Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs ac- trihydrochloride)} were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.

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Chronic lymphocytic leukemia (CLL) B cells express BR3, the specific receptor for the B cell-activating factor of tumor necrosis factor family (BAFF). CLL cells also express 2 other receptors for BAFF, namely B-cell maturation antigen (BCMA) and the transmembrane activator and calcium modulator and cyclophilin ligandinteractor (TACI), which also bind a proliferation-inducing ligand (APRIL). We found that signaling through BR3, but not BCMA or TACI, activated the alternative nuclear IntroductionThe B cell-activating factor of tumor necrosis factor (TNF) family (BAFF, also known as BlyS, TALL-1, zTNF4, and THANK) is a potent regulator of normal B-cell development and function. 1-3 A proliferation-inducing ligand (APRIL, also termed TALL-2 and TRAD-1) also is a member of the TNF family and shares significant homology with BAFF. APRIL has been found to stimulate the growth of tumor cells and the proliferation of primary lymphocytes. 3-5 BAFF and APRIL bind 2 receptors of the TNF superfamily, B-cell maturation antigen (BCMA) and transmembrane activator or the calcium modulator and cyclophilin ligandinteractor (TACI). 6-8 BAFF, but not APRIL, binds a third receptor known as BAFF receptor (BAFF-R or BR3). 9,10 BCMA, TACI, and BR3 are expressed on normal B lymphocytes. 9,11,12 Neoplastic B cells in chronic lymphocytic leukemia (CLL) also express these receptors for BAFF and APRIL, which, when ligated, can promote CLL cell survival in vitro. [13][14][15][16][17][18] Furthermore, nurselike cells (NLCs), which can protect CLL cells in vitro and presumably in vivo, 19,20 express high levels of BAFF and APRIL, accounting in part for their capacity to promote CLL cell survival in a paracrine fashion. 21 Kern et al 14 found that CLL cells themselves might express BAFF or APRIL, suggesting that these factors also might function in an autocrine fashion to promote leukemia cell survival. 14 Understanding the mechanisms whereby BAFF and APRIL support CLL survival could lead to the development of inhibitors to BAFF and APRIL signaling that may be therapeutic in patients with this disease.Many members of the TNF superfamily trigger the activation of nuclear factor of B (NF-B). Recent studies have revealed that 2 NF-B pathways, the canonical pathway and the alternative pathway, regulate the activity of NF-B. 22,23 Activation of the canonical NF-B pathway proceeds through the degradation of the inhibitor of NF-B␣ (IB␣), which is induced after its phosphorylation by the ␤ subunit of the IB kinase (IKK) complex (IKK␤). 24 Degradation of IB␣ leads to the nuclear translocation of active NF-B heterodimers (composed of p50, p65, and/or c-Rel), where they can affect changes in gene expression. Activation of the alternative NF-B pathway results from processing of NF-B2/ p100 to p52, which is triggered by the phosphorylation of NF-B2/p100 by the ␣ subunit of the IKK complex (IKK␣). 25 This allows for nuclear translocation of p52 along with RelB, where together they can influence the expression of genes that are distinct from those regulated b...

A general and stereospecific synthesis has been developed for the direct preparation of 2'-deoxy-/3-D-ribofuranosylpurine analogues including 2'-deoxyadenosine derivatives. The reaction of the sodium salt of 4-chloropyrrolo[2,3-if)pyrimidine (4) or 2,4-dichloropy rrolo [ 2,3-tf] pyrimidine (1) with l-chloro-2-deoxy-3,5-di-0-/Holuoyl-a-D-er>>r/¡ro-pentofuranose (25) provided the corresponding N-1,2'-deoxy-|8-D-ribofuranosyl blocked derivatives (5 and 2) which, on ammonolysis, gave 2'-deoxytubercidin (6) and 2-chloro-2'-deoxytubercidin (3), respectively, in good yield. This glycosylation also readily proceeds in the presence of a 2-methylthio group. Application of this glycosylation procedure to 4,6-dichloroimidazo[4,5-c]pyridine (10), 6-chloropurine ( 16), 2,6-dichloropurine ( 13), and 4-chloropyrazolo[ 3,4-t/] pyrimidine ( 19) gave 2-chloro-2'-deoxy-3-deazaadenosine ( 12), 2'-deoxyadenosine (18), 2-chloro-2'-deoxyadenosine (15), and 4-amino-l-(2-deoxy-/3-D-eryiAro-pentofuranosyl)pyrazolo-[3,4-t/] pyrimidine (21), respectively. Similarly, glycosylation and ammonolysis of 4,6-dichloro-1 /f-pyrrolo[3,2-c]pyridine ( 22) gave 4,6-dichloro-l-(2-deoxy-0-D-e/,>rAro-pentofuranosyl)pyrrolo[3,2-c]pyridine (24). This stereospecific attachment of the 2-deoxy-/3-D-ribofuranosyl moiety appears to be due to a Walden inversion at the C-l carbon of 25.

Activation of toll-like receptors (TLRs) on cells of the innate immune system initiates, amplifies, and directs the antigen-specific acquired immune response. Ligands that stimulate TLRs, therefore, represent potential immune adjuvants. In this study, a potent TLR7 agonist was conjugated to phospholipids, poly(ethylene glycol) (PEG), or phospholipid-PEG via a versatile benzoic acid functional group. Compared to the unmodified TLR7 agonist, each conjugate displayed a distinctive immunological profile in vitro and in vivo. In mouse macrophages and human peripheral blood mononuclear cells, the phospholipid TLR7 agonist conjugate was at least 100-fold more potent than the free TLR7 ligands, while the potency of PEG−phospholipid conjugate was similar to that of the unmodified TLR7 agonist. When administered systemically in mice, the phospholipid and phospholipid−PEG TLR7 conjugates induced prolonged increases in the levels of proinflammatory cytokines in serum, compared to the unmodified TLR7 activator. When the conjugates were used as adjuvants during vaccination, only the phospholipid TLR7 agonist conjugates induced both Th1 and Th2 antigen-specific immune responses. These data show that the immunostimulatory activity of a TLR7 ligand can be amplified and focused by conjugation, thus broadening the potential therapeutic application of these agents.

inosine monophosphate dehydrogenase ͉ IFN regulatory factor

The immunotherapeutic activity of Toll-like receptor (TLR) activators has been difficult to exploit because of side effects related to the release and systemic dispersion of proinflammatory cytokines. To overcome this barrier, we have synthesized a versatile TLR7 agonist, 4-[6-amino-8-hydroxy-2-(2-methoxyethoxy)purin-9-ylmethyl]benzaldehyde (UC-1V150), bearing a free aldehyde that could be coupled to many different auxiliary chemical entities through a linker molecule with a hydrazine or amino group without any loss of activity. UC-1V150 was covalently coupled to mouse serum albumin (MSA) at a 5:1 molar ratio to yield a stable molecule with a characteristically altered UV spectrum. Compared with the unconjugated TLR7 agonist, the UC-1V150/MSA was a 10-to 100-fold more potent inducer of cytokine production in vitro by mouse bone marrow-derived macrophage and human peripheral blood mononuclear cells. When administrated to the lung, the conjugate induced a prolonged local release of cytokines at levels 10-fold or more higher than those found in serum. Under the same conditions, the untethered TLR7 ligand induced quick systemic cytokine release with resultant toxicity. In addition, two pulmonary infectious disease models were investigated wherein mice were pretreated with the conjugate and then challenged with either Bacillus anthracis spores or H1N1 influenza A virus. Significant delay in mortality was observed in both disease models with UC-1V150/MSA-pretreated mice, indicating the potential usefulness of the conjugate as a localized and targeted immunotherapeutic agent.drug delivery ͉ influenza ͉ innate immunity T he Toll-like receptors (TLRs) are a set of conserved cellular receptors that play an important role in the recognition of microbial pathogens and in initiating the host innate immune response. These receptors recognize distinct molecular components of invading pathogens, such as cell wall structures and nucleic acids. The discovery that endogenous ligands as well as synthetic small molecules can activate certain TLR pathways has generated tremendous interest in the development of new therapeutics for diseases related to the immune response. TLR ligands control the activation of antigen-presenting cells, in particular dendritic cells, by triggering their maturation program, including up-regulation of the expression of HLA and costimulatory molecules and secretion of proinf lammatory cytokines, such as TNF-␣, IL-6, IL-12, and IFN-␣ (1). Recently, we reported that certain derivatives of guanine (2) and adenine can activate immune cells via TLR7 and can inhibit the replication of hepatitis C virus replicons in hepatocytes (3). However, the in vitro immunotherapeutic activities of TLR7 ligands have been difficult to translate in vivo, because systemic TLR activation can induce a rapid and potentially toxic cytokine syndrome (4, 5). Accordingly the major in vivo applications of TLR7 ligands have been as topically applied antiviral or antitumor agents or as immune adjuvants injected intramuscularly in small qu...

Current vaccines against influenza virus infection rely on the induction of neutralizing antibodies targeting the globular head of the viral hemagglutinin (HA). Protection against seasonal antigenic drift or sporadic pandemic outbreaks requires further vaccine development to induce cross-protective humoral responses, potentially to the more conserved HA stalk region. Here, we present a novel viral vaccine adjuvant comprised of two synthetic ligands for Toll-like receptor 4 (TLR4) and TLR7. 1Z105 is a substituted pyrimido[5,4-b]indole specific for the TLR4-MD2 complex, and 1V270 is a phospholipid-conjugated TLR7 agonist. Separately, 1Z105 induces rapid Th2-associated IgG1 responses, and 1V270 potently generates Th1 cellular immunity. 1Z105 and 1V270 in combination with recombinant HA from the A/Puerto Rico/8/1934 strain (rPR/8 HA) effectively induces rapid and sustained humoral immunity that is protective against lethal challenge with a homologous virus. More importantly, immunization with the combined adjuvant and rPR/8 HA, a commercially available split vaccine, or chimeric rHA antigens significantly improves protection against both heterologous and heterosubtypic challenge viruses. Heterosubtypic protection is associated with broadly reactive antibodies to HA stalk epitopes. Histological examination and cytokine profiling reveal that intramuscular (i.m.) administration of 1Z105 and 1V270 is less reactogenic than a squalene-based adjuvant, AddaVax. In summary, the combination of 1Z105 and 1V270 with a recombinant HA induces rapid, long-lasting, and balanced Th1-and Th2-type immunity; demonstrates efficacy in a variety of murine influenza virus vaccine models assaying homologous, heterologous, and heterosubtypic challenge viruses; and has an excellent safety profile. IMPORTANCENovel adjuvants are needed to enhance immunogenicity and increase the protective breadth of influenza virus vaccines to reduce the seasonal disease burden and ensure pandemic preparedness. We show here that the combination of synthetic Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic challenge models. Combining TLR4 and TLR7 ligands balances Th1-and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. The combined adjuvant has an attractive safety profile and the potential to augment seasonal-vaccine breadth, contribute to a broadly neutralizing universal vaccine formulation, and improve response time in an emerging pandemic. Influenza A and B viruses remain a substantial public health burden, with seasonal epidemics resulting in significant morbidity, mortality, and economic loss (1-3). Pandemic outbreaks occur when antigenically novel influenza A viruses emerge in a population with little preexisting immunity (4). Pandemic viruses spread more rapidly and cause more severe di...