Mycobacterial Codon Optimization Enhances Antigen
http://www.100md.com
病菌学杂志 2005年第14期
AIDS Research Center, National Institute of Infectious Diseases, Shinjuku, Tokyo 162-8640, Japan
Department of Pathobiology, School of Veterinary Medicine, Nihon University, Fujisawa, Kanagawa 252-8510, Japan
Department of Host Defense, Graduate School of Medicine, Osaka City University, Osaka, Osaka 545-8585, Japan
Department of Bacteriology, School of Dentistry, Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
ABSTRACT
Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.
INTRODUCTION
The Mycobacterium bovis bacille Calmette-Guérin (BCG) has been widely used as a live bacterial vaccine against Mycobacterium tuberculosis infection. Its recombinant form, rBCG, which has been used successfully to express foreign antigens and to induce immune responses, has been proposed as a vaccine candidate against a number of diseases (26, 32, 33), especially human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) (11, 13, 30). Moreover, mucosal immunization of rBCG has been found to elicit a long-term virus-specific immunity in animals (10, 14, 15), even in Th1- and Th2-deficient conditions (10). In short, an rBCG-based vaccine offers several clear advantages over other types of recombinant vector-based approaches in that it (i) induces cellular immune responses that are maintained for at least 1 to 2 years; (ii) is easy to administer, usually requiring only one or two immunizations; (iii) and is affordable because it can be easily and cheaply produced. These findings suggest that rBCG could be a potent vaccine against HIV-1 infection, one that is likewise capable of inducing safe, virus-specific immunity.
However, the results described above were obtained with high doses of rBCG, doses 10- to 100-fold larger than that needed for a practical BCG vaccination dose against tuberculosis in humans (7, 11). Therefore, the low immunogenicity seen in rBCG-inoculated animals is likely due to their inoculation with only a "normal," not a high, vaccination dose (15). Moreover, high doses of BCG administration in vivo may also act as the driving force for the replication of the immunodeficiency virus and its dissemination by hyperactivating T cells (6, 41).
We sought here to produce an rBCG vaccine that would be efficacious even in the low doses required for human vaccination. Because low-dose immunization of rBCG has been suggested to act as a prophylactic vaccination against HIV-1 (15, 28), we adopted the preferred codon of BCG to enhance the expression of the foreign HIV gene. In recombinant protein production, the potency of codon-optimized gene expression systems was demonstrated in Escherichia coli (39) and in mammalian cells (42). These results clearly show that codon-optimized recombinant genes induce vigorous expression by foreign genes in the host. Since 1998, many groups have reported that a sequence-modified DNA vaccine confers high immunogenicity against various foreign antigens, e.g., listeriolysin O of Listeria monocytogenes (37), HIV-1 Gag (43), Env (3), tetanus toxin (34), L1 protein of human papillomavirus (18), and merozoite surface protein 1 of Plasmodium falciparum (25). Most of these studies focused on demonstrating how mammalian codon usage bias efficiently enhanced the expression and immunogenicity of foreign antigens in DNA vaccination. However, although the effect of codon optimization in mammalian cells has been well documented, its effect in recombinant BCG vector-based vaccines has never been fully elucidated.
MATERIALS AND METHODS
Animals. Female BALB/c (H-2d) mice aged 6 to 8 weeks were purchased from Charles River Japan, Inc. Mice were maintained in the experimental animal facility under pathogen-free conditions and in a manner consistent with the institutional animal care and use guidelines of the National Institute of Infectious Diseases of Japan. The study was conducted in a biosafety level 2 facility with the approval of an institutional committee for biosafety and in accordance with the requirements of the World Health Organization.
Construction of an HIV antigen expression vector and transformation of BCG. We used E. coli HB101-competent cells (Takara Bio, Inc.) for gene manipulation and the BCGTokyo172 as a mycobacterial strain which does not accelerate disease progression in HIV-infected children (9). Middlebrook 7H9 broth containing albumin-dextrose complex (7H9-ADC; BBL Microbiology Systems) was used as the culture medium for rBCG. A DNA fragment encoding the hsp60 gene of BCG (36) was cloned into SmaI-SalI sites of pUC18 (pUC-hsp60). A synthetic DNA fragment corresponding to the multicloning site and terminator region of the hsp60 gene was cloned into the MunI-KpnI sites of pUC-hsp60. A KpnI linker was then inserted at the EcoRI site, giving rise to the pUC-hspK vector. The gag p24 gene of the subtype B NL4-3 virus was amplified by PCR from pNL4-3 plasmid using the primers AATggatccTATAGTGCAGAACCTC (forward, with lowercase letters indicating the BamHI site) and AATgggcccTTACAAAACTCTTGCTTTATGG (reverse, with lowercase letters indicating the ApaI site). The PCR product was cloned into BamHI-ApaI sites of pUC-hspK in frame (pUC-hspK-p24Wt). The whole p24 gene was also chemically synthesized with the preferred codons in BCG and then cloned into the same sites of the pUC-hspK vector (pUC-hspK-p24Mu). These vectors were digested with KpnI, and then small fragments containing p24 expression units were subcloned into a KpnI site of the stable E. coli-mycobacteria shuttle vector pSO246 (pSO-p24Wt and -p24Mu) (19). These plasmids and pSO246 were transformed into BCG by using a Gene-Pulser (Bio-Rad Laboratories, Inc.), and transformants were selected on Middlebrook 7H10 agar containing 20 μg of kanamycin/ml and supplemented with an OADC enrichment (BBL Microbiology Systems).
Western blot analysis. Transformants of rBCG were grown in 7H9-ADC broth for 2 weeks. A portion of the culture medium was periodically collected, sonicated, and subjected to immunoblot analysis with V107 monoclonal antibody (20) as described previously (11).
Lymphocyte proliferative assays. Single-cell suspensions from spleens of immunized animals were cultured with or without 25 μg of HIV-HXB2 Gag-overlapping peptide (NIH AIDS Research and Reference Reagent Program)/ml or 2.5 μg of tuberculin purified protein derivative (PPD)/ml. In the present study, the overlapping peptides p11 (LERFAVNPGLLETSE) through p35 (NIQGQMVHQAISPRT) covering the Gag p24 region were used for stimulation, either as a whole or in pools of 5. Proliferation was measured by determining the level of [3H]thymidine uptake (31).
Antigen-specific IFN- ELISPOT assay. P24- and PPD-specific IFN--secreting cells were assessed by using the mouse gamma interferon (IFN-) development module and the enzyme-linked immunospot assay (ELISPOT) blue color module (R&D Systems, Inc.). Briefly, single-cell suspensions were cultured in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum, 55 μM ?-mercaptoethanol, 50 U of penicillin/ml, and 50 μg of streptomycin/ml) with or without 25 μg of pooled Gag-overlapping peptide (p11-35)/ml, 5 μg of recombinant p24 protein (rp24; HIV-1IIIB p24; ImmunoDiagnostics, Inc.)/ml, or 2.5 μg of PPD/ml for 48 h at 37°C in a humidified 5% CO2 environment. After incubation, cells were transferred to anti-IFN- antibody-coated 96-well nitrocellulose plates (Millititer HA; Millipore Co.) at various concentrations and incubated for 16 h at 37°C in a humidified 5% CO2 environment before being developed according to manufacturer's instructions. Spot-forming cells (SFCs) were then quantified by using the KS ELISPOT compact system (Carl Zeiss) (23).
Assay for assessment on major histocompatibility complex class I-restricted CD8+-T-cell response. H-2d-restricted CD8+-T-cell responses were measured by ELISPOT assay using A9I (AMQMLKETI) peptide (27, 38). Single-cell suspensions were labeled with microbead-conjugated anti-CD8a monoclonal antibody (53-6.7; Miltenyi Biotec GmbH) and depleted labeled cells by using Auto MACS (Miltenyi Biotec GmbH). Whole splenocytes and CD8-depleted splenocytes from each mouse were used in an A9I-specific IFN- ELISPOT assay. The cells were incubated with or without A9I peptide at 50 μM for 24 h at 37°C in a humidified 5% CO2 environment, and the subsequent steps were as described above.
Serum antibody titration by HIV-1 Gag p24- and PPD-based ELISA. P24- and PPD-specific immunoglobulin G titers in plasma were determined by an endpoint enzyme-linked immunosorbent assay (ELISA) (10).
Statistical analyses. Statistical analyses were carried out by using the StatView program (version 3.0; SAS Institute). The lymphocyte proliferative activities and IFN- SFC counts of each group were compared by using the two-sided Student t test. A P value of <0.05 was considered significant.
RESULTS
Mycobacterial codon usage optimization of HIV-1 gag p24 gene and construction of an rBCG encoding the codon-optimized gene. In order to determine whether mycobacterial codon optimization could enhance the expression of the HIV gene in vitro, we first targeted the HIV-1 subtype B NL4-3 gag p24 gene for our research. Once we had designed the mycobacterial codon-optimized p24 gene, aligned it with the wild-type gene, and deduced the amino acid sequence (Fig. 1), we determined that the total G+C content of the coding region in the synthetic p24 gene was higher (67.4%) than that of the wild-type p24 gene from pNL4-3 (43.4%). (A translation table showing all 20 amino acids used in the present study is available [Table S1 in the supplemental material]). These two genes were initially cloned into the pUC-hspK vector (Fig. 2a) and subcloned into the pSO246 vector (Fig. 2b). Once these expression vectors were transformed into the BCGTokyo172 strain, rBCG-p24Mu (with optimal codon usage of the p24 gene) and rBCG-p24Wt (with wild-type codon usage) were selected for further experimentation.
Effects of codon usage modification on the expression levels of HIV-1 Gag p24 in vitro. We next sought to compare the expression levels of the p24 gene in the two types of BCG-HIV recombinants by studying the kinetics of the growth curve of the cultured rBCG cells and by measuring the levels of p24 protein to assess the production ability of the HIV antigen (Fig. 3). Using Western blot analysis at 2-week intervals, we observed that recombinant p24 protein in each of the lysates of rBCG-p24Wt and -p24Mu consistently appeared as a single band measuring ca. 24 kDa (lanes 1 and 2 of Fig. 3a, respectively). The p24 antigen expression level of rBCG-p24Mu was 37-fold higher (175.0 ± 25.1 ng/5 x 107 CFU of bacilli) than that of rBCG-p24Wt (4.7 ± 0.3 ng/5 x 107 CFU of bacilli) (Fig. 3b). Both rBCG-p24Mu and -p24Wt showed a more normal BCG growth curve than did the rBCG-pSO246 control transformant, and both peaked 21 days after cell culture (Fig. 3c), suggesting a correlation between p24 antigen generation and the growth rate of cultured rBCG-p24Mu. Thus, the codon-optimized BCG recombinant was successfully generated and found to express remarkable levels of p24 antigen, i.e., almost 200 ng of p24 antigen/5 x 107 CFU or 1 mg of bacilli.
Codon optimization of the HIV-1 Gag p24 antigen in rBCG generates strong HIV-specific immune responses in mice after intradermal immunization. We then analyzed how the modification of codon usage affected the immunogenicity of BCG vector-based vaccines encoding the HIV-1 gag p24 antigen gene. 35 BALB/c mice were divided into three experimental groups of 10 mice each, with the remaining five mice administered saline alone and used as normal healthy controls. Five mice from each experimental group were intradermally immunized with 0.01 mg, and five mice from each group were immunized with 0.1 mg of rBCG-p24Mu, -p24Wt, and -pSO246. At 10 weeks postinoculation (p.i.), we examined lymphocyte proliferation, IFN- ELISPOT cell generation, and antibody production in immunized animals. The same study was repeated three times, and all three results were summarized.
Significant lymphocyte proliferative responses (stimulation indices of 5.04 ± 1.09 and 4.02 ± 0.44) were obtained with pooled peptides p16-20 (pool 2) and pooled total p24 peptides p11-35 (pool 1-5) in mice immunized with 0.01 mg of rBCG-p24Mu. When this dosage was increased to 0.1 mg, the lymphocyte proliferative responses to pool 2 and pool 1-5 increased to 10.08 ± 2.40 and 8.05 ± 1.16, respectively (data not shown). In contrast, we could not detect any significant virus-specific proliferation in mice immunized with 0.01 or 0.1 mg of rBCG-p24Wt (Fig. 4). These in vivo differences in proliferative responses between rBCG-p24Mu and -p24Wt were statistically significant comparing pool 2 (P = 0.010) and pool 1-5 (P = 0.001). No p24-specific proliferation was detected in either rBCG-pSO246-immunized mice or normal healthy controls (data not shown). PPD-specific lymphocyte proliferations were obtained in all immunized animals similarly (stimulation indices were ca. 7).
In addition, p24-specific IFN--secreting cells were determined by ELISPOT assay. Both pooled p24 peptides (pool 1-5) and rp24-specific SFCs were detected in mice immunized with 0.1 mg of rBCG-p24Mu and -p24Wt but not in those immunized with the same dosage of rBCG-pSO246 (Fig. 5). In rBCG-p24Mu-immunized mice, stimulation with peptides resulted in 375 ± 202 SFC/106 splenocytes and stimulation with rp24 resulted in 483 ± 138 SFC/106 splenocytes—rates much higher than those observed for rBCG-p24Wt (93 ± 25 and 227 ± 120 SFC/106 splenocytes, respectively). These differences in response by groups immunized with rBCG-p24Mu and -p24Wt to peptides and to rp24 were also statistically significant (peptides, P = 0.033; rp24, P = 0.031). PPD-specific SFCs were strongly expressed in all mice receiving rBCGs (670 ± 180 SFC/106 splenocytes). Furthermore, similar levels of rp24-specific IFN- SFC activity were observed in splenocytes of rBCG-p24Mu-immunized animals even 6 months p.i. (402 ± 198 SFC/106 splenocytes, data not shown).
Furthermore, we studied whether these IFN- ELISPOT activities were attributed to major histocompatibility complex class I-restricted CD8+-T-cell response with 12 BALB/c mice immunized with 0.1 mg of rBCG-p24Mu (n = 4), -p24Wt (n = 4), or -pSO246 (n = 4). After 2 weeks p.i., the mice were sacrificed, and their spleens were used for the study. By peptide-antigen-specific IFN- ELISPOT assay, H-2d-restricted CD8+-T-cell responses specific for the CD8+-T-cell epitope A9I were detected in the two animal groups immunized with rBCG-p24Mu and -p24Wt (Fig. 6b). In rBCG-p24Mu-immunized mice, stimulation with 50 μM A9I resulted in 130 ± 16 SFC/106 splenocytes, activities significantly higher than that obtained by immunization with rBCG-p24Wt (70 ± 21 SFC/106 splenocytes, P = 0.011). Furthermore, by using magnetic cell sorting, the CD8+-T-cell-depleted cell fractions were purified to be >97% and >99% viable (Fig. 6a). The CD8+-T-cell response of immunized animal groups decreased significantly upon stimulation with A9I peptide compared to nonseparated splenocytes (rBCG-p24Mu immunized, 15 ± 12 SFC/106 cells, P = 0.001; rBCG-p24Wt immunized, 3 ± 3 SFC/106 cells, P = 0.006) ("CD8-depleted" in Fig. 6b). No A9I-specific IFN- responses were detected in rBCG-pSO246-immunized mice either whole or CD8-depleted splenocytes were used (data not shown).
Finally, sera from all animals immunized with 0.1 mg of rBCG-p24Mu, -p24Wt, and -pSO246 were assessed for specific antibody generation at 10 weeks p.i. by endpoint antibody-ELISA against rp24 and PPD (Fig. 7). Again, only low levels of antibodies against rp24 were generally elicited in animals immunized with rBCG-p24Mu and -p24Wt (antibody titers in sera of 102.41 and 102.03, respectively). Moreover, PPD-specific antibodies were similarly detected in all immunized animals at titers of ca. 103. In summary, virus-specific cell-mediated immunity was significantly induced during the initial immune response, but its antibody response was low.
DISCUSSION
In this study, we have clearly demonstrated that codon optimization is a useful strategy for enhancing foreign antigen expression in rBCG and for obtaining significant levels of foreign antigen-specific immune responses. This strategy is key to rBCG-HIV vaccine development, since low-dose immunization and/or intradermal immunization with 0.1 mg of codon-optimized rBCG has proven effective for induction of HIV-specific cellular immunity by (i) allowing for a smaller dosage of rBCG, one that is far more practicable for use in human tuberculosis vaccination than the 1 to 10-mg dose otherwise required, and by (ii) thereby reducing the risks associated with high-dosage cutaneous administration, including adverse local skin reactions, possible association with Th2-type immune responses, or exacerbation of retroviral infections. Given these results, rBCG is clearly poised to play a key role in the development of an HIV/AIDS vaccine.
When the mycobacterial codon usage of the p24 antigen gene of the HIV-1 gag was optimized, the codon-optimized rBCG expressed nearly 40-fold more antigen than did the wild-type rBCG. This enhancement of the Gag p24 expression level in rBCG is on a par with the 10- to 50-fold increase seen when DNA vaccine is codon optimized (3). Why was the mycobacterial codon optimization so effective? BCG is a high G+C gram-positive bacteria, with a genomic G+C content ca. 64.8%, and so has a strong bias toward C- and G-ending codons for every amino acid. Overall, the G+C content at the third position of codons is 81.0% (2). From the accumulated information on BCG genes (24), it should be noted that the AGA codon for Arg and the TTA codon for Leu make up only 0.9 and 1.6% of the total codons for Arg and Leu, respectively. In contrast, HIV-1 prefers the adenine or the thymidine at the third position of the codon (60.9%). In the coding sequence of the p24 gene of HIV-1 gag, 9 out of 11 Arg codons used AGA and 6 out of 18 Leu codons used TTA. Because it is generally accepted that codon preference correlates with the amount of aminoacyl tRNA in unicellular organisms (12), only low levels of aminoacyl tRNA for AGA and TTA codons would be expected in the BCG cell. These low levels of aminoacyl tRNA for AGA and TTA codons might help explain why the codon-optimized p24 gene was highly expressed in BCG.
Recombinant HIV-1 Gag p24 antigen expression in codon-optimized rBCG is 175 ng/mg of bacilli of BCGTokyo172 or ca. 5.3% of the total cytoplasmic rBCG protein, when calculated using the method of Langermann et al. (17). The previously reported production levels of recombinant HIV protein were all for non-codon-optimized BCG using a different expression system and a different BCG strain. The levels varied from 1% of cellular protein (HIV-1 Nef [40] and SIVmac251 Gag [22]) to 0.1% of the HIV-1 Gag protein (1), suggesting that codon-optimized recombinant HIV-1 protein induced responses 5- to 50-fold higher than those previously reported for non-codon-optimized rBCG. The codon-optimization of HIV Gag p24 is also effective in elicitation of antigen-specific CD8+-T-cell responses in animals. Since there is no difference in the growth/persistence in the various BCG (S. Yamamoto et al., unpublished data), the enhanced expression of the HIV protein by the recombinant construct suggests that it is responsible for the enhanced immunogenicity of the codon-optimized rBCG vaccine.
A successful preventive HIV vaccine must not only effectively protects against HIV-1 or SIV, a goal already achieved in nonhuman primate AIDS models using different vaccine modalities, but also will prove safe for use in humans. Instead of seeking to elicit sterilizing protection from the HIV infection, current vaccine research on HIV/AIDS is focused mainly on the induction of efficient cellular immune responses that may play a critical role in protective immunity.
One of the prospective measures is to evoke host immunity by delivering recombinant vector-based vaccines expressing recombinant antigens, e.g., modified vaccinia virus Ankara (4, 21), adenovirus type 5 (29), fowlpox virus (16), canarypox virus (8), and NYVAC (5). In combination with boosting or priming antigens, most of these recombinant vector-based vaccines effectively induce antiviral immunity. We also showed that rBCG could induce long-lasting anti-HIV-1 or -SIV specific immunity in small animals (14). In the present study, we have demonstrated the promise of a codon-optimized rBCG-HIV vaccine, one which could, even at low doses, elicit long-lasting cell-mediated immune responses without triggering humoral immunity.
Previous reports have demonstrated that a high-dose intravenous inoculation of BCG can induce disease progression, as it did, for example, with BCG-specific CD4+-T-cell activation in monkeys infected with SIV (6). Others have reported a correlation between the magnitude of T-cell activation of CDR3-restricted cells and the disease progression to AIDS in monkeys (41). These results suggest that these CD4+ T cells, once activated by a high dose of any live vaccine, may become infectious and even lead to the replication of the immunodeficiency virus at the coinfection stage. In this regard, our previous study indicated that high doses of BCG did indeed induce a remarkable expansion of Ia-positive activated T cells in guinea pigs but that intradermal inoculation with 0.1 mg of BCG, the common dose and route of BCG vaccination in humans, did not (35).
In showing that a low-dose vaccination with rBCG-HIV is both possible and practicable with the mycobacterial codon optimization of the foreign HIV gene, we offer here a way around this problem. Collectively, these results suggest that a novel vaccination strategy using a low dose of codon-optimized rBCG-HIV, one comparable to the common dosage used for BCG vaccination in humans, might promote stable cell-mediated immune responses and thereby help establish positive immunity against subsequent immunodeficiency virus infection.
ACKNOWLEDGMENTS
We thank Vijai Mehra and Patricia Fast of the International AIDS Vaccine Initiative and William Jacobs, Jr., of the Albert Einstein College of Medicine for helpful comments.
This work was supported in part by the Panel on AIDS of the U.S.-Japan Cooperative Medical Science Program, the Human Science Foundation of Japan, the Organization of Pharmaceutical Safety and Research, and the Japanese Ministry of Health, Labor and Welfare.
Supplemental material for this article may be found at http://jvi.asm.org/.
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Department of Pathobiology, School of Veterinary Medicine, Nihon University, Fujisawa, Kanagawa 252-8510, Japan
Department of Host Defense, Graduate School of Medicine, Osaka City University, Osaka, Osaka 545-8585, Japan
Department of Bacteriology, School of Dentistry, Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
ABSTRACT
Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.
INTRODUCTION
The Mycobacterium bovis bacille Calmette-Guérin (BCG) has been widely used as a live bacterial vaccine against Mycobacterium tuberculosis infection. Its recombinant form, rBCG, which has been used successfully to express foreign antigens and to induce immune responses, has been proposed as a vaccine candidate against a number of diseases (26, 32, 33), especially human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) (11, 13, 30). Moreover, mucosal immunization of rBCG has been found to elicit a long-term virus-specific immunity in animals (10, 14, 15), even in Th1- and Th2-deficient conditions (10). In short, an rBCG-based vaccine offers several clear advantages over other types of recombinant vector-based approaches in that it (i) induces cellular immune responses that are maintained for at least 1 to 2 years; (ii) is easy to administer, usually requiring only one or two immunizations; (iii) and is affordable because it can be easily and cheaply produced. These findings suggest that rBCG could be a potent vaccine against HIV-1 infection, one that is likewise capable of inducing safe, virus-specific immunity.
However, the results described above were obtained with high doses of rBCG, doses 10- to 100-fold larger than that needed for a practical BCG vaccination dose against tuberculosis in humans (7, 11). Therefore, the low immunogenicity seen in rBCG-inoculated animals is likely due to their inoculation with only a "normal," not a high, vaccination dose (15). Moreover, high doses of BCG administration in vivo may also act as the driving force for the replication of the immunodeficiency virus and its dissemination by hyperactivating T cells (6, 41).
We sought here to produce an rBCG vaccine that would be efficacious even in the low doses required for human vaccination. Because low-dose immunization of rBCG has been suggested to act as a prophylactic vaccination against HIV-1 (15, 28), we adopted the preferred codon of BCG to enhance the expression of the foreign HIV gene. In recombinant protein production, the potency of codon-optimized gene expression systems was demonstrated in Escherichia coli (39) and in mammalian cells (42). These results clearly show that codon-optimized recombinant genes induce vigorous expression by foreign genes in the host. Since 1998, many groups have reported that a sequence-modified DNA vaccine confers high immunogenicity against various foreign antigens, e.g., listeriolysin O of Listeria monocytogenes (37), HIV-1 Gag (43), Env (3), tetanus toxin (34), L1 protein of human papillomavirus (18), and merozoite surface protein 1 of Plasmodium falciparum (25). Most of these studies focused on demonstrating how mammalian codon usage bias efficiently enhanced the expression and immunogenicity of foreign antigens in DNA vaccination. However, although the effect of codon optimization in mammalian cells has been well documented, its effect in recombinant BCG vector-based vaccines has never been fully elucidated.
MATERIALS AND METHODS
Animals. Female BALB/c (H-2d) mice aged 6 to 8 weeks were purchased from Charles River Japan, Inc. Mice were maintained in the experimental animal facility under pathogen-free conditions and in a manner consistent with the institutional animal care and use guidelines of the National Institute of Infectious Diseases of Japan. The study was conducted in a biosafety level 2 facility with the approval of an institutional committee for biosafety and in accordance with the requirements of the World Health Organization.
Construction of an HIV antigen expression vector and transformation of BCG. We used E. coli HB101-competent cells (Takara Bio, Inc.) for gene manipulation and the BCGTokyo172 as a mycobacterial strain which does not accelerate disease progression in HIV-infected children (9). Middlebrook 7H9 broth containing albumin-dextrose complex (7H9-ADC; BBL Microbiology Systems) was used as the culture medium for rBCG. A DNA fragment encoding the hsp60 gene of BCG (36) was cloned into SmaI-SalI sites of pUC18 (pUC-hsp60). A synthetic DNA fragment corresponding to the multicloning site and terminator region of the hsp60 gene was cloned into the MunI-KpnI sites of pUC-hsp60. A KpnI linker was then inserted at the EcoRI site, giving rise to the pUC-hspK vector. The gag p24 gene of the subtype B NL4-3 virus was amplified by PCR from pNL4-3 plasmid using the primers AATggatccTATAGTGCAGAACCTC (forward, with lowercase letters indicating the BamHI site) and AATgggcccTTACAAAACTCTTGCTTTATGG (reverse, with lowercase letters indicating the ApaI site). The PCR product was cloned into BamHI-ApaI sites of pUC-hspK in frame (pUC-hspK-p24Wt). The whole p24 gene was also chemically synthesized with the preferred codons in BCG and then cloned into the same sites of the pUC-hspK vector (pUC-hspK-p24Mu). These vectors were digested with KpnI, and then small fragments containing p24 expression units were subcloned into a KpnI site of the stable E. coli-mycobacteria shuttle vector pSO246 (pSO-p24Wt and -p24Mu) (19). These plasmids and pSO246 were transformed into BCG by using a Gene-Pulser (Bio-Rad Laboratories, Inc.), and transformants were selected on Middlebrook 7H10 agar containing 20 μg of kanamycin/ml and supplemented with an OADC enrichment (BBL Microbiology Systems).
Western blot analysis. Transformants of rBCG were grown in 7H9-ADC broth for 2 weeks. A portion of the culture medium was periodically collected, sonicated, and subjected to immunoblot analysis with V107 monoclonal antibody (20) as described previously (11).
Lymphocyte proliferative assays. Single-cell suspensions from spleens of immunized animals were cultured with or without 25 μg of HIV-HXB2 Gag-overlapping peptide (NIH AIDS Research and Reference Reagent Program)/ml or 2.5 μg of tuberculin purified protein derivative (PPD)/ml. In the present study, the overlapping peptides p11 (LERFAVNPGLLETSE) through p35 (NIQGQMVHQAISPRT) covering the Gag p24 region were used for stimulation, either as a whole or in pools of 5. Proliferation was measured by determining the level of [3H]thymidine uptake (31).
Antigen-specific IFN- ELISPOT assay. P24- and PPD-specific IFN--secreting cells were assessed by using the mouse gamma interferon (IFN-) development module and the enzyme-linked immunospot assay (ELISPOT) blue color module (R&D Systems, Inc.). Briefly, single-cell suspensions were cultured in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum, 55 μM ?-mercaptoethanol, 50 U of penicillin/ml, and 50 μg of streptomycin/ml) with or without 25 μg of pooled Gag-overlapping peptide (p11-35)/ml, 5 μg of recombinant p24 protein (rp24; HIV-1IIIB p24; ImmunoDiagnostics, Inc.)/ml, or 2.5 μg of PPD/ml for 48 h at 37°C in a humidified 5% CO2 environment. After incubation, cells were transferred to anti-IFN- antibody-coated 96-well nitrocellulose plates (Millititer HA; Millipore Co.) at various concentrations and incubated for 16 h at 37°C in a humidified 5% CO2 environment before being developed according to manufacturer's instructions. Spot-forming cells (SFCs) were then quantified by using the KS ELISPOT compact system (Carl Zeiss) (23).
Assay for assessment on major histocompatibility complex class I-restricted CD8+-T-cell response. H-2d-restricted CD8+-T-cell responses were measured by ELISPOT assay using A9I (AMQMLKETI) peptide (27, 38). Single-cell suspensions were labeled with microbead-conjugated anti-CD8a monoclonal antibody (53-6.7; Miltenyi Biotec GmbH) and depleted labeled cells by using Auto MACS (Miltenyi Biotec GmbH). Whole splenocytes and CD8-depleted splenocytes from each mouse were used in an A9I-specific IFN- ELISPOT assay. The cells were incubated with or without A9I peptide at 50 μM for 24 h at 37°C in a humidified 5% CO2 environment, and the subsequent steps were as described above.
Serum antibody titration by HIV-1 Gag p24- and PPD-based ELISA. P24- and PPD-specific immunoglobulin G titers in plasma were determined by an endpoint enzyme-linked immunosorbent assay (ELISA) (10).
Statistical analyses. Statistical analyses were carried out by using the StatView program (version 3.0; SAS Institute). The lymphocyte proliferative activities and IFN- SFC counts of each group were compared by using the two-sided Student t test. A P value of <0.05 was considered significant.
RESULTS
Mycobacterial codon usage optimization of HIV-1 gag p24 gene and construction of an rBCG encoding the codon-optimized gene. In order to determine whether mycobacterial codon optimization could enhance the expression of the HIV gene in vitro, we first targeted the HIV-1 subtype B NL4-3 gag p24 gene for our research. Once we had designed the mycobacterial codon-optimized p24 gene, aligned it with the wild-type gene, and deduced the amino acid sequence (Fig. 1), we determined that the total G+C content of the coding region in the synthetic p24 gene was higher (67.4%) than that of the wild-type p24 gene from pNL4-3 (43.4%). (A translation table showing all 20 amino acids used in the present study is available [Table S1 in the supplemental material]). These two genes were initially cloned into the pUC-hspK vector (Fig. 2a) and subcloned into the pSO246 vector (Fig. 2b). Once these expression vectors were transformed into the BCGTokyo172 strain, rBCG-p24Mu (with optimal codon usage of the p24 gene) and rBCG-p24Wt (with wild-type codon usage) were selected for further experimentation.
Effects of codon usage modification on the expression levels of HIV-1 Gag p24 in vitro. We next sought to compare the expression levels of the p24 gene in the two types of BCG-HIV recombinants by studying the kinetics of the growth curve of the cultured rBCG cells and by measuring the levels of p24 protein to assess the production ability of the HIV antigen (Fig. 3). Using Western blot analysis at 2-week intervals, we observed that recombinant p24 protein in each of the lysates of rBCG-p24Wt and -p24Mu consistently appeared as a single band measuring ca. 24 kDa (lanes 1 and 2 of Fig. 3a, respectively). The p24 antigen expression level of rBCG-p24Mu was 37-fold higher (175.0 ± 25.1 ng/5 x 107 CFU of bacilli) than that of rBCG-p24Wt (4.7 ± 0.3 ng/5 x 107 CFU of bacilli) (Fig. 3b). Both rBCG-p24Mu and -p24Wt showed a more normal BCG growth curve than did the rBCG-pSO246 control transformant, and both peaked 21 days after cell culture (Fig. 3c), suggesting a correlation between p24 antigen generation and the growth rate of cultured rBCG-p24Mu. Thus, the codon-optimized BCG recombinant was successfully generated and found to express remarkable levels of p24 antigen, i.e., almost 200 ng of p24 antigen/5 x 107 CFU or 1 mg of bacilli.
Codon optimization of the HIV-1 Gag p24 antigen in rBCG generates strong HIV-specific immune responses in mice after intradermal immunization. We then analyzed how the modification of codon usage affected the immunogenicity of BCG vector-based vaccines encoding the HIV-1 gag p24 antigen gene. 35 BALB/c mice were divided into three experimental groups of 10 mice each, with the remaining five mice administered saline alone and used as normal healthy controls. Five mice from each experimental group were intradermally immunized with 0.01 mg, and five mice from each group were immunized with 0.1 mg of rBCG-p24Mu, -p24Wt, and -pSO246. At 10 weeks postinoculation (p.i.), we examined lymphocyte proliferation, IFN- ELISPOT cell generation, and antibody production in immunized animals. The same study was repeated three times, and all three results were summarized.
Significant lymphocyte proliferative responses (stimulation indices of 5.04 ± 1.09 and 4.02 ± 0.44) were obtained with pooled peptides p16-20 (pool 2) and pooled total p24 peptides p11-35 (pool 1-5) in mice immunized with 0.01 mg of rBCG-p24Mu. When this dosage was increased to 0.1 mg, the lymphocyte proliferative responses to pool 2 and pool 1-5 increased to 10.08 ± 2.40 and 8.05 ± 1.16, respectively (data not shown). In contrast, we could not detect any significant virus-specific proliferation in mice immunized with 0.01 or 0.1 mg of rBCG-p24Wt (Fig. 4). These in vivo differences in proliferative responses between rBCG-p24Mu and -p24Wt were statistically significant comparing pool 2 (P = 0.010) and pool 1-5 (P = 0.001). No p24-specific proliferation was detected in either rBCG-pSO246-immunized mice or normal healthy controls (data not shown). PPD-specific lymphocyte proliferations were obtained in all immunized animals similarly (stimulation indices were ca. 7).
In addition, p24-specific IFN--secreting cells were determined by ELISPOT assay. Both pooled p24 peptides (pool 1-5) and rp24-specific SFCs were detected in mice immunized with 0.1 mg of rBCG-p24Mu and -p24Wt but not in those immunized with the same dosage of rBCG-pSO246 (Fig. 5). In rBCG-p24Mu-immunized mice, stimulation with peptides resulted in 375 ± 202 SFC/106 splenocytes and stimulation with rp24 resulted in 483 ± 138 SFC/106 splenocytes—rates much higher than those observed for rBCG-p24Wt (93 ± 25 and 227 ± 120 SFC/106 splenocytes, respectively). These differences in response by groups immunized with rBCG-p24Mu and -p24Wt to peptides and to rp24 were also statistically significant (peptides, P = 0.033; rp24, P = 0.031). PPD-specific SFCs were strongly expressed in all mice receiving rBCGs (670 ± 180 SFC/106 splenocytes). Furthermore, similar levels of rp24-specific IFN- SFC activity were observed in splenocytes of rBCG-p24Mu-immunized animals even 6 months p.i. (402 ± 198 SFC/106 splenocytes, data not shown).
Furthermore, we studied whether these IFN- ELISPOT activities were attributed to major histocompatibility complex class I-restricted CD8+-T-cell response with 12 BALB/c mice immunized with 0.1 mg of rBCG-p24Mu (n = 4), -p24Wt (n = 4), or -pSO246 (n = 4). After 2 weeks p.i., the mice were sacrificed, and their spleens were used for the study. By peptide-antigen-specific IFN- ELISPOT assay, H-2d-restricted CD8+-T-cell responses specific for the CD8+-T-cell epitope A9I were detected in the two animal groups immunized with rBCG-p24Mu and -p24Wt (Fig. 6b). In rBCG-p24Mu-immunized mice, stimulation with 50 μM A9I resulted in 130 ± 16 SFC/106 splenocytes, activities significantly higher than that obtained by immunization with rBCG-p24Wt (70 ± 21 SFC/106 splenocytes, P = 0.011). Furthermore, by using magnetic cell sorting, the CD8+-T-cell-depleted cell fractions were purified to be >97% and >99% viable (Fig. 6a). The CD8+-T-cell response of immunized animal groups decreased significantly upon stimulation with A9I peptide compared to nonseparated splenocytes (rBCG-p24Mu immunized, 15 ± 12 SFC/106 cells, P = 0.001; rBCG-p24Wt immunized, 3 ± 3 SFC/106 cells, P = 0.006) ("CD8-depleted" in Fig. 6b). No A9I-specific IFN- responses were detected in rBCG-pSO246-immunized mice either whole or CD8-depleted splenocytes were used (data not shown).
Finally, sera from all animals immunized with 0.1 mg of rBCG-p24Mu, -p24Wt, and -pSO246 were assessed for specific antibody generation at 10 weeks p.i. by endpoint antibody-ELISA against rp24 and PPD (Fig. 7). Again, only low levels of antibodies against rp24 were generally elicited in animals immunized with rBCG-p24Mu and -p24Wt (antibody titers in sera of 102.41 and 102.03, respectively). Moreover, PPD-specific antibodies were similarly detected in all immunized animals at titers of ca. 103. In summary, virus-specific cell-mediated immunity was significantly induced during the initial immune response, but its antibody response was low.
DISCUSSION
In this study, we have clearly demonstrated that codon optimization is a useful strategy for enhancing foreign antigen expression in rBCG and for obtaining significant levels of foreign antigen-specific immune responses. This strategy is key to rBCG-HIV vaccine development, since low-dose immunization and/or intradermal immunization with 0.1 mg of codon-optimized rBCG has proven effective for induction of HIV-specific cellular immunity by (i) allowing for a smaller dosage of rBCG, one that is far more practicable for use in human tuberculosis vaccination than the 1 to 10-mg dose otherwise required, and by (ii) thereby reducing the risks associated with high-dosage cutaneous administration, including adverse local skin reactions, possible association with Th2-type immune responses, or exacerbation of retroviral infections. Given these results, rBCG is clearly poised to play a key role in the development of an HIV/AIDS vaccine.
When the mycobacterial codon usage of the p24 antigen gene of the HIV-1 gag was optimized, the codon-optimized rBCG expressed nearly 40-fold more antigen than did the wild-type rBCG. This enhancement of the Gag p24 expression level in rBCG is on a par with the 10- to 50-fold increase seen when DNA vaccine is codon optimized (3). Why was the mycobacterial codon optimization so effective? BCG is a high G+C gram-positive bacteria, with a genomic G+C content ca. 64.8%, and so has a strong bias toward C- and G-ending codons for every amino acid. Overall, the G+C content at the third position of codons is 81.0% (2). From the accumulated information on BCG genes (24), it should be noted that the AGA codon for Arg and the TTA codon for Leu make up only 0.9 and 1.6% of the total codons for Arg and Leu, respectively. In contrast, HIV-1 prefers the adenine or the thymidine at the third position of the codon (60.9%). In the coding sequence of the p24 gene of HIV-1 gag, 9 out of 11 Arg codons used AGA and 6 out of 18 Leu codons used TTA. Because it is generally accepted that codon preference correlates with the amount of aminoacyl tRNA in unicellular organisms (12), only low levels of aminoacyl tRNA for AGA and TTA codons would be expected in the BCG cell. These low levels of aminoacyl tRNA for AGA and TTA codons might help explain why the codon-optimized p24 gene was highly expressed in BCG.
Recombinant HIV-1 Gag p24 antigen expression in codon-optimized rBCG is 175 ng/mg of bacilli of BCGTokyo172 or ca. 5.3% of the total cytoplasmic rBCG protein, when calculated using the method of Langermann et al. (17). The previously reported production levels of recombinant HIV protein were all for non-codon-optimized BCG using a different expression system and a different BCG strain. The levels varied from 1% of cellular protein (HIV-1 Nef [40] and SIVmac251 Gag [22]) to 0.1% of the HIV-1 Gag protein (1), suggesting that codon-optimized recombinant HIV-1 protein induced responses 5- to 50-fold higher than those previously reported for non-codon-optimized rBCG. The codon-optimization of HIV Gag p24 is also effective in elicitation of antigen-specific CD8+-T-cell responses in animals. Since there is no difference in the growth/persistence in the various BCG (S. Yamamoto et al., unpublished data), the enhanced expression of the HIV protein by the recombinant construct suggests that it is responsible for the enhanced immunogenicity of the codon-optimized rBCG vaccine.
A successful preventive HIV vaccine must not only effectively protects against HIV-1 or SIV, a goal already achieved in nonhuman primate AIDS models using different vaccine modalities, but also will prove safe for use in humans. Instead of seeking to elicit sterilizing protection from the HIV infection, current vaccine research on HIV/AIDS is focused mainly on the induction of efficient cellular immune responses that may play a critical role in protective immunity.
One of the prospective measures is to evoke host immunity by delivering recombinant vector-based vaccines expressing recombinant antigens, e.g., modified vaccinia virus Ankara (4, 21), adenovirus type 5 (29), fowlpox virus (16), canarypox virus (8), and NYVAC (5). In combination with boosting or priming antigens, most of these recombinant vector-based vaccines effectively induce antiviral immunity. We also showed that rBCG could induce long-lasting anti-HIV-1 or -SIV specific immunity in small animals (14). In the present study, we have demonstrated the promise of a codon-optimized rBCG-HIV vaccine, one which could, even at low doses, elicit long-lasting cell-mediated immune responses without triggering humoral immunity.
Previous reports have demonstrated that a high-dose intravenous inoculation of BCG can induce disease progression, as it did, for example, with BCG-specific CD4+-T-cell activation in monkeys infected with SIV (6). Others have reported a correlation between the magnitude of T-cell activation of CDR3-restricted cells and the disease progression to AIDS in monkeys (41). These results suggest that these CD4+ T cells, once activated by a high dose of any live vaccine, may become infectious and even lead to the replication of the immunodeficiency virus at the coinfection stage. In this regard, our previous study indicated that high doses of BCG did indeed induce a remarkable expansion of Ia-positive activated T cells in guinea pigs but that intradermal inoculation with 0.1 mg of BCG, the common dose and route of BCG vaccination in humans, did not (35).
In showing that a low-dose vaccination with rBCG-HIV is both possible and practicable with the mycobacterial codon optimization of the foreign HIV gene, we offer here a way around this problem. Collectively, these results suggest that a novel vaccination strategy using a low dose of codon-optimized rBCG-HIV, one comparable to the common dosage used for BCG vaccination in humans, might promote stable cell-mediated immune responses and thereby help establish positive immunity against subsequent immunodeficiency virus infection.
ACKNOWLEDGMENTS
We thank Vijai Mehra and Patricia Fast of the International AIDS Vaccine Initiative and William Jacobs, Jr., of the Albert Einstein College of Medicine for helpful comments.
This work was supported in part by the Panel on AIDS of the U.S.-Japan Cooperative Medical Science Program, the Human Science Foundation of Japan, the Organization of Pharmaceutical Safety and Research, and the Japanese Ministry of Health, Labor and Welfare.
Supplemental material for this article may be found at http://jvi.asm.org/.
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