Fighting a Worm with Its Own Genome

Tiny parasitic hookworms infect nearly half a billion people worldwide—almost exclusively in developing countries—causing health problems ranging from gastrointestinal issues to cognitive impairment and stunted growth in children. By sequencing and analyzing the genome of one particular hookworm species, Caltech researchers have uncovered new information that could aid the fight against these parasites.  

The results of their work were published online in the March 2 issue of the journal Nature Genetics.

"Hookworms infect a huge percentage of the human population. Getting clean water and sanitation to the most affected regions would help to ameliorate hookworms and a number of other parasites, but since these are big, complicated challenges that are difficult to address, we need to also be working on drugs to treat them," says study lead Paul Sternberg, the Thomas Hunt Morgan Professor of Biology at Caltech and a Howard Hughes Medical Institute investigator.

Medicines have been developed to treat hookworm infections, but the parasites have begun to develop resistance to these drugs. As part of the search for effective new drugs, Sternberg and his colleagues investigated the genome of a hookworm species known as Ancylostoma ceylanicum. Other hookworm species cause more disease among humans, but A. ceylanicum piqued the interest of the researchers because it also infects some species of rodents that are commonly used for research. This means that the researchers can easily study the parasite's entire infection process inside the laboratory.

The team began by sequencing all 313 million nucleotides of the A. ceylanicum genome using the next-generation sequencing capabilities of the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech. In next-generation sequencing, a large amount of DNA—such as a genome—is first reproduced as many very short sequences. Then, computer programs match up common sequences in the short strands to piece them into much longer strands.

"Assembling the short sequences correctly can be a relatively difficult analysis to carry out, but we have experience sequencing worm genomes in this way, so we are quite successful," says Igor Antoshechkin, director of the Jacobs Laboratory. 

Their sequencing results revealed that although the A. ceylanicum genome is only about 10 percent of the size of the human genome, it actually encodes at least 30 percent more genes—about 30,000 in total, compared to approximately 20,000-23,000 in the human genome. However, of these 30,000 genes, the essential genes that are turned on specifically when the parasite is wreaking havoc on its host are the most relevant to the development of potential drugs to fight the worm.

Sternberg and his colleagues wanted to learn more about those active genes, so they looked not to DNA but to RNA—the genetic material that is generated (or transcribed) from the DNA template of active genes and from which proteins are made. Specifically, they examined the RNA generated in an A. ceylanicum worm during infection. Using this RNA, the team found more than 900 genes that are turned on only when the worm infects its host—including 90 genes that belong to a never-before-characterized family of proteins called activation-associated secreted protein related genes, or ASPRs.

"If you go back and look at other parasitic worms, you notice that they have these ASPRs as well," Sternberg says. "So basically we found this new family of proteins that are unique to parasitic worms, and they are related to this early infection process." Since the worm secretes these ASPR proteins early in the infection, the researchers think that these proteins might block the host's initial immune response—preventing the host's blood from clotting and ensuring a free-flowing food source for the blood-sucking parasite.

If ASPRs are necessary for this parasite to invade the host, then a drug that targets and destroys the proteins could one day be used to fight the parasite. Unfortunately, however, it is probably not that simple, Sternberg says.

"If we have 90 of these ASPRs, it might be that a drug would get rid of just a few of them and stop the infection, but maybe you'd have to get rid of all 90 of them for it to work. And that's a problem," he says. "It's going to take a lot more careful study to understand the functions of these ASPRs so we can target the ones that are key regulatory molecules."

Drugs that target ASPRs might one day be used to treat these parasitic infections, but these proteins also hold the potential for anti-A. ceylanicum vaccines—which would prevent these parasites from infecting a host in the first place, Sternberg adds. For example, if a person were injected with an ASPR protein vaccine before travelling to an infection-prone region, their immune system might be more prepared to successfully fend off an infection.

"A parasitic infection is a balance between the parasites trying to suppress the immune system and the host trying to attack the parasite," says Sternberg. "And we hope that by analyzing the genome, we can uncover clues that might help us alter that balance in favor of the host."

These findings were published in a paper titled, "The genome and transcriptome of the zoonotic hookworm Ancylostoma ceylanicum identify infection-specific gene families." In addition to Sternberg and Antoshechkin, other coauthors include Erich M. Schwarz of Cornell University; and Yan Hu, Melanie Miller, and Raffi V. Aroian from UC San Diego. Sternberg's work was funded by the National Institutes of Health and the Howard Hughes Medical Institute.

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Knocking Out Parasites with Their Own Genetic Code
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Caltech Professors Awarded 2015 Sloan Fellowships

Five Caltech faculty members have been named among the 2015 class of Sloan Research Fellows. The fellowships, awarded by the Alfred P. Sloan Foundation, honor "early-career scientists whose achievements and potential identify them as rising stars, the next generation of scientific leaders." This year, 126 young scientists were awarded fellowships in eight scientific and technical fields: chemistry, computer science, economics, mathematics, computational and evolutionary biology, neuroscience, ocean sciences, and physics. Candidates must be nominated by a department head or other senior researcher and are reviewed by a selection committee of three distinguished scientists in each field.

Viviana Gradinaru (BS' 05), an assistant professor of biology and the faculty director of the Beckman Institute Pilot Center for Optogenetics and CLARITY, received her fellowship in the area of neuroscience. The CLARITY technique, codeveloped by Gradinaru, is used to render tissues, organs, and even whole organisms transparent. Her research focuses on developing tools and methods for neuroscience as well as investigating the mechanisms underlying deep brain stimulation and its long-term effects on neuronal health, function, and behavior.

Mitchell Guttman, an assistant professor of biology, received the fellowship in the category of computational and evolutionary molecular biology. His work exploring unknown regions of the genome has led to the identification of genes that do not produce proteins, known as long noncoding RNAs (lncRNAs), which act as efficient administrators, gathering and organizing key proteins necessary for packaging genetic information and regulating gene expression. Guttman and his colleagues recently discovered that lncRNAs can shape chromosome structure to remodel the genome and pull in necessary target genes, unlike other proteins that must travel to their targets.

Gregg Hallinan, an assistant professor of astronomy, received his fellowship in the physics category. His group studies the universe at radio wavelengths, particularly examining the radio emissions produced by stars and their planets. His team recently completed construction of a new radio telescope at Caltech's Owens Valley Radio Observatory that can survey the entire sky instantaneously. This project aims to deliver the first detection of radio waves produced by the interaction of the magnetic field of an exoplanet—a planet outside our own solar system—with the stellar wind of its host star.

Heather Knutson, an assistant professor of planetary science, received the fellowship in the physics category. She studies the structure, chemistry, and atmospheric dynamics of extrasolar planets. These planets are often classified into broad categories based on their mass and radius. Knutson's research measuring exoplanet temperatures and characterizing atmospheric compositions adds detail to these classifications. She has helped develop many of the techniques that are now used to study exoplanet atmospheric dynamics.

Xinwen Zhu, an associate professor of mathematics, received the fellowship in the mathematics category. His research interests focus on geometric representation theory, in particular the geometric aspects of the Langlands program, a kind of "unified theory of mathematics" linking together many different mathematical fields of research. This research aims to provide a more intuitive visualization of prime numbers by relating the field to diverse topics such as geometry and quantum physics.

Also included among this year's class of fellows are six other Caltech alumni: Brandi Cossairt (BS '06), Jennifer A. Dionne (MS '05, PhD '09), Aaron Esser-Kahn (BS '04), Michael Kesden (PhD '05), Neal Mankad (PhD '10), and Stephanie Waterman (MS '02).

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New Frontiers in Biological Engineering

Genetically Engineered Antibodies Show Enhanced HIV-Fighting Abilities

Capitalizing on a new insight into HIV's strategy for evading antibodies—proteins produced by the immune system to identify and wipe out invading objects such as viruses—Caltech researchers have developed antibody-based molecules that are more than 100 times better than our bodies' own defenses at binding to and neutralizing HIV, when tested in vitro. The work suggests a novel approach that could be used to engineer more effective HIV-fighting drugs.

"Based on the work that we have done, we now think we know how to make a really potent therapeutic that would not only work at relatively low concentrations but would also force the virus to mutate along pathways that would make it less fit and therefore more susceptible to elimination," says Pamela Bjorkman, the Max Delbrück Professor of Biology and an investigator with the Howard Hughes Medical Institute. "If you were able to give this to someone who already had HIV, you might even be able to clear the infection."

The researchers describe the work in the January 29 issue of Cell. Rachel Galimidi, a graduate student in Bjorkman's lab at Caltech, is lead author on the paper.

The researchers hypothesized that one of the reasons the immune system is less effective against HIV than other viruses involves the small number and low density of spikes on HIV's surface. These spikes, each one a cluster of three protein subunits, stick up from the surface of the virus and are the targets of antibodies that neutralize HIV. While most viruses are covered with hundreds of these spikes, HIV has only 10 to 20, making the average distance between the spikes quite long.

That distance is important with respect to the mechanism that naturally occurring antibodies use to capture their viral targets. Antibodies are Y-shaped proteins that evolved to grab onto their targets with both "arms." However, if the spikes are few and far between—as is the case with HIV—it is likely that an antibody will bind with only one arm, making its connection to the virus weaker (and easier for a mutation of the spike to render the antibody ineffective).

To test their hypothesis, Bjorkman's group genetically engineered antibody-based molecules that can bind with both arms to a single spike. They started with the virus-binding parts, or Fabs, of broadly neutralizing antibodies—proteins produced naturally by a small percentage of HIV-positive individuals that are able to fight multiple strains of HIV until the virus mutates. When given in combination, these antibodies are quite effective. Rather than making Y-shaped antibodies, the Caltech group simply connected two Fabs—often from different antibodies, to mimic combination therapies—with different lengths of spacers composed of DNA.

Why DNA? In order to engineer antibodies that could latch onto a spike twice, they needed to know which Fabs to use and how long to make the connection between them so that both could readily bind to a single spike. Previously, various members of Bjorkman's group had tried to make educated guesses based on what is known of the viral spike structure, but the large number of possible variations in terms of which Fabs to use and how far apart they should be, made the problem intractable.

In the new work, Bjorkman and Galimidi struck upon the idea of using DNA as a "molecular ruler." It is well known that each base pair in double-stranded DNA is separated by 3.4 angstroms. Therefore, by incorporating varying lengths of DNA between two Fabs, they could systematically test for the best neutralizer and then derive the distance between the Fabs from the length of the DNA. They also tested different combinations of Fabs from various antibodies—sometimes incorporating two different Fabs, sometimes using two of the same.

"Most of these didn't work at all," says Bjorkman, which was reassuring because it suggested that any improvements the researchers saw were not just created by an artifact, such as the addition of DNA.

But some of the fabricated molecules worked very well. The researchers found that the molecules that combined Fabs from two different antibodies performed the best, showing an improvement of 10 to 1,000 times in their ability to neutralize HIV, as compared to naturally occurring antibodies. Depending on the Fabs used, the optimal length for the DNA linker was between 40 and 62 base pairs (corresponding to 13 and 21 nanometers, respectively).

Taking this finding to the next level in the most successful of these new molecules, the researchers replaced the piece of DNA with a protein linker of roughly the same length composed of 12 copies of a protein called tetratricopeptide repeat. The end product was an all-protein antibody-based reagent designed to bind with both Fabs to a single HIV spike.

"That one also worked, showing more than 30-fold average increased potency compared with the parental antibodies," says Bjorkman. "That is proof of principle that this can be done using protein-based reagents."

The greater potency suggests that a reagent made of these antibody-based molecules could work at lower concentrations, making a potential therapeutic less expensive and decreasing the risk of adverse reactions in patients.

"I think that our work sheds light on the potential therapeutic strategies that biotech companies should be using—and that we will be using—in order to make a better antibody reagent to combat HIV," says Galimidi. "A lot of companies discount antibody reagents because of the virus's ability to evade antibody pressure, focusing instead on small molecules as drug therapies. Our new reagents illustrate a way to get around that."

The Caltech team is currently working to produce larger quantities of the new reagents so that they can test them in humanized mice—specialized mice carrying human immune cells that, unlike most mice, are sensitive to HIV.

Along with Galimidi and Bjorkman, additional Caltech authors on the paper, "Intra-Spike Crosslinking Overcomes Antibody Evasion by HIV-1," include Maria Politzer, a lab assistant; and Anthony West, a senior research specialist. Joshua Klein, a former Caltech graduate student (PhD '09), and Shiyu Bai, a former technician in the Bjorkman lab, also contributed to the work; they are currently at Google and Case Western Reserve University School of Medicine, respectively. Michael Seaman of Beth Israel Deaconess Medical Center and Michel Nussenzweig of the Rockefeller University in New York are also coauthors. The work was supported by the National Institutes of Health through a Director's Pioneer Award and a grant from the HIV Vaccine Research and Design Program, as well as grants from the Collaboration for AIDS Vaccine Discovery and the Bill and Melinda Gates Foundation. Nussenzweig is also an investigator with the Howard Hughes Medical Institute.

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Getting a Better Grip on HIV
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