Th1 Cell-mediated Immune Response Research Articles

Repeated Exposure to Aspergillus fumigatus Conidia Results in CD4+T Cell-Dependent and -Independent Pulmonary Arterial Remodeling in a Mixed Th1/Th2/Th17 Microenvironment That Requires Interleukin-4 (IL-4) and IL-10

Pulmonary arterial remodeling is a pathological process seen in a number of clinical disease states, driven by inflammatory cells and mediators in the remodeled artery microenvironment. In murine models, Th2 cell-mediated immune responses to inhaled antigens, such as purified Aspergillus allergen, have been reported to induce remodeling of pulmonary arteries. We have previously shown that repeated intranasal exposure of healthy C57BL/6 mice to viable, resting Aspergillus fumigatus conidia leads to the development of chronic pulmonary inflammation and the coevolution of Th1, Th2, and Th17 responses in the lungs. Our objective was to determine whether repeated intranasal exposure to Aspergillus conidia would induce pulmonary arterial remodeling in this mixed Th inflammatory microenvironment. Using weekly intranasal conidial challenges, mice developed robust pulmonary arterial remodeling after eight exposures (but not after two or four). The process was partially mediated by CD4+ T cells and by interleukin-4 (IL-4) production, did not require eosinophils, and was independent of gamma interferon (IFN-γ) and IL-17. Furthermore, remodeling could occur even in the presence of strong Th1 and Th17 responses. Rather than serving an anti-inflammatory function, IL-10 was required for the development of the Th2 response to A. fumigatus conidia. However, in contrast to previous studies of pulmonary arterial remodeling driven by the A. fumigatus allergen, viable conidia also stimulated pulmonary arterial remodeling in the absence of CD4+ T cells. Remodeling was completely abrogated in IL-10-/- mice, suggesting that a second, CD4+ T cell-independent, IL-10-dependent pathway was also driving pulmonary arterial remodeling in response to repeated conidial exposure.

Selective down‐regulation of Th2 cell‐mediated airway inflammation in mice by pharmacological intervention of CCR4

The chemokine receptor CCR4 has been implicated in Th2 cell-mediated immune responses. However, other T cell subsets are also known to participate in allergic inflammation. The role of CCR4 in Th1, Th2, and Th17 cell-mediated allergic airway inflammation was investigated. We generated an allergic airway inflammation model by adoptive transfer of in vitro-polarized ovalbumin (OVA)-specific Th1, Th2, and Th17 cells. The effect of a low-molecular weight CCR4 antagonist, Compound 22, on this model was examined. Upon in vitro polarization of DO11.10 naïve T cells, Th1- and Th2-polarized cells dominantly expressed CXCR3 and CCR4, respectively, while Th17-polarized cells expressed CCR6 and CCR4. Intranasal OVA-challenge of mice transferred with each T cell subset induced accumulation of T cells in the lungs. Eosinophils were also massively accumulated in Th2-transferred mice, whereas neutrophils were preferentially recruited in Th1- and Th17-transferred mice. Compound 22, as well as anti-CCL17 or anti-CCL22 antibody selectively suppressed accumulation of Th2 cells and eosinophils in the lungs of Th2-transferred and OVA-challenged mice. Compound 22 also inhibited bronchial hyperresponsiveness but had little effect on goblet cell hyperplasia in Th2-transferred and OVA-challenged mice. There were notable differences in allergic lung inflammation mediated by different T cell subsets. CCR4 blockage was selectively effective for suppression of Th2-mediated allergic inflammation by blocking infiltration of Th2 cells.

Identification of M.tuberculosis-Specific Th1 Cells Expressing CD69 Generated in vivo in Pleural Fluid Cells from Patients with Tuberculous Pleurisy

Th1 cell-mediated immune responses at the site of active infection are important to restrict the growth of M.tuberculosis (MTB) and for the spontaneous resolution of patients with tuberculous pleurisy (TBP). In the present study, we found that without any stimulation, CD4+ T cells in pleural fluid cells (PFCs) from patients with TBP expressed significantly higher levels of CD69 than PBMCs from patients with tuberculosis (TB) or healthy donors. CD4+CD69+ T cells expressed T-bet and IL-12Rβ2. After stimulation with MTB-specific antigens, CD4+CD69+ T cells expressed significantly higher levels of IFN-γ, IL-2 and TNF-α than CD4+CD69− T cells, demonstrating that CD4+CD69+ T cells were MTB-specific Th1 cells. In addition, CD4+CD69+ T cells were mostly polyfunctional Th1 cells that simultaneously produced IFN-γ, IL-2, TNF-α and displayed an effector or effector memory phenotype (CD45RA−CCR7−CD62L−CD27−). Moreover, the percentages of CD4+CD69+ T cells were significantly and positively correlated with polyfunctional T cells. Interestingly, sorted CD4+CD69+ but not CD4+CD69− fractions by flow cytometry produced IFN-γ, IL-2 and TNF-α that were significantly regulated by CD4+CD25+ Treg cells. Taken together, based on the expression of CD69, we found a direct quantitative and qualitative method to detect and evaluate the in vivo generated MTB-specific polyfunctional CD4+ T cells in PFCs from patients with TBP. This method can be used for the potential diagnosis and enrichment or isolation of MTB-specific Th1 cells in the investigations.

Potentially functional polymorphisms in IL-23 receptor and risk of esophageal cancer in a Chinese population

Interleukin-23 (IL-23)/IL-23 receptor (IL-23R) is essential for Th17 cell-mediated immune response, involved in autoimmune diseases and cancer pathogenesis. Two potentially functional genetic single nucleotide polymorphisms (SNPs; IL-23R rs6682925 T>C and rs1884444 T>G) were found to contribute to cancer susceptibility. In our study, we conducted a case-control study including 1,645 patients with esophageal cancer and 1,694 cancer-free controls in a Chinese population to assess the association between the two SNPs and the risk of esophageal cancer. We found that IL-23R rs6682925 TC/CC and rs1884444 TG/GG variant genotypes were associated with significantly increased risk of esophageal cancer [rs1884444: adjusted odds ratio (OR) = 1.16, 95% confidence intervals (CIs) =1.01-1.33; rs6682925: adjusted OR = 1.23, 95% CIs = 1.07-1.42], compared to their corresponding wild-type homozygotes. Furthermore, the increased risks associated with the two SNPs were independent from smoking and alcohol drinking status. These findings indicated that genetic variants in IL-23R may contribute to esophageal cancer risk in our Chinese population.

Evaluation of a broadly protective Chlamydia–cholera combination vaccine candidate

The need to simultaneously target infections with epidemiological overlap in the population with a single vaccine provides the basis for developing combination vaccines. Vibrio cholerae ghosts (rVCG) offer an attractive approach for developing vaccines against a number of human and animal pathogens. In this study, we constructed a multisubunit vaccine candidate co-expressing the serovar D-derived Porin B and polymorphic membrane protein-D proteins of Chlamydia trachomatis and evaluated its ability to simultaneously induce broad-based chlamydial immunity and elicit a vibriocidal antibody response to the Vibrio carrier envelope. Intramuscular (IM) immunization with the vaccine candidate elicited high levels of antigen-specific genital mucosal and systemic Th1 cell-mediated and humoral immune responses against heterologous serovars and strains, including serovars E–H and L. Also, in addition to the multisubunit vaccine, the single subunit constructs conferred significant cross protection against the heterologous mouse strain, Chlamydia muridarum. Furthermore, all mice immunized with rVCG vaccine constructs responded with a significant rise in vibriocidal antibody titer, the surrogate marker for protection in cholera. These findings demonstrate the ability of the multisubunit vaccine to induce cross protective chlamydial as well as vibriocidal immunity and establish the possibility of developing a broadly efficacious Chlamydia–cholera combination vaccine.

ESAT‐6‐ and CFP‐10‐Specific Th1, Th22 and Th17 Cells in Tuberculous Pleurisy May Contribute to the Local Immune Response Against Mycobacterium Tuberculosis Infection

Th1 cell-mediated adaptive immune response is very important but may not be sufficient to control Mycobacterium tuberculosis (M. tuberculosis) infection. The roles of the various T cell subsets and cytokines in the inflammatory processes are not clearly elucidated. We investigated whether Th1, Th22 and Th17 cells mediated cellular immunity at the local site of M. tuberculosis infection in patients with tuberculous pleurisy (TBP). The results showed that the cytokines IFN-γ and IL-22 but not IL-17 were elevated in tubercular pleural fluid. Following stimulation with immune-dominant peptides of early secreted antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP-10) or Bacille Calmette-Guerin, pleural fluid mononuclear cells expressed high levels of cytokines IFN-γ, IL-22 and IL-17 as revealed by mRNA and protein measurements. In addition, we showed that cytokines IFN-γ, IL-22 and IL-17 were produced in M. tuberculosis-specific immune response by distinct subsets of CD4+ T cells with the phenotype of CD45RA-CD62L-CCR7+CD27+ . Our results demonstrated for the first time that ESAT-6- and CFP-10-specific Th1, Th22 and Th17 cells existed in the patients with TBP and might play an essential role against M. tuberculosis infection. The findings of this study raised the possibility of unravelling the critical targets for therapeutic intervention in chronic inflammatory diseases such as TBP.

B Lymphocyte Memory in X-Linked Lymphoproliferative Disease (XLP)

X-linked lymphoproliferative disease (XLP) is a severe immunodeficiency characterized by hypogammaglobulinemia, fulminant infectious mononucleosis, and/or lymphoma associated to mutations of the SH2D1A gene, encoding SAP (signaling lymphocytic activation molecule-associated protein). The initial encounter with Epstein Barr virus (EBV) triggers a massive response that leads to a fatal outcome in around 50% of the XLP individuals. Most surviving patients develop hypogammaglobulinemia and eventually B cell lymphoma. B lymphocyte development seems to be normal, but there is a marked reduction of memory B cells (CD27+ B lymphocytes). In addition, Th1 cell mediated immune responses predominate over Th2 responses. Hypogammaglobulinemia and failure to develop a long term humoral immune response can be explained because both the germinal center (GC) reaction and GC formation in secondary lymphoid organs are greatly impaired both in human XLP and in experimental SAP deficiency. Non switched memory B cells (IgM+, IgD+, CD27+ B lymphocytes) persist in XLP patients, suggesting that in spite of the lack of a GC reaction, some subgroups of memory B lymphocytes can play a role in immune homeostasis in these patients. In addition, their persistence in the presence of EBV infection, could perhaps be associated to late occurrence of extranodal B-cell lymphoma, which is another pathological condition associated to the absence of SAP in humans. Keywords: B cell memory, XLP, SAP, humoral response, CD27, long term memory.

Unraveling the complex genetic underpinnings of asthma and allergic disorders

Asthma and other allergic diseases are complex genetic disorders that result from interactions between multiple genes and environmental factors. In this review, we summarize findings from candidate gene analyses, discuss the recent success of genome-wide association (GWA) studies, and outline challenges facing the field. In the past year, five GWA studies have been reported for asthma, one for atopic dermatitis, and four for intermediate phenotypes using quantitative trait loci. These results have in general been more robust to replication than prior candidate gene studies, and have allowed the identification of novel loci for both asthma (i.e. 1q31, 9q21.31) and atopic dermatitis (11q13). The integration of results from recent GWA studies with careful analyses of candidate gene associations studies has confirmed the importance of immune detection and TH2-cell mediated immune responses in the pathogenesis of allergic disease, and has raised new interest in the role of epithelial barrier function and tissue-level responses. GWA studies appear to provide a robust way to identify novel gene loci contributing to disease susceptibility. Dissecting gene-gene and gene-environment interactions, and exploring the contribution of epigenetic phenomena to allergic disease susceptibility remain important challenges to understanding the complex nature of asthma and other allergic diseases.

Suppression of encephalitogenic T-cell responses by cilostazol is associated with upregulation of regulatory T cells

Cilostazol is a specific phosphodiesterase III inhibitor. Recent data show that cilostazol has anti-inflammatory effects and administration of cilostazol ameliorates experimental autoimmune encephalomyelitis (EAE). In this study, we used a mouse EAE model to explore the role of cilostazol in Th1 and Th17 cell-mediated immune responses. We found that cilostazol suppressed mitogen or antigen-induced T-cell responses and Th17 cell differentiation in vitro, which correlated with enhanced Treg-cell responses. Beginning of oral administration of cilostazol at the onset of EAE significantly inhibited encephalitogenic T cells, reduced the levels of inflammatory cytokines in the central nervous system, and ameliorated the severity of EAE. Moreover, administration of cilostazol markedly enhanced Treg-cell response in vivo. Cilostazol, therefore, may exert its therapeutic effects through upregulation of Treg-cell activity.

577: Th17 Cells and Their Role in Acute Rejection in Heart Transplant Patients

Th17 cells are known to have potent proinflammatory functions in human inflammatory and autoimmune diseases. Therefore, it is tempting to speculate that Th17 cells and their effector cytokines are significant in the inflammatory response after organ transplantation. These Th17 cell mediated immune responses are suppressed by FOXP3+ regulatory T cells. Here, we studied the involvement of IL-17 and FOXP3 in the rejection process after clinical heart transplantation. The traditional players in acute rejection IL-2 and IFN-γ were measured as a control.

Vitamin A deficiency alters splenic dendritic cell subsets and increases CD8 +Gr-1 + memory T lymphocytes in C57BL/6J mice

Vitamin A-deficient populations have impaired T cell-dependent antibody responses. Dendritic cells (DCs) are the most proficient antigen-presenting cells to naïve T cells. In the mouse, CD11b + myeloid DCs stimulate T helper (Th) 2 antibody immune responses, while CD8α + lymphoid DCs stimulate Th1 cell-mediated immune responses. Therefore, we hypothesized that vitamin A-deficient animals would have decreased numbers of myeloid DCs and unaffected numbers of lymphoid DCs. We performed dietary depletion of vitamin A in C57BL/6 J male and female mice and used multicolor flow cytometry to quantify immune cell populations of the spleen, with particular focus on DC subpopulations. We show that vitamin A-depleted animals have increased polymorphonuclear neutrophils, lymphoid DCs, and memory CD8 + T cells and decreased CD4 + T lymphocytes. Therefore, vitamin A deficiency alters splenic DC subpopulations, which may contribute to skewed immune responses of vitamin A-deficient populations.

A DNA adjuvant encoding a fusion protein between anti‐CD3 single‐chain Fv and AIMP1 enhances T helper type 1 cell‐mediated immune responses in antigen‐sensitized mice

T helper type 1 (Th1) cell-mediated immune responses contribute to host defences against intracellular pathogen infections and cancer. Previously, we found that aminoacyl tRNA synthetase-interacting multifunctional protein 1 (AIMP1) activated macrophages and dendritic cells to enhance Th1 responses. Herein, we manipulated this property to improve Th1 immune responses in vivo by constructing a mammalian expression plasmid (pAnti-CD3sFv/AIMP1) encoding AIMP1 fused to the anti-CD3 single-chain Fv (sFv), the smallest unit of the antibody that interacts with the CD3epsilon region of the T-cell receptor. Intramuscular injection of ovalbumin (OVA)-sensitized BALB/c mice with pAnti-CD3sFv/AIMP1 DNA adjuvant increased the OVA-specific, interferon-gamma production by their CD4(+) T cells and the levels of anti-OVA immunoglobulin G2a (IgG2a) isotype in their sera. Furthermore, the pAnti-CD3sFv/AIMP1 DNA adjuvant decreased interleukin-4 production and anti-OVA IgE levels in the OVA-injected mice. Importantly, the pAnti-CD3sFv/AIMP1 was more efficient than a mixture of pAnti-CD3sFv and pAIMP1 in inducing OVA-specific Th1 immune responses and also in inhibiting OVA-specific Th2 responses during antigen priming. These studies indicated that the pAnti-CD3sFv/AIMP1 fusion DNA adjuvant enhanced Th1 immune responses in antigen-sensitized mice.

Differential Regulatory Function of Resting and Preactivated Allergen-Specific CD4+CD25+ Regulatory T Cells in Th2-Type Airway Inflammation

Although CD4(+)CD25(+) regulatory T (Treg) cells are known to suppress Th1 cell-mediated immune responses, their effect on Th2-type immune responses remains unclear. In this study we examined the role of Treg cells in Th2-type airway inflammation in mice. Depletion and reconstitution experiments demonstrated that the Treg cells of naive mice effectively suppressed the initiation and development of Th2-driven airway inflammation. Despite effective suppression of Th2-type airway inflammation in naive mice, adoptively transferred, allergen-specific Treg cells were unable to suppress airway inflammation in allergen-presensitized mice. Preactivated allergen-specific Treg cells, however, could suppress airway inflammation even in allergen-presensitized mice by accumulating in the lung, where they reduced the accumulation and proliferation of Th2 cells. Upon activation, allergen-specific Treg cells up-regulated CCR4, exhibited enhanced chemotactic responses to CCR4 ligands, and suppressed the proliferation of and cytokine production by polarized Th2 cells. Collectively, these results demonstrated that Treg cells are capable of suppressing Th2-driven airway inflammation even in allergen-presensitized mice in a manner dependent on their efficient migration into the inflammatory site and their regulation of Th2 cell activation and proliferation.

Gene transfer of AIMP1 and B7.1 into epitope-loaded, fibroblasts induces tumor-specific CTL immunity, and prolongs the survival period of tumor-bearing mice

T helper type 1 (Th1) cell-mediated immune responses play various roles in cellular immunity, including inducing cytotoxic T lymphocytes (CTLs) and they have been shown to be crucial in cancer immunotherapy. Previously, we found that aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) stimulated antigen-presenting cells to secrete IL-12, leading to enhanced Th1 cell responses. In this study, as a way of enhancing antigen-specific Th1 responses, mouse fibroblasts (H-2 b) were genetically modified to express an AIMP1 and a costimulatory B7.1 (Fb/AIMP1/B7.1). Fb/AIMP1/B7.1 cells were then loaded with an ovalbumin epitope as a model antigen (Fb/AIMP1/B7.1/OVA), and tested to determine if they induced OVA-specific CTLs in C57BL/6 mice (H-2 b). Immunization with Fb/AIMP1/B7.1/OVA cells induced strong cytotoxic activities against OVA-expressing EG7 tumor cells, but not against other H-2 b tumor cells. The levels of the cytotoxic response in the immunized mice with Fb/AIMP1/B7.1/OVA cells were significantly higher than the responses in mice immunized with other cell constructs. CD8 + T cells were a major cell-type of OVA-specific antitumor immunity induced by Fb/AIMP1/B7.1/OVA cells. Furthermore, treatment with Fb/AIMP1/B7.1/OVA cells significantly prolonged the survival period of EG7 tumor-bearing mice. These results indicate that AIMP1-secreting, epitope-loaded fibroblasts efficiently induce antigen-specific CTL responses in mice.

TNF controls the infiltration of dendritic cells into the site of Leishmania major infection

TNF-negative C57BL/6 (B6.TNF(-/-)) mice are highly susceptible to Leishmania (L.) major infection and succumb rapidly to fatal leishmaniasis. A T helper type 1 (Th1) cell-mediated immune response is central for protective anti-leishmanial immunity. Therefore, the observed susceptibility of B6.TNF(-/-) mice to L. major parasites could be caused by a deficiency in mounting a Th1 response. Analysis of infected footpads revealed, that B6.TNF(-/-) mice exhibited a substantially diminished formation of DCs at the site of infection. Furthermore, Th1 cytokines such as IFN-gamma were reduced in footpads of infected B6.TNF(-/-) mice. Cutaneous reconstitution of B6.TNF(-/-) mice with either bone marrow derived DCs (BM-DCs) or recombinant TNF simultaneous to infection resulted in an increased expression of cytokines such as IFN-gamma and in an enhanced presence of Leishmania-antigen in skin draining lymph nodes. In addition, the individual time of survival was doubled. In conclusion we demonstrate that the expression of dermal TNF is necessary to provide an environment that initiates a local inflammatory response, but is not sufficient to induce protective immunity.

Intracellular production of IL-2, IL-4, IFN-γ, and TNF-α by peripheral blood CD3+ and CD4+ T cells in children with atopic dermatitis

The role of the type-2 T helper (Th2) cell-mediated immune response in the immunopathogenesis of atopic dermatitis (AD) is well documented. Whether polarized immunoresponse is confined to antigen-specific T cells or is distributed among all T cell subsets is still controversial. We investigated frequencies of interleukin-2 (IL-2), IL-4, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) producing CD3(+) and CD4(+) T cells in peripheral blood from children with atopic dermatitis and healthy subjects with and without in vitro stimulation. Children with severe AD had a significantly lower percentage of CD4(+) T cells spontaneously expressing IL-4 compared with healthy controls (p <0.01). Polyclonal stimulation significantly increased cytokine production in both AD patients and healthy individuals. Frequencies of CD3(+) and CD4(+) producing IL-2, IL-4, IFN-gamma, and TNF-alpha after in vitro stimulation with phorbol-12-myristate 13-acetate (PMA) + ionomycin were comparable in the AD and control groups. In response to PMA/ionomycin, children with AD and asthma symptoms had a significantly lower percentage of CD3(+) T cells producing TNF-alpha. We failed to demonstrate evidence of an imbalance with respect to type-2 cytokine productions in children with AD. Comparable induction of Th1 and Th2 cytokines in polyclonally stimulated peripheral CD3(+) and CD4(+)T cells from AD patients and controls puts into question the polarized Th2 immune response as a general characteristic of T cells in children with atopic dermatitis.

Inhibition of murine K-BALB and CT26 tumour growth using a Semliki Forest virus vector with enhanced expression of IL-18

The enhanced Semliki Forest virus vector (SFV10-E), an RNA-based suicide expression vector system, expresses foreign genes at levels up to 10x higher than the original SFV10 vector. This vector has been used previously to express interleukin-12 for a tumour treatment study in a BALB/c murine model. Interleukin-18, an IFN-gamma-inducing cytokine, plays a key role in the early induction of T helper1 (Th1) cell-mediated immune responses in addition to anti-angiogenic activity. In this study, the murine IL-18 gene along with an Ig-kappa leader sequence was cloned into the SFV10-E vector. The pSFV10-E-IL-18 construct was characterised in vitro for levels of expression and secretion, and the production of biologically active IL-18 was confirmed. An in vivo tumour treatment study using high titre rSFV10-E-IL-18 virus-like particles to treat subcutaneous K-BALB and CT26 tumours in BALB/c mice demonstrated therapeutic efficacy including the disappearance of tumour cells in a minority of treated animals. Tumour regression was associated with induction of avascular and suppurative necrosis.

Lipoteichoic acid-related molecule derived from the streptococcal preparation, OK-432, which suppresses atopic dermatitis-like lesions in NC/Nga mice

Bacterial stimulation may serve to control atopic disorders such as atopic dermatitis (AD) through inducement of Th1 cell-mediated immune response. The lipoteichoic acid (LTA)-related molecule (okLTA) from streptococcal preparation, OK-432, has been shown to be a potent Th1 inducer through the action of IL-12. Examination was made of the therapeutic effects of this okLTA injected intra- and/or subcutaneously into AD-like lesions in NC/Nga mice, particularly in the vicinity of the suppressor of cytokine signaling (SOCS) regulatory pathways. Using immunohistochemical staining with IL-4/IL-12p40 and phosphorylated STAT6/p-STAT4 and RT-PCR for IL-4/IL-12p40, STAT6/STAT4 and mRNA expression and in situ hybridization of SOCS3 and 5, evaluation was made of the immunoregulatory effects of this okLTA in the treatment of spontaneous AD-like lesions in NC/Nga mice. Following the injection of okLTA, remarkable improvement in the lesions of NC/Nga mice was noted. In okLTA-treated skin, IL-12p40/p-STAT4 positive cellular infiltration was extensive while IL-4/p-STAT6 positive cell infiltration was seen to diminish considerably, compared to untreated NC mice. SOCS3 in situ expression in okLTA-treated mice was noted to be significantly less compared to untreated NC mice, in which the expression was prominent. SOCS5 in situ expression was rather, though not significantly, strong in okLTA-treated mice. okLTA treatment is clearly shown to induce Th1 cellular response and down-regulate immune response in the Th2 pathway through SOCS3 reduction in AD-like lesions of NC/Nga mice. The present results demonstrate that bacterial wall components such as okLTA should serve as an effective new therapeutic approach for treating AD.

Selection of novel TB vaccine candidates and their evaluation as DNA vaccines against aerosol challenge

Putative TB vaccine candidates were selected from lists of genes induced in response to in vivo-like stimuli, such as low oxygen and carbon starvation or growth in macrophages, and tested as plasmid DNA vaccines for their ability to protect against Mycobacterium tuberculosis challenge in a guinea pig aerosol infection model. This vaccination method was chosen as it induces the Th1 cell-mediated immune response required against intracellular pathogens such as M. tuberculosis. Protection was assessed in the guinea pig model in terms of mycobacteria present in the lungs at 30 days post-challenge. Protection achieved by the novel candidates was compared to BCG (positive control) and saline (negative control). Four vaccines encoding for proteins such as PE and PPE proteins, a zinc metalloprotease and an acyltransferase, gave a level of protection that was statistically better than saline in the lungs. These findings have enabled us to focus on a sub-set of vaccine candidates for further evaluation using additional vaccination strategies.

Anti-CD3 single-chain Fv/interleukin-18 fusion DNA induces anti-mycobacterial resistance via efficient interferon-γ production in BALB/c mice infected with Mycobacterium avium

T helper type 1 (Th1) cell-mediated immune response performs a critical role in the induction of host defenses against a variety of intracellular pathogens. We recently demonstrated that the linkage between interleukin (IL)-18 and anti-CD3sFv genes confined the effect of IL-18 to the CD3 + T cells, resulting in the efficient induction of Thl immune responses. In this study, we attempted to determine whether the anti-CD3 single-chain Fv/IL-18 fusion DNA (pAnti-CD3sFv/IL-18) could stimulate resistance to Mycobacterium avium infection in genetically susceptible BALB/c mice. Immunization with pAnti-CD3sFv/IL-18 DNA during intranasal infection with M. avium resulted in a significant reduction in the bacterial load in the lung throughout the entire 10-week observation period. Immunization with pAnti-CD3sFv/IL-18 DNA induced and maintained significantly higher levels of cytotoxic activity and nitric oxide production by the lung cells, as compared with the injection of pAnti-CD3sFv DNA, or of a simple mixture of pAnti-CD3sFv DNA and pIL-18 DNA. Furthermore, the lung cells of the mice injected with pAnti-CD3sFv/IL-18 DNA exhibited persistent production of IFN-γ. Treatment with anti-IFN-γ antibody resulted in an abrogation of anti-mycobacterial effects in mice injected with pAnti-CD3sFv/IL-18 DNA. These results indicate that vaccination with the pAnti-CD3sFv/IL-18 fusion DNA induces significant resistance to M. avium infection, via the production of IFN-γ in CD4 + T cells.