Polymyxin B enhances ISS-mediated immune responses across

Cellular Immunology 229 (2004) 93–105

www.elsevier.com/locate/ycimm

Immunologyellular

Polymyxin B enhances ISS-mediated immune responsesacross multiple species

Jason D. Marshall,¤ Debbie Higgins, Christi Abbate, Priscilla Yee, Glen Teshima, Gary Ott, Tracy dela Cruz, David Passmore, Karen L. Fearon, Stephen Tuck, and Gary Van Nest

Dynavax Technologies Corporation, Berkeley, CA, USA

Received 15 December 2003; accepted 22 April 2004Available online 24 June 2004

Abstract

The immunostimulatory eVects of bacterial DNA on mammalian cells have been localized to unmethylated CpG motifs, and syn-thetic CpG-containing oligodeoxynucleotides that mimic these eVects are known as immunostimulatory sequences (ISS). We havefound that the polycationic antibiotic, polymyxin B (PMXB), associates with ISS and serum albumin in vitro and forms microparti-cles that greatly increase the activity of ISS on plasmacytoid dendritic cells (PDCs). SpeciWcally, ISS/PMXB greatly enhanced IFN-�production from PDCs and other activities downstream of IFN-�, including IFN-� secretion, NK lytic activity, and the expression ofgenes dependent upon IFN-�/IFN-�. This ampliWcation was speciWc for the IFN-� pathway since other ISS activities, including Bcell proliferation, B cell IL-6 secretion, and PDC maturation, were not aVected by PMXB. Both the polycationic peptide and lipo-philic fatty acid side chain domains of PMXB, as well as the presence of a third party stabilizing agent such as albumin or Tween 85,were required for particle formation and enhanced ISS activity. The ISS-enhancing activity of PMXB was observed across multiplespecies (human, primate, and mouse) and in vivo (primate, mouse). These data illustrate the usefulness of formulating ISS with a cat-ionic lipopeptide such as PMXB, which focuses and greatly ampliWes the ISS-induced pathway of IFN-�-mediated responses. 2004 Elsevier Inc. All rights reserved.

Keywords: CpG DNA; Plasmacytoid dendritic cells; Adjuvants

1. Introduction

The Wrst members of the family of antibiotics knownas polymyxins were isolated from strains of Bacillus poly-myxa and Bacillus colistinus. These approximately1.2 kDa molecules share a structural consistency deWnedby a decapeptide attached through an amide bond to afatty acid residue [1]. The peptide portion is remarkablyconserved between members and consists of a heptapep-tide ring with a further 1–3 residues branching oV line-arly. In polymyxin B (PMXB)1 and colistin, the peptideportion of the molecule is rich with positively charged

0008-8749/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.cellimm.2004.04.009

¤ Corresponding author. Present address: Dynavax Technologies CorpoE-mail address: [email protected] (J.D. Marshall).1 Abbreviations used: BSA, bovine serum albumin; CMS, colistin metha

rescein amidite; HBsAg, hepatitis B surface antigen; HSA, human serum alODN, oligodeoxynucleotide; PDC, plasmacytoid dendritic cell; PMXB, polphosphorothioate; SN, supernatant; TLR, Toll-like receptor.

2,4-diaminobutyric acids (DABs), which theoreticallycontribute a net charge of +5 to each molecule (Fig. 1).The cyclization of the peptide is conducted through the�-amino and carboxyl groups of one DAB residue, whichalso acts as the attachment point via the �-amino groupfor a tripeptide (in PMXB) that contains two moreDABs. A fatty acyl derivative is attached to the �-aminoterminus of the tripeptide and can be either 6-methyloc-tanoic acid (PMXB1) or 6-methylheptanoic acid(PMXB2). Other molecules with a similar basic structureas PMXB have been isolated from Bacillus species buthave not been clinically developed; these include circulin,

ration, Berkeley, CA, USA. Fax: 1-510-848-5694.

nesulfonate; cPLGA, cationic poly(D,L-lactide-co-glycolide); FAM, Xuo-bumin; ISS, immunostimulatory sequence; MSA, mouse serum albumin;ymyxin B; PMXB-9, polymyxin-B nonapeptide; PO, phosphodiester; PS,

mail to : [email protected]

94 J.D. Marshall et al. / Cellular Immunology 229 (2004) 93–105

octapeptin, brevistin, cerexin, polypeptin, and stendomy-cin [1].

Polymyxins demonstrate antibiotic activity against adiverse array of organisms including bacteria, yeasts, andprotozoa [1,2]. PMXB is especially active on gram-nega-tive bacteria and exerts toxicity through bacterial mem-brane permeabilization and the rapid eZux into theextracellular space of small charged or polar cytoplasmicmolecules such as nucleosides, bases, and ions [2]. Studieswith PMXB derivatives have shown that both the cyclicpeptide and acyl moiety are required for optimal mem-brane disruption. This disruption appears to be caused byinteraction between PMXB and bacterial phospholipidsand lipopolysaccharide in the outer membrane [1]. Thepolycationic peptide and fatty acid tail portions ofPMXB confer upon the molecule the amphipathic prop-erty of solubilizing in water or lipid membranes.Although both domains of PMXB are required for anti-bacterial activity, the peptide portion alone can bind bac-terial endotoxin [3], an activity routinely utilized as ameans for preventing LPS stimulation in mammalian cellcultures [2].

Much attention has been focused on the potential ofunmethylated CpG motif-containing immunostimulatorysequences (ISS) to enhance mammalian immunity. ISSactivates several immune functions through recognitionby the ISS-receptor TLR9, a member of the Toll-like

Fig. 1. Chemical structure of polymyxin B. PMXB is composed of adecapeptide, containing a heptapeptide ring linked to a linear tripep-tide which is attached to the fatty acid, 6-methyloctanoic acid, throughan amide bond. 2,4-Diaminobutyric acid (DAB) residues contribute anet charge of +5 to each molecule.

receptor family [4]. TLRs have evolved to recognizerepeating, invariant microbial motifs such as lipopep-tides, peptidoglycans, Xagellin, and double-strandedRNA. Known human TLR9-expressing cells includememory B cells and plasmacytoid dendritic cells (PDCs).Activation of TLR9 on PDCs promotes maturation andthe secretion of cytokines such as IFN-�, TNF-�, IL-6,and IL-8, while simultaneously sending anti-apoptoticsignals [5,6]. B cells respond to ISS by upregulating theexpression of several activation markers, including CD80,CD86, and CD40, proliferating, and secreting IL-6 andTNF-� [7–9]. The increased production of IFN-� byPDCs can also lead to downstream eVects such as theactivation of monocytes and the induction of lytic activ-ity and IFN-� production from NK cells [10,11]. ISS thusactivates the innate immune system to respond with anti-microbial defense measures while also contributing toadaptive immunity by priming PDCs to promote Th1development and cytotoxic T cells.

ISS are subdivided into three basic classes termed A,B, and C [12–14]. CpG-A are characterized by a centralCpG-containing palindromic phosphodiester (PO) coreXanked by phosphorothioate (PS) poly-guanosinesequences that enable these ISS to aggregate in large clus-ters, presumably through a combination of duplex for-mation and guanosine-tetrad stacking [14]. Thisaggregation is critical for CpG-A activity and promoteshighly elevated IFN-� secretion from PDCs andincreased NK cell activity. However, this ISS class exhib-its poor activity at promoting PDC maturation or acti-vating puriWed B cells. On the other hand, the CpG-Bclass does not form high-order structures and is generallyall PS with one or more CpG motifs and without strictXanking sequence requirements. CpG-B exhibit activitiesthat are largely mutually exclusive with CpG-A; theypotently activate B cells and promote PDC maturationbut are poor at eliciting IFN-� production from PDCs(reviewed in [15]). The CpG-C class are also PS ODNswhich require the following sequence characteristics: atleast one TCG motif at or close to the 5� end and a palin-dromic sequence that is optimally at least 12 bases longand contains at least two and preferably more CpG dinu-cleotides that can be separated by 0–3 bases. The func-tional capacity of CpG-C covers all known ISSresponses, including those of CpG-A and CpG-B [14,16].This, combined with their lack of structural complexity,makes CpG-C ISS leading candidates for many clinicalapplications.

Our laboratory has investigated various mechanismsfor enhancing the eVects of CpG-B and CpG-C ISS and,in particular, the induction of IFN-� from PDCs. Wepreviously determined that ODNs become tightlyadsorbed to the surface of cationic poly(D,L-lactide-co-glycolide) (cPLGA) microparticles through ionicinteractions [17]. Furthermore, cPLGA greatly enhancedthe uptake of ODNs into PDCs and B cells and also

J.D. Marshall et al. / Cellular Immunology 229 (2004) 93–105 95

ampliWed the various activities that ISS has been shownto exert in vitro, particularly IFN-�/� induction. Wesought to demonstrate whether another polycationicmolecule, PMXB, might also similarly associate with ISSODNs and whether this association would amplify ISSactivity. We found that PMXB does indeed complex withISS ODNs under deWned conditions to form microparti-cles and that this conWguration leads to greatly ampliWedIFN-� production by PDCs as well as immune eventsdownstream of IFN-�, including increased IFN-� secre-tion, cytokine/chemokine gene expression, and NK lyticactivity. Additionally, we show that ISS/PMXB enhancesthe immune activity of various species and demonstrateits eVectiveness in vivo in baboons and mice under cir-cumstances where other ISS-formulations such as ISS/cPLGA are poorly active.

2. Methods

2.1. Oligodeoxynucleotides

PS ODNs were synthesized as described [14]. AllODNs had 05 endotoxin units/mg of ODN, determinedby Limulus amebocyte lysate assay (Bio-Whittaker).Sequences: 1018 (CpG-B): 5�-TGACTGTGAACGTTCGAGATGA; C274 (CpG-C): 5�-TCGTCGAACGTTCGAGATGAT; 1040 (CpG-B control): 5�-TGACTGTGAACCTTAGAGATGA; C661 (CpG-C control): 5�-T GCTTGCAAGCTTGCAAGCA; C264 (CpG-A) 5�-GGtgcatcgatgcagGGGGG. Uppercase denotes PS, lower-case denotes PO, and CpG motifs are underlined.

2.2. ISS/PMXB formulations and other reagents

PMXB (Sigma) was administered in a concentrationrange of 1–500�g/ml. Optimal ISS enhancement wasobserved at 100�g/ml PMXB and all data reported belowwere generated using that dose. Colistin, colistinmethanesulfonate (CMS), polymyxin B nonapeptide(PMXB-9) (all from Sigma), and rENP (recombinantEndotoxin Neutralizing Protein; Cat. No. 910140, Sei-kagaku America) were also all used at 100�g/ml. Gener-ally, PMXB and ODNs were administered to cell culturesseparately in small volumes and therefore the PMXB(100�g/ml) complexed with ODN (20�g/ml) within thecell culture itself. In other cases, ISS, PMXB, and a thirdstabilizing component [human serum albumin (HSA),bovine serum albumin (BSA), mouse serum albumin(MSA), or keyhole limpet hemocyanin (KLH)] were pre-mixed at a w/w ratio of 1:5:20 (ISS:PMXB:stabilizer; allstabilizers from Sigma). Additionally, preparations ofISS/PMXB were generated using the non-ionic detergentTween 85 (Sigma) as a stabilizer, in which 2 mg/ml ISS,5 mg/ml PMXB, 4 mg/ml Tween 85, and 4 mg/ml oleicacid (Sigma) were pre-mixed. Preparations of ISS/PMXB

generated with 1018, 1040, or C274 were sized by laserlight scattering (Malvern Instruments, Worcestershire,UK) and proportions of ODN and PMXB free andbound were measured by HPLC (gel permeabilizationchromatography for ODN and reverse-phase chroma-tography for PMXB; Fig. 2). In the case of these pre-made formulations, the volume of particles required toget Wnal concentrations of 20 �g/ml ISS and 100 �g/mlPMXB was added to the culture. To obtain an ISS/PMXB preparation depleted of uncomplexed PMXB,ISS/PMXB was dialyzed for 18 h in a Slide-A-Lyzer cas-sette (Pierce) in buVer (0.4% Tween 85, 0.4% oleic acid in100 mM sodium phosphate, and 150 mM sodium chlo-ride, pH 7.5). A 71% reduction in free PMXB wasobtained as assayed by reversed-phase HPLC. ODN andcPLGA (Boehringer Ingelheim Chemicals) were also pre-mixed at Wnal concentrations of 20 and 100�g/ml, respec-tively, for 15 min at r.t. before they were added to the cul-ture, as described previously [17].

2.3. PBMC preparation

Peripheral blood was collected from healthy volun-teers by venipuncture using heparinized syringes. BuVycoats were obtained from the American Red Cross.PBMCs were isolated by centrifugation through a Ficoll(Pharmacia) density gradient and cultured in RPMI 1640(Bio-Whittaker) supplemented with 10% heat-inactivatedhuman AB serum (Gemini) plus 50 U/ml penicillin, 50�g/ml streptomycin, 300�g/ml glutamine, 1 mM sodiumpyruvate (Bio-Whittaker), 50 mM Hepes, and 1£ non-

Fig. 2. Physicochemical characterization of ISS/PMXB. Preparationsof ISS/PMXB were generated by addition of 2 mg/ml ODN (1018,1040, and C274), 5 mg/ml PMXB, 4 mg/ml Tween 85, and 4 mg/mloleic acid. ISS/PMXB preps were sized by laser light scattering anddata reported as volume average distribution of diameters (�m). Thepercentages of ODN and PMXB determined to be complexed oruncomplexed were calculated after dissolution of pellets/supernatantsfrom ISS/PMXB formulations centrifuged for 5 min at 12000g in alka-line:isopropanol:glycerol.


essential amino acids (Bio-Whittaker). For cytokinesecretion, PBMCs were cultured at 0.5 £ 106/well (2 £ 106/ml) in 96-well Xat-bottomed plates in duplicate with ISSODNs at a concentration range of 0.16–20�g/ml for 24 h,determined by previous studies to be the optimal time-point for ISS-induced cytokine secretion. Cell-free SNswere harvested and cytokine content was assayed byELISA. Culture conditions (including cell concentration,ODN concentration, and period of culture) to induceoptimal activity in each ISS functional assay were pre-determined.

2.4. Detection of cytokines by ELISA

Cell-free medium was harvested and assayed for cyto-kine content via commercial kits. IFN-� and IL-6 wereassayed via CytoSet antibody pairs (BioSource). Limitsof maximal/minimal detection were 4000/2 pg/ml for bothassays. IFN-� was assayed via ELISA kit (PBL Biomedi-cal Laboratories) and limit of maximal/minimal detectionwas 13160/102 pg/ml. All kits were used according tomanufacturers' instructions.

2.5. Statistical analysis

Statistical signiWcance was calculated using the Mann–Whitney test with unpaired and non-parametric values(GraphPad InStat). Symbols representing signiWcanceare: ***p 00.001; **p 0 0.01; *p 0 0.05; and ns, p 10.05.

2.6. Cell puriWcation and ISS functional assays

For PDC enrichment, PBMCs were depleted of CD3-,CD19-, and CD11b-expressing cells by MACS labelingand two passages over an LS column (Miltenyi Biotec).The resultant populations were 1–2% BCDA-2+ CD123+,indicating that the PDC population had been enrichedapproximately 5- to 10-fold. B cells were puriWed fromPBMCs through CD19-positive selection by MACS andused in proliferation assays as described [14]. NK lyticactivity was assayed through lysis of K562 target cells. Inbrief, PBMCs were stimulated with 10�g/mlISS§ 100�g/ml PMXB for 48 h then were co-culturedwith 51Cr-loaded K562 tumor target cells at a range ofeVector:target ratios for 4 h. 51Cr release was measured bya TopCount NXT scintillation counter (Packard), and %speciWc lysis was calculated as [(experimentalrelease¡ spontaneous release) /(maximumrelease¡ spontaneous release) £ 100]. All assays werepreviously optimized for ISS dose.

2.7. Gene expression assay and analysis

Human PBMCs were cultured with 5�g/mlISS§ 100�g/ml PMXB at 2 £106/ml for 4 h. Total RNAwas extracted and RT-PCR was performed as described

[14]. Primer sequences for ubiquitin, MIG, MCP-2, IP-10,IFN-�, and IFN-� have been reported [14]. Thresholdcycle (CT) values for each gene were normalized to ubiq-uitin. The negative control for each experiment, stimula-tion with medium alone, was assigned a value of 1 and alldata expressed as fold induction over the negative con-trol.

2.8. ISS uptake assay

ISS uptake into PDCs was measured as described else-where [17]. In brief, FAM-labeled 1018 was incubatedwith monocyte-depleted PBMCs (monocytes wereremoved because they act as a non-speciWc sink forODNs) for 2–4 h, then excess FAM-1018 was removed bywashing and cells were stained with BCDA-4-PE, thenanalyzed by FACScan.

2.9. In vivo hepatitis B surface antigen immunization and invitro activity assays for mouse and baboons

Mouse in vivo studies were performed at NorthviewBiosciences (Hercules, CA) using 8-week-old femaleBALB/c mice (Charles River). Ten mice/group wereinjected twice intramuscularly in the quadriceps at 2-week intervals with HBsAg alone (1�g), HBsAg + 1018(5�g), HBsAg + 1018/cPLGA (5�g/250�g DOTAP/PLGA), or HBsAg + 1018/PMXB or 1040/PMXB (5�g/25�g) stabilized with Tween 85. Blood was collectedretro-orbitally 2 weeks after each injection and serumwas prepared for antibody determination. Samples wereanalyzed for anti-HBsAg IgG1 and IgG2a content byELISA. Earlier studies on serum from naïve mice veriWedthat their anti-HBsAg levels are undetectable (0120).Plates were coated with 1�g/ml HBsAg, and goat anti-mouse IgG1 or IgG2a biotin-conjugated antibody(Southern Biotechnology) was used for detection. Thetiter is deWned as the reciprocal of the serum dilution thatgave an ELISA absorbance of 0.5 OD using four-param-eter analysis (Softmax Pro97, Molecular Devices).Splenocytes from immunized mice were harvested atweek 6 and used for cytokine secretion and CTL assays.To measure CTL activity, splenocytes were stimulatedwith 1�g/ml HBsAg MHC class I H-2d-restricted peptide(IPQSLDSWWTSL synthesized by Research Genetics)for 6 days, then co-cultured with 51Cr-labeled, peptide-loaded P815 target cells at a range of eVector:target ratiosfor 4 h. 51Cr release was measured by scintillationcounter. To characterize cytokine production, additionalsplenocytes were stimulated with HBsAg (5�g/ml) for 4days, then supernatants were harvested and analyzed forIFN-� and IL-5 content by ELISA. Baboon studies wereperformed at Southwest Foundation for BiomedicalResearch (San Antonio, TX). Five animals per group(male and female, 10–18 kg/animal) were immunized i.m.with HBsAg (20�g), HBsAg + 1018 (1 mg), or


HBsAg + 1018/PMXB (0.86 mg/3 mg) in volumes of 1 ml.Peripheral blood serum was collected at day 28 andanalyzed for anti-HBsAg conformational epitope viaAUSAB EIA (Abbott Laboratories), according to manu-facturer's protocols.

3. Results

3.1. PMXB enhances ISS activity on PBMCs

Previous studies have indicated that ISS ODNs caninduce variable amounts of IFN-� and IFN-� from

PBMCs, dependent upon the class of ISS [14]. CpG-B(e.g., 1018) induce low levels of IFN-� and IFN-� whileCpG-C (e.g., C274) induce slightly higher levels of IFN-�and very high levels of IFN-�. In the presence of 100�g/ml PMXB, however, induction of IFN-� by both classesof ISS is potently enhanced approximately 50- to 100-fold (Fig. 3A). IFN-� induction by 1018 is also markedlyincreased by PMXB at higher doses. The activity of thestrong IFN-�-inducer C274 was modestly increased(Fig. 3B). These eVects are CpG-speciWc as little or noactivity was observed from cells stimulated with the con-trol ODNs 1040/PMXB or C661/PMXB. CpG-A is anISS class that also provides an extremely potent stimula-

Fig. 3. PMXB greatly enhances IFN-�/IFN-�-inductive potential of ISS in CpG-speciWc manner. PBMCs were stimulated with 20, 4, 0.8, and 0.16 �g/ml ODN (open circles) or ODNs + 100 �g/ml PMXB (closed circles) for 24 h. The ODNs chosen are representatives of the CpG-B class (1018), CpG-C class (C274), and CpG-A class (C264). Also shown are the negative controls for class B (1040) and class C (C661). Supernatants were assayed for(A) IFN-� and (B) IFN-� levels via ELISA. Data are expressed as means of four donors § SEM and are representative of three experiments. Statisti-cal signiWcance was computed vs ODN alone.


tion for IFN-� production [12]. PMXB was observed togreatly enhance CpG-A-induced IFN-� but not IFN-�.Since the IFN-�-inducing capacity of CpG-A is depen-dent upon its super-aggregated structure [14], it is possi-ble that PMXB cannot associate in the same way withthis class of ISS as it does with monomeric CpG-B/C.

To determine whether the ISS-enhancing ability ofPMXB was dependent upon its LPS-inhibiting function,we analyzed activity of ISS in the presence of PMXB orrecombinant endotoxin neutralizing protein (rENP), aprotein originally isolated from Limulus that neutralizesendotoxin from gram-negative bacteria. Although rENPneutralized the LPS-mediated induction of IL-12 simi-larly to PMXB, it had no ability to alter ISS activity (datanot shown), suggesting that PMXB is not enhancing theeVects of ISS through the inactivation of endotoxin.

3.2. PMXB/ISS induces a similar gene pattern to ISS

ISS stimulation induces a well-deWned and speciWcpattern of gene activation in human PBMCs [14], includ-ing cytokines, chemokines, and antiviral factors. Wedetermined whether that pattern was altered duringPMXB enhancement by using TaqMan RT-PCR tech-niques to measure the mRNA expression of a panel ofgenes in PBMCs. As reported in Table 1, PMXB clearlyboosted the expression of various genes in ISS-speciWcfashion including IFN-� and IFN-�, and chemokinessuch as interferon-inducible protein-10 (IP-10 orCXCL10), monokine induced by �-interferon (MIG orCXCL9), and monocyte chemoattractant protein-2(MCP-2 or CCL8). Other genes not induced by ISS (e.g.,MCP-1, MIP-3�, and IL-1�) were also not enhanced inthe presence of PMXB (data not shown). Therefore,PMXB increases the ISS-transduced signal for gene acti-vation quantitatively but does not alter it qualitatively.

3.3. PMXB enhances ISS activity in PDCs, not B cells

The known ISS-responsive cells in human PBMCs arePDCs and B cells. We examined the eVect of ISS/PMXBformulations on enriched or puriWed cell populations to

determine whether the presence of PMXB enhances ISSactivity diVerently according to cell type. PBMCsenriched for PDCs by lineage depletion were stimulatedwith ISS§ PMXB for 24 h then analyzed for cytokinesecretion. As expected, Fig. 4A shows that stimulationwith either CpG-B 1018 or CpG-C C274 alone resulted in4- or 8-fold higher levels of IFN-� secretion, respectively,compared to non-depleted PBMCs (as presented inFig. 3B). This IFN-� production was conWrmed to bePDC-speciWc by intracellular co-staining for IFN-� andBCDA-2 and by speciWc PDC-depletion, which abro-gated IFN-� secretion (data not shown). The PDC-spe-ciWc IFN-� was elevated to even higher levels in thepresence of PMXB, especially in the case of the 1018.However, the presence of PMXB had no eVect on theability of ISS to enhance DC maturation by upregulationof CD86 (Fig. 4B) and CD80 (data not shown) on PDCs.Therefore, it appears that the enhancing eVect of PMXBis targeted speciWcally towards PDC secretion of IFN-�without altering other eVects of ISS on the PDC. NKcells are indirectly activated by ISS through signals trans-mitted from the PDC. The exact nature of this signal hasnot been yet identiWed, although IFN-� appears to play amajor role [13]. We sought to determine whether the lyticactivity of NK cells was also enhanced in the presence ofPMXB. Fig. 4C indicates that NK cells exhibit increasedlytic activity vs K562 target cells in the presence of 1018and that this activity is further ampliWed by PMXB.

When highly puriWed B cells were subjected to stimu-lation with ISS/PMXB, no improvement in B cell func-tions compared to ISS alone was observed. Neither ISS-enhanced proliferation (Fig. 4D) nor the induction of IL-6 secretion (data not shown) from B cells were found tobe increased by the presence of PMXB; in fact, the pres-ence of PMXB led to marked decreases in B cell prolifer-ation (constitutive and ISS-speciWc) but not IL-6production. Reduced B cell proliferation appeared to bedue to prolonged exposure of the B cells to non-com-plexed PMXB, since an ISS/PMXB preparation fromwhich unbound PMXB had been removed via dialysisshowed no reduction in B cell proliferation (Fig. 4E).These studies suggest that ISS stimulation activates at

Table 1PMXB enhances the induction of gene expression by ISS

PBMCs were stimulated with 5 �g/ml 1018, C274, or C661 § PMXB for 4 h and then RNA was extracted and analyzed via TaqMan RT-PCR.Gene expression was normalized to ubiquitin expression. Data are presented as the mean of fold induction over medium control (given the value of1.0) with SEM for four donors, and are representative of three separate experiments.

Stimulus IFN-� IFN-� IP-10 MIG MCP-2

Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM

1018 66.6 52.0 1.5 0.2 3.3 2.5 2.0 1.6 1.2 1.01018/PMXB 1484 962 46.3 24.5 14.7 7.2 17.0 12.2 4.7 2.4

C274 7271 3870 7.6 3.2 61.4 31.0 19.0 16.2 13.0 8.2C274/PMXB 19,705 8159 196.0 73.0 110.6 42.9 73.8 48.4 73.9 17.4

C661 1.9 0.8 1.1 0.2 0.8 0.2 0.5 0.1 0.1 0.0C661/PMXB 2.0 0.9 9.3 1.8 5.4 2.4 3.1 1.4 0.6 0.4


least two separate signaling pathways: one that results inelevated production of type I interferon by the PDC and,in turn, heightened downstream IFN-dependent eventssuch as NK activation and chemokine expression, whichcan be ampliWed by PMXB; and another that results inmany other known ISS functions including dendritic cellmaturation and B cell activation, which is unaltered byPMXB.

3.4. ISS/PMXB form particles that depend on the presenceof a stabilizing agent

We initially observed that ISS formulated with PMXBdid not exhibit enhanced activity on cells cultured inserum-free media and instead immediately formed anaggregated precipitate, which was not observed in cul-tures containing serum. Therefore, we hypothesized thata third component, found to be serum albumin, was

required to confer activity upon the ISS + PMXB formu-lation. Indeed, when ISS, PMXB, and HSA were com-bined at a 1:5:20 ratio in PBS, the result was a stablesolution of suspended particles (size range 100 nm–10�m,see Fig. 2) that exhibited ISS-enhancing activity onPBMCs. On the other hand, withholding the addition ofHSA resulted in immediate precipitation of theISS + PMXB, which became highly adsorptive to poly-propylene and exhibited poor ISS-enhancing activity. ToconWrm the requirement of albumin for ISS-enhancingactivity, ISS + PMXB preparations were generated usingalbumins of other species, BSA and MSA. AnISS + PMXB prep was also generated with a non-albu-min protein, KLH, which has an isoelectric point of 5.3,similar to that of HSA (5.8). ISS+ PMXB formulationsthat utilized HSA, BSA, MSA, or KLH demonstratedboth the capacity to form a stable suspension of particlesand comparable ISS-enhancing activity (data not shown).

Fig. 4. PMXB enhances the ISS-mediated induction of IFN-� from PDC-enriched cells and activation of lytic activity from NK cells. (A) PBMCswere enriched for PDCs through lineage depletion and cultured with 20 �g/ml ISS § PMXB for 24 h, then supernatants were assayed for IFN-� viaELISA. Data are expressed as mean of eight donors + SEM. (B) MACS-puriWed PDCs were stimulated with 5 �g/ml ISS § PMXB for 18 h, then ana-lyzed for CD86 expression by FACScan. The plot with Wlled-in area represents unstimulated PDCs. (C) PBMCs from two donors were cultured with10 �g/ml 1018 § 100 �g/ml PMXB for 48 h. These cells were then harvested and co-cultured with 51Cr-loaded K562 target cells at 50:1–6:1 E:T ratiosfor 4 h. Released 51Cr was then measured in supernatants via a scintillation counter; data are reported as (experimental value ¡ spontaneous release)/total release. This is one representative experiment of three. (D–E) MACS-puriWed B cells were cultured with 5 �g/ml ODNs § PMXB for 96 h. Pro-liferation was assessed by [3H]thymidine incorporation. Proliferation data are expressed as means of four donors + SEM.


We suspected that these proteins played the role of stabi-lizing agent in the ISS + PMXB formulations and couldbe substituted with another immunologically inert com-ponent. Tween 85 is a detergent that minimizes proteinaggregation and is commonly used as an excipient forpharmaceuticals. We combined ISS, PMXB, and Tween85/oleic acid and observed that, like HSA, the presence ofTween 85 allowed for the formation of particles of a sim-ilar size range. In comparison to ISS/PMXB/HSA preps,ISS/PMXB/Tween 85 preps displayed a similar ability toenhance IFN-�/IFN-� production from human PBMCs(data not shown). We further examined whether activitycould be localized to small or large particles through sep-aration of ISS/PMXB/HSA preparations by either centri-fugation at 2000 rpm for 5 min or by passage through a2.7�m Wlter. Both techniques result in a rough divisionbetween 01�m particles and 11�m particles. We foundthat both populations exerted comparable IFN-�/IFN-�-inducing activity on human PBMCs (data not shown),indicating that ISS-enhancing properties are notrestricted to one size of particle.

3.5. Both polycationic and hydrophobic domains of PMXBare required for optimal activity

Members of the polymyxin family (including PMXBand colistin) have two major domains: (1) a cyclic peptidethat contains Wve positively charged amino acid residuesand (2) an N-terminal lipophilic fatty acyl derivative. Inorder to determine whether one or both domains wererequired in order to enhance ISS activity, we examinedthe ISS-enhancing potential of two derivatives of thepolymyxin family, colistin methanesulfonate (CMS) andpolymyxin B nonapeptide (PMXB-9). CMS is producedby a sulfomethylation reaction in which the primaryamine groups of the 2,4-diaminobutyric acids are reactedwith formaldehyde and then sodium bisulWte [18]. Thisresults in a molecule in which the cationic amino acidsare now negatively charged but the hydrophobic tail is

intact. Conversely, the PMXB-9 molecule lacks thehydrophobic tail but retains the positively charged cyclicpeptide domain. Therefore, we used these two derivativesas a means of analyzing the importance of each domainfor the observed enhancement of ISS activity. Both CMS(which did not form particles with ISS) and PMXB-9(which did) exhibited substantially reduced ISS-enhanc-ing activity in comparison to PMXB or colistin (Fig. 5).These data indicated that both the polycationic and lipo-philic domains of the polymyxin molecule are requiredfor optimal ISS-enhancing activity.

3.6. PMXB does not enhance uptake of ISS into PDCs

To investigate whether the enhancement of ISS-induced IFN-� secretion by PMXB was attributable toan enhancement in ISS uptake by PDCs, we examineduptake of Xuoresceinated-ISS into PDCs in the presenceof cPLGA or PMXB (Fig. 6). As has been previously

Fig. 6. PMXB does not enhance uptake of ISS into PDCs. Monocyte-depleted PBMCs were cultured with 2 �g/ml FAM-conjugated1018 § 100 �g/ml PMXB or § 100 �g/ml cPLGA for 4 h, then washedthoroughly to remove surface-bound 1018. Cells were stained withBCDA-2-PE then analyzed via FACScan. Dead cells were gated outvia FSC vs SSC and double-positive cells (FL1-intracellular 1018,FL2-BCDA-2) acquired. This histogram is gated on the BCDA-2+

population. Data shown are from one representative donor of four.Lines for 1018 and 1018/PMXB are virtually superimposed.

Fig. 5. Both domains of the PMXB molecule are required for optimal ISS-enhancing activity. PBMCs were stimulated for 24 h with 20 �g/ml 1018 orC661 and 100 �g/ml PMXB, colistin, colistin methanesulfonate (CMS), or polymyxin B nonapeptide (PMXB-9). Supernatants were assayed for IFN-�/IFN-� levels via ELISA. Data are expressed as means of six donors + SEM. Numbers are for statistical relevance: IFN-�: 1 vs 2: *; 1 vs 3: **; 2 vs5: *; 3 vs 6: **; 1 vs 4: ns; 1 vs 5: ns. IFN-�: 1 vs 2: ***; 1 vs 3: *; 2 vs 5: ***; 3 vs 6: *; 1 vs 4: ns; 1 vs 5: ns.


shown [17], cPLGA greatly enhanced ISS uptake intoPDCs and this mechanism may account, in part, for itsability to enhance ISS activity by simply allowing moreISS molecules to enter the PDC and contact intracellu-larly expressed TLR9. In contrast, the presence of PMXBdid not enhance ISS uptake into PDCs and may haveslightly inhibited it. This suggests that PMXB may beenhancing IFN-� levels through a mechanism other thanincreasing the total amount of ISS entering the PDC. Notsurprisingly, B cells also demonstrated no enhanceduptake of ISS in the presence of PMXB (data not shown),similar to PDCs.

3.7. PMXB boosts ISS-induced hepatitis B surfaceantigen-speciWc IgG in vivo

To conWrm these in vitro Wndings, PMXB was exam-ined for ISS-enhancing activity in murine and baboon

in vivo models of immunogenicity with hepatitis B sur-face antigen (HBsAg). Mice were immunized on days 0and 14 with HBsAg, HBsAg + 1018, or HBsAg + a prep-aration of 1018 or 1040 complexed with PMXB andTween 85/oleate. HBsAg alone induced modest titers ofHBsAg-speciWc IgG1 levels 2 weeks post-second immu-nization, while the presence of 1018 elevated IgG1 andespecially IgG2a titers (Fig. 7B). Mice immunized in thepresence of 1018/PMXB, however, demonstrated amuch greater ampliWcation of IgG2a levels than wereobserved with 1018 alone, and this was even visible at 2weeks post-Wrst immunization (Fig. 7A), thus verifyingthat PMXB can greatly enhance and accelerate 1018activity in vivo as well as in vitro. Although 1040/PMXB control immunization led to an increase in IgG1levels (presumably due to CpG-unrelated eVects on Bcells as also observed in Fig. 4D), it led to no increase inIgG2a levels, an indication that the IgG2a eVect was

Fig. 7. PMXB enhances the ISS eVect on hepatitis B surface antigen-speciWc antibody production in immunized mice and baboons. (A–B) BALB/cmice were immunized in four groups of 10 animals each with HBsAg § 1018, 1040/PMXB, or 1018/PMXB. Immunizations (i.m.) took place on days0 and 14. Blood was collected for serum analysis of HBsAg-speciWc Ab titers at days 14 (A) and 28 (B). Naïve animals exhibited undetectable levels(0120) of anti-HBsAg Ab. Data are reported as means of 10 animals + SEM. Statistical signiWcance was computed vs HBsAg immunization. (C)Baboons were immunized with HBsAg § 1018 § PMXB and blood serum samples were analyzed for anti-HBsAg 4 weeks later. Data are reported asmeans of 5–10 animals. Numbers indicate animals sero-positive for anti-HBsAg (110 mIU/ml).


both CpG-speciWc and PMXB-dependent. Baboonsimmunized with HBsAg + 1018/PMXB also showedenhanced serum titers of anti-HBsAg IgG in compari-son with HBsAg + 1018 injection (Fig. 7C), further vali-dating the in vivo properties of ISS/PMXB as a vaccineadjuvant.

3.8. ISS/PMXB exhibits diVerential eVects on human andmouse immune activities in comparison to ISS/cPLGA

We have previously described the elevating eVects ofISS associated with cPLGA on IFN-� induction fromhuman PBMCs [17]. To compare the similar ISS-enhanc-

Fig. 8. ISS/PMXB induces immune activity equivalent to or greater than ISS/cPLGA formulations. (A) Human PBMCs were stimulated for 24 hwith 20 �g/ml ODNs § 100 �g/ml PMXB or cPLGA. Supernatants were assayed for IFN-�/IFN-� levels via ELISA. Data are expressed as means of8–12 donors + SEM. Statistical signiWcance for 1018 was computed vs medium and for all other conditions vs 1018. (B) Human B cells were culturedwith ODNs § PMXB or cPLGA for 96 h. The ISS/PMXB prep used in this assay had been previously dialyzed to remove free PMXB molecules. Pro-liferation was assessed by [3H]thymidine incorporation. Proliferation data are reported as percentage of 1018-induced proliferation and are expressedas means of four donors. Statistical signiWcance was computed vs 1018 for all conditions. (C) BALB/c mice were immunized in four groups of 10 ani-mals each with HBsAg § 1018, 1018/cPLGA, or 1018/PMXB. Immunizations (i.m.) took place on days 0 and 14. Blood was collected for serum anal-ysis of HBsAg-speciWc Ab titers at day 14. Data are reported as means of 10 animals + SEM. Statistical signiWcance was computed vs HBsAgimmunization. (D) Splenocytes were collected from these mice on day 42 and stimulated with HBsAg for 4 days after which IFN-�/IL-5 secretionwas measured by ELISA. Statistical signiWcance was computed vs HBsAg immunization. (E) Day 42 splenocytes were also stimulated with HBsAgpeptide for 6 days, then co-cultured with 51Cr-P815 target cells for 4 h to measure cytolytic activity. Data are reported as mean of Wve mice.


ing properties of ISS/PMXB, we utilized the two formu-lations in a variety of in vitro and in vivo models of ISSactivity. Although formulation with either PMXB orcPLGA greatly boosted the IFN-�/IFN-�-inducing prop-erties of 1018 upon human PBMCs (Fig. 8A), the formu-lations diverged in regards to B cell activation. While1018/cPLGA enhanced human B cell proliferation over1018 stimulation alone, 1018/PMXB does not signiW-cantly alter this activity (Fig. 8B). However, 1018/PMXBproved much more eVective at increasing HBsAg-speciWcIgG2a titers in HBsAg-immunized mice than did 1018/cPLGA and furthermore skewed the IgG1:IgG2a ratiotowards a much more pronounced IgG2a-dominant Th1-like proWle (Fig. 8C), a result that was continued follow-ing the second immunization as well (data not shown).This Th1 skew was also reXected in cytokine productionby splenocytes from HBsAg-immunized mice (Fig. 8D).Administration of 1018/PMXB maintained the IFN-�-dominant proWle established by 1018 alone, while cellsfrom mice treated with 1018/cPLGA reverted back tohigher levels of IL-5 and lower levels of IFN-�. Neitherwas 1018/cPLGA administration able to support spleno-cyte CTL activity from HBsAg-immunized mice, unlike1018/PMXB, which permitted the strong CTL activityinduced by 1018 (Fig. 8E). Therefore, ISS/PMXB isclearly superior to ISS/cPLGA in the mouse in vivomodel since it maintains or boosts the eVects of ISS, whilesuch activity is generally abrogated by association withcPLGA. We did not observe enhancement of IFN-�secretion or CTL activity by PMXB in comparison to1018 alone in this system (Figs. 8D and E), and this mayindicate that PMXB is less able to boost T cell-mediated,adaptive immunity compared to the potent enhancementof innate immune responses reported in Figs. 3–6 andTable 1.

4. Discussion

This study demonstrates that enhanced ISS potencycan be achieved by formulating ISS ODNs with PMXBand a stabilizing agent. ISS from the CpG-B and CpG-Cclasses exhibited the most dramatic PMXB-mediatedincrease in IFN-�/� activity, while less enhancement wasobserved with CpG-A, perhaps due to the diYculty informulating particles with a class of ISS known to bealready highly aggregated [14]. We observed that thoseimmune responses thought to be downstream of anddependent on IFN-� production were the same ones alsoenhanced by PMXB. These included enhancement ofIFN-�-inducible chemokine expression and NK cell acti-vation. Interestingly, we found that other measures ofPDC activation that are not dependent on IFN-�, i.e.,increased expression of the activation markers CD80/CD86, were not enhanced by PMXB. Furthermore, thesestudies indicated that the B cell arm of the immune

response to ISS was also not directly aVected by PMXB,although B cell responses might be aVected indirectly invivo through PDC-derived signals. These results suggestthat ISS/PMXB targets the PDC-derived IFN-� arm ofthe immune response for augmentation. The mechanismby which PMXB achieves these eVects does not appear tobe due to enhanced uptake of ISS ODNs by the PDC.

Studies were performed to elucidate the structure ofassociated ISS/PMXB. When ISS and PMXB are addedtogether in saline, precipitation immediately occurs,yielding a highly aggregated and insoluble substance thatlacks ISS activity when applied to cultured cells (data notshown). Since this precipitation is not observed when ISSand PMXB are added directly to cultured cells, we sur-mised that the presence of serum in the culture may berequired for allowing the appropriate conWguration tooccur and express activity. Indeed, when HSA is presentduring the mixing of ISS and PMXB in saline (1:5:20molar ratio ISS:PMXB:HSA), we observed the forma-tion of discrete particles conforming to a bimodal sizedistribution and ranging in size from 100 nm to 10�m(Fig. 2). These particles also showed enhanced ISS activ-ity for IFN-�/� production when added to PBMCs, simi-larly to adding ISS and PMXB separately to the culture.PS ODNs are known to bind serum albumins with micro-molar Kd [19], and thus HSA may assist the formation ofionic bonds between negatively charged ISS and posi-tively charged PMXB. In addition, HSA may act as a sta-bilizing agent that counteracts the tendency for large ISS/PMXB aggregates to form. The role of the stabilizingagent may be played by several molecules, includingother albumins, KLH, and even the surfactant Tween 85,an excipient commonly used in many pharmaceutical for-mulations to minimize protein–protein interactions.

The chemical properties of PMXB that are requiredfor optimal enhancing ability are its polycationic domainand its lipophilic fatty acid side chain. The ISS ODNsused herein have a net negative charge of 19–21 andshould tightly associate with +5 positively chargedPMXB molecules that are Wve times in molar excess. Thistight association leads to the formation of microparticles.Since the hydrophobic tail of PMXB is known to per-meabilize bacterial cell membranes [2], it is possible thatthe ISS/PMXB complex enters the PDC through thismeans. On the other hand, PMXB-9, which lacks a lipo-philic sidechain, is also known to have some cell mem-brane-permeabilizing activity [3], although it is unclearwhether it could accomplish this if its positively chargedregion were bound (and neutralized) by ISS. Sinceenhanced uptake of ISS does not occur when PMXB-for-mulated (Fig. 6), the partial activity enhancementobserved with PMXB-9 and the fact that PMXB-9 formsparticles with ISS (data not shown) point to clustered ISSpolyvalent presentation as the mechanism for elevatedIFN-� induction, rather than increased entry of ISS intothe cell interior through membrane permeabilization.


CMS is gradually hydrolyzed and converted to colistin inan aqueous solution of human plasma over several days[18] and this may explain its partial enhancement of ISSactivity in our system (Fig. 5).

Therefore, we propose that PMXB and related mole-cules complex with ISS via interaction between the poly-cationic domain of PMXB and the negatively chargedDNA oligonucleotides. Tight association between multi-ple molecules of both components occurs in such a waythat large aggregate microparticles are formed withsuper-aggregation and subsequent precipitation coun-tered by the presence of stabilizers such as HSA or Tween85. Such microparticles enter the interior of the PDC,possibly by permeabilization of the cell membrane usingremaining free polycationic charge or the lipophilic fattyacid side chain. Alternatively, we cannot rule out that thecomplex may be endocytosed by an unidentiWed receptoron the PDC cell surface. Once inside, the ISS/PMXBcomplex presents multiple ISS motifs in multimeric fash-ion, a conWguration observed to consistently induce veryelevated levels of IFN-� expression from PDCs.Although ISS complexed to PMXB is also able to enter Bcells, we see no increase in ISS-induced activity, indicat-ing that B cell-speciWc ISS pathways are not inXuenced bymultimeric presentation of ISS motifs.

We conducted studies to clarify the mechanism bywhich PMXB increases the potency of ISS. PMXB doesnot appear to be enhancing the eVect of ISS throughincreased ODN uptake into the PDC population;instead, the signal for PDC-internalized ISS after 4 hincubation in the presence of PMXB appears to be equiv-alent to or even somewhat less than that in the absence ofPMXB. Nor does PMXB appear to be shuttling ISSODNs to diVerential compartments within the PDC (J.Marshall, unpublished observations). This suggests analternative possibility to explain PMXB's mechanism ofaction: that the conWguration in which ISS is presentedmay be crucial for the enhanced eVect. Although theother ISS-enhancer we studied, cPLGA, does greatlyenhance ISS uptake into PDCs [17], the increased IFN-�levels induced by this formulation may not depend onthat process. cPLGA also presents numerous ISS ODNsin a multivalent display on the surface of the nanoparti-cle, which may share conWgurational similarities withISS/PMXB/HSA particles, and possibly it is this aggre-gated form that is critical to achieve enhanced IFN-�production in the case of both types of formulation. Fur-ther evidence supporting this hypothesis is provided byobservations concerning the activity of CpG-A [14] andshort heptameric ISS linked to Ficoll [20]. Both of theseformulations also present a large number of ISS motifs inmultimeric form, and both also induce high levels ofIFN-� from PDCs in a CpG-speciWc manner. Anothercommonality to these various forms of clustered ISS isthat they are not nearly as enhancing, if at all, for otherCpG-dependent functions such as PDC maturation,

TNF-�/IL-6 production, and B cell proliferation [14,17].This divergence of elevated IFN-� induction from otherISS functions implies separate pathways initiated by theISS receptor and that perhaps ISS presented in arrays oraggregate form crosslinks TLR9 and ampliWes only thepathway that leads to high IFN-� transcription.

PMXB, colistin, and related molecules have beenadministered as antibiotics to humans for over 40 years,especially to cystic Wbrosis patients [21]. We have shownthat in addition to its bactericidal properties, PMXB alsodemonstrates a unique ability to enhance the activity ofISS ODNs. This enhancement is speciWcally directed toIFN-� production from PDCs without aVecting otherISS functions, such as B cell activation. Although theactivity of ISS/PMXB formulations on human cells invitro is well duplicated by the previously described ISS/cPLGA microparticles [17], ISS/PMXB complexes areplainly much more eVective at carrying out ISS activitiesin vivo in mice (Fig. 8), while ISS/cPLGA particles areineVective, perhaps because they are sequestered at thesite of injection. Although the A-class of ISS is alsoknown for greatly amplifying PDC-derived IFN-�, CpG-A are not recognized by B cells, while ISS/PMXB candirectly induce both PDC and B cell activity. Further-more, CpG-A activity is dependent upon the formationof heterogeneous populations of aggregates comprised ofa variable number of CpG-A molecules [14], therebymaking GMP-level characterization diYcult. On theother hand, ISS/PMXB microparticles can be easily sepa-rated by size into much more homogeneous populations.Renal and neurological toxicities have been reportedstemming from use of polymyxins in humans [2,22]; how-ever, the dose required for antibacterial eYcacy is 2.5–5.0 mg/kg/day for several consecutive days [2]. We esti-mate a delivery of PMXB in ISS formulations of approx-imately 0.2 mg/kg, 1–2 times per week (or less often forvaccines), which should signiWcantly decrease its toxicityproWle. Furthermore, we have proved that ISS/PMXBformulations are fully functional when stabilized withTween 85, an approved pharmaceutical excipient. TheIFN-�-dominant proWle of activity induced by ISS/PMXB formulations might be particularly useful fortreating disorders such as asthma or B cell lymphomas, inwhich the Th1-inducing and antitumor eVects of IFN-�are desirable without the concurrent elevated polyclonalactivation of disease-mediating B cells. In addition, ourpreliminary studies with in vivo HBsAg immunization inmice and baboons also demonstrate the potent activity ofISS/PMXB as a vaccine adjuvant.

Acknowledgments

We are grateful to F. Barrat, E. Hessel, and H. Kanz-ler for helpful discussion; to Cindy Fressola and SherryKelly (Advanced Bioscience Resources) for phlebotomy


services; to Holli Aaron (CRL Molecular Imaging Cen-ter, University of California at Berkeley) for Xuorescentmicroscopy services; and to N. Khounchanh for techni-cal assistance.

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Technology

Polymyxin B enhances ISS-mediated immune responses across