Type 1 and 2 Immunity Following Vaccination Is Influenced by Nanoparticle Size:  Formulation of a Model Vaccine for Respiratory Syncytial Virus

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  Type 1 and 2 Immunity Following Vaccination Is Influenced by Nanoparticle Size:  Formulation of a Model Vaccine for Respiratory Syncytial Virus
  Type 1 and 2 Immunity Following Vaccination IsInfluenced by Nanoparticle Size: Formulation of a ModelVaccine for Respiratory Syncytial Virus Patricia L. Mottram, † David Leong, † Blessing Crimeen-Irwin, † Simone Gloster, † Sue D. Xiang, † Jayesh Meanger, ‡ Reena Ghildyal, ‡ Nicholas Vardaxis, § andMagdalena Plebanski* ,† The Burnet Institute at Austin (Austin Research Institute), Studley Road, Heidelberg 3084,VIC, Australia, The Burnet Institute, Commercial Road, Prahran, 3181, VIC, Australia, and  RMIT Uni V  ersity, Bundoora West Campus, GPO Box 2476V, Melbourne, 3001, VIC, Australia Received September 10, 2006; Revised Manuscript Received October 29, 2006; Accepted November 6, 2006 Abstract:  Previous studies compared uptake by dendritic cells (DC) of 20, 40, 100, 200, 500, 1000,and 2000 nm beads in vivo .  When beads were used as antigen carriers, bead size influenced antibodyresponses and induction of IFN- γ -producing CD4 and CD8 T cells. Beads of 40 - 50 nm were takenup preferentially by DC and induced particularly strong immunity. Herein, we examine immunity inducedby minute differences in nanobead size, specifically within a narrow viral-sized range (20, 40, 49, 67,93, 101, and 123 nm), to see if bead carrier size influenced the induction of type 1 or type 2 cells asdemonstrated by the production of IFN- γ  or IL-4. In vivo uptake by DC was assessed for selectedsizes in this range. Responses to whole ovalbumin (OVA) or the OVA-derived CD8 T cell peptideepitope (SIINFEKL) were tested. After one immunization with beads - OVA, IFN- γ  responses to bothOVA and SIINFEKL were significantly better with 40 and 49 nm beads than other sizes, while, incontrast, IL-4 responses to OVA were higher after immunization with OVA conjugated to larger beads(93, 101, and 123 nm). Thus IFN- γ  induction from CD8 T cells was limited to 40 - 49 nm beads, whileCD4 T cell activation and IL-4 were induced by 93 - 123 nm beads - OVA. After two immunizations,there were comparable high levels of IFN- γ  produced with 40 and 49 beads and IL-4 reactivity wasstill higher for larger beads (93, 101, 123 nm). Production of IgG1 was seen across the full range ofbead sizes, increasing after two immunizations. Since protection against respiratory syncytial virus(RSV) depends on strong IFN responses, while IL-4 responses are reported to cause asthma-likesymptoms, immunization with RSV antigens on the 49 nm carrier beads could provide the basis for asuitable vaccine. When the 49 nm beads were conjugated to RSV proteins G88 (surface) or M2.1(internal capsid), one immunization with G88 induced high levels of IFN- γ  and low levels of IL-4. IL-4increased with two immunizations. Beads - M2.1 induced only moderate levels of IFN- γ  and low titerantibody after two immunizations. Mice vaccinated once with G88-conjugated 49 nm beads andchallenged intranasally with RSV strain A2 subtype showed reduced viral titers and recovered fromweight loss more rapidly than mice immunized with M2.1-conjugated 49 nm beads or naive controlmice. These results show that precise selection of nanobead size for vaccination can influence thetype 1/type 2 cytokine balance after one immunization, and this will be useful in the development ofeffective vaccines against common human pathogens such as RSV. Keywords:  Type 1 and 2 immunity; nanobead size; ovalbumin; RSV vaccine; mice Introduction The difference between type 1 and type 2 immunity wassrcinally defined by functionally distinct subsets of CD4 Thelper cells, with type 1 (Th1) cells producing IFN- γ  andIL-2 and type 2 (Th2) cells secreting IL-4 and IL-10. Thisdefinition has since been modified in recognition of the othercell types involved in the response. 1,2 This polarization of responses is particularly important in vaccine development,where the most effective antiviral responses are type 1, with * Author to whom correspondence should be addressed. Mailingaddress: Vaccines and Infectious Diseases Unit, The BurnetInstitute, Austin Campus, Austin Hospital, Studley Road,Heidelberg, Victoria 3084, Australia. Tel: 61-3-92870643.Fax: 61-3-92870643. E-mail: mplebans@burnet.edu.au. † The Burnet Institute at Austin (Austin Research Institute),Heidelberg. ‡ The Burnet Institute, Prahran. § RMIT University. (1) Coffman, R. L. Origins of the T(H)1-T(H)2 model: a personalperspective.  Nat. Immunol.  2006 ,  7  , 539 - 541.(2) Mosmann, T. R.; Cherwinski, H.; Bond, M. W.; Giedlin, M. A.;Coffman, R. L. Two types of murine helper T cell clone. I.Definition according to profiles of lymphokine activities andsecreted proteins.  J. Immunol.  1986 ,  136  , 2348 - 2357. articles 10.1021/mp060096p CCC: $37.00 © XXXX American Chemical Society  VOL. XXXX NO. XXXX  MOLECULAR PHARMACEUTICS APAGE EST: 11.4 Published on Web 11/30/2006  IFN- γ  production and activation of CD8 T cells, whileallergic reactions (asthma, rhinitis) are type 2. 3,4 Thus optimalvaccine formulations would favor induction of primary type1 responses, leading to long-term CD4 and CD8 T cellmemory. At present, the most effective antiviral vaccinescontain attenuated or heat killed viral particles, 3 with recentlyavailable viral peptides and recombinant proteins being lesseffective. Coupling these new reagents to viral-sized par-ticles 5 may well provide vaccine formulations of greaterconsistency and with fewer side effects, such as unwantedtype 2 allergic responses.Endocytic uptake mechanisms vary depending on the sizeof the particle engulfed, thus larger beads ( > 500 nm) entervia phagocytosis and localize to macrophages, while virus-sized particles (20 - 200 nm) enter either by receptor-mediated endocytosis into clathrin-coated pits (particles < 150 nm) 6 or through caveolae (particles within 50 - 80 nm). 7,8 Smaller particles can enter via pathways used by viralpathogens, such as influenza and respiratory syncytialviruses. 9,10 Although solid polystyrene, gold, or silica beadshave previously been used as carriers for surface-absorbedantigen to stimulate CD8 T-cell responses, bead diametersof 500 - 1000 nm were commonly used. 11 Smaller antigen-carrying particles such as ISCOMS 12 and engineered virus-like particles (VLP) of 30 - 200 nm diameter usually alsoprovide “danger” signals, often through stimulation via Toll-like receptors (TLR), to induce activation of antigen present-ing cells. 13,14 Solid, synthetic, particulate vaccines, in contrast,can induce immune responses without added classical“danger signals”, 5,15,16 and our novel nanovaccine approach,using 40 - 50 nm polystyrene beads with covalently coupledtumor-specific antigen, has been shown to clear largeestablished tumor masses in mice within two weeks after asingle injection. 17 Thus, the size of the carrier bead could play a role indetermining the type of response induced, with virus-likeparticles inducing IFN- γ  cell mediated type 1 responses andlarger beads inducing type 2 responses. 18 In vaccine develop-ment, IFN- γ  responses are more likely to be protectiveagainst systemic intracellular pathogens such as viruses, 19,20 so 40 - 49 nm beads may be more effective antigen carriersthan beads of other sizes for antiviral vaccines. To test thishypothesis in a model viral infection in mice, 49 nm beadswere used in the second section of the study to formulate avaccine against respiratory syncytial virus, RSV. This virusis a major cause of lower respiratory tract disease in infantsand small children, infecting almost all children under theage of two. While most children recover and developimmunity as they mature, naturally acquired protection is“neither complete nor durable”, 21 and premature infants andthose with cardiovascular disease are at risk of fatal lung (3) Welsh, R. M.; Selin, L. K.; Szomolanyi-Tsuda, E. Immunologicalmemory to viral infections.  Annu. Re V  . Immunol.  2004 ,  22 , 711 - 743.(4) Connors, M.; Giese, N. A.; Kulkarni, A. B.; Firestone, C. Y.;Morse, H. C., 3rd; Murphy, B. R. Enhanced pulmonary histopa-thology induced by respiratory syncytial virus (RSV) challengeof formalin-inactivated RSV-immunized BALB/c mice is abro-gated by depletion of interleukin-4 (IL-4) and IL-10.  J. Virol. 1994 ,  68  , 5321 - 5325.(5) Xiang, S.; Scholzen, A.; Minigo, G.; David, C.; Apostolopoulos,V.; Mottram, P.; Plebanski, M. Pathogen recognition and develop-ment of particulate vaccines: does size matter?  Methods ,  2006 , 40  (1) (Sept), 1 - 9.(6) Daecke, J.; Fackler, O. T.; Dittmar, M. T.; Krausslich, H. G.Involvement of clathrin-mediated endocytosis in human immu-nodeficiency virus type 1 entry.  J. Virol.  2005 ,  79 , 1581 - 1594.(7) Chen, Y.; Norkin, L. C. Extracellular simian virus 40 transmits asignal that promotes virus enclosure within caveolae.  Exp. Cell Res.  1999 ,  246  , 83 - 90.(8) Norkin, L. C. Simian virus 40 infection via MHC class I moleculesand caveolae.  Immunol Re V  .  1999 ,  168  , 13 - 22.(9) Nunes-Correia, I.; Eulalio, A.; Nir, S.; Pedroso de Lima, M. C.Caveolae as an additional route for influenza virus endocytosisin MDCK cells.  Cell. Mol. Biol. Lett.  2004 ,  9 , 47 - 60.(10) Werling, D.; Hope, J. C.; Chaplin, P.; Collins, R. A.; Taylor, G.;Howard, C. J. Involvement of caveolae in the uptake of respiratorysyncytial virus antigen by dendritic cells.  J. Leukocyte Biol.  1999 , 66  , 50 - 58.(11) Robinson, H. L. DNA vaccines: basic mechanism and immuneresponses (Review).  Int. J. Mol. Med.  1999 ,  4 , 549 - 555.(12) Watson, D. L.; Watson, N. A.; Fossum, C.; Lovgren, K.; Morein,B. Interactions between immune-stimulating complexes (ISCOMs)and peritoneal mononuclear leucocytes.  Microbiol. Immunol.  1992 , 36  , 199 - 203.(13) Schwarz, K.; Storni, T.; Manolova, V.; Didierlaurent, A.; Sirard,J. C.; Rothlisberger, P.; Bachmann, M. F. Role of Toll-likereceptors in costimulating cytotoxic T cell responses.  Eur. J. Immunol.  2003 ,  33 , 1465 - 1470.(14) Maloy, K. J.; Donachie, A. M.; Mowat, A. M. Induction of Th1and Th2 CD4 + T cell responses by oral or parenteral immuniza-tion with ISCOMS.  Eur. J. Immunol.  1995 ,  25 , 2835 - 2841.(15) Allsopp, C. E.; Plebanski, M.; Gilbert, S.; Sinden, R. E.; Harris,S.; Frankel, G.; Dougan, G.; Hioe, C.; Nixon, D.; Paoletti, E.;Layton, G.; Hill, A. V. Comparison of numerous delivery systemsfor the induction of cytotoxic T lymphocytes by immunization.  Eur. J. Immunol.  1996 ,  26  , 1951 - 1959.(16) Plebanski, M.; Gilbert, S. C.; Schneider, J.; Hannan, C. M.; Layton,G.; Blanchard, T.; Becker, M.; Smith, G.; Butcher, G.; Sinden,R. E.; Hill, A. V. Protection from Plasmodium berghei infectionby priming and boosting T cells to a single class I-restrictedepitope with recombinant carriers suitable for human use.  Eur. J. Immunol.  1998 ,  28  , 4345 - 4355.(17) Fifis, T.; Mottram, P.; Bogdanoska, V.; Hanley, J.; Plebanski, M.Short peptide sequences containing MHC class I and/or class IIepitopes linked to nano-beads induce strong immunity andinhibition of growth of antigen-specific tumour challenge in mice. Vaccine  2004 ,  23 , 258 - 266.(18) Gamvrellis, A.; Leong, D.; Hanley, J. C.; Xiang, S. D.; Mottram,P.; Plebanski, M. Vaccines that facilitate antigen entry intodendritic cells.  Immunol. Cell Biol.  2004 ,  82 , 506 - 516.(19) Riedl, P.; Stober, D.; Oehninger, C.; Melber, K.; Reimann, J.;Schirmbeck, R. Priming Th1 immunity to viral core particles isfacilitated by trace amounts of RNA bound to its arginine-richdomain.  J. Immunol.  2002 ,  168  , 4951 - 4959.(20) Durbin, J. E.; Durbin, R. K. Respiratory syncytial virus-inducedimmunoprotection and immunopathology.  Viral Immunol.  2004 , 17  , 370 - 380.(21)  Acute respiratory infections: Respiratory syncytial V  irus (RSV) ;World Health Organization: 2006. http://www.who.int/vaccine_research/diseases/ari/en/index3.html (accessed November 22, 2006). articles  Mottram et al. B MOLECULAR PHARMACEUTICS  VOL. XXXX NO. XXXX  disease following infection. 21 Vaccine development has beenhampered by the failure of initial clinical trials usingformalin-inactivated virus, where induction of a type 2, IL-4response resulted in eosinophilia and acute respiratoryfailure. 21 More recently the availability of purified RSVprotein subunits (G, M, and F) has provided antigens that,with adjuvants, have entered clinical trials. Although thesevaccines have not produced IL-4 and adverse side effects,they have also not provided long lasting protection: annualvaccination will be required even with the most effective of these formulations, now in phase III trials. 21 There is a needtherefore to improve RSV vaccines, and designing suitableconjugates of recombinant antigens with nanobead-basedvaccine carriers that stimulate type 1 rather than type 2response may be useful in that context. Experimental Methods Protein and Peptide Antigens.  Ovalbumin (OVA) waspurchased from Sigma (grade III, Sigma-Aldrich, St Louis,MO). The minimum OVA CD8 T cell epitope, (SIINFEKL),was synthesized without flanking amino acids by Auspep(Melbourne, Australia). M2.1 proteins and pET-30a plasmidscontaining the coding sequence for RSV G88, tagged withpoly-histidine residues, were obtained from Jayesh Meangerand Reena Ghildyal, Burnet Institute, Melbourne, Austra-lia. 22,23 Small-scale production of recombinant RSV G88 wascarried out within our laboratory using standard expression,solubilization, and purification methods. Briefly, the plasmidswere induced to express in  Escherichia coli  BL-21 cells bythe addition of 1 mM IPTG. The cells were harvested bycentrifugation 3 - 4 h after IPTG induction. The cell pelletswere lysed by sonication in lysis buffer (50 mM Tris-Cl/2mM EDTA pH 8.0 + 1 mg/mL lysozyme and 0.1% TritonX-100). The resulting insoluble pellet was then solubilizedin buffer (8 M urea/0.1 mM Tris-Cl/0.1 M sodium phosphatebuffer), and the His-tagged recombinant proteins werepurified using a column of Ni 2 + NTA beads (Qiagen, ww-w.qiagen.com) and solubilization buffer at a lower pH. Thepurified proteins were then transferred into 2 M urea/PBSand centrifuged in Centricon concentration devices (Amicon,from Millipore, www.millipore.com). Routinely, recombinantRSV proteins of   > 95% purity were produced at ap-proximately 5 mg per liter of starting culture. Nanoparticle - Antigen Conjugation.  Carboxyl-modified20, 40, 49, 67, 93, 101, and 123 nm polystyrene beads fromMolecular Probes (Invitrogen), IDC/Polysciences (Poly-sciences, Inc., Warrington, PA), Sigma (St. Louis, MO) orPolyMicrospheres (PolyMicrospheres, Indianapolis) wereused (Table 1). There were no significant differences betweenbead activity as assessed in side-by-side experiments using20, 40, or 49 nm formulations from different manufacturers(not shown). Further, the standard error around the givenmean particle diameter size was in all cases (and for all sizestested) between 5% and 30%, as specified by the manufac-turers, as determined by Transmission Electron Microscopyand shown in Table 1.Conjugation to OVA was performed as previously de-scribed. 24 Briefly, carboxyl-modified polystyrene beads werediluted to a 2% slurry (wt:vol) and combined 1:1 (vol:vol)with 1 mg/mL OVA (grade III, Sigma-Aldrich, St. Louis,MO) to provide a 1% wt/vol bead preparation containing50  µ g of OVA/100  µ L. Conjugation was for 15 min withEDAC (4 mg/mL, 1-ethyl-3-(3-dimethylaminopropyl) car-bodiimide; Sigma) and 0.05 M MES ([2-  N  -morpholino]ethane sulfonic acid; Sigma) added; the pH was adjusted to6.5 with NaOH, and the mixture was rocked at roomtemperature for 2 h. Glycine (7 mg/mL; Sigma) was addedfor 30 min before overnight dialysis (12 - 14 kDa membranepore) against PBS. This did not remove unconjugated OVAfrom the conjugation mix. The volume was adjusted to 1%wt:vol solids. Conjugated particles were stored at 4  ° C forno longer than 3 days and sonicated for > 5 min in ice waterbefore use. To assess the amount of protein conjugated tobeads, 25  µ L from each conjugation was sampled and spunat 20000 g  in an ultracentrifuge for 20 - 30 min at 4 - 8  ° C,and the concentration of unconjugated protein in the super-natant was tested using a BCA assay kit (Pierce Biotech-nology, Rockford, IL) according to the manufacturer’sinstructions. This measured the amount of antigen notconjugated to beads and, with the known amount of antigenin the conjugation mix, allowed calculation of the % of antigen conjugated to the beads. At 500  µ g/mL of OVA with1% wt:vol beads, 96 - 100% of OVA was coupled to thebeads, regardless of bead size. In previous studies we have (22) Shields, B.; Mills, J.; Ghildyal, R.; Gooley, P.; Meanger, J.Multiple heparin binding domains of respiratory syncytial virusG mediate binding to mammalian cells.  Arch. Virol.  2003 ,  148  ,1987 - 2003.(23) Ghildyal, R.; Mills, J.; Murray, M.; Vardaxis, N.; Meanger, J.Respiratory syncytial virus matrix protein associates with nucleo-capsids in infected cells.  J. Gen. Virol.  2002 ,  83 , 753 - 757.(24) Fifis, T.; Gamvrellis, A.; Crimeen-Irwin, B.; Pietersz, G. A.; Li,J.; Mottram, P. L.; McKenzie, I. F.; Plebanski, M. Size-dependentimmunogenicity: therapeutic and protective properties of nano-vaccines against tumors.  J. Immunol.  2004 ,  173 , 3148 - 3154. Table 1.  Nanobead Specifications bead size(nm) ( SD bead source20 ( 6 Molecular Probes, Polysciences, PolyMicrospheres40 ( 4 PolyMicrospheres, Sigma, Molecular Probes49 ( 5 Polysciences, Sigma67 ( 4 PolyMicrospheres93 ( 7 PolyMicrospheres101 ( 10 PolyMicrospheres123 ( 6 PolyMicrospheres Type 1 and 2 Immunity, Nanoparticle Size, and RSV Vaccine  articles VOL. XXXX NO. XXXX  MOLECULAR PHARMACEUTICS C  also measured the amount of radioactive antigen coupled todifferent-sized beads, with similar results. 24 For RSV protein conjugation, carboxylated polystyrenenanobeads from Polysciences (49 nm) were conjugated asdescribed above to provide 1% wt:vol final solids with 20 - 80  µ g of RSV protein/mouse. Again, unconjugated proteinwas not removed from the immunizing dose of bead - antigen. BCA assays showed 100% of OVA conjugated tobeads at 20  µ g of protein and 80 - 90% at 40 and 80  µ g. Inpreparing injections of conjugates, all beads and solubleantigens were prepared in identical test tubes and syringes.For stored soluble antigen, protein concentrations wereroutinely checked by spectrophotometer and showed nosignificant decrease, at the concentrations used, due toabsorption to container surfaces. Animals and Immunizations.  H-2K b C57BL/6 and H-2K d BALB/c 6 - 8 week old mice were obtained from the Walterand Eliza Hall Institute, Melbourne, Australia. Antigen-conjugated beads, beads alone, or soluble protein alone wasadministered intradermally (ID) to the hind footpads of micein a final volume of 100  µ L (50  µ L in each footpad). Forone immunization experiments, mice were sacrificed 10 - 14days after vaccination. For two immunization experiments,two vaccinations at the same dose were given 14 days apartand mice were sacrificed at 10 - 14 days after the lastimmunization. Spleen cells were collected for cellular assaysat that time. In all of the experiments, regardless of the beadsize being tested, animals were immunized with 1% wt:volof polystyrene, i.e., the same total dose of polystyrenematerial regardless of bead size (1  µ g in 100  µ L). ELISPOT IFN- γ  and IL-4 Assays.  Spleen cells (0.2 - 1 × 10 6 ) were suspended in RPMI-1640 medium supplementedwith 5% heat inactivated fetal calf serum (JRH Biosciences,Lenexa, KS), 2 mM glutamine, 100  µ g/mL streptomycin and100 units/mL penicillin (all Gibco/Invitrogen,), or withSIINFEKL at 2.5  µ g/mL, OVA, 25  µ g/mL, or RSV recom-binant proteins G88 or M2.1, 20 - 25  µ g/mL, final concentra-tions, for 18 h in 96-well plates (MAHA, Millipore, Billerica,MA) coated with anti-murine IFN- γ  or anti-murine IL-4 mAb(551216 and 554387, respectively, Pharmingen, CA). Du-plicate or triplicate wells were set up for each condition. Cellswere then discarded and plates incubated for 2 - 4 h withanti-murine IFN- γ  mAb-biotin (554410, Pharmingen, CA),or anti-IL-4 mAb-biotin (554390, Pharmingen, CA), followedby extravidin-alkaline phosphatase (AP) (Sigma). Spots of activity were detected using a colorimetric AP kit (Biorad,Hercules, CA) and counted using an automated ELISPOTreader with manual checking. Extensive washing (5 × ) inPBS was performed between each of the incubation steps.Data are presented as mean spot forming units (SFU) permillion cells  (  standard deviation (SD, for single experi-ments) or error (for pooled experiments) of the mean (SE). ELISA.  Polyvinyl chloride microtiter plates precoated withOVA or recombinant RSV proteins G88 or M2.1 (10  µ g/ mL in 0.2 M NaHCO 3  buffer, pH 9.6) were blocked with2% (w/w) bovine serum albumin (BSA) in PBS for 1 h at37  ° C, washed 5 times with PBS containing 0.2% (w/w)Tween-20 (Sigma) and incubated for 2 h at room temperature(RT) with mouse sera, washed and then incubated withhorseradish-peroxidase (HRP)-conjugated sheep anti-mouseIgG or antibodies detecting mouse IgG1, 2a, or 2b (BD,Pharmingen) for 1 h at RT, and washed again before additionof developing buffer containing substrate (ABTS or TMB)for 30 min. Development was stopped by addition of 1 MHCl, and plates were read on an EL312e microplate reader(BMG Labtechnologies, Offenberg, Germany) at 450 nm.Data is presented as mean  (  standard deviation for eachdilution of sample, or as antibody titer, determined by themean and SD of all the naive wells (negative controls). Thusthe mean of naives  +  3  ×  SD was a reference numberspecific for an ELISA. The experimental titer for test sampleswas the dilution with a mean value higher than the referencenumber. FACScan Analysis of Bead Localization.  Popliteallymph node cells were prepared as single cell suspensionsand incubated with rat anti-mouse mAb hybridoma super-natants to MHC Class II, F4/80, or CD11c, with bindingdetected with PE-mouse anti-rat (Pharmingen, www.pharm-ingen.com) by FACScan (Becton & Dickinson, www.bd-biosciences.com). Beads used (40 and 67 nm from MolecularProbes) were fluorescent yellow-green and conformed toexcitation and emission spectra suitable for analysis throughchannel 1 by flow cytometry. RSV Infection of Mice.  BALB/c mice were immunizedonce with beads conjugated to protein (M2.1 or G88). After12 days, mice were challenged intranasally with 50  µ L(approximately 10 6 plaque forming units, pfu) of RSV strainA2 subtype (obtained from Greg Tannock, RMIT, Bundoora,VIC, Australia). Mice were anesthetized with ketamine/ xylazine prior to intranasal administration of RSV, weigheddaily from the day before challenge to the end of theexperiment, and sacrificed at day 4 post RSV infection.Weight loss is commonly used to assess the effects of respiratory viral load in mice. 25,26 The lungs were dissectedinto portions and either fixed in formalin or frozen and storedat - 70  ° C. Blood samples were taken prior to immunizationand at sacrifice.Viral titers were measured using standard techniques. 27 Briefly, frozen lungs were homogenized in sterile PBS,centrifuged at 10000 g  for 5 min at 4  ° C and the supernatantscollected. For the plaque assay, HEp-2 cells (2 × 10 5  /well) (25) Castro, S. M.; Guerrero-Plata, A.; Suarez-Real, G.; Adegboyega,P. A.; Colasurdo, G. N.; Khan, A. M.; Garofalo, R. P.; Casola,A. Antioxidant Treatment Ameliorates Respiratory SyncytialVirus-Induced Disease and Lung Inflammation.  Am. J. Respir.Crit. Care Med.  2006 .(26) Li, Y.; Li, C.; Liu, L.; Wang, H.; Wang, C.; Tian, G.; Webster,R. G.; Yu, K.; Chen, H. Characterization of an avian influenzavirus of subtype H7N2 isolated from chickens in northern China. Virus Genes  2006 ,  33 , 117 - 122.(27) Kong, X.; Hellermann, G. R.; Patton, G.; Kumar, M.; Behera,A.; Randall, T. S.; Zhang, J.; Lockey, R. F.; Mohapatra, S. S. Animmunocompromised BALB/c mouse model for respiratorysyncytial virus infection.  Virol. J.  2005 ,  2 , 3. articles  Mottram et al. D MOLECULAR PHARMACEUTICS  VOL. XXXX NO. XXXX  were plated in 2 mL/well of DM10 into 24-well plates andincubated overnight to 80% confluence. To each well,200  µ L of virus samples was added for 2 h at 37  ° C. TheRSV supernatants were then removed and the wells overlaidwith 1 mL of DMEM/F12 media (Gibco/Invitrogen, Carls-bad, CA). The cells were then incubated at 37  ° C for 5 days,the overlay was removed, and the cells were fixed in 80%methanol for 30 min, blocked with 5% powdered milk inPBS at 37  ° C for 1 h, rinsed with 0.05% Tween/PBS, thenincubated with goat-anti-RSV monoclonal antibody (Biode-sign, www.biodesign.com), and detected using rabbit anti-goat-HRP (P0449, Dako,Glostrup, Denmark). Plaques werecounted by microscopy. Statistics.  P  values for differences between the mean SFUby ELISPOT or mean of the inverse of titer by ELISA fordiversely immunized animal groups were calculated usingthe unpaired two-tailed equal variance Student’s  t   test. Results Immunogenicity of 20 - 123 nm OVA-Conjugated Beads. After one immunization with nanobeads conjugated to OVA,IFN- γ  responses to OVA were measured by ELISPOT usingspleen cells from immunized mice stimulated in vitro witheither the OVA CD8 T cell epitope, SIINFEKL (Figure 1A)or whole OVA protein (Figure 1B). IFN- γ  responses weresharply localized to a narrow bead size range, with 40 and Figure 1.  One immunization, spleen cells assayed at 10 days, after immunization with 50  µ g of OVA/mouse on beads of differentsizes: (A) IFN- γ  responses to SIINFEKL stimulation. (B) IFN- γ  response to OVA. (C) IL-4 response to OVA. (D - F) Responsefrom spleen cells of mice immunized with soluble OVA, 50  µ g/mouse or 40 nm beads alone (100  µ L of 1% vol/vol beads)compared with OVA (50  µ g) conjugated with 40 nm beads (40 nm - OVA, 100  µ L of 1% vol/vol beads) and naive controls: (D)IFN- γ  responses to SIINFEKL stimulation; (E) IFN- γ  responses to OVA stimulation; (F) IL-4 response to OVA. Darker gray barsand * indicate significant responses ( P   <  0.05) compared with naive controls: 4 - 12 mice/group, mean  (  SE. Type 1 and 2 Immunity, Nanoparticle Size, and RSV Vaccine  articles VOL. XXXX NO. XXXX  MOLECULAR PHARMACEUTICS E
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