To examine the usefulness of the pMyong2 vector system in the generation of rBCG for HIV-1 p24 Gag vaccination, we generated three types of rBCG strains expressing p24, i.e., rBCG-pMyong2-p24, rBCG-pAL-p24, and rBCG-pMV306-p24, using different Mycobacterium–E. coli shuttle vectors, i.e., pMyong2-TOPO, pAL-TOPO (39), and pMV306 (46), respectively (Figure 1). The growth rates of the three rBCG strains in the 7H9 broth (with 100 µg/ml of kanamycin) for 30 days were compared, and the rBCG and wild-type BCG strains showed a nearly identical growth rate (Figure S1 in Supplementary Material). Additionally, to examine the survival of these rBCG strains in the macrophages and DCs, the infected cells were lysed with 0.05% Triton X-100 (in PBS) and plated onto 7H10 agar plates. In both cells, the rBCG-pMyong2-p24 strain had fewer colony forming units (CFUs) than the other strains (i.e., rBCG-pAL-p24, -pMyong2-p24, and wild-type BCG strains) (Figure S2 in Supplementary Material) likely due to the bacterial burden by the enhanced p24 expression (40). To compare the expression levels of p24 in the bacteria of the three rBCG strains, we conducted enzyme-linked immunosorbent assay (ELISA) (Figure 2A) and Western blot (Figure 2B) analyses against p24 after lysis of the cultured bacteria. All rBCG strains could express the p24 protein. Similar to the rSmeg-pMyong2-p24 strain (40), the rBCG-pMyong2-p24 strain expressed approximately two or three times higher levels of p24 than the strains in the other vector systems. rBCG-pAL-p24 produced a slightly higher level of p24 than rBCG-pMV306-p24 (Figures 2A,B). To assess the stable expression of p24, the expression levels of p24 in rBCG-pMyong2-p24 in various passage points on 7H10 agar plates with or without kanamycin were also determined by Western blot analyses. The rBCG-pMyong2-p24 strain showed stable p24 expression even after 12 passages on the 7H10 agar plates with or without kanamycin (Figures S3 and S4 in Supplementary Material). Additionally, we examined the expression levels of p24 by ELISA in infected murine macrophages (J774A.1) and BMDCs infected with three rBCG strains. The trends observed were similar to those observed with the lysed rBCG strains (Figure 2C). Also, to compare the p24 expression levels according to the different M.O.I., BMDCs were infected with different M.O.I. (1 and 10 M.O.I.) of rBCG-pAL-p24 and rBCG-pMyong2-p24 for 1 and 3 days. The results showed that the increased M.O.I. of both strains affected the increased p24 expression. However, as presented above, rBCG-pMyong2-p24 induced more p24 expression than rBCG-pAL-p24 strain (Figure S8 in Supplementary Material). Altogether, compared to the other two rBCG strains, i.e., rBCG-pAL-p24 and rBCG-pMV306-p24, rBCG-pMyong2-p24 increased the production of p24 in infected antigen-presenting cells and bacteria.

To determine whether the rBCG-pMyong2-p24 showing enhanced p24 protein production improved the T cell proliferation capacity, we conducted a T cell proliferation assay in BMDCs infected with four different strains, i.e., a wild-type BCG (as a control), two types of rBCG (rBCG-pMyong2-p24 and rBCG-pAL-p24), and rSmeg-pMyong2-p24 (40), via mixed lymphocyte response using CFSE dilution methods (47). The rSmeg-pMyong2-p24 strain was also included to compare the capacity of inducing HIV-1 p24 Gag-specific immune responses between two different species using the same pMyong2 vector system, i.e., rBCG-pMyong2-p24 and rSmeg-pMyong2-p24. A schematic of the T cell proliferation assay is shown in (Figure 3A). All BMDCs infected with the two rBCG and one rSmeg strains induced significantly higher levels of CD4 and CD8 T cell proliferation than the BMDCs that were not infected. Notably, the BMDCs infected with rBCG-pMyong2-p24 induced significantly higher levels of CD4 and CD8 T cell proliferation than those infected with the other two recombinant strains, i.e., rBCG-pAL-p24 and rSmeg-pMyong2-p24 strains, and the wild-type BCG strain. However, no significant difference was observed between the BMDCs infected with rBCG-pAL-p24 and those infected with rSmeg-pMyong2-p24 in the proliferation of both CD4 and CD8 T cells (Figures 3B,C). The comparison of the IL-2 levels in stimulated CD4 and CD8 T cells also showed trends that were similar to those observed in T cell proliferation assays (Figure 3D).

To determine whether rBCG-pMyong2-p24 improved the T cell response after vaccination, splenocytes were isolated from the spleens of BALB/c mice (five mice/group) that were subcutaneously (s.c.) immunized with three different strains, i.e., two types of rBCG strains (rBCG-pMyong2-p24 and -pAL-p24), rSmeg-pMyong2-p24 (Figure 4A), and a wild type BCG strain as a control (~10⁶ CFU) and assayed for HIV-1 p24 Gag-specific T cell responses using IFN-γ ELISPOT assays. The splenocytes from the mice that were s.c. immunized with three recombinant strains showed significantly higher spot forming units (SFUs) than those from the mice that were immunized with the wild-type BCG strain. Notably, the splenocytes from mice immunized with rBCG-pMyong2-p24 (987.78 ± 195.11 SFUs/10⁶ splenocytes) yielded significantly higher SFUs than those from mice immunized with the other two strains, i.e., rBCG-pAL-p24 (479.56 ± 213.90 SFUs/10⁶ splenocytes) and rSmeg-pMyong2-p24 (647.00 ± 151.01 SFUs/10⁶ splenocytes) (Figure 4B). However, no significant difference was observed between the rBCG-pAL-24 and rSmeg-pMyong2-p24 strains in p24-specific IFN-γ SFUs from vaccinated mice (Figure 4B). Altogether, our data indicated that rBCG-pMyong2-p24 elicited an enhanced HIV-1 p24 Gag-specific production of IFN-γ, which is a Th-1 signature cytokine, suggesting its feasibility for enhancing vaccine efficacy by skewing the Th-1 type immune responses.

The splenocytes (five mice/group) obtained 4 weeks after the second immunization with the rBCG strains and rSmeg-pMyong2-p24 (Figure 4A) were stimulated in vitro in triplicate with purified p24 protein (5 µg/ml), and the induced cytokine productions of IL-2, IFN-γ, TNF-α, IL-6, and IL-10 in the cell culture supernatants were detected. In two Th1 type cytokines, i.e., IL-2 and IFN-γ, and one pro-inflammatory cytokine, i.e., IL-6, the rBCG-pMyong2-p24 strain always produced higher level of cytokines in splenocytes from vaccinated mice at all-time points (day 1 and 3) than the wild type or other two recombinant strains. (Figure 4C; Table S1 in Supplementary Material). Regarding the other pro-inflammatory cytokine, i.e., TNF-α, the rBCG-pMyong2-p24 strain produced higher level of cytokines than rBCG-pAL-p24, but an almost identical level of TNF-α was observed in both the rBCG-pMyong2-p24 and rSmeg-pMyong2-p24 strains. In the case of IL-10, which is a Th-2 cytokine, all three recombinant strains showed similar secretion levels (Figure 4C; Table S1 in Supplementary Material).

To determine whether rBCG-pMyong2-p24 elicits a Th1-biased humoral response in immunized mice, we analyzed the levels of HIV-1 p24 Gag-specific IgG2a and IgG1, which are known markers of Th1 and Th2 responses, respectively (48–50). As shown in Figure 5, the two rBCG and rSmeg-pMyong2-p24 strains elicited significantly higher levels of IgG2a isotype than the wild type. Regarding the IgG1 isotype, the three recombinant strains elicited similar level of IgG1; however, the result did not reach statistical significance. In the case of total IgG, rBCG-pMyong2-p24 showed significantly higher levers of total IgG than the other two recombinant strains (i.e., rBCG-pAL-p24 and rSmeg-pMyong2-p24) (Figure 5). Collectively, the IgG2a/IgG1 ratio, in which a higher ratio indicates a more Th1-biased humoral immune response (49), was higher in the sera from the mice immunized with rBCG-pMyong2-p24 (1.03 ± 0.02) than that in the sera from the mice immunized with the other strains (wild-type BCG = 0.91 ± 0.71; rBCG-pAL-p24 = 0.88 ± 0.21; rSmeg-pMyong2-p24 = 1.01 ± 0.17), suggesting that the rBCG-pMyong2-p24 strain can elicit an enhanced HIV-1 p24 Gag-specific Th1-biased humoral response in immunized mice.

To determine whether rBCG-pMyong2-p24 elicits an enhanced HIV-1 p24 Gag-specific cytotoxic T lymphocyte (CTL) response in immunized mice, we analyzed the CTL activity in splenocytes from mice immunized with two rBCG (i.e., rBCG-pMyong2-p24 and -pAL-p24), rSmeg-pMyong2-p24, or wild-type BCG strains via a lactate dehydrogenase (LDH) cytotoxicity assay. The immunization procedure is described in Figure 4A. The P815 cells (H-2^d) that were pulsed with the A9I peptide for 2 h served as the target cells, and the effector/target ratios were 10:1, 20:1, and 50:1 as previously described (40). As shown in Figure 6, at an E:T ratio of 50:1, the CTLs in the mice immunized with rBCG-pMyong2-p24 could elicit a significant higher level of HIV-1 p24 Gag-specific target cell lysis than those immunized with other strains (Figure 6). However, no significant difference was observed between the rBCG-pMyong2-p24 and rSmeg-pMyong2-p24 strains (Figure 6).

To compare the p24-specific immune responses between p24 protein and different CFUs of rBCG-pMyong2-p24 strains, we conducted an independent in vivo experiment with immunization groups of (i) PBS control, (ii) p24 protein (30 μg/mice) injection, (iii) rBCG-pMyong2-p24 (1 × 10⁶ CFU) injection, and (iv) rBCG-pMyong2-p24 (1 × 10⁷ CFU) injection (1 week interval, twice S.C. injection). The immunization procedure is described in Section “Materials and Methods.” After final immunization, we conducted p24-specific IFN-γ ELISPOT, IgG subtype analyses, and CTL analyses. In the case of IFN-γ ELISPOT analysis, the p24-specific IFN-γ SFUs were increased in a CFU-dependent manner. However, splenocytes from p24 protein injected mice could not induce the p24-specific IFN-γ SFUs (Figure 7A). Similarly, the p24-specific IgG2a antibodies in serum samples from each immunized mouse were also increased in a CFU-dependent manner. However, the p24-specific IgG2a antibody from serum of p24 protein injected mice showed lower levels, compared to those of rBCG-pMyong2-p24 injection groups (Figure 7B). Also, we compared the p24-specific CTL responses between p24 protein and different CFUs of rBCG-pMyong2-p24 strains. Our data showed that p24-specific CTL responses of rBCG-pMyong2-p24 were increased in a CFU-dependent manner and were always significantly higher than those of p24 protein (Figure 7C). Collectively, our data suggest that rBCG-pMyong2-p24 could elicit p24-specific Th1-biased cellular and humoral immune responses in a CFU-dependent manner and may have a merit as an HIV-1 vaccine regimen, compared to p24 protein vaccination module.

Female BALB/c mice (~25 g, 7 weeks old) were purchased from Orient-Bio (Seoul, South Korea) and used in the experiments at the age of 8 weeks. The mice were randomly divided into four groups of five mice per group.

For T cell proliferation assay, p24 protein was injected into two mice (BALB/c) through the tail vein (30 μg/mouse) and five mice (BALB/c) was used for preparation of BMDCs in each test.

For vaccination test, the BALB/c mice were subcutaneously immunized with wild type, two rBCG strains (i.e., rBCG-pAL-p24 and rBCG-pMyong2-p24), or the rSmeg-pMyong2-p24 strain twice (1 × 10⁶ CFU in the 100 µl PBS; at a 4-week interval) at the bottom of the tail. For negative control group, PBS was injected subcutaneously. After 4 weeks after the final immunization, the mice were euthanized at each time point by CO₂ inhalation, and their bloods and spleens were removed and used in the immunological assays, such as IFN-γ ELISPOT, cytokine determination, serum antibody detection (five mice/group), and CTL analysis (three mice/group).

Also, an independent in vivo test was conducted to compare the difference in immune responses induced by p24 protein treat and different bacterial number. In this case, with 1 week interval, the BALB/c mice (three mice/group) were subcutaneously injected p24 protein (30 μg/mice) and different number of rBCG-pMyong2-p24 strain (1 × 10⁶ and 1 × 10⁷ CFU) two times. For negative control group PBS was injected subcutaneously. One week after the final immunization, the mice were euthanized at each time point by CO₂ inhalation, and their bloods and spleens were removed and used in the immunological assays, such as IFN-γ ELISPOT, serum antibody detection, and CTL analysis.

All animal experiments were carried out in accordance with the recommendations of institutional guidelines and the protocol approved by the Institutional Animal Care and Use Committee (approval No. of SNU-160307-1-2) of the Institute of Laboratory Animal Resources at Seoul National University.

To generate three different types of rBCG strains expressing HIV-1 p24 Gag, BCG with the pMyong2-p24 plasmid (designated rBCG-pMyong2-p24), BCG with the pAL-p24 plasmid (designated rBCG-pAL-p24), and BCG with the pMV306-p24 plasmid (designated rBCG-pMV306-p24), the three constructed plasmids, i.e., pMV306-p24, pAL-p24, and pMyong2-p24 (40), were electroporated into the competent BCG strain (Tokyo 172) using the Gene Pulser II electroporation apparatus (Bio-Rad, Hercules, CA, USA) (58). Transformants were selected on Middlebrook 7H10 medium (Difco Laboratories, Detroit, MI, USA) supplemented with OADC containing 100 µg/ml of kanamycin. Typically, colonies of transformants were selected from the plates, transferred into 7H9 broth medium (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% glycerol, 0.05% Tween-80, 10% ADC and kanamycin and cultured for 3~4 weeks. The growth rate of the rBCG strains was determined by optical density (OD) at 600 nm.

Recombinant p24 proteins were purified from E. coli as previously described (59) with minor modification. For the expression and purification of the fusion protein, E. coli BL21 strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET23a-p24. Protein expression was induced by adding 0.4 mM isopropyl β-d-thiogalactoside (Duchefa Biochemie, Haarlem, Netherlands). Bacterial cells were harvested and disrupted by sonication on ice for 10 min. Sonicated lysates were centrifuged at 1,600 × g for 20 min at 4°C, and the pellets containing p24 protein were resuspended in binding buffer containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). The proteins were purified using Ni-NTA His binding resin (Merck, Darmstadt, Germany) and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 250 mM imidazole) containing 4 M urea. Purified proteins were dialyzed serially against the elution buffer to remove imidazole, urea, and residual salts. Purity of p24 protein was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel). The gel was visualized using Coomassie brilliant blue staining methods (60) (Figure S9 in Supplementary Material).

Bone marrow-derived dendritic cells were generated from the bone marrow (BM) of 8- to 12-week-old BALB/c mice as previously described (40). Briefly, the BM cells were flushed out of the femurs and tibias with serum-free Iscove’s modified Eagle medium (IMDM; Gibco Invitrogen, UK). The single cell suspension was seeded at a concentration of 1 × 10⁶ cells per well in a 24-well plate in final volume of 2 ml of complete IMDM supplemented with 10% fetal bovine serum (FBS) (Gibco Invitrogen), recombinant mouse GM-CSF (1.5 ng/ml; PeproTech, Rocky Hill, NJ, USA) and mouse IL-4 (1.5 ng/ml; PeproTech, USA), penicillin (100 units/ml; Gibco Invitrogen), streptomycin (100 µg/ml; Gibco Invitrogen), gentamicin (50 µg/ml; Gibco Invitrogen), l-glutamine (2 mM; Gibco Invitrogen), and β-mercaptoethanol (50 nM; Gibco Invitrogen). Half of the medium was replaced every other day with an equal volume of complete IMDM for 6 days. Five mice were used to prepare each experiment using BMDCs and five 24-well plates were used for differentiating the BMDCs.

The murine macrophage cell line J774.1 (American Type Culture Collection, TIB-67) was maintained at 37°C and 5% CO₂ in Dulbecco’s modified Eagle’s medium (Thermo Scientific, Rockford, IL, USA) supplemented with 10% (v/v) FBS, 2 mM glutamine, and essential amino acids. BMDCs were generated from mouse BM as previously described (40, 61) and maintained at 37°C and 5% CO₂ in Iscove’s modified Eagle medium (IMDM; Gibco Invitrogen, UK) supplemented with 10% FBS (Gibco Invitrogen), recombinant mouse GM-CSF (1.5 ng/ml; PeproTech, Rocky Hill, NJ, USA), mouse IL-4 (1.5 ng/ml; PeproTech, USA), penicillin (100 units/ml; Gibco Invitrogen), streptomycin (100 µg/ml; Gibco Invitrogen), gentamicin (50 µg/ml; Gibco Invitrogen), l-glutamine (2 mM; Gibco Invitrogen), and β-mercaptoethanol (50 nM; Gibco Invitrogen). The J774A.1 cells and BMDCs were infected with the rBCG strains, rBCG-pMyong2-p24, -pAL-p24, and -pMV306-p24 and wild type BCG strains (10 M.O.I.) (in triplicate) for 4 h, followed by three washes with PBS and culturing for 24 h with fresh media. After 24 h, the infected cells were lysed with 0.5% Triton X-100. The cell lysates were diluted with PBS and plated onto Middlebrook 7H10 agar plates supplemented with OADC for enumeration of the CFUs. All the infection groups were analyzed in triplicates in each experiment, and total two independent experiments were conducted.

To determine the p24 Gag expression levels in the rBCG strains, we conducted Western blot and ELISA analyses as previously described (40). Briefly, pellets of cultured rBCG strains were suspended in B-PER buffer (Thermo scientific, Rockford, IL, USA) supplemented with lysozyme (100 µg/ml), DNase (5 U/ml), and a proteinase inhibitor. Then, the suspensions were sonicated for 5 min (pulse: 0.3 s, stop: 0.7 s) on ice and centrifuged at 13,000 rpm at 4°C for 15 min. The same amount of proteins in the aqueous phase was used for the Western blot analysis. The expression levels of p24 in each rBCG strain were determined using a mouse anti-p24 monoclonal antibody (Abcam, Cambridge, MA, USA; 1:1,000 dilution). Mycobacterial Hsp65 (Abcam, 1:1,000 dilution) was used as an internal control to confirm that the protein concentrations were equal in all samples. To assess the stable expression of p24, the p24 expression level in the rBCG-pMyong2-p24 strain of various passage points (after 1, 4, 6, 8, 10, and 12 passages) was also determined. The passage process was conducted from plate to plate (7H10 agar plate with or without kanamycin), and the colonies from each passage were cultured in 7H9 broth medium for 3 weeks prior to performing each of the experiments. Additionally, the same amount of proteins was used for the detection of the p24 levels using the p24 ELISA kit (in triplicate well) (ABL, Rockville, MD, USA) as suggested by the manufacturer (62). All the groups were analyzed in two independent experiments.

For the rBCG infection, the J774.1 cells and BMDCs were seeded 5~10 × 10⁵ cells per well (24-well plate, in triplicate) and cultured for 18 h. The three different rBCG strains were infected into the cells at a multiplicity of infection (M.O.I) of 10. Also, different M.O.I (1 and 10 M.O.I) of rBCG-pMyong2-p24 strain was infected into BMDCs to compare the difference in p24 expression by different M.O.I. The J774.1 cells and BMDCs were incubated for 4 h to allow phagocytosis of the bacteria, and the extracellular bacteria were removed by washing with PBS three times. The infected J774.1 cells and BMDCs were incubated for 24 h and/or 72 h. For analysis the p24 expression in the cells, the total proteins in the cell pellets were prepared by suspension in RIPA lysis buffer and used for the determination of the p24 levels using the p24 ELISA kit (ABL) (in triplicate well) according to the manufacturer’s instructions. All the infection groups were analyzed in triplicates in each experiment, and total two independent experiments were conducted.

Two mice were injected intravenously with the p24 protein (30 μg/mouse). After 7 days, the splenocytes were washed with ice-cold FACS buffer [PBS containing 1% bovine serum albumin (BSA) and 1 mM EDTA] and blocked on ice for 30 min with a super block solution containing 10% rat sera (Sigma Aldrich), 10% goat sera (Gibco Invitrogen), 10% mouse sera (Sigma Aldrich), and 2.4G2 monoclonal antibody (10 µg/ml; BD Biosciences, San Diego, CA, USA). The cells were subsequently stained with BV421-conjugated anti-CD4 (Clone GK1.5, BD Biosciences) and PE-conjugated anti-CD8a (Clone 53-6.7, eBioscience, San Diego, CA, USA) for 30 min at 4°C and washed three times with ice-cold FACS buffer. The FACS AriaIII instrument (BD Biosciences) was used to sort the CD4 and CD8 T cell populations. One day before co-cultivation, immature BMDCs were also infected with the wild type, two rBCGs (i.e., rBCG-pMyong2-p24 and -pAL-p24) or rSmeg-pMyong2-p24 strains at an M.O.I. of 10 for 24 h. Proliferation assays were conducted using the fluorescent cytoplasmic tracking dye CFSE (Invitrogen, Carlsbad, CA, USA) as previously described (47). The sorted CD4 and CD8 T cells were stained with 5 µM CFSE for 4 min at 37°C and 4 min on ice. And then, the CFSE-labeled T cells and infected BMDCs were co-cultured for 4 days. Four days after co-cultivation of T cells and infected BMDCs, the co-cultured cells (in triplicate well) were blocked on ice for 30 min with a super block solution and stained with CD4 BV421-conjugated anti-CD4 (Clone GK1.5, BD Biosciences) and PE-conjugated anti-CD8a (Clone 53-6.7, eBioscience) for 30 min at 4°C. The cell cycle profiles were determined using FACS LSRFortessa (BD Biosciences) and analyzed using Flowjo software (Figure 3A). All the experiments were conducted in triplicate.

The amounts of murine IL-2 in the co-cultured supernatants (in triplicate well) from the above T cell proliferation assay were also determined using ELISA according to the manufacturer’s instructions (BioLegend, USA). All the experiments were conducted in duplicate.

Splenocytes from immunized mice (five mice/group) with wild type and rBCG strains were used to conduct an ELISPOT assay as previously described (63). In brief, 96-well ELISPOT plates (PVDF membrane) were coated with mouse IFN-γ (3 µg/ml, clone: AN-18) capture antibody (BD-Biosciences, San Diego, CA, USA) in PBS and incubated overnight at 4°C. The capture antibody was discarded, and the plates were washed with PBS containing 0.05% Tween-20 (PBST) and PBS (3 times each), and the plates were blocked with 200 µl of RPMI 1640 medium with 10% FBS for 3 h at 37°C. After blocking, 5 × 10⁵ cells of splenocytes from vaccinated mice were loaded into each well. For each treatment group, the cells were stimulated in triplicate with 5 µg/ml of purified p24 antigen or medium alone in a total volume of 200 µl. The plate was incubated at 37°C for 24 h. The cells were stimulated with 5 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) and 500 ng/ml of ionomycin (Sigma-Aldrich) as a positive control. After washing with PBST and PBS (three times each), each well was treated with the biotin-labeled mouse IFN-γ (3 µg/ml, clone: XMG1.2) detection antibody (BD-Biosciences) and the plate was incubated overnight at 4°C. The wells were washed again and horseradish peroxidase (HRP)-conjugated streptavidin was added to each well. The HRP reaction was developed using the 3-amino-9-ethylcarbazole substrate reagent set (BD-Biosciences). The number of SFUs per well was automatically counted using an ELISPOT reader (AID ELISPOT reader, Strasburg, Germany). All the groups were analyzed in triplicates and two independent experiments were conducted.

The splenocytes from the immunized mice (five mice/group) were adjusted to a concentration of 1 × 10⁶ cells/well (96-well microplate, 200 µl volume, in triplicate) in RPMI 1640 medium with 10% FBS, and purified p24 protein was added at a concentration of 5 µg/ml for the in vitro stimulation. The cells were cultured, and the supernatants were harvested for the IL-2 (BioLegend, San Diego, CA, USA), TNF-α (eBioscience, San Diego, CA, USA), IL-6 (eBioscience), IL-10 (eBioscience), and IFN-γ (BioLegend) cytokine determination using ELISA kits. All the groups were analyzed in triplicates and two independent experiments were conducted.

To detect the serum antibody ratio, serum samples were collected from the immunized mice (five mice/group) using the heart puncture method after euthanasia via hyperventilation of CO₂. The 96-well plate was coated overnight at 4°C with purified p24 protein (5 µg/ml) in 0.05 M carbonate-bicarbonate buffer (pH 9.6). The plate was washed three times with PBST and PBS and blocked at room temperature (RT) for 1 h with 5% BSA in PBST. The serum samples were diluted to a ratio of 1:10 in PBS and 100 µl were added to each well (in triplicate). The plate was incubated for 2 h at RT, washed three times with PBST and PBS, and incubated for 1 h with biotinylated rat anti-mouse IgG2a, IgG1 (BD Biosciences, 1:1,000 dilution), and total IgG (eBioscience, 1:1,000 dilution) antibodies. Then, the plate was washed again, incubated with HRP conjugated streptavidin (eBioscience) for 30 min at RT. After the final washing step, all the wells were reacted with the BD OptEIA substrate (BD Biosciences) for 10 min before the reaction was stopped using 1 N H₂SO₄. The OD was determined using a spectrometer at a wavelength of 450 nm (64).

The induced CTL responses were determined as previously described (65) with slight modifications. In brief, for the effector cells, the splenocytes from the mice in each immunized group were pulsed using the major histocompatibility complex class I-restricted p24 peptide A9I (AMQMLKETI) (10 µg/ml; Peptron, Daejeon, South Korea) (52) and incubated for 6 days with IL-2 (30 U/ml; PeproTech, Rocky Hill, NJ, USA) at 37°C in a 5% CO₂ incubator. The target cells, i.e., P815 cells (H-2^d), were prepared by an incubation with the A9I peptide (10 µg/ml) for 2 h before the co-culture of the effector and target cells. Cell cytotoxicity was evaluated using an LDH assay in U bottom 96-well plates according to the manufacturer’s protocol (CytoTox 96 Non-Radioactive Cytotoxicity Assay; Promega, Madison, WI, USA). In brief, the effector cells (splenocytes stimulated by antigens) were added to the target cells (p24 pulsed P815 cells) in triplicate at different effector/target (E/T) ratios (ranging from 10:1, 20:1 to 50:1) for 6 h; then, the values of the LDH released from the cultured supernatants were detected using a spectrometer at 490 nm. The percentage of specific cell lysis was calculated using the following formula: [(Experimental − Effector spontaneous − Target spontaneous)/(Target maximum − Target spontaneous)] × 100 (%). All the groups were analyzed in triplicates and two independent experiments were conducted.

All presented data are expressed as the mean ± SD. Student’s t-test was used to compare the variance using Microsoft Excel software, and the differences were considered statistically significant at probability values less than 0.05.