Attacking influenza: Scientists hope that new technologies for making vaccines will

lead to quicker availability of vaccines against the human strain of H1N1 that originated from the swine flu virus, shown here.
Credit: CDC

As new influenza strains emerge, researchers struggle to speed vaccine development.

MIT Technology Review, October 13, 2009, by Lauren Gravitz  —  Making a vaccine against seasonal influenza is a constant catch-up game. Scientists must predict which of the constantly mutating virus strains will be most virulent six months in the future, the amount of time it takes to manufacture the vaccine. The system has worked well enough for the regular flu. But when new, virulent strains emerge–including the current, rapidly spreading swine flu (H1N1)–the traditional approach falls short. Even as consumers clamored for a vaccine, it took seven months and around 48,000 confirmed U.S. cases before the first H1N1 vaccines were shipped to hospitals around the country.

Influenza vaccine production has not changed substantially since it was first introduced in the 1940s. The new H1N1 vaccine took so long to make because it was manufactured using the usual technique–vaccine specialists identify and isolate the most virulent strains, weaken them, genetically adapt them for growth in birds as well as in mammalian cells, and then inject them into fertilized chicken eggs, where the virus can reproduce without killing its host. Once inactivated, the viral proteins can then be made into a vaccine. Add quality control and distribution, “and it is a five-to-six-month process, at its best,” says Gregory Poland, director of the Mayo Clinic’s Vaccine Research Group, in Rochester, MN.

Researchers are working hard to develop faster production methods for seasonal flu vaccines as well as for “universal” vaccines that could guard against almost all influenza strains, including swine and avian. But both are a long way down the road. “There is really nothing in the pipeline that will guarantee the production of vaccine in much less than six months,” says Robert Webster, an infectious disease and viral immunology expert at St. Jude Children’s Research Hospital in Memphis, TN.

Some companies, including Novartis and Baxter International, are working on flu and other vaccines that could be grown in cell culture rather than in eggs–a method that has the potential to halve time to production. The time-consuming steps of tweaking the virus strains so they’ll grow in bird rather than mammalian cells, and weakening them so that they can reproduce without killing the egg, would no longer be required. And manufacturers would no longer be dependent on the available egg supply. “With cells, you can grow them up, freeze them, and bring them out when you need them,” Poland says. “You can make as much or as little as you want.”

Both Novartis and Baxter have clinical trials under way, and Baxter just received European marketing approval for its H1N1 vaccine. But the process could take much longer in the U.S. because the cell-culture method itself has not been approved by the Food and Drug Administration. Companies will have to go through testing and manufacturing inspections that will cost on the order of about $500 million each, says Poland.

Other researchers are looking beyond single strains of influenza and into the possibility of creating a vaccine that can protect against almost all versions of the virus. Polio and measles vaccines given in childhood confer a lifetime of immunity because the viruses they protect against change very little from year to year, but the flu virus mutates fast, changing its outer proteins almost completely every season. However, researchers have found a few stable regions on the virus that they believe could be used to create a vaccine that could guard its recipients against nearly all strains of influenza, including those most likely to cause a pandemic.

Exciting News – A Personalized Antibody Library

Theraclone Sciences, based in Seattle, has a proprietary technology that can create an entire immune history from a person’s blood sample. The end result is a personalized antibody library covering every ailment the individual has successfully fought off. The company has previously used this technology to identify antibodies against HIV and is now turning to influenza, examining blood from patients who successfully fought off some of the most lethal flu viruses. By studying how the patients’ antibodies react to H5N1 influenza, Theraclone scientists found that the most effective antibodies bound to a spot that appears conserved among all viral strains, a specific location on a known surface protein called M2.

Researchers will look at the crystal structures of these antibodies and then use them as templates to reverse-engineer a vaccine that would prompt the human immune system to produce them. “Finding these antibodies is a very important advance, and I think researchers are excited that they finally have the tools to be able to do the analytical work around the biology of these pathogens,” says David Fanning, Theraclone’s president and CEO. “We may be able to come up with immunogens that bind to the broadly neutralizing antibodies. But whether they’re capable of eliciting the same or similar antibodies on vaccination is really the big unknown right now.” Theraclone is beginning an $18 million collaboration with Tokyo-based Zenyaku Kogyo pharmaceutical company to look for conserved flu antibodies and develop subsequent vaccine candidates.

And………sequences of DNA that, when taken up by cells and expressed as proteins, prompt an immune response……………………using electroporation, where electric shock disrupts a cell membrane long enough for designer DNA fragments to slip through.

Perhaps the most exciting but challenging prospect for a universal vaccine lies in DNA-based vaccines–sequences of DNA that, when taken up by cells and expressed as proteins, prompt an immune response. DNA vaccines can be made and modified quickly, are cheap to produce, and have a long shelf life. The major hurdle in developing these vaccines is getting the right cells to take up enough DNA to elicit immunity. Inovio, a company based in Blue Bell, PA, is working to solve this problem through a process called electroporation, in which a small electric shock disrupts a cell membrane long enough for designer DNA fragments to slip through. Recent studies by the company have shown that consensus genes, synthetic sequences that look similar enough to certain components in a variety of viruses, can prompt a broad immune response against multiple strains of flu.

Despite the promise, vaccine researchers still have a long road to travel. “There’s lots of exciting things out there,” Webster says. “But the first thing with vaccines is safety. You must always be sure of their safety.”


Berkeley Researchers Get First Look at Gene-Silencing Human RISC-Loading Complex

Contact: Lynn Yarris,                (510)486-5375        , lcyarris@lbl.gov


Eva Nogales (left) and Jennifer Doudna produced the first images of a human RISC-loading complex, a trio of proteins containing snippets of RNA that help control whether genetic messages are silenced or expressed. (Photo by Roy Kaltschmidt, Berkeley Lab Public Affairs) 

Berkeley researchers have solved the structure of a protein complex that helps determine the fate of human cells. Called a RISC-loading complex, this structure consists of small RNA molecules that control whether genetic messages are silenced or expressed.

BERKELEY, CA – The molecular architecture of a protein complex that helps determine the fate of human cells has been imaged for the first time by researchers with the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab). Known as a human RISC-loading complex, this structure consists of snippets of ribonucleic acid (RNA) that control whether genetic messages are silenced or expressed.

From these new images, the research team, led by biochemist Jennifer Doudna and biophysicist Eva Nogales, has been able to propose a model of how RISC and other so-called “small RNA molecules” are able to target specific messenger RNA molecules for gene silencing and/or destruction. Their results have been published in the journal Nature Structure and Molecular Biology in a paper entitled: “Structural insights into RNA Processing by the Human RISC-Loading Complex.” RISC stands for RNA-Induced Silencing Complex.

Doudna and Nogales both hold joint appointments with Berkeley Lab, the University of California (UC) Berkeley, and the Howard Hughes Medical Institute (HHMI). Co-authoring the paper with them were Hong-Wei Wang, Cameron Noland, Bunpote Siridechadilok, David Taylor, Enbo Ma and Karin Felderer.

“We now know how the three main components of the RISC machinery – the Dicer and Argonaute enzymes and the TRBP binding protein – are arranged, and how they interact with one another and are likely to interact as a complex with messenger RNA,” says Doudna, an authority on RNA molecular structures. “Our work should help others in the design of mutants to test the mechanisms of the RNA binding and processing used by the gene-silencing RNA machinery in humans.”

Says Nogales, an expert on electron microscopy and image analysis, “Because of the relatively small size of RISC  and the added complications of its not being very stable and having highly mobile parts, imaging this complex was a challenge. We used negative-stain electron microscopy and sophisticated single particle analysis.”

Human versions of small RNA molecules consist of approximately 20 to 20 nucleotides – compared to the three billion nucleotides in a molecule of human DNA – and include small interfering RNAs (siRNA) and microRNAs (miRNA). Despite their tiny size, small RNA molecules are receiving a great deal of attention from the biomedical and biotechnology communities. While some small RNA molecules help carry out genetic instructions, siRNAs and miRNAs work to silence genes and prevent them from issuing instructions that would be harmful to the cell such as “turn cancerous.”

In humans, targeted gene silencing, also known as RNA interference, requires that messenger RNA, the RNA that carries DNA’s genetic coding instructions, be bound or loaded with the RISC protein group. RISC will then either cleave the messenger RNA or block its message from being translated. This process is used in a number of important activities, including viral defense, chromatin remodeling, genome rearrangement, developmental timing, brain morphogenesis and stem cell maintenance.

Doudna, Nogales and their colleagues have identified the  central component of the RISC-loading complex as the Dicer enzyme and that the human version is “L-shaped.” The short branch of the L-shaped human Dicer was shown to interact with RNA-binding protein TRBP, and the long branch of the L interacts with an Argonaute2 protein, which in turn binds with siRNA molecules (for cleaving messenger RNAs) or miRNA molecules (for blocking translation).


Electron microscopy structure of the human RISC-loading complex, with the L-shaped Dicer enzyme shown as a wire map and the Argonaute2 protein, shown in purple.

“Guide RNAs are generated from double-stranded RNA precursors by the enzyme Dicer, which binds directly to both Argonaute2 and TRBP,” says Doudna, whose earlier work showed how Dicer snips double-stranded RNA into segments of specific lengths. “Once loaded onto the Argonaute2 protein, a passenger strand is cleaved by Argonaute2 and dissociates from the complex, leaving behind a short single-stranded guide RNA that base-pairs with complementary messenger RNAs and targets them for catalyzed degradation or translational arrest by Argonaute2.”

Adds Nogales, “The results of this study indicate that the TRBP protein is flexibly linked to the Dicer enzyme, which has led to several specific hypotheses about the roles of these three proteins in binding and cleaving RNA during gene silencing by RNA interference.”

Doudna and her research group will be continuing this work with co-author by Hong-wei Wang, who carried out the microscopy and computation work for this study while he was a member of Nogales’ research group, and who is now an associate professor at Yale University.

“Hong-wei is investigating the dynamics of the proteins in the RISC-loading complex and the location of RNA in the complex at different steps in the RNA processing pathway,” says Doudna. “Together with higher-resolution structural studies, we should be able to provide even more new insights into the core machinery of RNA interference.”

This research was supported in part by grants from the U.S. National Institutes of Health and the Human Frontier Science Program.

Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California.  It conducts unclassified scientific research for DOE’s Office of Science and is managed by the University of California. Visit our Website at www.lbl.gov/

Additional Information

For more information about the Doudna research group see http://rna.berkeley.edu/

For more information about the Nogales research group see http://cryoem.berkeley.edu/

Posted on Monday, October 12th, 2009

A U.S. Department of Energy National Laboratory Operated by the University of California



Light cells: Researchers were able to get living cells (here dyed fluorescent green) to take in engineered capsules (dyed red) and treat them as though they were a normal peptide.
Credit: Raghavendra Palankar / Small

Drugs could be slipped into living cells using a light-sensitive capsule.


MIT Technology Review, October 13, 2009, by Rachel Kremen  —  Targeted drug delivery is a hot topic of research. Scientists around the world are working on different ways to get drugs into specific cells without negatively impacting the rest of the body.

Now researchers in England and Germany have created gold-studded polymer microcapsules that release compounds into cells by rupturing when exposed to ultraviolet light. The capsules could be useful for researchers studying the effects of drugs on cells, and eventually they could perhaps serve as a clinical tool for administering medication.

“You can keep the capsules in the body for a while, and then you switch [on] the light to release them,” says Gleb Sukhorukov, professor of biomaterials at Queen Mary University of London and a researcher on the project.

Sukhorukov says the capsules could be used for administering drugs at the site of surgery a few weeks after an operation, without having to open up the patient again. They could also prove useful for gene therapy, although a method for directing the capsules to the right cells has yet to be developed.

To create the capsules, polymer layers are wound around tiny silica particles. Gold nanoparticles are added to the walls of the capsule during this process, and the silica particles are later dissolved in acid, leaving hollow capsules behind. Sukhorukov says the capsules can be made anywhere from 200 nanometers to 10 microns in size. Once they have been produced, they are heated in a solution containing the compound that is to be delivered to cells. The capsules shrink as they are heated, trapping some of the compound inside. In experiments, the researchers put peptides inside the capsules, but in the future they hope to use drugs.

The capsules were inserted into living cells using electroporation, an existing technique in which a pulse of electricity is applied to the cell to make it temporarily more permeable. Once implanted in the cells, the capsules protect the substance inside from being metabolized. But when exposed to ultraviolet light, the gold nanoparticles in the walls of the capsule heat up, causing the drug to be released.

Getting the peptides into the cells was only the first step, however. Sebastian Springer, a professor of biochemistry and cell biology at Jacobs University Bremen in Germany, who also worked on the project, says the team wanted to prove that the peptides would properly interact with the cell. “We decided that we would see whether this peptide would get picked up by [the] immune system and treated like a normal intracellular peptide that was natural to the cell,” Springer says. Indeed, the peptides moved, as hoped, from a compartment in the cell known as the endoplasmic reticulum to the cell’s surface.

A paper published in the October issue of the journal Small outlines the research, which was conducted with other researchers from Jacobs University Bremen and the Max Planck Institute of Colloids, also in Germany.

Organic chemist Jean Fréchet and colleagues at the University of California, Berkeley, have created capsules that work using a similar approach–employing carbon nanotubes that heat up when exposed to laser light.

Gold nanoparticles have also been used before, notably by Naomi Halas, professor of chemistry and director of the Laboratory for Nanophotonics at Rice University. Halas describes the European microcapsule work as “very important research,” because it shows that peptides can be successfully delivered into living cells. “Peptides usually do not diffuse through cell membrane, so a cell usually only has the proteins that it makes,” she says.

In addition to drug delivery, Halas says the light-release approach could be useful in the lab. “It allows you to look at various cellular functions in a quantitative way,” she says. For example, researchers can carefully time the release of specific amounts of drugs and see what happens to the affected cell.

Robert Langer, an Institute Professor at MIT, agrees that as a tool for in vitro experiments, the microcapsule is “novel” and useful. But he notes that the research has a “tremendous” way to go in addressing safety concerns before it can be used in humans.

Recently, Halas began studying the mechanics of the light-induced release. “There’s a gentle heating that occurs,” she says, although in her experiments the heat wasn’t enough to kill the cell. The ambient temperature of the cell remains the same, and only the surface of the nanoparticle gets warmer.

Springer plans to repeat the experiment using different kinds of cells and peptides and characterizing what happens in greater detail. He is also working on a paper outlining another approach that allows the capsules to release the drugs in a predictable fashion but without the use of laser light. Such an approach could be particularly helpful for delivering drugs deep within the body, where light cannot easily penetrate.

Sukhorukov hopes to decrease the amount of light required to release the drug. The cells tend to survive the experiments, he says, “but the power of the light is a little bit too high, I think.”


Scientists are trying to design the last malaria control agent the world will ever need 

The-Scientist.com, GoogleNews.com, October 13, 2009, by Elie Dolgin  — Entomologist Simon Blanford attaches a spray nozzle onto the top of a jar of white-powdered fungus immersed in a concoction of mineral oils. He leans forward into a fume hood and applies an even coating of fungal spores onto cut-up strips of disposable coffee cups taped against the back wall.

The next morning, after the sopping wet strips have dried, Blanford, a senior research associate at Pennsylvania State University in State College, will return to put the cups back together. Then he’ll toss in a load of young Anopheles mosquitoes that have just eaten a malaria-ridden blood meal, cover the cups with a mesh lining, and wait. One week later, the vast majority of the mosquitoes will die, victims of the fungus that rubbed off on their bodies from the coated cups. At least, Blanford wants it to be 1 week later, which is just short enough to prevent the transmission of malaria, but long enough to potentially circumvent the evolution of insecticide resistance-indefinitely.

Malaria kills around a million people each year, and mosquitoes have developed resistance to nearly every chemical that public health officials have thrown at them. This has rendered most existing insecticides ineffective, so new practical alternatives are critically needed. With the fungus, “we’ve got a product that can break resistance to insecticide now but will also work in the long run,” says Andrew Read, a Penn State evolutionary biologist who is spearheading the project with his collaborator, ecological entomologist Matthew Thomas. Other scientists are trying to achieve the same feat-a new malaria treatment that also discourages resistance-using other biological control agents, such as a bacterium that shortens its host’s life, and through genetic engineering. “If you design the thing right from scratch you only need one product and it should last forever,” says Thomas.

But many scientists are less enthusiastic. Judging from past failures, they dismiss the Penn State researchers’ plans as lofty pipe dreams. Plus, to make this goal a reality, the scientists would have to release these “biopesticides” worldwide, raising red flags about feasibility issues and potential risks.

But it’s precisely the fact that this project is far-reaching and different that makes it exciting, says Thomas. “It is radical thinking,” he says. “It could be life changing. I genuinely think this will work.”


Seven years ago, Read and Thomas, then both working in the United Kingdom, began investigating ways of using fungal species purely as a cheap, green alternative to chemical pesticides for malaria control, not as any fundamentally new approach that would halt resistance. Previously, Thomas and his wife, mycologist Nina Jenkins, were part of an international team that had developed a commercially available fungal product to target locusts and grasshoppers. “So having done all that,” says Thomas, “we figured it’d be much easier to do it a second time.”

One of the problems with fungal-based control agents, however, is that they don’t kill insects right away. This is a sticking point with crop-eating pests, but “with malaria it doesn’t matter,” Thomas says, because mosquitoes take a while to become infectious.

After an Anopheles mosquito bites someone carrying malaria, the Plasmodium parasite responsible for the disease traverses the lining of the mosquito’s gut and starts multiplying. Many rounds of replication follow before the Plasmodium progeny migrate to the mosquito’s salivary glands. Only then can the mosquito infect another host. Importantly, this whole process takes between 10 to 14 days, so as long as the fungus kills the infected insects within that crucial 2-week window, it should effectively block malaria transmission.


Simon Blanford sprays his fungal concoction onto a pair of cut-up coffee cups (left), which are later taped back together and loaded with mosquitoes (right).  

In 2005, Read and Thomas showed that this could work. Using a rodent malaria model, they found that treating ice cream tubs (they’ve since switched to cheaper coffee cups) with the fungal pathogen Beauveria bassiana killed more than 90% of mosquitoes within 14 days, and reduced the number of insects able to transmit malaria by a factor of 80. 1 At the same time, Bart Knols, a medical entomologist now at the University of Amsterdam, found that a fungal killer could be delivered in the field. His team hung cloths impregnated with Metarhizium anisopliae spores on the ceilings of five traditional houses in a rural Tanzanian village. After 3 weeks, Knols and his colleagues collected mosquitoes and found that 23% of Anopheles gambiae females-the mosquitoes that transmit most human cases of malaria-became infected and died several days earlier than uninfected controls. 2 “This could have a massive impact on the transmission of the disease,” says Knols, also the managing director of K&S Consulting, a firm he cofounded that advises on infectious disease control.

A couple of years later, Read and Thomas finally realized the biggest advantage of the approach. They were preparing a review article focused on fungal pesticides for malaria control when “it dawned on us that actually maybe this [fungus] was not going to be imposing very strong selection for resistance just because of its late-life action,” Read says. After the mosquito is exposed to the fungus, it lives for up to 2 weeks-enough time for two to six cycles of mosquito egg production-which means the doomed animals could comfortably reproduce, and there would be little reproductive advantage for insects that developed resistance to the fungus.

“It’s a really outside-the-box kind of idea.” -Don Gardiner

If there’s hardly any selection for resistant insects, they realized, the vicious cycle of always needing to design new and better drugs might be halted-dead in the mosquitoes’ tracks, as it were. Then came the most important insight: “Wait, if that’s true for the fungus it must be true for anything else” that kills only mosquitoes that have been given time to reproduce, says Read. Plus, the same approach should work for other insect-borne diseases, such as dengue fever, filariasis, and Japanese encephalitis, which also infect short-lived hosts in which the pathogen takes a while to become infectious.


Day 0: After the mosquito sucks up the malaria parasites, it lands on a treated surface and the fungus sticks on; Day 1-9: Malaria parasite replicates before becoming infectious and the mosquito lays eggs. Fungus levels kill the mosquito; Day 10-14: Malaria Transmission Period.

Read and Thomas’s intuition stemmed from a decades-old idea that the force of natural selection acting on survival and fertility decreases with the age of an organism. Once individuals have passed their genes on to the next generation, there is little evolutionary pressure to keep them alive, so any beneficial mutations that act late in life generally confer less of an advantage than similar genetic changes operating at a young age. Thus, as long as the mosquitoes can feed and lay eggs, evolution will be largely blind to any modest differences in longevity.

After first proposing the idea in their 2007 review paper, 3 Read and Thomas then used mathematical models to show that if insecticides target older mosquitoes and if resistance to these insecticides poses a cost to individuals with that resistance, then these late-life acting agents might never be undermined by mosquito evolution. 4 Now that they had a theoretical concept, the duo could point to their earlier experimental data, which showed that it was possible to selectively kill only old, infected insects. “We proved it was practical before we had the idea, which is kind of an ass-backward way of showing it,” Read says.


Evolution-proofing  with a transgenic boost

In 2006, while trying to infect Anopheles mosquito cell lines with pathogenic Wolbachia bacteria, Johns Hopkins’s Jason Rasgon had an unexpected surprise. His postdoc Xiaoxia Ren was using PCR to test for the presence of Wolbachia and an aberrant band kept cropping up in her negative controls. Ren brought this to the attention of her boss, but “I said, ‘It’s junk, don’t worry about it, ignore it,'” recalls Rasgon. Ren didn’t listen. “I was just curious,” she says. So she sequenced the mystery PCR product anyway.

At first, Ren thought that Rasgon was right-it did just appear to be a garbage DNA sequence matching an unknown virus. Rasgon recalls, “She’s like, ‘Yeah, it was some virus,’ and she turned to walk away. And I said, ‘Wait, come back here. What virus?’ ‘Oh, some Aedes virus.’ I said, ‘OK, stop.'” They compared the phantom band’s DNA to other known sequences and found that it most closely matched a densonucleosis virus, or “densovirus,” that infects Aedes mosquitoes. But this cell line had never been anywhere near any Aedes species. Without realizing it, Ren had stumbled upon the first known densovirus naturally found in Anopheles gambiae (PLoS Pathog, 4:e1000135, 2008). The densovirus now provides another potential weapon in the much-needed arsenal against malaria. “It started off as a side project,” says Ren. “But then the more we looked into it, the more interesting it became.”

Densoviruses have been used as biological control agents against Aedes mosquitoes for decades, but never to target Anopheles. Part of the draw of the approach lies in its simple application: Densovirus-infected mosquito cell cultures can be ground up, sprinkled into the water where females lay their eggs, and the larvae rapidly become infected. The developing insects then continue to shed the virus into the water and pass on the parasite to their offspring, which allows both horizontal and vertical transmission. Although Rasgon’s Anopheles densovirus does not appear to harm the mosquitoes, he is now trying to introduce genes that will release a late-acting toxin to kill the insects before they can transmit malaria-yet another technique that might circumvent mosquitoes’ tendency to evolve resistance.

Bruce Hay, a geneticist at the California Institute of Technology in Pasadena, is also using genetic engineering to create what could provide another “evolution-proof process,” he says. He created a synthetic selfish genetic element called Medea that could be used to drive a life-shortening gene through mosquito populations (Science, 316:597-600, 2007). “You’d be converting the population through genetic means to a live fast-die young life history,” Hay says. “The logic behind all these approaches is exactly the same.”

Many researchers are hesitant to use any genetic modification for fear of a backlash from the general public, but if it’s a promising approach, it’s worth pursuing, Rasgon says. “The feedback we’ve gotten has been generally supportive.”

“It’s a really outside-the-box kind of idea,” says Don Gardiner, head of the Malaria Biology Laboratory at the Queensland Institute of Medical Research in Brisbane, Australia. “I would agree that you could essentially evolution-proof insecticides so that the mosquitoes don’t develop resistance to them.”

“It opens up the mind to new ways of searching for insecticides,” says Joachim Kurtz, an evolutionary parasitologist at the University of Münster in Germany. Rick Paul, who studies malaria transmission at the Pasteur Institute in Paris, France, adds, “It’s the first time we’ve ever used evolution properly at all in terms of any kind of tropical disease control.”

But many in the field remain deeply skeptical. Read and Thomas brazenly called their most recent theory paper: “How to make evolution-proof insecticides for malaria control.” Many researchers felt that the title was a bit over the top, especially for a paper with no experimental data. “No entomologist should ever use the term ‘evolution proof,'” says the University of California, Riverside’s Brian Federici. “This type of rhetoric receives a lot of press, but mosquito gene pools are large and diverse, and so far these vectors have overcome everything humans have thrown at them.”

“I’m not convinced that a late-acting insecticide would break the arms race” between mosquitoes and insecticides, agrees Martin Donnelly, who studies the evolution of insecticide resistance at the Liverpool School of Tropical Medicine. “Just because it’s late-acting doesn’t mean there aren’t any fitness costs at earlier stages during development.” Ary Hoffmann, an evolutionary biologist at the University of Melbourne, Australia, points out that the authors only considered the selective advantage of reproduction in their model, and there are other reasons why natural selection might favor long life. Read and Thomas’s model “is a little bit simplistic,” he says, “and the situation in nature is going to be far more unpredictable than the picture that they paint.”


A gust of warm, humid air blows into a cramped, walk-in insect chamber at the Johns Hopkins Malaria Research Institute in Baltimore, Md. Molecular entomologist Grant Hughes lifts the mesh cover from a tray of stagnant water swimming with mosquitoes of all life stages. With a deep breath, he sucks up all the flying adults that emerged overnight using a huge pipette resembling a turkey baster, and blows the insects into a pint-sized paper tub. He then grabs an eye-dropper, and transfers all the pupae from the tray into a small, oblong puddle of water on a plastic dish.

Last week, Hughes, a postdoc working with Jason Rasgon, injected female mosquitoes with Wolbachia, an inherited bacterium that infects more than half of all known insect species and can shorten life span, but is not known to naturally infect Anopheles mosquitoes, the main transmitters of malaria. Hughes mated the insects, gave them a blood meal, and let them lay eggs. Now he is collecting the offspring to check if the bacterial infection has taken hold. For the moment, the females are set aside. He’ll sacrifice the males and inspect their cells’ DNA for traces of the bacterium using molecular methods. “If the males are infected,” says Hughes, “then I’ll go and breed the females to start a population.” Establishing a population of Anopheles mosquitoes that stably transmits Wolbachia bacteria from one generation to the next is one of the “holy grails of the Wolbachia community,” says Rasgon. He has yet to get an infection to take hold, but he’s making progress.

Wolbachia are passed on from infected female hosts to their offspring, so if researchers can get a pathogenic strain to stick inside the mosquitoes that transmit malaria, they might provide a self-perpetuating control method. In 2003, Rasgon published a theoretical paper showing that Wolbachia that shorten life span should be able to spread and kill mosquitoes early enough to completely block malaria transmission. 5 Like Read and Thomas, without realizing it, Rasgon had independently devised a strategy for “evolution-proof” vector control, although he didn’t frame the idea as such at the time. In fact, late-acting lethality is integral to the approach, because Wolbachia intimately rely on the mosquito host to survive long enough to reproduce, and pass the infection on to offspring. “It’s built into the system to have that sort of dynamic,” Rasgon says.

A theory is one thing; getting it to work is another. Researchers have combed through more than 30 Anopheles species from four different continents to find Wolbachia symbionts, all with negative results. This led some to speculate that it was biologically impossible for Anopheles to be infected with the intracellular parasite, but Rasgon has forced Wolbachia into cell cultures, embryos, and adult A. gambiae mosquitoes. In May, he reported that a life-shortening strain from fruit flies-called popcorn -could be successfully injected into adults, although none of these infections ever relayed to the next generation.6Wolbachia is everywhere in that mosquito,” says Rasgon. “It’s in the fat body; it’s in the brain; it’s everywhere except the ovaries. So something is excluding it from the ovaries. Maybe this is why Anopheles isn’t infected in nature. It’s not that they can’t be infected but something blocks germ-line transmission. That’s sort of what we’re hypothesizing now.”

Rasgon has some ideas for how to get around that cellular block, but declines to comment because others are racing to solve the same problem. In February, an international consortium led by Steven Sinkins, a molecular geneticist at the University of Oxford, received a multimillion dollar European Commission grant to truncate Anopheles‘s life span using virulent Wolbachia. “I’m very confident that we’ll be able to get [Wolbachia] in [Anopheles],” says Sinkins. “It’s just a technically difficult thing to do.”

Scott O’Neill, a geneticist at the University of Queensland in Brisbane, Australia, and a member of Sinkins’s consortium, has already had some success, but with a different mosquito species. Earlier this year, O’Neill reported that he could infect Aedes aegytpi mosquitoes-the harbingers of dengue and yellow fever viruses-with the same life-shortening Wolbachia, which cut the insects’ life span in half.7


Jason Rasgon (left) and Grant Hughes (right) are trying to infect mosquitoes with a life-shortening bacterium.

O’Neill attributes his achievement to a combination of luck and perseverance. After failing for years to introduce Wolbachia into the mosquitoes, O’Neill put the life-shortening popcorn strain into a mosquito cell culture “and just left it there,” he says. A technician continued to maintain the cell line, transferring the culture medium every 4 days for 3 years before O’Neill “dusted off the project again.” Remarkably, the mosquito cell line-adapted Wolbachia took hold when injected into Ae. aegypti embryos, and were stably transferred from one generation to the next. Perhaps a similar brute-force approach will be needed with Anopheles, says Sinkins. “We anticipate that it will take some time.”

Evolution-proofing with a twist

Jacob Koella, an evolutionary biologist at Imperial College London, has his own ideas about how to make insecticides “evolution-proof.” Unlike Andrew Read and Matthew Thomas at Penn State, who aim to target older mosquitoes to break the cycle of resistance, Koella actively wants to encourage resistance in larvae. The two approaches “have different starting points,” says Koella, “but you can get similar types of effects.”

Koella studies single-celled, eukaryotic parasites called microsporidia that, like fungi, also form spores and infect insects. His idea is to use evolution to his advantage, and select for microsporidia-tolerant larvae that live to reproduce but become saddled with the pathogenic spores into adulthood. That way they die earlier and never transmit malaria. Already, some preliminary data support his theory.

Ford Denison, an evolutionary biologist at the University of Minnesota in St. Paul, is not convinced that targeting larval development will necessarily translate into shorter adult life spans. For example, resistance to microsporidia could lead to slower development times and ultimately prolonged life-the exact opposite of the desired effect, Denison suggests.

Koella admits that his idea needs more rigorous testing, but he has already found that microsporidia infections reduced lifespan, on average, by 10% under optimal food conditions and by 25% on restricted diets. What’s more, some microsporidian isolates were deadlier than others, and the most lethal strains for adults were also the least likely to kill larvae and vice versa-direct evidence of a larval-adult fitness tradeoff (Adv Parasitol, 68:315-27, 2009). An added bonus, Koella found, is that microsporidia infections appear to prime the immune system so that infected mosquitoes are less likely to harbor malaria eggs (PLoS ONE, 4:e4676, 2009).

Koella believes that his method of targeting larval resistance to shorten adult life spans shouldn’t just be restricted to microsporidia. “I’m studying the approach,” he says, “but I wouldn’t be surprised if other biopesticides or chemical insecticides with similar properties turn out to be more feasible.” Any control agent that kills mosquitoes after they’ve reproduced but before they become infectious should, at least in theory, bring about the same effects, he adds. Read agrees: “Anything that acts late in life should be easy to make evolution-proof.”

Wolbachia might not be as evolution-proof as the researchers imagine, however. Since the bacteria are vertically transmitted, Kurtz says, these “parasites” often evolve toward a more mutualistic relationship with their hosts, so eventually infection may no longer shorten life span. “This will always tend to be a problem,” he says. Indeed, Hoffman showed 2 years ago that this is exactly what happened with Wolbachia-infected fruit flies in California over the last 20 years. And in June, he reported that some popcorn-infected fruit flies artificially selected for either early or late reproduction responded by living longer.8 “When you have a bacterium that actually decreases life span then you’re going to potentially get a situation where the host genome will shift to counter the effect of the bacterium,” he says. “The costs of the insecticide resistance can evolve.”


At Penn State, thousands of red blood cells are dancing on a microscope slide, whose image is projected onto a computer screen. Minutes ago, Read’s research assistant Brian Chan withdrew a sample of blood from a malaria-infected mouse, and the drop in temperature from the rodent’s warm body to room temperature has triggered the Plasmodium parasite to become active. Countless cells shift in shape as the malaria parasites thrash around inside like cats in a bag. Some rupture, and the eelish male gametes zip off in search of their female counterparts. The Plasmodium is infectious; the mouse is ready. Now, days-old mosquitoes can feast upon the malaria-ridden rodent before they are exposed to the lethal fungus. A week later, they will drop dead.

For the moment, the fungus is still in the experimental stages. But Read and Thomas hope to have enclosed field trials up and running soon. In the meantime, they are working to find chemicals that can also target just the old mosquitoes, as well as continuing to characterize different fungal isolates and their modes of action. They also want to scale up their fungal production capabilities so that the approach can readily be applied on an industrial scale. That’s pretty straightforward, says Jenkins, who has adopted a production method used by commercial mushroom farmers. She grows the fungus in liquid culture, transfers spores to a cereal such as rice or barley, and then isolates a pure powder that can be stored indefinitely.

The Penn State researchers have also had discussions with an industrial partner, but declined to go into specifics, citing a confidential disclosure agreement. “We’re pleased to be taken seriously,” says Read. Many critics, however, continue to dismiss the approach. Some critics worry that if we select for mosquitoes to die earlier, then the Plasmodium parasite in turn will evolve to develop quicker. “We don’t lose too much sleep over [this criticism] because there’s already very strong natural selection for more rapidly developing malaria,” says Read. As it is, most mosquitoes don’t survive long enough in nature to transmit malaria. So if malaria could develop faster it would already have a huge advantage right now.

A bigger problem might be public acceptance. Imagine trying to convince people that the best way to combat malaria is to actually let the young, nontransmitting mosquitoes breed instead of trying to kill all insects as quickly as possible. That message runs counter to conventional wisdom, not to mention World Health Organization guidelines that encourage people to avoid (via bednets) and kill (via insecticide sprays) whenever possible.

Janet Hemingway, director of the Liverpool School of Tropical Medicine, says it will be “extraordinarily difficult” to sell the late-acting control strategy in endemic malarial regions. “Trying to tell an uneducated villager in rural Tanzania or elsewhere that [letting more mosquitoes survive] doesn’t matter is really not going to wash, I suspect.” Thomas Scott, director of the University of California Mosquito Research Laboratory in Davis, agrees. “Are people really going to buy a product that they don’t think is protecting them?”

Read and Thomas brush off this pessimistic attitude, saying that education and proper messaging are not insurmountable barriers. “What’s the plan B?” asks Thomas. “What do we do next? At the moment there is no plan B other than searching for another example that 5 years down the line will fail again. We can’t just do the same again. That’s why we still have malaria.”

Imagine trying to convince people that the best way to combat malaria is to actually let the young, nontransmitting mosquitoes breed instead of trying to kill all insects as quickly as possible .

This kind of response is typical “hand waving from people who are lab scientists,” says Hemingway, who also heads the Innovative Vector Control Consortium, a product development partnership funded by the Bill and Melinda Gates Foundation. “That’s where people who never work in the field really underestimate what it takes. They ought to do a couple months in the field and see the practicalities.”

O’Neill is doing just that. In 2005, he, Hoffmann, and others received a 5-year, $6.7 million Gates Foundation Grand Challenges in Global Health Initiative grant to test the feasibility of releasing Wolbachia-infected Aedes mosquitoes in the wild. In addition to monitoring outdoor enclosures, O’Neill is working with anthropologists to establish focus groups with dengue-affected people in three countries to gain a better understanding of community attitudes toward the control tactic. “Public acceptability seems very high to the approach,” he says.

Knols also received positive feedback with his fungus. “When we did the trial in Tanzania, the local population was crazy about this whole thing,” he says. “They loved it. They wanted more of the fungus.” And since the fungus still relies on treated bednets and indoor residual spraying-the cornerstones of existing control protocols-implementing the approach only requires a change in attitude, not a change of action. “In terms of what people actually get to see, there’s not that much difference in these control methods,” Knols says.

Read admits that his argument is “a hard sell,” but what’s the alternative? “An unsustainable approach now is going to do a lot of damage in the long run. That’s the lesson of history,” he says. “We can be smarter this time.”

-Orthopaedic Tissue Regeneration Research Presented at Bone-Tec Congress by Dr.Myron Spector- 

GoogleNews.com, October 13, 2009, ALPHARETTA, Ga.–(Business Wire)–

SANUWAVE, Inc., (OTC BB: RBME) (www.sanuwave.com), an emerging medical

technology company focused on the development and commercialization of

non-invasive, biological response activating devices in the regenerative

medicine area, reported that scientific findings titled “Extracorporeal Shock

Wave Stimulation of Osteoprogenitor Cells” were presented at the 2009

International Bone-Tissue-Engineering Congress (“Bone-Tec”) in Hannover, Germany, which was held October 9-11, 2009. Dr. Myron Spector, PhD, Professor of Orthopaedic Surgery (Biomaterials) at Harvard Medical School, Director of Orthopaedic Research at Brigham and Women’s Hospital and Director of Tissue Engineering at VA Boston Healthcare System, was an invited guest speaker at the Conference. The Bone-Tec Congress featured an international scientific forum to discuss progresses in modern bone tissue regeneration and extended a worldwide network to exchange findings on the latest developments.


Dr. Spector’s team employed SANUWAVE’s Pulsed Acoustic Cellular Expression

(PACE) technology in pre-clinical research to create autogenous sources of stem

cells for bone tissue engineering. Results support the proposition that PACE

could be employed as a non-invasive technique to cause proliferation and

thickening of the cambium layer of the femur’s periosteum for the subsequent

intraoperative harvesting of progenitor stem cells days later for bone or

cartilage regeneration.


PACE stimulated a dramatic proliferation and thickening (up to 10 fold) of

osteoprogenitor stem cells, precursors to bone and cartilage cells, in the

cambium layer of the periosteum in the femur of the adult rats within 4 days.

Neovascularization and new bone formation within the thickened periosteum were

also evident after 4 days.


Dr. Spector said, “This research has shown great potential. Through more study,

this technology could further advance tissue engineering autologous transplant

techniques towards clinical applications such as bone reconstruction and

cartilage defect repair.”


Christopher M. Cashman, President and CEO of SANUWAVE said, “We are excited

about the preliminary research that Dr. Spector and his team have conducted on

our PACE technology. The procedure could have meaningful use in clinical

applications, whereby a patient’s own osteoprogenitor cells could be harvested

and reimplanted for procedures such as bone fusion, joint reconstruction and

cartilage repair. In clinical application, stimulating a large amount of a

patient’s own cells for harvest and reuse elsewhere in the body may have the

added benefit of reducing the need for anti-rejection drug regimens. Further

studies are needed to confirm that the proliferated cambium stem cells maintain

their ability to differentiate into bone and cartilage cells.”


Mr. Cashman concluded, “Dr. Spector’s research is quite exciting and supports

our efforts to further develop our technology for multiple regenerative medicine

uses, in addition to SANUWAVE’s IDE clinical trial that is in progress for

diabetic foot ulcers.”


About SANUWAVE, Inc. SANUWAVE, Inc. (www.sanuwave.com) is an emerging medical

technology company focused on the development and commercialization of

non-invasive, biological response activating devices in the regenerative

medicine area for the repair and regeneration of tissue, musculoskeletal and

vascular structures. SANUWAVE’s portfolio of products and product candidates

activate biologic signaling and angiogenic responses, including new

vascularization and microcirculatory improvement, helping to restore the body’s

normal healing processes and regeneration. SANUWAVE intends to apply its Pulsed

Acoustic Cellular Expression (PACE) technology in wound healing,

orthopedic/spine, plastic/cosmetic and cardiac conditions. Its lead product

candidate for the global wound care market, dermaPACE, is CE marked for

treatment of the skin and subcutaneous soft tissue and is currently involved in

an FDA-approved Investigation Device Exemption trial in the U.S. for the

treatment of diabetic foot ulcers. SANUWAVE designs, manufactures, markets and

services its products worldwide and believes it has already demonstrated that

this technology is safe and effective in stimulating healing in chronic

conditions of the foot (plantar fasciitis) and the elbow (lateral epicondylitis)

through it’s U.S. Class III PMA approved Ossatron device and in the stimulation

of bone and chronic tendonitis regeneration in the musculoskeletal environment

through the utilization of its Ossatron and Evotron devices in Europe. For more

information about the dermaPACE trial, please visit www.dermapace.com.


Safe Harbor StatementThis press release may contain “forward-looking statements”

within the meaning of the Private Securities Litigation Reform Act of 1995, such

as statements relating to financial results and plans for future business

development activities, and are thus prospective. Forward-looking statements

include all statements that are not statements of historical fact regarding

intent, belief or current expectations of the Company, its directors or its

officers.Investors are cautioned that any such forward-looking statements are

not guarantees of future performance and involve risks and uncertainties, many

of which are beyond the Company’s ability to control.Actual results may differ

materially from those projected in the forward-looking statements.Among the key

risks, assumptions and factors that may affect operating results, performance

and financial condition are risks associated with the marketing of the Company’s

product candidates and products, unproven pre-clinical and clinical development

activities, regulatory oversight, the Company’s ability to manage its capital

resource issues, competition, and the other factors discussed in detail in the

Company’s periodic filings with the Securities and Exchange Commission.The

Company undertakes no obligation to update any forward-looking statement.


Barry Jenkins, CFO
Emily Browning, Marketing Manager

NewScientist.com  —  July 20, 1976. Gilbert Levin is on the edge of his seat. Millions of kilometers away on Mars, the Viking landers have scooped up some soil and mixed it with carbon-14-labelled nutrients. The mission’s scientists have all agreed that if Levin’s instruments on board the landers detect emissions of carbon-14-containing methane from the soil, then there must be life on Mars.

Viking reports a positive result. Something is ingesting the nutrients, metabolizing them, and then belching out gas laced with carbon-14.

So why no party?

Because another instrument, designed to identify organic molecules considered essential signs of life, found nothing. Almost all the mission scientists erred on the side of caution and declared Viking’s discovery a false positive. But was it?

The arguments continue to rage, but results from NASA’s latest rovers show that the surface of Mars was almost certainly wet in the past and therefore hospitable to life. And there is plenty more evidence where that came from, Levin says. “Every mission to Mars has produced evidence supporting my conclusion. None has contradicted it.”

Levin stands by his claim, and he is no longer alone. Joe Miller, a cell biologist at the University of Southern California in Los Angeles, has re-analyzed the data and he thinks that the emissions show evidence of a circadian cycle. That is highly suggestive of life.

Levin is petitioning ESA and NASA to fly a modified version of his mission to look for “chiral” molecules. These come in left or right-handed versions: they are mirror images of each other. While biological processes tend to produce molecules that favor one chirality over the other, non-living processes create left and right-handed versions in equal numbers. If a future mission to Mars were to find that Martian “metabolism” also prefers one chiral form of a molecule to the other, that would be the best indication yet of life on Mars.  Read more at: http://www.newscientist.com/article/mg18524911.600-13-things-that-do-not-make-sense.html?page=1