A Vaccine's Little Helper
As researchers discover more about the innate immune response, vaccine developers are broadening their understanding and use of adjuvants
By Andreas von Bubnoff
One of the most effective vaccines ever developed is the yellow fever vaccine. It protects more than 95% of vaccinees and induces balanced B- and T-cell responses that last several decades. Like other successful vaccines, such as those against measles, mumps, and rubella, the yellow fever vaccine is a live-attenuated version of the very pathogen it protects against.
But for HIV, using a live-attenuated version is considered too risky. Instead, HIV vaccine developers have focused on using HIV proteins as antigens. This is a much safer approach but also comes at a price—when the vaccine lacks many components of the actual virus, it is less effective because it doesn’t alert the immune system of a dangerous pathogen that it needs to mount an immune response against.
That’s where so-called adjuvants (from the Latin word adiuvare, to help) come into play. Adjuvants are substances added to vaccines to mimic the danger signals triggered by pathogens that activate the innate immune response, which in turn activates the later adaptive B- and T-cell immune responses. “Once you get further and further away from a living vaccine—an attenuated virus or bacterium—you become more and more dependent on adjuvants to essentially provide the innate immune trigger which we now recognize is so critical to getting good T- and B-cell responses,” says Bob Coffman, chief scientific officer for the biotech company Dynavax. “In a sense, the cleaner it is, the more you need to have an adjuvant to give you adequate responses and, just as importantly, adequate responses in a high percentage of people.”
Most currently licensed vaccines that contain an adjuvant contain alum, which consists of insoluble aluminum salts. Even though alum has been used as an adjuvant for over 80 years, its mechanism of action is still poorly understood (see The Mysteries of Alum). But as researchers gain a clearer understanding of how pathogens activate the innate immune response, they are better able to understand how existing adjuvants work and can use this information to develop new and improved adjuvants that can stimulate a more powerful immune response.
| The Mysteries of Alum |
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Even though alum, which consists of insoluble aluminum salts, has been used as an adjuvant for over 80 years, researchers are just beginning to understand how it works. According to Bob Coffman, chief scientific officer for the biotech company Dynavax, the original belief was that alum acts as a depot that holds immunogens in place so that they can be more efficiently taken up by antigen presenting cells, such as dendritic cells (DCs). But after a recent flurry of studies showed an immune stimulatory role of alum, “that’s pretty much out the window now,” Coffman says. One study involving a gene expression analysis in mice showed that alum induces many innate inflammatory genes, indicating that alum does stimulate the innate immune system (1). But exactly how alum stimulates an innate immune response remains unclear. In 2008, Stephanie Eisenbarth and Richard Flavell at Yale University reported that the stimulation of immune responses by alum in mice requires activation of an intracellular sensor called NLRP3, which is part of the inflammasome, a multiprotein complex inside the cell that activates inflammatory responses after detection of pathogens or cellular stress (2). Other researchers have confirmed that alum activates NLRP3, but did not find that this activation is required for the stimulation of immune responses. In addition, several recent studies have identified ways alum stimulates the innate immune response that are independent of inflammasome activation. One theory is that at least in macrophages, alum crystals are taken up by the cell, which then tries to digest it in phagolysosomes that eventually burst. This leads to the release of proteases—enzymes that can cleave proteins. Until recently, this enzyme release was thought to lead to the activation of the innate immune response by activation of the inflammasome, but earlier this year, Kuroda and colleagues showed that in mouse macrophages, this enzyme release can lead to Th2 type CD4+ T-cell responses and antibody production through a pathway that is independent of activation of the inflammasome (3). In addition, Yan Shi, an associate professor of microbiology at the University of Calgary, and colleagues recently reported that in DCs, alum can exert its immune stimulatory effects not only in the absence of an inflammasome, but even without entering the cell (4). They showed that alum crystals bind to certain lipids in the DC cell membrane more strongly than to others. As a result, certain lipids become more concentrated underneath the place where alum binds, which leads to a concentration of certain receptors associated with these lipids. These receptors can now interact with each other and start signaling, thereby activating the innate immune response in the DC. With so many different and sometimes conflicting results, it’s still far from clear how alum really works, says Coffman. “There are actually now too many explanations,” he says. —AvB |
Beyond Alum
Until two years ago, alum was the only adjuvant in licensed vaccines in the US. In 2009, the US Food and Drug Administration (FDA) approved Cervarix, a human papilloma virus (HPV) vaccine made by GlaxoSmithKline (GSK), which contains AS04, an adjuvant made by GSK that combines alum with monophosphoryl lipid A (MPL), a detoxified form of bacterial lipopolysaccharides (LPS).
But in Europe, alum lost its status as the only adjuvant in licensed vaccines much earlier. MF59, for example, an emulsion of a biodegradable oil called squalene in water—which was discovered in the early 1990s by Chiron (now Novartis)—was first licensed with the flu vaccine Fluad in Europe in 1997, and has since been licensed in flu vaccines in many other countries other than the US, according to Derek O’Hagan, the global head of vaccine delivery and formulation research at Novartis, where he also manages the adjuvant team.
Meanwhile, researchers have been accumulating evidence that suggests that many adjuvants are better than alum in their stimulation of the innate immune response. “Alum is kind of the baseline, it’s pretty weak and just about every other adjuvant you can talk about would be more potent,” says O’Hagan. But one reason vaccines with novel adjuvants are slow to get approval is that there are more safety data for alum, says Wolfgang Leitner, a program officer at the adjuvant discovery program of the division of allergy, immunology and transplantation (DAIT) at the US National Institute of Allergy and Infectious Diseases (NIAID), adding that regulatory authorities are more cautious in the US than in Europe. “The fear of adjuvants in the US is higher than in Europe, and in part it is a litigation issue,” Leitner says. “[There is] more suing and more threat of suing [for] adverse effects.”
But the recent approval of a vaccine that contains AS04 in the US has sparked hope that this will pave the way for the approval of vaccines that contain new adjuvants. “The success of the HPV vaccine with AS04 is obviously a potential jumping point for making the United States a little more relaxed about having new adjuvants,” says Carl Alving, the chief of the department of adjuvant & antigen research at the US Military HIV Research Program (MHRP).
The innate response
Adjuvants are thought to work by stimulating the innate immune response, often in dendritic cells (DCs), but also in other cells like macrophages. “Any vaccine that works, works by getting to dendritic cells,” says Sarah Schlesinger, an associate professor of clinical investigation at Rockefeller University. “Adjuvants are all supposed to get to dendritic cells one way or the other.” Once DCs are stimulated, they activate the later adaptive B- and T-cell immune responses by producing cytokines and presenting antigens to CD4+ or CD8+ T cells.
One key to understanding how adjuvants work has been the identification of receptors that innate immune cells such as DCs use to sense pathogens. The first such receptors researchers discovered were toll like receptors (TLRs), one of which, TLR4, senses bacterial LPS. “The field of innate immune receptors really started to get off the ground in the mid-1990s with the discovery of TLR4,” says Thomas Palker, a program officer for the adjuvant development program at NIAID’s DAIT.
Today, 10 functional TLRs have been identified in humans, and other types of innate immune receptors have been identified that can sense other pathogen-related stimuli, such as double-stranded RNA (dsRNA), or danger signals such as physiological changes that are the result of cell death or tissue damage, says Palker. “The definition first of all of the toll-like receptors and then many of the other innate immune receptors that followed along was probably the key intellectual and scientific breakthrough that allowed you to begin to understand how some of the adjuvants work,” Coffman says.
And the number of receptors continues to grow. Recently, Jeremy Luban and colleagues reported evidence that suggests that the host cell restriction factor TRIM5 is the first known pattern recognition receptor that specifically recognizes retroviruses, including HIV, and activates the innate immune response in DCs (5; see A Flurry of Updates from Keystone, IAVI Report, Mar.-Apr. 2011). Luban says this finding might lead to the development of more specific adjuvants for HIV vaccine candidates.
But Coffman isn’t so sure. “[What one] really needs to do is to be able to trigger the type of response you need to be protective,” Coffman says. “It doesn’t really matter whether it replicates some part of the normal recognition of the pathogen in any way. Given that natural HIV infection rarely, if ever, produces protective immunity, one might even suggest that TRIM5 is a bad candidate for an HIV vaccine adjuvant!”
Knowledge of the innate immune receptors activated by pathogens and adjuvants enables researchers to design adjuvants that can stimulate a combination of different receptors to see if this results in an improved stimulation of the innate immune system. Recently, Bali Pulendran, a professor of immunology at Emory University, and colleagues combined adjuvants that activate TLRs 4 and 7 with a nanoparticle and showed that the combination can lead to higher and more durable antibody and CD8+ T-cell responses in mice than nanoparticles with just one TLR ligand (6). They found that a combined delivery of MPL, a TLR4 ligand, and imiquimod, a TLR7 ligand, on a nanoparticle can synergistically increase the antibody titers to immunogens such as Ovalbumin delivered on a separate nanoparticle. Combined delivery of the two TLR ligands didn’t make a difference in the acute short-term antibody response, Pulendran says. But only the mice that received both TLR ligands developed a long term memory B-cell response that lasted 550 days, which is the life span of a mouse. Immunization with particles containing only a single TLR ligand didn’t develop such long lasting responses. “That was amazing,” Pulendran says. “When I saw this data my jaw dropped.” The researchers also showed that in nonhuman primates (NHPs), the nanoparticle vaccine could induce an antibody response to an H1N1 swine flu strain for at least 80 days.
Pulendran says these findings are relevant for HIV vaccine development because they suggest ways to make immune responses more persistent. “It’s important to get protection, but equally important to maintain it over time,” Pulendran says, referring to the RV144 trial where the initial protection observed waned after one year. “I don’t think anyone knew until this paper what role can adjuvants and the innate system play in the persistence of the immune responses,” he says. “There needs to be a very careful evaluation of TLR ligands in the context of HIV vaccines.” In collaboration with Juliana McElrath at the Fred Hutchinson Cancer Research Center, Pulendran plans to use the nanoparticle vaccine to look at immune responses to HIV Envelope antigens in NHPs.
Discovering new adjuvants
The better understanding of the receptors and pathways inside cells that are activated by adjuvants and pathogens also makes it possible to identify new adjuvants. One such effort is the adjuvant discovery program at NIAID’s DAIT, says Leitner, who is in charge of the program. “This [program] started with the recognition that there has to be more targeted systematic research to find new adjuvants,” he says. “Adjuvant research up to that point was really just a random process of chance discovery of compounds that happened to trigger inflammatory responses.”
The first round of the program started in 2003 with five contractors and the goal of identifying new TLR agonists. A US$60 million, 5-year second round was started in 2009. The six current contractors include academic groups and companies that do large scale screens of chemical libraries to identify compounds that can stimulate different elements of the innate immune response, not just TLRs. Once identified, the compounds are then narrowed down to ones that can activate the types of inflammatory signals that are most desirable for an adjuvant response, such as type I interferon. “You are selecting the compounds based on [the] specific pathways that they trigger,” says Leitner, adding that the program also funds approaches that aim to identify completely new innate immune receptors.
| Most Detailed 3-D Model of HIV Made to Date |
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This model of HIV is the most detailed 3D-model of the virus made to date. It summarizes the results from scientific publications in the fields of virology, X-ray analysis, and NMR spectroscopy. Model denotes the parts encoded by the virus’s own genome in orange, while grey shades indicate structures taken into the virus when it interacts with a human cell. A version of this image took first place in the illustrations category of the 2010 International Science & Engineering Visualization Challenge, sponsored by the National Science Foundation and the American Association for the Advancement of Science. Image courtesy of Ivan Konstantinov, Yury Stefanov, Alexander Kovalevsky, Yegor Voronin | Visual Science,www.vsci.us |
HIV vaccine adjuvants
In addition to the handful of adjuvants that are already in approved vaccines, many more are in preclinical development or early-stage clinical trials. But choosing the best adjuvants for HIV vaccine development is difficult because the correlates of protection from HIV are still unknown, and it’s unclear what kind of immune response a vaccine should induce. “Until you know what a protective response is, choosing a right adjuvant is almost a meaningless exercise,” Coffman says.
So far, the adjuvant of choice in most late-stage clinical trials of HIV vaccine candidates has been alum, which, when administered with a protein vaccine mostly stimulates CD4+ T-cell and antibody responses. Alum was used as an adjuvant in both the VAX003 and 004 trials of AIDSVAX, an HIV gp120 candidate that didn’t show any efficacy in protecting against HIV. Alum was also used in the AIDSVAX boost of the recent RV144 trial in Thailand. There was no adjuvant in the canarypox vector-based ALVAC prime in RV144 because viral vectors are believed to stimulate stronger innate immune responses than protein vaccines, according to Nelson Michael, director of MHRP, a key collaborator on RV144. This is also the reason why the adenovirus serotype 5 (Ad5) based MRKAd5 vaccine candidate that was used in the STEP trial did not contain an adjuvant. However, Michael adds, some researchers are just beginning to explore the use of adjuvants with viral vectors.
Michael says alum probably won’t be used in RV144 follow-up trials. Instead, the Phase IIb trial that will test a candidate vaccine regimen similar to RV144 in high-risk heterosexual men and women in South Africa will likely use Novartis’ oil in water adjuvant MF59 with the protein boost. For another efficacy trial in men who have sex with men slated to start in Thailand in 2014, Michael says “we are deliberating a switch to MF59 but need to look at immunogenicity in a Phase I [trial] before making a final decision.”
Evidence that MF59 is a more potent adjuvant than alum in humans has been building for some time. According to Alving, a Phase I clinical trial in the 1990s called AVEG 015 compared the immune responses of several adjuvants, including MF59, to alum, together with an HIV gp120 protein candidate vaccine. This trial suggested that alum induced the lowest antibody responses. “It wasn’t clear that there was a single winner, but it was clear that there was a single loser and that loser was alum,” Alving remembers (7).
Later, two Phase IIa trials suggested that a boost with MF59, when combined with the same prime as the one used in RV144, elicited better immune responses than a boost with alum (8; 9). Because the boost that was used with MF59 also contained a slightly different Env protein, it wasn’t clear whether the better immune responses were the result of the MF59 adjuvant or the different protein or both, Michael says. Still, at the time, this evidence, which was available before the start of RV144 in 2003, would have been enough to make a decision to choose the MF59 containing boost for RV144. However, Chiron (now Novartis), the company that made the MF59 adjuvanted boost, pulled out, Michael says.
More recent studies also have shown that MF59 is a more powerful inducer of innate inflammatory genes than alum (1). MF59 also has a dose sparing effect compared with alum, says Susan Barnett, senior director of vaccines research at Novartis Vaccines. That means that less of the vaccine is required for the same immune response. “For HIV it is a very, very urgent issue to get the dose of Envelope down because the yields are difficult and the protein is precious,” she says.
An adjuvant called PolyICLC—a synthetic dsRNA that binds to TLR3 and another receptor inside the cell called MDA5—is currently being tested in a Phase I clinical trial of an HIV vaccine candidate called DCVax-001, led by Ralph Steinman and Schlesinger at Rockefeller University (see Vaccine Briefs, IAVI Report, July-Aug. 2010). The vaccine contains an HIV Gag protein fused to a monoclonal antibody (mAb) that binds to a DC specific protein called DEC-205. “The monoclonal antibody brings the Gag p24 directly to the dendritic cells, which is where we believe it needs to get to to induce immunity,” Schlesinger says. The researchers chose PolyICLC because unlike alum, PolyICLC matures the DCs so they don’t just take up the antigen, but also present it to T cells to induce an adaptive immune response, says Schlesinger. Experiments in NHPs have shown that this DEC-205 targeted PolyICLC adjuvanted vaccine can induce both CD4+ and low level CD8+ T-cell responses, says Robert Seder, the chief of the cellular immunology section at the Vaccine Research Center (VRC) at NIAID, who led the studies (10). This is promising evidence that a protein vaccine platform can induce Th1 type CD4+ and CD8+ T-cell responses, Seder says. But he cautions that for now, vaccines that use viral vectors such as adenoviral vectors, are still more efficient in eliciting robust CD8+ T-cell immunity than protein based vaccines. Future studies using optimized DC targeting vectors may further enhance their ability to induce CD8 immunity.
The PolyICLC adjuvant induces the expression of similar innate immune response genes as the live-attenuated yellow fever vaccine when injected subcutaneously into humans, according to Rafick Sekaly, the co-director and chief scientific officer at the Vaccine and Gene Therapy Institute of Florida. Sekaly has been using microarrays to measure the innate immune response genes that are induced in response to subcutaneous injection of PolyICLC in collaboration with Steinman and Schlesinger. “Initially we did not expect that a very small molecule like PolyICLC would induce an innate immune response similar to a complex virus as yellow fever, but that’s what we saw, and it’s very encouraging,” Sekaly says.
Next, Sekaly plans to measure gene expression changes in volunteers from the DCVax001 trial, who were vaccinated with the DEC-205 vaccine with PolyICLC, and also in people injected with other adjuvants including MF59 and GLA, an adjuvant developed by the Seattle-based non-profit Infectious Disease Research Institute. GLA is a synthetic glycolipid based on MPL that activates the TLR4 pathway (see also An Immunological Rationale for Vaccines, IAVI Report, Nov.-Dec. 2010). Schlesinger and her colleagues also plan to test GLA in a Phase I trial of future versions of their DC directed vaccine, Schlesinger says.
Another non-alum adjuvant currently in a Phase I HIV vaccine trial is GSK’s AS01, which contains MPL and QS21, a saponin derived from the bark of the Quillaja saponaria Molina tree. GSK is currently collaborating with IAVI to test AS01 with an HIV Gag-Rev-Nef fusion protein called F4 in the B002 trial. In this trial, F4/AS01 is administered in a prime-boost regimen with an Ad35 vector-based vaccine candidate (see Vaccine Briefs, IAVI Report, Mar.-Apr. 2011).
AS01 does not induce CD8+ T-cell responses, but does induce a high titer of antibody responses and sustained and high level CD4+ T-cell responses, according to Gerald Voss, the head of the disease area program for emerging diseases and HIV at GSK. It does so better than alum, Voss adds, referring to a trial conducted more than ten years ago that showed that an earlier version of AS01 led to much better antibody and CD4+ T-cell responses than alum when combined with a gp120 HIV protein (11). In 1997, GSK also showed that the malaria vaccine candidate RTS,S (now in Phase III trials) protected against malaria in humans when administered with an adjuvant related to AS01 called AS02 (an oil in water emulsion which contains MPL and QS21), whereas with an oil in water emulsion alone or with an alum/MPL combination, it did not provide protection (12). AS01 was later shown to provide better protection against malaria and better antibody and CD4+ T-cell mediated immune responses than AS02 (13).
Preclinical studies
Researchers are also comparing immune responses to different combinations of adjuvants in NHPs. Seder and colleagues at the VRC are collaborating with Novartis to compare the types of CD4+ T cells and the resulting antibody responses induced by alum and other adjuvants with an HIV Env clade C trimer protein provided by Novartis. They want to see whether MF59 is better than alum, and whether adding the TLR4 ligand MPL or a TLR7 ligand can improve the alum or MF59 adjuvant effects, Seder says. These adjuvants are being studied because they have been used in humans, but the tests also include PolyICLC and an adjuvant called ISCOM (which is based on saponins), because they stimulate the innate immune response through different pathways. “Based on that, we can then narrow the scope to just maybe one or two adjuvant candidates that would be better than alum or perhaps even better than MF59,” says Seder. Already, some combinations appear to give a higher HIV Env clade C CD4+ T-cell and antibody response than alum, he adds.
While researchers don’t know what the ideal antibody and CD4+ T-cell response against HIV is, Seder hopes the different adjuvants he is testing in NHPs will provide insight into the type of response that will improve durability, magnitude, and ultimately neutralization ability of the immune responses. He says emphasis will be on how the adjuvants influence the induction of T follicular helper cells, which are believed to be important for affinity maturation of antibodies and therefore for the development of broadly neutralizing antibody responses.
Robert Johnston, executive director of the not-for-profit company Global Vaccines, and colleagues are developing an adjuvant that is designed to specifically target the induction or stimulation not only of systemic immunity, but also of mucosal immune responses, which are considered very important for protection against HIV. The adjuvant is based on alphavirus particles that only contain an RNA molecule with genes that enable it to make dsRNA copies of itself. Once inside a cell, the alphavirus particles therefore can’t spread to other cells, but instead only generate many dsRNA molecules, Johnston says.
In monkeys, he has shown that adding the alphavirus adjuvant to the commercially available killed flu vaccine results in 20 times more antibody. In mice, even an intramuscular vaccination results in mucosal immune responses, presumably because the adjuvant somehow induces types of B and T cells that migrate to the mucosal tissues. “In terms of the mucosal induction I think [the adjuvant] is unique,” says Johnston, who is now testing the alphavirus adjuvant with an HIV Env candidate vaccine in mice.
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