Jay T. Evans

Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents

Toll-like receptor (TLR) agonists are being developed for use as vaccine adjuvants and as stand-alone immunomodulators because of their ability to stimulate innate and adaptive immune responses. Among the most thoroughly studied TLR agonists are the lipid A molecules that target the TLR4 complex. One promising candidate, monophosphoryl lipid A, which is a derivative of lipid A from Salmonella minnesota, has proven to be safe and effective as a vaccine adjuvant in > 120,000 human doses. A new class of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates (AGPs), have been engineered specifically to target human TLR4 and are showing promise as vaccine adjuvants and as monotherapeutic agents capable of eliciting nonspecific protection against a wide range of infectious pathogens. In this review, the authors provide an update of the preclinical and clinical experiences with the TLR4 agonists, MPL® (Corixa Corporation) adjuvant and the AGPs.

Enhancement of antigen-specific immunity via the TLR4 ligands MPL adjuvant and Ribi.529.

MPL™(Corixa) adjuvant is a chemically modified derivative of lipopolysaccharide that displays greatly reduced toxicity while maintaining most of the immunostimulatory activity of lipopolysaccharide. MPL adjuvant has been used extensively in clinical trials as a component in prophylactic and therapeutic vaccines targeting infectious disease, cancer and allergies. With over 33,000 doses administered to date, MPL adjuvant has emerged as a safe and effective vaccine adjuvant. Recently, scientists at Corixa Corporation have developed a library of synthetic lipid A mimetics (aminoalkyl glucosaminide 4-phosphates) with demonstrated immunostimulatory properties. Similar to MPL adjuvant, these synthetic compounds signal through Toll-like receptor 4 to stimulate the innate immune system. One of these compounds, Ribi.529 (RC529), has emerged as a leading adjuvant with a similar efficacy and safety profile to MPL adjuvant in both preclinical and clinical studies.

The low‐toxicity versions of LPS, MPL® adjuvant and RC529, are efficient adjuvants for CD4+ T cells

Lipopolysaccharide (LPS) has long been known to enhance innate and adaptive immune responses; however, its extreme toxicity precludes its use in clinical settings. The combined toxicity and adjuvanticity of LPS have contributed to the view that immunological adjuvants need to be highly inflammatory to be maximally effective. Here, we compared the effects of LPS with its less‐toxic derivatives, monophosphoryl lipid A (MPL) and a chemical mimetic, RC529, on CD4+ T cell clonal expansion, long‐term survival, and T helper cell type 1 (Th1) differentiation. We found that LPS, MPL, and RC529 had similar effects on CD4+ T cell clonal expansion, cell division, and ex vivo survival. Analysis of the ability of activated CD4+ T cells to produce interferon‐γ following a 21‐day immunization and challenge protocol with LPS and MPL resulted in similar Th1 differentiation. In contrast, we found that LPS was more effective in promoting long‐term CD4+ T cell responses, as we recovered nearly sixfold more cells following immunization/challenge as compared with treatment with MPL. Our results indicate that low‐inflammation adjuvants, such as MPL and RC529, are capable of enhancing short‐term CD4+ T cell clonal expansion and Th1 differentiation, but inflammatory signaling aids in the long‐term retention of antigen‐specific T cells.

Human Cord Blood CD34+CD38- Cell Transduction via Lentivirus-Based Gene Transfer Vectors

The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.

Synthetic Toll-Like Receptor 4 Agonists Stimulate Innate Resistance to Infectious Challenge

ABSTRACT A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.

EFFICIENT TRANSDUCTION OF HUMAN LYMPHOCYTES AND CD34+ CELLS VIA HUMAN IMMUNODEFICIENCY VIRUS-BASED GENE TRANSFER VECTORS

The development of gene transfer systems for the efficient transduction of human primary cells including lymphocytes and CD34+ cells is a significant step in the advancement of gene therapy and cell marking protocols. Efficient gene transfer systems also represent useful tools for basic research. Here we show that human primary lymphocytes and CD34+ cells can be efficiently transduced using a VSV-G pseudotyped HIV-1-based gene transfer system. The enhanced green fluorescent protein (EGFP) was chosen as the marker transgene, because it can be easily visualized and quantitated using fluorescence microscopy and flow cytometry, thus eliminating the need for selection or PCR to score transduction. Vectors produced with this system did not generate replication-competent retroviruses (RCRs) and efficiently transduced human cell lines (40-90%), PBMCs (60%), mobilized CD34+ cells (39%), and CD34+ cells from umbilical cord blood (60%) as measured by flow cytometry. Cells treated with AZT prior to infection did not express EGFP, ruling out passive protein or plasmid DNA transfer. This was further confirmed in methylcellulose cultures, where expression in myeloid and erythroid colonies was maintained for at least 3 weeks. In addition, this HIV-based vector was able to efficiently transduce freshly isolated, not-prestimulated CD34+ cells (70% EGFP positive) in serum-free medium. Under these same conditions, a Moloney murine leukemia virus-based vector failed to transduce not-prestimulated CD34+ cells. These characteristics make this gene transfer system an excellent choice for both basic science and possible gene therapy applications.

Selective TRIF-dependent signaling by a synthetic toll-like receptor 4 agonist.

A single change in a synthetic lipid A mimetic may reduce the toxicity of this vaccine adjuvant. Biasing TLR4 Signaling Toward a Less Inflammatory Route Signaling downstream of Toll-like receptor 4 (TLR4) proceeds through two pathways that are dependent on distinct adaptor proteins. One of these pathways leads to immunostimulatory responses, whereas the other leads to the production of toxic proinflammatory cytokines. Compounds that could stimulate the beneficial arm rather than the proinflammatory arm would be useful as adjuvants for vaccines. Bowen et al. investigated two synthetic TLR4 agonists that differed only in their stereochemistry at a single point in their structure and found that whereas one agonist stimulated both arms of the TLR4 pathway, the other agonist stimulated only the noninflammatory branch. These findings have implications for the design of more effective vaccine adjuvants. In response to ligand binding to the Toll-like receptor 4 (TLR4) and myeloid differentiation-2 (MD-2) receptor complex, two major signaling pathways are activated that involve different adaptor proteins. One pathway depends on myeloid differentiation marker 88 (MyD88), which elicits proinflammatory responses, whereas the other depends on Toll–IL-1 receptor (TIR) domain–containing adaptor inducing interferon-β (TRIF), which elicits type I interferon production. Here, we showed that the TLR4 agonist and vaccine adjuvant CRX-547, a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic lipid A mimetics, displayed TRIF-selective signaling in human cells, which was dependent on a minor structural modification to the carboxyl bioisostere corresponding to the 1-phosphate group on most lipid A types. CRX-547 stimulated little or no activation of MyD88-dependent signaling molecules or cytokines, whereas its ability to activate the TRIF-dependent pathway was similar to that of a structurally related inflammatory AGP and of lipopolysaccharide from Salmonella minnesota. This TRIF-selective signaling response resulted in the production of substantially less of the proinflammatory mediators that are associated with MyD88 signaling, thereby potentially reducing toxicity and improving the therapeutic index of this synthetic TLR4 agonist and vaccine adjuvant.

Lentivirus Vector Mobilization and Spread by Human Immunodeficiency Virus

The rapid advancement of lentivirus-based gene transfer systems and their demonstrated utility in a variety of in vitro and in vivo settings has heightened the need for assays to evaluate the safety of these vectors prior to human clinical trials. Two major concerns relating to the use of lentivirus-based vectors in a clinical setting are the presence of contaminating replication-competent retroviruses in vector preparations and the efficiency of vector mobilization and spread by wild-type helper virus (rescue). This article describes an in vitro system to study the rescue of lentivirus-based vectors by wild-type HIV. We show that lentivirus-based vectors can be readily rescued from T cell lines and to a lesser extent from primary human lymphocytes by wildtype HIV, resulting in the spread of mobilized vector particles to previously untransduced cells. Furthermore, we show that vector mobilization can be prevented by antiretroviral drugs such as AZT. In contrast to recently published reports by Bukovsky et al. and An et al., the lentivirus vectors used in these studies had little or no effect on the replication and spread of HIV in transduced cells [Bukovsky et al. (1999). J. Virol. 73, 7087-7092; An et al. (1999). J. Virol. 73, 7671-7677]. Whereas vector spread is a significant concern for most gene therapy applications, in the context of gene therapy for HIV infection it may have beneficial effects.

Immunostimulatory activity of aminoalkyl glucosaminide 4-phosphates (AGPs): induction of protective innate immune responses by RC-524 and RC-529

Earlier we showed that the structural requirements for adjuvanticity among the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic immunostimulants may be less strict than those for other endotoxic activities, including the induction of nitric oxide synthase in murine macrophages and cytokine production in human whole blood. The known role of nitric oxide and pro-inflammatory cytokines in the activation of host defenses against infection prompted us to examine the ability of certain AGPs to enhance non-specific resistance in mice to Listeria monocytogenes and influenza infections as well as to stimulate the production of pro-inflammatory cytokines in mouse splenocytes, human PBMCs, and human U937 histiocytic lymphoma cells. Intranasal administration of RC-524 or RC-529 to mice 2 days prior to a lethal influenza challenge provided significant protection in each case. Similarly, the intravenous administration of these AGPs induced resistance to L. monocytogenes infection as measured by survival or reduction of bacteria in the spleen. Activation of the innate immune response by AGPs appears to involve activation of Toll-like receptor 4 (TLR4) because RC-524 failed to elicit a protective effect in C3H/HeJ mice which have a defect in TLR4 signaling or induce significant cytokine levels in C3H/HeJ splenocytes. Both AGPs also stimulated pro-inflammatory cytokine release in human cell cultures in a dose-dependent manner.

Intranasal Vaccination Promotes Detrimental Th17-Mediated Immunity against Influenza Infection

Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4+IL-17A+TNFα+). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development