Mycobacterial growth inhibition in murine splenocytes as a surrogate for protection against Mycobacterium tuberculosis (M. tb)

Marsay, Leanne; Matsumiya, Magali; Tanner, Rachel; Poyntz, Hazel C.; Griffiths, Kristin; Stylianou, Elena; Marsh, Philip; Williams, Ann; Sharpe, Sally; Fletcher, Helen A.; McShane, Helen

doi:10.1016/j.tube.2013.04.007

Cited by 39 publications

(56 citation statements)

References 29 publications

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“…A colony-forming unit (CFU)-based net growth value calculated relative to a control and stock standard curve permits measurement of mycobacterial growth. This assay has been adapted for measuring vaccine effect in whole blood and peripheral blood mononuclear cells (PBMC) from humans8 and in splenocytes from mice910. Here we show that there is no correlation between mycobacterial growth in human whole blood and PBMC MGIT assays, and investigate haemoglobin (Hb) and iron as potential contributing factors to this discrepancy.…”

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confidence: 74%

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The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays

Tanner

O’Shea

White

et al. 2017

Sci Rep

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The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays.

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confidence: 74%

“…We have evaluated the effect of Hb and iron on mycobacterial growth in vitro using the previously-described MGIT assay78910. This was investigated across different species commonly used in TB vaccine testing; human, mouse and non-human primates (NHPs).…”

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confidence: 99%

The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays

Tanner

O’Shea

White

et al. 2017

Sci Rep

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“…Therefore, a tempered DTH response-whether due to a deficiency in IL12R␤1⌬TM, IL-23, or tryptophan starvation-is manifested greater in these organs. Perhaps for this same reason, the functional inhibition of M. tuberculosis by mouse splenocytes is a useful surrogate for BCG-vaccine-mediated protection in mice (69).…”

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confidence: 99%

IL12Rβ1ΔTM Is a Secreted Product ofil12rb1That Promotes Control of Extrapulmonary Tuberculosis

et al. 2015

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bIL12RB1 is a human gene that is important for resistance to Mycobacterium tuberculosis infection. IL12RB1 is expressed by multiple leukocyte lineages, and encodes a type I transmembrane protein (IL12R␤1) that associates with IL12p40 and promotes the development of host-protective T H 1cells. Recently, we observed that il12rb1-the mouse homolog of IL12RB1-is alternatively spliced by leukocytes to produce a second isoform (IL12R␤1⌬TM) that has biological properties distinct from IL12R␤1. Although the expression of IL12R␤1⌬TM is elicited by M. tuberculosis in vivo, and its overexpression enhances IL12p40 responsiveness in vitro, the contribution of IL12R␤1⌬TM to controlling M. tuberculosis infection has not been tested. Here, we demonstrate that IL12R␤1⌬TM represents a secreted product of il12rb1 that, when absent from mice, compromises their ability to control M. tuberculosis infection in extrapulmonary organs. Furthermore, elevated M. tuberculosis burdens in IL12R␤1⌬TM-deficient animals are associated with decreased lymph node cellularity and a decline in T H 1 development. Collectively, these data support a model wherein IL12R␤1⌬TM is a secreted product of il12rb1 that promotes resistance to M. tuberculosis infection by potentiating T H cells response to IL-12.

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“…Recently, there has been a renewed interest in using in vitro mycobacterial growth inhibition assays to evaluate vaccine activity in clinical trials and to characterize the potency of newly manufactured vaccine preparations (17)(18)(19). To investigate whether MGIT TTD analyses are useful in quantifying mycobacteria for in vitro assays, coculture assays were generated with splenocytes recovered from vaccinated or naive mice and bone marrow macrophages infected with M. tuberculosis Erdman as previously described (17).…”

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Time to Detection of Mycobacterium tuberculosis Using the MGIT 320 System Correlates with Colony Counting in Preclinical Testing of New Vaccines

Kolibab

Yang

Parra

et al. 2013

Clin. Vaccine Immunol.

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Clinical studies have suggested that the enumeration of mycobacteria by using automated liquid systems is a faster and simpler alternative to quantitative cultures. Here, we show that the time to detection of M. tuberculosis growth as measured with the MGIT 320 liquid culture system inversely correlates with CFU determinations from culture on solid media and that mycobacterial quantification using the MGIT system is faster and easier to perform than CFU plating.T he limited effectiveness of the Mycobacterium bovis BCG vaccine in preventing adult pulmonary tuberculosis (TB) and the increasing incidence of deadly multidrug-resistant M. tuberculosis strains emphasize the need to develop improved vaccination strategies against TB (1-3). However, the recent disappointing results from the MVA85A TB vaccine trial in South Africa suggested that the search for new approaches to TB immunization is just beginning (4). Currently, most new vaccines against TB are evaluated in a multistep preclinical process that often includes testing in two or more animal models (5). Although established models are available for assessing novel TB vaccine preparations in mice, guinea pigs, rabbits, and nonhuman primates, testing new vaccines in these models is slow and often not reproducible in different laboratories. A time-consuming component of the vaccine evaluation protocols is the enumeration of postchallenge organ mycobacterial burdens by colony counting on solid media. Standard CFU determinations are labor-intensive, hampered by the tendency of mycobacteria to clump, and sometimes fail due to contamination (6). In many TB vaccine testing labs, CFU determinations can vary within different experiments because of the absence of standardized reagents for solid media preparation. Most importantly, CFU determinations are slow; usually about 3 weeks of incubation are required to detect colonies on mycobacterial growth plates (7).For more than a decade, the MGIT liquid media system has been increasingly employed to assess whether M. tuberculosis bacilli are present in clinical specimens (8-11). The MGIT system uses an oxygen-quenching fluorescent sensor and appropriate software algorithms to determine whether significant mycobacterial growth has occurred (12). The instrument reports this bacterial growth as the time to detection (TTD). Overall, the MGIT system has been shown to be highly sensitive and to shorten the time needed to detect M. tuberculosis in clinical samples by 1 to 3 weeks (relative to growth on solid media). Several multicenter studies have demonstrated that MGIT-based protocols for detecting drug-resistant M. tuberculosis strains are relatively rapid and highly reproducible between different laboratories (13-15). Interestingly, the TTD measurements obtained using the automated liquid systems have been shown to closely compare with the results of CFU counting on solid media, and therefore the MGIT system represents a viable alternative to CFU determinations in evaluating responses to TB chemotherapy (9).To potentially impro...

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Mycobacterial growth inhibition in murine splenocytes as a surrogate for protection against Mycobacterium tuberculosis (M. tb)

Cited by 39 publications

References 29 publications

The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays

The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays

IL12Rβ1ΔTM Is a Secreted Product ofil12rb1That Promotes Control of Extrapulmonary Tuberculosis

Time to Detection of Mycobacterium tuberculosis Using the MGIT 320 System Correlates with Colony Counting in Preclinical Testing of New Vaccines

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