This study aimed to investigate the ability of CpG7909 adjuvant to enhance immunogenicity and protective efficacy of a subunit vaccine composed of ESAT6-Ag85A fusion protein (Pe685a) of Mycobacterium tuberculosis.
This study aimed to investigate the ability of CpG7909 adjuvant to enhance immunogenicity and protective efficacy of a subunit vaccine composed of ESAT6-Ag85A fusion protein (Pe685a) of Mycobacterium tuberculosis.
ELISA was used to detect specific antibody and IFN-γ expression in sera; ELISPOT, to detect IFN-γ expression in splenocytes; MTT assay and FACS, to detect T-lymphocytes proliferation in spleens; and RT-PCR, to detect cytokines expression in lungs of mice after immunization. Bacterial load and histopathological lesions in lungs or spleens of mice challenged with Myco. tuberculosis H37Rv strain were analysed. Compared with incomplete Freund's adjuvant, CpG7909 induced more potent production of Pe685a-specific IgG2a/IgG1 antibody and higher expression of IFN-γ in sera, stimulated more generation of antigen-specific IFN-γ-secreting splenocytes, enhanced frequencies of CD3+CD4+ and CD3+CD8+ T-lymphocytes in spleen and increased transcription of TNF-α, IFN-γ, IL-6 and TLR9 in lung. However, lower bacterial load in lung and less severe lung pathology were not observed in CpG7909 group mice.
CpG7909 is able to enhance immunological effects of Pe685a subunit vaccine, but does not confer significant protective efficacy against Myco. tuberculosis infection.
CpG7909 as an adjuvant of subunit vaccine against Myco. tuberculosis is worthy of further investigation.
Tuberculosis (TB) caused by Mycobacterium tuberculosis is the most common infectious disease worldwide. The World Health Organization estimates that roughly one-third of the global population has been infected with TB, which is responsible for about 1 800 000 deaths annually (WHO 2009). Currently, Mycobacterium bovis Bacillus Calmette–Guerin (BCG) is regarded as the only available vaccine against TB administered annually with over 120 million doses (WHO 2004). However, the efficacy of BCG is controversial, particularly in preventing adults from pulmonary TB (Daniel 2006). To control this global threat, development of new effective vaccine and immunization strategies is urgently needed.
Subunit vaccine from potential immunogenic antigens of Myco. tuberculosis might be a promising direction (Sharma and Yoder 2011), combining the stable, nontoxic and harmless adjuvant as a carrier to exert their immune effects. Traditional adjuvants, such as incomplete Freund's adjuvant (IFA), which need to be emulsified with antigens, are toxic to humans. In recent years, CpG oligodeoxynucleotide (CpG ODN), the agonist of Toll-like receptor 9 (TLR9), has been found to be a potent adjuvant, which is able to enhance immune responses of many mammalian species including humans to a variety of antigens including peptides, soluble proteins and virus-like particles (Klinman 2006; Krieg 2006). CpG ODN directly or indirectly activates B lymphocytes, plasmacytoid dendritic cells (pDCs), macrophages and other monocytes to secrete cytokines as IL-2 and IFN-γ, resulting in strong Th1-type immune responses (Krieg et al. 1995; Ahmad-Nejad et al. 2002; Iwasaki and Medzhitov 2004; Suzuki et al. 2004). It is generally accepted that Th1-type cellular immune response plays a significant role in antituberculosis infection (Dietrich and Doherty 2009). Therefore, we suspect that CpG ODN might be a promising adjuvant of subunit vaccine to prevent from Myco. tuberculosis infection.
In this study, CpG7909, which belongs to B-type CpG and was shown to be a potent inducer of human innate immune responses (Krieg et al. 2004; Vollmer 2005; Stewart et al. 2008), was used to act as adjuvant for a recombinant ESAT-6 and Ag85A fusion protein (Pe685a), an efficient booster subunit vaccine against TB (Lu et al. 2011), to explore the possibility that CpG7909 develops into a novel adjuvant for subunit vaccine against Myco. tuberculosis.
Specific pathogen-free, six- to eight-week-old, female BALB/c mice (provided by Lab Animal of Tongji Medical College) were randomly divided into four groups, PBS group, CpG7909 group, CpG7909/Pe685a group and IFA/Pe685a group used as positive control (n = 10). 200 μl PBS, 50 μg CpG7909 (TCG TCG TTT TGT CGT TTT GTC GTT, synthesized by Sangon Biotech, Shanghai, China), mixture of 50 μg CpG7909 and 50 μg Pe685a protein (kindly provided by Prof. Xionglin Fan, Tongji Medical College, China) and emulsion of 100 μl IFA and 50 μg Pe685a were respectively injected intraperitoneally in the same area of mice. Immunization was repeated three times with 2 week intervals, and antigen dosage reduced to half at the last immunization. Five mice per group were sacrificed for immunoassay at 2 weeks after the last immunization. The remaining mice were used for challenge with Myco. tuberculosis H37Rv virulent strain.
Sera were collected from each mouse at 2 weeks after the last immunization, and levels of the anti-Pe685a antibodies were determined using enzyme-linked immunosorbent assay (ELISA). Plates were coated with Pe685a (2 μg) in a coating solution (15 mmol l−1 Na2CO3, 34·5 mmol l−1 NaHCO3, 0·02% NaN3, pH 9·6), incubated at 4°C overnight and then blocked with 2% BSA in PBS for 60 min at 37°C. Serum samples were applied in serial 10-fold dilutions from 1: 10–1: 106. After incubating the plates for 2 h at 37°C, biotin-conjugated anti-mouse IgG1 (RMG1-1, Biolegend, San Diego, CA, USA) and IgG2a (RMG2a-62, Biolegend) antibodies were added for the detection of specific antibodies and were incubated at room temperature for 45 min. To detect bound antibodies, the OPD substrate was added, and the reaction was stopped by the addition of 50 μl of 2 mol l−1 H2SO4. The optical density was measured at 492 nm.
Sera were collected from each mouse 2 weeks after immunization and diluted to appropriate concentrations. Mouse IFN-γ ELISA kit (DKW12-2000-048, Dakewe Biotech Company, Shenzhen, China) was used to determine the IFN-γ expression in sera following the manufacturers' instructions, using the provided standard curve reagents. Controls and samples were run in duplicate to assure consistency. Intrasample variability was less than 10%. The optical density was measured at dual wave length (405 and 630 nm).
Splenocytes were isolated as described below. Briefly, spleens were removed, and splenocyte suspension was filtered through a 200-mesh stainless steel sieve prior to being centrifuged at 1500 g for 5 min. Pellets were resuspended in 8 ml of red blood cell lysis buffer (Beyotime Institute Biotech, Haimen, China) and incubated at 37°C for 8 min. Splenocytes were collected with centrifuge at 1500 g for 5 min and resuspended at 1 × 106 cells ml−1 in complete culture medium (RPMI1640 supplemented with 10% FBS, 100 U ml−1 penicillin, 100 U ml−1 streptomycin). MTT assay was performed as per the manufacturers' instructions (Beyotime Institute Biotech). Briefly, splenocytes (2 × 104 well−1) were seeded into a 96-well microplate and were cultured at 37°C, 5% CO2 for 16 h with stimulation by Pe685a (2 μg well−1). MTT staining liquid (10 μl well−1, 5 mg ml−1) was added and incubated for 4 h. Formanzan dissolved liquid (100 μl well−1) was then added and incubated until the purple crystals dissolved. Controls and samples were set in duplicate to assure consistency. The optical density was measured at 570 nm. Stimulation index (SI) was introduced as evaluation index to estimate the proliferation of lymphocytes. The calculation method is as follows: SI = (samples OD570-blank controlled OD570)/(negative control OD570-blank controlled OD570). SI > 2 was considered as specific lymphocytes proliferation.
Of 2 × 104 well−1 splenocytes were seeded into a 96-well microplate and cultured at 37°C, 5% CO2 for 4 h with stimulation by Pe685a (2 μg well−1). Cells were removed into epoxy epoxide tubes, washed and stained with PE-CyTm7-labelled CD3e (anti-mouse), APC-labelled CD8 (anti-mouse) and PE-labelled CD4 (anti-mouse) (BD PharMingen, San Diego, CA, USA) for 30 min at 4°C in dark. Isotype controls were set up for every group. Cells were analysed by FACScan LRSII instrument (BD FACSCalibur flow cytometer, BD, San Jose, CA, USA).
Mouse IFN-γ ELISPOT kit (U-CyTech biosciences, Utrecht, the Netherlands) was used to determine the number of IFN-γ-expressing cells in splenocytes following the manufacturer's instructions as described previously (Shi et al. 2010). Briefly, the splenocytes were diluted to a concentration of 1 × 106 ml−1 with Lympho-Spot serum-free medium for rodent (Dakewe Biotech Company) containing an appropriate antigen (2 μg well−1 Pe685a). About 2 × 105 cells were added to the wells of the ELISPOT plate. IFN-γ spot-forming cells were enumerated using an ELISPOT reader (Biosys Bioreader 4000 PRO, Karben, Germany). For each animal, the number of spots in wells with medium alone was subtracted from the number of spots in test wells. The mean number of antigen-specific IFN-γ spot-forming cells (per million cells) for each group was determined.
Total RNA was extracted with TRIzol reagent (Invitrogen,Carlsbad, CA, USA) from about 100 mg of lung tissue. For RT-PCR, 2 μg RNA was reverse-transcribed with M-MLV Reverse Transcriptase (Invitrogen) to obtain cDNA samples. The resultant cDNA was stored at −70°C until PCR amplification. Cytokine-specific primers (Sangon Biotech) were listed as followed (Table 1). For a comparative internal standard, primers of β-actin were added to each PCR mixture according to the procedure described previously (Patten et al. 2003). The final PCR mixtures contained 1 μl cDNA, 10 μl 2 × PCR Mix (Dongsheng Biotech, Guangzhou, China), 2 μl sense and antisense primers (100 mmol l−1) and sterile water to 20 μl. PCR was performed by the following procedures: 95°C for 5 min, 94°C for 30 s, 55/60°C for 30 s, 72°C for 45 s, total 30 cycles, at last 72°C for 10 min. PCR products were visualized by UV light after electrophoresis. Analysis was performed using the Gene Genius Bio Image system and Gene Tools from syngene. β-actin amplification was performed on each individual sample as an internal positive control standard.
|Name||Sequences of primers||Length (bp)||Temperature (°C)|
Four weeks after the last immunization, BALB/c mice were challenged with 1 × 106 CFU of virulent Myco. tuberculosis H37Rv through a lateral tail vein. Four weeks postchallenge, five mice per group were sacrificed for efficacy comparison. The spleens and lungs were removed aseptically, homogenized and cultured for CFU of Myco. tuberculosis on Lowenstein–Jensen medium. Left lung lobes from different vaccinated mice were fixed in PBS-buffered formalin and embedded in paraffin. Tissue sections were prepared and stained with haematoxylin and eosin (H&E) stain. A pathologist with no prior knowledge of the experimental groups recorded the result under a light microscope and scored as described previously (Shi et al. 2010). Briefly, sections were evaluated according to the four histopathological parameters, including peribronchiolitis, perivasculitis, alveolitis and granuloma formation and noted as absent, minimal, slight, moderate, marked or strong, which was scored as 0, 1, 2, 3, 4 and 5 respectively; thus, the maximal sum was score 20.
One-way analysis of variance was used to compare the difference between groups of mice, and P-value <0·05 was considered statistically significant.
To determine what type of the immune response against Pe685a was induced by CpG7909 adjuvant, we first evaluated specific IgG2a and IgG1 antibody productions in sera of immunized mice. It was shown that both CpG7909 and IFA stimulated significant secretion of IgG2a and IgG1 against Pe685a (Fig. 1a). Nonspecific antibodies were found in PBS and CpG7909 control mice. However, the IgG2a level resulting from CpG7909-/Pe685a-immunized mice was significantly higher than that resulting from IFA-/Pe685a-immunized mice (Fig. 1a). IgG2a/IgG1 ratio was also established. Mice immunized with CpG7909/Pe685a presented higher IgG2a/IgG1 ratio than that did in IFA-/Pe685a-immunized mice (Fig. 1b). Antigen-specific IFN-γ-secreting splenocytes from immunized mice were further evaluated (Fig. 1c). Splenocytes from mice immunized with CpG7909, CpG7909/Pe685a and IFA/Pe685a produced a significant secretion of IFN-γ when compared with cells from nonimmunized mice. Splenocytes from CpG7909-immunized mice and IFA-/Pe685a-immunized mice produced similar quantities of IFN-γ. However, the splenocytes of mice immunized with CpG7909/Pe685a presented significantly more secretion of IFN-γ than those of other mice group did. The level of IFN-γ expression in sera from immunized mice was also evaluated, and highest concentration of IFN-γ production in sera was also observed in CpG7909-/Pe685a-immunized mice (Fig. 1d). The above results showed that apparent Th1-type immune response was induced in CpG7909-/Pe685a-immunized mice.
Proliferation of sensitized T-lymphocytes in splenocytes challenged by specific antigen was detected by MTT, and data were presented as SI (Stimulation index) value. With the stimulation by Pe685a (2 μg well−1), CpG7909 stimulated more potent production of specific T-lymphocytes induced by Pe685a than IFA (Fig. 2a). Furthermore, the proportion of CD3+CD4+ and CD3+CD8+ T-lymphocytes in spleen was measured with FACS. CpG7909 stimulated more proportion of both CD3+CD4+ and CD3+CD8+ T-lymphocytes in spleen induced by Pe685a, whereas IFA only enhanced proportion of CD3+CD4+ T-lymphocytes in spleen induced by Pe685a, which indicated that CpG7909 is able to stimulate higher frequencies of CD3+CD4+ and CD3+CD8+ T-lymphocytes than IFA (Fig. 2b,c).
Cytokine transcription in lungs was detected with semiquantitative reverse transcription PCR. Expression of target genes was normalized with β-actin transcription. The expression levels of TNF-α, IFN-γ, IL-6 and TLR9 in lungs of vaccinated mice were determined by grey value ratio. As shown in Fig. 3, compared with IFA-/Pe685a-immunized mice, mice vaccinated with CpG7909/Pe685a induced higher expression of TNF-α, IFN-γ, IL-6 and TLR9 (Fig. 3). The expression of IL-10 and IL-4 was also measured, and there was no statistical difference between experimental groups (data not shown). These results showed that CpG7909 induced stronger expression of TNF-α, IFN-γ, IL-6 and TLR9 than incomplete Freund's adjuvant.
Four weeks after virulent H37Rv challenge, mice were killed and bacterial load and histopathological lesions in lungs or spleens of mice were analysed. Lung tissues from different groups of infected mice were fixed, sectioned and stained with H&E stain for assessment of pathological changes (Fig. 4a). There were extensive pathological changes in the lung from mice of each group. PBS control mice had the highest score (score 12) of lung pathology, with marked peribronchiolitis, perivasculitis and alveolitis. Slight damage in alveolar tissues with aggregated, relatively large number of lymphocytes, peribronchiolitis and perivasculitis was also observed in lungs of mice vaccinated with CpG7909/Pe685a, which was assessed as score 7. Mice from IFA group had the lowest score (score 3), and pathological changes in lung tissue were much less, compared with PBS and CpG7909 control group and CpG7909/Pe685a group (Fig. 4b). The clear hierarchy of protective efficacy in different groups of vaccinated mice, evaluated with bacterial load in lungs and spleens, was consistent with the extent of histopathological lesions (Fig. 4c), which indicated that CpG7909 adjuvant was not able to confer more significant protective efficacy against Myco. tuberculosis infection than incomplete Freund's adjuvant.
CpG7909, the first CpG to enter clinical trials in 1999, has been shown to augment production of specific antibody as adjuvant for vaccine including hepatitis B, flu, anthrax and malaria vaccines (Amlie-Lefond et al. 2005; Vollmer 2005). CpG7909 is known to directly activate B cells and indirectly activate B cells through enhancing expression of cytokines such as IL-6 and IL-10 and then promote activated B cells to produce antibodies (Krieg et al. 1995; Jego et al. 2003; He et al. 2004). CpG ODN can also activate plasmacytoid dendritic cells (pDCs), macrophages and other monocytes to secrete cytokines as IL-2 and IFN-γ, resulting in strong Th1-type immune responses (Krieg et al. 1995; Ahmad-Nejad et al. 2002; Iwasaki and Medzhitov 2004; Suzuki et al. 2004; Thapa et al. 2012). In our study, higher level of IgG2a/IgG1 antibodies specific to ESAT6-Ag85A fusion protein, a subunit vaccine that has been confirmed to possess good immune protective effects on mice infected with Myco. tuberculosis (Lu et al. 2011), was detected in sera of CpG7909-/Pe685a-immunized mice, compared with IFA group mice, suggesting that CpG7909 might be able to activate more B cells than incomplete Freund's adjuvant, to increase immunological effect of mycobacterial subunit vaccine. Furthermore, higher level of IFN-γ in sera and more secretion of IFN-γ by spleen cells were detected in CpG7909-/Pe685a-immunized mice, compared with IFA-/Pe685a-immunized mice. These results showed that CpG7909 induced a strong Th1-type immune response against Pe685a in immunized mice, which is consistent with the previous reports.
The present work showed that CpG7909 enhanced the proliferation of spleen lymphocytes stimulated by ESAT6-Ag85A fusion protein, and it was found that higher proportion of CD3+CD4+ and CD3+CD8+ T-lymphocytes, especially CD3+CD4+ T cells, were induced in CpG7909-/Pe685a-immunized mice, compared with IFA-/Pe685a-immunized mice. Previous studies have shown that CpG7909 has the strong ability to induce specific CD8+ T-cell production (Rothenfusser et al. 2004; Amlie-Lefond et al. 2005). Our results of apparent proportion of CD3+CD4+ T-lymphocytes induced by CpG7909 might be related to ESAT6-Ag85A fusion protein, which has ever been confirmed to induce Th1-type response (Lu et al. 2011). Or other mechanisms might be involved in it such as activation of CD4+ T cells regulated by plasmacytoid dendritic cells stimulated by CpG7909 (Moseman et al. 2004; Jarnicki et al. 2008; Kuball et al. 2011). Therefore, CpG7909 effectively enhanced ESAT6-Ag85A fusion protein-mediated specific cellular immunity, by stimulating higher frequency of CD4+ T-lymphocytes.
We further detected transcription of TNF-α, IFN-γ, IL-6 and TLR9 in lung, the main target organ infected by Myco. tuberculosis. Previous studies have shown that CpG7909 enhanced TNF-α, IFN-γ and IL-6 expression through binding with TLR9, followed by B-cell activation and T-cell differentiation (Krieg et al. 1995; Jego et al. 2003; Gelman et al. 2006; Tross and Klinman 2008). It was found that CpG7909/Pe685a induced a significant higher expression of TNF-α, IFN-γ, IL-6 and TLR9, while IFA/Pe685a induced slight increase in TNF-α, IFN-γ and IL-6 expression in lungs. These cytokines played an important role in specific antibody production and CD4+ and CD8+ T-cell proliferation induced by CpG7909. IL-6 is involved not only in B-cell activation, but also in stimulation of T-cell proliferation and CTL activation (Jego et al. 2003). IFN-γ induces Th1-type T-cell differentiation (Liu et al. 2008). TNF-α is able to promote macrophage activation, cytokine expression and Th1-type T-cell differentiation (Zhang et al. 2002). Collectively, from above results, we suspect that CpG7909 might be able to enhance immunological responses induced by ESAT6-Ag85A fusion protein, through activation of a variety of cytokines, to prevent pneumonia infection from Myco. tuberculosis in some extent.
Finally, we evaluated CpG7909-protective effect on mice infected with Myco. tuberculosis. However, it was found that CpG7909 did not reduce bacterial load in lung and spleen and attenuate histopathological lesions in lungs. In previous studies, it has been reported that CpG1826 and CpG2041 could not confer significant protection against Myco. tuberculosis infection compared with IFA in mice, although both of them can enhance production of specific antibodies and IFN-γ as adjuvant to CFP antigen of Myco. tuberculosis (Fonseca et al. 2007). A mixture of Ag85B, HspX and fusion protein CFP-10/ESAT-6 (C/E) with a combination of CpG has also been found to elicit both humoral and cellular immune responses in mice, whereas it played only a minor role in control of disease progression in guinea pigs challenged with Myco. tuberculosis (Chen et al. 2010). However, another group reported that a low dose (1 μg) of CpG ODN-mixed MPT-1 antigen of Myco. tuberculosis has been found to play better protective role in mice against Myco. tuberculosis infection when compared with IFA (Silva et al. 2009). Contradictory mechanism of immunological and protective effect of CpG adjuvant remains to be further investigated.
Mice immunized with IFA/Pe685a displayed better protection from Myco. tuberculosis infection, despite poorer immune response indicated that comprehensive factors such as CpG decoration, immunization dose, antigen-binding proportion, immunization cycle and other factors might be considered. Incomplete Freund's adjuvant was confirmed to induce a mixed Th1-/Th2-type response that plays an important regulatory role in disease development (Hossain et al. 2001). In our research, lower level of IgG2a/IgG1 and IFN-γ in serum and less secretion of IFN-γ by spleen cells detected in IFA-/Pe685a-immunized mice might indicate the balance of Th1- and Th2-type immunological response induced by IFA, which might contribute to the protective efficacy of IFA/Pe685a immunization. How to develop CpG as an effective and safe adjuvant of subunit vaccine against Myco. tuberculosis requires further investigation. Furthermore, in the use of CpG as adjuvant, the immune pathological injury, which might be induced by CpG, needs further detailed research.
The authors would like to thank Prof. Xionglin Fan (Tongji Medical College, China) for providing ESAT6-Ag85A fusion protein and reviewing manuscript and Prof. Yanhong Liao (Tongji Medical College, China) for reviewing manuscript. This work was sponsored by Huazhong University of Science and Technology Research Fund Innovation to Hanju Huang (M2009041).
The authors declare that they have no conflict of interest related to the manuscript.