Phase 1 human trials are set to start later this year with a vaccine designed to prevent infection byLeishmania major, the parasite species that causes cutaneous leishmaniasis in tropical and subtropical regions of the Eastern Hemisphere.

The same research team, co-led by Abhay Satoskar, professor of pathology in The Ohio State University College of Medicine, has used CRISPR technology to develop a vaccine targetingLeishmania mexicana, the species of the parasite that is found in South, Central and North America.

In mice, the vaccine -- created with a mutated live parasite -- posed no threat of causing skin lesions and provided long-term infection prevention.

This "New World" species causes a more chronic infection that, unlike skin lesions generated byL. majorinfection, will not self-heal. Researchers expect theL. majormutant parasite vaccine to be effective against theL. mexicanaspecies, but have developed the second vaccine as a backup -- and to find out whether their technique could tame the more virulent organism.

"The main thing we wanted to see was if this approach, removing a specific gene, could break this tough cookie, which has always been a problem. It's not an easy parasite," said Satoskar, also a professor of microbiology at Ohio State. "Based on our experimental data, we should be able to use theL. majorvaccine in the New World. But if it doesn't work, we have a plan b."

这项研究发表在《华尔街日报》NPJ Vaccines.

In both vaccines, the team applied gene-editing technology to the century-old Middle Eastern practice of leishmanization -- deliberately introducing the live parasite to the skin to create a small infection that, once healed, leads to life-long immunity against further disease.

"One question we had was, is leishmanization even possible withL. mexicana, which normally doesn't self-resolve? We're talking about two leishmania species and they cause the same disease, but the clinical outcome and response to treatments differ," Satoskar said. "And even CRISPR might not have worked, because this species has different molecules, proteases and enzymes."

Using the precision technology to edit the genome ofL. mexicana, researchers deleted centrin, the gene that codes for a protein that supports cell division, and inactivated an antibiotic resistance marker gene that is introduced into the parasite as part of the process of knocking out centrin.

在正常情况下,这些寄生虫引起正无穷ection by hijacking immune cells, and use host cells to replicate indefinitely. Experiments in immune cell cultures showed that the mutant parasite could enter cells and make limited copies of itself, but not enough to cause symptoms.

The study showed in mice that the vaccine is safe, causing no skin lesions in animals that are susceptible to the disease. In further experiments, researchers vaccinated mice and exposed them six weeks later to theL. mexicanaparasite with a needle challenge to the ear -- a technique used to mimic a sand fly bite. In humans and animals, leishmania is transmitted through the bite of infected sand flies.

Unlike the unvaccinated control group, vaccinated mice remained clear of skin lesions and the number of parasites at the infection site was held at bay -- and the protection was sustained over 10 weeks.

The immune response produced by vaccination with the New World species differs substantially from the type of response generated by the previous vaccine, the researchers found. WithL. majorvaccination, pro-inflammatory proteins are increased to provide protection. TheL. mexicanavaccine had little effect on pro-inflammatory proteins, but suppressed anti-inflammatory proteins instead.

"The parasites are different, and the mechanism by which they confer protection is totally different," Satoskar said. "It's a balance. They both work."

Researchers are pursuing vaccines to provide an affordable way to prevent a disease that can lead to disfigurement, disability, social stigma and poverty. The team estimates that a vaccine would likely cost less than $5, compared to the $100 to $200 cost for treatment in the hardest-hit countries -- treatment that requires weeks of daily drug injections with unpleasant side effects, leading to poor patient compliance that allows parasites to develop resistance to the drugs.

Visceral leishmaniasis affects organs and is fatal if left untreated. While theL. majorvaccine may also prevent this more severe form of the disease, the team has used the CRISPR technique to work on a vaccine with theLeishmania donovaniparasite that causes visceral leishmaniasis.

This work was funded by the Global Health Innovative Technology Fund and the National Institutes of Health.

The study was co-led by Greg Matlashewski of McGill University and Hira Nakhasi of the FDA Division of Emerging and Transfusion Transmitted Diseases. Additional Ohio State co-authors include Greta Volpedo, Thalia Pacheco-Fernandez, Erin Holcomb, Blake Cox, Rebecca Fultz, Chelsea Mishan, Chaitenya Verma, Ryan Huston, Abigail Wharton and Steve Oghumu.