Current Protocols in Immunology

Laboratory of Mycobacterial Diseases and Cellular Immunology, Division of Bacterial, Parasitic, and Allergenic Products, CBER/U.S. FDA, Rockville, Maryland, USA.
Current protocols in immunology / edited by John E. Coligan ... [et al.] 04/2011; Chapter 14:Unit14.25. DOI: 10.1002/0471142735.im1425s93
Source: PubMed


Macrophages activated by T cell cytokines are a critical defense mechanism against intracellular bacterial pathogens. This unit presents two general methods for assessing the capacity of mouse macrophages, activated with either soluble cytokines or whole immune T lymphocytes, to control or reduce numbers of intracellular bacteria residing within them. "Measurement of killing" is inferred from a reduction in the number of colony-forming units (cfu) of bacteria at the end of a culture period, compared to the input numbers of cfu at initiation of culture, to the peak numbers of cfu measured during culture, or to a control group in which killing is expected to be poor.

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Available from: Karen L Elkins, Sep 18, 2014
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    ABSTRACT: In contrast with common human infections for which vaccine efficacy can be evaluated directly in field studies, alternative strategies are needed to evaluate efficacy for slowly developing or sporadic diseases like tularemia. For diseases such as these caused by intracellular bacteria, serological measures of antibodies are generally not predictive. Here, we used vaccines varying in efficacy to explore development of clinically useful correlates of protection for intracellular bacteria, using Francisella tularensis as an experimental model. F. tularensis is an intracellular bacterium classified as Category A bioterrorism agent which causes tularemia. The primary vaccine candidate in the U.S., called Live Vaccine Strain (LVS), has been the subject of ongoing clinical studies; however, safety and efficacy are not well established, and LVS is not licensed by the U.S. FDA. Using a mouse model, we compared the in vivo efficacy of a panel of qualitatively different Francisella vaccine candidates, the in vitro functional activity of immune lymphocytes derived from vaccinated mice, and relative gene expression in immune lymphocytes. Integrated analyses showed that the hierarchy of protection in vivo engendered by qualitatively different vaccines was reflected by the degree of lymphocytes' in vitro activity in controlling the intramacrophage growth of Francisella. Thus, this assay may be a functional correlate. Further, the strength of protection was significantly related to the degree of up-regulation of expression of a panel of genes in cells recovered from the assay. These included IFN-γ, IL-6, IL-12Rβ2, T-bet, SOCS-1, and IL-18bp. Taken together, the results indicate that an in vitro assay that detects control of bacterial growth, and/or a selected panel of mediators, may ultimately be developed to predict the outcome of vaccine efficacy and to complement clinical trials. The overall approach may be applicable to intracellular pathogens in general.
    PLoS Pathogens 01/2012; 8(1):e1002494. DOI:10.1371/journal.ppat.1002494 · 7.56 Impact Factor
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    ABSTRACT: Mucosa-associated invariant T (MAIT) cells are a unique population of αβ T cells in mammals that reside preferentially in mucosal tissues and express an invariant Vα paired with limited Vβ T-cell receptor (TCR) chains. Furthermore, MAIT cell development is dependent upon the expression of the evolutionarily conserved major histocompatibility complex (MHC) class Ib molecule MR1. Using in vitro assays, recent studies have shown that mouse and human MAIT cells are activated by antigen-presenting cells (APCs) infected with diverse microbes, including numerous bacterial strains and yeasts, but not viral pathogens. However, whether MAIT cells play an important, and perhaps unique, role in controlling microbial infection has remained unclear. To probe MAIT cell function, we show here that purified polyclonal MAIT cells potently inhibit intracellular bacterial growth of Mycobacterium bovis BCG in macrophages (MΦ) in coculture assays, and this inhibitory activity was dependent upon MAIT cell selection by MR1, secretion of gamma interferon (IFN-γ), and an innate interleukin 12 (IL-12) signal from infected MΦ. Surprisingly, however, the cognate recognition of MR1 by MAIT cells on the infected MΦ was found to play only a minor role in MAIT cell effector function. We also report that MAIT cell-deficient mice had higher bacterial loads at early times after infection compared to wild-type (WT) mice, demonstrating that MAIT cells play a unique role among innate lymphocytes in protective immunity against bacterial infection.
    Infection and immunity 07/2012; 80(9):3256-67. DOI:10.1128/IAI.00279-12 · 3.73 Impact Factor
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    ABSTRACT: Francisella tularensis (FT) is a highly virulent pathogen for humans and other mammals. Severe morbidity and mortality is associated with respiratory FT infection and there are concerns about intentional dissemination of this organism. Therefore, FT has been designated a category A biothreat agent and there is a growing interest in the development of a protective vaccine. In the present study, we determine the protective potential of a subunit vaccine comprised of the FT heat shock protein DnaK and surface lipoprotein Tul4 against respiratory infection with the live vaccine strain (LVS) of FT in mice. First, we establish an optimal intranasal immunization regimen in C57BL/6 mice using recombinant DnaK or Tul4 together with the adjuvant GPI-0100. The individual immunization regimens induced robust salivary IgA, and vaginal and bronchoalveolar IgA and IgG antigen-specific antibodies. Serum IgG1 and IgG2c antibody responses were also induced, indicative of a mixed type 2 and type 1 response, respectively. Next, we show that immunization with DnaK and Tul4 induces mucosal and systemic antibody responses that are comparable to that seen following immunization with each antigen alone. This immunization regimen also induced IFN-γ, IL-10 and IL-17A production by splenic CD4(+) T cells in an antigen-specific manner. Importantly, over 80% of the mice immunized with DnaK and Tul4, but not with each antigen alone, were protected against a lethal respiratory challenge with FT LVS. Protection correlated with reduced bacterial burden in the lung, liver and spleen of mice. This study demonstrates the potential of DnaK and Tul4 as protective antigens and lends support to the notion of combining distinct, immunodominant antigens into an effective multivalent tularemia vaccine.
    PLoS ONE 11/2012; 7(11):e50460. DOI:10.1371/journal.pone.0050460 · 3.23 Impact Factor
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