Working as a medical intern in his home country of India, Abhay Satoskar had seen many cases like the one in front of him. A young child had been rushed into the emergency room, having collapsed while playing on the playground. When the doctors found that the youth’s spleen had ruptured, there was very little they could do.
Satoskar and his fellow physicians soon discovered that the child had been suffering from visceral leishmaniasis, a deadly disease caused by the parasite Leishmania donovani, which is transmitted to humans via the bite of a blood-feeding sandfly.
“This was something preventable if you had the right interventions around,” said Satoskar, now an immunologist at Ohio State University. Drugs for leishmaniasis, however, can be toxic and are losing effectiveness due to increasing resistance. The real key to halting this deadly disease is vaccines, but so far, there are no approved vaccines for leishmaniasis.
Unapproved leishmaniasis vaccinations, on the other hand, have taken place for hundreds of years. Primarily occurring in rural tropical and sub-tropical areas where leishmaniasis is endemic, this practice, called leishmanization, involves a deliberate skin injection of live, non-lethal Leishmania major parasites. It often leads to skin sores, but protection against deadly L. donovani infection. While effective, safety and standardization concerns do not make leishmanization a feasible option for widespread vaccination.
Taking a cue from this hundreds-year-old practice, Satoskar and his colleagues developed a new leishmaniasis vaccine using live CRISPR-edited L. major parasites, which they recently reported in Communications Biology. The team formulated their attenuated parasite vaccine under Good Laboratory Practice (GLP) conditions, such that it can be easily produced at scale. They found that it protected hamsters from L. donovani infection via the bite of a sandfly, paving the way for human clinical trials.
Deleting L. major’s centrin gene enables the parasites to grow in a flask with no problem, but when they differentiate into their infective lifecycle stage in a mammalian host, they can no longer divide.
“It removes the pathogenic effects of the parasite,” Satoskar said, “and that gives you enough time to generate a good [immune] response.”
Unlike a standard vaccination into muscle, the team inject the mutated L. major parasites into the skin. The reason for this, Satoskar explained, is that the natural route of infection starts in the skin with the bite of a sandfly. The skin is also where the immune system mounts the best defense against the parasite.
“The skin is the organ where you have Langerhans cells,” Satoskar explained. These immature dendritic cells transport the parasites to the lymph nodes, where they trigger a robust T cell response.
“Antibodies do not have [a] major role in parasitic diseases like leishmania,” he added. “What you need is a CD4 T cell response, and that has been another reason why it’s been difficult to develop a vaccine. You can develop a vaccine and get a lot of antibodies, but they do not protect.”
Satoskar’s team found that their centrin knockout L. major parasites were safe in that they did not cause disease in healthy or immunocompromised hamsters, and none of the parasites reverted from mutant to wild type.
For Paul Kaye, an immunologist at the University of York who is also working on a leishmaniasis vaccine, but who was not involved in this study, the most important result was that the vaccine protected hamsters from a lethal L. donovani challenge administered via the bite of an infected sandfly.
“That's a really tough experiment to do,” he explained. “With sandfly transmission, you can't always determine exactly what the infection level will be … but it's part of the biology, unfortunately, to have that variability.”
While technically challenging, testing the vaccine in the context of a sandfly infection is important because when they bite, sandflies inject more than just parasites into their hosts.
“Sandfly challenge adds a little bit of infectivity or ‘umph’ to the infection,” Kaye explained. Along with leishmania parasites, sandflies transmit their own salivary proteins, parasite products, and some of their own microbiomes into hosts, likely affecting how the immune system reacts to the infection.
The strong protection provided by the vaccine to sandfly challenge surprised Satoskar.
“That was beyond what I expected, to be honest. I thought it would work, but I didn't think that it would work that well,” he said.
In anticipation of future human clinical trials, Satoskar and his team developed their centrin knockout L. major parasites under GLP conditions with Gennova Biopharmaceuticals. To produce their parasite vaccine on a commercial scale, they needed to change some of the ingredients in the parasite growth medium.
One of those ingredients was hemin, which is a source of iron for the parasites. Normally, researchers can buy hemin from commercial vendors, but because most companies derive hemin from pigs, its use in the formulation of a vaccine would not be halal compliant.
“If we use that in a product, this product may not be acceptable in the Middle East,” Satoskar explained. Instead, the researchers substituted porcine hemin with hemin derived from human red blood cells.
The GLP-grade parasites protected hamsters from a sandfly infection of L. donovani parasites, with results similar to the lab-reared parasites. Kaye added that he would have liked the researchers to continue to monitor the hamsters until all of the control hamsters succumbed to a fatal L. donovani infection, which the researchers did for the lab-grade parasite vaccine, but he thinks that this study is an important step forward in bringing a leishmania vaccine to the clinic.
“This is a paper that deserves a little bit of recognition,” he said. “It shows that you can move these [vaccine] candidates in the right direction. Hopefully, others in the field will be encouraged to move their candidates in a similar way.”
Satoskar’s team is gearing up to generate a Good Manufacturing Practices (GMP) grade version of their parasite vaccine. They plan to begin clinical trials with it by the end of 2022 or early 2023. They hope to place an effective vaccine into the hands of the people living in rural communities who are most affected by visceral leishmaniasis soon.
“There are many more children and other people who may be suffering with this [who] we don't even know about,” he said. While the world is overwhelmed by the COVID-19 pandemic right now, he added, “we want to make sure that we do not forget the diseases that are already here and affecting people.”
Reference
Karmakar, S. et al. Preclinical validation of a live attenuated dermotropic Leishmania vaccine against vector transmitted fatal visceral leishmaniasis. Commun Biol 4, 929 (2021).
