A comparison of IFNγ detection methods used in tuberculosis vaccine trials

doi:10.1016/j.tube.2008.06.005

Tuberculosis

Volume 88, Issue 6, November 2008, Pages 631-640

https://doi.org/10.1016/j.tube.2008.06.005 Get rights and content

Summary

Interferon gamma (IFNγ) is a critical component of the pro-inflammatory immune response that provides protection against Mycobacterium tuberculosis. In the absence of an immunological correlate of protection, antigen-specific production of IFNγ is a commonly used marker of a protective immune response. To facilitate the evaluation of tuberculosis candidate vaccines three different IFNγ detection methods were compared. The cultured whole blood ELISA, ex vivo IFNγ ELISpot and whole blood ex vivo intracellular cytokine staining (ICS) assays were performed head-to-head during a Phase I clinical trial using the candidate vaccine MVA85A. Whilst all three assays detected significant increases in IFNγ production immediately following vaccination, distinctions between the assays were apparent. Higher baseline IFNγ responses were detected using the cultured whole blood ELISA, whereas the ex vivo ELISpot assay was the most sensitive in detecting long-term (52 weeks) post-vaccination responses. The whole blood ex vivo ICS assay provided novel information by dissecting the IFNγ response into responding CD4, CD8 and γ/δ T cell subsets.

Future tuberculosis vaccine trials and immunology studies should ideally include a combination of ex vivo and cultured assays to ensure a thorough and multifaceted evaluation of the immune response is achieved.

Introduction

Tuberculosis is annually responsible for around 2 million deaths.¹ The greatest burden of morbidity and mortality occurs in the developing world, notably sub-Saharan Africa. In addition to strategic measures, considerable research efforts are underway to develop effective vaccines and novel therapeutic agents to control transmission and reduce disease prevalence.² The identification of surrogate markers of protective immunity would improve research efficiency and aid decision-making as to which candidate vaccines should be progressed into efficacy trials; however, no such marker has yet been clearly defined. IFNγ has an essential role in protection against tuberculosis3, 4, 5 and is likely to be a component of a protective biosignature. In the absence of more specific indicators, IFNγ remains a widely used immunological readout in tuberculosis research.6, 7, 8, 9

A number of tuberculosis vaccine candidates have entered Phase I clinical trials,10, 11, 12 and many more will shortly be tested in humans.13, 14 To expediate the evaluation of candidate TB vaccines it is important that the most informative immunological assays are used in these studies, and that data obtained from different vaccine trials can be compared. The comparability of IFNγ detection methods has been investigated in the context of diagnosis of tuberculosis disease,15, 16 however, no such studies have yet been performed in tuberculosis vaccine trials. To this end, various working groups led by the World Health Organisation Initiative for Vaccine Research and TB-VAC (EU 6th Framework), have been established to coordinate immunological assay harmonisation and standardisation across the different tuberculosis vaccine research groups.

The aim of this study was to extend these discussions by transferring technologies between research groups and performing a head-to-head comparison of three IFNγ detection methods during a Phase I trial of the candidate tuberculosis vaccine modified vaccinia Ankara expressing antigen 85A (MVA85A).

The three IFNγ detection methods examined in this study have each been previously used in tuberculosis vaccine trials in Africa and the UK; cultured whole blood ELISA,6, 17, 18ex vivo ELISpot9, 11 and ex vivo whole blood intracellular cytokine staining.19, 20 These assays provide information regarding antigen-specific IFNγ production by expanded peripheral blood leukocytes (PBL), ex vivo peripheral blood mononuclear cells (PBMC) and ex vivo α/β and/or γ/δ T cells, respectively. During this comparative study the three assays under investigation were performed in parallel on fresh blood or PBMC as appropriate. Recall responses to multifarious mycobacterial antigens (PPD and Mycobacterium bovis BCG) as well as those specific to the subunit antigen (antigen 85A) were measured, prior to and at various timepoints following the booster immunisation. The level of correlation between the assays was determined.

The harmonisation and standardisation of immunological assays will help expediate the assessment and selection of vaccine candidates, and ultimately the development of an effective tuberculosis vaccine. The information provided in this study is relevant to the planning of tuberculosis vaccine trials.

Section snippets

Vaccine study participants

BCG-vaccinated adults (maximum Heaf test reaction grade II), negative on ex vivo ELISpot for early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) were recruited under protocols approved by the Oxfordshire Research Ethics Committee, ClinicalTrials.gov ID NCT00395720. The age range for inclusion was 18–50; all subjects were required to be seronegative for HIV, hepatitis B virus and hepatitis C virus. Subjects received a single intradermal inoculation with MVA85A¹¹

Results

In total, 12 BCG-vaccinated subjects were recruited into this trial. The median interval between BCG vaccination and boosting with MVA85A was 11 years (range 1–30). For all assays antigen-specific IFNγ responses are shown at baseline (i.e., after BCG vaccination), at short-term timepoints following MVA85A boosting (weeks 1, 2 and 4), and at long-term timepoints following MVA85A boosting (week 52). The three assays (cultured whole blood ELISA, ex vivo ELISpot, and ex vivo whole blood ICS) were

Discussion

All three assays evaluated in this study detected mycobacteria-specific IFNγ responses in the BCG-vaccinated subjects, which were significantly amplified following immunisation with MVA85A.

Higher baseline (resting memory) responses were detected in the cultured whole blood ELISA assay compared to the two ex vivo assays (ex vivo ELISpot and whole blood ex vivo ICS). All subjects were previously vaccinated with BCG (the median interval between vaccinations was 11 years) and may well have been

Acknowledgements

We thank all subjects who took part in the study reported here. NERB holds a Medical Research Council Studentship. TJS is a Wellcome Trust Research Training Fellow (080929/Z/06/Z), HM is a Wellcome Trust Senior Clinical Fellow, AVSH is a Wellcome Trust Principle Research Fellow. This work was supported by the Oxford Biomedical Research Centre with funding from the UK Department of Health’s NIHR Biomedical Research Centres funding scheme. HM and AVSH are Jenner Institute Investigators. WAH is

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To optimize the TB control effort with accurate diagnostic tools, it is essential to better understand the interaction between Mycobacterium tuberculosis complex (MTBC) and the human immune system. Measurement of MTBC antigen-specific interferon-gamma (IFN-γ) remains the main immunological readout for TB vaccine and TB infection assessment (Beveridge et al., 2008). In addition to the human host immune status, the genetic diversity of MTBC plays a role in the host immune response and TB infection (Chae and Shin, 2018; Rutaihwa et al., 2019).
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Of the 24 cytokines studied, three were produced differentially in whole blood dependent on the infecting lineage of MTBC. Decreased production of IL-17 was observed in patients infected with modern lineages compared with patients infected with ancestral lineages (P < 0.01), and production of IFN-γ and IL-2 was significantly decreased in patients infected with lineage 4 strains compared with patients infected with lineage 3 strains (P < 0.05).
MTBC strains belonging to lineage 4 induced a decreased whole-blood PPD-stimulated pro-inflammatory cytokine response.
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Immune responses following Mycobacterium tuberculosis (Mtb) infection or vaccination are frequently assessed by measuring T-cell recognition of crude Mtb antigens, recombinant proteins, or peptide epitopes. We previously showed that not all Mtb-specific T cells recognize Mtb-infected macrophages. Thus, an important question is what proportion of T cells elicited by Mtb infection recognize Mtb-infected macrophages. We address this question by developing a modified elispot assay using viable Mtb-infected macrophages, a low multiplicity of infection and purified T cells. In C57BL/6 mice, CD4 and CD8 T cells were classically MHC restricted. Comparable frequencies of T cells that recognize Mtb-infected macrophages were determined using interferon-γ elispot and intracellular cytokine staining, and lung CD4 T cells more sensitively recognized Mtb-infected macrophages than lung CD8 T cells. Compared to the relatively high frequencies of T cells specific for antigens such as ESAT-6 and TB10.4, low frequencies of total pulmonary T cells elicited by aerosolized Mtb infection recognize Mtb-infected macrophages. Finally, we demonstrate that BCG vaccination elicits T cells that recognize Mtb-infected macrophages. We propose that the frequency of T cells that recognize infected macrophages could correlate with protective immunity and may be an alternative approach to measuring T-cell responses to Mtb antigens.
In vitro analysis of antigen induced T cell-monocyte conjugates by imaging flow cytometry
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The Th-1 cytokine, interferon gamma (IFN-γ) is considered a key protective cytokine based on earlier studies, in both mice and humans, which showed that defects in the IFN-γ signaling pathways provoked higher susceptibility to Mycobacterium tuberculosis (Mtb) infection and severe disease (Cooper et al., 1993; Newport et al., 1996; Flynn et al., 2003). Thus, many in vitro studies have focused on determining the frequency of Mtb specific T cells secreting IFN-γ alone, in combination with other Th-1 cytokines; IL-2 and TNFα measuring their level of secretion, and extent of T cell proliferation as markers of protection (McShane et al., 2004; Beveridge et al., 2008; Hoft et al., 2008; Smith et al., 2016). On the other hand, studies have shown that cytokine levels do not necessarily correlate with protective immunity nor predict risk of progression to active TB disease (Pai et al., 2004; Elias et al., 2005; Majlessi et al., 2006; Mittrucker et al., 2007; Panteleev et al., 2017).
There is a lack of suitable correlates of immune protection against Mycobacterium tuberculosis (Mtb) infection. T cells and monocytes play key roles in host immunity against Mtb. Thus, a method that allows assessing their interaction would contribute to the understanding of immune regulation in tuberculosis (TB). We have established imaging flow cytometer (IFC) based in vitro assay for the analysis of early events in T cell-monocyte interaction, upstream of cytokine production and T cell proliferation. This was achieved through short term stimulation of peripheral blood mononuclear cells (PBMC) from healthy Norwegian blood donors with Mycobacterium bovis Bacille Calmette-Guérin (BCG). In our assay, we examined the kinetics of BCG uptake by monocytes using fluorescently labeled BCG and T cell-monocyte interaction based on synapse formation (CD3/TCR polarization). Our results showed that BCG stimulation induced a gradual increase in the proportion of conjugated T cells displaying NF-κB translocation to the nucleus in a time dependent manner, with the highest frequency observed at 6 h. We subsequently tested PBMC from a small cohort of active TB patients (n = 7) and observed a similar BCG induced NF-κB translocation in T cells conjugated with monocytes. The method allowed for simultaneous evaluation of T cell-monocyte conjugates and T cell activation as measured by NF-κB translocation, following short-term challenge of human PBMC with BCG. Whether this novel approach could serve as a diagnostic or prognostic marker needs to be investigated using a wide array of Mtb specific antigens in a larger cohort of patients with different TB infection status.
T cell recall response of two hypothetical proteins (Rv2251 and Rv2721c) from Mycobacterium tuberculosis in healthy household contacts of TB – Possible subunit vaccine candidates
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The demonstrated variable efficacy of the only licensed TB vaccine Mycobacterium bovis bacillus Calmette–Guérin (M. bovis BCG) encourages the need for new vaccine candidates against TB. Antigen specific cellular immune response is often considered imperative during Mycobacterium tuberculosis (M. tuberculosis) infection and antigens that are strongly associated with the latent phase of infection are drawing increasing attention for anti-TB vaccine development. Here, we investigated the phenotypic and functional profiles of two novel mycobacterial antigens Rv2251 and Rv2721c during T cell recall response via multi-color flow cytometry. Healthy household contacts of TB (latent/HHC) and active pulmonary TB (PTB) patients were recruited to investigate the difference in antigen specific T cell recall response. These two antigens induced expansion of CD45RA⁻ CCR7⁺ central memory subtypes and CD45RA⁻ CCR7⁻ effector memory cells in latent population which suggests their possible association with HHC. Rv2251 and Rv2721c antigen specific IFN-γ, TNF-α and IL-2 response was also significantly high in HHC when compared to the PTB (p < 0.005, p < 0.05 and p < 0.05 respectively). The frequency of multifunctional T cells also was high in HHC compared to the PTB with statistical significance only for the antigen Rv2251. Often, the dominant Th1 immune response in HHC is correlated with the protection against the active TB disease. Collectively, we report the first insights into Rv2251 and Rv2721c antigen specific immune response in human donors of TB and provide the immunologic rationale for selecting them for vaccine development against TB.
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Cell-mediated immune responses are pivotal in the host's response to Mycobacterium tuberculosis (Mtb) infection and IFN-γ produced predominantly by CD4+ T lymphocytes is a crucial component of this response [1–4]. Production of IFN-γ in response to Mtb-specific antigens is commonly used as a marker of potentially protective immunity against Mtb [5–7]. The 20- to 40-fold increased risk of developing active TB among HIV-infected individuals, particularly those with low blood CD4+ T lymphocyte counts [8] underscores the importance of T cell-mediated adaptive immunity in protection against disease caused by Mtb.
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Homologous and heterologous parenteral prime–mucosal boost immunizations have shown great promise in combating mucosal infections such as tuberculosis and AIDS. However, their immune mechanisms remain poorly defined. In particular, it is still unclear whether T-cell and innate immunity may be independently affected by these immunization modalities and how it impacts immune protective outcome. Using two virus-based tuberculosis vaccines (adenovirus (Ad) and vesicular stomatitis virus (VSV) vectors), we found that while both homologous (Ad/Ad) and heterologous (Ad/VSV) respiratory mucosal boost immunizations elicited similar T-cell responses in the lung, they led to drastically different immune protective outcomes. Compared with Ad-based boosting, VSV-based boosting resulted in poorly enhanced protection against tuberculosis. Such inferior protection was associated with differentially imprinted innate phagocytes, particularly the CD11c⁺CD11b^+/− cells, in the lung. We identified heightened type 1 interferon (IFN) responses to be the triggering mechanism. Thus, increased IFN-β severely blunted interleukin-12 responses in infected phagocytes, which in turn impaired their nitric oxide production and antimycobacterial activities. Our study reveals that vaccine vectors may differentially imprint innate cells at the mucosal site of immunization, which can impact immune-protective outcome, independent of T-cell immunity, and it is of importance to determine both T-cell and innate cell immunity in vaccine studies.

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A comparison of IFNγ detection methods used in tuberculosis vaccine trials

Summary

Introduction

Section snippets

Vaccine study participants

Results

Discussion

Acknowledgements

Lancet

Vaccine

Tuberculosis (Edinb)

J Immunol Methods

J Immunol Methods

The global plan to stop TB, 2006–2015. Actions for life: towards a world free of tuberculosis

Int J Tuberc Lung Dis

An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection

J Exp Med

A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection

N Engl J Med

Heterogeneity in the granulomatous response to mycobacterial infection in patients with defined genetic mutations in the interleukin 12-dependent interferon-gamma production pathway

Int J Exp Pathol

Dynamic antigen-specific T-cell responses after point-source exposure to Mycobacterium tuberculosis

Am J Respir Crit Care Med

Dynamic relationship between IFN-gamma and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load

J Immunol

Direct ex vivo analysis of antigen-specific IFN-gamma-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment

J Immunol

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein

J Immunol