand with a small amount of the adjuvant. As a 2018 paper recounts, she required “a prolonged course of treatment” that lasted about a year and included two surgeries to cut away dead tissue. To tone down the mixture, researchers have omitted the TB bacteria, creating what’s known as incomplete Freund’s adjuvant (the original variety is now called complete Freund’s adjuvant). The milder version was incorporated into a few human vaccines in the 1950s and 1960s, but its side effects were still too severe for widespread use in people, limiting its applications to research and veterinary vaccines.

Researchers quickly discovered that Freund’s adjuvant could help them delve into the workings of the immune system. Two of the first scientists to harness it were immunologists Karl Landsteiner and Merrill Chase of the Rockefeller Institute. Landsteiner is best known for discovering the blood groups, a finding that earned him the 1930 Nobel Prize in physiology or medicine and the 1946 Albert Lasker Clinical Medical Research Award. In the early 1940s, the pair investigated skin reactions driven by the immune response to chemical exposure. The standard view was that antibodies were behind most of the immune system’s responses. However, Chase and Landsteiner’s experiments on guinea pigs implicated the actions of cells, not antibodies.

The duo had uncovered another branch of the immune system, unveiling what scientists now term cell-mediated immunity. “It’s a key mechanism,” Behr says. That defensive line is particularly important for fighting certain pathogens. And when it fails, such as in AIDS, patients become vulnerable to cancers and infections that the immune system normally defeats. Most vaccines also enlist cell-mediated immunity, and some, such as the yellow fever vaccine, induce a very strong cellular response.

Freund pioneered another research use for his adjuvant: probing autoimmune diseases, in which the immune system attacks the body’s own tissues. He and colleagues showed that adding fragments of tissue from the nervous system to his adjuvant and then injecting the mixture into rodents could spark a condition now known as experimental autoimmune encephalitis (EAE) that resembles MS. EAE remains the standard animal model for studying MS, and most researchers still rely on Freund’s adjuvant to trigger it. The adjuvant also is a key tool for probing other autoimmune illnesses. Reddy, who has been studying a type of heart inflammation, says that the adjuvant is indispensable in his lab.

“If complete Freund’s adjuvant is not there, we cannot investigate anything.”

Freund developed his eponymous adjuvant through trial and error. He didn’t know how it worked. Researchers have since learned that it’s “complex and dirty,” Behr says, containing components that trigger a range of effects that scientists still don’t completely understand. Freund’s animal experiments, for example, suggested that mineral oil alone yielded no benefit. But scientists have since discovered that the oil gives the immune system a kick in the pants, though the mechanism remains unclear. “Do we know the full story of how oils induce immune responses? I’d say we have a lot of information about how they cause inflammation but don’t fully understand why they are so effective at enhancing antibody responses,” Lavelle says.

Scientists have discovered other ways in which the adjuvant affects the immune system. Complete Freund’s adjuvant reshapes the metabolism of immune cells, as Reddy, his graduate student Kiruthiga Mone, and colleagues reported in 2025. And researchers have determined that the dead bacteria in complete Freund’s adjuvant stimulate pathogen-detecting molecules on innate immune cells, the body’s first line of defense against microbes. Although innate immune cells were known in Freund’s day, scientists didn’t understand how important that branch of the immune system was for mounting counterattacks against invading microbes. “The revolution in innate immunity gave the first intellectual foundations for how adjuvants might work,” says immunologist Bali Pulendran of Stanford University.

Adjuvant Research’s Rebirth
Even though Freund’s adjuvant proved unsuitable for human vaccines, research into other approaches languished for decades. Scientists—and drug companies—didn’t rush to develop alternatives because the aluminum-containing adjuvants already in use were safe and worked pretty well. In the United States, those compounds were the only adjuvants included in vaccines for more than 70 years, and they remain ingredients in the shots that protect against diphtheria, tetanus, whooping cough, hepatitis B, and other diseases.

In the last couple of decades, however, “there has been a revival in the adjuvant field,” Pulendran says. Some new adjuvants that have reached the market borrowed tricks from Freund’s formulation, including using oil or related compounds. MF59, an adjuvant in some flu vaccines, contains the oil squalene, for instance. And like Freund’s adjuvant, the AS01 adjuvant in the Shingrix vaccine for shingles targets pathogen receptors on innate immune cells. Because the immune system weakens with age, vaccines usually don’t work as well in the elderly. But thanks in part to AS01, Shingrix “gives fantastic protection for an older population,” Lavelle says.

Adjuvants such as MF59 and AS01 could be just the beginning. “Now, we are seeing there’s a lot of interest in developing additional adjuvants as well,” Pulendran says. For example, HogenEsch and colleagues are working to create a contraceptive vaccine for animals that contains a new adjuvant. An existing vaccine that incorporates proteins from a layer of material around the egg is one approach for curbing populations of wild horses, deer, and even elephants. That vaccine induces antibodies that glom onto the egg and prevent sperm from attaching, disrupting fertilization. But the vaccine contains a modified version of Freund’s adjuvant, which makes it inconvenient to administer, HogenEsch says. By contrast, the adjuvant in the new vaccine that he and his team are testing consists of tiny particles, known as nanoparticles, manufactured from corn. They deliver immune-stimulating proteins directly to cells. The adjuvant should make the vaccine simpler to deploy, and the researchers are now assessing it in the field, HogenEsch says.

Scientists say that they can deliver a new generation of adjuvants. “We have a better understanding of the immune system,” HogenEsch says. And powerful new techniques for comprehensively monitoring immune responses to vaccines in people “are revolutionizing the field,” Pulendran says. But adjuvant research and use face new obstacles: challenges from some U.S. government officials and scientists about the safety of aluminum-containing vaccines. Those concerns, Lavelle says, are not supported by “almost 100 years” of experience, during which “hundreds of millions of people” have received adjuvant-containing vaccines.

Even with the advances in adjuvant technology, Freund’s original mixture is holding on to its place in the lab, Reddy says. His group tested whether some of the new adjuvants for human vaccines could induce EAE in animals. Freund’s adjuvant was superior, he says. “Nothing can beat it in terms of immune-stimulating properties.”

By Mitchell Leslie