Article

Three protein cocktails mediate delayed-type hypersensitivity responses indistinguishable from that elicited by purified protein derivative in the guinea pig model of Mycobacterium tuberculosis infection.

Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.
Infection and immunity (Impact Factor: 4.16). 02/2011; 79(2):716-23. DOI: 10.1128/IAI.00486-10
Source: PubMed

ABSTRACT Purified protein derivative (PPD) is a widely used reagent for the diagnosis of Mycobacterium tuberculosis infection. Recently, the molecular composition of PPD was defined, with hundreds of mycobacterial protein representatives making up PPD. Which, if any, of these specific products drive the potency of PPD remains in question. In this study, two proteins (DnaK and GroEL2) previously identified as dominant proteins in PPD were tested for the capacity to induce delayed-type hypersensitivity (DTH) responses in H37Rv-infected or BCG-vaccinated guinea pigs. These two proteins were used in pull-down assays to identify interacting PPD products. Six proteins were identified as interacting partners with DnaK and GroEL2, i.e., Rv0009, Rv0475, Rv0569, Rv0685, Rv2626c, and Rv2632c. These six proteins were tested alone and in combination with DnaK and GroEL2 for the capacity to induce a DTH response in the guinea pig model. From these studies, two cocktails, DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685, were found to induce DTH responses in H37Rv-infected or BCG-vaccinated guinea pigs that were indistinguishable from DTH responses driven by a PPD injection. The mechanism by which DTH responses were induced was elucidated by histologic examination, analysis of activated CD4(+)/CD8(+) T cells, and cytokine mRNA expression at the site of the DTH response. PPD and the protein cocktails tested induced strong DTH responses in H37Rv-infected guinea pigs. Ex vivo phenotyping of T cells at the DTH site indicated that this response is mediated by activated CD4(+) and CD8(+) T cells, with increases in gamma interferon and tumor necrosis factor alpha, but not interleukin-10, at the site of the DTH response. Our results demonstrate for the first time that the PPD response can be mimicked at the molecular level with defined protein cocktails. The use of this defined product will allow a more thorough understanding of the DTH response and may provide a platform for more rapid and sensitive second-generation skin test reagents for the diagnosis of M. tuberculosis infection.

1 Follower
 · 
117 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis (Mtb) currently infects billions of people; many of whom are latently infection and at risk for reactivation. Mycobacterium bovis Bacille Calmette-Guerin (BCG) while approved as a vaccine, is unable to prevent reactivation of latent tuberculosis infection (LTBI). Subunit vaccines boosting BCG or given alone are being tested for efficacy in LTBI models. Alpha-crystallin (Acr, HspX), is a latency associated protein and subunit vaccine candidate. In this report, three HspX formulas (native and two recombinant variants) were used as vaccines in the guinea pig model of tuberculosis; none were protective during challenge with WT Mtb. However, recombinant HspX was protective in animals challenged with a strain of Mtb lacking hspX (X4-19), indicating protection was driven by molecules co-purifying with HspX or an adjuvant effect of recombinant HspX in this system. Mtb X4-19 was significantly less virulent than WT Mtb. Quantitative PCR and whole genome sequencing identified several genes (Rv2030c-Rv2032, Rv1062, Rv1771, Rv1907, and Rv3479) with altered expression that may contribute to loss of virulence. Physiological differences required for the establishment of Mtb infection in different hosts may affect the potential of subunit vaccines to elicit protection, supporting the need for rigorous biochemical and modeling analyses when developing tuberculosis vaccines.
    02/2014; 71(3). DOI:10.1111/2049-632X.12147
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.
    PLoS Pathogens 05/2014; 10(5):e1004132. DOI:10.1371/journal.ppat.1004132 · 8.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The tuberculin skin test (TST) is a simple and inexpensive test to determine whether individuals have been exposed to Mycobacterium tuberculosis (Mtb). This test is not always reliable, however, in people previously immunized with BCG and/or who have been exposed to environmental mycobacterial species due to a reaction to purified protein derivative (PPD) used in the skin test. An issue with BCG, therefore, is that the resulting sensitization to PPD in some individuals compromises the diagnostic use of the skin test. The ability to induce protective immune responses without sensitizing to the tuberculin skin test will be important properties of next generation tuberculosis (TB) vaccine candidates. Here we show that guinea pigs immunized with the candidate TB vaccine ID93/GLA-SE, currently in clinical trials, do not react to intradermal PPD administration. In contrast, positive DTH responses to both ID93 and components thereof were induced in ID93/GLA-SE-immunized animals, indicating robust but specific cellular responses were present in the immunized animals. Non-interference with the TST is an important factor for consideration in the development of a vaccine against Mtb.
    Clinical and vaccine Immunology: CVI 07/2014; DOI:10.1128/CVI.00372-14 · 2.37 Impact Factor