Helen A. Fletcher

The health of the planet is one objective of the United Nations’ Sustainable Development Goals. Vaccines can affect not only human health but also planet health by reducing poverty, preserving microbial diversity, reducing antimicrobial resistance, and preventing an increase in pandemics that is fueled partly by climate change.

Over 1 million children develop tuberculosis (TB) each year, with a quarter dying. Multiple factors impact the risk of a child being exposed toMycobacterium tuberculosis(Mtb), the risk of progressing to TB disease, and the risk of dying. However, an emerging body of evidence suggests that coinfection with cytomegalovirus (CMV), a ubiquitous herpes virus, impacts the host response toMtb, potentially influencing the probability of disease progression, type of TB disease, performance of TB diagnostics, and disease outcome. It is also likely that infection withMtbimpacts CMV pathogenesis. Our current understanding of the burden of these 2 diseases in children, their immunological interactions, and the clinical consequence of coinfection is incomplete. It is also unclear how potential interventions might affect disease progression and outcome for TB or CMV. This article reviews the epidemiological, clinical, and immunological literature on CMV and TB in children and explores how the 2 pathogens interact, while also considering the impact of HIV on this relationship. It outlines areas of research uncertainty and makes practical suggestions as to potential studies that might address these gaps. Current research is hampered by inconsistent definitions, study designs, and laboratory practices, and more consistency and collaboration between researchers would lead to greater clarity. The ambitious targets outlined in the World Health Organization End TB Strategy will only be met through a better understanding of all aspects of child TB, including the substantial impact of coinfections.

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates isin vivopreclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulentM.tbin a ‘challenge’ experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as ‘Moderate’ in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe anin vitroprotocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using anin vitroassay in place ofin vivo M.tbchallenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward toin vivoefficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.

The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates isin vivopreclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulentM.tbin a ‘challenge’ experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as ‘Moderate’ in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe anin vitroprotocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using anin vitroassay in place ofin vivo M.tbchallenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward toin vivoefficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

Tuberculosis (TB) vaccine candidates must be tested for safety and efficacy using preclinical challenge models prior to advancement to human trials, because of the lack of a validated immune correlate or biomarker of protection. New, unbiased tools are urgently needed to expedite the selection of vaccine candidates at an early stage of development and reduce the number of animals experimentally infected with virulent Mycobacterium tuberculosis ( M . tb ). In recent years, there has been a concerted effort to develop standardised functional ex vivo mycobacterial growth inhibition assays (MGIAs) as a potential surrogate read-out of vaccine efficacy. We have previously described a direct MGIA for use with mouse splenocytes. In the current study, we set out to systematically compare co-culture conditions for the murine direct splenocyte MGIA with respect to both intra-assay repeatability and inter-site reproducibility. Common sample sets were shared between laboratory sites and reproducibility and sensitivity to detect a BCG-vaccine induced response were assessed. Co-culturing 5×10 ⁶ splenocytes in 48-well plates resulted in improved reproducibility and superior sensitivity to detect a vaccine response compared with standing or rotating sealed 2ml screw-cap tubes. As the difference between naïve and BCG vaccinated mice was not consistently detected across both sample sets at both sites, we sought to further improve assay sensitivity by altering the multiplicity of infection (MOI). Cell viability at the end of the co-culture period was improved when splenocyte input number was reduced, with the highest viability for the condition of 3×10 ⁶ splenocytes in 48-well plates. This cell input was also associated with the greatest sensitivity to detect a BCG vaccine-mediated MGIA response using an M . tb inoculum. Based on our findings, we recommend optimal co-culture conditions in a move towards aligning direct MGIA protocols and generating a cross-species consensus for early evaluation of TB vaccine candidates and biomarker studies.

We present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

In the absence of a correlate(s) of protection against human tuberculosis and a validated animal model of the disease, tools to facilitate vaccine development must be identified. We present an optimised ex vivo mycobacterial growth inhibition assay (MGIA) to assess the ability of host cells within the lung to inhibit mycobacterial growth, including Bacille Calmette–Guérin (BCG) and Mycobacterium tuberculosis (MTB) Erdman. Growth of BCG was reduced by 0.39, 0.96 and 0.73 log₁₀ CFU following subcutaneous (s.c.) BCG, intranasal (i.n.) BCG, or BCG s.c. + mucosal boost, respectively, versus naïve mice. Comparatively, a 0.49 (s.c.), 0.60 (i.n.) and 0.81 (s.c. + mucosal boost) log₁₀ reduction in MTB CFU was found. A BCG growth inhibitor, 2-thiophenecarboxylic acid hydrazide (TCH), was used to prevent quantification of residual BCG from i.n. immunisation and allow accurate MTB quantification. Using TCH, a further 0.58 log₁₀ reduction in MTB CFU was revealed in the i.n. group. In combination with existing methods, the ex vivo lung MGIA may represent an important tool for analysis of vaccine efficacy and the immune mechanisms associated with vaccination in the organ primarily affected by MTB disease.