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

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,3, ZHAO Xiu Qin,WAN Kang Lin, LIU Hai Can,#, and FENG Wen Hai

1.State Key Laboratory of Agrobiotechnology,Department of Microbiology and Immunology,College of Biological Sciences,China Agricultural University,Beijing 100193,China;2.State Key Laboratory for Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;3.Department of Stomatology,The Second Xiangya Hospital of Central South University,Changsha 410000,Hunan,China

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.

Key words:Mycobacterium intracellulare;Mycobacterium tuberculosis; Cross-reactive immune response;Vaccine;Antigen

INTRODUCTION

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 withMycobacterium tuberculosis[1].Furthermore,co-infection ofM.tuberculosisand 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 ofM.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 bothM.bovisandM.tuberculosisin humans[4].Therefore,scientists have hypothesized that identifying new antigens from non-tuberculous mycobacteria (NTM) such asM.vaccaeis 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. intracellulareinfection usually causes lung damage in the host.M.intracellulare,M.avium,and M.tuberculosishave been shown to induce greater,albeit similar,levels of chemokines compared to those induced byM.smegmatisandM.abscessus,whereasM.intracellulareshowed higher levels of phagosomelysosome fusion and apoptosis thanM.tuberculosisin macrophagesin vitro[14].However,studies on the immune mechanisms induced byM.intracellulareinfection are required,especially to characterize the cross-immunity withM.tuberculosis.

In this study,we aimed to identify antigens that induce cross-immunity betweenM.intracellulareandM.tuberculosis.First,we used whole bacterial protein extracts fromM.intracellulareto immunize mice and evaluated the antigen performance using cellular and humoral immunoassays and the capability of macrophages to control intracellularM.tuberculosisgrowth.We then determined cross-reactive antigens betweenM.intracellulare andM.tuberculosisby comparative genomic analysis and immunoproteome microarray hybridization.The results of this study will help identify effective antigens amongM.intracellulareproteins and may provide potential candidates for the development of new types of TB vaccines and immunotherapies againstM.intracellulareandM.tuberculosisinfection.

MATERIALS AND METHODS

Ethics Statement

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.

Bacterial Strains and Protein Preparation

M.intracellulare(ATCC13950) was cultured in L?wenstein-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.

Immunological Evaluation of Whole M.intracellulare Protein Extracts in Mice

Mouse ImmunizationFemale 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 wholeM.intracellulareprotein 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.

Detection of CytokinesOn 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 ScepterTM2.0 (merckmillipore,Germany).

For each well,splenocytes (2 × 106cells/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.

Detection of Immunoglobulins in the SerumTen 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.

Ability of Bone Marrow-derived Macrophages to Inhibit M.tuberculosis Growth ex vivo

1) Isolation of bone marrow-derived macrophageBone 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 modelBMDMs (2 × 106cells)were plated in 24-well plates (Nunc,Denmark) in complete DMEM (10% FBS,1% penicillinstreptomycin) per well and left to adhere for 12 h.BMDMs were then infected with log phaseM.tuberculosisH37Rv for 4 h (day 0) and 3 and 5 days in a BSL-3 laboratory.M.tuberculosisH37Rv 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 intracellularM.tuberculosiswas determined after 3 weeks.

Comparative Genomic Analysis

The complete genome sequences and coding sequences (CDSs) ofM.intracellulare(NC_016946)andM.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].

Immune Proteomics

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 antimouse 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.

Statistical Analysis

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

RESULTS

Mycobacteria Intracellulare Proteins Predominantly Induce Th1-type Immunity in the Host

After immunizing mice three times withM.intracellulareprotein extracts or adjuvants only,splenic lymphocytes were isolated and stimulated withM.intracellulareproteins.M.intracellulareprotein extracts induced significantly higher levels of IFN-γ,IL-2,IL-12,and IL-6 (P＜ 0.0001,P＜ 0.001,P＜0.05,andP＜ 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).

Mycobacterium Intracellulare Protein Extracts Markedly Enhanced Antibody Responses

As shown in Figure 2,the prime and two primeboost immunizations withM.intracellulareprotein extracts induced strong and significant increase in IgG,IgG1,IgM,and IgG2a titers compared with preimmunization and the adjuvant alone group at each detection point (Pvalues were all ＜ 0.001).

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.

Immunization Significantly Improved the Ability of Bone Marrow-derived Macrophage to Inhibit M.tuberculosis Growth ex vivo

BMDMs isolated from mice immunized withM.intracellulareprotein 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 intracellularM.tuberculosisin 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).

Comparative Genomic Analysis of Mycobacterium

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.

Identification of Common Antigens

In the serum ofM.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.

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.

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

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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).

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.

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 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.

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.

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.

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.

Table 1.Annotation information for the two genomic sequences

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

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).

Figure 3.Immunization with M.intracellulare protein extracts enhanced the ability of bone marrowderived 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 twotailed Student's t test.

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

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DISCUSSION

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].SeveralM.tuberculosissecretory proteins have been used in newly designed anti-TB vaccines,and some of them showed excellent immune responses and protective efficacy againstM.tuberculosisinfection[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 NTMM.vaccaeused in vaccines had promising protective effects against TB[12].Several vaccines based onM.vaccaeantigens,such as Vaccae?,DAR-901,and SRL172,have entered different phases of clinical trials[27].SRL172,an inactivated,whole-cell vaccine prepared fromM.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 HIVinfected adults who had received BCG at birth[29].The excellent immune effect ofM.vaccaeindicates 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 ofM.intracellularewhole bacterial protein extracts in BALB/c mice to explore the possibility of usingM.intracellulareas a source of immune effective antigens for developing anti-TB vaccines.Our results showedthat M.intracellularebacterial 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 withM.intracellularebacterial 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 againstM.tuberculosisin 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 inM.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 againstM.tuberculosisinfection,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 thatM.intracellulareproteins could induce a Th1/Th2 balance shift toward Th1 in BALB/c mice,which favors the control ofM.tuberculosisinvivo.

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,includingM.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 thatM.intracellularebacterial 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 againstM. tuberculosisinfection[46,47].During its long-term interaction with macrophages,M. tuberculosisdevelops 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 fromM.intracellulare-immunized mice had a significant ability to inhibit the growth ofM.tuberculosiscompared to those from control micein vitro,indicating thatM.intracellularebacterial proteins can effectively change the host immune system environment to impairM.tuberculosissurvival.One explanation for this result may be that bacterial protein immunization upregulates the expression of specific genes in BMDMs and thus helps to impedeM.tuberculosisgrowth.Therefore,more specific experiments,including transcriptome analysis or other methods,are required to reveal the relevant mechanisms.Our results also suggest the possibility of usingM.intracellulareantigenic proteins for the development of TB vaccines.

To gain insights into the antigens expressed among theM.intracellularebacterial proteins that changed the immune properties of mice to preventM.tuberculosisinfection,a protein microarray coated with 4,219 proteins ofM.tuberculosiswas used to identify the antigen repertoire by cross-reacting with IgG and IgM in the sera of mice immunized with wholeM.intracellulareproteins.A total of 478M.tuberculosisproteins 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 betweenM.intracellulareandM. tuberculosis,we primarily obtained an insight into the main biological processes and pathways involved in shared antigens betweenM.tuberculosisandM.intracellulare.In the proteinprotein 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 crossimmune vaccines for bothM.intracellulareandM.tuberculosisin the future.

In summary,the present study demonstrates the possibility of whole bacterial protein extracts ofM.intracellulareto serve as antigens against TB vaccines and reveals a number of proteins with cross-reactivity betweenM.intracellulareandM.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 againstM.tuberculosisinfection.Further studies are required to cautiously choose immunodominant antigens,which are crucial for developing novel vaccines for the control of TB.

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS' CONTRIBUTIONS

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 theM.intracellularestrain.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.

ACKNOWLEDGEMENTS

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.