a rapid and broadened anamnestic response to subsequentboosting with PA83. Cynomolgus macaques were immunizedintranasally on days 0 and 14 with CVD 908-htrA expressingClyA-PA83 (n=12) or CVD 908-htrA carrying an empty plasmid(n=8). Three months after priming, monkeys of both groupswere evenly divided to be boosted either with 85 µg of rPA83plus alum or 0.5 mL of Biothrax (the currently licensedanthrax vaccine). All monkeys elicited serum IgG anti-S.Typhi LPS in response to the live vector priming. One weekafter the boost, all animals primed with S. Typhi(ClyA-PA83)exhibited remarkably high levels of anthrax toxin neutraliza-tion antibodies (TNA), regardless of the boosting agent(rPA83-alum or Biothrax). On the contrary, unprimed animalslacked detectable TNA responses (pb0.05). TNA titerspeaked on day 113 in animals primed with CVD 908-htrA(ClyA-PA83). This is the first report describing effectivemucosal immune responses to B. anthracis PA83 observed innon-human primates using an S. Typhi-based prime-boostapproach.

doi:10.1016/j.clim.2008.03.389

Su.39. Loss of Virus-specific Memory T Cells inCoxsackievirus B3 and B4-Infected MiceLu Li, Alfred Dufour. U.S. EPA, Cincinnati, OH

There are two major types of enteroviruses: poliovirusesand non-polio enteroviruses. While vaccines have effectivelyeliminated poliovirus infections, no vaccine is currentlyavailable for the non-polio enteroviruses. Generation oflong-term surviving, pathogen specific,memory cells is criticalfor the development of a good vaccine. To investigate whetherlong-termmemory Tcells are produced in response to infectionby non-polio enteroviruses, coxsackieviruses B3 (CVB3) andcoxsackieviruse B4 (CVB4) were intraperitoneally inoculatedinto adult BABL/c mice. Virus-specific memory T cells wereassayed for proliferation and release interferon gamma (IFN-γ)under ex vivo stimulationwith the viral pathogens. The level ofIFN-γ was measured by antibody-capture chemiluminescentELISA. Four days after stimulation, there were no detectablechanges in the levels of IFN-γ from mice 17 months post-exposure to CVB3 and CVB4 compared with negative controlmice inoculated onlywith sterilizedPBSbuffer. In contrast, thelevels of IFN-γweremarkedly increased after stimulation withspecific viral pathogens in mice 21/2 months post-infectionwith CVB3 and CVB4. These results suggest that non-polioenteroviruses might generate only short-lived virus-specificmemory T cells. This would imply that vaccines for non-polioenterviruses might not provide long term protection againstthese viruses. This study might explain why people canrepeatedly be infected by non-polio enteroviruses.

doi:10.1016/j.clim.2008.03.390

Su.40. Differential Antigen Processing Activities ofPBMC Subsets Targeted by HIV Modulate HIVEpitope ProductionEstibaliz Lazaro,1 Sasha Blue Godfrey,1 Pamela Stamegna,1

Jeremy Ho,1 Bruce Walker,2 Sylvie Le Gall. 1 1MGH and

Harvard Medical School, Charlestown, MA; 2HHMI, ChevyChase, MD

Background: Immune recognition of HIV-infected cellsrequires peptide presentation by major histocompatibilitycomplex I (MHC-I) at the cell surface. These peptides resultfrom a cascade of proteolytic events involving proteasomeand aminopeptidases. Although HIV infects several cellulartypes, the capacity of each of them to process epitopes isunknown. Here we compare epitope processing activities ofprimary CD4 Tcells and monocytes. Methods: CD4 Tcells andmonocytes were enriched by magnetic immunosorting fromperipheral blood monocytic cells (PBMC) of 20 healthydonors. Proteasome and aminopeptidase activities weremeasured for each cell subset using specific fluorogenicsubstrates. The capacity of the extracts to produce epitopeswas assessed during the in vitro degradation of long HIV Gagor Pol peptides. Kinetics, identity and amount of digestionproducts were identified by HPLC and mass spectrometry.Their antigenicity was measured by 51Cr release assay withepitope-specific CTL. Results: Proteasome and aminopepti-dase activities were significantly higher in monocytes than inPBMC or CD4 T cells. These differences affected the identity,amount and kinetics of peptides produced. Monocytesextracts yielded the largest and fastest production of optimalepitopes or extended antigenic peptides and consequently theantigenicity of the degradation peptides produced in mono-cyte extracts was 5-fold higher than that of CD4 T cells.Conclusion: Differential antigen processing activities in twoPBMC subsets targeted by HIVaffects the identity, amount andantigenicity of peptides produced. This may alter the capacityof CD8 T cells to recognize infected cells.

doi:10.1016/j.clim.2008.03.391

Su.41. Mucosal Salmonella Typhi Prime Followedby Parenteral Subunit Vaccine Boost Can Serve asan Effective Vaccine Strategy for Early LifeImmunizationKarina Ramirez, Liliana Rodriguez, Katherine Davis, JamesGalen, Marcela Pasetti. University of Maryland School ofMedicine, Baltimore, MD

Neonates have a limited capacity to generate protectiveimmunity in response to vaccination. We have shown thatSalmonella-based live vector vaccines are excellent candi-dates to induce immune responses against a foreign antigenat very early life stages. Recognizing that biodefensevaccines will be needed for high-risk groups such as younginfants and children, we investigated the immunogenicity ofthe licensed attenuated S. Typhi vaccine Ty21a expressingBacillus anthracis protective antigen (Ty21a-PA). Newbornmice were immunized intranasally with Ty21a-PA on day 7and 14 after birth, and boosted intramuscularly 14 days laterwith PA-alum. Control groups received Ty21a alone or PBSfollowed by PA-alum. Newborns primed with Ty21a-PA andboosted with PA-alum induced high levels of PA-specific IgGand toxin neutralizing antibodies that surpassed those of theunprimed group. Live vector priming elicited strong cell-mediated immunity (Tcell proliferation and IFN-γ secretion)

S137Abstracts

and mucosal antibody secreting cells during the neonatalperiod. Unprimed animals elicited modest antibodyresponses but failed to induce cell-mediated and mucosalimmunity. We hypothesized that one reason for the enhancedresponses in live-vector primed newborns was the capacity ofSalmonella to enhance maturation of neonatal DC and T cellstimulation. Bone-marrow derived neonatal DC exposed toTy21a-PA showed increased expression of CD80 and CD40 andstrong proliferation of PA-specific T cells as opposed to DCincubated with PA or untreated DC. We conclude that S. Typhiexpressing anthrax protective antigen followed by parent-eral subunit vaccine boost could serve as an effectivebiodefense vaccine strategy for young hosts.

doi:10.1016/j.clim.2008.03.392

Su.42. Oligoribonucleotides ContainingArabinonucleosides Act as Potent Agonistsof Toll-like Receptors 7 and 8Tao Lan, Lakshmi Bhagat, Meiru Dai, Daqing Wang, EkambarKandimalla, Sudhir Agrawal. Idera Pharmaceuticals, Inc.,Cambridge, MA

Single-stranded viral RNA sequences have been shown tobe natural ligands for Toll-like receptors (TLR) 7 and 8. Wehave developed stabilized immune modulatory RNA (SIMRA)compounds, which act as agonists of TLR7 and 8 dependingon the nucleotide composition and chemical modificationsincorporated (Lan, T., et al., Proc. Natl. Acad. Sci. USA, 104,13750, 2007). In continuation of our efforts in identifyingnovel modifications of nucleosides to incorporate in SIMRAcompounds to act as potent TLR7 and 8 agonists, we havestudied arabinonucleosides as substitutes for ribonucleosidesat various positions in SIMRA compounds. SIMRA compoundscontaining ara-G, ara-C, ara-A, or ara-U in place of G, C, A, orU, respectively, were synthesized and purified followingstandard protocols and evaluated for their ability to act asagonists of TLR7 and 8 in HEK293 cells expressing humanTLR7 or 8, and human PBMCs and plasmacytoid dendritic cellcultures. The results of these studies suggest that SIMRAcompounds containing ara-G, ara-A, or ara-U act as agonistsof TLR8 and SIMRA compounds containing ara-C act asagonists of both TLR7 and TLR8. Furthermore, ara-C contain-ing SIMRA compounds induced IL-12 production in vivo inmice. For the first time, these studies indicate that RNAcompounds containing arabinonucleoside substitutions act asagonists of TLR7 and 8.

doi:10.1016/j.clim.2008.03.393

Su.43. Neutrophils: Key Players in Systemic-onsetJuvenile Idiopathic Arthritis PathogenesisFlorence Allantaz Frager, Vicky Cantrell, Benoit Coudert,Jacques Banchereau, M. Virginia Pascual. Baylor Institutefor Immunology Research, Dallas, TX

Juvenile idiopathic arthritis (JIA) is the most commonrheumatic disease in childhood and includes a heterogeneousgroup of chronic inflammatory arthritis. Among them,

Systemic onset JIA (SoJIA) is unique in terms of clinicalmanifestations, prognosis and response to therapy. Wepreviously showed the importance of IL-1b in thepathogenesis of SoJIA as well as the beneficial effects ofanti-IL1 use in controlling the clinical manifestations ofthe disease. Because the gene expression profiles of SoJIApatient's PBMCs are remarkably similar to those of patientswith Gram positive bacterial infections, we sought todetermine if exposure to bacteria might contribute to theIL-1 dysregulation in this disease. Gene expression changesinduced by Gram positive bacterial stimulation of healthyas well as SoJIA patient whole blood in vitro wereanalyzed using Illumina Human 6v2 chips. SoJIA bloodcells had an increased and more prolonged expression ofinnate immunity genes, in particular members of the IL-1family, in response to bacteria. To ascertain the contribu-tion of innate immunity cells to this response, the geneexpression profiles of freshly isolated neutrophils fromactive SoJIA patients were analyzed using the samemicroarray platform. Neutrophils appeared indeed to bea major source of IL-1b transcripts, and several membersof the IL-1 family were among the most upregulated genesin these cells. Taking together, these results suggest thatSoJIA may arise from an inappropriate response of theinnate immune system to bacterial challenge and thatneutrophils may be key players in the pathogenesis of thedisease.

doi:10.1016/j.clim.2008.03.394

Su.44. IL-10-Producing HIV-specific T Cells HaveSuppressive PropertiesEirik Torheim,1 Lishomwa Ndhlovu,2 Aashish Jha,2 FrankPettersen,3 Dag Kvale,3 Douglas Nixon,2 Kjetil Taskén,1 EinarAandahl.1 1University of Oslo, Oslo, Norway; 2San FranciscoGeneral Hospital, San Francisco, CA; 3Ulleval UniversityHospital, Oslo, Norway

Recent studies have indicated that regulatory T cells(Treg) may contribute to HIV-1-related immune suppression.However, it is not clear whether T cells with suppressiveproperties reside within the HIV-1-specific T-cell population.Here, PBMC from HIV-infected individuals were stimulatedwith a 15-mer gag peptide-pool, and HIV-1-specific IL-10-secreting cells were enriched using immunomagnetic cellsorting (MACS). The isolated IL-10-secreting cells did notexpress FOXP3, yet potently suppressed CD3/CD28-inducedproliferative T-cell responses. Furthermore, as shown byintracellular cytokine-staining, most IL-10-producing cellsdid not simultaneously produce IFN-γ, suggesting thepresence of a regulatory T-cell population distinct fromIFN-γ-secreting effector T cells. Interestingly, a majority ofIL-10-producing cells expressed CD4 rather than CD8,whereas IFN-γ-producing cells were predominantly CD8-positive. Our data suggest that HIV-1-specific T cells harbourpotent immunoregulatory properties that may contribute tothe HIV-associated T-cell dysfunction.

doi:10.1016/j.clim.2008.03.395

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