Boosting Immune Response to Hepatitis B DNA Vaccine by Coadministration of Prothymosin {alpha}-Expressing Plasmid

DNA vaccines induce protective humoral and cell-mediated immuneresponses in several animal models. However, compared to conventionalvaccines, DNA vaccines usually induce poor antibody responses.In this study, we report that coadministration of a hepatitisB virus (HBV) DNA vaccine with prothymosin ${alpha}$ as an adjuvant improvesantibody responses to HBV S antigen. We also observed higherseroconversion rates and higher antibody titers. Prothymosin ${alpha}$ appears to increase the number and affinity of hepatitis Bsurface antigen-specific, gamma interferon-secreting T cellsand to enhance cellular immune response to the PreS2S DNA vaccine.Interestingly, administering the DNA separately from the prothymosin ${alpha}$ plasmid abrogated the enhancement of DNA vaccine potency. Theresults suggest that prothymosin ${alpha}$ may be a promising adjuvantfor DNA vaccines.

INTRODUCTION

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Introduction

Immunization with naked DNA can be defined as the in vivo deliveryof an antigen-encoding expression vector into a given tissuefor the purpose of the induction of immune responses. This novelimmunization approach results in de novo production of correctlyfolded and glycosylated protein antigens and in certain respectsmimics the actions of live attenuated or recombinant virus vaccines.As a consequence, this mode of immunization may mimic naturalinfection and induce protective immune responses. In animalmodels, such genetic immunization induces both humoral and cellularimmune responses against a range of viral pathogens, such ashepatitis B virus (31), influenza A virus (37, 43), herpes simplexvirus (5, 27, 28), rabies virus (48), and human immunodeficiencyvirus type 1 (6, 45). In spite of the existence of safe andefficacious vaccines, hepatitis B virus (HBV) infection is oneof the most common infectious diseases, with an estimated 350million chronic HBV carriers worldwide (12, 22). Patients withchronic hepatitis B are at high risk of developing liver cirrhosis,which is associated with a high rate of mortality due to thedevelopment of hepatocellular carcinoma or noncarcinomatouscomplications of cirrhosis (portal hypertension and liver failure)(19). In addition, these chronically HBV-infected carriers representa reservoir of HBV. Prophylactic immunization against HBV infectionscan be achieved with recombinant subunit HBV vaccines (10, 30,44); however, a small number of individuals do not develop protectiveimmunity even after more than the recommended three hepatitisB surface antigen (HBsAg) inoculations. The incidence of nonresponsein immunocompetent young adults is around 5% but increases upto 30% with age of vaccination or even more in immunocompromisedindividuals (32). Other problems arising from the use of currentHBV vaccines are escape variants with mutations within HBsAgand the requirement of at least three injections to achieveprotection. For control of HBV infections and liver disease,efficacious but inexpensive DNA vaccines may thus be promisingtools (18). Recent reports have demonstrated the efficacy ofDNA-based HBV vaccines. Immunization of animals with plasmidDNA vectors encoding the small or middle viral envelope proteinselicits a stronger and longer-lasting humoral and cell-mediatedimmunity than does immunization with recombinant small and middleenvelope protein particles, which are presently in use as vaccines(9). Moreover, DNA-mediated immunization has been shown to breakimmune tolerance in an HBV transgenic mouse model, indicatingthat DNA vaccines may also be beneficial for immunotherapy ofchronic HBV infections (26).

DNA vaccines can induce cytotoxic T lymphocyte (CTL) responsesthat do not usually occur after immunization with protein subunitvaccines. But compared with conventional vaccines, DNA vaccinesare often less potent in the induction of antibody responses.Alternative forms of immunization for DNA vaccines have beenreported to increase the immunogenicity of encoded antigens.These approaches include particle bombardment using DNA-coatedgold beads (40); coadministration of DNA vaccines with plasmidDNA expressing cytokines (21, 47), chemokines (42), or costimulatorymolecules (7, 20, 29); formulation of DNA with cationic lipids(14, 41); or adding experimental adjuvants, such as monophosphoryllipid A (38).

Prothymosin ${alpha}$ is a highly acidic polypeptide, originally isolatedfrom the rat thymus (15), and is the putative precursor of thymosin ${alpha}$ 1, an immunomodulatory peptide derived from thymus (25). Prothymosin ${alpha}$ is reported to be more effective than thymosin ${alpha}$ 1 in protectionagainst Candida albicans infection in mice (16). Because ofits immunostimulating effects in cell-mediated immunity, prothymosin ${alpha}$ is a promising therapeutic agent for immunodeficient and cancerpatients. We found that the potency of DNA vaccines encodingHBsAg can be increased substantially with coadministration ofthe HBV DNA vaccine and a plasmid encoding prothymosin ${alpha}$ .

MATERIALS AND METHODS

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Materials and Methods

Animals. Female BALB/c and C57BL/6 mice (6 to 8 weeks old) were purchasedfrom the Animal Center of Academy of Military Medical Science,Beijing, People's Republic of China. All studies were approvedby the Institutional Animal Welfare Committee.

Plasmid expressing prothymosin ${alpha}$ . The gene encoding prothymosin ${alpha}$ was amplified from the plasmidby using the following primers: the forward primer sequencewas 5'-CGCGGATCCATGTCTGATGCAGCTGTAGATACC-3', and the reverseprimer sequence was 5'-CCGGAATTCGTCATCCTCGTCGGTCTTCTG-3' (thebold nucleotides indicate the restriction endonuclease recognitionsite). The primers contained a flanking sequence recognizedby restriction endonuclease enzymes BamHI and EcoRI, respectively,for the convenience of cloning manipulation. The PCR fragmentand plasmid vector pcDNA3/flag (containing a Flag tag; Invitrogen,CA) were cleaved by BamHI and EcoRI (New England BioLabs, MA).The cleaved products were ligated using T4 ligase. Escherichiacoli DH5 ${alpha}$ cells were transformed using the ligated products,and the correct recombinant plasmid was confirmed by endonucleasecleavage followed by DNA sequencing.

Construction of HBV DNA vaccines (PreS2S) and prothymosin ${alpha}$ -linker-PreS2S constructs. DNA fragments containing HBV PreS1, PreS2, and S genes wereamplified by PCR using the adw genome-containing plasmid (agenerous gift from Zongde Chen from Zhengzhou University) asa template with the following primers. The forward primers wereS1, 5'-GGAAGATCTCAGGCCATGCAGTGGAATT-3'(BglII), and S2, 5'-CCCAAGCTTCAGGCCATGCAGTGGAATT-3'(HindIII).The reverse primer was SR, 5'-AGGGGGCCCAGCCCATGAAGTTTAGGGAA-3'(ApaI).Bold nucleotides indicate the restriction endonuclease recognitionsite. The PCR fragments of S1 and SR or S2 and SR were clonedinto plasmid vector pcDNA3/flag by using BglII-ApaI (New EnglandBioLabs) or HindIII-ApaI (New England BioLabs, MA).

A vector expressing the fusion protein of HBV S antigen andprothymosin ${alpha}$ was constructed by the same approaches. A linker(5'-GAATTCTTCGGTGGCGGCTCCGGTGGGGGCA, encoding SGGGSGGG) wasinserted between the HBV S gene and the human prothymosin ${alpha}$ gene.A schematic representation is shown in Fig. 1.

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FIG. 1. Flag-HBV PreS2S, Flag-HBV PreS2S-prothymosin ${alpha}$ , and Flag-prothymosin ${alpha}$ were expressed in P815 cells. P815 cells were transfected with Flag-PreS2S, Flag-PreS2S-prothymosin ${alpha}$ , Flag-prothymosin ${alpha}$ , or pcDNA3/flag alone. Twenty-four to 48 h after incubation, the cells were lysed in LAC buffer, and the supernatant was separated by SDS-PAGE and analyzed by immunoblotting with an anti-Flag Western blot.

HBs protein antigen. The HBsAg used here was derived from recombinant yeast containingthe gene for the adw subtype of HBsAg as prepared in the Temervac-HB(Beijing Tiantan Biological Products Co. Ltd.). In certain experiments,the HBsAg was formulated with an aluminum hydroxide adjuvantat 10 µg HBsAg per 1 mg aluminum per ml.

HBsAg-specific antibody assay. Serum samples were collected by tail bleeding at different times,and the presence of HBsAg-specific antibody was analyzed byenzyme-linked immunosorbent assay according to the manufacturer'sprotocol (Sino-American Biological Co. Ltd., Luoyang, People'sRepublic of China).

DNA immunization in mice. BALB/c mice (10 mice per group) were immunized with the indicatedvaccine in 100 µl of sterile phosphate-buffered saline(PBS) via intramuscular (i.m.) injection into the left-thighquadriceps muscle at a 2-week interval.

Transient expression of the proteins. P815 (mastocytoma) cells were maintained in RPMI 1640 (GIBCO)with 10% fetal bovine serum in a six-well tissue culture plateat 37°C in a 5% CO₂ humidified atmosphere and then transfectedwith Flag-PreS2S, Flag-PreS2S-prothymosin ${alpha}$ , Flag-prothymosin ${alpha}$ , or pcDNA3/flag vector by using Lipofectamine 2000 (Invitrogen,CA). For each transfection, 10 µg of plasmid and 15 µlof Lipofectamine 2000 were mixed in 0.2 ml of OPTI-MEM I reducedserum medium and incubated for 30 min. The DNA-Lipofectaminecomplexes were overlaid onto the cells. After incubating for12 h, the medium was replaced with normal medium. After 24 to48 h of incubation, the cells were collected in LAC buffer (20mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 9 mM CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate},25% [vol/vol] glycerol) containing 0.5 mM dithiothreitol, 2.5mM EDTA, 5 mg/ml aprotinin, 5 mg/ml leupeptin, and 0.2 mM phenylmethylsulfonylfluoride and lysed by sonication. The supernatant was collectedand analyzed as follows: proteins were separated by 12% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)and transferred to polyvinylidene difluoride membranes (Millipore)followed by blocking for 2 h in PBS containing 5% (wt/vol) nonfatdry milk at room temperature. For detection of HBsAg or prothymosin ${alpha}$ -HBsAg, the membranes were incubated with horseradish peroxidase-conjugatedanti-Flag antibody (A8592; Sigma, MO) at a dilution of 1:5,000.The blots were visualized by enhanced luminol reagent and oxidizingreagent (Renaissance; NEN, MA).

Enzyme-linked immunospot (ELISPOT) assay for cytokine production. Spleen cells from mice 1 week following the booster immunizationwere subjected to an assay of secreted gamma interferon (IFN- ${gamma}$ )in vitro by restimulation with antigenic peptides. Briefly,96-well polyvinylidene difluoride-backed plates (U-CyTech, B.V.,The Netherlands) were coated with antibody to IFN- ${gamma}$ , washed threetimes with PBS, and then blocked with RPMI 1640 medium containing10% heat-inactivated fetal bovine serum (FBS). Cells were culturedat 5 x 10⁵ per well in 0.1 ml of medium for restimulation withdefined peptides corresponding to known HBsAg T-cell epitopeS28-29 (IPQSLDSWWTSL) (1, 4, 24). After 18 to 24 h of incubationat 37°C, the plates were washed 10 times with PBS containing0.005% Tween 20 and then incubated with detector antibodies(biotinylated anti-mouse IFN- ${gamma}$ ). The plates were washed 10 moretimes before the addition of GABA ( ${phi}$ -labeled antibiotin antibodies).After three washes with PBS-Tween and three washes with PBS,spots were developed with Activator I mixed with Activator II.Spots were counted using a stereomicroscope.

Cytotoxic T lymphocyte assay. BALB/c mice were immunized i.m. with 100 µg of PreS2Smixed with 100 µg of pcDNA3 or 100 µg of prothymosin ${alpha}$ and boosted at the same dosage by the same route 21 days afterthe first inoculation. Mice immunized with 100 µg of pcDNA3vector were employed as negative control. Spleens were harvestedfrom immunized mice 3 weeks after the final immunization. Single-splenocytesuspensions from each mouse were prepared separately in RPMI1640 (GibcoBRL) supplemented with 10% (vol/vol) FBS, 2 mM L-glutamine(GibcoBRL), antibiotics, and 5 x 10^–5 M 2-mercaptoethanol(Invitrogen, CA) and restimulated in vitro with specific peptides(HBsAg28-39, 10 µg/ml). Effector cells were pooled 6 daysafter restimulation and then used in a standard ⁵¹Cr releaseassay. Peptide-pulsed P815 cells (H-2d, mastocytoma cell line)were used as target cells against hepatitis B surface antigen-specificeffector cells. The P815 cells were preincubated with 50 µgof specific peptides for 60 min at room temperature and thenlabeled with ⁵¹Cr for another 60 min at 37°C and 5% CO₂.These cells were washed three times with RPMI 1640 medium containing2% (vol/vol) FBS and resuspended at a concentration of 5 x 10⁵/mlin RPMI 1640 medium containing 10% (vol/vol) FBS. Serial dilutionsof effector cells were incubated with ⁵¹Cr-labeled target cells(5 x 10³) for 4 h in a 200-ml volume in round-bottom 96-wellplates at the indicated effector/target cell ratio. After 4h of incubation, 100 ml of supernatant was removed and examinedfor ⁵¹Cr release in a gamma counter (Perkin-Elmer, MA). Thepercent specific ⁵¹Cr release was calculated with the followingequation: (experimental counts – spontaneous counts)/(totalcounts – spontaneous counts) x 100. Net percent specificlysis was defined as the difference between the percent specificchromium release of the peptide-unpulsed target and that ofthe target cell pulsed with specific CTL peptide. Spontaneousrelease of ⁵¹Cr from labeled cells was determined by incubatingtarget cells with medium alone and was less than 15% of thetotal release in all experiments. The total release of radioactivitywas obtained by lysing the target cells with 5% Triton X-100.Lysis at each effector/target cell ratio was determined in triplicateand repeated at least twice.

RESULTS

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Results

Transient expression of the antigen and prothymosin ${alpha}$ . After P815 cells were transfected with recombinant plasmids(PreS2S and PreS2S-prothymosin ${alpha}$ ), the supernatants of lysedcells were separated by SDS-PAGE and transferred to a nitrocellulosemembrane. The protein expressed was analyzed by immunoblottingwith anti-Flag. As shown in Fig. 1, PreS2S, prothymosin ${alpha}$ , andthe fusion protein of PreS2S-prothymosin ${alpha}$ were all expressedin P815 cells. As control, the P815 cells not transfected withrecombinant plasmids were negative for any HBsAg antigen andprothymosin ${alpha}$ .

Humoral response to HBsAg in mice injected with DNA vaccines. Anti-HBs antibody response was monitored in the sera of miceinjected with a mixture of 100 µg of pcDNA3 and 100 µgof PreS2S, a mixture of 100 µg of prothymosin ${alpha}$ and 100µg of PreS2S, or 100 µg of PreS2S-prothymosin ${alpha}$ inthe left-thigh quadriceps muscle (i.e., under conditions inducingthe strongest CTL response) (see Fig. 6). As shown in Fig. 2,after booster injection, inoculation with PreS2S-prothymosin ${alpha}$ generated the highest responses in both BALB/c and C57BL/7mice strains; a four- to fivefold increase in antibody titerswas observed when PreS2S was coadministered with prothymosin ${alpha}$ compared to PreS2S alone (P < 0.01). Similarly, mice inoculatedwith PreS2S-prothymosin ${alpha}$ generated greater responses than thoseinoculated with PreS2S alone (Fig. 2A and B). To investigatewhether the adjuvant effect of prothymosin is anti-HBs specificor causes a polyclonal B-cell activation, serum total immunoglobulinG (IgG) was assessed, and an increase (less than onefold) wasobserved. It indicates that the adjuvant effect of prothymosin ${alpha}$ is partially anti-HBs specific, and a polyclonal B-cell activationalso contributes to the effect (Fig. 2C). CD4 and CD8 T cellswere also analyzed by flow cytometry; a slight increase of CD4T cells and a decrease of CD8 T cells were observed, but thedifference is not statistically significant.

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FIG. 6. Prothymosin ${alpha}$ boosts cytotoxic T-cell response in mice immunized by Flag-PreS2S. Eight BALB/c mice in each group were immunized with the indicated plasmid in the left-thigh quadriceps muscle. Spleen cells were obtained from immunized mice 3 weeks after boost injection and assayed for specific cytolytic activity against P815 cells pulsed with the peptide S28-39. The results represent mean specific lysis values ± SE at the indicated effector-to-target ratio.

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FIG. 2. Anti-HBs responses to PreS2S DNA vaccines. (A) Eight BALB/c mice in each group were immunized. The mice were boosted once at the same dosage at day 14. Results are demonstrated as the endpoint geometric mean titer ± SE. ${blacktriangleup}$ , 100 µg Flag-PreS2S plus 100 µg Flag-prothymosin ${alpha}$ ; x, 100 µg Flag-Pre S2S-prothymosin ${alpha}$ ; ${square}$ , 100 µg Flag-PreS2S plus pcDNA3; ${blacklozenge}$ , 200 µg pcDNA3 vector. (B) Eight C57BL/6 mice in each group were immunized with 100 µg of Flag-PreS2S plasmid formulated with (hatched bar) or without (dotted bar) Flag-prothymosin ${alpha}$ or immunized with 100 µg of Flag-PreS2S-prothymosin ${alpha}$ plasmid (open bar). Antibodies were assayed as described in Materials and Methods. The star indicates a statistically significant higher vlaue (P < 0.01). (C) Total IgG in BALB/c mice was assayed.

Predominance of anti-HBs antibodies of the IgG2a isotype in response to the adjuvanted DNA vaccine. To determine whether the prothymosin ${alpha}$ adjuvant changes the isotypeprofile of the response to DNA immunization, responses to theHBs DNA vaccine PreS2S formulated with or without prothymosin ${alpha}$ were quantified. The result (Fig. 3) shows that the responseto the PreS2S vaccine formulated with prothymosin ${alpha}$ was three-to fivefold higher in the anti-HBs titers of both IgG1 (P <0.05) and IgG2a isotypes; furthermore, the ratio of IgG1 toIgG2a showed that IgG2a antibodies predominated. Thus, prothymosin ${alpha}$ can increase the overall anti-HBs response to the PreS2S DNAvaccine while maintaining a TH1 isotype profile.

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FIG. 3. Prothymosin ${alpha}$ boosts the elucidation of IgG2a and IgG1 isotype in mice. Eight BALB/c mice in each group were immunized at 0 and 14 days with the indicated plasmid(s). Both IgG2a (open bar) and IgG1 (hatched bar) isotype antibodies against HBV S antigen were assayed at day 36. Results are expressed as the geometric mean titer ± SE.

Prothymosin ${alpha}$ increases the anti-HBs seroconversion rate. As shown in Fig. 4, all BALB/c and C57BL/6 mice immunized withPreS2S-prothymosin ${alpha}$ generated anti-HBs antibodies 4 weeks afterbooster immunization; the anti-HBs seroconversion rate was 100%.Similarly, seven of eight mice BALB/c mice or C57BL/6 mice immunizedwith PreS2S-prothymosin ${alpha}$ generated anti-HBs antibodies at thesame time. In contrast, only three of the eight BALB/c mice(37.5%) and two of eight C57BL/6 mice immunized with PreS2Salone generated anti-HBs antibodies. These results indicatethat prothymosin ${alpha}$ significantly increases the anti-HBs seroconversionrate when coadministered with PreS2S DNA vaccine.

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FIG. 4. Anti-HBs seroconversion rates were increased by coadministration of prothymosin ${alpha}$ . Eight BALB/c mice (A) or eight C57BL/6 mice (B) in each group were immunized with 100 µg of Flag-PreS2S formulated with 100 µg Flag-prothymosin ${alpha}$ ( ${blacktriangleup}$ ) or pcDNA3 vector ( ${square}$ ), 100 µg Flag-PreS2S-prothymosin ${alpha}$ (x), or 200 µg pcDNA3 vector as control ( ${blacklozenge}$ ). The mice were boosted once on day 14. Anti-HBs antibodies were determined 4 weeks after the first immunization.

Prothymosin ${alpha}$ -expressing plasmid boosts cytokine-secreting T-cell responses to the HBs DNA vaccine. An ELISPOT assay was employed to determine the number of cellssecreting IFN- ${gamma}$ upon in vitro restimulation with antigenic peptides.As shown in Fig. 5, both BALB/c and C57BL/6 mice immunized withDNA encoding PreS2S and prothymosin ${alpha}$ generated $~$ 3-fold more IFN- ${gamma}$ ELISPOTs than did mice immunized with PreS2S alone (P < 0.01).Furthermore, the affinity of T cells appeared to be increased,because detectable responses could be elicited with 5- to 10-fold-lowerconcentrations of peptide in mice immunized with PreS2S-prothymosin ${alpha}$ than in mice immunized with PreS2S alone. Interestingly, theadjuvant also improved the readability of the ELISPOTs: boththe size and intensity of spots were increased for IFN- ${gamma}$ T-cellresponses to the HBV DNA vaccine. Interestingly, the level ofELISPOT background in mice vaccinated with coadministrationof prothymosin was higher (mean ± standard error [SE],10.45 spot-forming cells [SPF] ± 3.17 SPF) than thatwithout coadministration of prothymosin (mean ± SE, 7.65SPF ± 4.39 SPF), although it was not statistically significant(P > 0.05).

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FIG. 5. Prothymosin ${alpha}$ boosts HBs peptide-specific cytokine responses to Flag-PreS2S. Six BALB/c mice (top) or C57BL/6 mice (bottom) in each group were immunized with 100 µg PreS2S with 100 µg Flag-prothymosin ${alpha}$ ( ${blacktriangleup}$ ) or 100 µg pcDNA3 vector ( ${square}$ ) on days 0 and 21 or 200 µg pcDNA3 vector as a control ( ${blacklozenge}$ ). Spleen cells were harvested 8 days after boost immunization, and IFN- ${gamma}$ -producing cells were assayed by ELISPOT assay. The results are demonstrated as the mean ± SE of SPF per 10⁵ spleen cells in each mouse.

Prothymosin ${alpha}$ boosts the CTL activity induced by PreS2S. To assess whether the CTL-stimulating activity of a DNA vaccinecan be modulated by the simultaneous expression of prothymosin ${alpha}$ , BALB/c mice (six per group) were inoculated i.m. with PreS2S-prothymosin ${alpha}$ plasmid or PreS2S plasmid alone. The splenocytes from eachgroup of mice were analyzed for CTL activity 21 days after thebooster inoculation. As shown in Fig. 6, mice coimmunized withPreS2S and prothymosin ${alpha}$ exhibited greater cellular immune responsesto HBsAg than those immunized with PreS2S alone, as determinedby CTL activity of the splenocytes. Therefore, coexpressionof prothymosin ${alpha}$ increased the cellular immune responses inducedby the PreS2S DNA vaccine, which may result in a higher protectiveefficacy.

DISCUSSION

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Discussion

Despite recombinant HBV protein being used successfully as avaccine for a long time, chronic viral hepatitis is still asignificant health problem in China and other developing countries(8, 12, 49). People in these countries are at risk of developingchronic hepatitis leading to liver cirrhosis and hepatocellularcarcinoma. Up to now, vaccination is the main approach in theprevention of HBV infection (11, 13, 23, 36).

DNA immunization represents a novel means of expressing antigensin vivo for the generation of both humoral and cellular immuneresponses. Direct injection in either skeletal muscles or skinhas demonstrated the ability of either to take up and expressDNA-encoded sequences without any specific delivery system (39,46). Thus, i.m. injection of plasmid DNA has been widely usedfor DNA vaccination. However, the apparent potency of a DNAvaccine, as measured by antibody response, is often poorer thanthat of a corresponding protein-based vaccine.

To develop vaccines with clinical relevance, numerous ways havebeen evaluated to increase the potency of DNA vaccines, includingcoadministration with experimental adjuvants such as saponins,cytokine genes, CpG motifs, and prothymosin ${alpha}$ . In this study,we found that prothymosin ${alpha}$ significantly increases the potencyof the HBV DNA vaccine.

Prothymosin ${alpha}$ is a highly acidic and small protein of only 111amino acids with an unusual primary structure. Although no secretorypathway for prothymosin ${alpha}$ has been described, it has been detectedin human serum, representing 10% of the total prothymosin ${alpha}$ contentin blood (34). Rat prothymosin ${alpha}$ was demonstrated early on tobe protective against Candida albicans infection in mice (17)and to stimulate in vivo the release of migration inhibitoryfactor (MIF) (33) to a degree 10 to 20 times higher than itsderivative thymosin ${alpha}$ 1. In an experimental tumor model, animalspretreated with prothymosin ${alpha}$ prolonged their survival in 40to 60% of cases. Prothymosin ${alpha}$ induces tumoricidal peritonealmacrophages, natural killer (NK) cells, lymphokine-activatedkiller activity in splenocytes, production of interleukin-2and tumor necrosis factor alpha, and tumor-specific cytotoxic(CD8⁺) and helper (CD4⁺) T-cell activation when administeredsimultaneously with tumoral cells (2, 3, 35).

In this report, we show that mice vaccinated with the plasmidencoding the middle protein of HBsAg (PreS2S) elicited humoraland cellular immune responses. Coadministration of the plasmidencoding prothymosin ${alpha}$ boosts specific humoral and cellular immuneresponses elicited by PreS2S in mice. Coexpression of prothymosin ${alpha}$ and PreS2S as a fusion protein can also boost the specifichumoral and cellular immune responses elicited by PreS2S inmice. Specifically, prothymosin ${alpha}$ increases the seroconversionrate of anti-HBs antibodies in mice immunized with PreS2S-prothymosin ${alpha}$ or PreS2S-prothymosin ${alpha}$ gene fusions compared to the rate thatresults from PreS2S alone. Therefore, prothymosin ${alpha}$ may be apromising adjuvant for DNA vaccines.

ACKNOWLEDGMENTS

This investigation was supported by grant 2004AA215230 from"High-tech Research and Development Program of China (863 program)."

We acknowledge Zongde Chen (Zhengzhou University) for providingthe plasmid pcDNA3-HBV2.

None of us have a financial interest with this work.

FOOTNOTES

* Corresponding author. Mailing address: P.O. Box 130(8), 27 Taiping Rd., Beijing 100850, China. Phone: 86 10 6815 5151. Fax: 86 10 6385 3882. E-mail: caoc{at}nic.bmi.ac.cn.

REFERENCES

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