HSP70 Domain II of Mycobacterium tuberculosis Modulates Immune Response and Protective Potential of F1 and LcrV Antigens of Yersinia pestis in a Mouse Model

No ideal vaccine exists to control plague, a deadly dangerous disease caused by Yersinia pestis. In this context, we cloned, expressed and purified recombinant F1, LcrV antigens of Y. pestis and heat shock protein70 (HSP70) domain II of M. tuberculosis in E. coli. To evaluate the protective potential of each purified protein alone or in combination, Balb/C mice were immunized. Humoral and cell mediated immune responses were evaluated. Immunized animals were challenged with 100 LD₅₀ of Y. pestis via intra-peritoneal route. Vaccine candidates i.e., F1 and LcrV generated highly significant titres of anti-F1 and anti-LcrV IgG antibodies. A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals. Significantly increased percentages of CD4+ and CD8+ T cells producing IFN-γ in spleen of vaccinated animals were observed in comparison to control group by flow cytometric analysis. We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes. Signs of histopathological lesions noticed in lung, liver, kidney and spleen of immunized animals on 3^rd day post challenge whereas no lesions in animals that survived to day 20 post-infection were observed. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3^rd day post-infection whereas no bacteria was observed on day 20 post-infection in surviving animals in LcrV, LcrV+HSP70(II), F1+LcrV, and F1+LcrV+HSP70(II) vaccinated groups. A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group. Three combinations that included LcrV+HSP70(II), F1+LcrV or F1+LcrV+HSP70(II) provided 100% protection, whereas LcrV alone provided only 75% protection. These findings suggest that HSP70(II) of M. tuberculosis can be a potent immunomodulator for F1 and LcrV containing vaccine candidates against plague.     

二硝基氟苯修饰的黑色素瘤细胞激活树突状细胞诱导特异性T细胞反应

目的:探索半抗原二硝基氟苯(DNP)修饰的恶性黑色素瘤细胞(恶黑)激活树突状细胞(DC)后,在体外诱导特异性T细胞反应的抗肿瘤效应。方法:采用DNP修饰恶黑细胞M3(H-2d),然后在体外激活BALB/c小鼠(H-2d)外周血来源的DC,用于激发自体的T细胞,观察对T细胞的增殖和特异性T细胞的杀伤功能。结果:经DNP修饰的M3细胞激活的DC,其诱发的T细胞增殖能力和对M3细胞的特异性杀伤效应均明显高于未修饰的M3细胞组和DC组。结论:DNP修饰M3所激活的DC可以诱导更强的恶黑特异性T细胞效应。     

The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models

Background

It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.

Methodology/Principal Findings

By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates.

Conclusions/Significance

These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made.     

B Cells Regulate Neutrophilia during Mycobacterium tuberculosis Infection and BCG Vaccination by Modulating the Interleukin-17 Response

We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization.     

Mycobacterium tuberculosis Strains Are Differentially Recognized by TLRs with an Impact on the Immune Response

Tuberculosis associates with a wide spectrum of disease outcomes. The Beijing (Bj) lineage of Mycobacterium tuberculosis (Mtb) is suggested to be more virulent than other Mtb lineages and prone to elicit non-protective immune responses. However, highly heterogeneous immune responses were reported upon infection of innate immune cells with Bj strains or stimulation with their glycolipids. Using both in vitro and in vivo mouse models of infection, we here report that the molecular mechanism for this heterogeneity may be related to distinct TLR activations. Among this Mtb lineage, we found strains that preferentially activate TLR2, and others that also activate TLR4. Recognition of Mtb strains by TLR4 resulted in a distinct cytokine profile in vitro and in vivo, with specific production of type I IFN. We also uncover a novel protective role for TLR4 activation in vivo. Thus, our findings contribute to the knowledge of the molecular basis underlying how host innate immune cells handle different Mtb strains, in particular the intricate host-pathogen interaction with strains of the Mtb Bj lineage.     

Characterization of NLRP12 during the In Vivo Host Immune Response to Klebsiella pneumoniae and Mycobacterium tuberculosis

The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1β production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12^−/− mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12^−/− mice showed a slight decrease in IL-1β and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12^−/− mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12^−/− mice. Likewise, Nlrp12^−/− mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.     

Differential Expression of a Mycobacterium tuberculosis Protein Recognized by a Mouse Monoclonal Antibody

Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme-linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over-expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications.     

Initiation of the Primary Immune Response to Sheep Red Blood Cells in the Dissociated Mouse Spleen Cell Culture

From a suspension of mouse spleen cells were separated two functionally different cell types on the basis of their ability or inability to adhere to plastic dishes during a short period of incubation. Morphological observations of cells of these two fractions were made with the aid of histochemical methods. The majority of cells in the adherent fraction possessed β-glucuronidase, which is one of the lysosomal enzymes rich in macrophages or phagocytic cells. In contrast, almost all cells in the non-adherent fraction were devoid of this enzyme activity and identified morphologically as small lymphocytes. The adherent and non-adherent cells were found to associate in cell clusters during the cultivation. In the cultures stimulated with sheep red blood cells, some of the non-adherent cells which were located peripherically in the cell clusters began to show alkaline phosphatase activity in their cytoplasm. This may perhaps indicate that antibody synthesis is going on in these alkaline phosphatase-positive cells, since in an in vivo study such cells were found to arise in the lymph nodes of immunized animals concomitantly with the appearance of specific serum antibody.     

Recombinant Herpesvirus Glycoprotein G Improves the Protective Immune Response to Helicobacter pylori Vaccination in a Mouse Model of Disease

Alphaherpesviruses, which have co-evolved with their hosts for more than 200 million years, evade and subvert host immune responses, in part, by expression of immuno-modulatory molecules. Alphaherpesviruses express a single, broadly conserved chemokine decoy receptor, glycoprotein G (gG), which can bind multiple chemokine classes from multiple species, including human and mouse. Previously, we demonstrated that infection of chickens with an infectious laryngotracheitis virus (ILTV) mutant deficient in gG resulted in altered host immune responses compared to infection with wild-type virus. The ability of gG to disrupt the chemokine network has the potential to be used therapeutically. Here we investigated whether gG from ILTV or equine herpesvirus 1 (EHV-1) could modulate the protective immune response induced by the Helicobacter pylori vaccine antigen, catalase (KatA). Subcutaneous immunisation of mice with KatA together with EHV-1 gG, but not ILTV gG, induced significantly higher anti-KatA IgG than KatA alone. Importantly, subcutaneous or intranasal immunisation with KatA and EHV-1 gG both resulted in significantly lower colonization levels of H. pylori colonization following challenge, compared to mice vaccinated with KatA alone. Indeed, the lowest colonization levels were observed in mice vaccinated with KatA and EHV-1 gG, subcutaneously. In contrast, formulations containing ILTV gG did not affect H. pylori colonisation levels. The difference in efficacy between EHV-1 gG and ILTV gG may reflect the different spectrum of chemokines bound by the two proteins. Together, these data indicate that the immuno-modulatory properties of viral gGs could be harnessed for improving immune responses to vaccine antigens. Future studies should focus on the mechanism of action and whether gG may have other therapeutic applications.     

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.     

Kinetics and Immunoglobulin Nature of the Antibody Response to Mycobacterium tuberculosis

Antibody production after injection of Mycobacterium tuberculosis was studied by use of the single cell plaque assay technique. Maximal numbers of antibody-forming cells were found 9 days after a single injection of M. tuberculosis, about 1 day before the appearance of maximal circulating antibody. Immunoelectrophoretic studies and 2-ME studies on 19S and 7S globulin fractions obtained by gel filtration show that the hemagglutination and hemagglutination-lysis antibody activity is associated with 19S immunoglobulin.     

A Broad Profile of Co-Dominant Epitopes Shapes the Peripheral Mycobacterium tuberculosis Specific CD8+ T-Cell Immune Response in South African Patients with Active Tuberculosis

We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.     

Differential Response of B and T Cells to ALS revealed by Electrophoretic Mobility Determinations

SINCE their discovery anti-lymphocyte antisera (ALS) have been widely investigated in order to clarify their mode of action and to determine their usefulness in clinical practice. Briefly, it seems that the efficacy of a particular ALS depends on its mode of production and on the method adopted for testing its immunosuppressive effect. It also seems likely that many ALS have a harmful effect on a population of thymus-dependent (T) lymphocytes¹. It is the intention of this paper to confirm this notion and to indicate that other lymphocytes (B cells) are likely to be less affected by ALS.     

Protective and Pathologic Roles of the Immune Response to Mouse Hepatitis Virus Type 1: Implications for Severe Acute Respiratory Syndrome

Intranasal mouse hepatitis virus type 1 (MHV-1) infection of mice induces lung pathology similar to that observed in severe acute respiratory syndrome (SARS) patients. However, the severity of MHV-1-induced pulmonary disease varies among mouse strains, and it has been suggested that differences in the host immune response might account for this variation. It has also been suggested that immunopathology may represent an important clinical feature of SARS. Little is known about the host immune response to MHV-1 and how it might contribute to some of the pathological changes detected in infected mice. In this study we show that an intact type I interferon system and the adaptive immune responses are required for controlling MHV-1 replication and preventing morbidity and mortality in resistant C57BL/6J mice after infection. The NK cell response also helps minimize the severity of illness following MHV-1 infection of C57BL/6J mice. In A/J and C3H/HeJ mice, which are highly susceptible to MHV-1-induced disease, we demonstrate that both CD4 and CD8 T cells contribute to morbidity during primary infection, and memory responses can enhance morbidity and mortality during subsequent reexposure to MHV-1. However, morbidity in A/J and C3H/HeJ mice can be minimized by treating them with immune serum prior to MHV-1 infection. Overall, our findings highlight the role of the host immune response in contributing to the pathogenesis of coronavirus-induced respiratory disease.Severe acute respiratory syndrome (SARS) is caused by a zoonotic coronaviral infection that reached epidemic proportions beginning in late 2002 (37, 52, 55, 76, 84, 86). The etiologic agent, SARS-coronavirus (CoV), is a novel group 2 CoV that emerged in the human population exposed to infected animals that were present in wet markets in various provinces of southern China (16, 22, 35, 45, 57, 61). Although the outbreak was quickly contained by the application of aggressive public health measures, it highlighted the deadly potential of this novel pathogen as more than 8,000 people in more than 25 countries were affected, and almost 800 infected individuals died (37, 76, 84, 86). Although there have not been additional outbreaks of this disease in the general population since 2003, due to the continued presence of related viruses in bats and other animals and to cultural practices prevalent in the local population in southern China, the reemergence of this pathogen in the human population may occur in the future (40).Currently, there are no rigorously tested efficacious prophylactic or therapeutic agents targeting this pathogen. Given the lethal potential of this virus, it is imperative to develop specific antiviral therapies that can be rapidly and universally applied. One of the serious drawbacks in the field is the paucity of appropriate animal models that faithfully reproduce the clinical features of SARS (52, 60). Although a mouse-adapted strain of this virus is available, studies with this strain need to be performed in biosafety level 3 facilities (48, 59). Logistical issues associated with such requirements hamper the rapidity and ease with which one can perform a comprehensive and detailed systemic examination of the dynamics of host-pathogen interactions. Recently, it was reported that intranasal infection of certain strains of mice with a related group 2 respiratory CoV, mouse hepatitis virus type 1 (MHV-1), induced pulmonary disease that was very similar to that observed in human subjects infected with SARS-CoV (11). In addition to the phylogenetic proximity of MHV-1 and SARS-CoV, they also share similarities in genome organization and in mechanisms of replication (63, 68). Hence, it is likely that the pathophysiology observed in MHV-1-infected mice mimics important pathological features associated with SARS-CoV infection in humans. A dysregulated immune response characterized by aberrant cytokine production is postulated to contribute to clinical disease in patients with SARS (8, 26, 55, 58, 72, 75, 82, 83). MHV-1 infection of susceptible strains of mice is also associated with an altered cytokine profile, and published reports suggest that the host immune response to the virus is an important contributor to the pathology observed in susceptible strains of mice (11). Examination of the immune response to a pathogen is critical for the purpose of designing rational and effective vaccination approaches. In addition, it also helps identify potentially deleterious effects of the immune response that can subsequently be manipulated to the advantage of the host, thereby maximizing recovery and minimizing morbidity.In the present study we have carried out a comprehensive analysis of the immune response to MHV-1 following intranasal infection of both resistant and susceptible strains of inbred mice. Our observations in alpha/beta interferon (type I IFN) receptor-knockout (IFN-αβR-KO) mice and NK cell-depleted mice shed light on the protective role of these components of the innate immune response in resistant C57BL/6J (B6) mice. And our examination of the adaptive immune responses to MHV-1 shows that they function as a double-edged sword, mediating protection in resistant strains and contributing to pathology in susceptible strains of mice.     

Immune Response Suppression by an Inhibitor in Normal and Immune Mouse Serum

CONTROL mechanisms responsible for determining the intensity and duration of the immune response have been studied extensively. Jerne¹ and Uhr and Möller² described the phenomenon of feedback inhibition whereby the production of a 7S class of antibodies terminates the formation of antibodies with the same specificity. An immunosuppressive effect has also been observed in antigenic competition, in which the administration of one antigen results in a reduced response to a subsequently administered second antigen³. Recently, several investigations have focused on the mechanism of antigenic competition^4,5. A humoral rather than cellular mechanism is generally believed to be responsible for this phenomenon^4–6, but attempts to identify the humoral mediator have been unsuccessful. We have also searched for a humoral substance responsible for the immunosuppressive effect in antigenic competition and found in the sera of adult untreated mice an immunosuppressive agent whose concentration was substantially increased after antigenic stimulation.     

Differential immune response of adipocytes to virulent and attenuated Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tb) takes advantage of various cell types, allowing it to remain in the host for long periods. Because adipocytes have been proposed as niches for dormant M. tb in the latent state, understanding the interaction of virulent M. tb with adipocytes is important. We compared changes in cytokine secretion from 3T3-L1 murine adipocytes infected with virulent M. tb H37Rv (V-M. tb) and attenuated M. tb H37Ra (A-M. tb) strains. Both strains maintained non-replicating states within adipocytes until 10 days post-infection. Adipocytes infected with V-M. tb secreted lower levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12p40, IL-6, and IL-17, and lower levels of nitric oxide than those infected with A-M. tb. In contrast, the anti-inflammatory cytokines, IL-10 and IL-4, were markedly induced in V-M. tb-infected adipocytes versus those infected with A-M. tb at an early time point. Heat-killed or formalin-fixed bacteria induced lower levels of cytokines and no difference was observed between strains. Moreover, V-M. tb induced a high level of necrosis versus A-M. tb in conjunction with increased levels of LHD. These results suggest that V-M. tb regulates cytokine expression in its favor, increasing cytokines necessary for immune evasion and decreasing those required for protective immunity.     

Tuberculin Skin Test Reversion following Isoniazid Preventive Therapy Reflects Diversity of Immune Response to Primary Mycobacterium tuberculosis Infection

Rationale

Healthy household contacts (HHC) of individuals with Tuberculosis (TB) with Tuberculin Skin Test (TST) conversions are considered to harbor latent Mycobacterium tuberculosis (Mtb), and at risk for TB. The immunologic, clinical, and public health implications of TST reversions that occur following Isoniazid preventive therapy (IPT) remain controversial.

Objectives

To measure frequency of TST reversion following IPT, and variation in interferon-gamma (IFN-γ) responses to Mtb, in healthy Ugandan TB HHC with primary Mtb infection evidenced by TST conversion.

Methods

Prospective cohort study of healthy, HIV-uninfected, TST-negative TB HHC with TST conversions. Repeat TST was performed 12 months following conversion (3 months following completion of 9 month IPT course) to assess for stable conversion vs. reversion. Whole blood IFN-γ responses to Mtb antigen 85B (MtbA85B) and whole Mtb bacilli (wMtb) were measured in a subset (n = 27 and n = 42, respectively) at enrollment and TST conversion, prior to initiation of IPT.

Results

Of 122 subjects, TST reversion was noted in 25 (20.5%). There were no significant differences in demographic, clinical, or exposure variables between reverters and stable converters. At conversion, reverters had significantly smaller TST compared to stable converters (13.7 mm vs 16.4 mm, respectively; p = 0.003). At enrollment, there were no significant differences in IFN-γ responses to MtbA85B or wMTB between groups. At conversion, stable converters demonstrated significant increases in IFN-γ responses to Ag85B and wMtb compared to enrollment (p = 0.001, p<0.001, respectively), while there were no significant changes among reverters.

Conclusions

TST reversion following IPT is common following primary Mtb infection and associated with unique patterns of Mtb-induced IFN-γ production. We have demonstrated that immune responses to primary Mtb infection are heterogeneous, and submit that prospective longitudinal studies of cell mediated immune responses to Mtb infection be prioritized to identify immune phenotypes protective against development of TB disease.