Variability between countries in cytokine responses to BCG vaccination: what impact might this have on protection?

Keywords::

• BCG
• biomarkers
• immunological assays
• infants
• tuberculosis
• vaccine efficacy

In 2010, there were an estimated 8.8 million incident cases of TB and 1.4 million deaths globally despite the widespread use of BCG [101]. With the aim of elimination of TB by 2050, there is a concerted drive to combine interventions in order to improve diagnosis, prevent infection, treat active disease and reduce the risk factors for TB [102]. With the rising burden of drug-resistant strains of TB and with TB susceptibility in those with HIV/AIDS, there is an urgent need for new effective vaccines that work for all forms of TB in pre- and post-exposed individuals of all ages in all parts of the world.

Whilst there are promising signs in TB vaccine development [1], there are still many unanswered questions, in particular as to what constitutes a protective immune response. Geographic variations in protective immunity induced by the current vaccine, BCG [2], are still poorly understood, and without biomarkers of protection, there are many challenges ahead to identify and test potential vaccines for TB globally.

Smaller immunological studies have shown variability in cytokine responses to BCG vaccination between countries, including between BCG-vaccinated adolescents and infants in Malawi and the UK [3–5]. Whereas UK adolescents made strong IFN-γ responses to Mycobacterium tuberculosis (Mtb) purified protein derivative (PPD) post-vaccination, Malawian adolescents and young adults who were presensitized to PPD failed to show a significant increase [3]. Malawian infants also produced weaker IFN-γ responses following BCG vaccination than infants in the UK [4,5]. Three questions arise from these observations: are the differences real, are they important and what implications might they have on protection induced by BCG and future vaccines?

The need for harmonized assays

In order to determine whether geographic variations in cytokine responses are real, it is essential to consider the methods employed to measure them in each setting. A variety of approaches to measure cytokine responses have been used in different locations. Although studies have documented that complex cytokine profiles are induced following BCG vaccination of infants [5,6], the effect of a particular geographical setting can only be assessed where identical protocols have been rigorously employed in at least two settings [3–5].

Groups from different settings have used different protocols, assays and markers at different time points post-vaccination. In order to combine data from different locations to determine the local environmental effects on the response to BCG vaccination, harmonization of approaches is required, as advocated for TB vaccine trials [7]. Until recently, the measurement of IFN-γ alone was often used as an indicator of mycobacterial immunity, but although IFN-γ may be necessary for protection, it does not appear to be sufficient or to correlate quantitatively with protection [1,8,9]. IFN-γ release combined with Mtb-specific stimulants can provide a simple yes/no answer to the question of whether or not an individual has been infected with Mtb, but not of whether that individual is protected from developing active TB disease. For that, an expanded cytokine profile or biosignature is required and multiplex readouts are well placed to provide this.

Many different systems and protocols have been used with multiplex bead arrays or with flow cytometry. What the latter sacrifices in multiple measurement capacity, it gains in its ability to identify the cellular sources of different cytokines, including the numerous cell types that may contribute to mycobacterial immunity (e.g., CD4⁺ Th1, Th17, CD8⁺ and γδ T cells) or that may prevent the immunopathology characterizing active TB disease (Treg cells, T-follicular helper cells and B cells). However, even the most advanced flow cytometers can measure only a handful of cytokines simultaneously, so rational design is required to select an optimal antibody panel. In the same way that a quantitative measure of IFN-γ does not provide an indicator of protective immune status, the same may soon apply to four, five or six cytokine profiles that are restricted by our incomplete understanding of mycobacterial immunity [10]. Multiplex bead array assays substantially expand upon what it is possible to measure by flow cytometry, but an even greater expansion is possible when assessing transcriptional gene profiles. Targeted transcriptional analysis using multiplex techniques, such as multiplex ligation-dependent probe amplification [11] or to a much greater extent microarray [12], can assess many hundreds to tens of thousands of biomarkers expressed at a given time or in response to a particular antigen stimulus. When combined with modern bioinformatic analysis, the potential of these approaches to reveal informative biosignatures is immense. Using premanufactured kits, bead sets, probe sets or chips enables standardization of protocols across different studies and sites. Once a common approach to biomarker measurement has been agreed, the challenge then becomes one of ensuring that groups are not comparing apples with oranges. For example, biomarker profiles measured in 7-day stimulated peripheral blood mononuclear cell samples will be distinct from those measured in freshly isolated peripheral blood. Harmonization of protocols requires decisions on the study cohort, when to sample, what stimulant to use (if any) and which clinical samples to use. Clearly, with testing of new vaccines in progress, now is the time to ensure such harmonization across sites and vaccines.

Are geographic differences in cytokine responses important?

While it may be difficult to directly compare results from different settings, it would be unwise not to reflect on their implications. The timing of vaccination is one example in which the results from three African countries differ. A delay in administering BCG from birth to 2–4 months of age can result in greater immunogenicity (South Africa [13]), reduced immunogenicity (Gambia [14]) or no difference (Malawi [Ben-Smith A, Unpublished Data]). While it is true that ‘immunogenicity’ was measured in different ways and that the timings of delay and testing were different, perhaps even at such young ages infants in these three countries have different immunological set-points and thus respond differently to BCG vaccination.

Using the example of the UK–Malawi studies where stringent quality controls and assay harmonization were used, there are major differences in immune responses following vaccination. Previously unvaccinated Malawian adolescents were presensitized and did not show enhanced IFN-γ production in diluted whole blood cultures stimulated with Mtb PPD 12 months following BCG vaccination, while UK adolescents showed a large increase in IFN-γproduction [3]. Subsequent studies in infants showed an entirely different profile of cytokines following BCG vaccination in the UK compared with Malawi, with enhanced Th2 and IL-10, innate proinflammatory cytokine and growth factor production [5], as well as reduced IFN-γ responses [4,5]; another study in Indonesia showed marked induction of IL-5 and IL-13 in BCG-vaccinated infants tested at 5 months of age [15]. While we do not know what constitutes a protective biosignature, these results suggest that efficacy in infants could be different in these populations as has been shown in adolescents [2].

Future work needed

The implications of these differing cytokine responses following BCG vaccination across different countries are huge. It is possible that different vaccines, vaccine doses or immunization schedules will be required for different settings and in different populations. Perhaps the most important lesson is that we cannot limit testing of vaccines to a few sites, but that more comprehensive testing strategies should be used where harmonized protocols are carried out in multicentered trials, particularly in settings where it has previously been shown that BCG vaccination fails to induce protection from TB disease.

It would also be useful for all future TB vaccine test sites to profile the biosignatures induced by BCG vaccination in their setting. Whether such cytokine profiles have an impact on the protective efficacy of the BCG vaccine, and if so, whether the new candidate TB vaccines in development would also be affected is not known. But without this knowledge, or an explanation for the causes of variability (e.g., nutrition, infections, maternal immunity, Expanded Program on Immunization vaccine schedules and other environmental effects), it will be difficult to interpret the results from new TB vaccine trials. It would be ideal if TB vaccine trials could be performed at sites conducting multidisciplinary life-course studies examining the development of immunological footprints and their consequences throughout life [16]. Although the clinical trials of the efficacy of BCG vaccination in infants have all shown protection against the disseminated forms of TB in childhood [17], none were performed in Africa. Geographical variation in vaccine efficacy has been reported for other vaccines; the causes of such variation are also unknown, but may include differences in gut microbiota, which are likely to be different in European and African infants, with associated effects on systemic as well as mucosal immunity [18].

Expectations are high that a new generation of vaccines will improve upon the variable efficacy of BCG and give consistent protection against TB no matter where or to whom they are administered. However, for this to happen, it is essential that we understand what it is in these different environments that leads to variations in immune responses to BCG and also how these variations might contribute to differences in efficacy. Without this knowledge, we have no way to ensure that new vaccines will not be affected by whatever factors determine the variable efficacy of BCG vaccination.

Financial & competing interests disclosure

HM Dockrell’s laboratory has received financial support from the Wellcome Trust, the EU-funded TBVAC, NewTBVAC and TRANSVAC Consortia, and the Bill and Melinda Gates Foundation Grand Challenge 6–74 Consortium for these studies.The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.