Can what have we learnt about BCG vaccination in the last 20 years help us to design a better tuberculosis vaccine?

The BCG vaccine will, in 2021, have been in use for 100 years. Much remains to be understood, including the reasons for its variable efficacy against pulmonary tuberculosis in adults. This review will discuss what has been learnt about the BCG vaccine in the last two decades, and whether this new information can be exploited to improve its efficacy, by enhancing its ability to induce either antigen-specific and/or non-specific effects. Many factors affect both the immunogenicity of BCG and its protective efficacy, highlighting the challenges of working with a live vaccine in man, but new insights may enable us to exploit better what BCG can do

Modulation of NKG2D expression in human CD8(+) T cells corresponding with tuberculosis drug cure.

BACKGROUND: Biomarkers predicting tuberculosis treatment response and cure would facilitate drug development. This study investigated expression patterns of the co-stimulation molecule NKG2D in human tuberculosis and treatment to determine its potential usefulness as a host biomarker of tuberculosis drug efficacy. METHODS: Tuberculosis patients (n = 26) were recruited in Lahore, Pakistan, at diagnosis and followed up during treatment. Household contacts (n = 24) were also recruited. NKG2D expression was measured by qRT-PCR in RNA samples both ex vivo and following overnight mycobacterial stimulation in vitro. Protein expression of NKG2D and granzyme B was measured by flow cytometry. RESULTS: NKG2D expression in newly diagnosed tuberculosis patients was similar to household contacts in ex vivo RNA, but was higher following in vitro stimulation. The NKG2D expression was dramatically reduced by intensive phase chemotherapy, in both ex vivo blood RNA and CD8(+) T cell protein expression, but then reverted to higher levels after the continuation phase in successfully treated patients. CONCLUSION: The changes in NKG2D expression through successful treatment reflect modulation of the peripheral cytotoxic T cell response. This likely reflects firstly in vivo stimulation by live Mycobacterium tuberculosis, followed by the response to dead bacilli, antigen-release and finally immunopathology resolution. Such changes in host peripheral gene expression, alongside clinical and microbiological indices, could be developed into a biosignature of tuberculosis drug-induced cure to be used in future clinical trials

Circulating B-lymphocytes as potential biomarkers of tuberculosis infection activity.

Accurate biomarkers of Mycobacterium tuberculosis infection activity would significantly improve early diagnosis, treatment and management of M. tuberculosis infection. We hypothesised that circulating B-lymphocytes may be useful biomarkers of tuberculosis (TB) infection status in highly TB-endemic settings. Ex-vivo and in-vitro mycobacteria-specific B-cell ELISPOT assays were used to examine the plasmablast (PB) and memory B-cell (MBC) responses in the peripheral blood of adult, healthy, community controls (n = 151) and of active TB patients (n = 48) living in Uganda. Frequencies of mycobacteria-specific PBs were markedly higher in active TB patients compared to healthy controls, and, conversely, MBCs were markedly higher in the healthy controls compared to active TB patients. In addition, the community controls with evidence of latent TB infection had higher peripheral blood PB and MBC responses than those without evidence of TB infection. These data demonstrate that peripheral blood B-cell responses are differentially modulated during latent and active M. tuberculosis infection, and suggest that the PB to MBC ratio may be a useful biomarker of TB infection activity

Increased activated memory B-cells in the peripheral blood of patients with erythema nodosum leprosum reactions.

B-cells, in addition to antibody secretion, have emerged increasingly as effector and immunoregulatory cells in several chronic inflammatory diseases. Although Erythema Nodosum Leprosum (ENL) is an inflammatory complication of leprosy, the role of B- cell subsets has never been studied in this patient group. Therefore, it would be interesting to examine the contribution of B-cells in the pathogenesis of ENL. A case-control study design was used to recruit 30 untreated patients with ENL and 30 non-reactional lepromatous leprosy (LL) patient controls at ALERT Hospital, Ethiopia. Peripheral blood samples were obtained before, during and after treatment from each patient. Peripheral blood mononuclear cells (PBMCs) were isolated and used for immunophenotyping of B- cell subsets by flow cytometry. The kinetics of B-cells in patients with ENL before, during and after Prednisolone treatment of ENL was compared with LL patient controls as well as within ENL group. Total B-cells, mature B-cells and resting memory B-cells were not significantly different between patients with ENL reactions and LL controls before treatment. Interestingly, while the percentage of naive B-cells was significantly lower in untreated ENL patients than in LL patient controls, the percentage of activated memory B-cells was significantly higher in these untreated ENL patients than in LL controls. On the other hand, the percentage of tissue-like memory B-cells was considerably low in untreated ENL patients compared to LL controls. It appears that the lower frequency of tissue-like memory B-cells in untreated ENL could promote the B-cell/T-cell interaction in these patients through downregulation of inhibitory molecules unlike in LL patients. Conversely, the increased production of activated memory B-cells in ENL patients could imply the scale up of immune activation through antigen presentation to T-cells. However, the generation and differential function of these memory B-cells need further investigation. The finding of increased percentage of activated memory B-cells in untreated patients with ENL reactions suggests the association of these cells with the ENL pathology. The mechanism by which inflammatory reactions like ENL affecting these memory cells and contributing to the disease pathology is an interesting area to be explored for and could lead to the development of novel and highly efficacious drug for ENL treatment

PD-1, PD-L1 and PD-L2 Gene Expression on T-Cells and Natural Killer Cells Declines in Conjunction with a Reduction in PD-1 Protein during the Intensive Phase of Tuberculosis Treatment.

BACKGROUND: The PD-1 axis is a cell intrinsic immunoregulatory pathway that mediates T cell exhaustion in chronic infection particularly in some viral infections. We hypothesized that PD-1, PD-L1 and PD-L2 would be highly expressed in untreated tuberculosis patients compared to controls due to their chronic infection and would decrease with successful TB treatment. MATERIALS AND METHODS: Untreated tuberculosis patients (n = 26) were recruited at diagnosis and followed up during treatment. Household contacts (n = 24) were recruited to establish baseline differences. Blood gene expression ex vivo was investigated using qRT-PCR. Flow cytometry was performed to establish protein expression patterns. RESULTS: PD-L1 gene expression was found to be elevated in active TB disease; however, this was not observed for PD-1 or PD-L2. The intensive phase of TB treatment was associated with a significant decline in PD-1, PD-L1 and PD-L2 gene expression. PD-1 protein expression on the surface of NK cells, CD8+ and CD4+ T cells was similar in patients with active TB disease compared to controls but declined with successful TB treatment, with the greatest decline occurring on the NK cells followed by CD8+ T cells and then CD4+ T cells. Granzyme B/PD-1 co-expression declined with successful intensive phase treatment. CONCLUSION: Modulation of PD-1/PD-L1 pathway through TB treatment indicates changes in the peripheral T cell response caused by live Mycobacterium tuberculosis (Mtb) followed by the response to dead bacilli, antigen-release and immuno-pathology resolution. The PD-1 axis could be a host drug target for immunomodulatory treatments in the future

Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from mycobacterium tuberculosis

A number of different interferon-c ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-c spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen

Post-immunization leucocytosis and its implications for the management of febrile infants.

AIMS: Clinical guidelines for management of infants with fever but no evident focus of infection recommend that those aged 1-3 months with a white cell count >15 × 109/l have a full septic screen and be admitted for parenteral antibiotics. However, there is limited information about leucocyte changes following routine immunization, a common cause of fever. We investigated white cell counts shortly after routine immunization in Ugandan infants under 3 months of age. METHODS: White cell counts were measured in 212 healthy infants following routine immunizations (DTwP-HepB-Hib, oral polio and pneumococcal conjugate 7 vaccines) received prior to 3 months of age. RESULTS: Mean leucocyte counts increased from 9.03 × 109/l (95% confidence interval 8.59-9.47 × 109/l) pre-immunizations to 16.46 × 109/l (15.4-17.52 × 109/l) at one-day post-immunizations at 6 weeks of age, and 15.21 × 109/l (14.07-16.36 × 109/l) at one-day post-immunizations at 10 weeks of age. The leucocytosis was primarily a neutrophilia, with neutrophil percentages one-day post-immunization of 49% at 6 weeks of age and 46% at 10 weeks of age. White cell parameters returned to baseline by two-days post-immunization. No participant received antibiotics when presenting with isolated fever post-immunization and all remained well at follow-up. CONCLUSIONS: In our study almost half the children <3 months old presenting with fever but no evident focus of infection at one-day post-immunization met commonly used criteria for full septic screen and admission for parenteral antibiotics, despite having no serious bacterial infection. These findings add to the growing body of literature that questions the utility of white blood cell measurement in identification of young infants at risk of serious bacterial infections, particularly in the context of recent immunizations, and suggest that further exploration of the effect of different immunization regimes on white cell counts is needed. This observational work was nested within a clinical trial, registration number ISRCTN59683017

Screening vaccine formulations for biological activity using fresh human whole blood.

Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models. Furthermore, data for immunological functionality lag formulation design by months, making it cumbersome to relate back to formulations in real-time. It is also likely that animal testing may not accurately reflect the response in humans. For a more effective formulation screen, a human whole blood (hWB) approach can be used to assess immunological functionality. The functional activity relates directly to the human immune response to a complete formulation (adjuvant/antigen) and includes adjuvant response, antigen response, adjuvant-modulated antigen response, stability, and potentially safety. The following commentary discusses the hWB approach as a valuable new tool to de-risk manufacture, formulation design, and clinical progression