Subunit Vaccine Consisting of Multi-Stage Antigens Has High Protective Efficacy against Mycobacterium tuberculosis Infection in Mice

To search for more effective tuberculosis (TB) subunit vaccines, antigens expressed in different growth stages of Mycobacterium tuberculosis (M. tuberculosis), such as RpfE (Rv2450c) produced in the stage of resuscitation, Mtb10.4 (Rv0288), Mtb8.4 (Rv1174c), ESAT6 (Rv3875), Ag85B (Rv1886c) mainly secreted by replicating bacilli, and HspX (Rv2031c) highly expressed in dormant bacilli, were selected to construct six fusion proteins: ESAT6-Ag85B-MPT64_190-198-Mtb8.4 (EAMM), Mtb10.4-HspX (MH), ESAT6-Mtb8.4, Mtb10.4-Ag85B, ESAT6-Ag85B, and ESAT6-RpfE. The six fusion proteins were separately emulsified in an adjuvant composed of N,N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA), polyribocytidylic acid (poly I:C) and gelatin to construct subunit vaccines, and their protective effects against M. tuberculosis infection were evaluated in C57BL/6 mice. Furthermore, the boosting effects of EAMM and MH in the adjuvant of DDA plus trehalose 6,6''-dimycolate (TDM) on BCG-induced immunity were also evaluated. It was found that the six proteins were stably produced in E. coli and successfully purified by chromatography. Among them, EAMM presented the most effective protection against M. tuberculosis. Interestingly, the mice that received EAMM+MH had significantly lower bacterial counts in the lungs and spleens than the single protein vaccinated groups, and had the same effect as those that received BCG. In addition, EAMM and MH could improve BCG-primed protective efficacy against M. tuberculosis infection in mice. In conclusion, the combination of EAMM and MH containing antigens from both replicating and dormant stages of the bacilli could induce robust immunity against M. tuberculosis infection in mice and may serve as promising subunit vaccine candidate.     

A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells with Higher Protective Efficacy against Tuberculosis

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that is a major public health problem. The vaccine used for TB prevention is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine in vivo was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials.     

The fbpA/sapM double knock out strain of Mycobacterium tuberculosis is highly attenuated and immunogenic in macrophages

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.     

BCG vaccine mediated reduction in the MHC-II expression of macrophages and dendritic cells is reversed by activation of Toll-like receptors 7 and 9

Tuberculosis is a major cause of death in mankind and BCG vaccine protects against childhood but not adult tuberculosis. BCG avoids lysosomal fusion in macrophages decreasing peptides required for activating CD4 T cells and Th1 immunity while suppressing the expression of MHC-II by antigen presenting cells (APCs). An in vitro model of antigen presentation showed that ligands for TLR-9, 7, 4 and 1/2 increased the ability of APCs to present antigen-85B of BCG to CD4 T cells, which correlated with an increase in MHC-II expression. TLR-activation led to a down-regulation of MARCH1 ubiquitin ligase which prevents the degradation of MHC-II and decreased IL-10 also contributed to an increase in MHC-II. TLR-activation induced up-regulation of MHC-II was inhibited by the blockade of IRAK, NF-kB, and MAPKs. TLR-7 and TLR-9 ligands had the most effective adjuvant like effect on MHC-II of APCs which allowed BCG vaccine mediated activation of CD4 T cells.     

Evaluation of the anti-tuberculosis activity generated by different multigene DNA vaccine constructs

Development of multigenic constructs expressing Mycobacterium tuberculosis (Mtb) antigens may be a strategy to obtain improved DNA vaccines against tuberculosis (TB). Several multigenic constructs expressing two or three Mtb antigens as fusion proteins were developed, both as tPA- and ubiquitin-fusion proteins. To demonstrate proper protein expression and intracellular turnover all multiantigens were tagged with the HA epitope and constructs were used to transfect rhabdomyosarcoma (RD) cells. Antigen expression was demonstrated by immunofluorescence using anti-HA antibodies. C57Bl/6 mice were immunized with selected constructs and protective activity was assessed following aerogenic challenge with Mtb. Several of these constructs induced a significant level of protection in the lung and in the spleen. Immunization with the construct expressing tPA85B-ES6 induced a level of protection that approached that provided by BCG. Immunization with a combination of these constructs induced levels of protection that were not superior to those elicited by a single combination, and immunization with a construct expressing five Mtb antigens could not provide an improved level of protection compared to tPA85B-ES6. We conclude that the activity of a DNA vaccine based on tPA85B-ES6 cannot be enhanced by broadening the antigen repertoire with other highly immunogenic secreted Mtb proteins.     

Immune responses and protective efficacy of the gene vaccine expressing Ag85B and ESAT6 fusion protein from Mycobacterium tuberculosis

Genetic immunity is a new promising approach for the development of novel tuberculosis vaccines. In this study, it is shown that DNA vaccines expressing the fusion protein of antigen 85B (Ag85B) and early secreted antigenic target 6-kDa antigen (ESAT6) can induce high levels of specific IgG2a antibody subtype in the mice. With the prolongation of postimmunization time, the levels of IgG2a antibody decrease gradually. Although a high-level specific IgG2a antibody subtype is also elicited by classical BCG, the ratio of antibody subtypes IgG2a to IgG1 changes 4 weeks after immunization, and IgG1 is gradually shifted to the main antibody subtype. DNA vaccines also elicit cellular immunity as shown by specific spleen lymphocytes proliferation to Ag85B or ESAT6 protein and the production of high levels of IFN-gamma and IL-2, which is similar to that elicited by BCG. Vaccination of mice with DNA vaccines expressing the fusion protein Ag85B-ESAT6 results in a significant level of protection against the subsequent high-dose challenge with virulent Mycobacterium tuberculosis (MTB) H37Rv. Dramatic reduction in the number of MTB colony-forming units in the spleens and lungs is observed. Pathological examination showed that recombinant plasmid and BCG groups have only minor damage and organizational structures that are kept relatively complete, while in the control group, spleens and lungs are damaged seriously. Therefore, although the reducing degree of mycobacterial loads in the organ of mice immunized with recombinant plasmid is not more than that of BCG, through the analysis of pathological changes, we may conclude that the protective effect provided by DNA vaccine expressing the Ag85B-ESAT6 fusion protein is equivalent to that afforded by the classical BCG.     

Low Cost Tuberculosis Vaccine Antigens in Capsules: Expression in Chloroplasts,Bio-Encapsulation,Stability and Functional Evaluation In Vitro

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts.     

Cancer Associated Aberrant Protein O-Glycosylation Can Modify Antigen Processing and Immune Response

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/− glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.     

Prime-boost vaccination with rBCG/rAd35 enhances CD8⁺ cytolytic T-cell responses in lesions from Mycobacterium tuberculosis-infected primates

To prevent the global spread of tuberculosis (TB) infection, a novel vaccine that triggers potent and long-lived immunity is urgently required. A plasmid-based vaccine has been developed to enhance activation of major histocompatibility complex (MHC) class I–restricted CD8+ cytolytic T cells using a recombinant Bacille Calmette-Guérin (rBCG) expressing a pore-forming toxin and the Mycobacterium tuberculosis (Mtb) antigens Ag85A, 85B and TB10.4 followed by a booster with a nonreplicating adenovirus 35 (rAd35) vaccine vector encoding the same Mtb antigens. Here, the capacity of the rBCG/rAd35 vaccine to induce protective and biologically relevant CD8⁺ T-cell responses in a nonhuman primate model of TB was investigated. After prime/boost immunizations and challenge with virulent Mtb in rhesus macaques, quantification of immune responses at the single-cell level in cryopreserved tissue specimen from infected organs was performed using in situ computerized image analysis as a technological platform. Significantly elevated levels of CD3⁺ and CD8⁺ T cells as well as cells expressing interleukin (IL)-7, perforin and granulysin were found in TB lung lesions and spleen from rBCG/rAd35-vaccinated animals compared with BCG/rAd35-vaccinated or unvaccinated animals. The local increase in CD8⁺ cytolytic T cells correlated with reduced expression of the Mtb antigen MPT64 and also with prolonged survival after the challenge. Our observations suggest that a protective immune response in rBCG/rAd35-vaccinated nonhuman primates was associated with enhanced MHC class I antigen presentation and activation of CD8+ effector T-cell responses at the local site of infection in Mtb-challenged animals.     

Multi-Stage Tuberculosis Subunit Vaccine Candidate LT69 Provides High Protection against Mycobacterium tuberculosis Infection in Mice

Effective tuberculosis (TB) vaccine should target tubercle bacilli with various metabolic states and confer long-term protective immunity. In this study, we constructed a novel multi-stage TB subunit vaccine based on fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-HspX (LT69 for short) which combined early expressed antigens and latency-associated antigen. The fusion protein was mixed with an adjuvant being composed of N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA) and polyriboinosinic polyribocytidylic acid (PolyI:C) to construct subunit vaccine, whose immunogenicity and protective ability were evaluated in C57BL/6 mice. The results showed that LT69 had strong immunogenicity and high protective effect against Mycobacterium tuberculosis (M. tuberculosis) H37Rv aerosol challenge. Low-dose (2 μg) of LT69 generated long-term immune memory responses and provided effective protection, which was even higher than traditional vaccine BCG did at 30 weeks post the last vaccination. In conclusion, multistage subunit vaccine LT69 showed high and long-term protection against M. tuberculosis infection in mice, whose effect could be enhanced by using a relative low dosage of antigen.     

Study of systemic fever reaction of the delayed type hypersensitivity

The possibility of elaborating a model of fever reaction to a simple protein antigen, ovalbumin, was investigated. Administration of the antigen in adjuvants into the foot-pads or intravenously proved unsatisfactory and did not sensitize the animal to induction of a fever reaction to a challenging dose of antigen. Sensitization by the simultaneous intravenous administration of ovalbumin together with living BCG vaccine yielded positive results. The fever response to ovalbumin is specific, since rabbits sensitized with BCG vaccine only did not respond to the administration of ovalbumin. The degree of fever reactions to tuberculin and ovalbumin in the individual rabbits was more or less proportional. The given model is reproducible and is useful for experimental studies. Further experiments will be necessary, however, for detailed characterization and for analysis of the mechanism (antibody-mediated or delayed type hypersensitivity).     

Live antigen carriers as tools for improved anti-tuberculosis vaccines

Recombinant (r) Mycobacterium bovis BCG strains have been constructed which secrete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes. In human and murine macrophage-like cell lines, intracellular persistence of these r-BCG strains was reduced as compared to the parental BCG strain. By immunogold labelling Hly was detected in membrane structures and within the phagosomal space of macrophages. Hly fusions consistently co-localized with a lysosome-associated membrane glycoprotein (LAMP-1) suggesting that membrane attack conformation of Hly was not altered. Although r-BCG microorganisms apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of co-phagocytosed soluble ovalbumin as compared with wild-type BCG microbes. These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells. Because CD8 T cells play a major role in protection against tuberculosis such Hly-secreting r-BCG constructs are anti-tuberculosis vaccine candidates. In addition, we report on our r-Salmonella typhimurium expression system combined with the HlyB/HlyD/ TolC export machinery for delivering the prominent mycobacterial antigen Ag85B for immune recognition.     

三重表达肺结核DNA疫苗在细胞水平的检测

目的 检测共表达siRNA、抗原基因与hIL-12的新型肺结核DNA疫苗在人胚肾293细胞中表达。方法 在已构建含有靶向抗调亡基因Mcl-1L的siRNA、结核分枝杆菌抗原基因Ag85B-ESAT6 (PVAE)、hIL-12的新型肺结核DNA疫苗pVAX-siRNA-PVAE-IL-12基础之上,将抗原基因与增强型绿色荧光蛋白（EGFP）基因融合,得到真核表达重组质粒pVAX1-siRNA-PVAE-EGFP-hIL12。并将siRNA单元用靶向EGFP基因的siEGFP代替,得到pVAX1-siEGFP-PVAE-EGFP-hIL12重组表达载体。用重组质粒转染人胚肾细胞293,分别观察融合抗原基因与siEGFP的表达。以ELISA测定培养细胞上清中hIL-12的表达。结果 经酶切鉴定和测序证实肺结核DNA疫苗改造成功。转染细胞中检测到绿色荧光,证实抗原表达。对照组检测到siEGFP的表达。转染48 h后检测出细胞上清中hIL-12的量为1571.63 pg/mL;72 h后检测出细胞上清中hIL-12的量为2392.25pg/mL。结论 已构建的肺结核质粒DNA疫苗能在真核细胞中有效表达siRNA、抗原基因与hIL-12。为进一步研究该DNA疫苗抗肺结核的免疫治疗和基因治疗效果打下基础。     

Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.     

ESAT6 differentially inhibits IFN-γ-inducible class II transactivator isoforms in both a TLR2-dependent and -independent manner

Mycobacterium tuberculosis (Mtb) downregulates the surface expression of major histocompatibility class II (MHC II) molecules on macrophages via modulating class II transactivator (CIITA) protein of the host cell. This results in decreased effector function of CD4(+) T cells. In macrophages, CIITA is transcribed by the promoters I (pI) and IV (pIV) and the corresponding gene products are referred to as type I and type IV CIITA, respectively. Earlier studies have mainly focused on CIITA transcribed by pIV; however, these studies also showed that type IV CIITA expression was transient and dispensable for MHC II expression. In the present study, we observed that the Mtb 6-kDa, early secreted antigen (ESAT6) inhibited interferon (IFN)-γ-induced type I as well as type IV CIITA, but, interestingly, inhibition of type I CIITA was found to be independent of Toll-like receptor-2 (TLR2), whereas that of type IV was TLR2 dependent. Moreover, we also present evidence to show that ESAT6-mediated inhibition was regulated via remodeling of the chromatin. We found that ESAT6 caused a decrease in the IFN-γ-stimulated methylation of the histone H3K4, as well as in the levels of histone acetylation at the CIITA pI locus in macrophages. We also found the involvement of mitogen-activated protein kinases ERK1/2 and p38 in the regulation of CIITA by ESAT6. In conclusion, our studies suggest that ESAT6 could inhibit the expression of type I and type IV CIITA through different pathways. Furthermore, ESAT6 could signal through putative receptors other than TLR2, and that the inhibition of IFN-γ-stimulated CIITA by ESAT6 was regulated at the chromatin level.     

Epitope-driven TB vaccine development: a streamlined approach using immuno-informatics, ELISpot assays, and HLA transgenic mice

New vaccine candidates that might better control the worldwide prevalence of Mycobacterium tuberculosis (Mtb) have yet to be described. Strong CD4+ T cell-mediated immune response (CMI) is correlated with protection from the development of TB disease; however, the selection of suitable vaccine antigens has been thwarted by the size and complexity of the (Mtb) proteome, and by the relative difficulty of delivering these antigens in the right immunological context. One possible solution is to develop immunotherapeutic vaccines for TB that are based on T cell epitopes representing multiple antigens. This text illustrates the stepwise development of epitope-driven vaccines from in silico epitope mapping to testing the vaccine in a live Mtb challenge model. First, we used the whole genome Mtb microarray to identify bacterial proteins expressed under the conditions thought to model Mtb survival and replication in human macrophages. Eighteen of these proteins were also found by Behr et al. to be absent from at least one strain of BCG; the sequences of these eighteen proteins were then screened for T-cell epitopes using the immuno-informatics algorithm, EpiMatrix. Of the seventeen representative epitopes evaluated in ELISpot assays, all seventeen were confirmed to elicit interferon (IFN)-gamma secretion by PBMC from Mtb-exposed subjects. A parallel live Mtb challenge study in mice showed prototype epitope-based TB vaccines to be robustly immunogenic but not as effective as BCG. These experiments illustrate the use of immuno-informatics tools for vaccine development and describe a pathway for the development of a more effective, epitope-driven, immunotherapeutic vaccine for TB.     

Immunological Memory Transferred with CD4 T Cells Specific for Tuberculosis Antigens Ag85B-TB10.4: Persisting Antigen Enhances Protection

Background

High levels of death and morbidity worldwide caused by tuberculosis has stimulated efforts to develop a new vaccine to replace BCG. A number of Mycobacterium tuberculosis (Mtb)-specific antigens have been synthesised as recombinant subunit vaccines for clinical evaluation. Recently a fusion protein of TB antigen Ag85B combined with a second immunodominant TB antigen TB10.4 was emulsified with a novel non-phospholipid-based liposomal adjuvant to produce a new subunit vaccine, investigated here. Currently, there is no consensus as to whether or not long-term T cell memory depends on a source of persisting antigen. To explore this and questions regarding lifespan, phenotype and cytokine patterns of CD4 memory T cells, we developed an animal model in which vaccine-induced CD4 memory T cells could transfer immunity to irradiated recipients.

Methodology/Principal Findings

The transfer of protective immunity using Ag85B-TB10.4-specific, CD45RB^low CD62L^low CD4 T cells was assessed in sub-lethally irradiated recipients following challenge with live BCG, used here as a surrogate for virulent Mtb. Donor T cells also carried an allotype marker allowing us to monitor numbers of antigen-specific, cytokine-producing CD4 T cells in recipients. The results showed that both Ag85B-TB10.4 and BCG vaccination induced immunity that could be transferred with a single injection of 3×10⁶ CD4 T cells. Ten times fewer numbers of CD4 T cells (0.3×10⁶) from donors immunised with Ag85B-TB10.4 vaccine alone, transferred equivalent protection. CD4 T cells from donors primed by BCG and boosted with the vaccine similarly transferred protective immunity. When BCG challenge was delayed for 1 or 2 months after transfer (a test of memory T cell survival) recipients remained protected. Importantly, recipients that contained persisting antigen, either live BCG or inert vaccine, showed significantly higher levels of protection (p<0.01). Overall the numbers of IFN-γ-producing CD4 T cells were poorly correlated with levels of protection.

Conclusions/Significance

The Ag85B-TB10.4 vaccine, with or without BCG-priming, generated TB-specific CD4 T cells that transferred protective immunity in mice challenged with BCG. The level of protection was enhanced in recipients containing a residual source of specific antigen that could be either viable or inert.     

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205⁺ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4⁺ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ⁺ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205⁺ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.     

结核分枝杆菌抗原重组酿酒酵母免疫诱导小鼠特异性免疫应答

近年来基于重组酿酒酵母全细胞的新型疫苗研究报道不断出现。以结核杆菌重要保护抗原ESAT6和Ag85B为对象,采用pHR酿酒酵母表达系统,构建了两种分别表达ESAT6-Ag85B(EA)和IFN-γ-ESAT6-Ag85B(IEA)融合抗原的重组酿酒酵母Yeast-EA和Yeast-IEA。重组酵母以皮下注射方式免疫小鼠后,小鼠产生高水平Ag85B特异性抗体,淋巴细胞分泌IFN-γ、IL-2等细胞因子,无IL-4产生,发生Th1型细胞免疫应答,其中Yeast-IEA效应更强,优于传统的BCG疫苗。实验证实重组酵母能够刺激树突状细胞的成熟分化。研究结果显示结核分枝杆菌抗原重组酿酒酵母全细胞疫苗具有发展成为新型抗结核疫苗的潜力。     

Cloning and expression of MPT83 gene from Mycobacterium tuberculosis in E. coli BL21 as vaccine candidate of tuberculosis: A preliminary study

The appearance of Mycobacterium tuberculosis strains leading to drug resistance has caused new problems in TB treatment in various parts of the world and forces WHO to declare TB as a global emergency. With the increase of TB drug resistance, it is convinced that a more effective vaccine development will stop the epidemic of TB. Some M. tuberculosis antigens, one of which is MPT83, have been examined as TB vaccine candidate. MPT83 antigen, which is very immunogenic in lipoprotein micro bacteria, is identified as surface cell interrelated to antigen with cytometry circulation. Having TB resistance from BCG vaccine, MPT83 is considered TB vaccine candidate that can protect people against TB at adult age. The purpose of this research is to conduct amplification of MPT83 antigen cloning, and expression of its antigen on E. coli bacteria. From the result of the research, it is expected that raw material to produce TB vaccine as well as a high-quality antigen can be obtained. The band of DNA in PCR product is 660?bp, while the one in pGEMT-Easy-Mpt83 recombinant plasmid is 3678?bp. This is expressed in E. coli BL21 strain and produces 48?kDa protein as well as GST-MPT83 fusion protein.