Volume 34 Issue 7
Jul.  2021
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XIAO Shi Qi, XU Da, DUAN Hong Yang, FAN Xue Ting, LI Gui Lian, ZHANG Wen, LI Ma Chao, HAN Na, LI Xin Yao, LI Na, ZHAO Li lan, ZHAO Xiu Qin, WAN Kang Lin, LIU Hai Can, FENG Wen Hai. Immunogenicity of Whole Mycobacterium intracellulare Proteins and Fingding on the Cross-Reactive Proteins between M. intracellulare and M. tuberculosis[J]. Biomedical and Environmental Sciences, 2021, 34(7): 528-539. doi: 10.3967/bes2021.073
Citation: XIAO Shi Qi, XU Da, DUAN Hong Yang, FAN Xue Ting, LI Gui Lian, ZHANG Wen, LI Ma Chao, HAN Na, LI Xin Yao, LI Na, ZHAO Li lan, ZHAO Xiu Qin, WAN Kang Lin, LIU Hai Can, FENG Wen Hai. Immunogenicity of Whole Mycobacterium intracellulare Proteins and Fingding on the Cross-Reactive Proteins between M. intracellulare and M. tuberculosis[J]. Biomedical and Environmental Sciences, 2021, 34(7): 528-539. doi: 10.3967/bes2021.073

Immunogenicity of Whole Mycobacterium intracellulare Proteins and Fingding on the Cross-Reactive Proteins between M. intracellulare and M. tuberculosis

doi: 10.3967/bes2021.073
Funds:  This work was supported by National Science and Technology Major Project of China [2018ZX10731301-002]
More Information
  • Author Bio:

    XIAO Shi Qi, female, 1993, PhD, majoring in pathogenesis and immunology of tuberculosis

  • Corresponding author: FENG Wen Hai, Professor, PhD, Tel: 86-13910576731, E-mail: whfeng@cau.edu.cn; LIU Hai Can, Associate Professor, PhD, Tel: 86-13811073052, 86-10-58900778, E-mail: liuhaican@icdc.cn
  • Received Date: 2020-06-23
  • Accepted Date: 2021-02-01
  •   Objectives  To evaluate the immunogenicity of Mycobacterium intracellulare proteins and determine the cross-reactive proteins between M. intracellulare and M. tuberculosis.  Methods  Protein extracts from M. intracellulare were used to immunize BALB/c mice. The antigens were evaluated using cellular and humoral immunoassays. The common genes between M. intracellular and M. tuberculosis were identified using genome-wide comparative analysis, and cross-reactive proteins were screened using immunoproteome microarrays.  Results  Immunization with M. intracellulare proteins induced significantly higher levels of the cytokines interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-6 (IL-6) and immunoglobulins IgG, IgG1, IgM, and IgG2a in mouse serum. Bone marrow-derived macrophages isolated from mice immunized with M. intracellulare antigens displayed significantly lower bacillary loads than those isolated from mice immunized with adjuvants. Whole-genome sequence analysis revealed 396 common genes between M. intracellulare and M. tuberculosis. Microchip hybridization with M. tuberculosis proteins revealed the presence of 478 proteins in the serum of mice immunized with M. intracellulare protein extracts. Sixty common antigens were found using both microchip and genomic comparative analyses.  Conclusion  This is the advanced study to investigate the immunogenicity of M. intracellulare proteins and the cross-reactive proteins between M. intracellulare and M. tuberculosis. The results revealed the presence of a number of cross-reactive proteins between M. intracellulare and M. tuberculosis. Therefore, this study provides a new way of identifying immunogenic proteins for use in tuberculosis vaccines against both M. intracellulare and M. tuberculosis in future.
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Immunogenicity of Whole Mycobacterium intracellulare Proteins and Fingding on the Cross-Reactive Proteins between M. intracellulare and M. tuberculosis

doi: 10.3967/bes2021.073
Funds:  This work was supported by National Science and Technology Major Project of China [2018ZX10731301-002]
  • Author Bio:

  • Corresponding author: FENG Wen Hai, Professor, PhD, Tel: 86-13910576731, E-mail: whfeng@cau.edu.cn LIU Hai Can, Associate Professor, PhD, Tel: 86-13811073052, 86-10-58900778, E-mail: liuhaican@icdc.cn

Abstract:   Objectives  To evaluate the immunogenicity of Mycobacterium intracellulare proteins and determine the cross-reactive proteins between M. intracellulare and M. tuberculosis.  Methods  Protein extracts from M. intracellulare were used to immunize BALB/c mice. The antigens were evaluated using cellular and humoral immunoassays. The common genes between M. intracellular and M. tuberculosis were identified using genome-wide comparative analysis, and cross-reactive proteins were screened using immunoproteome microarrays.  Results  Immunization with M. intracellulare proteins induced significantly higher levels of the cytokines interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-6 (IL-6) and immunoglobulins IgG, IgG1, IgM, and IgG2a in mouse serum. Bone marrow-derived macrophages isolated from mice immunized with M. intracellulare antigens displayed significantly lower bacillary loads than those isolated from mice immunized with adjuvants. Whole-genome sequence analysis revealed 396 common genes between M. intracellulare and M. tuberculosis. Microchip hybridization with M. tuberculosis proteins revealed the presence of 478 proteins in the serum of mice immunized with M. intracellulare protein extracts. Sixty common antigens were found using both microchip and genomic comparative analyses.  Conclusion  This is the advanced study to investigate the immunogenicity of M. intracellulare proteins and the cross-reactive proteins between M. intracellulare and M. tuberculosis. The results revealed the presence of a number of cross-reactive proteins between M. intracellulare and M. tuberculosis. Therefore, this study provides a new way of identifying immunogenic proteins for use in tuberculosis vaccines against both M. intracellulare and M. tuberculosis in future.

XIAO Shi Qi, XU Da, DUAN Hong Yang, FAN Xue Ting, LI Gui Lian, ZHANG Wen, LI Ma Chao, HAN Na, LI Xin Yao, LI Na, ZHAO Li lan, ZHAO Xiu Qin, WAN Kang Lin, LIU Hai Can, FENG Wen Hai. Immunogenicity of Whole Mycobacterium intracellulare Proteins and Fingding on the Cross-Reactive Proteins between M. intracellulare and M. tuberculosis[J]. Biomedical and Environmental Sciences, 2021, 34(7): 528-539. doi: 10.3967/bes2021.073
Citation: XIAO Shi Qi, XU Da, DUAN Hong Yang, FAN Xue Ting, LI Gui Lian, ZHANG Wen, LI Ma Chao, HAN Na, LI Xin Yao, LI Na, ZHAO Li lan, ZHAO Xiu Qin, WAN Kang Lin, LIU Hai Can, FENG Wen Hai. Immunogenicity of Whole Mycobacterium intracellulare Proteins and Fingding on the Cross-Reactive Proteins between M. intracellulare and M. tuberculosis[J]. Biomedical and Environmental Sciences, 2021, 34(7): 528-539. doi: 10.3967/bes2021.073
    • Tuberculosis (TB) is a serious infectious disease and the ninth leading cause of death worldwide. Therefore, it remains one of the most important public health problems in the world. According to the Global TB Report 2019, there were approximately 10 million active cases of TB worldwide in 2018, of which approximately 1.24 million died of the disease. In the meantime, a quarter of the world’s population has been latently infected with Mycobacterium tuberculosis[1]. Furthermore, co-infection of M. tuberculosis and human immunodeficiency virus (HIV), prevalence of multidrug-resistant TB (MDR-TB) and emergence of extensively drug-resistant TB (XDR-TB), controlling the current TB epidemic remains a severe challenge[2]. Vaccination is the most efficient method for controlling communicable diseases; the Bacillus Calmette-Guérin (BCG) vaccine has been the first and only licensed vaccine used for TB prevention since 1921 globally. It was developed after long-term subculture of M. bovis, whereby the cells almost completely lost all pathogenicity to humans, but maintained strong immunogenicity, making it an ideal vaccine at the time[3]. According to current statistics, more than 3 billion people have been vaccinated with the BCG vaccine worldwide; however, its immune protection effect has always been a controversial topic. It is generally considered that the BCG vaccine is effective for the prevention and control of TB, especially tuberculous meningitis in infants, while its immune protection against adult TB is inferior or even ineffective[4]. Therefore, there is an urgent need to develop new and effective TB vaccines to end the epidemic, and researchers worldwide are working towards this goal using different approaches[5-7]. The key to these different approaches depends on finding new and effective antigens[8-11]. The effectiveness of the BCG vaccine against TB depends on the cross-reactivity of immune responses induced by both M. bovis and M. tuberculosis in humans[4]. Therefore, scientists have hypothesized that identifying new antigens from non-tuberculous mycobacteria (NTM) such as M. vaccae is a promising approach for determining suitable antigens for use in vaccines[12, 13]. M. intracellulare, named by Runyon in the 1960s, is one of the most common pathogenic NTM and is widely distributed worldwide. M. intracellulare infection usually causes lung damage in the host. M. intracellulare, M. avium, and M. tuberculosis have been shown to induce greater, albeit similar, levels of chemokines compared to those induced by M. smegmatis and M. abscessus, whereas M. intracellulare showed higher levels of phagosome-lysosome fusion and apoptosis than M. tuberculosis in macrophages in vitro[14]. However, studies on the immune mechanisms induced by M. intracellulare infection are required, especially to characterize the cross-immunity with M. tuberculosis.

      In this study, we aimed to identify antigens that induce cross-immunity between M. intracellulare and M. tuberculosis. First, we used whole bacterial protein extracts from M. intracellulare to immunize mice and evaluated the antigen performance using cellular and humoral immunoassays and the capability of macrophages to control intracellular M. tuberculosis growth. We then determined cross-reactive antigens between M. intracellulare and M. tuberculosis by comparative genomic analysis and immunoproteome microarray hybridization. The results of this study will help identify effective antigens among M. intracellulare proteins and may provide potential candidates for the development of new types of TB vaccines and immunotherapies against M. intracellulare and M. tuberculosis infection.

    • All animal experiments conducted in the study were approved by the Ethics Committee and the Animal Experimental Ethical Committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention.

    • M. intracellulare (ATCC13950) was cultured in Löwenstein–Jensen medium (ENCODE, China) at 37 °C. The bacterial colonies were washed in PBS buffer three times before being harvested into lysis buffer (10 mmol/L Tris-HCl + 100 mmol/L NaH2P04 + 8 mol/L urea + 50 mmol/L IAA + 1× protease inhibitor cocktail). The harvested bacteria were then shock-crushed with magnetic beads four times (6.5 m/s, 2 min, ice for 1 min) to release the whole bacterial protein extract. Protein concentration was determined using a BCA kit (TransGen Biotech, China). All protein products were stored at −80 °C until use.

    • Female BALB/c mice at 6–8 weeks were used in this study. Five mice were included in both experimental group and control group, and at least three mice were included to ensure reliability and statistical analysis. The experimental group was immunized with a 200 μL mixture containing 100 μg whole M. intracellulare protein extract, 50 μL 0.5 mg/mL PolyI:C, and 100 μL 2.5 mg/mL Dimethyl-dioctyldecylammonium bromide (DDA) as adjuvants. The control group was immunized with adjuvant mixture only (50 μL 0.5 mg/mL PolyI:C and 100 μL 2.5 mg/mL DDA). Each mouse was immunized 3 times, with subcutaneous injections at ten-day intervals.

    • On the 10th day after the last vaccination, spleens from immunized mice were surgically removed, crushed with a syringe plunger into Roswell Park Memorial Institute (RPMI) 1,640 medium (with 1% penicillin-streptomycin) (Gibco, USA), and filtered into centrifuge tubes with a cell strainer. Cell suspensions were centrifuged at 1,000 rpm (r = 8.5 cm) for 5 min, and erythrocytes were lysed with ACK lysis buffer (Solarbio, China). Finally, the splenocytes were collected and quantified using Millipore ScepterTM 2.0 (merckmillipore, Germany).

      For each well, splenocytes (2 × 106 cells/mL, 500 μL) were co-cultured with 10 μg of corresponding bacterial antigens for 48 h. RPMI 1640 + ConA (5 μg/mL) or just RPMI 1640 were used as positive and negative controls, respectively. The BD OptEIA ELISA kits were used for detection of cytokines, including IFN-γ, IL-2, IL-4, IL-6, and IL-12, based on absorbance at 450 nm.

    • Ten days after the first, second, and third immunization, 120 μL mouse blood was collected from the orbital vein. Blood samples were stored at 4 °C overnight and then centrifuged at 2,000 rpm (r = 8.5 cm) for 10 min. The sera were collected and stored at −80 °C. ELISA was performed as follows: The 96-well ELISA plates were coated with antigens (the whole bacterial protein extract) at a final concentration of 10 μg/mL at 4 °C overnight. Blocking was achieved by adding 200 μL 3% BSA (Saibao, China) in each well, and extracts were incubated at 37 °C for 2 h. Sera (diluted 1:100, 1:1,000, 1:10,000, 2:10,000, 4:10,000, 8:10,000, and 1:100,000) were added and incubated for 1 h. Next, 100 μL 1:3,000 diluted secondary antibody (Southern Biotech, USA) was added and incubated at 37 °C for 50 min. TMB substrate (100 μL/well) (InnoReagents, China) was then added and the reaction was terminated by adding 2 mol/L H2SO4 (50 μL/well). Absorbance was read at 450 nm after 10 min.

    • 1) Isolation of bone marrow-derived macrophage Bone marrow-derived macrophages (BMDMs) were isolated from mouse femurs at day 10 after the last immunization. First, the bone was cut at the hip joint, the entire leg was removed, and the skin and muscle were removed very carefully. Then, the bone was cut with scissors at the knee joint. Femurs were crushed using sterilized mortar pestle in 5 mL complete Dulbecco’s Modified Eagle Media (DMEM) containing 10% FBS and 1% penicillin-streptomycin (Gibco, USA) and the cells were cultured in complete DMEM (with 10 μg/mL M-CSF) to differentiate BMDMs at 37 °C in 5% CO2. Mature macrophages could be observed under the microscope by checking the morphology of cells after approximately 10 days.

      2) H37Rv infection model BMDMs (2 × 106 cells) were plated in 24-well plates (Nunc, Denmark) in complete DMEM (10% FBS, 1% penicillin-streptomycin) per well and left to adhere for 12 h. BMDMs were then infected with log phase M. tuberculosis H37Rv for 4 h (day 0) and 3 and 5 days in a BSL-3 laboratory. M. tuberculosis H37Rv infected BMDMs were lysed with 1 mL ddH2O and serially diluted (1:10, 1:100, and 1:1,000). Then, 100 µL from each preparation were inoculated on 7H10 media containing 50 µg/mL cycloheximide, 25 µg/mL polymixin B, 50 µg/mL carbenicillin, and 20 µg/mL trimethoprim and incubated at 37 °C. BMDMs isolated from adjuvant-immunized mice were used as controls. The multiplicity of infection (MOI) was set as 3 and 5. The count of intracellular M. tuberculosis was determined after 3 weeks.

    • The complete genome sequences and coding sequences (CDSs) of M. intracellulare (NC_016946) and M. tuberculosis (NC_000962) were downloaded from the National Center of Biotechnology Information (NCBI) genome website. BLASTN in the NCBI BLAST+ software package (Version 2.6.0+) was used to determine the sequence similarity (parameters were set as follows: -perc_identity 85.00, -qcov_hsp_perc 90.00, -outfmt 5, -num_threads 32, and -evalue 1e-5) between the two species. The genes that were common between the two species were classified into functional categories according to S. T. Cole’s classification[15].

    • Antigens common between the species were determined using protein microarrays (CapitalBio, China) spotted with 3791 H37Rv proteins and 428 CCDC1551 proteins. First, 3 mL serum sample diluted 1:200 with PBST was overlaid on the arrays and incubated at room temperature (RT) for 1 h. After washing three times with PBST, goat anti-mouse IgG (H+L) antibodies with Alexa-Fluor 532 or IgM with Alexa-Fluor 635 (diluted 1:1,000 with PBST) were added and incubated at RT for 1 h. Finally, arrays were washed with PBST, dried in a SlideWasher (CapitalBio, China), and scanned with GenePix 4200A (Molecular Devices, USA). Data were analyzed using GenePix 6.0 (Molecular Devices, USA). The signal-to-noise ratio (SNR) of each spot was defined as the ratio of the foreground to the background median intensity. To eliminate the systematic error between the protein arrays and different serum samples, quantile normalization was employed between the arrays. Spots with IgG-SNR > 3 or IgM-SNR > 5 were determined as positive. Gene ontology (GO) enrichment analysis and KEGG pathway analysis were performed at http://geneontology.org/ and https://www.genome.jp/kegg/tool/map_pathway2.html, respectively. Protein-protein interaction analysis was performed using STRING 11.0 online (http://string-db.org/). Venn diagrams were generated using the R VennDiagram package https://rdrr.io/bioc/limma/man/venn.html.

    • All statistical analyses were performed using GraphPad Prism8 (GraphPad software, USA). The immunological data were compared using a two-tailed t-test. A two-sided P value ≤ 0.05 was used to determine statistical significance in all analyses.

    • After immunizing mice three times with M. intracellulare protein extracts or adjuvants only, splenic lymphocytes were isolated and stimulated with M. intracellulare proteins. M. intracellulare protein extracts induced significantly higher levels of IFN-γ, IL-2, IL-12, and IL-6 (P < 0.0001, P < 0.001, P < 0.05, and P < 0.01, respectively), compared with control mice; however, IL-4 levels were similar in these mice (P > 0.05). Further, IL-4 concentrations obtained using both the immunization methods were low (< 30 pg/mL) (Figure 1).

      Figure 1.  Cellular immune responses in immunized BALB/c mice. Lymphocytes isolated from BALB/c mice immunized with M. intracellulare protein extracts or adjuvants only were stimulated with M. intracellulare protein extracts, and the concentration of five different cytokines (A) IFN-γ, (B) IL-2, (C) IL-12, (D) IL-4, and (E) IL-6 were determined after 48 h. Capped line with asterisk * indicates significant difference between the two immunization methods, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Columns indicate the mean from at least three samples, and error bars denote the standard deviation (SD)

    • As shown in Figure 2, the prime and two prime-boost immunizations with M. intracellulare protein extracts induced strong and significant increase in IgG, IgG1, IgM, and IgG2a titers compared with pre-immunization and the adjuvant alone group at each detection point (P values were all < 0.001).

      Figure 2.  Antibody level monitoring in mice that had received three interval immunizations with M. intracellulare protein extracts or adjuvants. The change trends of titer levels of IgG, IgG1, IgM, and IgG2a are shown in (A), (B), (C), and (D), respectively. Dotted lines show the antibody titer levels in BALB/c mice immunized with M. intracellulare bacterial proteins + PolyI:C and DDA as an adjuvant; lines with squares show the antibody titer levels in BALB/c mice immunized with PolyI:C and DDA. The antibody titer levels were monitored at day 0, day 10 (10 days after the first immunization), day 20 (10 days after the second immunization), and day 30 (10 days after the third immunization). Points indicate the mean of at least three different serum samples in a group, and error bars denote standard deviation (SD).

      By day 30, after the second boost immunization, the IgG titer reached 1:800,000 and the titer of IgG1 also reached a high level of 1:400,000, while the titer of IgM was 1:32,000 and that of IgG2a was only 1:4,000 (Figure 2). All IgG, IgG1, IgM, and IgG2a titers were significantly higher than those acquired from the prime and the first boost immunization.

    • BMDMs isolated from mice immunized with M. intracellulare protein antigens were found to display significantly lower bacterial loads than those isolated from mice immunized only with adjuvants (control group) and inhibited the growth of intracellular M. tuberculosis in a dose- and time-dependent manner (Figure 3). Figure 3A shows that in MOI = 3 with 2.62 CFUs/mL (log10) H37Rv in day 0, the colony counts of the immunized group increased to 2.68 CFUs/mL (log10) when BMDMs were infected with H37Rv for 3 days, which was less than those of the control group [2.97 CFUs/mL (log10)] by 0.28 CFU/mL (log10) (P < 0.01); when BMDMs were infected with H37Rv for 5 days, the colony counts of the immunized group increased to 2.76 CFUs/mL (log10), which was significantly less than those obtained for 3.20 CFUs/mL (log10) of the control group by 0.45 CFUs/mL (log10) (P < 0.01). Figure 3B shows that in MOI = 5 with 3.01 CFUs/mL (log10) on day 0, the colony counts of the immunized group increased to 3.13 CFUs/mL (log10) when BMDMs were infected with H37Rv for 3 days, which was less than those of the control group [3.54 CFUs/mL (log10)] by 0.40 CFUs/mL (log10) (P < 0.05). When BMDMs were infected with H37Rv for 5 days, the colony counts of the immunized group increased to 3.37 CFUs/mL (log10), significantly less than those of the control group, which was 3.74 CFUs/mL (log10) by 0.3 CFUs/mL (log10) (P < 0.01).

      Figure 3.  Immunization with M. intracellulare protein extracts enhanced the ability of bone marrow-derived macrophages to inhibit M. tuberculosis growth ex vivo. (A) BMDMs were infected with H37Rv at MOI = 3. (B) BMDMs were infected with H37Rv at MOI = 5. Colony counts were obtained for 0, 3, and 5 days of BMDM infection were compared with those obtained for H37RV. Points indicate the mean from triplicate cultures, and error bars denote standard deviation (SD). P-values were determined with two-tailed Student's t test.

    • Basic information regarding the two downloaded genomic sequences is shown in Table 1. In total, 369 common genes were found between these two genomes; the classification of these genes is shown in Table 2.

      CharactersNC_000962*NC_016946**
      Genome size (Mb)4.415.4
      GC (%)65.6068.10
      No. of proteins3,9065,000
      No. of rRNA33
      No. of tRNA4546
      No. of other RNA223
      No. of genes4,0085,104
      No. of pseudogenes3052
        Note. *https://www.ncbi.nlm.nih.gov/genome/166?genome_assembly_id=159857. **https://www.ncbi.nlm.nih.gov/genome/1703?genome_assembly_id=171542.

      Table 1.  Annotation information for the two genomic sequences

      No.FunctionNumber
      1Virulence, detoxification, adaptation10
      2Lipid metabolism35
      3Information pathways73
      4Cell wall and cell processes39
      5Intermediary metabolism and respiration143
      6Regulatory proteins24
      7Conserved hypotheticals41

      Table 2.  Functional classification of the 369 common genes found in the genomes of M. tuberculosis and M. intracellulare

    • In the serum of M. intracellulare-immunized mice, 478 proteins were recognized by IgG and IgM antibodies in the protein microarray. GO enrichment analysis based on the Gene Ontology database revealed the top 15 GO terms to be in three categories according to the GO classification, of which “binding” “catalytic activity” “metabolic process” “cellular process” “cell” “cell part” were dominant, as shown in Figure 4.

      Figure 4.  Top 15 GO terms identified based on the proteins found in the serum of M. intracellulare immunized mice. The results include three main categories: red bars represent biological processes, green bars represent molecular functions, and blue bars represent cellular components. The x-axis shows the percentage of the specific GO term in each category. GO, gene ontology.

      We also conducted KEGG pathway analysis on these microarray-screened proteins. The names, functions, and pathways of 478 proteins are listed in Supplementary Table S1, available in www.besjournal.com. The top 15 enrichment pathways classified based on the screened proteins are displayed in Figure 5. Of the enriched pathways, “metabolic pathways” was the predominant pathway with 30 proteins, followed by “biosynthesis of secondary metabolites” with 19 proteins and “microbial metabolism in diverse environments” with 12 proteins.

      IDKEGG pathwayNo. of genesGenes mapped
      mtu01100Metabolic
      pathways
      30mtu:Rv0091 mtn; 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase mtu:Rv0500 proC; pyrroline-5-carboxylate reductase mtu:Rv0511 hemD; uroporphyrin-III C-methyltransferase mtu:Rv0673 echA4; enoyl-CoA hydratase EchA4 mtu:Rv0769 oxidoreductase mtu:Rv0848 cysK2; cysteine synthase CysK mtu:Rv0952 sucD; succinyl-CoA ligase subunit alpha mtu:Rv0956 purN; phosphoribosylglycinamide formyltransferase PurN mtu:Rv1092c coaA; pantothenate kinase mtu:Rv1164 narI; nitrate reductase subunit gamma mtu:Rv1257c oxidoreductase mtu:Rv1263 amiB2; amidase AmiB mtu:Rv1306 atpF; ATP synthase subunit B mtu:Rv1308 atpA; ATP synthase subunit alpha mtu:Rv1448c tal; transaldolase mtu:Rv1530 adh; alcohol dehydrogenase mtu:Rv1604 impA; inositol-monophosphatase ImpA mtu:Rv1609 trpE; anthranilate synthase component I mtu:Rv2195 qcrA; ubiquinol-cytochrome C reductase rieske iron-sulfur subunit mtu:Rv2215 dlaT; pyruvate dehydrogenase E2 component dihydrolipoamide acyltransferase mtu:Rv2350c plcB; membrane-associated phospholipase B mtu:Rv2377c hypothetical protein mtu:Rv2465c rpiB; ribose-5-phosphate isomerase B mtu:Rv2996c serA1; D-3-phosphoglycerate dehydrogenase mtu:Rv3009c gatB; aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit B mtu:Rv3042c serB2; phosphoserine phosphatase SerB mtu:Rv3106 fprA; NADPH-ferredoxin reductase FprA mtu:Rv3465 rmlC; dTDP-4-dehydrorhamnose 3,5-epimerase mtu:Rv3600c type III pantothenate kinase mtu:Rv3703c etgB; iron(II)-dependent oxidoreductase EgtB
      mtu01110Biosynthesis of
      secondary
      metabolites
      19mtu:Rv0500 proC; pyrroline-5-carboxylate reductase mtu:Rv0511 hemD; uroporphyrin-III C-methyltransferase mtu:Rv0673 echA4; enoyl-CoA hydratase EchA4 mtu:Rv0769 oxidoreductase mtu:Rv0952 sucD; succinyl-CoA ligase subunit alpha mtu:Rv0956 purN; phosphoribosylglycinamide formyltransferase PurN mtu:Rv1086 (2Z,6E)-farnesyl diphosphate synthase mtu:Rv1257c oxidoreductase mtu:Rv1448c tal; transaldolase mtu:Rv1530 adh; alcohol dehydrogenase mtu:Rv1604 impA; inositol-monophosphatase ImpA mtu:Rv1609 trpE; anthranilate synthase component I mtu:Rv2215 dlaT; pyruvate dehydrogenase E2 component dihydrolipoamide acyltransferase mtu:Rv2350c plcB; membrane-associated phospholipase B mtu:Rv2377c hypothetical protein mtu:Rv2465c rpiB; ribose-5-phosphate isomerase B mtu:Rv2996c serA1; D-3-phosphoglycerate dehydrogenase mtu:Rv3042c serB2; phosphoserine phosphatase SerB mtu:Rv3465 rmlC; dTDP-4-dehydrorhamnose 3,5-epimerase
      mtu01120Microbial
      metabolism in
      diverse
      environments
      12mtu:Rv0511 hemD; uroporphyrin-III C-methyltransferase mtu:Rv0673 echA4; enoyl-CoA hydratase EchA4 mtu:Rv0952 sucD; succinyl-CoA ligase subunit alpha mtu:Rv1164 narI; nitrate reductase subunit gamma mtu:Rv1257c oxidoreductase mtu:Rv1263 amiB2; amidase AmiB mtu:Rv1448c tal; transaldolase mtu:Rv1530 adh; alcohol dehydrogenase mtu:Rv2215 dlaT; pyruvate dehydrogenase E2 component dihydrolipoamide acyltransferase mtu:Rv2465c rpiB; ribose-5-phosphate isomerase B mtu:Rv2996c serA1; D-3-phosphoglycerate dehydrogenase mtu:Rv3042c serB2; phosphoserine phosphatase SerB
      mtu01230Biosynthesis of
      amino acids
      7mtu:Rv0091 mtn; 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase mtu:Rv0500 proC; pyrroline-5-carboxylate reductase mtu:Rv1448c tal; transaldolase mtu:Rv1609 trpE; anthranilate synthase component I mtu:Rv2465c rpiB; ribose-5-phosphate isomerase B mtu:Rv2996c serA1; D-3-phosphoglycerate dehydrogenase mtu:Rv3042c serB2; phosphoserine phosphatase SerB
      mtu01200Carbon
      metabolism
      6mtu:Rv0952 sucD; succinyl-CoA ligase subunit alpha mtu:Rv1448c tal; transaldolase mtu:Rv2215 dlaT; pyruvate dehydrogenase E2 component dihydrolipoamide acyltransferase mtu:Rv2465c rpiB; ribose-5-phosphate isomerase B mtu:Rv2996c serA1; D-3-phosphoglycerate dehydrogenase mtu:Rv3042c serB2; phosphoserine phosphatase SerB
      mtu01240Biosynthesis of
      cofactors
      4mtu:Rv0511 hemD; uroporphyrin-III C-methyltransferase mtu:Rv0769 oxidoreductase mtu:Rv1092c coaA; pantothenate kinase mtu:Rv3600c type III pantothenate kinase
      mtu02024Quorum sensing4mtu:Rv0732 secY; preprotein translocase SecY mtu:Rv1609 trpE; anthranilate synthase component I mtu:Rv2350c plcB; membrane-associated phospholipase B mtu:Rv3676 crp; cAMP receptor protein
      mtu00190Oxidative
      phosphorylation
      4mtu:Rv1306 atpF; ATP synthase subunit B mtu:Rv1308 atpA; ATP synthase subunit alpha mtu:Rv2195 qcrA; ubiquinol-cytochrome C reductase rieske iron-sulfur subunit mtu:Rv3628 ppa; inorganic pyrophosphatase
      mtu02020Two-component
      system
      4mtu:Rv1164 narI; nitrate reductase subunit gamma mtu:Rv2234 ptpA; protein-tyrosine-phosphatase mtu:Rv3132c devS; two component sensor histidine kinase DevS mtu:Rv3676 crp; cAMP receptor protein
      mtu00270Cysteine and
      methionine
      metabolism
      3mtu:Rv0091 mtn; 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase mtu:Rv0848 cysK2; cysteine synthase CysK mtu:Rv2996c serA1; D-3-phosphoglycerate dehydrogenase
      mtu00380Tryptophan
      metabolism
      3mtu:Rv0673 echA4; enoyl-CoA hydratase EchA4 mtu:Rv1263 amiB2; amidase AmiB mtu:Rv2215 dlaT; pyruvate dehydrogenase E2 component dihydrolipoamide acyltransferase
      mtu03018RNA
      degradation
      3mtu:Rv0350 dnaK; chaperone protein DnaK mtu:Rv0440 groEL2; molecular chaperone GroEL mtu:Rv3417c groEL1; chaperonin GroEL
      mtu05152Tuberculosis3mtu:Rv0350 dnaK; chaperone protein DnaK mtu:Rv0440 groEL2; molecular chaperone GroEL mtu:Rv3417c groEL1; chaperonin GroEL
      mtu03430Mismatch repair2mtu:Rv0002 dnaN; DNA polymerase III subunit beta mtu:Rv2413c hypothetical protein
      mtu01212Fatty acid
      metabolism
      2mtu:Rv0673 echA4; enoyl-CoA hydratase EchA4 mtu:Rv0769 oxidoreductase

      Table S1.  Names, functions, and pathways of 478 proteins identified in the sera of M. intracellulare-immunized mice by protein microarrays

      Figure 5.  Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis for screened antigens. Blue bars represent each category with the name labeled in the left; x-axis shows the number of antigens in each specific KEGG term in each category.

      The results from the protein-protein interaction analysis are shown in Figure 6. Of the proteins recognized by the microchip, three main interaction network clusters were found. The cluster with the highest score contained 13 proteins (Rv1308, Rv2925c, Rv2909c, Rv0041, Rv1306, Rv0732, Rv0006, Rv0440, Rv0053, Rv0684, Rv3417c, Rv3457c, Rv1650) and belonged to the cell composition cluster (GO:0044464). The second cluster contained seven proteins (Rv2460c, Rv2299c, Rv0350, Rv2703, Rv2534c, Rv3628, Rv0685) and belonged to the cytoplasmic composition modification (GO:0005737) related cluster. The third cluster contained four proteins (Rv2830c, Rv2546, Rv3321c, Rv0657c) and belonged to the toxin-antitoxin system (kw-1277) related cluster.

      Figure 6.  Protein-protein interaction network. The protein interaction network was constructed based on the String database. (A) Map of the interaction network of all screened antigenic proteins. (B) Cell composition (GO:0044464) correlated cluster. (C) Cytoplasmic composition modification (GO:0005737) correlated cluster. (D) Toxin-antitoxin system (kw-1277) correlated cluster. Lines represent specific protein-protein associations. GO, gene ontology.

      The results from the comparative analysis of antigens in protein microarray and comparative genomic analysis are shown in Figure 7. Of the 478 genes and 369 proteins found by microchip and comparative genomic analysis, 60 shared common antigens were found. In total, 418 exclusive antigens were revealed from protein microarray analysis and 309 from comparative genomic analysis. The names of the proteins in each group are listed in Supplementary Table S2, available in www.besjournal.com.

      Figure 7.  Comparative analysis of antigens in protein microarray and comparative genomic analysis. Venn diagram showing the distribution of common antigens and exclusive antigens between protein microarrays (A) and comparative genomic analysis (B). The numbers shown in the diagram represent the number of proteins in each group.

      No.GenomeBioChipCommon
      1Rv0009MT0066.1Rv0009
      2Rv0011cMT0066.2Rv0046c
      3Rv0019cMT0116.1Rv0053
      4Rv0046cMT0250Rv0337c
      5Rv0053MT0270.1Rv0350
      6Rv0055MT0383Rv0384c
      7Rv0126MT0392Rv0423c
      8Rv0130MT0407Rv0440
      9Rv0137cMT0470Rv0491
      10Rv0156MT0487Rv0551c
      11Rv0157MT0543Rv0667
      12Rv0158MT0555Rv0672
      13Rv0189cMT0610Rv0673
      14Rv0211MT0946Rv0684
      15Rv0230cMT0968.1Rv0685
      16Rv0236AMT1029Rv0732
      17Rv0238MT1040.1Rv0861c
      18Rv0242cMT1055Rv0984
      19Rv0244cMT1083.1Rv1013
      20Rv0267MT1172.1Rv1070c
      21Rv0321MT1264.1Rv1080c
      22Rv0337cMT1305.1Rv1086
      23Rv0350MT1329Rv1092c
      24Rv0352MT1479.1Rv1257c
      25Rv0357cMT1534Rv1292
      26Rv0363cMT1555.1Rv1308
      27Rv0380cMT1775Rv1458c
      28Rv0384cMT1849.1Rv1654
      29Rv0391MT2068Rv1829
      30Rv0407MT2113Rv2115c
      31Rv0411cMT2138.2Rv2241
      32Rv0423cMT2142Rv2346c
      33Rv0430MT2283Rv2374c
      34Rv0440MT2291Rv2457c
      35Rv0458MT2316Rv2460c
      36Rv0465cMT2330.1Rv2465c
      37Rv0467MT2334.1Rv2477c
      38Rv0491MT2361.1Rv2534c
      39Rv0498MT2405Rv2558
      40Rv0500AMT2455Rv2697c
      41Rv0510MT2488.1Rv2711
      42Rv0527MT2501Rv2754c
      43Rv0548cMT2502Rv2788
      44Rv0551cMT2520.1Rv2795c
      45Rv0566cMT2554.1Rv2909c
      46Rv0634BMT2625Rv3009c
      47Rv0636MT2626Rv3028c
      48Rv0639MT2637.1Rv3118
      49Rv0640MT2721Rv3221c
      50Rv0641MT2779Rv3248c
      51Rv0642cMT2871Rv3412
      52Rv0647cMT2958.1Rv3442c
      53Rv0651MT3139.1Rv3457c
      54Rv0652MT3270.1Rv3551
      55Rv0655MT3279Rv3583c
      56Rv0667MT3284Rv3609c
      57Rv0668MT3289Rv3628
      58Rv0672MT3290.2Rv3676
      59Rv0673MT3532.1Rv3710
      60Rv0682MT3573.12Rv3791
      61Rv0683MT3631
      62Rv0684MT3770
      63Rv0685MT3858
      64Rv0691AMT3876
      65Rv0693MT3878
      66Rv0700MT4026.1
      67Rv0701Rv0002
      68Rv0702Rv0006
      69Rv0703Rv0009
      70Rv0704Rv0022c
      71Rv0705Rv0025
      72Rv0707Rv0028
      73Rv0708rv0036
      74Rv0709Rv0040c
      75Rv0714Rv0041
      76Rv0716Rv0043c
      77Rv0717Rv0045c
      78Rv0718Rv0046c
      79Rv0719Rv0053
      80Rv0721Rv0063
      81Rv0723Rv0076c
      82Rv0732Rv0089
      83Rv0733Rv0091
      84Rv0737Rv0095c
      85Rv0753cRv0098
      86Rv0803Rv0100
      87Rv0808Rv0110
      88Rv0814cRv0119
      89Rv0815cRv0145
      90Rv0820Rv0150c
      91Rv0821cRv0155
      92Rv0859Rv0180c
      93Rv0861cRv0185
      94Rv0889cRv0187
      95Rv0896Rv0226c
      96Rv0903cRv0232
      97Rv0946cRv0248c
      98Rv0958Rv0250c
      99Rv0974cRv0264c
      100Rv0975cRv0277c
      101Rv0981Rv0281
      102Rv0984Rv0285
      103Rv1013Rv0287
      104Rv1017cRv0288
      105Rv1019Rv0290
      106Rv1023Rv0295c
      107Rv1038cRv0299
      108Rv1070cRv0301
      109Rv1074cRv0308
      110Rv1077Rv0333
      111Rv1080cRv0337c
      112Rv1086Rv0350
      113Rv1092cRv0369c
      114Rv1095Rv0379
      115Rv1098cRv0384c
      116Rv1099cRv0385
      117Rv1151cRv0387c
      118Rv1177Rv0398c
      119Rv1187Rv0423c
      120Rv1197Rv0429c
      121Rv1198Rv0437c
      122Rv1211Rv0440
      123Rv1213Rv0446c
      124Rv1240Rv0459
      125Rv1248cRv0466
      126Rv1257cRv0489
      127Rv1262cRv0491
      128Rv1292Rv0500
      129Rv1298Rv0508
      130Rv1305Rv0511
      131Rv1308Rv0514
      132Rv1310Rv0518
      133Rv1311Rv0521c
      134Rv1315Rv0546c
      135Rv1321Rv0551c
      136Rv1331Rv0561c
      137Rv1380Rv0571c
      138Rv1381Rv0577
      139Rv1383Rv0579
      140Rv1384Rv0580c
      141Rv1388Rv0598c
      142Rv1392Rv0600c
      143Rv1415Rv0603
      144Rv1423Rv0604
      145Rv1436Rv0606
      146Rv1447cRv0612
      147Rv1458cRv0635
      148Rv1474cRv0657c
      149Rv1475cRv0659c
      150Rv1479Rv0666
      151Rv1481Rv0667
      152Rv1483Rv0672
      153Rv1484Rv0673
      154Rv1488Rv0678
      155Rv1493Rv0684
      156Rv1547Rv0685
      157Rv1589Rv0699
      158Rv1601Rv0730
      159Rv1611Rv0731c
      160Rv1617Rv0732
      161Rv1627cRv0750
      162Rv1630Rv0764c
      163Rv1633Rv0766c
      164Rv1638Rv0769
      165Rv1641Rv0772
      166Rv1642Rv0784
      167Rv1643Rv0790c
      168Rv1654Rv0793
      169Rv1655Rv0801
      170Rv1657Rv0810c
      171Rv1658Rv0819
      172Rv1659Rv0828c
      173Rv1729cRv0837c
      174Rv1730cRv0848
      175Rv1731Rv0857
      176Rv1783Rv0861c
      177Rv1793Rv0865
      178Rv1821Rv0869c
      179Rv1827Rv0882
      180Rv1829Rv0887c
      181Rv2050Rv0937c
      182Rv2062cRv0950c
      183Rv2097cRv0952
      184Rv2111cRv0954
      185Rv2112cRv0956
      186Rv2115cRv0970
      187Rv2122cRv0984
      188Rv2134cRv1008
      189Rv2146cRv1012
      190Rv2150cRV1013
      191Rv2156cRv1046c
      192Rv2166cRv1056
      193Rv2178cRv1070c
      194Rv2193Rv1080c
      195Rv2204cRv1083
      196Rv2218Rv1086
      197Rv2220Rv1092c
      198Rv2222cRv1097c
      199Rv2225Rv1109c
      200Rv2241Rv1112
      201Rv2244Rv1113
      202Rv2245Rv1124
      203Rv2247Rv1132
      204Rv2256cRv1153c
      205Rv2259Rv1164
      206Rv2346cRV1181
      207Rv2347cRv1202
      208Rv2362cRv1208
      209Rv2367cRv1239c
      210Rv2374cRv1257c
      211Rv2375RV1259
      212Rv2402Rv1263
      213Rv2404cRV1264
      214Rv2406cRv1275
      215Rv2412RV1282C
      216Rv2420cRv1284
      217Rv2421cRv1292
      218Rv2426cRv1306
      219Rv2428Rv1308
      220Rv2441cRv1309
      221Rv2442cRV1312
      222Rv2448cRV1328
      223Rv2457cRv1371
      224Rv2460cRv1373
      225Rv2461cRv1377c
      226Rv2465cRV1395
      227Rv2466cRV1404
      228Rv2477cRv1414
      229Rv2502cRv1427c
      230Rv2511Rv1428c
      231Rv2534cRv1448c
      232Rv2539cRv1451
      233Rv2540cRv1458c
      234Rv2558Rv1462
      235Rv2572cRv1463
      236Rv2583cRv1476
      237Rv2592cRv1501
      238Rv2603cRv1530
      239Rv2605cRv1531
      240Rv2606cRv1536
      241Rv2674Rv1544
      242Rv2676cRv1584c
      243Rv2692Rv1604
      244Rv2697cRv1609
      245Rv2699cRV1650
      246Rv2708cRv1654
      247Rv2710Rv1677
      248Rv2711Rv1685c
      249Rv2713Rv1692
      250Rv2720Rv1693
      251Rv2725cRv1695
      252Rv2733cRv1708
      253Rv2744cRv1710
      254Rv2754cRv1717
      255Rv2764cRV1718
      256Rv2783cRv1719
      257Rv2788Rv1724c
      258Rv2795cRv1742
      259Rv2831Rv1770
      260Rv2840cRv1791
      261Rv2861cRv1806
      262Rv2868cRv1828
      263Rv2882cRv1829
      264Rv2890cRV1837c
      265Rv2901cRv1848
      266Rv2904cRv1875
      267Rv2909cRv1876
      268Rv2911Rv1893
      269Rv2919cRv1894c
      270Rv2927cRv1896c
      271Rv2965cRv1898
      272Rv2975aRV1912C
      273Rv2987cRv1936
      274Rv3003cRv1959c
      275Rv3009cRv1968
      276Rv3011cRv1988
      277Rv3012cRv1992c
      278Rv3028cRv2021c
      279Rv3029cRv2042c
      280Rv3043cRv2043c
      281Rv3048cRv2049c
      282Rv3051cRV2089C
      283Rv3053cRv2098c
      284Rv3102cRv2099c
      285Rv3105cRv2102
      286Rv3117Rv2104c
      287Rv3118Rv2107
      288Rv3146Rv2115c
      289Rv3148Rv2130c
      290Rv3150Rv2135c
      291Rv3155Rv2140c
      292Rv3219Rv2145c
      293Rv3221cRv2158c
      294Rv3240cRv2163c
      295Rv3246cRv2195
      296Rv3248cRv2198c
      297Rv3270Rv2215
      298Rv3280Rv2226
      299Rv3303cRv2229c
      300Rv3318Rv2233
      301Rv3319Rv2234
      302Rv3339cRv2239c
      303Rv3340Rv2241
      304Rv3356cRv2258c
      305Rv3362cRv2293c
      306Rv3368cRv2299c
      307Rv3370cRv2321c
      308Rv3396cRv2324
      309Rv3409cRv2346c
      310Rv3410cRv2350c
      311Rv3411cRv2351c
      312Rv3412Rv2360c
      313Rv3418cRv2368c
      314Rv3432cRv2371
      315Rv3436cRv2374c
      316Rv3442cRv2376c
      317Rv3443cRv2377c
      318Rv3457cRv2413c
      319Rv3458cRv2431c
      320Rv3459cRv2436
      321Rv3460cRV2438C
      322Rv3461cRv2445c
      323Rv3462cRv2452c
      324Rv3464Rv2457c
      325Rv3489Rv2460c
      326Rv3501cRv2465c
      327Rv3515cRV2467
      328Rv3516Rv2471
      329Rv3526Rv2473
      330Rv3534cRv2477c
      331Rv3535cRv2493
      332Rv3543cRv2499c
      333Rv3550Rv2505c
      334Rv3551Rv2517c
      335Rv3553Rv2518c
      336Rv3556cRv2528c
      337Rv3557cRv2534c
      338Rv3559cRv2546
      339Rv3560cRv2555c
      340Rv3562Rv2558
      341Rv3567cRv2561
      342Rv3568cRv2564
      343Rv3570cRv2576c
      344Rv3574Rv2579
      345Rv3583cRv2595
      346Rv3586Rv2614A
      347Rv3592Rv2631
      348Rv3596cRv2638
      349Rv3597cRv2641
      350Rv3609cRv2654c
      351Rv3610cRv2666
      352Rv3620cRv2680
      353Rv3628Rv2684
      354Rv3648cRv2685
      355Rv3676Rv2697c
      356Rv3678ARv2703
      357Rv3692Rv2711
      358Rv3708cRv2731
      359Rv3709cRv2754c
      360Rv3710Rv2775
      361Rv3715cRv2788
      362Rv3753cRv2795c
      363Rv3783Rv2806
      364Rv3789Rv2830c
      365Rv3791Rv2835c
      366Rv3809cRv2837c
      367Rv3842cRv2841c
      368Rv3856cRv2863
      369Rv3859cRv2878c
      Rv2885c
      Rv2889c
      Rv2903c
      Rv2907c
      Rv2909c
      Rv2914c
      Rv2925c
      Rv2928
      Rv2937
      Rv2944
      Rv2945c
      RV2951C
      Rv2975c
      Rv2984
      Rv2996c
      Rv3002c
      Rv3007c
      Rv3009c
      Rv3013
      Rv3019c
      Rv3020c
      Rv3022c
      Rv3024c
      Rv3028c
      Rv3042c
      Rv3050c
      Rv3061c
      Rv3071
      Rv3072c
      Rv3076
      Rv3089
      Rv3106
      Rv3115
      Rv3118
      Rv3132c
      Rv3160c
      Rv3169
      Rv3179
      Rv3196
      Rv3198c
      RV3213C
      Rv3218
      Rv3221c
      Rv3232c
      Rv3237c
      Rv3248c
      Rv3257c
      Rv3283
      RV3284
      Rv3285
      Rv3300c
      Rv3309c
      Rv3311
      Rv3315c
      Rv3321c
      Rv3322c
      Rv3341
      Rv3349c
      Rv3385c
      Rv3395c
      Rv3404c
      Rv3406
      Rv3412
      Rv3416
      Rv3417c
      Rv3437
      Rv3442c
      Rv3446c
      RV3455C
      Rv3457c
      Rv3465
      Rv3477
      RV3503C
      Rv3519
      Rv3551
      Rv3555c
      Rv3572
      Rv3575c
      Rv3583c
      Rv3600c
      Rv3609c
      Rv3614c
      Rv3628
      Rv3653
      Rv3672c
      Rv3676
      Rv3688c
      Rv3703c
      Rv3710
      Rv3717
      Rv3733c
      Rv3735
      Rv3749c
      Rv3755c
      Rv3756c
      Rv3760
      Rv3768
      Rv3788
      Rv3791
      Rv3799c
      Rv3836
      Rv3841
      Rv3855
      Rv3862c
      Rv3872
      Rv3882c
      Rv3890c
      Rv3908
      Rv3918c

      Table S2.  Names of common antigens identified by microchip and comparative genomic analysis

    • TB is an ancient infectious respiratory disease that seriously endangers human health. Almost a quarter of the world’s population is estimated to have latent TB infection (LTBI). Numerous potential risk factors may promote the progression of LTBI to active TB once the efficiency of the immune system declines[16]. Therefore, there is an imperative need to develop new improved vaccines that protect against both active TB and LTBI[17]. To date, vaccination continues to be the most effective method to prevent and control TB[18]. However, the protection capacity of traditional BCG vaccines is limited. As a result, new strategies to improve the effectiveness of vaccination are required.

      Bacterial proteins are important potential candidates for the development of new anti-TB vaccines because they can interact with host cells as key cell antigens[19]. Several M. tuberculosis secretory proteins have been used in newly designed anti-TB vaccines, and some of them showed excellent immune responses and protective efficacy against M. tuberculosis infection[20-22]. For example, Ag85, EAST-6, CFP10, and Rv1886c are excellent antigens; one or more of these proteins have been used in subunit and viral-vector vaccines, some of which have successfully entered phases I–III clinical trials and showed better protection ability than BCG[23-26].

      A recent study showed that mycobacterial extracts from the rapidly growing NTM M. vaccae used in vaccines had promising protective effects against TB[12]. Several vaccines based on M. vaccae antigens, such as Vaccae™, DAR-901, and SRL172, have entered different phases of clinical trials[27]. SRL172, an inactivated, whole-cell vaccine prepared from M. vaccae, was safe, well-tolerated, and immunogenic in humans[13,28]. A randomized controlled phase III trial in Tanzania demonstrated that boosting with SRL172 could protect against culture-confirmed TB in HIV-infected adults who had received BCG at birth[29]. The excellent immune effect of M. vaccae indicates that NTMs could also serve as a good source for designing new effective anti-TB vaccines. In the present study, we evaluated the immunogenicity and protective efficacy of M. intracellulare whole bacterial protein extracts in BALB/c mice to explore the possibility of using M. intracellulare as a source of immune effective antigens for developing anti-TB vaccines. Our results showed that M. intracellulare bacterial proteins promote high-level production of IFN-γ, IL-2, IL-6, and IL-12 in mice, while IL-4 remained at a very low level, thus proving that immunization with M. intracellulare bacterial proteins predominantly elicited Th1-type cytokine production in BALB/c mice. Although the immune mechanism of vaccine protection from TB remains unclear, high expression levels of IFN-γ have been associated with enhanced protection against mycobacterial infection[30, 31], and studies have shown that IFN-γ responses against multiple mycobacterial antigens could predict protection against TB[32, 33]. IL-2 is also an important indicator in most clinical trials for evaluating TB-vaccine efficacy; high IL-2 levels indicate high concentrations of vaccine-induced activated CD4 and/or CD8 cells. These cells are necessary, although not sufficient, for protective immunity against M. tuberculosis in both animal models and humans[34,35]. IL-6 is a well-known inflammatory marker cytokine. Although several studies have confirmed that the level of IL-6 could be used to monitor the progress of infection and infer the risk of progression to active TB, its role in vaccine-mediated immune protection against TB is still unclear[36]. A previous study showed that IL-6 plays a major role in priming but not in late Th1 response to a TB vaccine, thus regulating the phenotype of the immune response[37]. The high concentration of IL-6 detected in this study was possibly induced by the antigenic proteins in M. intracellulare, which are also critical for immunity[38]. IL-12 is a key cytokine that mediates the immune response of Th1 cells. Its main immunomodulatory effect is to induce differentiation of early T helper cells into Th1 cells and promote the development and proliferation of Th1 cells. High levels of IL-12 indicate cellular immunity rather than humoral immunity[39]. A recent study reported that IL-12 production could also inhibit intracellular mycobacterial growth by enhancing autophagy[40]. Another cytokine we detected is IL-4, which is a central cytokine produced by Th2 cells and promotes B cell proliferation[41]. We found that IL-4 was at a very low concentration in both groups, and there was no significant difference between the two groups.

      Data from previous studies suggested that both Th1 and Th2 immune responses play important roles in host protection against M. tuberculosis infection, and Th1 is essential against this intracellular pathogen. Current attempts to generate a vaccine against TB are generally based on the assumption that it must drive a Th1 response[42]. Our results showed that M. intracellulare proteins could induce a Th1/Th2 balance shift toward Th1 in BALB/c mice, which favors the control of M. tuberculosis in vivo.

      While the role of cell-mediated immunity in the protective immune response against TB has been well established, the role of B cells in this process is not clearly understood[43]. Emerging evidence suggests that B cell-dominated humoral immunity can modulate the host immune response to various intracellular pathogens, including M. tuberculosis, by regulating the level of granulomatous reaction, cytokine production, and T cell response[44,45]. Therefore, we also monitored antibody production in our animal model. The levels of the four antibodies (IgG, IgG1, IgM, and IgG2a) were measured in a time- and dose-dependent manner; results showed an increase in antibody titer levels. After three immunizations, they all reached a significantly higher quantity than that in the negative control group mice, suggesting that M. intracellulare bacterial proteins potentially improve the humoral immune response in mice. The results also indicated that PolyI:C and DDA function well as subcutaneous adjuvants to promote the production of antibodies without eliciting immune responses of their own.

      Macrophages are the first line of defense of the host immune system against M. tuberculosis infection[46,47]. During its long-term interaction with macrophages, M. tuberculosis develops many effective strategies to avoid elimination, while surviving and proliferating inside macrophages[48]. Although a series of complex interactions between the host and pathogen ultimately determine the outcome of infection, the mechanisms of macrophage-bacillus interactions are complicated and still under investigation. The improved clearance ability of macrophages is essential for the host to fight TB. Our results showed that BMDMs from M. intracellulare-immunized mice had a significant ability to inhibit the growth of M. tuberculosis compared to those from control mice in vitro, indicating that M. intracellulare bacterial proteins can effectively change the host immune system environment to impair M. tuberculosis survival. One explanation for this result may be that bacterial protein immunization upregulates the expression of specific genes in BMDMs and thus helps to impede M. tuberculosis growth. Therefore, more specific experiments, including transcriptome analysis or other methods, are required to reveal the relevant mechanisms. Our results also suggest the possibility of using M. intracellulare antigenic proteins for the development of TB vaccines.

      To gain insights into the antigens expressed among the M. intracellulare bacterial proteins that changed the immune properties of mice to prevent M. tuberculosis infection, a protein microarray coated with 4,219 proteins of M. tuberculosis was used to identify the antigen repertoire by cross-reacting with IgG and IgM in the sera of mice immunized with whole M. intracellulare proteins. A total of 478 M. tuberculosis proteins were recognized by the serum antibodies IgG and IgM. Through GO enrichment analysis and KEGG pathway analysis of the 478 proteins as well as the comparative genomic analysis between M. intracellulare and M. tuberculosis, we primarily obtained an insight into the main biological processes and pathways involved in shared antigens between M. tuberculosis and M. intracellulare. In the protein-protein interaction analysis, we selected specific antigens that formed three particular clusters: cell composition, cytoplasmic composition modification, and toxin-antitoxin system-related cluster. The strong interactions between these proteins suggest that they may work together to regulate the host immune profile. These protein combinations may therefore be potential targets for the development of anti-TB vaccines. In addition, we compared the antigens detected by combinational genomics and proteomics; among 478 genes and 369 proteins identified in protein microarray and comparative genomic analysis, 60 common antigens were found. These antigens may possibly be potential cross-reactive antigens that may be used for the subsequent development of cross-immune vaccines for both M. intracellulare and M. tuberculosis in the future.

      In summary, the present study demonstrates the possibility of whole bacterial protein extracts of M. intracellulare to serve as antigens against TB vaccines and reveals a number of proteins with cross-reactivity between M. intracellulare and M. tuberculosis. One limitation of the present study is that we still cannot screen for more specific antigens with clear immunogenicity to provide candidates for TB vaccines that can effectively protect against M. tuberculosis infection. Further studies are required to cautiously choose immunodominant antigens, which are crucial for developing novel vaccines for the control of TB.

    • The authors declare that they have no competing interests.

    • FENG Wen Hai, LIU Hai Can, and LI Gui Lian designed this study, revised the manuscript, and edited the manuscript. XIAO Shi Qi prepared the first draft of the manuscript. XIAO Shi Qi, XU Da, DUAN Hong Yang, FAN Xue Ting, LI Xin Yao, LI Na, LI Ma Chao, and ZHAO Li Lan performed experiments. ZHAO Xiu Qin cultured the strains. ZHANG Wen and HAN Na performed genome sequencing of the M. intracellulare strain. WAN Kang Lin provided analysis support. All authors provided suggestions on the preparation of the manuscript and read and approved the final draft of the manuscript.

    • The authors thank all the staff working at the Laboratory Animal Center, the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention for their support of this research.

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