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Boosting Our Best

al orr a C rin Ma s by Shot ration Illust work by training the immune system to target pathogens, but many types of shots need added substances called adjuvants to elicit a robust response. Despite the power of adjuvants, only one, called alum, is approved in the US. Charlotte Schubert looks at recent discoveries that could translate into a wider range of adjuvants and perhaps help provide future protection against diseases ranging from malaria to H1N1 ‘swine’ flu.

© All rights reserved. 2009 Inc. Nature America, Max Theiler never thought it would be easy to particularly good at bumping up the immune FDA is poised to give the green light to such vanquish one of the biggest killers of his time. response. By comparison, most vaccines adjuvanted vaccines, lined up for approval in Yellow fever had already stumped a previous developed today rely on bits of microbes, such , remains unclear (see sidebar). generation of microbe-hunters. And in the as short protein sequences—and they don’t For many years, researchers such as Theiler early 1900s it killed subjects who volunteered work quite so well on their own. To elicit an moved their candidates forward with for experiments in which they received bites immune response, these vaccines typically little mechanistic understanding of how they from mosquitoes, proving that the insects need a jolt from an adjuvant, a substance worked. That empirical approach is ending, transmit the disease. named from the Latin ‘adjuvans’, which says Bali Pulendran, an immunologist at the Theiler’s work was painstaking. He spent means ‘to help’. But only one adjuvant, based Emory Vaccine Center in Atlanta, and it’s a more than a decade isolating the yellow on aluminum salts, is approved for use in the boon to the development of new adjuvants. fever virus, growing different strains in test US. Dubbed alum, it spikes common vaccines “What we are now seeing in the adjuvant tubes, mouse brains and embryos, such as shots for hepatitis B and tetanus. field is immunology and vaccinology coming and testing various imperfect versions of the New adjuvants, say researchers, have the together,” says Pulendran. Research in his lab, vaccine in animal experiments. Ultimately, potential not only to improve existing vaccines for instance, is finally beginning to unravel his persistence paid off. In 1937, he finally but also to quell diseases that, more than half how the builds such a hit upon a version of the yellow fever virus a century after Theiler conquered yellow fever, marvelous immunological defense without an that did not itself cause disease but could still lack an effective jab. And adjuvants may adjuvant—work that could lead to new ways be injected into people as a vaccine. The help provide protection against the H1N1 to augment other vaccines. Other researchers achievement won Theiler a Nobel Prize, and ‘swine flu’ virus—as vaccine are applying findings from immunology to the vaccine is still one of the most effective production faces limitations, adjuvants based intelligent design of adjuvants. And just last ones known: one shot will protect against on oil and water emulsions (such as one made December, malaria researchers made a splash yellow fever for more than 30 years. by Novartis dubbed MF59) have the potential with positive results in a trial of the RTS,S Despite his arduous path, Theiler was lucky. to increase vaccine potency and also to stretch vaccine, which contains a carefully chosen His vaccine is based on a live virus—one the supply by reducing dosage. Whether the adjuvant1,2.

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Scientists are getting steadily closer to their using a live, attenuated version of HIV might ultimate goal of making adjuvants into ideal generate decent protection, such a live vaccine helpers, intelligently tailored to the specific is unlikely to receive regulatory approval needs of each vaccine. “The dark ages are because of safety concerns. So vaccine coming to the end,” says Pulendran. developers generally err on the side of caution and create vaccines using building blocks such Safety first as lifeless protein fragments from microbes, Despite the excitement about the emerging which are also generally cheaper and easier to science of how adjuvants work, researchers manufacture in a standardized way. developing new vaccines face major regulatory The catch is that most nonliving vaccines hurdles. “The FDA [US Food and Drug are too weak on their own and therefore need Administration] is risk averse—particularly in an adjuvant. The parts of the vaccine based on their response to the safety of vaccines,” says the offending microbe, the , instruct Ripley Ballou, who currently serves as deputy the body to attack the microbe. But it’s the director for vaccines at the Bill and Melinda adjuvant—mixed with antigen, welded to it Gates Foundation and previously researched or positioned together with the antigen in a and the the RTS,S . tiny, spherical particle—that can amplify the i m m u n e But, paradoxically, it is because of safety immune response and make the vaccine more system may turn concerns that vaccines often require an potent. on the body, fostering adjuvant. For example, although laboratory Herein lies the rub: adjuvants work by autoimmune disease. experiments suggest that a vaccine built boosting the immune system: Boost it too much Regulatory agencies outside the US have approved a handful of vaccines with newer adjuvants, such as a seasonal Illuminating alum flu vaccine in Europe mixed with Novartis’ adjuvant MF59, an emulsion of oil in water. Alum, the world’s most widely used thought to subsequently bump up the MF59 boosts protection against flu in older adjuvant, got its start in the 1920s body’s production of antibodies. people, who have relatively frail immune when vaccinologists found that mixing The new findings from Flavell and systems, although it’s still unclear exactly how it into their preparations gave a boost his colleagues, along with other studies it achieves this. to the . Researchers implicating the inflammasome, potentially The FDA’s view toward new adjuvants will be then proposed that it worked by explain why alum is good at prompting an tested as early as this fall, when initial clinical glomming onto vaccine components, antibody response. A separate study in data on adjuvanted pandemic H1N1 vaccine causing them to be released slowly into 2008 suggested that alum may initiate this becomes available (sidebar). An advisory group the bloodstream. response partly through the induction of for the agency is also scheduled to consider on That theory held for 80 years. It is cellular damage. Cells damaged by alum 9 September whether to recommend approval only now falling by the wayside, as were shown to release uric acid, a known of , a cervical available immunologists get their hands on alum, trigger of the inflammasome (J. Exp. Med. in Europe that is produced by GlaxoSmithKline © All rights reserved. 2009 Inc. Nature America, a term for various immune-activating 4, 869–882; 2008). (GSK). That vaccine contains an adjuvant aluminum salts. But subsequent research has developed by GSK dubbed AS04, containing “Alum has been used in billions of muddied the waters. For instance, other alum plus monophosphoryl lipid A (MPL), doses since 1920,” says Rino Rappuoli, immunologists, such as Philippa Marrack at which stimulates the immune system in a head of research for Novartis Vaccines the University of Colorado Health Sciences distinct way. in Sienna, Italy, “and, up until about Center in Denver, have found evidence that Last year, the FDA put on hold clinical a year ago, we had no clue about the alum works just fine as an adjuvant in the trials of a version of the mechanism of action.” absence of inflammasome activity (Eur. containing an experimental adjuvant, dubbed A 2008 study by Richard Flavell J. Immunol. 38, 2085–2089, 2008; Nat. CpG oligonucleotides. One subject in a clinical at Yale University in New Haven, Rev. Immunol. 4, 287–293; 2009). trial of the vaccine, developed by Dynavax Connecticut and his colleagues helped “In trying to understand the molecular Technologies Corporation, a biotechnology to crack open alum’s secrets. Their mechanism of adjuvants, we will find a company based in Berkeley, California, research suggests that alum activates lot of redundant mechanisms, so I’m not developed a severe autoimmune disease— a complex of proteins in immune surprised there is a controversy,” says Wegener’s granulomatosis, in which blood cells dubbed the ‘inflammasome’. Rappuoli. vessels become inflamed. Mice lacking the inflammasome did Marrack and other immunologists are More than 2,500 people have received not respond to alum, although other using standardized reagents to help work Dynavax’s experimental vaccine in clinical adjuvants still worked (Nature 453, out the discrepancies, which may have trials, and the illness may be due to statistical 1122–1126; 2008). arisen because of differences in alum chance. Nonetheless, the FDA is reviewing In response to alum, the formulation and the analysis of the immune the full analysis of the event before deciding inflammasome seems to cleave and response. Ultimately, researchers may be whether trials can proceed. Notably, Dynavax’s activate certain immune-stimulating able to design more effective alum-based vaccine improves on an already available proteins, such as interleukin-1β and adjuvants, she says. vaccine for hepatitis B, setting a high bar for interleukin-18. These proteins are Charlotte Schubert, Washington DC FDA approval. “It enters in the inevitable risk-

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Their trials also showed that recipients require certain pairs of Toll-like receptors can more only two shots, in contrast with the three for the than double the effect. current vaccine3. Pulendran has applied this principle most Now, says Coffman, “we have backfilled recently to create experimental vaccines against our understanding.” That understanding has the H1N1 ‘swine flu’ virus and the H5N1 ‘bird emerged largely from years of basic research flu’ virus. by immunologists revealing how the body His lab created an adjuvant containing two responds to microbes. TLR activators: MPL and a synthetic TLR7 CpG oligonucleotides bind and activate Toll- ligand. They also paid attention to what benefit analysis at the FDA,” says like receptor 9 (TLR9), a member of a family vaccinologists call the ‘delivery vehicle’. They Robert Coffman, chief scientific of receptors that recognize the components of encapsulated the vaccine in a nanoparticle, officer at Dynavax. microbes. These receptors stud the membranes using polylactic glycolic acid, which aids in the of immune cells such as dendritic cells, which immune response. Putting the pieces together alert the body to foreign pathogens. Dendritic By combining two TLR activators, the When company scientists helped cells activate the immune response and help researchers could radically decrease the design the first clinical trials of tailor it to the type of microbe—whether virus, dose of H5N1 antigen required in mice. In Dynavax’s hepatitis B vaccine bacterium or parasite. as yet unpublished work, 0.1 microgram in the mid-1990s, they weren’t People have at least 11 Toll-like receptors, of antigen, when combined with their sure precisely how CpG each one recognizing distinct substances. For experimental adjuvant, had the same effect oligonucelotides worked. instance, MPL, part of the adjuvant in Cervarix, as 10 micrograms of antigen combined with What they did know activates TLR4. alum. The experimental adjuvant “beat alum, was that the particular In fact, says Pulendran, TLRs are also what hands down,” says Pulendran. He adds that in oligonucleotides they give the yellow fever vaccine much of its power. preliminary experiments it also elicited a more selected, containing a In 2006, Pulendran and his colleagues showed robust, longer-lasting response than alum. type of DNA prevalent that the yellow fever vaccine activates four Other researchers have observed a similar in bacteria, somehow different Toll-like receptors, which, in turn, synergistic effect with multiple TLR activators, bumped up ‘innate’ activate a range of dendritic cell subtypes, including Steve Reed, head of research and immunity, the arm of each with its own distinct booster effect on the development at the Infectious Disease Research the immune response that acts a first responder immune system4. Institute, a Seattle-based nonprofit that fosters to microbes. “What we find is that there is this remarkable vaccine development for diseases such as The extra boost from the adjuvant resulted synergy in the immune response,” says tuberculosis, HIV/AIDs and malaria. in a more rapid and potent antibody response Pulendran. Activating one Toll-like receptor “We are pioneering the next generation of than the currently approved hepatitis B vaccine. tweaks the immune response, but activating TLR ligands,” says Reed, “it’s a very aggressive

Swine flu agitates the adjuvant debate

© All rights reserved. 2009 Inc. Nature America, US regulatory agencies will face a big emulsions—such as Novartis’ MF59, which up to submit applications for pandemic decision, as early as this fall, as to is used in seasonal flu vaccines for the H1N1 vaccine under this accelerated whether to approve the use of adjuvants elderly in Europe. process. for pandemic H1N1 flu vaccine on an There is clearly pressure to stretch the The tone at the US Food and Drug emergency basis. supply when it comes to pandemic H1N1 Administration (FDA) seems to be more Adjuvants used in some seasonal flu vaccines. Ballou says his ‘back of cautious. One reason the US treads more vaccines in Europe have the potential to the envelope’ calculations suggest the US carefully than Europe is that the climate boost the effectiveness of pandemic H1N1 is snapping up 40% to 50% of the world’s in the US tends to be “more litigious, vaccine and also substantially reduce supply, an estimate other public health with a very active antivaccine lobby,” says dosage—thereby stretching supplies. If experts say is reasonable. That is based on Amesh Adalja, a fellow at the Center for clinical trials underway bear out such a worst-case scenario, in which two shots Biosecurity in Pittsburgh, Pennsylvania and advantages, the US will have to balance its are required of a standard 15-microgram a physician at the University of Pittsburgh concerns for safety against the benefits, dose without adjuvant. Medical Center. particularly given the short supply of Given the potential of adjuvants to shrink The FDA has the authority to approve vaccine worldwide. the dosage, The World Health Organization ‘emergency use authorization’ with input “If they decide to not use adjuvants, has recommended their use in pandemic from other agencies such as the US National there is no doubt it will have a big impact H1N1 flu vaccine. Institutes of Health (NIH). But in a July on the supply of H1N1 vaccines globally,” The European Medicines Agency has a meeting of the committee that advises the says Ripley Ballou, deputy director for mechanism to quickly approve adjuvanted FDA on vaccines, Norman Baylor, director vaccines at the Bill and Melinda Gates vaccines with novel , such as of the agency’s Office of Vaccines Research Foundation, based in Seattle, Washington. H1N1, on the basis of presubmitted and Review said than an unadjuvanted The only adjuvant currently used in the US efficacy and safety data of vaccines H1N1 shot is “the most expeditious pathway is alum, whereas the adjuvants typically with other antigens. Novartis and for providing a safe and effective vaccine to paired with flu vaccines are oil and water GlaxoSmithKline, for instance, are lined the public.”

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program.” Researchers there have combined oil- has been tested in more than 4,000 infants volunteers. They injected volunteers with the in-water emulsions with various TLR activators, and children; recently, a study of 340 infants adjuvant mixed with RTS,S, which is based on such as one that activates TLR4 and another showed a safety profile comparable to the a malaria peptide, and then exposed them to that activates TLR7 and TLR8. In unpublished standard hepatitis B shot1. mosquitoes infected with the parasite. (That’s experiments, Reed says, they have found that the Developing the right adjuvant also was an approach Ballou had subjected himself to in combinations of TLR activators give extra zip central to getting a vaccine that, after more than a previous study, where he unfortunately ended to experimental vaccines for tuberculosis and two decades of development, has begun to look up getting infected and having to down a dose malaria (tested in primates) and leishmaniasis promising. In his first years working on malaria of antimalaria pills). (tested in dogs). vaccines, says Ballou, he and his colleagues In 1995, the researchers found that one Vaccine researchers praise the Seattle institute could barely get an immune response. Alum adjuvant, dubbed AS02, worked particularly for a program it operates in collaboration with just didn’t cut it. well—it protected six out of seven volunteers the World Health Organization that makes Alum is good at fostering an antibody bitten soon after their shot. And it produced standardized adjuvants, ready for clinical use, response, but antibodies, found Ballou, were not only a strong antibody response but also a available to outside researchers and vaccine not sufficient to thwart the malaria parasite. robust T cell response5. manufactures in developing countries. More Antibodies are decent at eliminating microbes That adjuvant consisted of an oil-in-water than 40 researchers have used the service, which in the bloodstream, but the malaria parasite emulsion, which bumped up the antibody bypasses the difficulties some face in getting worms its way to the liver, where it buries itself response, along with two substances—MPL and adjuvant from companies, says Reed. inside cells. another immune-stimulating agent, QS21— But getting new adjuvants such as Reed’s To get at the buried parasite, Ballou and his that not only further enhanced the antibody and Pulendran’s, with two TLR activators, colleagues at the Walter Reed Army Institute of response but also promoted the robust activity into vaccines for everyday use is a long shot, Research reasoned that they had to bump up the of T cells. That original adjuvant has since been cautions Dynavax’s Coffman. “There is always activity of T cells, which can kill parasites inside updated; the emulsion was replaced with lipid the potential for complications,” he says. “You cells. The breakthrough came in 1995, when vesicles called liposomes, creating a particle might synergize with regards to efficacy but you the researchers began testing experimental that gives the adjuvant an extra boost. This might also synergize with regards to side effects. adjuvants produced by GSK, which had begun adjuvant, dubbed AS01, was used in phase 2 If you can get the type of response you want to take these substances seriously. trials of the RTS,S vaccine recently conducted with a single agent, you might be better off.” “GSK had made a conscious decision in . that one of the new technologies that would Using this updated vaccine, Ballou and his Moving ahead with malaria open the potential for new vaccines was the colleagues last fall reported an approximately Safety has not been an issue to date with RTS,S, development of new adjuvant platforms,” 50% reduction in incidence of malaria in an experimental vaccine against malaria that Ballou says. “This was the very beginning.”After children who received the shot compared to is formulated with new-generation adjuvants, screening nine GSK adjuvants in animals, those who did not2. This formulation of the such as oil-and-water emulsions. The vaccine he and his colleagues tested three on human vaccine is now in phase 3 trials.

Still, the US government is clearly keeping Vaccines in Sienna, Italy. But the deliberation been acquired in older adults. Given that, the option of adjuvants open: in May and of long-term safety will have to incorporate the US will consider using adjuvanted © All rights reserved. 2009 Inc. Nature America, July, the country’s Department of Health and data from other vaccines, such as seasonal pandemic flu vaccine in children in “only Human Services arranged to buy nearly $500 flu jabs containing MF59 and AS03. extreme circumstances,” in a scenario million worth of MF59 from Novartis and Both of these adjuvants “have an excellent in which the unadjuvanted vaccine does more than $200 million of a similar adjuvant, safety profile in literally millions of doses,” not work, says , director of AS03, from GlaxoSmithKline. (If approved, says Steve Reed, head of research and the US National Institute of Allergy and the adjuvants would be mixed with the flu development at the Infectious Disease Infectious Diseases. antigen at the site of vaccine administration.) Research Institute in Seattle. MF59, for Charlotte Schubert, Washington, DC Dozens of studies with for instance, has been tested in have also shown a strong more than 26,000 people ‘dose sparing’ effect. Experiments with AS03 in more than 44 clinical suggest that it could reduce the dose thought trials, to be needed for protection in people from says 90 micrograms to as little as 3.8 micrograms Reed. (Curr. Opin. Mol. Ther. 3, 337­–345; 2009). Adjuvanted flu vaccines can also provide Although stronger, longer-lasting protection. vaccines The decision of whether to use adjuvants adjuvanted will depend partly on results of such trials. with AS03 The NIH, for instance, is initiating two and MF59 clinical trials of H1N1 vaccine using AS03; have been Novartis, meanwhile, began trials in July found to be safe in with MF59 and expects early results in children and young late September or early October, says Rino adults, the bulk of Rappuoli, head of research for Novartis the clinical data has

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to create a more specific immune Immunologists such as Pulendran are response. For example, malaria optimistic that some as yet undiscovered vaccines need to elicit a strong molecule could help researchers come closer T cell response, and HIV to the vision expressed by the chairman of the vaccines may need to Nobel Committee, Hilding Bergstrand, when elicit an immune he introduced Max Theiler before his Nobel response in the lecture in 1951: mucosa of the “Dr. Theiler’s discovery gives new hope that rectum and the in this manner we shall succeed in mastering vagina, where the other virus diseases, many of which have a virus first contacts devastating, effect and against which we are the body. Vaccines still entirely powerless.” containing smartly chosen combinations of immune-stimulating Charlotte Schubert is senior editor for News “This is a great example of the approach agents could guide the immune response in the and Views and is the Washington, DC for a new generation of vaccines,” says Reed. right direction. correspondent for Nature Medicine. “RTS,S would not have worked with alum or Researchers caution that there is a long way emulsions alone.” to go before vaccinology is a precise science. “I 1. Abdulla, S. et al. New Engl. J. Med. 359, 2533–2544 think we are not nearly as smart as we like to (2008). 2. Bejon, P. et al. New Engl. J. Med. 359, 2521–2532 Back to the basics think at predicting what type of adjuvant will (2008). Scientists are only just beginning to understand work best for a type of vaccine,” says Coffman. 3. Halperin, S.A. et al. Vaccine 24, 20–26 (2006). 4. Querec, T. et al. J. Exp. Med. 203, 413–424 (2006). how commonly used adjuvants such as alum “There is still a lot of trial and error,” says 5. Stoute, J.A. et al. New Engl. J. Med. 336, 86-91 work (sidebar). Meanwhile, some researchers Ballou. But he adds that things are getting (1997). have gone back to the basics. They emphasize better. “We are still relatively crude at this, but 6. Querec, T. et al. Nat. Immunol. 10, 116–125 (2009). that potent, traditional vaccines such as yellow we are much better at it than we were, say, ten 7. Gaucher, D. et al. J. Exp. Med. 205, 3119–3131 fever still have a lot to teach us. Ultimately, years ago,” says Ballou. (2008). understanding how simple vaccines can work without adjuvants might shed light on the type Key TLR-independent adjuvants in development for prophylatic vaccines of immune response that adjuvants need to Name Company Indication create. Alum various various Last winter, Pulendran and other researchers AS03 GSK Pandemic influenza applied large-scale gene analysis technology to MF59 Novartis Influenza come up with a gene ‘signature’ showing which genes are activated in people with an effective Provax Biogen Idec N/A immune response to the yellow fever vaccine6,7. Montanide Seppic SA, Bioven, Cancervax Malaria, cancer The studies are a “landmark in this field,” says TiterMax CytRx N/A Coffman. Advax Vaxine Pty various, including Hepatitis B, influenza, rabies, © All rights reserved. 2009 Inc. Nature America, Genes for several other immune cell receptors, and cancer in addition to Toll-like receptors, popped up QS21 Antigenics various, including , malaria, HIV in the analysis and were found to mediate the Quil A Statens Serum Institute various response to the vaccine. Pulendran’s group also Iscom various, e.g. CSL, Isconova various, including influenza found that the vaccine turned on the ‘stress Liposomes various, e.g. Crucell, Nasvax various response’, which readies the body for trouble such as oxygen deprivation or viral . Key TLR-dependent adjuvants in development for prophylactic vaccines “It was surprising,” says Pulendran. “I spent Ampligen Hemispherx Pandemic influenza hours on PubMed reading up on this stress AS01 GlaxoSmithKline Malaria, tuberculosis response protein I had never before heard of.” AS02 GlaxoSmithKline Malaria (Mosquirix), tuberculosis, HBV, HIV Perhaps, he says, clues to the next generation of AS04 GlaxoSmithKline various, including Fendrix (HBV) and Cervarix adjuvants could emerge from such data. Other (HPV) clues could also arise from even more basic MPL RC-529 Dynavax Supervax (HBV) research into how the immune system deals E6020 Eisai/ N/A with microbes. TLR-technology VaxInnate Influenza Pulendran is currently applying for funds PF-3512676 Coley/Pfizer, partnered with various, including HBV and influenza (Novartis) to the NIH to be part of a project to generate (CpG 7909) Novartis for some indications databases on how various vaccines and ISS Dynavax HBV (Heplisav), influenza adjuvants affect the human immune system, IC31 Intercell various, including influenza, tuberculosis, malaria, using in part large-scale genomics approaches, meningitis, and cancer indications now being ramped up in many vaccine labs. APC, antigen-presenting cells; EBV, Epstein-Barr virus; HBV, hepatitis B virus; HPV, human In the future, Pulendran and other papillomavirus; HSV, herpes simplex virus; MPL, monophosphoryl lipid A; N/A, not available; NSCLC, researchers envision, vaccinologists may be able non small cell ; O/W, oil-in-water; RSV, respiratory syncytial virus; TLR, toll-like receptor to exactly tailor the adjuvant to each vaccine Source: Vaccine Adjuvants-Uncertainties Rule, Datamonitor, MedTRACK (2008), Thomson Pharma (2008)

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