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[(Biotechnology)-539.5(Associates,)-532.2(SBA\).)-537.4(The)-531.1(antibody)-533.7(titers)-531.2(were)]TJ T* [(determined)-561.4(according)-559.7(to)-559.7(the)-560.2(optical)-554.2(density)-559.8(\(OD450)-167.8(nm\).)]TJ T* [(Finally,)-335.1(the)-338.9(relative)-337.1(ratio)-335.8(of)-332(IgG)]TJ 6.7246 0 0 5.9768 439.5967 620.1636 Tm (2a)Tj 8.9663 0 0 8.9663 449.3479 621.6943 Tm [(to)-332(IgG)]TJ 6.7246 0 0 5.9768 474.803 620.1636 Tm (1)Tj 8.9663 0 0 8.9663 481.1526 621.6943 Tm [(was)-337.6(calculated.)]TJ /F2 1 Tf -18.7474 -2.447 TD [(Lymphocytes)-335.1(proliferation)-338.6(assay)]TJ /F4 1 Tf 1.1128 -1.8336 TD [(Mice)-324(were)-324.6(sacriÞced)-325.7(3)-315.7(weeks)-323.3(after)-329.8(the)-319.9(last)-321(immunization.)]TJ -1.1128 -1.2203 TD [(Spleens)-324(from)-311.3(each)-318.2(group)-314.1(were)-318.3(pooled)-316.8(and)-315.7(analyzed.)-319.2(Th-cell)]TJ 0 -1.2266 TD [(proliferation)-439.9(assay)-436(was)-432.4(performed)-436.3(as)-437.1(previously)-436.9(described)]TJ 0 -1.2203 TD (\(Wang)Tj /F5 1 Tf 3.446 0 TD [(et)-494.4(al.)]TJ /F4 1 Tf 2.1624 0 TD [(,)-496.1(2003\).)-494.6(Brießy,)-498(the)-496.9(isolated)-500.9(spleen)-496.3(cells)-500.8(were)]TJ -5.6084 -1.2203 TD [(resuspended)-438.8(to)-426.9(a)-429.5(concentration)-436.2(of)-433.2(5)]TJ /F7 1 Tf 16.5913 0 TD (·)Tj /F4 1 Tf .7208 0 TD (10)Tj 6.7246 0 0 5.9768 477.2408 543.4581 Tm (6)Tj 8.9663 0 0 8.9663 484.4408 539.4896 Tm [(cells/mL.)-431.4(A)-431.4(vol-)]TJ -19.1142 -1.2266 TD [(ume)-270.7(of)-268.8(100)]TJ /F6 1 Tf 5.0077 0 TD (m)Tj /F4 1 Tf .5501 0 TD [(L)-268.6(o)0(f)-268.8(cell)-265.5(suspension)-272.6(was)-268(added)-267.7(to)-268.8(96-well)-273.7(plates)]TJ -5.5578 -1.2203 TD [(and)-309.4(the)-313.6(MPT64)-305.7(protein)-309.3(\(Wang)]TJ /F5 1 Tf 13.8282 0 TD [(et)-311(al.)]TJ /F4 1 Tf 1.9727 0 TD [(,)-306.4(2004\))-308.3(was)-312.3(added)-305.6(to)-306.7(the)]TJ -15.801 -1.2203 TD [(wells)-332.4(in)-329.4(triplicate)-328.4(at)-328.8(the)-326.2(Þnal)-331.9(concentration)-335(of)-325.7(5)]TJ /F6 1 Tf 21.2576 0 TD (m)Tj /F4 1 Tf .5564 0 TD [(g/mL.)-327.1(The)]TJ -21.814 -1.2203 TD [(plates)-360.2(were)-356.2(incubated)-356.3(at)-354.1(37)]TJ /F7 1 Tf 12.3107 0 TD ()Tj /F4 1 Tf .3983 0 TD [(C)-354.3(i)0(n)-354.7(a)0(n)-350.7(atmosphere)-361(of)-351(5%)-355.2(CO)]TJ 6.7246 0 0 5.9768 548.844 494.1353 Tm (2)Tj 8.9663 0 0 8.9663 313.0582 484.6676 Tm [(for)-364.3(66)-169.7(h.)-360.4(Then,)-360.4(the)-364.2(proliferation)-364.1(responses)-366(were)-362.5(detected)-360(by)]TJ 0 -1.2203 TD [(MTT)-415.8([3-\(4,)-414.3(5-dimethylthiazol-2-yl\))-423.1(2,)-413.4(5-diphenyltetrazolium)]TJ T* [(bromide])-575.1(\(5)-170.7(mg/mL;)-564.9(Sigma\))-573(method)-576.2(and)-568.6(the)-572.8(stimulation)]TJ 0 -1.2266 TD [(index)-397.7(\(SI\))-393.6(was)-388.2(calculated.)-391.7(The)-392(SI)-392.6(was)-394.5(determined)-390.7(from)-393.5(the)]TJ 0 -1.2203 TD [(formula:)-356.6(SI)]TJ /F7 1 Tf 5.1279 0 TD (=)Tj /F4 1 Tf .7145 0 TD [(experimental)-359.7(OD/negative)-360.7(OD.)-355.1(To)-353.5(assure)-351.8(that)]TJ -5.8424 -1.2203 TD [(cells)-266.8(were)-255(healthy,)-264.1(10)]TJ /F6 1 Tf 9.5097 0 TD (m)Tj /F4 1 Tf .5501 0 TD [(g/mL)-254.6(ConA)-260.6(was)-261.7(used)-258.5(as)-260.1(a)-258.7(polyclonal)]TJ -10.0598 -1.2266 TD [(stimulator)-340(for)-332.7(positive)-336.1(control.)]TJ /F2 1 Tf 0 -2.4406 TD [(Evaluation)-339.5(of)-330.4(cytokines)-334.3(production)]TJ /F1 1 Tf 15.6176 0 TD [(in)-330.2(vitro)]TJ /F4 1 Tf -14.5048 -1.8336 TD [(Single)-398.9(splenocyte)-395.7(suspension)-399(from)-399.8(immunized)-395(mice)-392.3(was)]TJ -1.1128 -1.2203 TD [(diluted)-356.2(in)-348.4(10%)-354.7(bovine)-349.9(calf)-350.6(serum-supplemented)-362.1(RPMI)-354.3(1640)]TJ 0 -1.2266 TD [(to)-534.4(5)]TJ /F7 1 Tf 2.0676 0 TD (·)Tj /F4 1 Tf .7208 0 TD (10)Tj 6.7246 0 0 5.9768 347.0172 362.6078 Tm (6)Tj 8.9663 0 0 8.9663 355.1243 358.6393 Tm [(cells/mL.)-538.9(About)-535.2(100)]TJ /F6 1 Tf 9.731 0 TD (m)Tj /F4 1 Tf .5564 0 TD [(L)-534.1(o)0(f)-534.4(cell)-531(suspension)-538.1(was)]TJ -14.979 -1.2203 TD [(added)-299.3(to)-294.1(96-well)-292.6(plates)-296.9(\(Costar\))-296.7(and)-296.8(the)-294.6(MPT64)-293(protein)-296.6(was)]TJ T* [(added)-368.8(to)-370(the)-370.5(wells)-370.3(in)-373.6(triplicate)-372.7(at)-366.7(the)-370.5(Þnal)-369.8(concentration)-372.9(of)]TJ 0 -1.2266 TD (5)Tj /F6 1 Tf .6702 0 TD (m)Tj /F4 1 Tf .5501 0 TD [(g/mL.)-617.9(After)-616.3(72)-163.4(h)-616.7(o)0(f)-616.6(incubation,)-612.6(cell-free)-621(supernatants)]TJ -1.2203 -1.2203 TD [(were)-305.6(harvested)-312.4(and)-303.1(were)-305.6(screened)-310.9(for)-301.1(the)-307.3(presence)-309.2(of)-306.7(IFN-)]TJ /F6 1 Tf 26.1705 0 TD (g)Tj /F4 1 Tf -26.1705 -1.2203 TD [(and)-695.1(interleukin)-688(\(IL\)-4)-695.2(with)-685.6(an)-692.1(ELISA)-690.9(detection)-689.3(system)]TJ 0 -1.2266 TD [(\()-126.9(Jingmei;)-426.7(Biotech\))-430.1(according)-426.9(to)-426.9(the)-427.4(manufacturerÕs)-431(instruc-)]TJ 0 -1.2203 TD (tions.)Tj /F2 1 Tf 0 -2.447 TD [(Intracellular)-340(IFN-)]TJ /F12 1 Tf 7.4294 0 TD (c)Tj /F2 1 Tf .7714 0 TD [(measurement)-332.1(using)-332.2(ßow)-335.5(cytometry)]TJ /F4 1 Tf -7.088 -1.8273 TD [(Splenocytes)-811.5(from)-810.8(vaccinated)-811.6(mice)-809.6(were)-805.1(cultured)-812.6(at)]TJ -1.1128 -1.2266 TD (2.5)Tj /F7 1 Tf 1.4163 0 TD (·)Tj /F4 1 Tf .7208 0 TD (10)Tj 6.7246 0 0 5.9768 341.1779 236.5795 Tm (6)Tj 8.9663 0 0 8.9663 344.5794 232.611 Tm [(/mL)-269.1(in)-272.5(24-well)-280(tissue)-273.4(culture)-278.5(plates)-271.6(\(Nunclon\))-280.2(in)-272.5(the)]TJ -3.5155 -1.2203 TD [(presence)-328.2(of)-325.7(5)]TJ /F6 1 Tf 5.9941 0 TD (m)Tj /F4 1 Tf .5501 0 TD [(g)-326.9(o)0(f)-319.4(MPT64)-324.6(protein/mL)-331.8(for)-320.1(3)-328.3(days.)-321(Brefeldin)]TJ -6.5442 -1.2203 TD [(A)-355.5(\(Sigma\))-358.5(was)-356.5(added)-356.2(to)-357.3(the)-351.5(cultures)-362(for)-358(the)-357.8(last)-352.6(5)-170.2(h)-351.1(to)-357.3(pre-)]TJ 0 -1.2267 TD [(vent)-320.6(secretion)-321.1(of)-319.4(the)-313.6(intracellular)-322.5(cytokine.)-318.3(One)-316.2(million)-321.4(cells)]TJ 0 -1.2203 TD [(from)-418.8(each)-419.4(group)-421.6(were)-419.4(Þrst)-416.5(incubated)-425.8(with)-420(ßuorescein)-419.8(iso-)]TJ T* [(thiocyanate-conjugated)-561(anti-CD4)-552.2(Ab)-549.6(\(clone)-553.1(RM4;)-549.2(4)-549.6(Phar-)]TJ 0 -1.2266 TD [(Mingen\))-570.3(or)-566.2(CD8)-570.1(Ab)-568.6(for)-566.7(30)-163.4(min)-567.2(at)-569(4)]TJ /F7 1 Tf 17.369 0 TD ()Tj /F4 1 Tf .3983 0 TD [(C.)-565.8(Cells)-564.9(were)-571.2(then)]TJ -17.7674 -1.2203 TD [(washed,)-498.5(Þxed)-494.9(with)-495.9(4%)-494.3(paraformaldehyde,)-496.4(and)-499.1(permeabi-)]TJ T* [(lized)-376.2(with)-375.7(phosphate)-380.2(buffered)-376.9(saline)-371.6(containing)-377.8(0.1%)-370.3(sapo-)]TJ 0 -1.2266 TD [(nin.)-417.9(To)-416.8(label)-409.2(intracellular)-423.6(IFN-)]TJ /F6 1 Tf 13.8851 0 TD (g)Tj /F4 1 Tf .4995 0 TD [(,)-413.9(cells)-412.2(were)-419.4(incubated)-413.2(with)]TJ -14.3846 -1.2203 TD [(phycoerythrin-conjugated)-749.1(anti-IFN-)]TJ /F6 1 Tf 16.155 0 TD (g)Tj /F4 1 Tf 1.233 0 TD [(Ab)-733(\(clone)-742.8(XMG1.2;)]TJ -17.388 -1.2203 TD [(PharMingen\))-619.5(for)-617.3(1)-170.2(h)-610.4(at)-613.3(room)-617.1(temperature,)-618.1(washed,)-618.7(and)]TJ T* [(acquired)-391.1(on)-385.5(a)-385.2(cytoßuorometer)-389.1(\(FACSCALIBUR;)-389.9(BD\).)-387.2(Lym-)]TJ 0 -1.2266 TD [(phocytes)-449.2(were)-444.7(gated)-449.9(by)-445.1(their)-444.3(forward)-445.1(and)-442.2(side)-450.4(light)-445.3(scat-)]TJ 0 -1.2203 TD [(tering)-549.9(properties,)-552.4(and)-549.7(100,000)-543.8(cells)-551.3(were)-545.9(acquired)-549.1(in)-544.4(the)]TJ T* [(lymphocyte)-541.9(gate.)-544.7(Analysis)-536.9(was)-539.9(performed)-543.8(by)-533.6(Cell)-544.6(Quest)]TJ 0 -1.2267 TD (software.)Tj /F8 1 Tf -27.9978 77.2217 TD (490)Tj 47.9403 0 TD [(WANG)-332.6(ET)-338.7(AL.)]TJ ET endstream endobj 54 0 obj <>stream /GS1 gs BT /F2 1 Tf 8.9663 0 0 8.9663 59.7543 725.7825 Tm 0 0 0 1 k 0 Tc 0 Tw [(Cytotoxicity)-336.7(assay)-336.2(of)-330.4(T)-334.4(cells)]TJ /F4 1 Tf 1.1128 -1.8336 TD [(Spleen)-411.2(cells)-412.2(adjusted)-405.5(to)-407.9(a)-410.5(concentration)-410.9(of)-407.9(10)]TJ 6.7246 0 0 5.9768 254.1543 713.3101 Tm (7)Tj 8.9663 0 0 8.9663 257.4991 709.3416 Tm [(/mL)-408.2(from)]TJ /F5 1 Tf -22.0543 -1.2203 TD [(in)-259.2(vivo)]TJ /F4 1 Tf 2.7884 0 TD [(-primed)-253.1(mice)-259.5(were)-255(cocultured)-258.2(with)-255.6(mitomycin)-252.8(\(10)]TJ /F6 1 Tf 22.1744 0 TD (m)Tj /F4 1 Tf .5501 0 TD (g/)Tj -25.5129 -1.2266 TD [(mL\)-treated)-258.4(target)-252.1(cells)-247.8(\(P815-MPT64,)-252.5(5)]TJ /F7 1 Tf 17.4259 0 TD (·)Tj /F4 1 Tf .7145 0 TD (10)Tj 6.7246 0 0 5.9768 231.3637 691.4266 Tm (5)Tj 8.9663 0 0 8.9663 234.7653 687.4014 Tm [(/mL\))-250.6(in)-247.2(a)-252.4(1)0(0)-163.4(m)0(L)]TJ -19.5188 -1.2203 TD [(cell)-328.7(suspension)-329.5(in)-323.1(RPMI)-329(1640)-320.5(for)-326.4(5)-328.3(days)-324.4(at)-322.4(37)]TJ /F7 1 Tf 20.2902 0 TD ()Tj /F4 1 Tf .3983 0 TD .3231 Tc [(Ci)323.1(n5)323.1(%C)323.1(O)]TJ 6.7246 0 0 5.9768 293.2723 674.9856 Tm 0 Tc (2)Tj 8.9663 0 0 8.9663 296.6172 676.4597 Tm (.)Tj -26.4171 -1.2203 TD [(Twenty)-493.3(units)-490.6(per)-495.1(milliliter)-487.7(recombinant)-495.5(murine)-487(IL-2)-492(\(Bio-)]TJ 0 -1.2266 TD [(source\))-391.5(was)-388.2(also)-392.8(added)-387.8(to)-388.9(the)-389.5(cell)-391.9(solution)-392.2(for)-389.6(5)-385.2(days.)-390.5(The)]TJ 0 -1.2203 TD [(P815)-335(cell)-335(was)-331.2(used)-340.7(as)-329.6(a)-334.6(negative)-334.3(control.)]TJ 1.1128 -1.2203 TD [(To)-227.1(measure)-227.9(the)-231.4(speciÞc)-229(lysis)-229.1(of)-224.5(the)-231.4(target)-226.8(cells,)-225.5(we)-229.1(used)-226.9(the)]TJ -1.1128 -1.2203 TD [(lactate)-318.8(dehydrogenase)-316.5(\(LDH\))-315.1(release)-314.6(assay,)-312.5(which)-314.7(yields)-312.9(re-)]TJ 0 -1.2266 TD [(sults)-204(similar)-199.4(to)-205.6(the)-199.8(standard)-200.6(chromium)-202.2(release)-207.1(assay,)-198.7(but)-202.3(does)]TJ 0 -1.2203 TD [(not)-253.2(require)-250.8(the)-244(use)-249.2(of)-249.8(radioisotopes.)-253.5(In)-245.8(96-well)-248.4(round-bottom)]TJ T* [(plates,)-382.1(effector)-383.3(cells)-374.3(were)-381.5(incubated)-381.6(with)-375.7(target)-378.5(cells)-380.6(at)-379.4(dif-)]TJ 0 -1.2266 TD [(ferent)-428.5(E/T)-426.3(ratio)-424.3(for)-427.6(4h)-426.5(in)-424.2(phenol)-431.6(red-free)-428.7(RPMI)-423.8(1640)-421.7(con-)]TJ 0 -1.2203 TD [(taining)-530.4(2%)-525.9(BSA,)-524.3(2)-163.9(m)0(M)-527.5(glutamine,)-522.4(and)-524.4(1%)-525.9(penicillin)-530.8(and)]TJ T* [(streptomycin.)-214.1(After)-211.6(centrifuging)-214.5(the)-212.4(plates)-208.4(at)-208.6(250)]TJ /F5 1 Tf 21.3588 0 TD (g)Tj /F4 1 Tf .7082 0 TD [(for)-206.3(10)-169.7(min,)]TJ -22.067 -1.2266 TD (100)Tj /F6 1 Tf 1.6692 0 TD (m)Tj /F4 1 Tf .5501 0 TD [(L)-236.9(per)-235.9(well)-236.2(of)-237.2(the)-237.7(supernatant)-239(was)-236.4(then)-234.8(transferred)-244(to)-237.2(96-)]TJ -2.2193 -1.2203 TD [(well)-451.2(plates,)-457.9(and)-448.5(lysis)-450.4(was)-451.4(determined)-454(by)-451.4(measuring)-448(LDH)]TJ T* [(release)-377.8(using)-374.4(a)-372.6(cytotocxicity)-376.9(detection)-379.4(kit)-375.1(\(Roche)-376.8(Molecular)]TJ 0 -1.2266 TD [(Biochemicals\).)-374.1(The)-366.7(released)-368.2(LDH)-371.1(converted)-370.6(the)-370.5(added)-368.8(sub-)]TJ 0 -1.2203 TD [(strate)-267.9(\(tetrazolium)-269(salt\))-264.5(into)-269.4(a)-258.7(red)-269.2(formazan)-262.7(product,)-264.8(and)-265.1(the)]TJ T* [(amount)-302.2(of)-300.4(color)-299.9(is)-295.7(proportional)-301.1(to)-300.4(the)-300.9(number)-298.2(of)-300.4(lysed)-296.7(cells.)]TJ T* [(The)-379.3(absorbance)-375.6(values)-373.1(from)-374.6(supernatants)-379.4(were)-375.2(recorded)-376(at)]TJ 0 -1.2266 TD (OD)Tj 6.7246 0 0 5.9768 73.7008 466.7527 Tm (492)Tj 8.9663 0 0 8.9663 86.7401 468.2267 Tm [(nm)-323.4(on)-328.6(an)-325.4(ELISA)-324.1(microplate)-331.1(reader.)-329.1(The)-322.4(percent)-333.8(lysis)]TJ -3.0097 -1.2203 TD [(was)-464(calculated)-464.6(as)-462.4(follows:)-467.9(\(sample)-465.2(release)]TJ /F7 1 Tf 19.9488 0 TD (-)Tj /F4 1 Tf 1.1824 0 TD (spontaneous)Tj -21.1312 -1.2203 TD (release\))Tj /F7 1 Tf 3.4839 0 TD (·)Tj /F4 1 Tf .7145 0 TD [(100%/\(total)-263.3(release)]TJ /F7 1 Tf 8.909 0 TD (-)Tj /F4 1 Tf .9801 0 TD [(spontaneous)-270.5(release\))]TJ /F7 1 Tf 9.282 0 TD (·)Tj /F4 1 Tf .7145 0 TD (100%.)Tj /F2 1 Tf -24.084 -2.447 TD [(Statistical)-336.1(analysis)]TJ /F4 1 Tf 1.1128 -1.8336 TD [(The)-290.8(statistical)-297.5(signiÞcance)-292.4(of)-287.8(differential)-296.6(Þndings)-293.9(between)]TJ -1.1128 -1.2203 TD [(experimental)-290.1(groups)-290.1(was)-280.7(determined)-289.6(by)-280.7(StudentÕs)-288.5(test.)-289.6(Data)]TJ 0 -1.2266 TD [(were)-337.2(considered)-339.3(statistically)-337(signiÞcant)-338(at)]TJ /F5 1 Tf 18.8802 0 TD (p)Tj /F7 1 Tf .6702 0 TD (<)Tj /F4 1 Tf .7208 0 TD (0.05.)Tj /F8 1 Tf -20.2713 -3.054 TD (Results)Tj /F2 1 Tf 0 -1.8336 TD [(Construction)-339.9(of)-330.4(pcDNA3-MPT64)]TJ 0 -1.2203 TD [(and)-335.7(UbGR-MPT64)-339.1(fusion)-329.4(DNA)-338.6(vaccine)]TJ /F4 1 Tf 1.1128 -1.8336 TD [(The)-252.9(recombinant)-255.2(pcDNA3-MPT64)-258.5(plasmid)-250.1(was)-255.4(conÞrmed)]TJ -1.1128 -1.2203 TD (by)Tj /F5 1 Tf 1.5871 0 TD (Hin)Tj /F4 1 Tf 1.5934 0 TD [(dIII)-483.6(and)]TJ /F5 1 Tf 4.2427 0 TD (Xba)Tj /F4 1 Tf 1.6123 0 TD [(I)-482.7(digestion)-483.9(\(Qingmin)]TJ /F5 1 Tf 10.0598 0 TD [(et)-481.7(al.)]TJ /F4 1 Tf 2.1435 0 TD [(,)-483.5(2003\).)-482(The)]TJ -21.2387 -1.2266 TD [(UbGR-MPT64)-428.8(fusion)-423.8(DNA)-420.1(vaccine)-422.9(was)-426.1(conÞrmed,)-423.5(respec-)]TJ 0 -1.2203 TD [(tively,)-426.5(by)]TJ /F5 1 Tf 4.6726 0 TD (Hin)Tj /F4 1 Tf 1.5934 0 TD [(dIII)-420.3(and)]TJ /F5 1 Tf 4.1225 0 TD (Xba)Tj /F4 1 Tf 1.6187 0 TD (I,)Tj /F5 1 Tf 1.0053 0 TD (Bam)Tj /F4 1 Tf 1.8084 0 TD [(HI)-425.7(and)]TJ /F5 1 Tf 3.6799 0 TD (Xba)Tj /F4 1 Tf 1.6123 0 TD (I,)Tj /F5 1 Tf 1.0117 0 TD (Hin)Tj /F4 1 Tf 1.5934 0 TD [(dIII,)-423.3(and)]TJ /F5 1 Tf -22.7182 -1.2203 TD (Xba)Tj /F4 1 Tf 1.6187 0 TD [(I)-634.4(digestion.)-644.9(Finally,)-638.6(the)-642.4(sequences)-643.1(of)-641.9(the)-636(two)-643.5(DNA)]TJ -1.6187 -1.2267 TD [(vaccine)-713.8(plasmids)-713(were)-710.3(conÞrmed)-711.5(by)-710.7(sequencing.)-713(After)]TJ 0 -1.2203 TD [(large-scale)-600.1(preparation,)-604.1(the)-598.1(plasmids)-599.2(were)-596.5(suspended)-602.6(in)]TJ T* [(endotoxin-free)-579.2(PBS.)-568.5(DNA)-565.5(was)-571.5(quantiÞed)-568.7(by)-565.3(spectropho-)]TJ T* [(tometry)-284.5(at)-284.5(260)-162.9(nm)-285.5(and)-277.8(the)-282(Þnal)-281.3(concentration)-290.7(of)-281.4(the)-282(solution)]TJ 0 -1.2266 TD [(was)-337.6(adjusted)-336(to)-332(1)]TJ /F6 1 Tf 8.0238 0 TD (m)Tj /F4 1 Tf .5501 0 TD (g/)Tj /F6 1 Tf 1.1571 0 TD (m)Tj /F4 1 Tf .5501 0 TD [(L)-331.8(o)0(f)-338.4(DNA)-331.6(in)-335.7(PBS.)]TJ /F2 1 Tf -10.2811 -2.4406 TD [(The)-330.6(stable)-335.7(expression)-329.5(of)-330.4(MPT64)-330.2(protein)-330.1(in)-330.2(P815)-333.6(cells)]TJ /F4 1 Tf 1.1128 -1.8336 TD [(The)-328.8(purpose)-331.2(of)-325.7(transfection)-337.7(experiment)-331.3(was)-324.9(to)-325.7(obtain)-330.2(the)]TJ -1.1128 -1.2266 TD [(speciÞc)-361.8(target)-353.2(cells)-361.6(for)-351.7(cytotoxicity)-365(assay.)-350.4(After)-363.3(selection)-355.5(by)]TJ 0 -1.2203 TD [(G418)-357.7(\(800)]TJ /F6 1 Tf 4.6031 0 TD (m)Tj /F4 1 Tf .5564 0 TD [(g/mL\),)-352.8(15)-353.1(clones)-357.9(were)-356.2(obtained)-352.1(and)-353.7(5)-353.6(clones)-357.9(of)]TJ -5.1595 -1.2203 TD [(transfected)-539.5(cells)-538.7(were)-533.2(randomly)-533.4(chosen)-541.2(and)-530.7(screened)-538.5(for)]TJ 0 -1.2266 TD [(MPT64-mRNA)-391.5(by)-388.2(RT-PCR.)-389.4(After)-388.6(electrophoresis,)-390.5(a)-391.5(speciÞc)]TJ 0 -1.2203 TD [(single)-338.6(band)-326.5(about)-337.3(0.7)-166.3(kb)-325(in)-335.7(length)-331.2(was)-331.2(observed)-335.2(in)-329.4(clones)-339(I,)]TJ T* [(II,)-272.7(III,)-268.5(IV,)-267.1(and)-271.5(V.)-265(Expression)-272.4(of)-275.1(MPT64)-267.7(was)-268(further)-272.5(examined)]TJ T* [(in)-506.4(clone)-514.7(I)-501.6(b)0(y)-508.3(immunocytochemistry.)-513.5(The)-505.8(immunostaining)]TJ 0 -1.2266 TD [(was)-350.2(restricted)-350.7(to)-351(the)-351.5(cytoplasm)-351.7(of)-344.7(the)-351.5(cells)-349(transfected)-356.2(with)]TJ 0 -1.2203 TD [(pcDNA3-MPT64)-574.6(plasmid.)-575.5(However,)-571.3(no)-575.2(staining)-569.6(was)-571.5(de-)]TJ T* [(tected)-363.4(in)-361(P815)-354(cells.)-364.6(No)-356.8(signals)-358.4(were)-362.5(detected)-360(with)-363.1(the)-357.8(sera)]TJ 0 -1.2267 TD [(from)-507.3(healthy)-507.8(people,)-504.9(which)-504.4(indicated)-508.8(that)-503.3(the)-509.6(staining)-506.4(is)]TJ 28.0042 74.5598 TD [(speciÞc.)-579.6(Those)-585.8(results)-584(demonstrated)-587.4(that)-579.2(MPT64)-583.9(antigen)]TJ 0 -1.2203 TD [(could)-338.7(be)-345.8(expressed)-344.7(stably)-342.5(in)-342(P815)-341.3(cells,)-345.6(and)-341(that)-345.3(the)-345.2(clone)-344(I)]TJ 0 -1.2266 TD [(could)-332.4(be)-333.1(used)-334.4(as)-335.9(target)-334.3(cells)-336.3(in)-335.7(the)-332.5(cytotoxicity)-339.7(assay.)]TJ /F2 1 Tf 0 -2.4406 TD [(Production)-313(of)-311.5(antibodies)-314.4(induced)-309.4(by)-313.8(different)-311.9(vaccines)]TJ /F4 1 Tf 1.1128 -1.8336 TD [(To)-385.2(determine)-387.7(the)-383.1(level)-393.3(of)-382.6(MPT64-speciÞc)-389.7(IgG)-384.7(elicited)-392.5(by)]TJ -1.1128 -1.2266 TD [(different)-296.4(vaccines,)-288.1(mice)-291.1(of)-294.1(different)-290(groups)-296.4(were)-286.7(immunized)]TJ 0 -1.2203 TD [(three)-390(times)-388.5(at)-385.7(3)-385.2(weeks)-386.5(intervals.)-389.1(Three)-386.3(weeks)-392.9(after)-386.7(the)-389.5(last)]TJ T* [(immunization,)-423.7(the)-421.1(sera)-425.1(from)-425.1(mice)-417.6(were)-425.8(collected)-422.7(by)-419.8(retro-)]TJ T* [(orbital)-571.4(bleeding)-572.2(and)-568.6(antigen-speciÞc)-579.2(antibodies)-571.9(were)-577.5(de-)]TJ 0 -1.2266 TD [(tected)-350.8(by)-356.6(ELISA.)-352.4(As)-350.5(shown)-353.9(in)-354.7(Figure)-349.4(1,)-356.5(compared)-351.8(with)-350.4(the)]TJ 0 -1.2203 TD [(pcDNA3)-527.7(vector)-528.7(group)-529.1(or)-528.3(pcDNA3-ub)-527.7(group,)-532(the)-522.2(MPT64)]TJ T* [(DNA)-578.2(vaccine)-581(elicited)-582.2(a)-574.9(signiÞcantly)-579.9(higher)-584.4(level)-576.7(of)-578.6(IgG)]TJ 0 -1.2266 TD (\()Tj /F5 1 Tf .4869 0 TD (p)Tj /F7 1 Tf .6702 0 TD (<)Tj /F4 1 Tf .7145 0 TD [(0.01\).)-238.8(However,)-242.5(the)-244(IgG)-239.2(level)-241.6(in)-240.9(the)-237.7(UbGR-MPT64)-245.4(fusion)]TJ -1.8716 -1.2203 TD [(DNA)-451.7(vaccine)-460.9(group)-453.2(was)-451.4(lower)-457.5(than)-455.7(that)-452.8(in)-455.8(MPT64)-451.1(DNA)]TJ T* [(vaccine)-334.4(group)-333.1(\()]TJ /F5 1 Tf 7.0817 0 TD (p)Tj /F7 1 Tf .6639 0 TD (<)Tj /F4 1 Tf .7208 0 TD (0.05\).)Tj -7.3535 -1.2266 TD [(The)-423.6(IgG)-422.6(subclasses)-424.2(give)-425.9(an)-426.5(indication)-427.1(of)-420.6(the)-427.4(Th)]TJ 6.7246 0 0 5.9768 517.266 543.4581 Tm (1)Tj 8.9663 0 0 8.9663 524.466 544.9321 Tm (versus)Tj -23.8247 -1.2203 TD (Th)Tj 6.7246 0 0 5.9768 321.392 532.5164 Tm (2)Tj 8.9663 0 0 8.9663 328.7054 533.9904 Tm [(nature)-447.9(of)-445.8(the)-446.4(immune)-445.9(response.)-449.8(We)-441.5(also)-449.7(detected)-442.2(the)]TJ -1.9917 -1.2203 TD [(relative)-305.5(ratio)-304.2(of)-306.7(IgG)]TJ 6.7246 0 0 5.9768 389.0267 521.5179 Tm (2a)Tj 8.9663 0 0 8.9663 395.7731 523.0487 Tm (/IgG)Tj 6.7246 0 0 5.9768 415.9558 521.5179 Tm (1)Tj 8.9663 0 0 8.9663 419.3007 523.0487 Tm [(.)-306.4(A)0(s)-306.2(shown)-303.3(in)-304.1(Figure)-305.2(2,)-305.9(although)]TJ -12.0957 -1.2266 TD [(the)-244(IgG)-245.6(level)-241.6(decreased)-246.8(in)-247.2(the)-244(Ub)-246.1(fusion)-246.8(DNA)-243(vaccine)-245.9(group,)]TJ 0 -1.2203 TD [(the)-300.9(relative)-299.1(ratio)-304.2(of)-294.1(IgG)]TJ 6.7246 0 0 5.9768 403.8235 499.6345 Tm (2a)Tj 8.9663 0 0 8.9663 410.5699 501.1085 Tm (/IgG)Tj 6.7246 0 0 5.9768 430.696 499.6345 Tm (1)Tj 8.9663 0 0 8.9663 436.7621 501.1085 Tm [(increased)-301.4(signiÞcantly)-301.7(in)-304.1(the)]TJ -14.0432 -1.2203 TD [(fusion)-461.8(DNA)-458(vaccine)-460.9(\()]TJ /F5 1 Tf 10.2115 0 TD (p)Tj /F7 1 Tf .6702 0 TD (<)Tj /F4 1 Tf .7145 0 TD [(0.05\),)-460.1(compared)-459.3(with)-457.9(the)-459(MPT64)]TJ -11.5962 -1.2203 TD [(DNA)-331.6(vaccine)-340.8(group.)]TJ /F2 1 Tf 0 -2.447 TD [(Lymphocyte)-329.3(proliferation)-338.6(and)-335.7(cytokine)-334.8(production)]TJ 0 -1.2203 TD [(by)-332.7(splenocytes)-330.5(from)-334.2(vaccinated)-337.3(mice)]TJ /F4 1 Tf 1.1128 -1.8336 TD [(T)-540.5(helper)-552.5(cells)-545(play)-546.3(an)-546.7(important)-547.2(role)-544.3(in)-544.4(eliciting)-552.7(both)]TJ -1.1128 -1.2266 TD [(humoral)-489.4(and)-486.4(cellular)-485.6(immune)-490.2(responses)-486.2(via)-490.7(expansion)-491.5(of)]TJ ET q 1 i 319.011 395.773 m 541.701 395.773 l 541.701 184.649 l 319.011 184.649 l W n q 223.158 0 0 211.124 319.011 184.649 cm /Im1 Do Q Q BT /F9 1 Tf 8.9663 0 0 8.9663 310.8472 167.074 Tm [(FIG.)-406.4(1.)]TJ /F4 1 Tf 4.1668 0 TD -.0149 Tc [(The)-324.6(M)-1(PT64-s)-14.9(p)7.7(eciÞc)-324(I)-14.9(g)16.5(G)-323.5(titer)-319.1(i).5(n)-328.1(m).2(ice)-318.3(i)-14.9(m)9.3(m).2(unized)-321.4(by)]TJ -4.1668 -1.1128 TD -.0138 Tc [(diff)4.7(er)-13.8(e)17.2(n)-13.8(t)-643.6(D)3.8(NA)-653.8(v)-13.8(a)17.6(ccines)-13.8(.)-637.7(Each)-649.5(gr)-13.8(o)14.3(u)-13.8(p)-642.5(o)2.4(f)-652.9(m)1.3(ice)-652.3(\()]TJ /F5 1 Tf 22.0923 0 TD 0 Tc (n)Tj /F7 1 Tf .7018 0 TD (=)Tj /F4 1 Tf .7018 0 TD [(8)13.1(\))-639.1(wa)30.5(s)]TJ -23.4959 -1.1128 TD [(i)15.4(m)15.1(m)15.1(u)17.6(ni)24.7(z)19.5(e)12.7(d)16(,)-502.4(re)24.7(s)17.7(p)0(e)30.4(c)13.4(t)12.2(i)15.4(ve)31(l)15.4(y)14.5(,)-502.4(b)14.5(y)-503.9(t)12.2(h)15.6(e)-499.4(b)14.6(l)15.4(a)13.1(n)15.6(k)-503.9(v)0(e)31(c)13.4(t)12.2(o)16.2(r,)-490.5(p)17.6(c)13.4(DN)30.2(A3)24.4(-)18.5(u)0(b)25.8(,)]TJ T* [(p)17.6(c)13.4(DN)30.2(A3)24.4(-)18.5(M)13.9(PT)27.3(6)13.1(4)-208.2(D)17.6(N)12.7(A)-210(v)18.2(a)13.1(c)13.4(c)13.4(i)15.4(n)15.6(e)12.7(,)-205.2(o)0(r)-193.2(U)0(b)25.8(G)13.9(R)14.7(-)12.2(M)20.2(P)0(T)27.3(6)13.1(4)-208.2(D)17.6(N)12.7(A)-210(va)31.4(c)13.4(c)13.4(i)15.4(n)15.6(e)]TJ 0 -1.1065 TD -.0143 Tc [(a)5.2(t)-343.6(0,)-333.4(3,)-339.7(and)-339.8(6)-336.3(weeks.)-339.7(Mice)-336.7(w)-14.3(e)15.8(r)-14.3(e)-324.7(bl)-14.3(e)13.9(d)-339.8(at)-337.2(3)-342.6(w)3(ee)4.8(ks)-344.4(a)5.2(fte)4.8(r)-343.8(t)4.2(he)-343(last)]TJ 0 -1.1128 TD -.014 Tc [(immu)3.6(n)-14(i)10.7(z)5.5(a)-.9(tion,)-371(a)-.9(nd)-371.1(MP)-14(T)13.3(6)-.9(4)5.5(-)-1.8(s)3.7(p)-14(e)10(c)5.7(i)-14(Þ)11.1(c)-367.4(I)-14(g)11.1(G)-366.8(titer)-368.8(w)3.3(as)-369.4(d)-14(e)14.7(tect)4.5(ed)-371.1(by)]TJ T* -.0154 Tc 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[(0)13.1(.)16.1(0)13.1(1)-239.8(p)17.6(c)13.4(DN)23.9(A)17.6(3)13.1(-)18.5(M)13.9(PT)27.3(6)13.1(4)-239.8(v)18.2(e)12.8(rs)29.6(us)-230.3(p)17.6(c)13.4(D)17.6(N)12.7(A3)-228.5(g)14.6(r)18.3(ou)27.5(p)-241.6(o)16.2(r)-241(p)0(c)24.7(D)17.6(N)12.7(A)0(3)30.7(-)]TJ -2.0929 -1.1128 TD [(u)17.6(b)-649.4(g)14.6(r)18.3(ou)27.5(p;)-636.6(*)]TJ /F5 1 Tf 5.6337 0 TD (p)Tj /F7 1 Tf .6513 0 TD (<)Tj /F4 1 Tf .7082 0 TD -.0158 Tc [(0.05)-666.6(pcDNA)-15.8(3)14.9(-)-3.6(M)-1.9(PT)-15.8(6)13.3(4)-666.6(ver)-15.8(s)13.8(u)-15.8(s)-650.8(UbGR-)-3.6(M)-1.9(PT)-15.8(6)13.3(4)]TJ -6.9931 -1.1065 TD 0 Tc [(g)14.5(r)18.3(ou)27.5(p.)-219.2(T)16(h)0(e)-224.6(e)12.7(x)16.8(p)0(e)30.4(r)0(i)27.4(m)15.1(e)12.8(n)15.6(t)-234.4(w)0(a)30.5(s)-235.3(re)24.7(p)17.6(e)12.7(a)13.1(t)18.5(e)12.7(d)-236.9(t)18.5(h)0(r)27.5(e)12.8(e)-233.8(t)12.2(i)15.4(m)15.1(e)12.7(s)17.7(.)]TJ /F8 1 Tf -28.0042 77.2028 TD 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[(and)-334.7(IL-4)-333.9(were)-337.2(examined.)]TJ 1.1128 -1.2266 TD [(As)-369.4(demonstrated)-378.7(in)-367.3(Figure)-374.7(4,)-369.2(the)-376.8(level)-368(of)-376.3(IFN-)]TJ /F6 1 Tf 20.9921 0 TD (g)Tj /F4 1 Tf .8726 0 TD [(was)-369.2(sig-)]TJ -22.9775 -1.2203 TD [(niÞcantly)-267.1(higher)-268.2(in)-266.2(the)-269.3(MPT64)-261.4(DNA)-268.3(vaccine)-264.9(group)-269.8(than)-266(that)]TJ T* [(in)-253.5(the)-256.7(pcDNA3)-255.8(group)-257.2(or)-256.4(in)-253.5(pcDNA3-ub)-262.1(group.)-253.8(The)-259.2(secretion)]TJ T* [(of)-667.1(IFN-)]TJ /F6 1 Tf 3.5598 0 TD (g)Tj /F4 1 Tf 1.1634 0 TD [(signiÞcantly)-662.1(increased)-668.1(in)-664.5(UbGR-MPT64)-662.7(fusion)]TJ -4.7232 -1.2266 TD [(DNA)-363.2(vaccine)-359.7(group)-364.7(\()]TJ /F5 1 Tf 9.8574 0 TD (p)Tj /F7 1 Tf .6702 0 TD (<)Tj /F4 1 Tf .7145 0 TD [(0.01\))-362.3(compared)-358.2(with)-363.1(MPT64)-362.6(DNA)]TJ -11.2421 -1.2203 TD [(vaccine)-366(group.)-367.6(However,)-362.6(the)-364.2(level)-368(of)-363.6(IL-4)-365.5(was)-362.9(lower)-362.6(in)-367.3(the)]TJ T* [(fusion)-392.2(DNA)-394.8(vaccine)-391.3(group)-390(than)-392.5(that)-389.5(in)-392.6(the)-389.5(nonfusion)-395.4(vac-)]TJ 0 -1.2266 TD [(cine)-309.6(group)-301.5(\()]TJ /F5 1 Tf 5.5262 0 TD (p)Tj /F7 1 Tf .6639 0 TD (<)Tj /F4 1 Tf .7208 0 TD [(0.01\).)-302(In)-302.7(the)-307.3(MPT64)-305.7(DNA)-306.3(vaccine)-309.1(group,)-304.4(the)]TJ -6.9109 -1.2203 TD [(level)-330.1(of)-332(IFN-)]TJ /F6 1 Tf 5.6147 0 TD (g)Tj /F4 1 Tf .8346 0 TD [(was)-324.9(higher)-337.8(than)-329.2(that)-332.6(of)-332(IL-4,)-330.5(which)-333.7(indicated)]TJ -6.4494 -1.2203 TD [(the)-623.4(MPT64)-628.1(DNA)-622.4(vaccine)-625.3(elicited)-626.5(a)-619.2(Th1-proÞle)-631(immune)]TJ 0 -1.2266 TD [(response.)-411.9(The)-404.6(Ub)-404.2(fusion)-404.9(DNA)-407.4(vaccine)-410.3(increased)-408.9(the)-402.1(secre-)]TJ 27.9978 28.118 TD [(tion)-433.8(of)-426.9(IFN-)]TJ /F6 1 Tf 5.4693 0 TD (g)Tj /F4 1 Tf .9231 0 TD [(and)-429.5(decreased)-423.8(the)-427.4(level)-431.2(of)-426.9(IL-4,)-425.3(which)-428.5(dem-)]TJ -6.3925 -1.2203 TD [(onstrated)-289.7(that)-282(the)-275.6(Ub)-284(fusion)-284.7(enhanced)-277.3(the)-282(Th1-type)-284.4(immune)]TJ 0 -1.2266 TD (response.)Tj /F2 1 Tf 0 -3.054 TD (IFN-)Tj /F12 1 Tf 1.9475 0 TD (c)Tj /F2 1 Tf .7714 0 TD [(intracellular)-339.8(staining)-337.2(for)-332.5(splenocytes)]TJ -2.7189 -1.2203 TD [(in)-336.5(vaccinated)-337.3(mice)]TJ /F4 1 Tf 1.1128 -1.8336 TD [(The)-303.5(role)-297.8(of)-300.4(CD4)]TJ /F7 1 Tf 6.7246 0 0 5.9768 387.4959 236.8062 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 395.0361 232.8377 Tm [(and)-296.8(CD8)]TJ /F7 1 Tf 6.7246 0 0 5.9768 431.603 236.8062 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 439.1432 232.8377 Tm [(T)-300.2(cell)-297.1(in)-304.1(secreting)-303.8(IFN-)]TJ /F6 1 Tf 10.0597 0 TD (g)Tj /F4 1 Tf .7967 0 TD (was)Tj -24.9186 -1.2266 TD [(investigated)-500.7(by)-495.7(intracellular)-493.2(staining)-500.1(since)-494.3(IFN-)]TJ /F6 1 Tf 21.9215 0 TD (g)Tj /F4 1 Tf .9927 0 TD [(is)-491.7(a)-492.7(key)]TJ -22.9142 -1.2203 TD [(molecule)-595.9(in)-588.6(the)-591.8(anti-tuberculosis)-596.4(response.)-595.3(As)-584.4(shown)-594.2(in)]TJ T* [(Figure)-400(5,)-400.8(the)-395.8(frequency)-399.5(of)-401.6(IFN-)]TJ /F6 1 Tf 14.1823 0 TD (g)Tj /F7 1 Tf 6.7246 0 0 5.9768 445.8329 203.9244 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 454.2235 199.9559 Tm [(CD4)-393.1(T)-401.3(cells)-399.6(and)-391.6(IFN-)]TJ /F6 1 Tf 9.7626 0 TD (g)Tj /F7 1 Tf 6.7246 0 0 5.9768 547.3699 203.9244 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 313.0582 189.0141 Tm [(CD8)-399.4(T)-401.3(cells)-399.6(was)-394.5(higher)-401(in)-398.9(the)-402.1(MPT64)-394.2(DNA)-401.1(vaccine)-397.7(group)]TJ 0 -1.2267 TD [(than)-303.9(that)-301(in)-297.8(the)-300.9(pcDNA3)-300.1(vector)-301(group)-295.1(or)-300.6(in)-297.8(the)-300.9(pcDNA3-ub)]TJ 0 -1.2203 TD [(group.)-557.3(The)-556.4(frequency)-563.9(of)-553.3(IFN-)]TJ /F6 1 Tf 14.0305 0 TD (g)Tj /F7 1 Tf 6.7246 0 0 5.9768 444.4724 171.0425 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 454.3369 167.074 Tm [(CD8)-551.2(T)-559.4(cells)-551.3(was)-558.9(much)]TJ -15.7567 -1.2203 TD [(higher)-344.1(in)-342(the)-338.9(spleens)-339.5(of)-344.7(the)-338.9(UbGR-MPT64)-340.2(fusion)-341.6(DNA)-344.2(vac-)]TJ 0 -1.2266 TD [(cine)-486.6(group)-478.5(than)-481(those)-482.1(in)-481.1(the)-478(MPT64)-482.7(DNA)-477(vaccine)-479.9(group)]TJ 0 -1.2203 TD (\()Tj /F5 1 Tf .4932 0 TD (p)Tj /F7 1 Tf .6639 0 TD (<)Tj /F4 1 Tf .7208 0 TD [(0.01\).)-498(The)-493.2(frequency)-500.7(of)-496.4(IFN-)]TJ /F6 1 Tf 13.1959 0 TD (g)Tj /F7 1 Tf 6.7246 0 0 5.9768 453.8267 138.1606 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 463.1243 134.1921 Tm [(CD4)-494.3(T)-496.2(cells)-494.4(was)-495.6(also)]TJ -16.7367 -1.2203 TD [(higher)-306.2(in)-304.1(the)-307.3(UbGR-MPT64)-308.6(fusion)-303.7(DNA)-306.3(vaccine)-302.8(group)-307.8(than)]TJ 0 -1.2266 TD [(that)-364.3(in)-354.7(the)-357.8(MPT64)-362.6(DNA)-356.9(vaccine)-359.7(group)-358.4(although)-360.3(to)-357.3(a)-359.9(lesser)]TJ 0 -1.2203 TD [(extent)-297.1(\()]TJ /F5 1 Tf 3.446 0 TD (p)Tj /F7 1 Tf .6702 0 TD (<)Tj /F4 1 Tf .7145 0 TD [(0.05\).)-289.4(Overall,)-292.1(UbGR-MPT64)-296(fusion)-291(DNA)-287.3(vaccine)]TJ -4.8307 -1.2203 TD [(induced)-462.4(more)-456.2(antigen-speciÞc)-465.3(CD8)]TJ /F7 1 Tf 6.7246 0 0 5.9768 457.7951 94.337 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 466.6959 90.3685 Tm [(T)-458.3(cells)-462.8(than)-455.7(CD4)]TJ /F7 1 Tf 6.7246 0 0 5.9768 537.8455 94.337 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 546.7463 90.3685 Tm (T)Tj -26.063 -1.2266 TD [(cells.)-295(These)-292(results)-293.1(indicated)-293.8(that)-288.4(UbGR-MPT64)-296(fusion)-291(DNA)]TJ 0 -1.2203 TD [(vaccine)-252.2(activated)-240.4(CD4)]TJ /F7 1 Tf 6.7246 0 0 5.9768 401.3857 72.3968 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 408.4156 68.4283 Tm [(and)-246.2(CD8)]TJ /F7 1 Tf 6.7246 0 0 5.9768 444.529 72.3968 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 451.5022 68.4283 Tm [(T)-243.3(cells,)-250.7(particularly)-245.5(CD8)]TJ /F7 1 Tf 6.7246 0 0 5.9768 547.3699 72.3968 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 313.0582 57.4866 Tm [(T)-338.1(cells.)]TJ ET q 190.028 0 0 226.998 86.627 505.247 cm /Im2 Do Q BT /F9 1 Tf 8.9663 0 0 8.9663 62.022 487.6723 Tm [(FIG.)-494.9(2.)]TJ /F4 1 Tf 4.2553 0 TD [(The)-493.2(ratio)-493.9(of)-490.1(IgG)]TJ 6.7246 0 0 5.9768 168.8314 486.1416 Tm (2a)Tj 8.9663 0 0 8.9663 179.9999 487.6723 Tm [(titer)-491.9(to)-496.4(IgG)]TJ 6.7246 0 0 5.9768 227.2818 486.1416 Tm (1)Tj 8.9663 0 0 8.9663 235.0488 487.6723 Tm [(titer)-498.3(in)-493.8(different)]TJ -19.2975 -1.1128 TD [(groups.)-318.3(The)-322.4(mice)-316.4(were)-318.3(immunized)-319.2(as)-317(described)-318.5(in)-316.7(Figure)-317.8(1.)]TJ T* [(The)-347.7(titers)-354.1(of)-351(IgG)]TJ 6.7246 0 0 5.9768 128.3527 466.2424 Tm (2a)Tj 8.9663 0 0 8.9663 138.2173 467.7164 Tm [(and)-347.3(IgG)]TJ 6.7246 0 0 5.9768 171.0992 466.2424 Tm (1)Tj 8.9663 0 0 8.9663 177.6189 467.7164 Tm [(were)-343.6(detected,)-356.6(and)-347.3(the)-351.5(ratio)-348.4(of)]TJ -12.8924 -1.1065 TD (IgG)Tj 6.7246 0 0 5.9768 76.7622 456.2645 Tm (2a)Tj 8.9663 0 0 8.9663 83.5086 457.7952 Tm (/IgG)Tj 6.7246 0 0 5.9768 103.6913 456.2645 Tm (1)Tj 8.9663 0 0 8.9663 110.7212 457.7952 Tm [(was)-413.4(obtained)-415.3(by)-407.2(dividing)-417.2(the)-414.7(titer)-409.7(of)-414.2(IgG)]TJ 6.7246 0 0 5.9768 280.7999 456.2645 Tm (2a)Tj 8.9663 0 0 8.9663 291.2314 457.7952 Tm (by)Tj -25.5635 -1.1128 TD [(that)-339(of)-338.4(IgG)]TJ 6.7246 0 0 5.9768 105.959 446.3432 Tm (1)Tj 8.9663 0 0 8.9663 109.3606 447.8172 Tm .338 Tc (.*)Tj /F5 1 Tf .9737 0 TD 0 Tc (p)Tj /F7 1 Tf .6639 0 TD (<)Tj /F4 1 Tf .7208 0 TD [(0.05)-336.6(UbGR-MPT64)-340.2(group)-339.4(versus)-340.4(pcDNA3-)]TJ -7.6381 -1.1128 TD [(MPT64)-337.3(group.)-329.7(The)-335.1(experiment)-344(was)-331.2(repeated)-339.8(three)-333.1(times.)]TJ ET q 228.408 0 0 232.668 318.501 499.805 cm /Im3 Do Q BT /F9 1 Tf 8.9663 0 0 8.9663 313.0582 482.2298 Tm [(FIG.)-368.4(3.)]TJ /F4 1 Tf 4.1225 0 TD [(The)-366.7(proliferation)-364.1(of)-363.6(splenocytes)-365.4(after)]TJ /F5 1 Tf 16.8695 0 TD [(in)-360.4(vitro)]TJ /F4 1 Tf 3.4586 0 TD (stim-)Tj -24.4507 -1.1065 TD [(ulation)-368.6(with)-369.4(MPT64)-368.9(protein.)-369.1(Different)-370.6(group)-364.7(of)-370(mice)-360.7(\()]TJ /F5 1 Tf 24.4128 0 TD (n)Tj /F7 1 Tf .7145 0 TD (=)Tj /F4 1 Tf .7208 0 TD (8\))Tj -25.8481 -1.1128 TD [(were,)-258(respectively,)-264.5(immunized)-255.9(by)-255.4(pcDNA3)-262.2(vector,)-259.7(pcDNA3-)]TJ T* [(ub,)-381.7(pcDNA3-MPT64,)-387.9(or)-376.5(UbGR-MPT64)-384.5(DNA)-382.2(vaccines)-380(three)]TJ T* [(times)-356.9(at)-360.4(3)-353.6(weeks)-354.9(interval.)-356.1(Three)-361(weeks)-354.9(after)-355.1(the)-357.8(last)-358.9(immu-)]TJ 0 -1.1065 TD [(nization,)-379.4(spleen)-376.1(cells)-374.3(from)-368.2(sacriÞced)-376.3(mice)-373.3(were)-375.2(pooled)-373.7(and)]TJ 0 -1.1128 TD [(stimulated)-250.9(with)-249.3(5)]TJ /F6 1 Tf 7.8341 0 TD (m)Tj /F4 1 Tf .5501 0 TD [(g/mL)-248.3(MPT64)-242.4(protein,)-249(or)-250.1(10)]TJ /F6 1 Tf 12.2601 0 TD (m)Tj /F4 1 Tf .5501 0 TD [(g/mL)-248.3(ConA)]TJ -21.1944 -1.1128 TD [(as)-443.4(a)-435.8(positive)-437.3(control.)-439.5(After)-445.5(66)-163.4(h)-439.7(stimulation,)-437.7(samples)-440.8(were)]TJ 0 -1.1065 TD [(assayed)-483.2(in)-481.1(triplicate.)-483.1(The)-480.5(ConA)-481.9(control)-487.1(sample)-477.4(showed)-486.2(a)]TJ 0 -1.1128 TD [(stimulation)-295.7(index)-283.9(of)-287.8(7.02)]TJ /F7 1 Tf 11.0651 0 TD (…)Tj /F4 1 Tf .7208 0 TD [(1.6.)-283.1(Values)-293(and)-284.1(bars)-284.1(represent)-292.2(the)]TJ -11.7859 -1.1128 TD [(mean)-392.4(and)-385.3(S.D.)-385.1(This)-390.6(experiment)-388.2(was)-388.2(repeated,)-387(with)-388.4(the)-389.5(ex-)]TJ T* [(pected)-781.6(results.)-776.6(**)]TJ /F5 1 Tf 8.3526 0 TD (p)Tj /F7 1 Tf .6639 0 TD (<)Tj /F4 1 Tf .7208 0 TD [(0.01)-772.9(pcDNA3-MPT64)-777(group)-775.7(versus)]TJ -9.7373 -1.1065 TD 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-1.2203 TD [(strategies.)-469.7(RodriguezÕs)-471.6(report)-467.5(\(1997\))-460.6(demonstrated)-473.6(that)-465.4(an)]TJ T* [(ubiquitinated)-471.5(DNA)-470.7(vaccine)-467.2(targeted)-469.9(to)-471.1(the)-465.3(protein)-473.7(degra-)]TJ 0 -1.2266 TD [(dation)-303.5(pathway)-301.1(enhanced)-302.6(cytotoxic)-299.7(T-lymphocyte)-305.1(induction)]TJ 0 -1.2203 TD [(and)-562.3(abrogated)-558.5(antibody)-559(induction.)-561.1(However,)-565(in)-557(VadlinÕs)]TJ T* [(study)-516.4(\(1999\),)-520.4(when)-514.1(Ub)-518(was)-514.6(fused)-518.2(with)-521.2(hepatitis)-519.3(C)-512.3(virus)]TJ 0 -1.2266 TD [(\(HCV\))-332.7(core)-325.1(antigen,)-333.7(an)-325.4(undetectable)-331.3(antibody)-331.3(response)-326.8(and)]TJ 0 -1.2203 TD [(no)-429.8(increase)-431.2(in)-430.6(CTL)-423.5(activity)-435.6(were)-425.8(observed)-430.1(compared)-434(with)]TJ T* [(the)-433.7(nonfusion)-433.4(vaccine.)-432.2(In)-429.1(our)-428.5(study,)-430.8(the)-433.7(humoral)-432.5(immune)]TJ T* [(responses)-498.8(were)-489(not)-493.5(completely)-493.3(abrogated.)-491.8(Those)-497.2(different)]TJ 0 -1.2266 TD [(results)-375.3(may)-368.2(correlate)-373.5(with)-369.4(the)-370.5(different)-372.2(antigenicities)-380.3(of)-370(the)]TJ 0 -1.2203 TD [(proteins)-335.9(and)-334.7(different)-340.6(dependence)-335.5(of)-338.4(the)-332.5(antigens)-338.4(on)-334.9(Ub.)]TJ 1.1128 -1.2203 TD [(In)-252.1(conclusion,)-257.5(the)-256.7(data)-255.4(presented)-262.8(above)-253.9(suggested)-256.4(that)-256.8(the)]TJ -1.1128 -1.2267 TD [(fusion)-354.3(of)-351(UbGR)-356.6(to)-351(our)-352.6(DNA)-350.5(vaccine)-353.4(signiÞcantly)-358.6(increased)]TJ 27.9978 74.5598 TD [(the)-269.3(antigen-speciÞc)-275.7(cellular)-270.6(immune)-268.9(response.)-272.8(Infection)-268.2(with)]TJ /F5 1 Tf 0 -1.2203 TD [(M.)-501.2(tuberculosis)]TJ /F4 1 Tf 7.0058 0 TD [(is)-498(largely)-502.9(intracellular;)-502.4(thus,)-509(the)-503.3(protective)]TJ -7.0058 -1.2266 TD [(immune)-294.2(response)-288.8(against)]TJ /F5 1 Tf 11.5393 0 TD [(M.)-286.2(tuberculosis)]TJ /F4 1 Tf 6.5695 0 TD [(infection)-294.9(involves)-287.8(a)]TJ -18.1088 -1.2203 TD [(cell-mediated)-279.2(response)-269.9(rather)-270.2(than)-272.3(a)-271.4(humoral)-268.1(response.)-272.8(Both)]TJ T* (CD4)Tj /F7 1 Tf 6.7246 0 0 5.9768 331.9369 685.9274 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 341.2346 681.9589 Tm [(and)-492.8(CD8)]TJ /F7 1 Tf 6.7246 0 0 5.9768 379.559 685.9274 Tm (+)Tj /F4 1 Tf 8.9663 0 0 8.9663 388.8566 681.9589 Tm [(T)-489.9(cells)-500.7(and)-492.8(the)-496.9(ability)-493.6(to)-496.4(respond)-499.3(with)]TJ -8.4537 -1.2266 TD [(Th1-type)-385.6(cytokines,)-382.8(particularly)-378.3(IFN-)]TJ /F6 1 Tf 16.6103 0 TD (g)Tj /F4 1 Tf .4995 0 TD [(,)-382.3(are)-373.2(involved.)-380.9(Taken)]TJ -17.1098 -1.2203 TD [(together,)-369(our)-358.9(results)-362.7(demonstrated)-359.7(that)-364.2(the)-357.8(fusion)-366.9(of)-357.3(UbGR)]TJ T* [(to)-313.1(MPT64)-312(DNA)-312.6(vaccine)-309.1(could)-313.4(be)-314.1(a)-309.3(new)-308.4(strategy)-314.7(to)-313.1(improve)]TJ 0 -1.2266 TD [(the)-338.9(efÞcacy)-334.7(of)-332(TB)-334.8(DNA)-337.9(vaccines.)]TJ /F8 1 Tf 0 -2.4406 TD (Acknowledgments)Tj /F4 1 Tf 1.1128 -1.8336 TD [(We)-498.4(thank)-491.7(Dr.)-498.2(Xiao)-490.6(An)-498(for)-490.8(providing)-497.7(us)-495.9(the)-497(sera)-494.6(from)]TJ -1.1128 -1.2203 TD [(patients)-604.3(infected)-604.3(with)]TJ /F5 1 Tf 10.7679 0 TD [(M.)-596.1(tuberculosis.)]TJ /F4 1 Tf 7.4421 0 TD [(This)-605.6(research)-599(was)]TJ -18.21 -1.2266 TD [(funded)-403.1(by)-394.5(the)-395.8(Natural)-397.1(Science)-402.1(Fund)-391.5(of)-401.6(Shanghai)-395(\(Number)]TJ 0 -1.2203 TD [(08ZR1405600\))-333.3(and)-328.4(the)-332.6(National)-335.2(Natural)-327.5(Science)-332.6(Foundation)]TJ T* [(\(Number)-339.5(31070121\).)]TJ /F8 1 Tf 0 -2.447 TD [(Disclosure)-336.7(Statement)]TJ /F4 1 Tf 1.1128 -1.8336 TD [(No)-337.9(competing)-335.2(Þnancial)-337.6(interests)-333.5(exist.)]TJ /F8 1 Tf -1.1128 -2.8643 TD (References)Tj /F4 1 Tf 8.4682 0 0 8.4682 313.0582 475.7668 Tm [(Abou-Zeid,)-308.9(C.,)-305(Gares,)-307.3(M.P.,)-304.2(Inwald,)-302.5(J.,)]TJ /F5 1 Tf 17.3395 0 TD [(et)-303.9(al.)]TJ /F4 1 Tf 2.2695 0 TD [(\(1997)-7.8(\).)-298.3(Induction)-310.9(of)]TJ -18.6651 -1.2318 TD [(a)-504.2(type)-508.6(1)-504.2(immune)-513.6(response)-512.7(to)-502.7(a)-510.9(recombinant)-506.9(antigen)-515.6(from)]TJ /F5 1 Tf 0 -1.2385 TD [(Mycobacterium)-470(tuberculosis)]TJ /F4 1 Tf 12.0104 0 TD [(expressed)-465.5(in)-465.9(Mycobacterium)-470.9(vac-)]TJ -12.0104 -1.2318 TD [(cine.)-335.2(Infect)-337.7(Immun)]TJ /F9 1 Tf 8.7233 0 TD (65,)Tj /F4 1 Tf 1.58 0 TD (1856Ð1862.)Tj -11.2472 -1.2385 TD [(Ande)-9.5(rsen,)-312.4(P.,)-312.9(and)-312.4(Doherty,)-317.6(T.M.)-308.9(\(2005\).)-312.8(The)-317.6(success)-318.4(and)-312.4(failure)]TJ .944 -1.2318 TD [(of)-482.7(BCGÑimplications)-478.9(for)-486(a)-477.4(novel)-474.9(tuber)-9.8(culosis)-476.1(vaccine.)-483.1(Nat)]TJ 0 -1.2385 TD (Rev)Tj /F9 1 Tf 2.0352 0 TD (3,)Tj /F4 1 Tf 1.0779 0 TD (656Ð662.)Tj -4.057 -1.2318 TD [(Arun)-9.8(,)-332.4(T.K.,)-331.4(Carl,)-333(G.F.,)-334.7(Murdo,)-338.7(M.,)-337.1(Helen,)-332.4(B.,)-335.4(and)-339.1(Warwick,)-334.6(J.B.)]TJ .944 -1.2385 TD [(\(1999\).)-500.3(Differential)-511.1(protective)-508.2(efÞcacy)-502.7(of)-502.7(DNA)-496.7(vaccin)-9.4(es)-495.1(ex-)]TJ 0 -1.2318 TD [(pressing)-307.1(secreted)-301.6(proteins)-305(of)]TJ /F5 1 Tf 12.9008 0 TD [(Mycobacterium)-309.3(tuberculosis)]TJ /F4 1 Tf 11.3878 0 TD [(.)-299(Infect)]TJ -24.2887 -1.2385 TD (Immun)Tj /F9 1 Tf 3.5817 0 TD (67,)Tj /F4 1 Tf 1.5867 0 TD (1702Ð1707.)Tj -6.1123 -1.2318 TD [(Bode,)-358.2(C.,)-351.9(Zhao,)-356.5(G.,)-346.8(Steinhagen,)-359.2(F.,)]TJ /F5 1 Tf 15.7194 0 TD [(et)-350.8(al.)]TJ /F4 1 Tf 2.37 0 TD [(\(2007\).)-353(CpG)-355.8(DNA)-356.1(as)-349.3(a)]TJ -17.1453 -1.2385 TD [(vaccine)-338.1(adjuvant.)-340.1(Expert)-331.5(Rev.)-336.9(Vaccine)]TJ /F9 1 Tf 17.3127 0 TD (10)Tj /F4 1 Tf 1.0042 0 TD [(,)-332.5(499Ð511.)]TJ -19.2609 -1.2318 TD [(Brandt,)-378.2(L.,)-375.5(Elhay,)-374.2(M.,)-370.6(Rosenkrands,)-384.6(I.,)-372.1(Lindblad,)-371.4(E.B.,)-378.5(and)-372.6(An-)]TJ .944 -1.2385 TD [(dersen,)-425.4(P.)-429(\(2000\).)-426.6(ESAT-6)-429.1(subunit)-426.8(vaccination)-431.2(against)]TJ /F5 1 Tf 24.6904 0 TD (Myco-)Tj -24.6904 -1.2318 TD [(bacterium)-339.9(tuberculosis)]TJ /F4 1 Tf 9.1384 0 TD [(.)-332.4(Infect)-337.7(Immun)]TJ /F9 1 Tf 6.9559 0 TD (68,)Tj /F4 1 Tf 1.5867 0 TD (791Ð795.)Tj -18.6249 -1.2385 TD [(Brandsma)-9.4(,)-526.6(J.L.,)-540(Shlyankevich,)-535.6(M.,)-538(Zelterman,)-535.4(D.,)-530.9(and)-540(Su,)-531.6(Y.)]TJ .944 -1.2318 TD [(\(2007\).)-493.6(Therapeutic)-494.4(vaccination)-491.4(of)-489.3(rabbits)-493.4(with)-489.2(a)-490.8(ubiquitin-)]TJ 0 -1.2385 TD [(fused)-261.5(papillomavirus)-260.5(E1,)-259.5(E2,)-259.5(E6)-255(and)-258.8(E7)-255(DNA)-262.4(vaccine.)-262.2(Vaccine)]TJ /F9 1 Tf 0 -1.2318 TD (25,)Tj /F4 1 Tf 1.5867 0 TD (6158Ð6163.)Tj -2.5306 -1.2385 TD [(Cardona,)-351.9(P.J,)-352.4(Cooper,)-350.1(A.,)-350.1(Luquin,)-351(M.,)]TJ /F5 1 Tf 17.219 0 TD [(et)-350.8(al.)]TJ /F4 1 Tf 2.3633 0 TD [(\(1999\).)-353(The)-351.1(intrave-)]TJ -18.6383 -1.2318 TD [(nous)-294.8(model)-290.5(of)-288.5(murine)-293.9(tuberculosis)-298.5(is)-287.1(less)-293.5(pathogenic)-296.9(than)-290.1(the)]TJ 0 -1.2385 TD [(aerogenic)-301.6(model)-303.9(owing)-300.6(to)-301.9(a)-296.7(more)-301.8(rapid)-301.5(induction)-302.6(of)-301.9(systemic)]TJ 0 -1.2318 TD [(immunity.)-339.8(Scand)-332.8(J)-334.1(Immunol)]TJ /F9 1 Tf 12.9678 0 TD (49,)Tj /F4 1 Tf 1.5867 0 TD (362Ð366.)Tj -15.4984 -1.2385 TD [(Cendron,)-251.6(D.,)-249.7(Ingoure,)-249.2(S.,)-245.3(Martino,)-249(A.,)]TJ /F5 1 Tf 16.6298 0 TD [(et)-250.4(al.)]TJ /F4 1 Tf 2.1624 0 TD [(\(2007\).)-245.9(A)-240.9(tuber)-9.8(culosis)]TJ -17.8483 -1.2318 TD [(vaccine)-532.3(based)-526.2(on)-527.2(phosphoantigens)-542.9(and)-526.6(fusion)-528.7(proteins)-532.6(in-)]TJ 0 -1.2385 TD [(duces)-360.8(distinct)-366.8(gammadelta)-360.9(and)-359.2(alphabeta)-364.9(T)-360.1(cell)-360.7(responses)-364.5(in)]TJ T* [(primates.)-335.3(Eur)-339.4(J)-334.1(Immun)]TJ /F9 1 Tf 10.5845 0 TD (37,)Tj /F4 1 Tf 1.5867 0 TD (549Ð565.)Tj -13.1151 -1.2318 TD [(Chou)-9.5(,)-238.7(B.,)-235(Hiromatsu)-11.6(,)-238.7(K.,)-241.5(Hisaeda,)-242.8(H.,)-237.9(Duan,)-240.6(X.,)-246.6(Imai,)-238.1(T.,)-241.6(Murata,)]TJ .944 -1.2385 TD [(S.,)-339(Tanaka,)-338(K.,)-341.9(and)-339.1(Himeno,)-341.8(K.)-337.5(\(2010)-7.8(\).)-338.5(Genetic)-340.7(immunization)]TJ 0 -1.2318 TD [(based)-472.7(on)-473.7(the)-479.1(ubiquitin-fusion)-477.8(degradation)-482.1(pathway)-472.9(against)]TJ /F5 1 Tf 0 -1.2385 TD [(Trypanosoma)-393.9(cruzi)]TJ /F4 1 Tf 7.8932 0 TD [(.)-386(Biochem)-392.9(Biophys)-397.6(Res)-391.5(Commun)]TJ /F9 1 Tf 15.2641 0 TD (392,)Tj /F4 1 Tf 2.1356 0 TD (277Ð)Tj -25.2929 -1.2318 TD (282.)Tj -.944 -1.2385 TD [(Cotten,)-418.8(M.,)-410.8(Baker,)-414.3(A.,)-410.4(Saltik,)-413.8(M.,)-410.8(Wagner,)-417.1(E.,)-409(and)-412.8(Buschle,)-416(M.)]TJ .944 -1.2318 TD [(\(1994\).)-761.4(Lipopolysaccharide)-766.7(is)-762.5(a)-758.6(frequent)-763.4(contaminant)-765.6(of)]TJ 0 -1.2385 TD [(plasmid)-634.3(DNA)-637.3(preparations)-643.4(and)-633.7(can)-640(be)-633.6(toxic)-633.8(to)-636.6(primary)]TJ 0 -1.2318 TD [(human)-576.8(cells)-574(in)-579.7(the)-572.8(presence)-584.8(of)-576.4(adenovirus.)-578.8(Gene)-577.3(Ther)]TJ /F9 1 Tf 26.5448 0 TD (1,)Tj /F4 1 Tf -26.5448 -1.2385 TD (239Ð246.)Tj -.944 -1.2318 TD [(Danko,)-403.4(I.,)-392.1(Fritz,)-401.3(J.D.,)-394.1(Jiao,)-395.6(S.,)-399.3(Hogan,)-398.3(K.,)-395.4(Latendresse,)-398.4(J.S.,)-396.4(and)]TJ .944 -1.2385 TD [(Wolff,)-510.9(J.A.)-516.9(\(1994\).)-513.7(Pharmacological)-516.8(enhancement)-514(of)]TJ /F5 1 Tf 24.2552 0 TD [(in)-510.3(vivo)]TJ /F4 1 Tf -24.2552 -1.2318 TD [(foreign)-340.2(gene)-336.7(expression)-337.6(in)-332(muscle.)-342.1(Gene)-336.3(Ther)]TJ /F9 1 Tf 20.6601 0 TD (1,)Tj /F4 1 Tf 1.0846 0 TD (114Ð121.)Tj /F8 1 Tf 8.9663 0 0 8.9663 62.022 749.6502 Tm (494)Tj 47.9403 0 TD [(WANG)-332.6(ET)-338.7(AL.)]TJ ET endstream endobj 77 0 obj <>stream /GS1 gs BT /F4 1 Tf 8.4682 0 0 8.4682 59.7543 725.7825 Tm 0 0 0 1 k 0 Tc 0 Tw [(Dobano,)-324.4(C.,)-318.4(Rogers,)-323.8(W.O.,)-321(Gowda,)-321.4(K.,)-315.1(and)-319.1(Doolan,)-327.6(D.L.)-315.2(\(2007\).)]TJ .944 -1.2318 TD [(Targeting)-410.2(antigen)-415.1(to)-409(MHC)-411.8(Class)-410.5(I)-410.1(and)-406.1(Class)-410.5(II)-411.9(antigen)-408.5(pre-)]TJ 0 -1.2385 TD [(sentation)-458.1(pathways)-458.6(for)-452.5(malaria)-453.9(DNA)-456.5(vaccines.)-455.4(Immun)-454.2(Lett)]TJ /F9 1 Tf 0 -1.2318 TD (111,)Tj /F4 1 Tf 2.0821 0 TD (92Ð102.)Tj -3.026 -1.2385 TD [(Faludi,)-346.7(I.,)-345.3(Szabo)]TJ 6.7617 .0134 TD («)Tj .4352 -.0134 TD [(,)-345.8(A.M.,)-341.4(Buria)]TJ 5.3491 .0134 TD («)Tj .4151 -.0134 TD [(n,)-342.9(K.,)-341.9(Endre)]TJ 4.9876 .0134 TD («)Tj .3749 -.0134 TD [(sz,)-340.3(V.,)-348.6(and)-339.1(Micza)]TJ 7.3308 .0134 TD («)Tj .4151 -.0134 TD [(k,)-342.8(A.)]TJ -25.1255 -1.2318 TD [(\(2011\).)-426.6(Recombinant)]TJ /F5 1 Tf 9.5401 0 TD [(Mycobacterium)-429.8(smegmatis)]TJ /F4 1 Tf 11.3477 0 TD [(vaccine)-431.8(candi-)]TJ -20.8877 -1.2385 TD [(dates.)-338.9(Acta)-334.3(Microbiol)-336.6(Immunol)-335.2(Hung)]TJ /F9 1 Tf 17.1186 0 TD (58,)Tj /F4 1 Tf 1.58 0 TD (13Ð22.)Tj -19.6425 -1.2318 TD [(Harries,)-335.8(A.D.,)-334.4(and)-332.4(Dye,)-336.7(C.)-327.4(\(2006\).)-332.9(Tuberculosis.)-342.2(Ann)-335.5(Trop)-334(Med)]TJ .944 -1.2385 TD (Parasitol)Tj /F9 1 Tf 4.1575 0 TD (100,)Tj /F4 1 Tf 2.0821 0 TD (415Ð431.)Tj -7.1835 -1.2318 TD [(Huygen,)-522.5(K.)-518.2(\(2006\).)-513.7(DNA)-516.8(vaccines)-524.6(against)-519.1(mycobacterial)-517(dis-)]TJ .944 -1.2385 TD [(eases.)-334(Future)-340.7(Microbiol)]TJ /F9 1 Tf 10.6782 0 TD (1,)Tj /F4 1 Tf 1.0846 0 TD (63Ð73.)Tj -12.7067 -1.2318 TD [(Kamath,)-594.5(A.T.,)-587.4(Feng,)-591.9(C.G.,)-587.1(Macdonald,)-591.8(M.,)-584.8(Briscoe,)-594.7(H.,)-586(and)]TJ .944 -1.2385 TD [(Britton,)-485.5(W.J.)-481(\(1999)-7.8(\).)-479.1(Differential)-491(protective)-488.1(efÞcacy)-482.7(of)-489.3(DNA)]TJ 0 -1.2318 TD [(vaccines)-377.4(expressing)-373.5(secreted)-381.9(proteins)-371.9(of)]TJ /F5 1 Tf 18.2232 0 TD [(Mycobacterium)-376.3(tuber-)]TJ -18.2232 -1.2385 TD (culosis)Tj /F4 1 Tf 2.7114 0 TD [(.)-332.4(Infect)-337.7(Immun)]TJ /F9 1 Tf 6.9559 0 TD (67,)Tj /F4 1 Tf 1.5867 0 TD (1702Ð1707.)Tj -12.1979 -1.2318 TD [(Okada,)-588.9(M.)-580.4(\(2006\).)-587.3([Novel)-592.4(vaccines)-584.9(against)-586(M.)-587.1(tuberculosis].)]TJ .944 -1.2385 TD (Kekkaku)Tj /F9 1 Tf 4.2713 0 TD (81,)Tj /F4 1 Tf 1.5867 0 TD (745Ð751.)Tj -6.8019 -1.2318 TD [(Palma,)-281.5(C.,)-271.6(Iona,)-281.1(E.,)-275.1(Giannoni,)-283.7(F.,)]TJ /F5 1 Tf 14.5545 0 TD [(et)-277.1(al.)]TJ /F4 1 Tf 2.216 0 TD [(\(2007)-7.8(\).)-271.5(The)-277.4(Ag85B)-280.8(protein)]TJ -15.8265 -1.2385 TD (of)Tj /F5 1 Tf 1.2921 0 TD [(Mycobacterium)-429.8(tuberc)-9.3(ulosis)]TJ /F4 1 Tf 11.9368 0 TD [(may)-429.6(turn)-431(a)-423.9(protective)-434.6(immune)]TJ -13.2289 -1.2385 TD [(response)-425.7(induced)-431.1(by)-415.7(Ag85B-DNA)-428.7(vaccine)-425.1(into)-426.3(a)-423.9(potent)-422.3(but)]TJ 0 -1.2318 TD [(non-protective)-375.8(Th1)-359.2(immune)-366.3(response)-372.1(in)-365.5(mice.)-360.2(Cell)-370.9(Microbiol)]TJ /F9 1 Tf 0 -1.2385 TD (9,)Tj /F4 1 Tf 1.0846 0 TD (1455Ð1465.)Tj -2.0285 -1.2318 TD [(Rodriguez,)-361.9(F.,)-360.6(Zhang,)-362.6(J.,)-356.3(and)-359.2(Whitton,)-364.4(J.L.)-361.5(\(1997\).)-359.7(DNA)-356.1(immu-)]TJ .944 -1.2385 TD 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