Tag: Parasitology

A protein found in the saliva of ticks may prove to be an attractive target for a new type of Lyme disease vaccine. In studies in mice, Howard Hughes Medical Institute researchers at Yale University produced an antiserum against a protein in tick saliva that significantly reduced the likelihood that mice could be infected with the tick-borne bacterium that causes Lyme disease. Lyme disease first manifests in humans as a rash that may pass unnoticed. As the infection worsens, symptoms may include fever and chills, joint swelling, numbness, weakness, and even heart problems. The findings suggest a new way forward for Lyme disease vaccine development. “For vector-borne diseases, where the bacteria are transmitted by a tick or a mosquito, we wanted to know: Is it possible there is something that is not pathogen-based that can be targeted?” Vaccines have traditionally targeted unique proteins found on the surface of pathogens. In the new studies, published in the November 19, 2009, issue of Cell Host & Microbe, the researchers show that it is possible to target molecules carried by a disease vector – not the pathogen itself. This could be an effective strategy to prevent Lyme disease, as well as malaria, dengue fever, and other diseases carried by arthropods such as ticks and mosquitoes, said senior author Erol Fikrig, a Howard Hughes Medical Institute investigator at Yale University.

When the bacterium that causes Lyme disease is transmitted to a mammal via a tick’s bite, the bacterium wraps itself in a protein cloak that makes it invisible to the host’s immune system. That cloak is made from a protein found in tick saliva, which the Lyme bacterium, Borrelia burgdorferi, causes the tick to produce in excess. In Cell Host & Microbe, Fikrig and his colleagues describe a way to turn this cloak of invisibility into a vulnerability. Fikrig, who is chief of infectious diseases at the Yale School of Medicine, said vaccine development — even as far back as Louis Pasteur’s discoveries in the 1880s — has historically relied on using a weakened form of the pathogen, or a component of it, to evoke an immune response that would protect against later encounters with the same microbe. “For vector-borne diseases, where the bacteria are transmitted by a tick or a mosquito, we wanted to know: Is it possible there is something that is not pathogen-based that can be targeted?” Fikrig said.

“The tick isn’t just a syringe,” Fikrig said. Tick saliva contains a variety of unsavory ingredients that help the insect’s five- or six-day blood meal proceed unnoticed by the host, and the presence of the pathogen actually changes the composition of the tick’s saliva. For example, the saliva contains anesthetics that keep the bite from stinging and blood thinners to prevent clotting.

In 2005, Fikrig and his colleagues found that tick saliva also harbors Salp15, a protein that shields the tick from mammalian immune cells known as T-cells. The Lyme disease bacterium drives the tick to overproduce Salp15, so that it can use that protein to remain invisible to the host’s immune system. “For us, a central question was, if the spirochete requires the tick protein for infection, and it’s coated with it, can we actually target this protein?” Fikrig said. To test the efficacy of Salp15 as an immunizing agent, researchers injected a few mice with an antiserum against Salp15. Other mice, to be used as controls, were injected with an inactive serum. The following day, both groups of mice were injected with B. burgdorferi coated by the cloaking Salp15 protein. When the mice were examined a week later, all of the control mice showed signs of Lyme disease, but only half of the mice treated with the Salp15-antiserum were sick. When infection did occur in Salp15-injected mice, the antiserum was still protective: It helped to reduce the total amount of Borrelia burgdorferi bacteria in the body compared to control mice. After three weeks, 40 percent of the mice given Salp15 antiserum remained Lyme-free. Those that showed infection had lower levels of the Lyme bacteria in their hearts and joints than control mice.

One value of creating a vaccine against a protein produced by the vector, Fikrig said, is that it might enhance the effectiveness of a traditional pathogen-based vaccine. “Take a disease like dengue virus or malaria, for which there is no highly efficacious vaccine,” he said. The vaccines that do exist follow the traditional model — that is, they are based on components of the pathogen that infects the host. Fikrig suspects that including a vector protein like Salp15 in a vaccine that also targets a pathogen component could boost the vaccine’s ability to prevent disease. “Let’s say the vector targets are 50 percent efficacious and some pathogen components 40 to 50 percent. Combine the two and you might have a very good vaccine.” To test that idea, Fikrig’s team combined the Salp15 antiserum with an existing pathogen-based vaccine against Lyme disease. That vaccine, which Fikrig helped develop in the early 1990s, promotes immunity to a protein on the surface of the Borrelia burgdorferi pathogen called outer-surface protein A, or OspA. The vaccine was on the U.S. market from 1998 to 2002, when the manufacturer, GlaxoSmithKline, withdrew it, blaming poor sales. In an article that Fikrig wrote earlier this year for the journal Future Microbiology, he noted that concerns about potential side effects may have been a factor in the drug company’s decision to withdraw the Lyme disease vaccine.

In another set of experiments reported in Cell Host & Microbe, Fikrig and his colleagues combined low-dose Salp15 antiserum with a lower-than-effective dose of OspA monoclonal antibody and administered the mix to one group of mice. Other mice received either protective doses or low doses of just one of the two vaccine components. Each mouse was then exposed to 10 ticks carrying the Lyme-disease bacteria. Mice treated with a combination of low-dose OspA and Salp15 fared better than mice treated with either agent alone at low dose. Only 25 percent of mice treated with both agents showed signs of Lyme infection. In addition, only 20 percent developed signs of arthritis, and their inflammation was low compared to the inflammation of controls. These mice also showed the lowest spirochete burden. In contrast, 90 percent of mice treated with low-dose OspA alone and 100 percent of untreated mice were infected with Borrelia burgdorferi. These results eased one of the researchers’ concerns about combining the two approaches, Fikrig said. Since Salp15 normally mutes the body’s immune response to the spirochete invader, there was concern that it might blunt the immune response to OspA, and fail to produce any combined benefit. But their experiments ruled out that possibility. “We immunized with both together and found out that wasn’t the case,” he said.

In additional tests, mice were inoculated with a form of Salp15 that the researchers produced in the lab. These mice developed antibodies that protected them against infection when they were exposed to Lyme-carrying ticks. Three weeks after tick exposure, 40 percent of the inoculated mice showed no signs of infection, while 95 to 100 percent of controls were infected. Further research suggested that Salp15 probably protected the mice from infection by flagging the cloaked Borrelia burgdorferi spirochetes, which led immune cells to ingest the invaders, Fikrig said. Mice inoculated with both Salp15 and OspA fared even better— 80 percent remained uninfected. The combined impact of the two vaccines suggests other areas of research, and Fikrig says his group has already begun investigating whether a similar vaccine strategy might be effective in preventing malaria.

Howard Hughes Medical Institute
December 1, 2009

Original web page at Howard Hughes Medical Institute

The parasites that cause leishmaniasis use a quirky trick to convince the immune system to effectively invite them into cells for dinner, according to a new study published today in PLoS Pathogens. The researchers, from Imperial College London, say their findings improve understanding of the way Leishmania parasites establish an infection and could aid the search for a vaccine against this neglected tropical disease. Leishmania parasites are transmitted by sand flies. After the parasites infect a sand fly, they make a sticky gel so that when the fly bites a human, it regurgitates this gel into the body. Today’s research, which was funded by the Wellcome Trust, shows that the gel persuades immune cells known as macrophages to feed the parasites, rather than killing them. Leishmaniasis is an infection caused by Leishmania parasites that affects around 12 million people per year, mainly in tropical and sub-tropical countries. Symptoms include disfiguring and painful skin ulcers and in severe cases the infection can also spread to the internal organs. Patients with the infection often suffer from social exclusion because of their disfigurement. There is currently no vaccine to protect against infection and although treatments are available, they are not always effective and access to drugs is limited in many areas.

Leishmania-infected sand flies carry the parasites in their midgut. The parasites produce a gel that turns into a plug, stopping anything from passing in or out of the fly’s gut. The fly must regurgitate the gel plug before it can feed on human blood. When the fly bites a person, its barbed mouth parts tear the skin so when it regurgitates parasites along with the gel plug, the skin becomes infected. Today’s study shows that the gel’s work doesn’t stop there – it also helps the parasites to establish an infection by enticing macrophages to the bite site. Macrophages usually kill invading pathogens by eating and digesting them. However, according to the new research, the gel persuades macrophages to engulf the parasites and feed them rather than digest them. This happens within the first few days following infection, enabling the parasites to establish themselves and infect the skin. Previous research suggested that the sand fly’s saliva could be involved in manipulating the immune system. Today’s study suggests that the gel has an even bigger effect than the saliva on establishing infection.

Dr Matthew Rogers, lead author of the study from the Division of Investigative Science at Imperial College London, said: “Leishmaniasis is a very debilitating disease, yet we know comparatively little about the way the parasites are transmitted by sand flies. This is because when scientists study the disease they usually inject the parasite into tissues without including the gel or the sand fly’s saliva. Our new research shows that we must consider the way the parasites enter the body – along with the gel and saliva – if we are to recreate infection and get an accurate picture of what is going on. “Our new research shows that Leishmania parasites are very cunning – they make their own gel to control the human immune system so they can establish a skin infection. There is more work to be done here – our previous work in mice has suggested that injecting a synthetic version of the gel into people might provide them with some protection against infection and we would like to explore this further,” added Dr Rogers.

The researchers looked at Leishmania infection in mice and found that the gel, called promastigote secretory gel (PSG) enticed macrophages to the site of entry. They compared the effect of PSG with the effects of saline and sand fly saliva on the number of macrophages recruited to a bite site, 4-72 hours after the bite. In the experiment, PSG recruited 108 times more macrophages to the bite than saline and five times more than sand fly saliva. The researchers also found that PSG persuaded macrophages to feed, rather than kill, the parasites. When macrophages want to kill a pathogen, they produce nitric oxide. However, the researchers’ experiments showed that PSG influences the immune cells to produce food, in the form of polyamines, for the parasites instead. Finally, the researchers looked at the effect of PSG on parasite survival in vitro. They infected macrophage cells with Leishmania parasites with and without PSG. They found that more parasites survived in the first 48 hours following infection when PSG was added; both the proportion of infected cells and the number of parasites in the cells increased by up to 8-fold with PSG. The parasite infection declined after 48 hours in cells both with and without PSG, suggesting an early window of time in which PSG helps the parasites establish an infection.

Science Daily
September 8, 2009

Original web page at Science Daily

Every day 2000 children die from malaria in Africa alone. The infection is transmitted from human to human by biting mosquitoes and remains one of the world’s most devastating diseases. Despite many years of effort a vaccine is still not available but is urgently needed, if we are to make an impact on this enormous problem. Continual exposure can generate protection against malaria and can be acquired through an exposure to a high number of infectious mosquito bites. Parasites that are injected by a mosquito first migrate to the liver where they mature and then are released into the blood circulation and it is only here that they cause disease and fatal complications. A very promising method for vaccination is to sufficiently weaken parasites such that they invade liver cells but then are not able to develop any further. It is, however, required that these attenuated parasites are still able to stimulate a good immune response in the liver. This can be achieved by irradiating the parasites or by genetically inactivating individual parasite genes that are active during the parasites growth in the liver. Researchers from Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands and Leiden University Medical Centre, Leiden, the Netherlands, have now characterized a large number of parasite proteins (‘proteome’) that are present only during liver stage development and therefore are potential targets for inactivation.

The research groups had previously shown that protection in mice can be achieved by vaccinating mice with a rodent malaria which had one of these liver stage genes removed, specifically p36p. Moreover, the protection was long lasting and virtually complete. Now, these same researchers from Nijmegen and Leiden have succeeded in making the first critical transition from the rodent system to humans by inactivating the equivalent gene (p52) in the most important human malaria parasite, P. falciparum. Similar to the results with the rodent parasite, these human parasites are unable to develop in liver cells. This is the first time that genetic modification of a human parasite results in its growth arrest in a liver cell, opening up exciting possibilities for its use as a human vaccine.

Science Daily
November 25, 2008

Original web page at Science Daily

An effective vaccine against a parasite-borne disease called neosporosis may be a few steps closer to development, thanks to Agricultural Research Service (ARS) scientists and cooperators. Neosporosis, caused by the parasite Neospora caninum, affects cattle worldwide. Infected animals often abort their calves and develop other debilitating complications that can cost producers millions of dollars every year. Animal scientist Wenbin Tuo and microbiologist Mark Jenkins work at the ARS Animal Parasitic Diseases Laboratory in Beltsville, Md. They collaborated with ARS Molecular Plant Pathology Laboratory molecular biologist Yan Zhao and National Institutes of Health researcher Daming Zhu to test a new neosporosis vaccine in a mouse model.

The team created the vaccine using the parasite’s own proteins. One of these proteins, called Neospora caninum cyclophilin (NcCyP), regulates the response of the host immune system that limits the survival of the parasite in the host after infection. The other protein–called NcSRS2– helps the parasite attach to and invade host cells. The researchers tested three different vaccine cocktails containing these proteins. One group of mice received a formulation of NcCyP. A second group received a formulation of NcSRS2. A third group was immunized with a mix of both proteins. After vaccination, the mice were inoculated with the parasite. The researchers found mice that received the vaccine formulated with NcCyP alone exhibited the highest levels of protection against the disease. On average, only 13 percent of the mice in this group had detectable levels of N. caninum in brain tissue following infection. In contrast, more than 80 percent of the non-vaccinated mice were infected after challenge.

The scientists found that the serum antibody levels against the protein correlated well with the levels of protection. They also observed that the vaccine containing both NcCyP and NcSRS2 was no more effective than the vaccine that just contained NcCyP. However, more work is needed to evaluate the efficacy of these proteins in protecting cattle–the host animal–against the disease.

Science Daily
September 2, 2008

Original web page at Science Daily

A new drug that will soon enter clinical trials for treatment of malaria also appears to be 10 times more effective than the key medicine in the current gold-standard treatment for toxoplasmosis, a disease caused by a related parasite that infects nearly one-third of all humans–more than two billion people worldwide. A research team based at the University of Chicago Medical Center shows that the drug, known as JPC-2056, is extremely effective against Toxoplasma gondii, the parasite that causes toxoplasmosis, without toxicity. “JPC-2056 has the potential to replace the standard treatment of pyrimethamine and sulfadiazine,” said infectious disease specialist Rima McLeod, professor of ophthalmology at the University of Chicago and senior author of the study. “The drug, taken by mouth, is easily absorbed, bioavailable, and relatively nontoxic. In tissue culture and in mice, it was rapidly effective, markedly reducing numbers of parasites within just a few days.” Untreated mice injected with the parasite “appeared ill,” four days after the injection, the authors note, “with ruffled fur and hunched shoulders.” Treated mice remained well.

“Studies in tissue culture found no evidence of the parasite or the plaques they produce 52 days after four days of treatment,” said co-author Ernest Mui, a researcher in McLeod’s laboratory. “The absence of growth,” the authors write, “indicates that this compound is ‘cidal’ and not merely ‘static’ for the active form of T. gondii. The drug inhibits the action of an enzyme, dihydrofolate reductase (DHFR), produced by the family of parasites that includes those that cause toxoplasmosis and malaria. It is structurally distinct from the human DHFR. “The drug’s effect on the malaria and Toxoplasma enzymes is robust,” said McLeod. “It has much less effect on the human enzyme.” The new drug was effective against all malaria parasites, even those with multiple mutations that make them resistant to other anti-folate medicines, suggesting that “this family of parasites, including not just Toxoplasma but also various malaria parasites, will not easily develop resistance,” she said. Toxoplasma infection is “probably the most common parasitic infection in the world, causing very significant disease in those who have immature immune systems or who are immune-compromised,” McLeod said. “New medications are urgently needed.” The standard medicines to treat the infection can cause severe side effects and many patients become hypersensitive to them. There are no medicines that can eliminate certain latent stages of the parasite’s life cycle. There is no vaccine.

T. gondii infects humans through three principal routes: a newly infected pregnant woman passing the infection to her fetus; consumption of undercooked, infected meat; and ingestion of T. gondii oocysts in food, through accidental contamination from cat litter. “An infected cat can excrete up to 20 million oocysts over two weeks,” McLeod said. “Even a single oocyst is infectious and they can remain infectious in water for up to six months and in warm moist soil for up to a year.” Congenital toxoplasmosis, which occurs in an estimated 1 per 5,000 births a year in the United States, can cause severe vision loss, brain damage and even death. The annual cost of caring for these children may exceed $1 billion. Also at increased risk are people with compromised immune systems, such as those with cancer, autoimmune disease, AIDS or transplant recipients. Even people with normal immune systems can suffer major organ damage from chronic infections. Eye disease leading to loss of sight is caused both during the primary infection and as a result of infection transmitted from mother to child. Recent epidemics in Surinam and French Guiana have been lethal even for young healthy victims. Studies have also found an association between chronic brain infection with Toxoplasma and diseases such as schizophrenia and epilepsy, although cause-and-effect relationships have not been proven.

JPC-2056 was developed in the late 1980s by teams led by Wilbur Milhous and Dennis Kyle of the Walter Reed Army Institute for Research in Silver Spring Maryland (both now at the University of South Florida), and David Jacobus of Jacobus Pharmaceutical Company. The original version was quite toxic, but the researchers found ways to reduce the toxicity and developed an oral version of the drug. Clinical trials using JPC-2056 to treat malaria are scheduled to begin later this year. This new class of medicine holds “considerable promise for significant advances in the treatment of toxoplasmosis, which damages the eye and the brain,” said McLeod, “as well as malaria, which kills one million children each year.”

Science Daily
March 17, 2008

Original web page at Science Daily