Protective Enterotoxigenic Escherichia coli Antigens in a Murine Intranasal Challenge Model

Enterotoxigenic Escherichia coli (ETEC) is an endemic health threat in underdeveloped nations. Despite the significant effort extended to vaccine trials using ETEC colonization factors, these approaches have generally not been especially effective in mediating cross-protective immunity. We used quantitative proteomics to identify 24 proteins that differed in abundance in membrane protein preparations derived from wild-type vs. a type II secretion system mutant of ETEC. We expressed and purified a subset of these proteins and identified nine antigens that generated significant immune responses in mice. Sera from mice immunized with either the MltA-interacting protein MipA, the periplasmic chaperone seventeen kilodalton protein, Skp, or a long-chain fatty acid outer membrane transporter, ETEC_2479, reduced the adherence of multiple ETEC strains differing in colonization factor expression to human intestinal epithelial cells. In intranasal challenge assays of mice, immunization with ETEC_2479 protected 88% of mice from an otherwise lethal challenge with ETEC H10407. Immunization with either Skp or MipA provided an intermediate degree of protection, 68 and 64%, respectively. Protection was significantly correlated with the induction of a secretory immunoglobulin A response. This study has identified several proteins that are conserved among heterologous ETEC strains and may thus potentially improve cross-protective efficacy if incorporated into future vaccine designs.     

Heterologous Prime Boost Regimes with N-terminal Peptides of Ag85B Induces Better Protection than Ag85B and BCG in Murine Model of Tuberculosis

Limited experimental evidences are available on the use of peptides as vaccines to boost BCG induced immunity for protection against tuberculosis. The present study therefore evaluated protective efficacy of booster dose of N-terminal peptides of Ag85B, using prime boost approaches in murine model of tuberculosis. Using earlier established subcutaneous murine model of TB in our laboratory, we compared the protective vaccination efficacy of peptides of Ag85B with that of booster dose of whole Ag85B and BCG by evaluating both antibody and cell-mediated immune response. Groups of mice primed by BCG and boosted with Ag85B peptides showed limited pulmonary bacillary burden and reduced lung pathology after challenge with virulent dose of Mycobacterium tuberculosis in mice. Significant levels (p < 0.001) of BCG specific antibodies (anti-BCG, anti-PPD) and T cell-specific cytokines were observed in Ag85B peptides boosted mice compared to Ag85B and BCG. Ag85B and BCG boosted mice however showed significant protection compared to single BCG dose and unvaccinated control groups. Our result suggests that prime boost strategy using N-terminal peptides of Ag85B may improve immunogenicity of BCG against TB. Such peptides may be attractive candidates for boosting waning BCG induced immune response in near future. However study demands further work including improvisation in experimental designs to justify the results.     

Evaluation of the Protective Immunity of a Novel Subunit Fusion Vaccine in a Murine Model of Systemic MRSA Infection

Staphylococcus aureus is a common commensal organism in humans and a major cause of bacteremia and hospital acquired infection. Because of the spread of strains resistant to antibiotics, these infections are becoming more difficult to treat. Therefore, exploration of anti-staphylococcal vaccines is currently a high priority. Iron surface determinant B (IsdB) is an iron-regulated cell wall-anchored surface protein of S. aureus. Alpha-toxin (Hla) is a secreted cytolytic pore-forming toxin. Previous studies reported that immunization with IsdB or Hla protected animals against S. aureus infection. To develop a broadly protective vaccine, we constructed chimeric vaccines based on IsdB and Hla. Immunization with the chimeric bivalent vaccine induced strong antibody and T cell responses. When the protective efficacy of the chimeric bivalent vaccine was compared to that of individual proteins in a murine model of systemic S. aureus infection, the bivalent vaccine showed a stronger protective immune response than the individual proteins (IsdB or Hla). Based on the results presented here, the chimeric bivalent vaccine affords higher levels of protection against S. aureus and has potential as a more effective candidate vaccine.     

Influence of Maternal Gestational Treatment with Mycobacterial Antigens on Postnatal Immunity in an Experimental Murine Model

Background

It has been proposed that the immune system could be primed as early as during the fetal life and this might have an impact on postnatal vaccination. Therefore, we addressed in murine models whether gestational treatment with mycobacterial antigens could induce better immune responses in the postnatal life.

Methods/Findings

BALB/c mice were treated subcutaneously (s.c.) at the second week of gestation with antigen (Ag)85A or heparin-binding hemagglutinin (HBHA) in the absence of adjuvant. Following birth, offspring mice were immunized intranasally (i.n.) with the same antigens formulated with the adjuvant cholera toxin (CT) at week 1 and week 4. One week after the last immunization, we assessed antigen-specific recall interferon gamma (IFN-γ) responses by in vitro restimulation of lung-derived lymphocytes. Protection against infection was assessed by challenge with high dose Mycobacterium bovis Bacille Calmette-Guérin (BCG) given i.n. We found that recall IFN-γ responses were higher in the offspring born to the treated mother compared to the untreated-mother. More importantly, we observed that the offspring born to the treated mother controlled infection better than the offspring born to the untreated mother. Since the gestational treatment was done in absence of adjuvant, essentially there was no antibody production observed in the pregnant mice and therefore no influence of maternal antibodies was expected. We hypothesized that the effect of maternal treatment with antigen on the offspring occurred due to antigen transportation through placenta. To trace the antigens, we conjugated fluorescent nanocrystals with Ag85A (Qdot-ITK-Ag85A). After inoculation in the pregnant mice, Qdot-ITK-Ag85A conjugates were detected in the liver, spleen of pregnant females and in all the fetuses and placentas examined.

Conclusion

The fetal immune system could be primed in utero by mycobacterial antigens transported through the placenta.     

Protective Immunity against Trichinella spiralis Infection Induced by a Multi-Epitope Vaccine in a Murine Model

Trichinellosis is one of the most important food-borne parasitic zoonoses throughout the world. Because infected pigs are the major source of human infections, and China is becoming the largest international producer of pork, the development of a transmission-blocking vaccine to prevent swine from being infected is urgently needed for trichinellosis control in China. Our previous studies have demonstrated that specific Trichinella spiralis paramyosin (Ts-Pmy) and Ts-87 antigen could provide protective immunity against T. spiralis infection in immunized mice. Certain protective epitopes of Ts-Pmy and Ts-87 antigen have been identified. To identify more Ts-Pmy protective epitopes, a new monoclonal antibody, termed 8F12, was produced against the N-terminus of Ts-Pmy. This antibody elicited significant protective immunity in mice against T. spiralis infection by passive transfer and was subsequently used to screen a random phage display peptide library to identify recognized epitopes. Seven distinct positive phage clones were identified and their displayed peptides were sequenced. Synthesized epitope peptides conjugated to keyhole limpet hemocyanin were used to immunize mice, four of which exhibited larval reduction (from 18.7% to 26.3%, respectively) in vaccinated mice in comparison to the KLH control. To increase more effective protection, the epitope 8F7 that was found to induce the highest protection in this study was combined with two other previously identified epitopes (YX1 from Ts-Pmy and M7 from Ts-87) to formulate a multi-epitope vaccine. Mice immunized with this multi-epitope vaccine experienced a 35.0% reduction in muscle larvae burden after being challenged with T. spiralis larvae. This protection is significantly higher than that induced by individual-epitope peptides and is associated with high levels of subclasses IgG and IgG1. These results showed that a multi-epitope vaccine induced better protective immunity than an individual epitope and provided a feasible approach for developing a safer and more effective vaccine against trichinellosis.     

结核杆菌Ag85B基因疫苗的免疫保护效果研究

为研究结核杆菌Ag85B基因疫苗的免疫原性和免疫保护效果,将雌性C57BL/6N小鼠32只,随机分为4组,即结核杆菌Ag85B基因疫苗组、BCG组、pcDNA3.1( )组和PBS组.取各免疫组小鼠脾细胞培养上清检测细胞因子水平;同时进行CTL杀伤活性检测;进一步用结核杆菌H37Rv国际标准强毒株静脉注射攻击小鼠,计数肺和脾组织中的结核杆菌菌落数,对小鼠部分肺和脾组织作病理切片,HE染色观察组织病变程度,Z-N染色检查抗酸杆菌,观察该疫苗对小鼠结核杆菌感染的免疫保护效果.结果表明,结核杆菌Ag85B基因疫苗对小鼠结核杆菌感染有一定的免疫保护效果,能够诱导较强的抗原特异性Th1型细胞免疫应答,细胞因子IFN-γ和IL-1分泌增加,IL-4分泌减少,CTL特异性活性增加;使小鼠肺和脾组织中的结核杆菌菌落数较空载体组显著减少,组织病变明显减轻.     

Nitric Oxide Releasing Nanoparticles Are Therapeutic for Staphylococcus aureus Abscesses in a Murine Model of Infection

Staphylococcus aureus (SA) is a leading cause of a diverse spectrum of bacterial diseases, including abscesses. Nitric oxide (NO) is a critical component of the natural host defense against pathogens such as SA, but its therapeutic applications have been limited by a lack of effective delivery options. We tested the efficacy of a NO-releasing nanoparticle system (NO-np) in methicillin-resistant SA (MRSA) abscesses in mice. The results show that the NO-np exert antimicrobial activity against MRSA in vitro and in abscesses. Topical or intradermal NO-np treatment of abscesses reduces the involved area and bacterial load while improving skin architecture. Notably, we evaluated pro- and anti-inflammatory cytokines that are involved in immunomodulation and wound healing, revealing that NO-np lead to a reduction in angiogenesis preventing bacterial dissemination from abscesses. These data suggest that NO-np may be useful therapeutics for microbial abscesses.     

The Role of Scavenger Receptor B1 in Infection with Mycobacterium tuberculosis in a Murine Model

Background

The interaction between Mycobacterium tuberculosis (Mtb) and host cells is complex and far from being understood. The role of the different receptor(s) implicated in the recognition of Mtb in particular remains poorly defined, and those that have been found to have activity in vitro were subsequently shown to be redundant in vivo.

Methods and Findings

To identify novel receptors involved in the recognition of Mtb, we screened a macrophage cDNA library and identified scavenger receptor B class 1 (SR-B1) as a receptor for mycobacteria. SR-B1 has been well-described as a lipoprotein receptor which mediates both the selective uptake of cholesteryl esters and the efflux of cholesterol, and has also recently been implicated in the recognition of other pathogens. We show here that mycobacteria can bind directly to SR-B1 on transfected cells, and that this interaction could be inhibited in the presence of a specific antibody to SR-B1, serum or LDL. We define a variety of macrophage populations, including alveolar macrophages, that express this receptor, however, no differences in the recognition and response to mycobacteria were observed in macrophages isolated from SR-B1^−/− or wild type mice in vitro. Moreover, when wild type and SR-B1^−/− animals were infected with a low dose of Mtb (100 CFU/mouse) there were no alterations in survival, bacterial burdens, granuloma formation or cytokine production in the lung. However, significant reduction in the production of TNF, IFNγ, and IL10 were observed in SR-B1^−/− mice following infection with a high dose of Mtb (1000 CFU/mouse), which marginally affected the size of inflammatory foci but did not influence bacterial burdens. Deficiency of SR-B1 also had no effect on resistance to disease under conditions of varying dietary cholesterol. We did observe, however, that the presence of high levels of cholesterol in the diet significantly enhanced the bacterial burdens in the lung, but this was independent of SR-B1.

Conclusion

SR-B1 is involved in mycobacterial recognition, but this receptor plays only a minor role in anti-mycobacterial immunity in vivo. Like many other receptors for these pathogens, the loss of SR-B1 can be functionally compensated for under normal conditions.     

Persistent Salmonellosis Causes Pancreatitis in a Murine Model of Infection

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b⁺ F4/80⁺, CD11b⁺ Ly6C^int Ly6G⁺ and CD11b⁺ Ly6C^hi Ly6G⁻ cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma.     

Low-Density Granulocytes Are Elevated in Mycobacterial Infection and Associated with the Severity of Tuberculosis

Tuberculosis remains a global health problem caused by infection with Mycobacterium tuberculosis. Numerous studies have established a close correlation between the development of tuberculosis and the roles of neutrophils. Recently, a distinct population of CD15⁺ granulocytes was found to be present in the peripheral blood mononuclear cell (PBMC) fraction in humans. This population of granulocytes, termed low-density granulocytes (LDGs), was reported to be elevated and associated with disease activity or severity in a number of different conditions including SLE, asthma and HIV infection. However, both the frequency and clinical significance of LDGs associated with tuberculosis are unclear. Here we determined LDG levels and made comparisons between subjects with active pulmonary tuberculosis (PTB) and healthy controls, between PTB patients with mild-to-moderate disease and patients with advanced disease, and among PTB patients following anti-tuberculous therapy of varying durations. The direct correlation between M. tuberculosis infection and LDG levels was confirmed by in vitro infection of whole peripheral blood and isolated granulocytes with mycobacteria. Our results demonstrated that PBMCs in PTB patients contained significantly elevated percentages of LDGs compared with control subjects. LDGs in tuberculosis expressed higher levels of activation markers compared to normal-density granulocytes (NDGs). M. tuberculosis induced the generation of LDGs in both whole blood and isolated NDGs from control subjects, which suggests that LDGs associated with M. tuberculosis infection are likely to originate from in situ activation. Furthermore, our results revealed that the frequency of LDGs is associated with the severity of tuberculosis.     

Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model

Infection with a replication-competent bovine leukemia virus structural gene vector (BLV SGV) is an innovative vaccination approach to prevent disease by complex retroviruses. Previously we developed BLV SGV that constitutively expresses BLV gag, pol, and env and related cis-acting sequences but lacks tax, rex, RIII, and GIV and most of the BLV long terminal repeat sequences, including the cis-acting Tax and Rex response elements. The novel SGV virus is replication competent and replicates a selectable vector to a titer similar to that of the parental BLV in cell culture. The overall goal of this study was to test the hypothesis that infection with BLV SGV is nonpathogenic in rabbits. BLV infection of rabbits by inoculation of cell-free BLV or cell-associated BLV typically causes an immunodeficiency-like syndrome and death by 1 year postinfection. We sought to evaluate whether in vivo transfection of BLV provirus recapitulates pathogenic BLV infection and to compare BLV and BLV SGV with respect to infection, immunogenicity, and clinical outcome. Three groups of rabbits were subjected to in vivo transfection with BLV, BLV SGV, or negative control DNA. The results of our 20-month study indicate that in vivo transfection of rabbits with BLV recapitulates the fatal BLV infection produced by cell-free or cell-associated BLV. The BLV-infected rabbits exhibited sudden onset of clinical decline and immunodeficiency-like symptoms that culminated in death. BLV and BLV SGV infected peripheral blood mononuclear cells and induced similar levels of seroconversion to BLV structural proteins. However, BLV SGV exhibited a reduced proviral load and did not trigger the immunodeficiency-like syndrome. These results are consistent with the hypothesis that BLV SGV is infectious and immunogenic and lacks BLV pathogenicity in rabbits, and they support the use of this modified proviral vector delivery system for vaccines against complex retroviruses like BLV.     

c-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.     

Cytokine Profile of a Self-Healing Fonsecaea pedrosoi Infection in Murine Model

Chromoblastomycosis is a chronic infectious disease of the skin and subcutaneous tissue. However, the host-defence response to this fungal infection has not been investigated thoroughly. This study was carried out to analyse the sequential events and the change of local cytokine release in a murine model infected with Fonsecaea pedrosoi in footpad. The anti-inflammatory Th2 cytokine IL-10 demonstrated an upward trend up to 7 days post infection followed by a steady decline. The titers of TNF-α (a pro-inflammatory Th1 cytokine) increased up to 7 days post infection followed by a relatively steady-state until full recovery. The anti-inflammatory cytokine IL-4 showed a similar pattern as TNF-α. The pro-inflammatory cytokine IFN-γ did not increased until 7 days post infection, while demonstrated an upward trend up to 30 days when the mice reached a full recovery from infection.     

Genetic Determinants of Reovirus Pathogenesis in a Murine Model of Respiratory Infection

Many viruses invade mucosal surfaces to establish infection in the host. Some viruses are restricted to mucosal surfaces, whereas others disseminate to sites of secondary replication. Studies of strain-specific differences in reovirus mucosal infection and systemic dissemination have enhanced an understanding of viral determinants and molecular mechanisms that regulate viral pathogenesis. After peroral inoculation, reovirus strain type 1 Lang replicates to high titers in the intestine and spreads systemically, whereas strain type 3 Dearing (T3D) does not. These differences segregate with the viral S1 gene segment, which encodes attachment protein σ1 and nonstructural protein σ1s. In this study, we define genetic determinants that regulate reovirus-induced pathology following intranasal inoculation and respiratory infection. We report that two laboratory isolates of T3D, T3D^C and T3D^F, differ in the capacity to replicate in the respiratory tract and spread systemically; the T3D^C isolate replicates to higher titers in the lungs and disseminates, while T3D^F does not. Two nucleotide polymorphisms in the S1 gene influence these differences, and both S1 gene products are involved. T3D^C amino acid polymorphisms in the tail and head domains of σ1 protein influence the sensitivity of virions to protease-mediated loss of infectivity. The T3D^C polymorphism at nucleotide 77, which leads to coding changes in both S1 gene products, promotes systemic dissemination from the respiratory tract. A σ1s-null virus produces lower titers in the lung after intranasal inoculation and disseminates less efficiently to sites of secondary replication. These findings provide new insights into mechanisms underlying reovirus replication in the respiratory tract and systemic spread from the lung.     

Ag85B DNA vaccine suppresses airway inflammation in a murine model of asthma

Background

In allergic asthma, Th2 lymphocytes are believed to play important roles in orchestrating airway eosinophilia and inflammation. Resetting the Th1/Th2 imbalance may have a therapeutic role in asthma. The mycobacterium tuberculosis 30-kilodalton major secretory protein (antigen 85B, Ag85B) can protect animals from M. tuberculosis infection by inducing a Th1-dominant response.

Methods

In this study, the Ag85B gene was cloned into pMG plasmids to yield the pMG-Ag85B plasmid. The expression of Ag85B gene in murine bronchial epithelia cells was detected by Western blotting and immunohistochemical staining after intranasal immunization with reconstructed pMG-Ag85B plasmids. The protective effect of pMG-Ag85B plasmids immunization in airway inflammation was evaluated by histological examination and bronchoalveolar lavage (BAL). IL-4 and IFN-γ levels in the BAL and supernatant from splenocyte culture were determined using ELISA kits.

Results

The Ag85B gene was successfully expressed in murine bronchial epithelia cells by intranasal immunization with reconstructed pMG-Ag85B plasmids. Using a murine model of asthma induced by ovalbumin (OVA), pMG-Ag85B immunization significantly inhibited cellular infiltration across the airway epithelium with a 37% decrease in the total number of cells (9.6 ± 2.6 × 10⁵/ml vs. 15.2 ± 3.0 × 10⁵/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 ± 0.2 × 10⁵/ml vs. 5.4 ± 1.1 × 10⁵/ml, p < 0.01) compared with the OVA-sensitized control group. There was no difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group. IL-4 production was significantly decreased in the BAL fluid (32.0 ± 7.6 pg/ml vs. 130.8 ± 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 ± 1.6 pg/ml vs. 10.1 ± 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-γ production was increased in the BAL fluid (137.9 ± 25.6 pg/ml vs. 68.4 ± 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 ± 5.4 pg/ml vs. 11.3 ± 3.2 pg/ml, p < 0.05).

Conclusion

In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration. This protective effect was associated with decreased IL-4 and increased IFN-γ production in the BAL fluid and in the supernatant of cultured splenocytes.     

Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection

The Bacillus Calmette - Guerin (BCG) vaccine provides a critical but limited defense against Mycobacterium tuberculosis (M.tb). More than 60 years after the widespread introduction of BCG, there is an urgent need for a better vaccine. A large body of pre-clinical research continues to support ongoing clinical trials to assess whether viral vectors expressing M.tb antigens that are shared by BCG and M.tb, can be used alongside BCG to enhance protection. A major focus involves using multiple unique viral vectors to limit anti-vector immunity and thereby enhance responses to the insert antigen delivered. The successful introduction of viral vector vaccines to target M.tb and other pathogens will be reliant on reducing the costs when using multiple vectors and inhibiting the development of unwanted anti-vector responses that interfere with the response to insert antigen. This study examines methods to reduce the logistical costs of vaccination by mixing different viral vectors that share the same insert antigen in one vaccine; and whether combining different viral vectors reduces anti-vector immunity to improve immunogenicity to the insert antigen. Here we show that a homologous prime-boost regimen with a mixture of MVA (Modified Vaccinia virus Ankara) and Ad5 (human adenovirus type 5) vectors both expressing Ag85A in a single vaccine preparation is able to reduce anti-vector immunity, compared with a homologous prime-boost regimen with either vector alone. However, the level of immunogenicity induced by the homologous mixture remained comparable to that induced with single viral vectors and was less immunogenic than a heterologous Ad5 prime-MVA-boost regimen. These findings advance the understanding of how anti-vector immunity maybe reduced in viral vector vaccination regimens. Furthermore, an insight is provided to the impact on vaccine immunogenicity from altering vaccination methods to reduce the logistical demands of using separate vaccine preparations in the field.     

结核分枝杆菌CFP10、ESAT6、Ag85A和Ag85B抗原真核共表达载体的构建与表达

CFP10、ESAT6、Ag85A和Ag85B是结核分枝杆菌的主要免疫优势抗原。为了构建一种可同时表达这4种抗原的真核多基因共表达载体pcDNA-CFP10-ESAT6-Ag85A-Ag85B(pcDNA-CEAB),并利用HEK 293T细胞对其体外表达进行检测。采用酶切、连接的方法,将CFP10和ESAT6编码基因以(Gly4Ser)3蛋白Linker连接,插入至质粒pcDNA3.1(+)多克隆位点CMV启动子与加尾信号BGH pA之间,使两者融合表达,将Ag85A和Ag85B编码基因以内部核糖体进入位点(Internal ribosome entry site,IRES)序列连接,并赋予RSV启动子和加尾信号BGH pA,使两者在RSV启动子作用下独立表达。重组质粒经酶切及测序验证后转染至HEK 293T细胞中进行体外表达实验,48 h后提取总蛋白,利用CFP10、ESAT6、Ag85A和Ag85B特异性抗体进行Western blotting检测。结果显示多基因共表达载体pcDNA-CEAB在真核细胞HEK 293T中得到表达,且CFP10、ESAT6、Ag85A和Ag85B抗原能被相应的特异性抗体所识别,表明质粒pcDNA-CEAB构建正确,这为进一步研究其免疫原性和免疫保护效果奠定了基础。     

Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.     

Bactofection with Toll-Like Receptor 4 in a Murine Model of Urinary Tract Infection

The role of innate immunity in the prevention of urinary tract infection is well-documented. Toll-like receptor 4 (TLR4) is a major determinant of innate immune response. In an animal model of urinary tract infection, bactofection-mediated gene transfer of TLR4 was tested in a preventive approach. Bactofection with TLR4 reduced the colonization with uropathogenic Escherichia coli by 91% in the kidney and by 41% in the bladder. Reduced colonization was associated with lower oxidative stress and expression of monocyte chemoattractant protein-1 and myeloperoxidase in the kidney. Bactofection with TLR4 was successful in the prevention of ascending pyelonephritis. Further studies should focus on long-term effects, the dose response and the potential therapeutic use in models of chronic urinary tract infection.     

共表达结核杆菌Ag85A-ESAT-6融合抗原与IL-21核酸疫苗的构建及免疫效果研究

目的：构建结核杆菌Ag85A-ESAT-6融合抗原与细胞因子IL-21共表达核酸疫苗pIRES-IL-21-Ag85A-ESAT-6,研究其对小鼠免疫功能的影响。方法：用PCR方法分别扩增出Ag85A和IL-21编码基因,并先后插入质粒pIRES-ESAT-6中,构成共表达核酸疫苗pIRES-IL-21-Ag85A-ESAT-6;免疫小鼠后,通过CTL和NK细胞杀伤活性和脾细胞增殖试验,以及小鼠血清抗体、IFN-γ和IL-4的检测,评价该核酸疫苗的免疫效果。结果：pIRES-IL-21-Ag85A-ESAT-6核酸疫苗免疫鼠的CTL活性、脾细胞增殖反应、IFN-γ水平及血清抗体产生明显高于对照组,差异有显著性意义。结论：pIRES-IL-21-Ag85A-ESAT-6核酸疫苗能够诱导小鼠产生有效的免疫反应,可作为新型结核疫苗的候选组分。