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[(described)-350.2(previously)-348.4(\(Wang)]TJ
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[(et)-342.6(al.)]TJ
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[(,)-344.4(2003\),)-342.9(using)-349.1(recombinant)]TJ
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[(MPT64)-381.5(protein)-378.8(\(1)]TJ
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(m)Tj
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[(g)-377.5(per)-381.3(well\))-375.8(\(Wang)]TJ
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8.6434 0 TD
[(et)-380.5(al.)]TJ
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[(,)-376(2004\))-377.9(and)-379(anti-)]TJ
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[(mouse)-692.1(IgG,)-691.1(IgG)]TJ
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(1)Tj
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[(,)-685.8(o)0(r)-692.7(IgG)]TJ
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(2a)Tj
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[(coupled)-694.1(to)-686.1(HRP)-694.8(\(Southern)]TJ
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[(Biotechnology)-539.5(Associates,)-532.2(SBA\).)-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
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(2a)Tj
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[(to)-332(IgG)]TJ
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(1)Tj
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[(was)-337.6(calculated.)]TJ
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[(Lymphocytes)-335.1(proliferation)-338.6(assay)]TJ
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[(Mice)-324(were)-324.6(sacri辌ed)-325.7(3)-315.7(weeks)-323.3(after)-329.8(the)-319.9(last)-321(immunization.)]TJ
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[(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
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[(proliferation)-439.9(assay)-436(was)-432.4(performed)-436.3(as)-437.1(previously)-436.9(described)]TJ
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(\(Wang)Tj
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[(et)-494.4(al.)]TJ
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[(,)-496.1(2003\).)-494.6(Brie遹,)-498(the)-496.9(isolated)-500.9(spleen)-496.3(cells)-500.8(were)]TJ
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[(resuspended)-438.8(to)-426.9(a)-429.5(concentration)-436.2(of)-433.2(5)]TJ
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16.5913 0 TD
(?Tj
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(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
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[(ume)-270.7(of)-268.8(100)]TJ
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(m)Tj
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[(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
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[(and)-309.4(the)-313.6(MPT64)-305.7(protein)-309.3(\(Wang)]TJ
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13.8282 0 TD
[(et)-311(al.)]TJ
/F4 1 Tf
1.9727 0 TD
[(,)-306.4(2004\))-308.3(was)-312.3(added)-305.6(to)-306.7(the)]TJ
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[(wells)-332.4(in)-329.4(triplicate)-328.4(at)-328.8(the)-326.2(辬al)-331.9(concentration)-335(of)-325.7(5)]TJ
/F6 1 Tf
21.2576 0 TD
(m)Tj
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[(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
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[(C)-354.3(i)0(n)-354.7(a)0(n)-350.7(atmosphere)-361(of)-351(5%)-355.2(CO)]TJ
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(2)Tj
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[(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
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[(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
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[(formula:)-356.6(SI)]TJ
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(=)Tj
/F4 1 Tf
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[(experimental)-359.7(OD/negative)-360.7(OD.)-355.1(To)-353.5(assure)-351.8(that)]TJ
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[(cells)-266.8(were)-255(healthy,)-264.1(10)]TJ
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9.5097 0 TD
(m)Tj
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[(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
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-14.5048 -1.8336 TD
[(Single)-398.9(splenocyte)-395.7(suspension)-399(from)-399.8(immunized)-395(mice)-392.3(was)]TJ
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[(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
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(10)Tj
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(6)Tj
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[(cells/mL.)-538.9(About)-535.2(100)]TJ
/F6 1 Tf
9.731 0 TD
(m)Tj
/F4 1 Tf
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[(L)-534.1(o)0(f)-534.4(cell)-531(suspension)-538.1(was)]TJ
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[(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*
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(m)Tj
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[(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
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26.1705 0 TD
(g)Tj
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[(and)-695.1(interleukin)-688(\(IL\)-4)-695.2(with)-685.6(an)-692.1(ELISA)-690.9(detection)-689.3(system)]TJ
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[(\()-126.9(Jingmei;)-426.7(Biotech\))-430.1(according)-426.9(to)-426.9(the)-427.4(manufacturer誷)-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
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[(measurement)-332.1(using)-332.2(遫w)-335.5(cytometry)]TJ
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[(Splenocytes)-811.5(from)-810.8(vaccinated)-811.6(mice)-809.6(were)-805.1(cultured)-812.6(at)]TJ
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(2.5)Tj
/F7 1 Tf
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(?Tj
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(10)Tj
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(6)Tj
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[(/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
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[(presence)-328.2(of)-325.7(5)]TJ
/F6 1 Tf
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(m)Tj
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[(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
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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
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[(from)-418.8(each)-419.4(group)-421.6(were)-419.4(農st)-416.5(incubated)-425.8(with)-420(遳orescein)-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(辺ed)-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
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[(,)-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
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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(cyto遳orometer)-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
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[(/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(\(10)]TJ
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(m)Tj
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(g/)Tj
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[(mL\)-treated)-258.4(target)-252.1(cells)-247.8(\(P815-MPT64,)-252.5(5)]TJ
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17.4259 0 TD
(?Tj
/F4 1 Tf
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(10)Tj
6.7246 0 0 5.9768 231.3637 691.4266 Tm
(5)Tj
8.9663 0 0 8.9663 234.7653 687.4014 Tm
[(/mL\))-250.6(in)-247.2(a)-252.4(1)0(0)-163.4(m)0(L)]TJ
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[(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
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.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
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(.)Tj
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[(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(speci辌)-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(signi辌ance)-292.4(of)-287.8(differential)-296.6(辬dings)-293.9(between)]TJ
-1.1128 -1.2203 TD
[(experimental)-290.1(groups)-290.1(was)-280.7(determined)-289.6(by)-280.7(Student誷)-288.5(test.)-289.6(Data)]TJ
0 -1.2266 TD
[(were)-337.2(considered)-339.3(statistically)-337(signi辌ant)-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(con農med)]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(con農med,)-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(con農med)-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(quanti辝d)-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(辬al)-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
[(speci辌)-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(speci辌)]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
[(speci辌.)-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-speci辌)-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-speci辌)-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(signi辌antly)-579.9(higher)-584.4(level)-576.7(of)-578.6(IgG)]TJ
0 -1.2266 TD
(\()Tj
/F5 1 Tf
.4869 0 TD
(p)Tj
/F7 1 Tf
.6702 0 TD
(<)Tj
/F4 1 Tf
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[(0.01\).)-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(signi辌antly)-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(eci辌)-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(?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
[(enzyme-linked)-410.4(i)0(mmun)-15.4(o)10.1(absor)-15.4(b)11.1(ent)-407.9(a)-2.3(ss)-15.4(a)9.1(y)-.8(.)-404(O)-15.4(p)11.8(t)-3.2(ical)-411(dens)-15.4(i)11.4(t)-3.2(y)-405.5(w)-15.4(a)15.1(s)]TJ
0 -1.1065 TD
-.0139 Tc
[(mea)5.6(s)-13.9(u)15(r)-13.9(e)10.8(d)-276.1(at)-273.6(450)-165.2(nm.)-276.1(D)-13.9(a)10.5(t)4.6(a)-279(s)-13.9(h)13(o)2.3(wn)-282.9(r)4.4(e)-1.2(p)-13.9(r)15.7(es)-13.9(e)16.5(n)1.7(ted)-276.1(g).7(eometr)4.4(ic)-278.7(mean)]TJ
0 -1.1128 TD
0 Tc
[(t)18.5(i)0(t)27.6(e)12.7(rs)-375.1(\()12.2(G)13.9(M)13.9(T)16(\))-392.5(a)13.1(n)15.6(d)-388.7(st)29.8(a)13.1(n)15.6(d)16(a)13.1(rd)-376.8(e)12.8(r)18.3(ro)28.1(rs)-381.4(f)18.5(o)0(r)-376.5(e)12.8(a)13.1(c)13.4(h)-389.1(g)14.6(ro)28.1(u)17.6(p)-393.4(o)16.2(f)-392.5(a)13.1(n)15.6(i)15.4(m)15.1(a)13.1(l)15.4(s.)]TJ
T*
-.0183 Tc
(**)Tj
/F5 1 Tf
.7398 0 TD
0 Tc
(p)Tj
/F7 1 Tf
.6513 0 TD
(<)Tj
/F4 1 Tf
.7018 0 TD
[(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
[(IMPROVED)-336.4(IMMUNE)-339.1(RESPONSE)-333.3(BY)-335.8(A)-337.9(FUSION)-331.9(DNA)-337.2(VACCINE)-23215.7(491)]TJ
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26.1705 0 TD
(g)Tj
/F4 1 Tf
-26.1705 -1.2203 TD
[(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
[(ni辌antly)-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
[(signi辌antly)-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-pro辧e)-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-speci辌)-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(sacri辌ed)-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
[(pcDNA3)-723.7(group)-718.8(or)-718(pcDNA3-ub)-723.7(group;)-721.7(*)]TJ
/F5 1 Tf
19.7465 0 TD
(p)Tj
/F7 1 Tf
.6639 0 TD
(<)Tj
/F4 1 Tf
.7208 0 TD
[(0.05)-715.9(UbGR-)]TJ
-21.1312 -1.1128 TD
[(MPT64)-368.9(group)-371(versus)-372.1(pcDNA3-MPT64)-372.3(group.)-367.6(Eight)-368.9(spleens)]TJ
T*
[(were)-337.2(pooled)-335.8(and)-334.7(the)-338.9(experiment)-337.7(was)-331.2(repeated)-339.8(three)-333.1(times.)]TJ
/F8 1 Tf
-27.9978 45.3795 TD
(492)Tj
47.9403 0 TD
[(WANG)-332.6(ET)-338.7(AL.)]TJ
ET
endstream
endobj
62 0 obj
<>stream
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/GS1 gs
BT
/F2 1 Tf
8.9663 0 0 8.9663 310.8472 725.7825 Tm
0 0 0 1 k
0 Tc
0 Tw
[(Improved)-334.3(CTL)-335.4(response)-333.1(by)-332.7(UbGR-MPT64)]TJ
0 -1.2203 TD
[(fusion)-335.8(DNA)-332.3(vaccine)]TJ
/F4 1 Tf
1.1128 -1.8336 TD
[(Cytotoxic)-313.2(T-cell)-313.2(responses)-315.5(were)-311.9(determined)-314.8(with)-312.5(an)-312.7(LDH)]TJ
-1.1128 -1.2266 TD
[(release)-428.4(assay,)-426.3(after)]TJ
/F5 1 Tf
8.8963 0 TD
[(in)-429.9(vitro)]TJ
/F4 1 Tf
3.5914 0 TD
[(restimulation)-429(against)-429.5(the)-427.4(target)]TJ
-12.4878 -1.2203 TD
[(cell)-518.4(line)-522.5(P815-MPT64,)-523.9(which)-523.4(stably)-519.5(expressed)-521.7(the)-522.2(MPT64)]TJ
T*
[(protein.)-242.7(P815)-246.5(cells)-247.8(were)-242.4(used)-245.8(as)-241.1(a)-246.1(negative)-245.8(control.)-249.8(As)-243(shown)]TJ
0 -1.2266 TD
[(in)-361(Figure)-368.4(6,)-356.5(DNA)-369.5(immunization)-363.9(with)-363.1(MPT64)-362.6(DNA)-369.5(vaccine)]TJ
0 -1.2203 TD
[(resulted)-395.9(in)-392.6(a)-391.5(strong)-392.4(CTL)-391.9(response,)-392.9(which)-396.9(was)-388.1(signi辌antly)]TJ
T*
[(above)-835.6(the)-838.4(blank)-837.2(vector)-838.5(or)-838.1(pcDNA3-ub)-837.5(immunization)]TJ
T*
(\()Tj
/F5 1 Tf
.4869 0 TD
(p)Tj
/F7 1 Tf
.6702 0 TD
(<)Tj
/F4 1 Tf
.7145 0 TD
[(0.01\).)-472.7(Ub)-467.4(fusion)-474.4(DNA)-470.7(vaccine)-473.5(enhanced)-473.3(the)-471.6(cytotoxic)]TJ
-1.8716 -1.2266 TD
[(T-cell)-553.5(response,)-557.3(compared)-554.2(with)-559.1(MPT64)-552.3(DNA)-552.9(inoculation)]TJ
0 -1.2203 TD
(\()Tj
/F5 1 Tf
.4869 0 TD
(p)Tj
/F7 1 Tf
.6702 0 TD
(<)Tj
/F4 1 Tf
.7145 0 TD
[(0.05\).)-529.6(The)-524.8(blank)-527.4(vector)-535(or)-528.3(pcDNA3-ub)-527.7(immunization)]TJ
-1.8716 -1.2203 TD
[(did)-351(not)-354.4(induce)-358.2(CTL)-353.9(response.)-361.3(The)-354(spontaneous)-359(release)-358.8(was)]TJ
0 -1.2266 TD
[(below)-334.7(10%.)]TJ
/F8 1 Tf
0 -2.4406 TD
(Discussion)Tj
/F4 1 Tf
1.1128 -1.8336 TD
[(DNA)-1103(vaccines)-1100.8(preferentially)-1108.8(induce)-1104.3(Th1-dominant)]TJ
-1.1128 -1.2266 TD
[(immune)-313.1(response.)-310.7(The)-316.1(exact)-311.1(mechanism)-315.3(driving)-310.2(Th1)-309.4(or)-313.3(Th2)]TJ
0 -1.2203 TD
[(type)-353.9(response)-358.4(is)-352.6(not)-354.4(well)-356.4(known,)-353.6(but)-354(it)-351.8(has)-352(been)-355.4(suggested)]TJ
T*
[(that)-364.2(CpG)-373.3(motifs)-366.5(from)-368.2(a)-366.2(bacterial)-364.9(plasmid)-370.2(might)-369.8(be)-364.7(respon-)]TJ
T*
[(sible)-366.8(for)-370.7(driving)-367.1(immune)-370(responses)-372.4(toward)-368.5(Th1-type)-366.6(\(Bode)]TJ
/F5 1 Tf
0 -1.2266 TD
[(et)-538.6(al.)]TJ
/F4 1 Tf
2.2067 0 TD
[(,)-534(2007\).)-545.2(Th1-type)-543.6(response)-541.8(correlates)-545.5(with)-540.1(protective)]TJ
-2.2067 -1.2203 TD
[(immunity)-340.6(in)-348.4(certain)-341.9(tumor,)-290.3(bacterial,)-292(and)-290.4(viral)-292.5(infections,)-292.8(as)]TJ
T*
[(well)-305.8(as)-310.6(some)-311.4(parasitic)-312.7(diseases.)-309.9(Protective)-318.9(immunity)-309(against)]TJ
0 -1.2266 TD
[(tuberculosis)-386.7(mainly)-385(depends)-384.9(on)-385.5(cellular)-384.4(immune)-382.7(responses)]TJ
0 -1.2203 TD
[(and)-593.9(some)-595.9(Th1-type)-600.5(cytokines,)-597.8(such)-593.4(as)-595.2(IFN-)]TJ
/F6 1 Tf
20.8403 0 TD
(g)Tj
/F4 1 Tf
.4995 0 TD
[(.)-597.3(Hence,)-599.7(to)]TJ
ET
q
232.767 0 0 173.424 63.042 558.822 cm
/Im4 Do
Q
BT
/F9 1 Tf
8.9663 0 0 8.9663 59.7543 541.2471 Tm
[(FIG.)-450.6(4.)]TJ
/F4 1 Tf
4.2111 0 TD
[(Production)-454.9(of)-452.2(IFN-)]TJ
/F6 1 Tf
8.6687 0 TD
(g)Tj
/F4 1 Tf
.9548 0 TD
[(and)-448.5(interleukin-4)-454.1(from)-450.4(sple-)]TJ
-13.8345 -1.1065 TD
[(nocytes)-422.5(of)-414.2(different)-422.8(groups.)-413.2(Different)-421.2(group)-415.3(of)-420.6(mice)-411.3(\()]TJ
/F5 1 Tf
24.4064 0 TD
(n)Tj
/F7 1 Tf
.7208 0 TD
(=)Tj
/F4 1 Tf
.7208 0 TD
(8\))Tj
-25.8481 -1.1128 TD
[(were)-255(immunized)-249.6(by)-242.8(the)-250.3(blank)-249.2(vector,)-253.4(pcDNA3-ub,)-252.4(pcDNA3-)]TJ
T*
[(MPT64)-343.6(DNA)-344.2(vaccine,)-343.7(or)-344.9(UbGR-MPT64)-346.6(DNA)-344.2(vaccine)-340.7(at)-347.7(a)-340.9(3)]TJ
T*
[(week)-593.9(interval.)-596.4(Three)-601.2(weeks)-588.9(after)-601.7(the)-591.8(last)-592.9(immunization,)]TJ
0 -1.1065 TD
[(spleen)-502.6(cells)-500.7(from)-501(sacri辌ed)-502.7(mice)-493.5(were)-501.6(pooled)-500.2(and)-499.1(stimu-)]TJ
0 -1.1128 TD
[(lated)-411.1(with)-413.7(5)]TJ
/F6 1 Tf
5.6716 0 TD
(m)Tj
/F4 1 Tf
.5564 0 TD
[(g/mL)-406.3(MPT64)-413.2(protein)-410.5(for)-414.9(72)-163.4(h.)-411(Samples)-412.7(were)]TJ
-6.2281 -1.1128 TD
[(assayed)-337.8(in)-335.7(triplicate.)-331.3(Values)-337.2(and)-334.7(bars)-334.7(represent)-336.4(the)-332.5(mean)-335.5(of)]TJ
0 -1.1065 TD
[(released)-425.1(cytokine)-422.8(concentrations)-431.2(and)-423.2(S.D.)-423(**)]TJ
/F5 1 Tf
20.0436 0 TD
(p)Tj
/F7 1 Tf
.6639 0 TD
(<)Tj
/F4 1 Tf
.7208 0 TD
[(0.01)-418.8(UbGR-)]TJ
-21.4283 -1.1128 TD
[(MPT64)-368.9(group)-371(versus)-372(pcDNA3-MPT64)-372.3(group.)-367.6(Eight)-368.9(spleens)]TJ
T*
[(were)-394.1(pooled)-386.4(and)-391.6(the)-389.5(experiment)-388.2(was)-388.2(repeated)-390.4(three)-390(times.)]TJ
T*
(IFN-)Tj
/F6 1 Tf
2.036 0 TD
(g)Tj
/F4 1 Tf
.4995 0 TD
[(,)-331.7(interferon-gamma.)]TJ
ET
q
230.849 0 0 208.63 64.063 174.444 cm
/Im5 Do
Q
BT
/F9 1 Tf
8.9663 0 0 8.9663 59.7543 156.8692 Tm
[(FIG.)-330.5(5.)]TJ
/F4 1 Tf
4.0909 0 TD
[(Flow)-328.5(cytometry)-332.5(analysis)-326.3(of)-332(MPT64-speci辌)-326.5(IFN-)]TJ
/F6 1 Tf
21.4157 0 TD
(g)Tj
/F7 1 Tf
6.7246 0 0 5.9768 294.0661 160.8945 Tm
(+)Tj
/F4 1 Tf
8.9663 0 0 8.9663 59.7543 146.948 Tm
[(CD4)-304.6(or)-300.6(CD8)]TJ
/F7 1 Tf
6.7246 0 0 5.9768 110.0976 150.9165 Tm
(+)Tj
/F4 1 Tf
8.9663 0 0 8.9663 117.6378 146.948 Tm
[(T)-300.2(cells.)-301.3(Mice)-305(were)-299.3(inoculated)-307.6(as)-298(before)-305.3(\()]TJ
/F5 1 Tf
17.7042 0 TD
(n)Tj
/F7 1 Tf
.7208 0 TD
(=)Tj
/F4 1 Tf
.7145 0 TD
(8\).)Tj
-25.5951 -1.1128 TD
[(Three)-417.9(weeks)-411.8(after)-412(the)-414.7(last)-409.5(immunization,)-417.4(spleen)-414(cells)-412.2(from)]TJ
T*
[(sacri辌ed)-445.8(mice)-436.6(were)-444.7(pooled)-443.3(and)-442.2(stimulated)-440.6(with)-439(5)]TJ
/F6 1 Tf
23.4833 0 TD
(m)Tj
/F4 1 Tf
.5501 0 TD
(g/mL)Tj
-24.0334 -1.1065 TD
[(MPT64)-463.8(protein)-467.4(for)-465.5(72)-163.4(h.)-467.9(The)-461.5(frequency)-469.1(of)-464.8(IFN-)]TJ
/F6 1 Tf
21.6749 0 TD
(g)Tj
/F7 1 Tf
6.7246 0 0 5.9768 259.7102 121.0393 Tm
(+)Tj
/F4 1 Tf
8.9663 0 0 8.9663 268.6676 117.0708 Tm
[(CD4)-462.7(or)]TJ
-23.2999 -1.1128 TD
[(CD8)-355.2(T)-350.8(cells)-349(was)-350.2(detected)-353.7(by)-350.3(遫w)-355.2(cytometry.)-348(Samples)-355.7(were)]TJ
T*
[(assayed)-337.8(in)-335.7(triplicate.)-331.3(Values)-337.2(and)-334.7(bars)-334.7(represent)-336.4(the)-332.5(mean)-335.5(of)]TJ
T*
[(frequency)-545(and)-537(S.D.)-536.8(**)]TJ
/F5 1 Tf
10.2052 0 TD
(p)Tj
/F7 1 Tf
.6702 0 TD
(<)Tj
/F4 1 Tf
.7145 0 TD
[(0.01)-538.9(UbGR-MPT64)-542.6(group)-535.4(versus)]TJ
-11.5899 -1.1065 TD
[(pcDNA3-MPT64)-460.8(group;)-456.1(*)]TJ
/F5 1 Tf
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[(The)-442.6(Ub衟roteasome)-447.9(pathway)-446.6(is)-441.1(the)-446.4(main)-440.3(source)-447.9(for)-440.2(in-)]TJ
-1.1128 -1.2266 TD
[(tracellular)-264.9(protein)-265(turnover.)-272.2(MHC)-264.5(class)-264.2(I)-261.4(most)-261.4(often)-266.2(presents)]TJ
0 -1.2203 TD
[(peptides)-453.4(derived)-453.4(from)-450.4(endogenously)-453.8(synthesized)-450.1(proteins,)]TJ
T*
[(which)-333.7(are)-335.2(degraded)-329.6(by)-331.3(the)-332.5(proteasome.)-338(Hence,)-334.2(higher)-337.8(rates)]TJ
0 -1.2266 TD
[(of)-294.1(intracellular)-303.5(antigen)-292.8(turnover)-300.9(should)-293.6(increase)-298.4(the)-294.6(number)]TJ
0 -1.2203 TD
[(and)-404.2(variety)-407.6(of)-407.9(fragments)-406.1(and)-404.2(peptides)-409.2(available)-406.2(for)-408.6(MHCI)]TJ
T*
[(binding,)-458.6(which)-466.5(may)-456.7(result)-462.5(in)-462.2(an)-458.1(increase)-462.8(of)-458.5(cell-mediated)]TJ
T*
[(response)-415.3(to)-407.9(the)-408.4(expressed)-407.9(antigens.)-410.9(To)-410.4(this)-407(point,)-412.8(conjuga-)]TJ
0 -1.2266 TD
[(tion)-339(of)-338.4(the)-338.9(antigen)-337.1(with)-337.8(Ub)-340.9(should)-337.8(target)-340.6(the)-338.9(endogenously)]TJ
0 -1.2203 TD
[(synthesized)-355.2(antigens)-351(to)-351(the)-351.5(proteasome)-354(pathway)-351.7(and)-347.3(result)]TJ
T*
[(in)-316.7(an)-319(enhanced)-321.6(cellular)-321.2(immune)-319.5(response.)-323.4(Some)-321.2(researchers)]TJ
0 -1.2266 TD
[(have)-370.3(optimized)-365.7(the)-370.5(ef辌acy)-366.3(of)-370(DNA)-369.5(vaccines)-367.4(by)-369.2(increasing)]TJ
0 -1.2203 TD
[(antigen)-469.9(degradation)-469.1(\(Brandsma)]TJ
/F5 1 Tf
14.7766 0 TD
[(et)-469.1(al.)]TJ
/F4 1 Tf
2.1371 0 TD
[(,)-464.5(2007;)-462.5(Dobano)]TJ
/F5 1 Tf
7.3725 0 TD
[(et)-469.1(al.)]TJ
/F4 1 Tf
2.1308 0 TD
(,)Tj
-26.4171 -1.2203 TD
[(2007;)-405.6(Sharma)]TJ
/F5 1 Tf
6.4114 0 TD
[(et)-405.8(al.)]TJ
/F4 1 Tf
2.0676 0 TD
[(,)-407.6(2009;)-399.3(Chou)]TJ
/F5 1 Tf
6.1269 0 TD
[(et)-405.8(al.)]TJ
/F4 1 Tf
2.0739 0 TD
[(,)-401.3(2010\).)-406.1(There)-405.2(are)-404.8(two)]TJ
-16.6798 -1.2266 TD
[(methods)-438.4(of)-439.5(fusing)-444.4(Ub)-435.8(to)-439.5(the)-433.7(protein)-442.1(of)-439.5(interest.)-442.5(One)-436.3(is)-441.1(to)]TJ
0 -1.2203 TD
[(mutate)-347(the)-351.5(C-terminal)-344.4(residue)-351.1(of)-344.7(Ub)-347.3(from)-349.3(glycine)-347.3(\(G\))-347.5(to)-344.7(al-)]TJ
T*
[(anine)-483.9(\(A\),)-479.5(resulting)-486.5(in)-481.1(a)-480.1(stable)-477(Ub-protein)-486.3(\(UbAAg\).)-484.8(This)]TJ
T*
[(stable)-508.7(Ub-protein)-505.3(can)-508(be)-503.8(polyubiquitinated)-515.2(and)-499.1(degraded)]TJ
0 -1.2266 TD
[(quickly)-394(by)-394.5(the)-389.5(proteasome.)-401.2(The)-392(other)-392.9(method)-399.1(is)-390.5(to)-395.3(add)-391.2(an)]TJ
0 -1.2203 TD
[(arginine)-265(\(R\))-264.4(to)-262.5(the)-263(C-terminus)-268.1(of)-262.5(Ub,)-261.6(resulting)-271.5(in)-259.8(an)-262.1(unstable)]TJ
T*
[(Ub-protein)-435.7(\(UbGR-Ag\).)-436.3(This)-434.9(fusion)-430.1(protein)-435.7(can)-432.1(be)-434.3(quickly)]TJ
0 -1.2266 TD
[(recognized)-294.6(and)-290.4(degraded)-291.6(by)-287(the)-294.6(Ub)-290.3(system)-295.8(according)-287.8(to)-294.1(the)]TJ
0 -1.2203 TD
[(N-rule,)-337.6(also)-335.9(resulting)-334.8(in)-335.7(promoted)-337.3(protein)-334.6(degradation.)]TJ
1.1128 -1.2203 TD
[(Based)-335.5(on)-334.9(the)-338.9(Ub)-334.6(paradigm,)-341.3(we)-330.3(fused)-341.2(UbGR)-337.6(with)-337.8(MPT64)]TJ
-1.1128 -1.2266 TD
[(antigen)-362.4(from)]TJ
/F5 1 Tf
6.1079 0 TD
[(M.)-355.8(tuberculosis)]TJ
/F4 1 Tf
6.3545 0 TD
[(.)-357(The)-354(change)-357.6(of)-357.3(the)-357.8(immune)-357.4(re-)]TJ
-12.4625 -1.2203 TD
[(sponse)-402.1(elicited)-398.8(by)-400.9(UbGR-MPT64)-403.5(fusion)-398.5(DNA)-401.1(vaccine)-397.7(indi-)]TJ
T*
[(rectly)-299.2(showed)-296.5(the)-294.6(change)-294.4(of)-294.1(MPT64)-299.4(degradation.)-295(Compared)]TJ
0 -1.2266 TD
[(with)-407.4(the)-402.1(MPT64)-400.5(DNA)-407.4(immunization,)-404.7(UbGR-MPT64)-403.5(fusion)]TJ
0 -1.2203 TD
[(DNA)-382.2(vaccine)-385(resulted)-383.2(in)-386.3(an)-375.9(lower)-387.9(antibody)-381.9(IgG,)-381.3(enhanced)]TJ
T*
[(lymphocytes)-391.5(proliferation,)-398.6(a)-385.2(stronger)-399.4(Th1)-385.3(type)-391.8(immune)-389(re-)]TJ
T*
[(sponse,)-348.1(and)-341(enhanced)-346.9(cytotoxicity)-352.4(of)-344.7(CTLs.)-339.2(The)-347.7(IFN-)]TJ
/F6 1 Tf
23.7615 0 TD
(g)Tj
/F4 1 Tf
.8409 0 TD
(level)Tj
-24.6024 -1.2266 TD
[(in)-405.3(UbGR-MPT64)-403.5(was)-400.8(higher)-407.3(than)-405.1(that)-402.2(in)-405.3(the)-402.1(MPT64)-406.8(DNA)]TJ
0 -1.2203 TD
[(vaccine)-454.6(group.)-449.8(The)-448.9(increased)-453.1(level)-450.2(of)-445.8(IFN-)]TJ
/F6 1 Tf
19.6073 0 TD
(g)Tj
/F4 1 Tf
.9484 0 TD
[(resulted)-452.8(from)]TJ
-20.5558 -1.2203 TD
[(both)-390.4(CD4)]TJ
/F7 1 Tf
6.7246 0 0 5.9768 102.2173 324.2834 Tm
(+)Tj
/F4 1 Tf
8.9663 0 0 8.9663 110.5511 320.3149 Tm
[(T)-388.7(and)-385.3(CD8)]TJ
/F7 1 Tf
6.7246 0 0 5.9768 156.8125 324.2834 Tm
(+)Tj
/F4 1 Tf
8.9663 0 0 8.9663 165.1464 320.3149 Tm
[(T)-388.7(cells,)-389.9(particularly)-391(from)-387.2(CD8)]TJ
/F7 1 Tf
6.7246 0 0 5.9768 287.3763 324.2834 Tm
(+)Tj
/F4 1 Tf
8.9663 0 0 8.9663 295.7101 320.3149 Tm
(T)Tj
-26.063 -1.2266 TD
[(cells.)-465.7(Interestingly,)-473.1(the)-465.3(ubiquitination)-472(strategy)-466.4(designed)-467.4(to)]TJ
0 -1.2203 TD
[(improve)-297.4(MHC)-296.1(I-mediated)-300.3(cellular)-295.9(responses)-302.8(also)-298(resulted)-301(in)]TJ
T*
[(improved)-344.7(cytokine)-347(and)-341(proliferative)-345.9(responses)-347.1(mediated)-342(by)]TJ
0 -1.2266 TD
(CD4)Tj
/F7 1 Tf
6.7246 0 0 5.9768 80.9008 280.4597 Tm
(+)Tj
/F4 1 Tf
8.9663 0 0 8.9663 88.2141 276.4345 Tm
[(T)-268.6(cells.)-276(It)-274.5(could)-281.8(be)-269.9(that)-275.7(increasing)-278.2(protein)-277.7(degradation)]TJ
-2.9212 -1.2203 TD
[(by)-375.6(the)-376.8(proteasome)-379.3(also)-380.2(yields)-376.1(peptides)-377.6(that)-383.2(could)-376.7(be)-371(taken)]TJ
T*
[(up)-369.4(by)-375.6(MHC)-365.7(II)-371(molecules.)-378.8(Our)-371.8(result)-374(is)-365.3(consistent)-382.6(with)-369.4(Do-)]TJ
T*
(ban)Tj
1.1571 .0063 TD
(?Tj
.4679 -.0063 TD
[(o誷)-695.2(report)-701.4(\(2007\),)-697.4(which)-694.1(showed)-701.2(that)-699.4(immunization)]TJ
-1.625 -1.2266 TD
[(with)-337.8(DNA)-337.9(vaccine)-334.4(encoding)-342.2(PyHEP17)-337.9(fused)-334.8(to)-338.4(Ub)-334.6(induced)]TJ
0 -1.2203 TD
[(higher)-495.9(IFN-)]TJ
/F6 1 Tf
5.3555 0 TD
(g)Tj
/F4 1 Tf
.4995 0 TD
[(,)-489.8(cytotoxic,)-492.3(and)-492.8(proliferative)-497.7(T)-489.9(cell)-493.1(responses)]TJ
-5.855 -1.2203 TD
[(than)-367.2(those)-361.9(of)-363.6(unmodi辝d)-365.3(vaccines.)-370.3(However,)-362.6(no)-366.6(effect)-367.2(was)]TJ
0 -1.2267 TD
[(seen)-409.2(for)-408.6(another)-408.5(antigen)-406.6(\(PyCSP\))-411.4(using)-406(the)-408.4(same)-409.3(targeting)]TJ
0 -1.2203 TD
[(strategies.)-469.7(Rodriguez誷)-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(Vadlin誷)]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(signi辌antly)-358.6(increased)]TJ
27.9978 74.5598 TD
[(the)-269.3(antigen-speci辌)-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(ef辌acy)-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(辬ancial)-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
(1856?862.)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(BCG裪mplications)-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
(656?62.)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(ef辌acy)-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
(1702?707.)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(499?11.)]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
(791?95.)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
(6158?163.)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
(362?66.)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
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