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A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis
© The Author(s). 2016
Received: 8 March 2016
Accepted: 3 August 2016
Published: 12 August 2016
In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed.
We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen.
Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3–0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay.
We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms.
Tuberculosis (TB) is a world-wide public health problem and the biggest cause of death due to a single pathogen. It is estimated that in 2014, 9.6 million people developed the disease, and 1.5 million died from it . Bacillus Calmette-Guérin (BCG) is the only available vaccine. It confers reliable protection against severe TB in infants and children, but its efficacy in adults is extremely variable (0–80 %), and a new vaccine is urgently needed to control the spread of the infection [2, 3]. Progress towards this goal has been slow. There are several reasons for this stagnation. Mycobacterium tuberculosis (Mtb) can persist in infected persons for years without causing disease or symptoms. Vaccine-mediated protection is therefore difficult to measure, resulting in a need for lengthy and costly clinical trials to establish vaccine efficacy. Additionally, only a handful of new vaccine candidates are currently under development, and funders have become more reluctant to provide large investments due to a risk of vaccine failure.
There is currently no validated immune correlate that predicts efficacy (such as antibody titres used for other infections) and this severely hampers the development and pre-clinical and clinical testing of TB vaccine candidates. Additionally, as highlighted by McShane and Williams  we do not have a single validated animal model for the screening of TB vaccine candidates, although head-to-head testing of TB vaccine candidates across a range of animal models can be reasonably used to demonstrate that vaccines are efficacious. Further, Henao-Tamayo et al.  argue that TB vaccines should be tested against different clinical isolates of Mtb, since the BCG Pasteur vaccine used in their study displayed varying levels of protection against different Mtb isolates in mouse and guinea pig models. A novel tool that would allow vaccine screening in different species may be more time- and cost-efficient than in vivo challenge experiments and could therefore help to accelerate vaccine development. The ability to test several different isolates or lineages of Mtb using cells from the same animal would reduce the number of animals needed, and the cost involved in these experiments.
Here, we present an optimized ex vivo mycobacterial growth inhibition assay (MGIA) for assessment of the summative vaccine-mediated host capacity to control mycobacterial growth. Several variations of mycobacterial growth inhibition assays have been described previously [6–9]. The assay described here involves direct co-culture of mouse splenocytes with mycobacteria, and subsequent measurement of mycobacterial growth inhibition. This particular variation of the assay has previously been shown to distinguish between naïve and immunized individuals in mice as well as humans by use of PBMC; however, the differences in mycobacterial burden between those two groups were small [10, 11]. To allow detection of a range of efficacies from different vaccine candidates, we aimed to achieve a difference of >0.5 log10 colony forming units (CFU) between BCG-naïve and BCG-immunized groups in our mouse model of TB. Our optimized assay produces a difference of up to 0.8 log10 CFU in our hands. Growth inhibition was also observed when using fast-growing Mycobacterium smegmatis (Msm) as the target bacteria in the MGIA. An in vivo challenge with the Mtb Erdman laboratory strain resulted in a ~0.7 log10 reduction in mycobacterial burden in the lungs and a 1 log10 reduction in the spleens of immunized animals, compared to unimmunized controls. We further demonstrate that an MGIA can show the importance of non-vaccine mediated interferon gamma (IFNγ)-dependent activity by using IFNγ-deficient (IFNγ-/-) mice. Collectively, our data suggest that MGIAs could be a promising tool for screening vaccine candidates pre-clinically, as well as determine the underlying immune mechanisms.
For immunization experiments, female C57BL/6 mice were acquired from Charles River UK at 5–7 weeks of age. Animals were acclimatized for at least 5 days before the start of any experimental procedure. Female B6.129S7-Ifng tm1Ts /J (IFNγ-/-) and C57BL/6 wild type (WT) controls bred in-house were used at 10–12 weeks of age. Group sizes of 5–6 mice were used as indicated throughout the manuscript.
Mycobacteria and culture conditions
BCG SSI and BCG Pasteur Aeras strains were obtained from Aeras (Rockville, MD, USA) as frozen aliquots. These were stored at -80 °C until needed. M. smegmatis was grown in 7H9 media with 10 % OADC, 0.5 % Glycerol and 0.05 % Tween80. At late log phase, bacteria were washed once with phosphate-buffered saline (PBS) + 0.05 % Tween80, and resuspended in PBS + 10 % Glycerol. Aliquots were frozen and stored at -80 °C until needed.
Bacteria were thawed at room temperature and diluted to a final concentration of 2–5 × 106 CFU/ml in physiological saline solution for irrigation (Baxter Healthcare, Newbury, UK). Each animal received a subcutaneous injection of 100 μl BCG (immunized groups) or physiological saline solution (control groups) into the left or right leg flap. Animals were rested for 6 weeks (unless indicated otherwise) before an ex vivo mycobacterial growth inhibition assay or infection with M. tuberculosis was carried out.
Ex vivo Mycobacterial Growth Inhibition Assay (MGIA)
Six weeks after immunization (unless specified otherwise), spleens were removed aseptically and single cell suspensions of splenocytes isolated by mechanical disruption of spleens through a 100 μm cell strainer. After lysis of red blood cells, single cell suspensions containing the desired number of total splenocytes per 300 μl were made up in antibiotic-free media (RPMI-1640 (Sigma-Aldrich, Dorset, UK) + 10 % heat-inactivated FBS (Labtech International Ltd, Uckfield, UK) + 2 mM L-Glutamine (Fisher Scientific, Loughborough, UK), and 300 μl aliquots were added to 2 ml screw cap tubes (Sarstedt, Nümbrecht, Germany). Mycobacteria were diluted in sufficient volume for all samples in the same media to a concentration of 90 to 3800 CFU per 300 μl as indicated for individual experiments. 300 μl aliquots of bacteria were added to the splenocytes, and the splenocyte-mycobacteria co-culture was then incubated on 360° tube rotators (VWR International, Lutterworth, UK) at 37 °C for 4 days.
After 4 days of incubation, the 2 ml screw cap tubes were centrifuged at 12,000 rpm in a bench-top microcentrifuge. The supernatants were removed (except 100 μl), and 400 μl of water was added to each tube to lyse host cells. The tubes were then vortexed briefly three times, with an incubation at room temperature for 5 min between each of the vortex steps. Each cell lysate (approx. 500 μl total) was then added to a MGIT tube (BD, Oxford, UK) and incubated in a BACTEC MGIT liquid culture system (BD) until registered positive.
To convert time to positivity (TTP) to bacterial numbers (CFU), a standard curve was used. To produce the standard curve, 500 μl of 10-fold dilutions of the mycobacterial strains were inoculated into the MGIT tubes, and TTP was plotted against CFU obtained from plating aliquots of the mycobacteria on 7H11 agar plates containing 10 % OADC supplement (Yorlab, York, UK) and 0.5 % glycerol. A linear regression analysis was carried out using GraphPad Prism version 6, and the resulting equation was used to convert TTP to CFU. Data are presented here as total number of CFUs per sample, as determined by use of a standard curve (Additional file 1: Figure S1). The difference between the medians of respective groups is described in the text and figures as Δ X log, and was calculated by subtracting the median of the test group (immunized or IFNγ-deficient mice) from the median of the control group (unimmunized or wild type mice).
Infection of mice with M. tuberculosis
Female C57BL/6 mice were infected intranasally with M. tuberculosis Erdman (BEI Resources, Manassas, VA, USA) 6 weeks after immunization and kept in isolators under BSL-3 containment. Frozen aliquots as received from BEI Resources were thawed at room temperature, and diluted in saline to a concentration of 1.4x104 CFU/ml. Mice were anaesthetized by an intraperitoneal injection of a combination of Ketamine (50 mg/kg; Ketalar, Pfizer Itd, Kent, UK) and Xylazine (10 mg/kg; Rompun; Berkshire, UK) in saline. Each animal then received 50 μl of the inoculum, estimated to contain 700 CFU. The number of bacteria in the inoculum was confirmed by plating aliquots on 7H11 agar plates containing 10 % OADC and 0.5 % glycerol.
Four weeks after infection, animals were killed by cervical dislocation. Lungs and spleens were removed aseptically and homogenized by mechanical disruption in sterile PBS. A series of 10-fold dilutions of tissue homogenates in PBS with 0.05 % Tween 80 were plated onto 7H11 agar plates with 10 % OADC supplement and 0.5 % glycerol. Plates were incubated at 37 °C and colonies counted after 3 weeks.
Statistical analysis was carried out using GraphPad Prism software Version 6 (GraphPad, La Jolla, CA, USA). The specific test used is indicated in each figure legend.
Comparison of BCG SSI and BCG Pasteur in an ex vivo MGIA
The number of target bacteria and the number of host cells both influence MGIA outcome
BCG-immunized and control groups can be distinguished in an MGIA by using fast-growing Mycobacterium smegmatis
MGIA reflects non-vaccine mediated, interferon gamma-dependent responses that are important for control of mycobacterial growth
The first step towards optimization of an ex vivo mycobacterial growth inhibition assay for pre-clinical vaccine testing was to compare two different BCG strains, BCG SSI and BCG Pasteur Aeras (Fig. 1). We found that using BCG Pasteur Aeras as both the strain for immunization and the strain for assessment of ex vivo growth inhibition led to larger differences between immunized and control groups (Fig. 1b), compared to when BCG SSI was used as the MGIA inoculum and vaccine (Fig. 1a). Importantly, this assay assesses growth inhibition ex vivo, and thus provides a snapshot of the ability of the immune system to control mycobacterial growth at one specific time point. The effect of BCG sub-strains on the elicited host immune response and conferred protection has been investigated previously. Irwin and colleagues find differences after immunization of mice with 3 BCG sub-strains in the number of IFNγ-producing splenic and lung T cells . However, this did not translate to differences in protection from an in vivo infection. Interestingly, the immune profile changes over time, but these changes were not translated to in vivo protection . This is in accordance with our data, as no difference in lung or spleen bacterial burden was found after Mtb challenge in mice immunized with BCG Pasteur Aeras or BCG SSI (Fig. 3). The MGIA using BCG SSI was carried out 4 weeks after immunization, whilst the assay using BCG Pasteur Aeras was carried out 6 weeks after immunization (Fig. 1). It is possible that a difference in immune responses at different time points would be apparent in an ex vivo MGIA, which might contribute towards the differences seen in Fig. 1, but not after challenge in vivo. We observed a faster growth rate in vitro of BCG Pasteur Aeras compared to BCG SSI (data not shown). This could be an additional reason for the larger differences between control and immunized groups seen with this strain ex vivo, if faster growth of bacteria is found in the control groups with comparable growth inhibition in the immunized groups. The immune response to BCG immunization was not characterized here, and it is therefore difficult to conclude which factor is the main driver behind the observed differences. The MGIA at this stage is not meant to exactly reproduce in vivo effects, but is a step that provides additional information between assays determining immunogenicity and in vivo challenge models. As a better indication of immunization-mediated growth inhibition was seen using the faster growing BCG Pasteur Aeras, this strain was used for subsequent experiments.
We reasoned that vaccine-mediated growth inhibition would depend on the presence of mycobacterial antigen-specific T cells. As total splenocytes are used in this assay, the proportion of antigen specific T cells in the total population is not known, and the total number may be too low to have an effect on growth inhibition when using low numbers of splenocytes. Similarly, the ratio of monocytes to mycobacteria may influence the outcome as mycobacteria can survive in these cells. In order to maximize growth inhibition in immunized compared to control splenocytes, we investigated several combinations of total host cell numbers (1, 3, or 5 × 106) and inocula of BCG Pasteur Aeras (2000, 500, or 100 CFU; Fig. 2). This confirmed that a lower number of bacteria leads to a greater difference between the groups, regardless of the number of host cells. We also observed that a higher number of host cells increased the difference between groups. Overall, we determined that 5 × 106 splenocytes and 100 CFU result in a comparatively large and statistically significant difference between medians of 0.81 log10 CFU whilst variability is acceptable (Coefficient of Variation = 22.63 % in the BCG group; 8.06 % in the control group; Fig. 2d). A significant difference was detectable in three out of four independent experiments, indicating that some variability remains and that controls of BCG immunized and naïve animals should be included when testing experimental TB vaccine candidates (Additional file 2: Figure S2). Analysis of variance of the data shown in Fig. 2a-c suggests that sample sizes of 6–8 are required to achieve statistical power to detect an effect size of >0.5 log10 CFU. Therefore increasing group sizes from the numbers used here (5–6 per group) may increase overall reproducibility.
Using fast growing mycobacteria such as M. smegmatis also led to growth inhibition ex vivo after immunization with BCG Pasteur Aeras (Fig. 4). The effect size seen here was similar to the one found when using BCG Pasteur Aeras as the target bacteria. We argued that the faster growth rate of Msm would lead to a shorter study time, that the non-virulent nature of the bacteria would ease handling and allow this assay to be carried out in a wider range of laboratories, and that a faster growth rate may lead to an accentuation of the effect size of growth inhibition. The overall experiment time was reduced by approx. 7 days. However, we did not find more pronounced growth inhibition. This may be because the non-pathogenic nature of the bacteria does not allow it to grow proportionately faster intracellularly in cells from non-immunized mice, or there may not be enough shared antigens between the BCG strain used for immunization and the target of growth inhibition (Msm).
Importantly, both assays result in inhibition of mycobacterial growth by up to 0.7–0.8 log10 CFU. This is within the range of the reduction of bacterial burden seen in our own challenge with Mtb Erdman, where we found a reduction of approx. 0.7 log10 CFU in lungs and 1 log10 CFU in spleens of immunized animals (Fig. 3). Others have found varying degrees of reduction in bacterial burden depending on the Mtb challenge strain used (0.75–1.26 log10 CFU in lung, 0.48–1.32 log10 CFU in spleen) .
We did not carry out any assessment of ex vivo growth inhibition using Mtb as the target bacteria, and it is therefore uncertain how representative these conditions are of an infection with virulent Mtb. However, using a growth inhibition assay that involves co-culture of mycobacteria-infected bone marrow-derived macrophages with non-adherent splenocytes from mice vaccinated with different vaccines, Kolibab and colleagues show that in vitro growth inhibition of BCG correlates with in vitro growth inhibition of Mtb, as well as with in vivo challenge with Mtb . We are presenting here a proof-of-concept study to enable further development of this assay. The advantage of using BCG as the target bacteria is that no specialized containment facilities are needed, making the MGIA much more accessible for a wide range of laboratories.
Using splenocytes from IFNγ-/- mice, we examined whether the MGIA can be used to assess the impact of immune factors such as IFNγ-dependent activity on growth control of mycobacteria. We found that splenocytes from IFNγ-/- mice are more permissive to mycobacterial growth than their WT counterparts (Fig. 5). This is in accordance with a wide array of literature describing the crucial role of IFNγ during tuberculosis [12, 13, 19, 20].
The main factor driving the outcome of the MGIA is currently unclear. However, given that the ex vivo MGIA directly assesses the summative ability of the host immune system to inhibit mycobacterial growth, the vaccine-mediated immune mechanism that underlies growth control does not need to be known a priori. In fact, this assay could help to determine underlying immune mechanisms of protection by investigating common factors in samples with efficient growth inhibition. As an ex vivo assay, it is also easily manipulated, for example by adding or depleting sub-populations of cells or cytokines. A further strength lies in the fact that several Mtb strains could be tested by using one single group of immunized mice and incubating aliquots of splenocytes with the different Mtb strains. This would significantly reduce the number of animals needed in comparison to in vivo challenge experiments, which need one group of animals for each Mtb strain to be tested – an ethical consideration that is regarded a priority within the UK research community. This approach may further allow the comparison of immune mechanisms elicited by the different strains.
We have taken several steps to optimize an existing mycobacterial growth inhibition assay in order to provide a potential tool that could help accelerate TB vaccine development. We titrated both host splenocyte numbers and bacterial inocula, and found three conditions that fulfill our criteria of delta >0.5 log CFU, Coefficient of Variance < 50 %, and required sample size < 10 per group (Fig. 2d). Whilst this is an improvement over the previously published 0.2 log10 CFU difference , variability and reproducibility could be further optimized. However, this assay could be useful in pre-clinical testing of vaccine candidates, as an intermediate step between immunogenicity testing and in vivo challenge experiments. It is both more rapid and economic than in vivo challenge experiments, and would also significantly reduce the potential harm experienced by the animals during such experiments. With an MGIA vaccine candidates could be screened in multiple doses, with different adjuvant formulations or against different lineages of Mtb before proceeding to in vivo efficacy testing. One of the strengths of this assay is its potential to be translated to other species, and therefore the possibility to test the same vaccine candidate in several animal models without the need for a pathogenic challenge experiment, or in humans.
The ex vivo MGIA is an important tool for the TB vaccine community and we encourage others to include this assay into their studies, and to contribute to the characterization and optimization of parameters will drive the development of the MGIA forward.
BCG, Bacillus Calmette-Guérin; CFU, colony forming unit; IFNγ, interferon gamma; MGIA, mycobacterial growth inhibition assay; Msm, Mycobacterium smegmatis; Mtb, Mycobacterium tuberculosis; PBS, phosphate buffered saline; TB, tuberculosis; TTP, time to positivity; WT, wild type
We are very grateful to Nathalie Cadieux and Megan Fitzpatrick (Aeras, Rockville) for provision of the BCG SSI and BCG Pasteur Aeras stocks. We would like to thank all staff at the Biological Services Facility at the London School of Hygiene and Tropical Medicine for their expert technical assistance.
Funding for this work was provided by Aeras and the European Union Horizon2020 program (TBVAC2020).
Availability of data and materials
Data will be shared upon request.
Author contributions are listed under the following categories and against each author’s initials: Data acquisition (DAc); data analysis (DAn); study design (SD); manuscript written (MW); manuscript revision (MR). AZ – DAc, DAn, SD, MW, MR. RT – DAn, SD, MR. ES - DAn, SD, MR. TD – DAc, DAn, MR. SM – SD, MR. AI – SD, MR. AW – SD, MR. SS – SD, MR. IP – SD, MR. BW – SD, MR. DAH – SD, MR. HM – DAn, SD, MR. MB – DAn, SD, MR. HF – DAn, SD, MW, MR. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All animal work was carried out in accordance with the Animals (Scientific Procedures) Act 1986 under a license granted by the UK Home Office (PPL 70/8043), and approved by the LSHTM Animal Welfare and Ethics Review Body.
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