Protective Immune Responses Elicited by Fusion Protein Containing PsaA and PspA Fragments

Streptococcus pneumoniae is an important pathogen accounting for a large number of deaths worldwide. Due to drawbacks of the current polysaccharide-based vaccine, the most promising way to generate an improved vaccine may be to utilize protection-eliciting pneumococcal proteins. Pneumococcal surface adhesin A (PsaA) and pneumococcal surface protein A (PspA) are two vaccine candidates which have been evaluated against S. pneumoniae infection in animal models or human clinical trials with encouraging results. In this study, the efficacy of the fusion protein PsaA–PspA, which includes PsaA part and PspA part, in inducing immunoprotective effects against fatal pneumococcal challenge was evaluated in an animal model. PspA part of PsaA–PspA fusion protein contains both family1 N-terminal region and family 2 N-terminal clade-defining region of PspA. Immunization with the PsaA–PspA fusion protein induced high levels of antibodies against both PsaA and PspA, which could bind to intact S. pneumoniae strains bearing different PspAs. Ex vivo stimulation of splenocytes from mice immunized with PsaA–PspA induced IL-17A secretion. Mice immunized with PsaA–PspA showed reduced S. pneumoniae levels in the blood and lungs compared with the PBS group after intranasal infection. Finally, mice immunized with PsaA–PspA fusion proteins were protected against fatal challenge with pneumococcal strains expressing different PspAs regardless of the challenge route. These results support the PsaA–PspA fusion protein as a promising vaccine strategy, as demonstrated by its ability to enhance the immune response and stimulate production of high titer antibodies against S. pneumoniae strains bearing heterologous PspAs, as well as confer protection against fatal challenge with PspA family 1 and family 2 strains.     

Antibodies against Secreted and Non-Secreted Antigens in Mice after Infection with Live Mycobacterium tuberculosis

Mice from four different inbred strains were infected with live Mycobacterium tuberculosis and the immune response to M. tuberculosis was followed for 24 weeks, using Western blotting. Nearly all mice, irrespective of H-2 type, reacted with the 38-kDa protein band. Antibodies against this secreted 38-kDa protein were the first to appear, 4 weeks after infection. Thereafter the secreted 19-kDa protein and non-secreted antigens, such as the 65-kDa and 33-kDa proteins, were recognized. The immune response against the non-secreted antigens was influenced by the mouse strain. However, the 33-kDa protein band was recognized by all mouse strains after a second injection with live M. tuberculosis. The specificity of the antibodies was analysed in Western blot using sonicates of M. tuberculosis, M. kansasii, M. avium, M. terrae, M. gordonae and Escherichia coli. Antibodies against the 38-kDa and 33-kDa protein bands seemed to be specific for M. tuberculosis, while antibodies against the 19-kDa protein band showed limited cross-reactivity. Antibodies against the 65-kDa protein were strongly cross-reactive. These results suggest that the 38-kDa protein is secreted in vivo and, therefore, may be available to the humoral immune system at an early stage of infection. The non-secreted 33-kDa protein is only recognized by all mouse strains after prolonged contact with M. tuberculosis.     

Intranasal Administration of Mycobacterium bovis BCG Induces Superior Protection against Aerosol Infection with Mycobacterium tuberculosis in Mice

Despite the widespread use of Mycobacterium bovis BCG, the only licensed vaccine against tuberculosis (TB), TB remains a global epidemic. To assess whether more direct targeting of the lung mucosa by respiratory immunization would enhance the potency and longevity of BCG-induced anti-TB protective immunity, the long-term impact of intranasal (i.n.) BCG vaccination was compared to conventional subcutaneous (s.c.) immunization by using a mouse model of pulmonary tuberculosis. Although significantly improved protection in the lung was seen at early time points (2 and 4 months postvaccination) in i.n. BCG-immunized mice, no differences in pulmonary protection were seen 8 and 10 months postvaccination. In contrast, in all of the study periods, i.n. BCG vaccination induced significantly elevated protective splenic responses relative to s.c. immunization. At five of nine time points, we observed a splenic protective response exceeding 1.9 log₁₀ protection relative to the s.c. route. Furthermore, higher frequencies of CD4 T cells expressing gamma interferon (IFN-γ) and IFN-γ/tumor necrosis factor alpha, as well as CD8 T cells expressing IFN-γ, were detected in the spleens of i.n. vaccinated mice. Using PCR arrays, significantly elevated levels of IFN-γ, interleukin-9 (IL-9), IL-11, and IL-21 expression were also seen in the spleen at 8 months after respiratory BCG immunization. Overall, while i.n. BCG vaccination provided short-term enhancement of protection in the lung relative to s.c. immunization, potent and extremely persistent splenic protective responses were seen for at least 10 months following respiratory immunization.     

Immune Responses in Cattle Inoculated with Mycobacterium bovis,Mycobacterium tuberculosis,or Mycobacterium kansasii

Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare the antigen-specific immune responses to various patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis infection) and colonization without pathology (M. tuberculosis infection) to no colonization or pathology (M. kansasii infection). Delayed-type hypersensitivity and gamma interferon responses were elicited by each mycobacterial inoculation; however, the responses by the M. bovis- and M. tuberculosis-inoculated animals exceeded those of the M. kansasii-inoculated animals. Specific antibody responses were detected in all M. tuberculosis- and M. bovis-inoculated cattle 3 weeks after inoculation. From 6 to 16 weeks after M. tuberculosis inoculation, the antibody responses waned, whereas the responses persisted with M. bovis infection. With M. kansasii inoculation, initial early antibody responses waned by 10 weeks after inoculation and then increased 2 weeks after the injection of purified protein derivative for the skin test at 18 weeks after challenge. These findings indicate that antibody responses are associated with the antigen burden rather than the pathology, cellular immune responses to tuberculin correlate with infection but not necessarily with the pathology or bacterial burden, and exposure to mycobacterial antigens may elicit an antibody response in a presensitized animal.Tuberculosis (TB) in humans and animals may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedi, M. microti, M. caprae, or M. canetti [8]). Despite their ∼99.95% sequence identity (12), M. bovis and M. tuberculosis exhibit distinct differences in virulence and host adaptation. Compared to M. tuberculosis, experimental M. bovis infection of mice or rabbits results in a more severe pathology and shorter mean survival times (9, 17, 18). Mycobacterium tuberculosis is primarily a human pathogen that demonstrates a high level of attenuation in cattle (as reviewed by Francis in 1947 [10]), whereas M. bovis has a wider host range and affects many domesticated and free-ranging mammals as well as humans. Prior to the initiation of control and eradication campaigns in the early to mid-1900s, M. bovis infection accounted for up to 30% of human tuberculosis cases, with M. bovis being transmitted to humans primarily by the consumption of unpasteurized dairy products and contact with infected livestock. Control efforts, including slaughter surveillance and test/cull campaigns, have dramatically reduced the prevalence of M. bovis infection in domestic cattle herds, thereby reducing the spread of M. bovis to humans. However, in developing countries, M. bovis infection of humans persists as a serious and relatively common zoonosis (16).Although Mycobacterium kansasii is not a member of the M. tuberculosis complex, it may cause disease in otherwise healthy humans, albeit infrequently, that is clinically indistinguishable from M. tuberculosis infection (1, 3). As with humans, M. kansasii infection of cattle is uncommon; however, it is occasionally associated with granulomatous lesions within lymph nodes and the respiratory tract of cattle (B. Harris, unpublished observations). Of particular relevance for the diagnosis of tuberculosis, M. kansasii infection/sensitization may elicit responses to antigens generally considered to be tuberculosis specific, such as ESAT-6, CFP-10, and MPB83 (2, 30, 35).With experimental M. bovis infection of cattle, the levels of MPB83-specific antibody correlate with disease severity, bacterial burden, and specific cell-mediated immune responses (15, 33). With this particular scenario, disease severity (i.e., pathology) and bacterial burden are intimately linked; thus, it is difficult to define a potential correlation of a particular immune response to either readout independently. Prior studies have demonstrated that virulent and attenuated strains of M. bovis induce similar delayed-type hypersensitivity responses in cattle; however, only the virulent M. bovis strain induces a persistent gamma interferon (IFN-γ), interleukin-2 (IL-2), and antibody response (34). The objective of the present study was to compare mycobacterium-specific immune responses to the patterns of mycobacterial disease expression in which the mycobacterial burden is uncoupled from pathological changes. Disease expression patterns included persistent colonization with an associated pathology (i.e., M. bovis infection), colonization without an associated pathology (i.e., M. tuberculosis infection), and no colonization or pathology (i.e., M. kansasii infection). Antigen-specific immune responses were evaluated for their correlation to manifestations of disease expression.     

Early Pulmonary Cytokine and Chemokine Responses in Mice Immunized with Three Different Vaccines against Mycobacterium tuberculosis Determined by PCR Array

In this study, the early pulmonary cytokine and chemokine responses in mice immunized with either BCG vaccine, a ΔsecA2 mutant of Mycobacterium tuberculosis, or a DNA vaccine expressing an ESAT6-antigen 85B fusion protein and then aerogenically challenged with a low dose of M. tuberculosis were evaluated by PCR array. The cellular immune responses at day 10 postchallenge were essentially equivalent in the lungs of mice immunized with either the highly immunogenic BCG vaccine or the ΔsecA2 M. tuberculosis mutant strain. Specifically, 12 immune biomolecules (including gamma interferon [IFN-γ], interleukin-21 [IL-21], IL-27, IL-17f, CXCL9, CXCL10, and CXCL11) were differentially regulated, relative to the levels for naïve controls, in the lungs of vaccinated mice at this time point. Although the vaccine-related immune responses evoked in mice immunized with the DNA vaccine were relatively limited at 10 days postinfection, upregulation of IFN-γ RNA synthesis as well as increased expression levels of CXCL9, CXCL10, and CXCL11 chemokines were detected.     

Immune Responses Induced by Heterologous Boosting of Recombinant Bacillus Calmette-Guerin with Ag85B-ESAT6 Fusion Protein in Levamisole-Based Adjuvant

In the present study, we evaluated the effectiveness of a levamisole-based adjuvant (ADL) to enhance the ability of the Ag85B-ESAT6 fusion protein to boost immune responses after primary vaccination with recombinant bacillus Calmette-Guerin (rBCG) in Balb/c mice. The results were compared with that of the control adjuvant formulation of dimethyl dioctadecylammonium bromide (DDA) and monophosphoryl lipid A (MPL), which has previously been shown to induce T-helper type 1 (Th1)-biased responses. Enzyme-linked immunospot (ELISPOT) assay with Ag85B and ESAT6 derived peptides corresponding to CD4+ and CD8+ T cell restricted epitopes and cell surface immunostaining indicated that Ag85B-ESAT6/ADL predominantly triggered activation of CD4+ T cells. Functional CD8+ T cells with interferon (IFN)-γ production or cytotoxicity were undetectable all vaccinated mice. The ADL adjuvant modified T-helper (Th) subtypes by up-regulating multiple signature cytokines. Furthermore, profiles of the immunoglobulin G (IgG) subtypes indicated ADL enhanced the secretion of Th1-associated IgG2a antibodies and decreased the yield of Th2-associated IgG1 subtype. These observations suggest that the ADL adjuvant formulated with a protein booster may induce Th1-biased cellular and humoral immune responses to primary vaccination with a live attenuated bacterial TB vaccine.     

结核杆菌Hsp65与EGFP融合基因的构建及DC疫苗的制备

目的: 构建结核杆菌H37Rv株Hsp65与增强型绿色荧光蛋白 (EGFP)的融合基因pEGHsp65, 并以其转染小鼠的树突状细胞 (DC), 制备抗结核的DC疫苗。方法: 采用PCR技术, 从培养的结核杆菌H37Rv株中抽提Hsp65基因,克隆到含有EGFP基因的质粒pEGFP- C1中, 构建pEGHsp65融合基因。以其转染Hela细胞, 在共聚焦激光扫描荧光显微镜下观测不同时间荧光表达的强弱, 并用RT- PCR检测Hsp65mRNA的表达。以pEGHsp65融合基因转染小鼠骨髓细胞经GM- CSF和IL -4诱导分化的DC, 用MTT比色法检测DC疫苗刺激未致敏脾细胞的增殖。结果: 用EcoRⅠ和BglⅡ双酶切鉴定证实, H37Rv株Hsp65DNA已插入重组表达载体pEG -FP- C1中。将融合基因转染入Hela细胞, 48h转染率最高;用RT- PCR在mRNA水平上可检测到Hsp65mRNA的表达。用MTT比色法检测表明, 融合基因转染的DC能激活并引起未致敏的脾细胞增殖。结论: 成功地构建pEGHsp65融合基因和以其制备的DC疫苗, 为进一步观察其治疗结核病的效应奠定了基础。     

Characterization of Protective Mucosal and Systemic Immune Responses Elicited by Pneumococcal Surface Protein PspA and PspC Nasal Vaccines against a Respiratory Pneumococcal Challenge in Mice

Pneumococcal surface protein A (PspA) and PspC are virulence factors that are involved in the adhesion of Streptococcus pneumoniae to epithelial cells and/or evasion from the immune system. Here, the immune responses induced by mucosal vaccines composed of both antigens as recombinant proteins or delivered by Lactobacillus casei were evaluated. None of the PspC vaccines protected mice against an invasive challenge with pneumococcal strain ATCC 6303. On the other hand, protection was observed for immunization with vaccines composed of PspA from clade 5 (PspA5 or L. casei expressing PspA5) through the intranasal route. The protective response was distinguished by a Th1 profile with high levels of immunoglobulin G2a production, efficient complement deposition, release of proinflammatory cytokines, and infiltration of neutrophils. Intranasal immunization with PspA5 elicited the highest level of protection, characterized by increased levels of secretion of interleukin-17 and gamma interferon by lung and spleen cells, respectively, and low levels of tumor necrosis factor alpha in the respiratory tract.Pneumococcal diseases kill more than 1 million children worldwide every year. The situation is worse in developing countries, where 90% of deaths occur. In Latin America, there are at least 1.6 million cases of pneumococcal disease every year, killing 18,000 children (53). While appropriate treatment, including the use of antibiotics and good nutrition, lowers the incidence of pneumococcal diseases, vaccines are the most efficacious way of preventing them. The existing pneumococcal conjugate vaccine dramatically reduces diseases, disabilities, and deaths, but elevated cost and protection restricted to included serotypes have prevented its implementation in large-scale immunization programs in developing countries. For these reasons, there is considerable interest in using conserved pneumococcal protein antigens as vaccines to provide cost-effective broad protection in all age groups. A number of leading candidates have been shown to elicit protection in mice (10, 51); among these antigens, two of the most promising candidates are pneumococcal surface protein A (PspA) and PspC.An additional concern in the development of cost-effective vaccines against pneumococcal disease is the route of immunization. Human vaccines are traditionally administered intramuscularly by needle inoculation, which brings the risk of transmitting blood-borne pathogens such as human immunodeficiency virus and hepatitis viruses (20). Furthermore, the cost of equipment and well-trained personnel for delivering vaccines by parenteral routes is several times higher than the cost of the vaccines themselves. This aspect is extremely important for vaccine implementation in large-scale immunization programs for developing countries. Mucosal delivery of pediatric vaccines has become an explicit goal of the WHO (20). Immunization via mucosal surfaces would greatly increase the ease of vaccination and would be more readily acceptable than parenteral immunization in many populations. Therefore, the move from injection to mucosal application would be very positive from economical, logistical, and safety standpoints. Mucosal immune responses are also important for the prevention of many infectious diseases because they represent the first barrier from the hosts that pathogens must evade.Research into the host immune response to pneumococcal diseases has focused primarily on the role of innate and adaptative humoral immune responses. However, in the last few years, attention has been drawn to cellular immune responses against Streptococcus pneumoniae, with interesting results. The majority of these studies analyzed cellular aspects of innate immunity and proposed that lymphocytes, neutrophils, and macrophages orchestrate effective immune responses without the presence of specific antibodies. In this context, proinflammatory cytokines promote an adequate milieu for pneumococcal clearance (22, 24, 25, 31, 34, 50, 54). A Th1-biased immune response has also been shown to be engaged in the resolution of pneumococcal infection in humans (21). Nevertheless, inflammatory cell influx into the lung and mucosal responses must be regulated to avoid exacerbated tissue injury. This is evidenced in recent studies of the role of γδ T cells and/or anti-inflammatory cytokines, such as interleukin-10 (IL-10), in pneumococcal infection (26, 42, 55).Protective immune responses against invasive pneumococcal disease and colonization were shown using pneumococcal whole-cell vaccines (28, 46) or recombinant proteins as mucosal vaccines (2, 6, 7, 9, 40). In recent approaches, lactic acid bacteria (3, 11, 18, 37), which are able to activate and modulate the innate immune system (35, 42), were used for pneumococcal antigen presentation, with promising results.Very few works compared pulmonary and systemic immune responses induced by pneumococcal antigens using parenteral and mucosal immunizations (13). The present study aims at investigating local and systemic cellular and humoral immune responses required for protection against invasive intranasal (i.n.) challenge with S. pneumoniae strain ATCC 6303 using PspA and PspC antigens administered by both routes, without the use of adjuvants, or presented by Lactobacillus casei through the nasal route.     

Efficacy of Influenza Haemagglutinin and Nucleoprotein as Protective Antigens against Influenza Virus Infection in Mice

Influenza nucleoprotein (NP)-specific cytotoxic T lymphocytes (CTL) stimulated by immunization of mice with VV-PR8-NP6, a recombinant vaccinia virus expressing A/PR/8/34 NP, did not protect mice against challenge with A/PR/8/34 4 days later. Neither were secondary NP-specific CTL stimulated by reimmunization able to protect mice. These results contrast with the ability of transferred, in vitro-cultured and stimulated, NP-specific CTL to protect recipient mice from challenge with A/PR/8/34. Immunization of mice with a recombinant vaccinia virus expressing A/PR/8/34 HA protected mice challenged 4 days later, either via the small amount of antibody already present, or via HA-specific CTL that would have to be more efficient than NP-specific CTL in either trafficking to the infected lung or in effector function.     

Analysis of Immune Responses against a Wide Range of Mycobacterium tuberculosis Antigens in Patients with Active Pulmonary Tuberculosis

Characterizing host immune responses to molecular targets of Mycobacterium tuberculosis is essential to develop effective immunodiagnostics and better vaccines. We investigated the immune response against a large series of M. tuberculosis antigens, including 5 classical and 64 nonclassical (39 DosR regulon-encoded, 4 resuscitation-promoting factor [RPF], and 21 reactivation-associated) antigens in active-pulmonary-tuberculosis (TB) patients. Whole blood from TB patients (n = 34) was stimulated in vitro with M. tuberculosis antigens. Gamma interferon (IFN-γ) was measured after 7 days of stimulation, using an enzyme-linked immunosorbent assay (ELISA). The majority of the study participants responded to the classical M. tuberculosis antigens TB10.4 (84.8%), early secreted antigenic target-6 kDa (ESAT-6)/CFP-10 (70.6%), and purified protein derivative (PPD) (55.9%). However, only 26.5% and 24.2% responded to HSP65 and Ag85A/B, respectively. Of the 64 nonclassical antigens, 23 (33.3%) were immunogenic (IFN-γ levels, >62 pg/ml) and 8 were strong inducers of IFN-γ (IFN-γ levels, ≥100 pg/ml). The RPF antigens were the most immunogenic. In addition, we observed distinct cytokine expression profiles in response to several M. tuberculosis antigens by multiplex immunoassay. Tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10), and IL-6 were commonly detected at high levels after stimulation with 4/15 latency antigens (Rv0081, Rv2006, Rv2629, and Rv1733c) and were found especially in supernatants of the three strong IFN-γ inducers (Rv2629, Rv1009, and Rv2389c). IL-8, IL-6, and IL-17 were exclusively detected after stimulation with Rv0574c, Rv2630, Rv1998, Rv054c, and Rv2028c. In conclusion, in active-pulmonary-TB patients, we identified 23 new immunogenic M. tuberculosis antigens. The distinct expression levels of IFN-γ, TNF-α, IL-6, and IL-10 in response to specific subsets of M. tuberculosis antigens may be promising for the development of immunodiagnostics.     

Host Responses and Antigens Involved in Protective Immunity to Mycobacterium tuberculosis

Tuberculosis (TB) is the largest single infectious cause of human mortality. The incidence of TB has remained high in most of the developing world and the disease has recently re-emerged as a public health problem in industrialized countries. The development of a new improved TB vaccine is a highly prioritized international research area, which has been further stimulated by the appearance of multi-drug resistant strains of Mycobacterium tuberculosis . The present status of the attempts to characterize the protective immune response to TB will be reviewed with special emphasis on recent progress in the identification and characterization of target molecules recognized by protective cells. This paper will focus on proteins released from live bacteria and discuss their role in the host–pathogen interaction and the ongoing attempts to use these molecules in TB subunit vaccines.     

Infection with Mycobacterium kansasii and efficacy of vaccination against tuberculosis.

The purpose of the present study was to examine further the recent hypothesis that subcutaneous infection with Mycobacterium kansasii resulted in the generation of a cell-mediated hypersensitivity reaction ('Koch' reaction) which could, it was argued, subsequently interfere with the generation of acquired immunity following vaccination of the animal with BCG. The results of the present study were unable to confirm this hypothesis in that they show, firstly, that subcutaneous M. kansasii infection was associated with the development of substantial Arthus-like reactivity which masked the detection of any subsequent delayed response, and that furthermore, attempts to adoptively transfer this form of delayed reaction by means of passive transfer of cells were unsuccessful. Furthermore, the results show that, despite the presence of the M. kansasii infection, BCG-vaccinated animals were fully resistant to subsequent aerosol-delivered challenge with virulent M. tuberculosis.     

Immunoglobulin A (IgA) and IgG Immune Responses against P-90 Antigen for Diagnosis of Pulmonary Tuberculosis and Screening for Mycobacterium tuberculosis Infection

The purpose of the present study was to evaluate the usefulness of detection of serum immunoglobulin A (IgA) and IgG antibodies directed against the mycobacterial P-90 antigen for the diagnosis of active pulmonary tuberculosis (PTB) among symptomatic individuals and for the detection of Mycobacterium tuberculosis infections among close contacts of PTB patients. Two commercially available enzyme immunoassay (EIA) kits (IgA EIA-TB [EIA-IgA] and IgG EIA-TB [EIA-IgG]; Kreatech Diagnostics) were evaluated in a blinded fashion by using stored serum samples from 268 individuals, including 69 patients with PTB, 41 patients with diseases other than tuberculosis (TB), 12 subjects with healed PTB, 39 close contacts of PTB patients, and 107 healthy volunteers. For the EIA-IgA, the sensitivity was 74% and the specificity was 68% when a cutoff determined by a receiver operator characteristic curve was used. For the EIA-IgG, the sensitivity was 69% and the specificity was 64%. The EIA-IgA was positive for 54% of healthy close contacts of PTB patients but only 8% of healthy controls without contact with a PTB patient or a prior personal history of TB (P < 0.001). The relatively low sensitivities and specificities of these serologic tests make them poor tools for the diagnosis of PTB among patients with suspected PTB. However, the relatively high prevalence of positive EIA-IgA results among healthy close contacts of PTB patients warrants further evaluation of this test with close contacts and other populations at risk for recent M. tuberculosis exposure and development of disease.     

Humoral response against Mycobacterium bovis Hsp65 derived fragments in children and young people with various disorders

Using Western blotting, we investigated IgG antibodies against Mycobacterium bovis heat shock protein 65 (MB-Hsp65) fragments produced by cleavage with cyanogen bromide (CNBr) in 10 healthy controls, 11 patients with juvenile idiopathic arthritis (JIA), and 10 children with various diseases before haematopoietic stem cell transplantation (HSCT). CNBr cleaved MB-Hsp65 to three larger fragments: P1-163, P191-285, and P290-534. Sera of JIA patients and those before HSCT reacted with individual MB-Hsp65 fragments P1-163 and P290-534 significantly more frequently when compared with healthy controls. These results suggested that the key B-cell epitopes of MB-Hsp65 might be located on the aforementioned sequences.     

Induction of Cell-Mediated Immunity against Mycobacterium tuberculosis Using DNA Vaccines Encoding Cytotoxic and Helper T-Cell Epitopes of the 38-Kilodalton Protein

Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed DNA vaccines encoding cytotoxic T lymphocytes (CTL) and T helper cell (Th) epitopes of the 38-kDa lipoglycoprotein of Mycobacterium tuberculosis and analyzed and compared their immunogenicities with that of pXJ38, a DNA vaccine encoding the entire 38-kDa protein (X. Zhu, N. Venkataprasad, H. S. Thangaraj, M. Hill, M. Singh, J. Ivanyi, and H. M. Vordermeier, J. Immunol. 158:5921-5926, 1997). Plasmid DNAs encoding a CTL epitope, P3 (pP3), a Th epitope (vTh), or both the Th and the P3 epitopes (pThP3) were prepared and tested in C57BL6/J (H-2(b)) mice. Our results confirmed that DNA immunization with pXJ38 induces strong CD8(+) CTL and Th1 responses (high gamma interferon [IFN-gamma], low interleukin-4 [IL-4]). Coadministration of plasmid DNAs encoding a Th epitope with those encoding a CTL epitope (vTh+pP3) elicited both antigen-specific CD8(+) CTL and Th1 responses. High levels of IFN-gamma were secreted by spleen cells from all plasmid DNA-vaccinated mice after in vitro stimulation with the recombinant 38-kDa protein. Small or undetectable amounts of IL-4 were observed, which indicates the induction of a Th1-like response. Multiple-epitope vaccination by vTh+pP3 or pThP3 resulted in a broader Th1 response to peptide or epitopes than the single-epitope plasmid DNAs. Antigen-specific immunoglobulin G2a was only detected in sera from mice immunized with the plasmid pXJ38, and not in mice immunized with the epitope-based DNA vaccines. Thus, the absence of an antibody response after immunization with epitope plasmid DNAs and their ability to trigger only a specific cellular immune response may prove to be important advantages for a vaccine against tuberculosis.     

Rectal and Intranasal Immunizations with Recombinant Urease Induce Distinct Local and Serum Immune Responses in Mice and Protect against Helicobacter pylori Infection

To determine the optimal inductive sites for immunization against Helicobacter pylori infection, the protective efficacy of recombinant urease (rUre) was assessed for mice given the vaccine by either the oral (p.o.), intranasal (i.n.), or rectal route. When mice were immunized with rUre (25 μg p.o. or rectally or 10 μg i.n.) plus heat-labile toxin from Escherichia coli as the mucosal adjuvant, all routes afforded protection against challenge with H. pylori, as indicated by a significant reduction in gastric urease activity (P < 0.0005) compared to that of sham-immunized controls. Quantitative H. pylori culture of stomach tissue demonstrated a >97% reduction in bacterial burden in mice immunized by all routes (P < 0.05). Induction of antiurease immunoglobulin A (IgA) levels in gastric luminal secretions after p.o. immunization was greater than after i.n. administration (means, 6.0 and 1.02 ng/ml, respectively) and was dependent upon challenge with H. pylori. However, immunization by the rectal route resulted in the generation of the highest levels of gastric antiurease IgA (mean, 40.89 ng/ml), which was detectable prior to challenge with H. pylori. Immunohistochemical staining of stomach tissue for cells secreting urease-specific antibody and CD4⁺ T cells showed levels of recruitment to be dependent upon challenge with H. pylori and equivalent for all routes. These results identify both the rectum and nasal passages as suitable inductive sites for urease immunization.     

地塞米松对刀豆蛋白A引起肝损伤小鼠的保护作用

用刀豆蛋白Ａ（ＣｏｎＡ）诱导小鼠建立实验性肝损伤动物模型，观察了该模型血浆丙氨酸转氨酶（ＡＬＴ）、肿瘤坏死因子－α（ＴＮＦ－α）及肝超微结构的变化，证明血浆ＴＮＦ－α与肝损伤密切相关，肝超微结构的改变突出表现为肝细胞凋亡和坏死。     

Evaluation of Gamma Interferon Immune Response Elicited by the Newly Constructed PstS-1(285-374):CFP10 Fusion Protein To Detect Mycobacterium tuberculosis Infection

The PstS1 antigen is highly immunogenic, principally when combined with CFP10 during both latent and active TB infection. In the present study, a selected pstS1 gene fragment was cloned, fused with CFP10, and expressed in Escherichia coli. The product [PstS-1(285-374):CFP10] was compared to the recombinant fused RD1 (region of deletion 1) protein (ESAT-6:CFP10) in detecting Mycobacterium tuberculosis infection in 108 recent contacts of pulmonary tuberculosis (TB) cases, considering a positive tuberculin skin test (TST) to be the baseline. The release of gamma interferon (IFN-γ) in 22-h whole-blood and 5-day lymphocyte stimulation assays primed with each antigen was determined. All contacts were clinically followed for up to 1 year, and 87% of the tuberculin skin test-positive (TST^positive) patients accepted preventative treatment. Concerning the IFN-γ response to PstS-1(285-374):CFP10 in the 22-h and 5-day assays, a slight increase in contact-TST^positive detection was observed (23/54 and 26/54) compared to the level seen with the RD1 protein (18/54 and 24/54) whereas in the TST^negative group, similarly lower numbers (≤5/48) of responders were achieved for both antigens, except for RD1 in the 5-day assay (8/48). By combining the IFN-γ responders to both antigens in the 5-day assays, slightly higher increases in positivity were found in the TST^positive (32/54) and TST^negative (10/48) groups. Two of 12 untreated TST^positive contacts progressed to active TB and were concordantly positive in all assays, except for one contact who lacked positivity in the RD1 5-day assay. We demonstrated for the first time that PstS-1(285-374):CFP10 slightly increased contact positivity and detection of active disease progression, suggesting its potential application as a TB infection marker.     

Identification and Characterization of Protective T Cells in hsp65 DNA-Vaccinated and Mycobacterium tuberculosis-Infected Mice

Immunization by intramuscular injection of plasmid DNA expressing mycobacterial 65-kDa heat shock protein (hsp65) protects mice against challenge with virulent Mycobacterium tuberculosis H37Rv. During infection or after immunization, CD4⁺/CD8⁻ and CD8⁺/CD4⁻ hsp65-reactive T cells increased equally in spleens. During infection, the majority of these cells were weakly CD44 positive (CD44^lo) and produced interleukin 4 (IL-4) whereas after immunization the majority were highly CD44 positive (CD44^hi) and produced gamma interferon (IFN-γ). In adoptive transfer of protection to naive mice, the total CD8⁺/CD4⁻ cell population purified from spleens of immunized mice was more protective than that from infected mice. When the cells were separated into CD4⁺/CD8⁻ and CD8⁺/CD4⁻ types and then into CD44^hi and CD44^lo types, CD44^lo cells were essentially unable to transfer protection, the most protective CD44^hi cells were CD8⁺/CD4⁻, and those from immunized mice were much more protective than those from infected mice. Thus, whereas the CD44^lo IL-4-producing phenotype prevailed during infection, protection was associated with the CD8⁺/CD44^hi IFN-γ-producing phenotype that predominated after immunization. This conclusion was confirmed and extended by analysis of 16 hsp65-reactive T-cell clones from infected mice and 16 from immunized mice; the most protective clones, in addition, displayed antigen-specific cytotoxicity.

Tuberculosis is a classic example of an infectious disease in which the disease process is caused by the immune response directed at the infectious agent. The bacteria and their products are, in themselves, not very toxic, and the extensive tissue damage, wasting, and death of the diseased individual largely constitute the immunopathology of the cell-mediated immune response. Nevertheless, it is also the cell-mediated response that protects against the disease by arresting, killing, and removing the multiplying bacteria. Whether this protective effect occurs early or late, and temporarily or permanently, determines disease progression by regulating the supply of antigen that drives the immunopathology. An important question that arises from this balance between the protective and harmful effects of the immune response is whether the antigens and immune responses that protect can be distinguished from those that harm. If so, they might be separately manipulated in new vaccines or in immunotherapy of the disease.The T lymphocytes that regulate cellular immunity can be divided not only into the CD4⁺/CD8⁻ and CD8⁺/CD4⁻ phenotypes that primarily recognize exogenous and endogenous antigens, respectively (8), or into activated (memory) and nonactivated cells according to highly CD44-positive (CD44^hi) and weakly CD44-positive (CD44^lo) expression (5) but also into two major functionally distinct types on the basis of the profiles of cytokines that they produce. Type 1 cells (Th1 or TC1) favor development of cellular immunity (typified by gamma interferon [IFN-γ], interleukin 2 [IL-2], and IL-12 production). Type 2 cells (Th2 or TC2) favor development of antibody response (typified by IL-4, IL-6, and IL-10 production). Each type promotes differentiation of precursors into the same phenotype and inhibits development of the other phenotype (2, 27), and in consequence the type of response initiated in a microenvironment tends to be self-sustaining. IFN-γ is essential for the development of protective immunity (38) and is probably the most important factor that activates macrophages for antimycobacterial action, at least in mice (13, 34, 35). Therefore protection would be expected to be associated with an immune response in which the type 1 cytokine profile predominated. We have found that immunization procedures that present mycobacterial hsp65 to the immune system as an endogenous antigen generate strong protection against tuberculosis challenge and that this is associated with the presence of a splenic T-cell population in which CD8⁺/CD44^hi IFN-γ-producing cytotoxic cells are prominent (28a). However, cells with a type 2 profile are also present in substantial numbers following infection (22, 39) or Mycobacterium bovis BCG vaccination (24), and the question of what contribution the other phenotypes make to protection arises. To help to answer this question we have used here the combined approaches of comparing the frequency of the different phenotypes in spleens of hsp65 DNA-vaccinated and Mycobacterium tuberculosis-infected mice and testing the different phenotypes, either as purified subpopulations or as T-cell clones, in adoptive transfer of protection.