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The Journal of Immunology, 2005, 175: 2541-2547.
Copyright © 2005 by The American Association of Immunologists

Ag85B of Mycobacteria Elicits Effective CTL Responses through Activation of Robust Th1 Immunity as a Novel Adjuvant in DNA Vaccine1

Shiki Takamura*,{dagger}, Kazuhiro Matsuo{ddagger},¶, Yutaka Takebe§ and Yasuhiro Yasutomi2,{dagger}

* Japanese Foundation for AIDS Prevention, Tokyo, Japan; {dagger} Department of Bioregulation, Mie University School of Medicine, Mie, Japan; {ddagger} Vaccine Research and Development Group and § Laboratory of Molecular Virology and Epidemiology, AIDS Research Center, National Institute of Infectious Disease, Tokyo, Japan; and Japan Science and Technology, Saitama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD4+ T cells play a crucial role in CTL generation in a DNA vaccination strategy. Several studies have demonstrated the requirement of CD4+ T cells for the induction of a sufficient immune response by coadministrating DNAs. In the present study we investigated the effectiveness of Ag85B of mycobacteria, which is known to be one of the immunogenic proteins for Th1 development, as an adjuvant of a DNA vaccine. HIV gp120 DNA vaccine mixed with Ag85B DNA as an adjuvant induced HIV gp120-specific Th1 responses, as shown by delayed-type hypersensitivity, cytokine secretion, and increasing HIV-specific CTL responses. Moreover, these responses were enhanced in mice primed with Mycobacterium bovis bacillus Calmette-Guérin before immunization of HIV DNA vaccine mixed with Ag85B DNA. Furthermore, these immunized mice showed substantial reduction of HIV gp120-expressing recombinant vaccinia virus titers compared with the titers in other experimental mice after recombinant vaccinia virus challenge. Because most humans have been sensitized by spontaneous infection or by vaccination with mycobacteria, these findings indicate that Ag85B is a promising adjuvant for enhancing CTL responses in a DNA vaccination strategy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The use of adjuvant in vaccination is thought to be useful for enhancing the immune responses to various pathogens and tumors. One of the major advantages of plasmid DNA vaccination is the induction of MHC class I-restricted CTL responses through endogenous production of an Ag similar to viral infection (1). However, plasmid DNA immunization does not fully elicit cellular immune responses against infectious pathogens in some cases. Unlike viral infection, generation of CTLs after DNA vaccination appears to be critically dependent on functions of CD4+ T cells, such as secretion of Th1 cytokines, which facilitate CTL expansion and activity (2), and activation of professional APCs through CD40-CD40L interaction to increase the expression of costimulatory molecules (3, 4, 5). Accordingly, simultaneous activation of CD4+ T cells, especially Th1 cells, during priming is a promising strategy for the generation of substantial CTL responses when using a noninflammatory Ag expression system. In many cases, some helper epitopes are already present in a DNA vaccine, and epitope-specific CD4+ Th cell responses are induced after vaccination. However, because CD4+ T cell help for CTL generation does not require a pathogen including a CTL epitope, pathogen-specific CD4+ T cell responses are not necessary for eliciting pathogen-specific CTL immunity (6, 7). This prompted us to use an appropriate molecule as an adjuvant for the induction of an effective CTL response by the activation of CD4+ T cells.

Mycobacterium bovis bacillus Calmette-Guérin (BCG),3 a currently available vaccine to prevent tuberculosis, is thought to have powerful immunogenic adjuvant activity that augments cell-mediated immune responses by induction of several Th1 cytokines (8). It is also well known that CFA, which contains heat-killed mycobacteria, augments immune responses by activating Th cells. However, the specific proteins that elicit Th1 immunity of BCG are not clear. One immunogenic protein that can induce a strong Th1-type immune response in hosts sensitized by BCG is Ag85B (also known as {alpha} Ag or MPT59) (9). Ag85B belongs to the Ag85 family, which participates in cell wall mycolic acid synthesis (10). Moreover, Ag85B is one of most dominant protein Ags secreted from all mycobacterial species, shows extensive cross-reactivity between different species, and has been shown to induce substantial Th cell proliferation and vigorous Th1 cytokine production in humans and mice infected with mycobacterial species, including individuals vaccinated with BCG (9). We previously showed that inoculation of Ag85B-transfected tumor cells enhances the immunogenicity of tumor-associated Ags and elicits a strong tumor-specific CTL response (11). In the present study we evaluated the effectiveness of Ag85B from Mycobacterium kansassi as an adjuvant for enhancing cellular immune responses induced by DNA vaccine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

In mice, unlike humans, studies using inbred and congenic strains have demonstrated different fastness against BCG infection among each strain (12). To give resistance to BCG infection, BALB/c (BCG-susceptible strain) x C3H/HeN (BCG-resistant strain; CC3HF1, H-2d/k) female mice were used in this study. The mice were housed at the Laboratory Animal Center of Mie University School of Medicine.

Plasmid

A highly efficient mammalian expression vector, pJW4303, was used for efficient expression of HIV env gp120 of the NL432 strain (pJWNL432) (13). The Ag85B expression vector pcDNA-Ag85B has been constructed by cloning a PCR product that possesses an Ag85B of M. kansasii open reading frame lacking a signal sequence into KpnI-ApaI sites of pcDNA 3.1 (11).

Peptide synthesis

The peptides used in this study were an HIV-1 env helper epitope (315–329; RIQRGPGRAFVTIGK; p18) and CTL epitope (318–327; RGPGRAFVTI; p18-I10) in association with the class II MHC molecule I-Ad and the class I MHC molecule H-2Dd, respectively (14).

Priming to BCG

Six- to 8-wk-old female mice were primed to BCG by i.p. inoculation of 0.01 mg (dry weight) of BCG (Japan BCG Laboratory).

Immunization

Four weeks after BCG priming, groups of mice were i.m. injected four times with 100 µg of pJWNL432 mixed with or without 100 µg of pcDNA-Ag85B, and then the site of inoculation was immediately given an electric pulse by an Electric Square Porator (T820; BTX) to express both Ags of Ag85B and HIV gp120 in the same tissue, as previously described (15). Pulses were delivered to the muscle using a pair of electrode needles. Eight electric pulses of 50 V were delivered at a rate of one pulse per second. Each electric pulse was 99 ms in duration. Resistance was monitored with a graphic pulse analyzer (Optimizer 500; BTX). To test the dose dependency of Ag85B as an adjuvant, mice primed with BCG were coadministered various doses of pcDNA-Ag85B. Insufficiency of the amount of DNA by reduction of pcDNA-Ag85B was compensated for by mock DNA pcDNA3.1, the original expression vector of pcDNA-Ag85B, to equalize the total volume of administered DNA.

Immunohistochemical analysis

Immunized leg muscles were examined immunohistochemically for in vivo expression of HIV gp120 and Ag85B. Three days after injection, the tibialis anterior muscle was removed, fixed with 4% paraformaldehyde in PBS, and embedded in paraffin wax. Serial sections were prepared and deparaffinized and then incubated with proteinase K for 5 min at room temperature (gp120) or heated by microwave oven three times for 5 min each time (Ag85B) to reactivate the Ag. After incubation with 3% H2O2/methanol for 30 min to quench endogenous peroxidase activity, the sections were blocked with normal serum and incubated with anti-HIV gp120 Ab (OEM Concepts) diluted 1/100 or rabbit anti-Ag85B antiserum (16) diluted 1/250 for 30 min at room temperature. Subsequently, the sections were additionally incubated with a biotinylated secondary Ab and HRP-labeled avidin-biotin complex (ABC-peroxidase staining kit Elite; Vector Laboratories). They were then reacted with 0.5% 3.3'-diaminobenzidine tetrachloride and 0.01% H2O2 to visualize the bound Abs. Sections incubated with an irrelevant Ab instead of the primary Ab were used as negative controls. Sections were slightly counterstained with hematoxylin.

Delayed-type hypersensitivity (DTH) responses

DTH responses to HIV were elicited by injecting 5 µg of p18 peptide into the footpad of each immunized mouse. The degree of footpad swelling 24 h after the injection was measured using a micrometer and was expressed as the mean increment ± SE of three mice per group (11).

Determination of cytokine production

Spleen cells from immunized mice (5 x 106) were cultured with 2.5 x 106 mitomycin C (MMC)-treated autologous spleen cells labeled with p18 peptide in 24-well culture plates at a volume of 2 ml. After incubation at 37°C in a humidified incubator (5% CO2) for 48 h, culture supernatants were collected and analyzed for IFN-{gamma} (BioSource International) or IL-4 (Quantikine; R&D Systems) production by an ELISA according to the manufacturer’s protocol.

RT-PCR

Total RNA was isolated from leg muscles of the site of immunization using TRIzol (Invitrogen Life Technologies), then reverse transcribed to cDNAs using a SuperScript system (Invitrogen Life Technologies). The resulting cDNA was amplified using TLR sequence-specific primers for 30 cycles of PCR (94°C for 1 min, 58°C for 1 min, and 72°C for 1 min). The following primers corresponding to each TLR were used: 5'-ATGGCAGAAGATGTGTCCG-3' and 5'-GTCACCATGGCCAATGTAGG-3' for TLR2, 5'-TGGATTCTTCTGGTGTCTTCC-3' and 5'-AGTTCTTCACTTCGCAACGC-3' for TLR3, 5'-CTGGCATCATCTTCATTGTCC-3' and 5'-GCTTAGCAGCCATGTGTTCC-3' for TLR4, 5'-CAGAACCTTCCTGGCTATTGC-3' and 5'-AGAGGTTGACCAGACCTTGG-3' for TLR9, and 5'-AGAAGAGCTATGAGCTGCCTGACG-3' and 5'-CTTCTGCATCCTGTCAGCAATGCC-3' for {beta}-actin.

Generation of CTL effector cells

Effector cells were derived from spleen cells as precursor CTLs. CD8+ T cells were purified with anti-CD8 magnetic beads (Miltenyi Biotec) by positive selection according to the recommended protocol (MACS system). Aliquots of 1 x 106 CD8+ T cells were cocultured with 5 x 106 MMC-treated autologous spleen cells labeled with p18-I10 peptide at 37°C in a 5% CO2 atmosphere. Two days after stimulation, human rIL-2 (Shionogi) was added to all wells at a final concentration of 5 ng/ml. The effector cells generated were harvested after 5 days of culture.

Cytotoxicity assay

MHC-matched (A20.2j) and unmatched (FBL-3) target cells (2 x 106) were incubated at 37°C in a 5% CO2 atmosphere with or without 10 µg/ml p18-I10 peptide for 16 h. Then the target cells were washed and labeled with 51Cr. The 51Cr-labeled target cells were incubated for 5 h with effector cells. Spontaneous release varied from 5 to 10%. Percent lysis was calculated as [(experimental release – spontaneous release)/(100% release – spontaneous release)] x 100. All experiments were performed more than three times, and each group consisted of three mice.

Blocking of cytolysis

Blocking of cytolysis was performed by a method previously described (13). 51Cr-labeled target cells were preincubated at 37°C for 20 min with anti-H-2 Kd, Dd, or Ld mAb (Meiji Institute of Health Science), and effector cells were then added. In a separate experiment, effector cells were preincubated with anti-CD4 mAb (GK1.5) or anti-CD8 mAb (Lyt2.2) at a 1/50 dilution with complement (Sigma-Aldrich) for 20 min at 37°C, and then labeled target cells were added. Blocking of cytolytic activities by these mAbs was assessed by a 5-h 51Cr release assay.

Evaluation of HIV gp120-specific CD8+ T cells by ELISPOT assay

The number of gp120-specific, IFN-{gamma}-secreting cells was determined by ELISPOT assay. Briefly, 96-well nitrocellulose plates (Millipore) were each coated with 8 µg/ml anti-mouse IFN-{gamma} mAb R4-6A2 (BD Pharmingen) in 100 µl of PBS. After overnight incubation at 4°C, the wells were washed three times with PBS. Then 100 µl of complete medium supplemented with 10% FCS was added to each well, and the plates were incubated at 37°C for 1 h. Triplicate samples of CD8+ T cells separated from the spleen were plated in 2-fold dilutions from 5 x 105 to 6.25 x 104 cells/well. The p18-I10-labeled MMC-treated P815 cells were used as APCs. APCs (1 x 105) were added to each well, and the plates were incubated for 24 h in a 37°C incubator with a 5% CO2 atmosphere. After stimulation, plates were washed intensively with PBS containing 0.05% Tween 20 and incubated overnight at 4°C with a solution of 2 µg/ml biotinylated anti-mouse IFN-{gamma} mAb XMG1.2 (BD Pharmingen). Afterward, plates were washed with PBS containing 0.05% Tween 20 and 100 µl of streptavidin-alkaline phosphatase (Mabtech) at a 1/1000 dilution was added to each well. Spots were visualized using alkaline phosphatase color development buffer (Bio-Rad) and counted using KS ELISPOT (Zeiss).

Study of protection from vaccinia virus expressing HIV env gp120

The protective ability in immunized mice against systemic infection of recombinant vaccinia virus (rVV) was analyzed by real-time detection PCR as described previously (16). Twelve weeks after the first immunization, mice were challenged i.p. with 5 x 107 PFU of rVV carrying the HIV IIIB gp120 gene (rVV-HIV gp120). Five days after the challenge, the ovaries were harvested and homogenized, and DNA was isolated using a Genomic DNA Isolation kit (Promega). Primers (forward, 5'-GTTCCTTCGCCAACAGGTTAA-3'; reverse, 5'-ACTCGCGATCCTCAAAATGC-3') and a TaqMan probe (5'-FAM-TTGGAAGCGCCACGGTTACATTCACT-3') were selected from the core 4b gene of vaccinia virus. Amplification and detection were performed using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). After incubation at 50°C for 2 min, amplification was begun at 95°C for 10 min, followed by 45 two-step cycles of 95°C for 15 s and 60°C for 60 s.

Statistical analysis

Statistical analysis was performed using Mann-Whitney’s U test and the Kruskal-Wallis test. Values are expressed as the mean ± SD. A 95% confidence limit was taken as significant (p < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In vivo expression of HIV gp120 and Ag85B

It has been reported that CD4+ T cells play a critical role in the generation of CTLs at the site of Ag presentation (17). To induce the effect of Ag85B as an adjuvant for augmentation of cellular immune responses, two DNA vaccines, encoding HIV gp120 and Ag85B, were mixed and administered simultaneously using in vivo electroporation. Three days after immunization, transgene expression was assessed by immunohistochemical analysis in serial sections. Except for slight transient inflammation, no pathological changes were detected in muscles after DNA injection and in vivo electroporation (data not shown) (15). Both HIV gp120 (Fig. 1A) and Ag85B (Fig. 1B) were observed inside bundles of muscle cells and connective tissue among the muscle fascicles in the same area. Transgene expressions were only seen in the area between the electrode needles.



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  		FIGURE 1. Immunostaining of serial sections of muscle tissue from a mouse 3 days after electric administration of pJWNL432 mixed with pcDNA-Ag85B. HIV gp120 (A) and Ag85B (B) were observed in muscle cells and connective tissue among the muscle fascicles in the same area. Bars represent 100 µm.

 
Coadministration of Ag85B DNA induces strong HIV gp120-specific Th1-type immune responses

As in cases of tuberculosis, one of the important markers of Th1-mediated acquired immunity (not synonymous with protection) is the DTH response. To confirm the ability of Ag85B to induce Th1 responses against coadministered Ag, immunized mice were injected with HIV env helper epitope p18 into footpads, and HIV gp120-specific DTH responses were assessed. As shown in Fig. 2, mice coadministered pcDNA-Ag85B showed greater footpad swelling than mice not administered pcDNA-Ag85B. The effectiveness of Ag85B for inducing Th1-type immune responses to vaccine Ag was augmented by BCG sensitization. In contrast, no significant responses were observed in nonimmunized mice and immunized mice injected with a control peptide (data not shown).



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  		FIGURE 2. Anti-HIV gp120 DTH responses in immunized mice. BCG-primed or unprimed mice were immunized with pJWNL432 with or without pcDNA-Ag85B. The helper epitope peptide of HIV gp120 (p18) was injected into the footpads of immunized mice. The degree of footpad swelling was measured 24 h after the challenge. The results are expressed as the mean footpad increment ± SE of five mice per group. *, p < 0.01; **, p < 0.001.

 
Next, to determine the effect of Ag85B on the patterns of Th1/Th2 cytokine production, we quantified the production of IFN-{gamma} and IL-4 by ELISA in supernatants obtained from 48-h cocultures of peptide-pulsed syngeneic APCs with spleen cells of immunized mice. The production of IL-4 from spleen cells stimulated by p18 in mice immunized with both pcDNA-Ag85B and pJWNL432 was much less than that in mice immunized with pJWNL432 alone. Relatively high levels of HIV gp120-specific IFN-{gamma} production were observed in mice coadministered pcDNA-Ag85B. Furthermore, these Th1-type immune responses were clearly observed when mice were sensitized by BCG inoculation before DNA immunization (Fig. 3). These results are in accordance with the results for DTH responses against HIV gp120 in in vivo experiments and indicate that predominant HIV gp120-specific Th1 responses were induced by coadministration of pcDNA-Ag85B.



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  		FIGURE 3. Induction of HIV gp120-specific Th1 immune responses by spleen cells obtained from immunized mice. Spleen cells obtained from BCG-primed or unprimed mice immunized with pJWNL432 with or without pcDNA-Ag85B were stimulated with p18-labeled spleen cells, and supernatants were assessed for cytokine concentrations. The results are expressed as the mean concentration ± SE of five mice per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
Alteration of TLR mRNA expression after Ag85B DNA administration

Various proteins derived from pathogens promote Th1 responses through stimulation of TLRs and subsequently through secretion of cytokines (18). We therefore compared TLR mRNA expression profiles at the site of DNA injection with electroporation in pcDNA-Ag85B-immunized mice and mice immunized with pcDNA alone using RT-PCR. Ag85B stimulated the expression of TLR2, TLR3, and TLR4 mRNA, whereas mock immunization with electroporation resulted in only modest increases in the levels of these TLR mRNAs (Fig. 4). TLR9 mRNA was not detected in either group of mice (Fig. 4). Plasmid pcDNA3.1 has immunostimulatory sequence (six 5'-GACGTC-3'), whereas TLR9 mRNA was not detected in either group of mice under these conditions (Fig. 4). Positive reactions, however, were observed in both Ag85B-injected tissues and control tissues using high cycles (>60) of PCR (data not shown). This positive reaction was not thought to be the effect of Ag85B. These results suggested that Ag85B immunization plays a role in enhancement of the expression of these TLRs, although the possibility of indirect responses by cytokine production cannot be ruled out (18).



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  		FIGURE 4. TLR mRNA expression profiles of the DNA injection site with electroporation in mice immunized with pcDNA-Ag85B or pcDNA3.1 alone. Total RNA was isolated 3 and 7 days after injection and was analyzed by RT-PCR for TLR2, TLR3, TLR4, and TLR9 mRNA expression. Equality of the RT reaction of isolated RNA between samples was confirmed by amplification of {beta}-actin. Data are representative of three independent experiments.

 
Ag85B enhances anti-HIV gp120-specific CTL responses

CD8+ cells from BCG-primed mice and unprimed mice immunized with pJWNL432 with or without pcDNA-Ag85B were stimulated in vitro with p18-I10-pulsed syngenic splenocytes, and the lytic activities of the cells against p18-I10-pulsed target cells were assessed. Effector cells from mice immunized with pJWNL432 and pcDNA-Ag85B showed higher levels of p18-I10-specific lytic activity than effector cells from BCG-unprimed mice. Moreover, these cytolytic activities were clearly enhanced by BCG sensitization in mice immunized with pJWNL432 mixed with pcDNA-Ag85B (Fig. 5A). These effector cells from immunized mice were cultured in a medium containing anti-CD4 or anti-CD8 mAb during the 51Cr release assay. Anti-CD8 mAb inhibited cytolysis against target cells pulsed with the peptide, whereas anti-CD4 mAb did not affect this effector cell function (Fig. 5B). Therefore, these results indicate that effector cells expressed CD8 and used this molecule to recognize the target cells. Furthermore, lytic activities of peptide-specific effector cells from immunized mice against MHC-matched or mismatched target cells labeled with the peptide were assessed. These p18-I10-specific effector cells lysed MHC-matched, H-2d target cells, but not mismatched, H-2b target cells pulsed with the peptide (Fig. 5C). Moreover, the functions of these p18-I10-specific effector cells were inhibited by anti-H-2Dd mAb, but not by anti-H-2Kd mAb or anti-H-2Ld mAb (Fig. 5D). These results indicated that effector cells elicited in immunized mice were CD8+ and MHC class I-restricted CTLs and suggested that Ag85B has potent adjuvant activities for enhancement of CTL responses by being mixed with DNA vaccine Ag.



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  		FIGURE 5. Spleen cells from BCG-primed mice coadministered pJWNL432 and pcDNA-Ag85B showed high levels of HIV gp120-specific MHC class I-restricted lytic activity. A, CD8+ T cells were purified from spleens of BCG-primed or unprimed mice immunized with pJWNL432 with or without pcDNA-Ag85B, and the cells were then stimulated with p18-I10-labeled spleen cells and assessed for lytic activities against p18-I10-pulsed target cells. B, Lytic activities of effector cells were assessed in the presence of anti-CD4 mAb, anti-CD8 mAb with complement, or medium. The E:T cell ratio was 40:1. C, Lytic activities of effector cells against p18-I10-pulsed H-2d or H-2b target cells were assessed. The E:T cell ratio was 40:1. D, Effector cells were examined for p18-I10-specific lytic activities in the presence of anti-H-2Kd, anti-H-2Dd, or anti-H-2Ld mAb. The E:T cell ratio was 40:1. Each value is the mean percentage of the specific lysis values obtained from five mice. *, p < 0.01.

 
Ag85B increase the number of HIV gp120-specific, IFN-{gamma}-secreting, CD8+ T cells

ELISPOT assays were performed to compare the numbers of HIV gp120-specific, IFN-{gamma}-secreting, CD8+ cells in immunized mice. CD8+ T cells purified from spleens of immunized mice were stimulated with peptide-pulsed P815 cells in ELISPOT filter plates coated with an IFN-{gamma} capture mAb for 24 h. The numbers of spots were counted automatically using a KS ELISPOT system. In BCG-unprimed mice, the number of p18-I10-specific IFN-{gamma}-secreting CD8+ T cells was slightly increased in mice coadministered pcDNA-Ag85B (20.3 ± 10.0/106 cells) compared with that in mice immunized with pJWNL432 alone (14.0 ± 3.6/106 cells). In BCG-primed mice, however, the number of p18-I10-specific IFN-{gamma}-secreting CD8+ T cells was ~3.7-fold greater in mice coadministered pcDNA-Ag85B (96.7 ± 13.3/106 cells) than in mice immunized pJWNL432 alone (26.3 ± 5.1/106 cells; Fig. 6A). To confirm whether the improved CTL responses strictly depend on the presence of Ag85B, BCG-primed mice were coadministered various doses of pcDNA-Ag85B, and the frequency of anti-p18-I10-specific IFN-{gamma}-secreting CD8+ T cells was determined by ELISPOT assay. The number of anti-p18-I10-specific, IFN-{gamma}-secreting, CD8+ T cells was gradually increased by coadministration of Ag85B in a dose-dependent manner (Fig. 6B). In addition, dose dependency in improving the anti-p18-I10-specific response was not found in mice coadministered a control plasmid, which expresses an unrelated protein constructed by the same expression vector (data not shown). These results clearly indicate that the anamnestic response to Ag85B could enhance the simultaneously induced CTL responses. These data also support the results for CTL responses and suggest that coadministration of pcDNA-Ag85B, especially in BCG-primed mice, induces high frequency, Ag-specific, responding CD8+ T cells.



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  		FIGURE 6. pcDNA-Ag85B coadministration in BCG-primed mice enhances HIV gp120-specific, IFN-{gamma}-secreting cell frequency. CD8+ T cells were purified from spleens of BCG-primed or unprimed mice immunized with pJWNL432 with or without pcDNA-Ag85B (A) or with various doses of pcDNA-Ag85B (B) and were examined for IFN-{gamma} production in an ELISPOT assay after stimulation with p18-I10-labeled P815 cells. Data are presented as the mean number of p18-I10-specific spots per 106 CD8+ spleen cells ± SE of five mice per group. *, p < 0.02; **, p < 0.01; ***, p < 0.001.

 
Ag85B enhances protective immunity against rVV-HIVenv infection

Finally, to determine the functional relevance of HIV gp120-specific CTLs generated by immunization with these DNA vaccines to eliminate the virus infection, immunized mice were challenged with 5 x 106 PFU of rVV-HIV gp120. Five days after the challenge, the mice were killed, and the ovaries were harvested and used for estimation of the vaccinia virus titer by RT-PCR, because the ovary is the organ in which the vaccinia virus preferentially replicates. The titers of rVV-HIV gp120 in mice coadministered pcDNA-Ag85B were much lower than those in mice immunized with pJWNL432 alone. Moreover, this inhibitory effect on replication of rVV gp120 was clearly demonstrated in mice primed with BCG before immunization (Fig. 7). These results indicated that immunization of mice with pJWNL432 mixed with pcDNA-Ag85B resulted in the generation of an effector T cell response capable of recognizing endogenously processed viral protein, and that DNA immunization inhibited the replication of rVV-expressing HIV gp120 in vivo.



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  		FIGURE 7. pcDNA-Ag85B coadministration in BCG-primed mice enhances HIV gp120-specific protective immunity. BCG-primed or unprimed mice immunized with pJWNL432 with or without pcDNA-Ag85B were challenged with 5 x 107 PFU of rVV-HIV-gp120. The bars show the virus as the log of the number of virus copies in ovaries of mice. The data represent the mean copies of virus obtained from five mice. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
DNA vaccination is a practical and effective way to induce cellular immune responses, especially a CTL response, and has shown great promise for initiating cellular immune responses that are regulated for the prevention of various disease such as tumors, HIV, tuberculosis, hepatitis C virus, and malaria. In humans and large animal models, however, immune responses induced by DNA vaccination are not sufficient for prevention or control of infection. Thus, there is a need to increase the potency of DNA vaccines for use in humans. In the present study we investigated the effectiveness of a novel approach for enhancing the ability of a DNA vaccine to induce cellular immune responses by using previously experienced immunogenic proteins that induce a strong Th1-dominant immune response.

CD4+ T cells play a critical role in the generation and maintenance of CTL responses in a noninflammable vaccination strategy including conventional plasmid DNA vaccination. Convincing evidence that dendritic cells (DCs) are the principal cells for priming CD8+ T cells in DNA vaccination through direct transfection of DNA (19, 20, 21) or cross-presentation of a vaccine-derived Ag has been presented (22, 23, 24). Activation of CD4+ T cells is required for maturation of DCs through CD40 signaling (3, 4, 5); however, this requirement is not sufficient in many cases of DNA vaccine immunization (25, 26). Various studies on compensation for the insufficiency of CD4+ T cell help by coadministration of costimulatory molecules, such as B7-1 or B7-2 (27, 28), or by treatment with a CD40 agonist during immunization have therefore been conducted (26). Another CD4+ T cell-mediated helper effect for induction of CTL by DNA vaccine is thought to be the production of cytokines for enhancement of activity and proliferation of CTLs (29). Cognate CD4+ T cell help is important for inducing pathogen-specific CTLs (30), and cognate CD4+ T cell help should be induced to elicit HIVgp120-specific CTLs by injection of DNA vaccine. The Ag85B in our system enhances this cognate manner and polarizes Th1-type immune responses as a vaccine adjuvant. Numerous studies have focused on the effectiveness of coadministration of Th1 cytokine-encoding plasmids, such as IL-2, IL-12, IL-15, IL-18, IL-23, and IFN-{gamma}, for enhancing CTL responses (31, 32). However, adjuvant effects of cytokines on CTL generation are different (33, 34), suggesting that it is necessary to augment the immune responses by these strategies for administration not only of the combination of cytokines codelivered, but also for the timing of administration (35, 36). The expression of both Ag85B and HIV Ag in the same tissues provides this advantage, because Ag85B is thought to be a strong potentiator of Th1-type cytokines. In fact, our results showed production of IFN-{gamma} from spleen cells after stimulation with HIV Ag (Fig. 3).

BCG is an important clinical tool because of its strong immunostimulatory properties. Humans as well as resistant mouse strains infected with BCG predominantly exhibit a Th1 cytokine secretion profile (37). Although specific Ags eliciting Th1 cell responses in mycobacteria are not yet known, a recent study suggested that one of the immunogenic proteins for Th1 development is Ag85B (9). Apparently, strong Th1 responses have been elicited in vitro from purified protein derivative-positive asymptomatic individuals using purified Ag85B (38, 39, 40). Furthermore, vaccination of mice with plasmid DNA encoding Ag85B induced strong Ag85B-specific CD4 T cell proliferation and vigorous IFN-{gamma} secretion, resulting in the protection of further Mycobacterium tuberculosis infection (41). We have also shown that Ag85B-specific recall responses enhance tumor-specific cellular immune responses in Ag85B gene-transfected tumor cell immunization (11). One possible reason for Th1 domination by Ag85B is that the immunogenic Th1-inducing helper epitope, known as peptide-25, is included in Ag85B protein (42, 43). Peptide-25 was able to stimulate proliferation and a high amount of IFN-{gamma} production in M. tuberculosis-primed cells (42). It remains unclear why peptide-25 can induce potent Th1 responses; however, several recent studies have suggested that the avidity of the peptide for its specific TCR may be strong enough to induce Th1 development (9, 44). It is now generally accepted that MHC class II-dependent activation of CD4+ T cells, mainly Th1-polarized cells, potently enhances concomitantly existing unrelated CTL responses (7, 44). According to this line of reasoning, coadministration of Ag85B DNA is a promising tool for enhancement of CTL responses through Ag85B-specific Th cell proliferation and Th1 polarization in a DNA vaccination strategy.

The roles of some proteins and peptides in the polarized development of Th1 cells have been reported, and Ag85B is considered to be one such protein. In fact, we found therapeutic effects of Ag85B on Th2-type allergic disease, asthma, and atopic dermatitis (unpublished observations). The mechanisms, however, are still not clear. Various products with adjuvant activities, such as LPS, CpG motif, or polyinosinic-polycytidylic acid, involve TLRs and show augmentation of Th1-type immune responses (18). Bacterial components, mycobacterial lipoprotein, bacterial peptidoglycan, and flagellin, also associate with TLRs (18). A correlation between the adjuvant activities of Ag85B and TLRs has not been found. Mycobacteria can bind some TLRs and show typical Th1-type immune responses (45). In a transfection model using Chinese hamster ovary cells (which are relatively deficient in TLRs), the expression of TLR2 or TLR4 conferred responsiveness to both virulent and attenuated M. tuberculosis (46). Lipoarabinomannan, a major mycobacterial cell wall component, appears to resemble the cell wall component of Gram-negative bacterial LPS. TLR2 was shown to be necessary for signaling of mycobacterial LPS lipoarabinomannan (47). An undefined, heat-labile, cell-associated, mycobacterial factor was found to be the ligand for TLR4 (47). Ag85B might be included in one of these factors, if it is involved in innate immunity through TLRs. In fact, our results showed enhancement of the expression of TLR2, TLR3, and TLR4 in Ag85B DNA-injected mice (Fig. 4). Because it has been reported that not only microbial components, but also several cytokines regulate the expression of TLRs, there is the possibility of secondary responses for the expression of TLRs by induction of cytokine (18).

Another important biological role of Ag85B is binding of fibronectins (FNs) (48, 49, 50). FNs are a family of high molecular weight glycoproteins found in plasma and tissues and are involved in cell motility and adhesion, regulation of cell morphology, phagocytic function, and wound healing (51). Many integrin-binding sites have been identified in amino acid sequences of FNs (52), and adhesion of FN-binding proteins to FNs helps the phagocytosis of proteins into integrin-expressing APCs, especially monocytes, macrophages, and DCs (53). Binding of FNs to human monocytes enhances the phagocytic function of monocytes for bacilli (51), and inhibition of FN-integrin receptor interaction can prevent M. kansasii phagocytosis (54). Moreover, Ag85B from M. tuberculosis and FNs synergistically stimulate TNF-{alpha} expression in human monocytes (55), suggesting that the binding ability of Ag85B with FNs influences not only the enhancement of incorporation of Ags into phagocytic cells, but also the construction of the Th1 milieu at the site of injection.

The results of the present study suggest that coadministration of Ag85B DNA has several potential advantages over other genetic adjuvants due to the existence of multiple mechanisms for elicitation of CTL responses by a DNA vaccine. The results also showed the effectiveness of mycobacterial sensitization for enhancing adjuvanticity of Ag85B. Because most humans have been sensitized by spontaneous infection or by vaccination with mycobacteria, this finding is valuable for the possible use of Ag85B as a genetic adjuvant of a DNA vaccine. The results of this study have provided evidence of the potential utility of Ag85B for the development of a DNA vaccination strategy for successful human use.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Health Science Research Grants from the Ministry of Health, Labor, and Welfare of Japan and the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Back

2 Address correspondence and reprint requests to Dr. Yasuhiro Yasutomi, Department of Bioregulation, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. E-mail address: yasutomi{at}doc.medic.mie-u.ac.jp Back

3 Abbreviations used in this paper: BCG, Mycobacterium bovis bacillus Calmette-Guérin; DC, dendritic cell; DTH, delayed-type hypersensitivity; FN, fibronectin; MMC, mitomycin C; rVV, recombinant vaccinia virus. Back

Received for publication February 25, 2005. Accepted for publication May 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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