Taking toll: lipid A mimetics as adjuvants and immunomodulators

Taking toll: lipid A mimetics as adjuvants and immunomodulators

Review | A TRENDS Guide to Infectious Diseases Trends in Microbiology Vol. 10 No. 10 (Suppl.), 2002 Taking toll: lipid A mimetics as adjuvants and...

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A TRENDS Guide to Infectious Diseases

Trends in Microbiology Vol. 10 No. 10 (Suppl.), 2002

Taking toll: lipid A mimetics as adjuvants and immunomodulators David H. Persing, Rhea N. Coler, Michael J. Lacy, David A. Johnson, Jory R. Baldridge, Robert M. Hershberg and Steven G. Reed Vaccine adjuvants based on the structure of lipid A, such as monophosphoryl lipid A (MLA), have proven to be safe and effective in inducing immune responses to heterologous proteins in animal and human vaccines. Recent work on the development of a recombinant vaccine for leishmaniasis has demonstrated that a clinical grade MLA formulation – MPL adjuvant – is essential in the development of a protective response. Preliminary evidence suggests that MLA and a chemically distinct family of lipid A mimetics – the aminoalkyl glucosaminide 4-phosphates – act on Toll-like receptor 4 (TLR4). As TLR4 agonists, they have potent immunomodulatory effects when used both as vaccine adjuvants and as stand-alone products. Novel approaches to vaccine development could benefit from taking full advantage of the effects of these compounds on innate and adaptive responses.

*David H. Persing, Robert M. Hershberg Steven G. Reed Corixa, Suite 200, 1124 Columbia Street, Seattle, WA 98104, USA, and The Infectious Disease Research Institute, Suite 600, 1124 Columbia Street, Seattle, WA 98104, USA. *e-mail: [email protected]

Rhea N. Coler The Infectious Disease Research Institute.

Michael J. Lacy, David A. Johnson Jory R. Baldridge Corixa, 553 Old Corvallis Road, Hamilton, MT 59840, USA.

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In recent years, significant advances in our understanding of the mechanisms of innate immunity have evolved from initial observations of fruit-fly developmental mutants to the discovery of a family of 10-or-more distinct receptors collectively called human Toll-like receptors (TLRs) [1].Tolllike receptors recognize structural components of pathogens and, through this interaction, propel the immune system into an activated state [2,3]. Within minutes of recognizing pathogen components, an array of cell types initiate defensive actions that mediate protection against the invading pathogen, including the production of reactive oxygen intermediates and secretion of inflammatory cytokines, including interleukin (IL)-1, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-12. These cytokines activate natural killer cells and also initiate a cascade of signals to cells of the adaptive immune response, preparing them for the development of antigen-specific immune responses. Lipopolysaccharide (LPS) exposure also results in production of defensins, which comprise several distinct families of antibacterial, antifungal and antiviral peptides [4]. There is substantial interest in the development of agonists and antagonists of TLRs because pharmacological manipulation of innate immune responses might lead to more effective vaccines and novel therapeutic approaches to autoimmune, atopic, malignant and infectious diseases [5]. Recently, it has become clear that most vaccine adjuvants and stand-alone immunomodulators act on various members of the Toll-like receptor family [6]. The first microbial product discovered to be a Toll-like receptor agonist was LPS, a bacterial membrane component specific to Gram-negative bacteria, which activates Toll-like receptor 4 (TLR-4) [7–9].

Although LPS is a potent immunomodulatory agent, its medicinal use is limited because of its extreme toxicity, including the induction of a sepsis-like systemic inflammatory response syndrome. In the 1980s, scientists at Ribi Immunochem (now Corixa; http://www.corixa.com/) studied methods of chemically modifying lipid A with the intention of uncoupling its toxic effects from potentially useful immunomodulatory effects. This resulted in the identification of monophosphoryl lipid A (MLA), an acylated diglucosamine derivative of lipid A from Salmonella minnesota RC595. Although it displays greatly reduced toxicity, MLA retains many of the immunomodulatory properties of the parent LPS molecule. Refinement of MLA by selective hydrolysis of the B-hydroxymyristoyl residue attached to the 3-position of the reducing (so-called ‘right handed’) sugar, further reduced the pyrogenicity of the molecule without destroying its adjuvant properties; Fig. 1 shows a congener that corresponds to the main active component of Corixa’s MPL adjuvant in its various formulations. In numerous preclinical and clinical studies, MPL adjuvant has proven to be a potent yet apparently non-toxic vaccine adjuvant when administered with heterologous antigens; it has been used extensively as an adjuvant in human vaccine trials for several infectious disease and cancer indications. To date, over 33 000 doses have been administered to over 12 000 individuals, with a side-effect profile similar to that of alum [10–14]. This review provides an update on the adjuvant effects of lipid A mimetics, and provides further evidence that the modeof-action of these compounds is through TLR4-dependent induction of the innate immune response.

1471-4914/02/$ – see front matter ©2002 Elsevier Science Ltd. All rights reserved. PII: S0966-842X(02)02426-5

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Trends in Microbiology Vol. 10 No. 10 (Suppl.), 2002

Evaluation of adjuvants in leishmaniasis vaccine development Human leishmanial infections can result in a wide range of clinical manifestations, from a self-healing cutaneous lesion, to a disseminated visceral form that is often fatal [15–17]. Chemotherapeutic regimens are of limited effectiveness and are associated with significant side-effects. The development of safe and effective vaccines has been a goal since the first realization that individuals who recover from infection are immune to re-infection [15]. Several years ago, scientists at the Infectious Disease Research Institute (http://www.idri.org) and Corixa embarked on a campaign to discover leishmanial antigens for the development of a recombinant subunit vaccine. Many candidate antigens were identified on the basis of serological and T-cell expression cloning [18–21], three of which (TSA, LmSTI1 and LeIF) were incorporated into a single recombinant polyprotein (Leish-111f). Leish-111f is the basis of a new investigational vaccine for leishmaniasis, which is scheduled to begin clinical trials this year. A good example of the importance of adjuvants in vaccine development is provided in our experience of developing the Leish-111f vaccine. We have focused on adjuvants that have: (1) the ability to stimulate Th1 responses, which are known to generate protective responses in animal models; and (2) a documented record of safety in humans. Little or no protection was gained with the vaccine in alum alone (unpublished results). We thus evaluated and compared the immunogenic and protective efficacies of Leish-111f with MPL-SE, which consists of MPL adjuvant formulated as an oil-in-water emulsion. Recombinant Leish-111f was formulated with 5, 10 or 20 µg of MPL-SE and administered three times over a nine-week period. Three weeks after the last immunization, mice were challenged with 2 × 105 Leishmania major metacyclic promastigotes by injection into the left footpad. Lesion development was scored weekly and expressed as mm of footpad swelling. In the these studies, leishmanial infections progressed similarly in vaccinated and control groups for the first two weeks. Thereafter, lesion size progressed at a more rapid rate in mice injected with saline or adjuvant alone, compared with mice immunized with Leish-111f in combination with 5, 10 or 20 µg of MPL-SE. These findings correlated with immunogenicity data wherein mice that developed a protective immune response had substantially higher levels of IgG2a antibody titers and IFN-γ production from T lymphocytes, compared with non-protected or non-healing mice.The most effective combination was Leish-111f formulated with 20 µg of MPL-SE, wherein protection was observed for at least 14 weeks (Fig. 2). Interestingly, we also observed that the two higher doses of MPL-SE alone, without Leish-111f, resulted in a substantial reduction in footpad swelling. This effect http://www.trends.com

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Figure 1. Chemical structure of the main active component of MPL® adjuvant

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Monophosphoryl lipid A is extracted from Salmonella minnesota as a mixture of tetra-, penta- and hexa-acylated forms. Each congenger has been synthetically reproduced and tested for individual reactivity. Shown here, is the most active congener, the synthetic form of which is referred to as RC528.

(C14) (C14) (C12)

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might have been caused by enhancement of innate immune responses by MPL adjuvant in our model (see later). From these experiments, we found that Leish-111f has a relatively low inherent immunogenicity, despite the strong intrinsic adjuvant effect of one of its protein components (LeIF) [18]. Changing the challenge dose from 2 × 105 to 4 × 105 L. major metacyclic promastigotes affected the kinetics of footpad swelling, but did not change the protective effect of Leish-111f with MPL-SE. Additional experiments showed that similar protection results were achieved when mice were given two rather than three subcutaneous immunizations of the Leish-111f vaccine, followed by challenge with 2 × 105 L. major promastigotes or 8 × 103 L. major amastigotes, and that protective responses persisted for at least three months post-vaccination (unpublished results). Finally, we have tested the Leish-111f–MPL-SE vaccine for its ability to protect against another species, Leishmania amazonensis. Studies to-date indicate that infections progress similarly in vaccinated and control groups for the first nine weeks. Thereafter, lesion size progressed at a more rapid rate in mice injected with saline or adjuvant alone, whereas the majority of the animals (3 out of 5) receiving the vaccine showed evidence of lesion regression within the next few weeks. Mucosal immunity Preclinical studies indicate that MPL adjuvant is an unusually effective mucosal adjuvant [13]. When applied to intranasal [13] and oral [22] mucosal surfaces, MPL adjuvant promotes antigen-specific immune responses at proximal and distal mucosal sites, as well as systemic immunity. In a representative set of experiments, ICR mice were vaccinated with a

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A TRENDS Guide to Infectious Diseases

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Trends in Microbiology Vol. 10 No. 10 (Suppl.), 2002

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Figure 3. Chemical structure of RC-544

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RC-544 is a member of the aminoalkyl glucosaminide-4phosphate family of lipid A mimetics. Members of this family of synthetic molecules have a wide range of immunomodulatory properties mediated by their interaction with the Toll-like receptor 4 signaling complex.

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Figure 2. Immunization against leishmaniasis using Leish-111f and MPL-SE BALB/c mice were immunized three times, three weeks apart with Leish-111f in combination with 5, 10 or 20 µg of MPL-SE or with the adjuvant alone. Three weeks after the last immunization, mice were challenged with 2 × 105 Leishmania major metacyclic promastigotes into the left footpad. Lesion development was scored weekly and is expressed as the mean ± S.E.M. of footpad swelling. These data reveal a significant prophylaxis against Leishmanial infection following immunization with recombinant Leish-111f protein administered with MPL-SE. In addition, these data demonstrate limited, dosedependent protection against Leishmanial infection with the MPL-SE adjuvant alone.

detergent-split influenza vaccine applied intranasally. The equivalent of 0.1 µg of influenza hemagglutinin plus 10 µg of MPL adjuvant or a synthetic lipid A mimetic RC-544

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(Fig. 3) was administered on days 0, 14 and 28. Serum and vaginal wash samples were harvested 14 days after the final vaccination and evaluated for anti-influenza antibodies. The remaining cohorts in each group were challenged by intranasal delivery of a lethal dose of infectious influenza A/HK/68 35 days after the final vaccination. Influenza vaccine formulated in dipalmitoyl-phosphatidylcholine, which was used in the aqueous formulation of the lipid A mimetics, was used as a control formulation. We found that intranasal administration of influenza vaccines adjuvanted with either MPL adjuvant or RC-544 resulted in the development of both systemic and mucosal immune responses (Table 1).The presence of IgA was documented in vaginal washes, even though the vaccine was administered intranasally. More importantly, enhanced protection against infectious influenza virus challenge was observed. The responses mediated by these adjuvants were equivalent to those elicited by the more classical mucosal adjuvant, wild-type heat-labile toxin (LT) from E. coli. Although LT might be considered too toxic to be used for mucosally-delivered vaccines, MPL adjuvant and the aminoalkyl glucosaminide 4-phosphates (AGPs) might provide safe and effective mucosal adjuvants for vaccines delivered to mucosal surfaces. Additional studies with other model antigens confirm the results found in the influenza model. Furthermore, we have found that cell-mediated responses – including cytotoxic T-lymphocytes (CTL) – are induced following mucosal administration of vaccines adjuvanted with MPL adjuvant, RC-544 or other AGPs. http://www.trends.com

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Mechanisms-of-action of MLA The evidence in-hand that MLA operates by specific activation of TLRs is indirect but, nonetheless, compelling. Interferon-γ is produced following intravenous administration of MLA in C57Bl/6 mice. However, in the LPS non-responsive mouse strain C3H/HEJ, no IFN-γ is produced after intravenous exposure of MLA. C3H/HEJ mice harbor a single-point mutation resulting in an amino acid change in TLR-4; because of this mutation the mice are hyporesponsive to LPS and lipid A [7,23]. We tested MPL adjuvant and its hexaacyl, pentaacyl and tetracyl congeners alongside LPS and CpG in a mouse model using cytokine production and nonspecific B-cell proliferation as a biological endpoint. To demonstrate the latter, we used a bioassay for quantitative detection of anti-hapten IgM in culture supernatants of mouse splenocytes stimulated in vitro. Lipopolysaccharide and MPL adjuvant induced littleor-no IgM anti-fluorescein antibody in C3H/HEJ mice, in contrast to BALB/C mice, where florid production of antihapten antibody was produced from splenocytes (Table 2). By contrast, a CpG oligonucleotide with an immunostimulatory motif from bacterial DNA was reactive in both strains of mice, consistent with an independent mode-of-action on a different Toll-like receptor,TLR9 [24,25].These mechanistic studies suggest that the primary site-of-action of MPL adjuvant is TLR-4, as is the case for lipid A and LPS. As TLR-4 has been shown to be present on tracheobronchial epithelial cells and antigen-presenting cells, one explanation for the immunomodulatory and adjuvant effects of MPL after mucosal administration is the local stimulation of TLR4 at the site of antigen deposition at these crucial locations. Modulation of innate immunity to enhance resistance to infectious challenge Several studies have been published describing the immunostimulatory effects of MLA after systemic administration [14]. However, little work has been done to show whether MLA has immunostimulatory effects when administered as a stand-alone-agent to the airways.The effectiveness of lipid A mimetics for airway protection against infectious challenge would probably be a function of: (1) the presence of the therapeutic target on epithelial cells, Langerhans cells, and other cells at the site of deposition; (2) the local concentration of lipid A mimetic at the site of activation of innate immune responses; and (3) the ability of target cells to respond in a protective fashion. In one representative experiment, a mouse dose of 20 µg MLA was given to cohorts of 10 female BALB/c mice by intranasal administration, either two days before or the day of lethal influenza virus challenge. All mice were challenged with approximately 2 lethal dose (LD)50 infectious influenza A/HK/68 administered intranasally. Mortality was monitored for 21 days following http://www.trends.com

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Table 1. Mucosal adjuvant effects of lipid A mimetics Adjuvant

a

Serum titers (IgG)

VW titers (IgA)

Percentage protection

Non-immune

<400

<20

0

AF-vehicle

102 400

160

60

MPL-AF

>2 048 000

2560

100

RC-544-AF

>2 048 000

10 240

90

Non-immune

<400

<20

22

AF-vehicle

102 400

160

50

Experiment 1

Experiment 2

RC-544-AF

>819 200

2560

100

LT

>819 200

2560

100

ICR mice were vaccinated three times by intranasal administration with 0.1 µg influenza HA and 10 µg adjuvant. Serum was collected 14 days following the third vaccination. Vaginal wash (VW) samples were collected 14 days after the third vaccination. Mice were then challenged 35 days after the third vaccination with infectious influenza A/HK/68. Abbreviations: AF, aqueous formulation; HA, hemaglutinin; IgA, immunoglobulin A; IgG, immunoglobulin G; LT, heat labile toxin from E. coli; MPL, monophosphorylated lipid A; RC, Ribi compound. a

influenza challenge (Fig. 4).Whereas 60% of untreated mice were dead at 21 days, the mortality rate was halved for mice treated with 20 µg MLA the same day as the virus challenge. Strikingly, 100% of mice treated prophylactically were protected against lethal influenza virus challenge – equivalent to the most effective vaccination regimens. The protective effects of MPL are not limited to viral infections; similar results have been shown after airway delivery of Listeria monocytogenes (unpublished results). AGPs: the next generation of lipid A mimetics Drawing from our experience with MPL and other bacterial cell-wall components, a library of novel synthetic

Table 2. Nonspecific B-cell proliferation measured by anti-hapten antibody production in response to a lipid A mimetics Compound

BALB/c

C3H/HEJ

Monophosphoryl lipid A

+++

none

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+++

none

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none

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none

+

none

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++

Hexa-acyl MPL



Penta-acyl MPL Tetra-acyl MPL CpG



a

Monophosphoryl lipid A is a family of molecules possessing various degrees of ocylation. The major species, hexa-, penta-, and tetra-acyl, were isolated and compared.

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100 90 80 Percent survival

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Figure 4. Nonspecific protection against lethal influenza challange MPL in an aqueous formulation administered via intranasal application 2 days before influenza infection (red squares) conferred 100% protection against viral challenge. The same dose, route, and formulation administered on the day of infection (green triangles) had an intermediate protective effect compared with vehicle alone (blue circles). These data are representative of more than 10 experiments, with highly reproducible results.

glycolipids – the AGPs – has been developed [26]. These molecules are acylated monosaccharides that are structurally related to lipid A; they incorporate a spacer arm that carries acyl side-chains in a configuration similar to the

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Figure 5. Structure-based approach to AGP design The twist-boat conformation of a biologically active component of MPL is the energetically favored form that is mimicked by the aminoalkyl glucosaminide 4-phosphates (AGPs).

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‘twist-boat’ conformation of MPL component RC-528 (Fig. 5), thereby enabling energetically favored close-packing of the fatty acyl chains [27–29]. The AGPs exhibit immunostimulatory activity comparable to or better than MLA in preclinical studies. Additionally, several of the AGPs demonstrate mucosal adjuvant activity. A significant advantage of the AGPs is that they were designed so that their chemical moieties can be modified for improved performance. These modifications could result in enhanced biological activity, enhanced stability with increased resistance to enzymatic and chemical degradation, and/or improved safety profiles. Because they can be synthesized organically in large-scale, high-purity batches that are free of the trace biological contaminants found in MLA, we believe that, in certain scenarios, AGPs could have advantages over MLA as vaccine adjuvants, such as in pediatric immunization protocols where adjuvant pyrogenicity must be minimized. The AGP, RC-529 (Fig. 6), is a good example. Extensive preclinical studies at Corixa have already demonstrated that RC-529 is as effective a vaccine adjuvant as MPL, and has a substantially better safety profile. For example, rabbit pyrogenicity testing shows that RC-529 is much less pyrogenic than MPL, and thus might be the preferred compound for pediatric application. As a result, RC-529 (now called Ribi.529) has become Corixa’s lead synthetic vaccine adjuvant for the development of next-generation vaccines. Discussion It has been more than two decades since Edgar Ribi and colleagues embarked on studies to understand the adjuvant effects of LPS and, in subsequent studies, uncouple the adjuvant effects of this molecule from its toxicity. Lipopolysaccharide toxicity, it was later discovered, was largely caused by the massive, coordinated release of cytokines such as IL-1, IL-10, IL-12, and TNF-α. However, the actual molecular mechanisms by which LPS and its relatives signal cytokine release from antigen-presenting cells via a tri-molecular complex (CD14–MD2–TLR-4) would take years to elucidate. During the past decade, much has been learned about the adjuvant activity of lipid A and its chemical cousins, most notably MLA and the AGPs. MPL adjuvant has now been produced on an industrial scale and injected into thousands of individuals as an essential component of several next-generation vaccines. These studies, beyond demonstrating the powerful adjuvant effects of lipid A mimetics, have shown MPL to be safe. One of the most striking and unexpected breakthroughs has been the finding that MPL can enhance Th1 and CTL responses in animals and humans. The prospect for defined subunit vaccines against leishmaniasis and tuberculosis has increased dramatically with the discovery that safe and effective vaccine adjuvants exist that are capable of eliciting the latter responses. http://www.trends.com

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Concluding remarks The full potential of lipid A mimetics as adjuvants and stand-alone immunomodulators is only now beginning to be realized. The discovery of the AGP family, with its potential to provide a large number of chemically distinct TLR agonists and antagonists, could enable custom design of adjuvant molecules that can induce very different types of immune responses. Armed with an increased understanding of TLR-4 signaling, we can now create compounds that are capable of providing antigenspecific and nonspecific immune enhancement or specific blockade of the proinflammatory effects of LPS. Novel vaccine approaches can be considered now that take full advantage of the innate and adaptive immune responses. For example, an intranasal subunit vaccine for influenza virus formulated in MPL might be predicted to have a nearly immediate effect on reducing susceptibility to viral infection by activating both arms of the immune response. Activation of innate immune responses by intranasal MPL adjuvant can provide protection against airway challenge by influenza virus within 8–24 h of administration, therefore, a period of reduced susceptibility could be maintained if intranasal booster doses of the vaccine are administered weekly. The nonspecific protective effect could theoretically be maintained until durable protection develops via an antigenspecific mucosal immune response against the protein component of the vaccine. Natural infection by viruses for which there are no effective vaccines – such as rhinoviruses and respiratory syncitial virus – within a Th1biased immunological environment, might also have long-lasting immunological consequences. The paradigm shift promoted by a fundamental understanding of innate immunity ushers in a new era of designer adjuvants, therapeutic and prophylactic vaccines, and standalone immunomodulatory therapy for a variety of human diseases. For immunologists and vaccine developers alike, it is truly time to take Toll. References 1 Rock, F.L. et al. (1998) A family of human receptors structurally related to Drosophila Toll. Proc. Natl. Acad. Sci. U. S. A. 95, 588–593 2 Ulevitch, R.J. (1999) Toll gates for pathogen selection. Nature 401, 755–756 3 Ulevitch, R.J. (2000) Molecular mechanisms of innate immunity. Immunol. Res. 21, 49–54 4 Ouellette, A.J. et al. (2000) Characterization of luminal paneth cell α-defensins in mouse small intestine: attenuated antimicrobial activities of peptides with truncated amino termini. J. Biol. Chem. 275, 33969–33973 5 Han, J. and Ulevitch, R.J. (1999) Emerging targets for antiinflammatory therapy. Nat. Cell Biol. 1, E39–E40 6 Kaisho, T. and Akira, S. (2002) Toll-like receptors as adjuvant receptors. Biochim. Biophys. Acta 1589, 1–13 7 Poltorak, A. et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282,2085–2088 8 Poltorak, A. et al. (2000) Physical contact between lipopolysaccharide and toll-like receptor 4 revealed by genetic complementation. Proc. Natl. Acad. Sci. U. S. A. 97, 2163–2167

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Figure 6. Corixa’s lead synthetic adjuvant: RC-529 RC-529 is a completely synthetic, hexacylated aminoalkyl glucosaminide-4phosphate with all six acyl chains having a length of 14 carbons (myristic acid). This compound has been extensively evaluated in human clinical trials and has been demonstrated to be an efficacious adjuvant with an excellent safety profile.

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9 Ulevitch, R.J. (1999) Endotoxin opens the Tollgates to innate immunity. Nat. Med. 5, 144–145 10 Thoelen, S. et al. (2001) A prophylactic hepatitis B vaccine with a novel adjuvant system.Vaccine 19, 2400–2403 11 Bienzle, U. et al. (2002) Successful hepatitis B vaccination in patients who underwent transplantation for hepatitis B virus-related cirrhosis: preliminary results. Liver Transpl. 8, 562–564 12 Baldridge, J.R. and Crane, R.T. (1999) Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines. Methods 19, 103–107 13 Baldridge, J.R. et al. (2000) Monophosphoryl lipid A enhances mucosal and systemic immunity to vaccine antigens following intranasal administration. Vaccine 18, 2416–2425 14 Ulrich, J.T. and Myers, K.R. (1995) Monophosphoryl lipid A as an adjuvant. Past experiences and new directions. Pharm. Biotechnol. 6, 495–524 15 Reed, S.G. (2001) Leishmaniasis vaccination: targeting the source of infection. J. Exp. Med. 194, F7–F9 16 Choi, C.M. and Lerner, E.A. (2002) Leishmaniasis: recognition and management with a focus on the immunocompromised patient. Am. J. Clin. Dermatol. 3, 91–105 17 Ashford, R.W. (2000) The leishmaniases as emerging and reemerging zoonoses. Int. J. Parasitol. 30, 1269–1281 18 Skeiky, Y.A. et al. (1998) LeIF: a recombinant Leishmania protein that induces an IL-12-mediated Th1 cytokine profile. J. Immunol. 161, 6171–6179 19 Webb, J.R. et al. (1998) Human and murine immune responses to a novel Leishmania major recombinant protein encoded by members of a multicopy gene family. Infect. Immun. 66, 3279–3289 20 Webb, J.R. et al. (1997) Molecular characterization of the heat-inducible LmSTI1 protein of Leishmania major. Mol. Biochem. Parasitol. 89, 179-193 21 Webb, J.R. et al. (1996) Molecular cloning of a novel protein antigen of Leishmania major that elicits a potent immune response in experimental murine leishmaniasis. J. Immunol. 157, 5034–5041 22 Doherty, T.M. et al. (2002) Oral vaccination with subunit vaccines protects animals against aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 70, 3111–3121 23 Beutler, B. and Poltorak, A. (2000) Positional cloning of LPS, and the general role of toll-like receptors in the innate immune response. Eur. Cytokine Netw. 11, 143–152 24 Hemmi, H. et al. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 25 Bauer, S. et al. (2001) Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. U. S.A. 98, 9237–9242 26 Johnson, D.A. et al. (1999) Synthesis and biological evaluation of a new class of vaccine adjuvants: aminoalkyl glucosaminide 4phosphates (AGPs). Bioorg. Med. Chem. Lett. 9, 2273–2278 27 Brandenburg, K. et al. (1999) Investigation into the acyl chain packing of endotoxins and phospholipids under near physiological conditions by WAXS and FTIR spectroscopy. J. Struct. Biol. 128, 175–186 28 Brandenburg, K. et al. (1995) Conformation and fluidity of endotoxins as determinants of biological activity. Prog. Clin. Biol. Res. 392, 167–182 29 Seydel, U. et al. (1993) Phase behavior, supramolecular structure, and molecular conformation of lipopolysaccharide. Immunobiology 187, 191–211

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