•Real-time RT-PCR assays for measles wild-type and vaccine strain adapted for use on a high-throughput, random access, continuous loading molecular platform.
In 2019, Aotearoa New Zealand (NZ) ...experienced its worst measles outbreak since 1997. Due to declining childhood vaccination rates since the beginning of the SARS-CoV-2 pandemic, NZ is at serious risk of another major measles outbreak. Our laboratory provides diagnostic services to NZ's Southern region. In 2019 the Southern region experienced the greatest number of cases outside of Auckland and Northland, however we did not have a validated measles PCR assay in our laboratory.
We sought to develop reverse transcription real-time polymerase chain reaction (RT-PCR) assays for measles on the Hologic Panther Fusion® System by utilising its open access function.
Previously published real-time RT-PCR assays were modified and optimised to detect wild-type measles virus (LDT-Mea), and the vaccine strain of measles virus (LDT-MeaVacA), on the Hologic Panther Fusion® System. The assays were clinically validated.
The LDT-Mea assay has a limit of detection (LoD) of 0.1 CCID50, while the LDT-MeaVacA assay is less sensitive with a LoD of 1 CCID50. Using 27 samples, the clinical sensitivity and specificity was 100% for both assays. Other common respiratory viruses were found not to cross-react with either the LDT-Mea or LDT-MeaVacA assays.
We have successfully adapted and validated for diagnostic use on the Hologic Panther Fusion® System previously published assays to detect wild-type and vaccine strains of the measles virus. The implementation of measles testing on this system will greatly improve the turn-around time for measles testing, and better support the measles public health response, for our region.
•MAIT cells recognise vitamin-B2 metabolites via their semi-invariant TCR.•TCR-independent cytokine-mediated activation extends roles to anti-viral defence.•MAIT and iNKT cells share common ...transcriptional programmes.•Gene set enrichment analyses implicate MAIT cells in novel tissue repair roles.•MAIT cells are highly activated by SARS-CoV2 virus in Covid-19.
Mucosal associated invariant T (MAIT) cells have a recognised innate-like capacity for antibacterial host defence, consequent on the specificity of their T cell receptor (TCR) for small molecule metabolites produced by a range of prokaryotic and fungal species, their effector memory phenotype, and their expression of cytotoxic molecules. However, recent studies have identified at least two other important functions of MAIT cells in antiviral immunity and in tissue homeostasis and repair. Each are related to distinct transcriptional programmes, which are activated differentially according to the specific immune context. Here we discuss these diverse functions, we review the evidence for the newly identified role of MAIT cells in promoting tissue repair, and we discuss emerging data pointing to the future directions of MAIT cell research including roles in cancer, in antiviral immunity and recent studies in the immune response to SARS-CoV-2 infection. Overall these studies have made us aware of the potential for pleiotropic roles of MAIT cells and related cell populations in micee and humans, and have created a simple and attractive new paradigm for regulation in barrier tissues, where antigen and tissue damage are sensed, integrated and interpreted.
Mucosal‐associated invariant T (MAIT) cells are an abundant innate‐like T lymphocyte population that are enriched in liver and mucosal tissues. They are restricted by MR1, which presents antigens ...derived from a metabolic precursor of riboflavin synthesis, a pathway present in many microbial species, including commensals. Therefore, MR1‐mediated MAIT cell activation must be tightly regulated to prevent inappropriate activation and immunopathology. Using an in vitro model of MR1‐mediated activation of primary human MAIT cells, we investigated the mechanisms by which it is regulated. Uptake of intact bacteria by antigen presenting cells (APCs) into acidified endolysosomal compartments was required for efficient MR1‐mediated MAIT cell activation, while stimulation with soluble ligand was inefficient. Consistent with this, little MR1 was seen at the surface of human monocytic (THP1) and B‐cell lines. Activation with a TLR ligand increased the amount of MR1 at the surface of THP1 but not B‐cell lines, suggesting differential regulation in different cell types. APC activation and NF‐κB signaling were critical for MR1‐mediated MAIT cell activation. In primary cells, however, prolonged TLR signaling led to downregulation of MR1‐mediated MAIT cell activation. Overall, MR1‐mediated MAIT cell activation is a tightly regulated process, dependent on integration of innate signals by APCs.
MR1 presents a microbial ligand to MAIT cells, activating them. We found that efficient MR1‐mediated MAIT cell activation requires uptake of intact bacteria and depends upon antigen presenting cell (APC) activation. However, prolonged TLR stimulation of APCs inhibited activation of MAIT cells. Therefore MR1‐mediated MAIT cell activation is tightly regulated.
Abstract
Background
Early clearance of Mycobacterium tuberculosis is the eradication of infection before an adaptive immune response develops. We aimed to identify host factors associated with early ...clearance.
Methods
Indonesian household contacts patients with smear-positive tuberculosis (TB) had an interferon-γ release assay (IGRA) at baseline and 14 weeks later. Early clearance was defined as a persistently negative IGRA. Contact characteristics, exposure, and disease phenotype were assessed for association with a positive IGRA at each time point.
Results
Of 1347 contacts of 462 TB cases, 780 (57.9%) were IGRA positive and 490 (36.3%) were IGRA negative. After 14 weeks, 116 of 445 (26.1%) initially negative contacts were IGRA converters; 317 (71.2%) remained persistently negative. BCG vaccination reduced the risk of a positive baseline IGRA (relative risk RR, 0.89 95% confidence interval {CI} .83–.97; P = .01), and strongly reduced the risk of IGRA conversion (RR, 0.56 95% CI, .40–.77; P < .001). BCG protection decreased with increasing exposure (P = .05) and increasing age (P = .004). Risk of IGRA conversion was positively associated with hemoglobin concentration (P = .04).
Conclusions
A quarter of household TB case contacts were early clearers. Protection against M. tuberculosis infection was strongly associated with BCG vaccination. Lower protection from BCG with increasing M. tuberculosis exposure and age can inform vaccine development.
A quarter of household tuberculosis case contacts from Indonesia were persistently interferon-γ release assay (IGRA) negative at 14 weeks. BCG vaccination reduced risk of IGRA conversion, but this protection was reduced in older and heavily exposed contacts.
A S. capitis strain called NRCS-A (S. capitis NRCS-A) has emerged as a cause of bloodstream infections and sepsis in neonatal intensive care units (NICUs) worldwide.
To identify risk factors for ...S. capitis NRCS-A colonisation among neonates, Dunedin Hospital NICU, Dunedin, New Zealand, from September 2013 through March 2015.
Weekly axillary swabs categorised eligible neonates as a case or a control. A case was defined as a week ending with a neonate's first positive swab for S. capitis NRCS-A and a control as a week in which a neonate remained negative. Weekly exposures were abstracted from hospital medical records. Analyses were performed using conditional logistic regression.
The median (range) gestational age at birth of participants was 32.7 (23.1–41.3) weeks. Participants contributed 26 weeks of case data and 177 weeks of control data. On adjusted analysis compared with matched controls, cases had higher odds of requiring invasive mechanical ventilation (OR 3.6, 95% CI: 1.1–11.6, p=0.035) and of a patent ductus arteriosus (PDA) (OR 3.0, 95% CI: 1.0–9.0, p=0.044). Cases had lower odds of being part of a multiple birth (OR 0.24, 95% CI 0.08–0.73, p=0.001), having an area of inflamed skin (OR 0.31, 95% CI: 0.13–0.75, p=0.009), and specifically an area of inflamed axillary skin (OR 0.08, 95% CI: 0.01–0.50, p=0.006).
We found that premature neonates with invasive mechanical ventilation and PDA had greater odds for S. capitis NRCS-A colonisation. Transmission may be mediated by increased staff contact, but prospective research is needed to confirm this.
Abstract
Background
A proportion of tuberculosis (TB) case contacts do not become infected, even when heavily exposed. We studied the innate immune responses of TB case contacts to understand their ...role in protection against infection with Mycobacterium tuberculosis, termed “early clearance.”
Methods
Indonesian household contacts of TB cases were tested for interferon-γ release assay (IGRA) conversion between baseline and 14 weeks post recruitment. Blood cell populations and ex vivo innate whole blood cytokine responses were measured at baseline and, in a subgroup, flow cytometry was performed at weeks 2 and 14. Immunological characteristics were measured for early clearers, defined as a persistently negative IGRA at 3 months, and converters, whose IGRA converted from negative to positive.
Results
Among 1347 case contacts, 317 were early clearers and 116 were converters. Flow cytometry showed a resolving innate cellular response from 2 to 14 weeks in persistently IGRA-negative contacts but not converters. There were no differences in cytokine responses to mycobacterial stimuli, but compared to converters, persistently IGRA-negative contacts produced more proinflammatory cytokines following heterologous stimulation with Escherichia coli and Streptococcus pneumoniae.
Conclusions
Early clearance of M. tuberculosis is associated with enhanced heterologous innate immune responses similar to those activated during induction of trained immunity.
In household tuberculosis case contacts from Indonesia, a persistently negative interferon-γ release assay was associated with resolving innate immune cell response over time. Stronger heterologous cytokine responses were also seen, similar to those activated during induction of trained innate immunity.
Abstract
There is increased recognition that complex health challenges at the human-animal-environmental interface require a transdisciplinary, “
whole-of-society
” approach. This philosophy is ...particularly pertinent in Aotearoa-New Zealand because of the country’s relatively isolated island ecosystem, economic reliance on agriculture and its intensification, and existing indigenous worldview that emphasises holism and interconnectivity between humans, animals and the environment. In New Zealand, the One Health Aotearoa (OHA) alliance was established in order to better connect researchers and to address a growing number of infectious diseases challenges. The emphasis of OHA is to bring together and facilitate interactions between people from diverse disciplines, link to stakeholders and communities, and engage with policy-makers, government operational agencies, and funders, thus providing a holistic and integrative systems-thinking approach to address priority questions and achieve desired outcomes in One Health. The initial focus of OHA has been on infectious diseases, but there is increasing recognition of the potential benefits of the alliance to address broader complex issues. Greater involvement and overlap of the environmental sciences, human and animal health sciences, social science, and indigenous kaupapa Māori research is particularly critical for ensuring its success within the New Zealand context. Given the economic and cultural importance of New Zealand’s “
clean, green
” image, a One Health approach that draws strongly on the environmental sciences makes particular sense. Furthermore, as the global environment becomes increasingly stressed by anthropogenic pressures our research may hold potential solutions for similar challenges elsewhere.
Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells that can be stimulated either via their TCR or by innate cytokines. The MAIT cell TCR recognises a pyrimidine ligand, ...derived from riboflavin synthesising bacteria, bound to MR1. In infection, bacteria not only provide the pyrimidine ligand but also co‐stimulatory signals, such as TLR agonists, that can modulate TCR‐mediated activation. Recently, type I interferons (T1‐IFNs) have been identified as contributing to cytokine‐mediated MAIT cell activation. However, it is unknown whether T1‐IFNs also have a role during TCR‐mediated MAIT cell activation. In this study, we investigated the co‐stimulatory role of T1‐IFNs during TCR‐mediated activation of MAIT cells by the MR1 ligand 5‐amino‐6‐d‐ribitylaminouracil/methylglyoxal. We found that T1‐IFNs were able to boost interferon‐γ and granzyme B production in 5‐amino‐6‐d‐ribitylaminouracil/methylglyoxal‐stimulated MAIT cells. Similarly, influenza virus‐induced T1‐IFNs enhanced TCR‐mediated MAIT cell activation. An essential role of T1‐IFNs in regulating MAIT cell activation by riboflavin synthesising bacteria was also demonstrated. The co‐stimulatory role of T1‐IFNs was also evident in liver‐derived MAIT cells. T1‐IFNs acted directly on MAIT cells to enhance their response to TCR stimulation. Overall, our findings establish an important immunomodulatory role of T1‐IFNs during TCR‐mediated MAIT cell activation.
Type I interferons enhance activation and effector responses of TCR‐stimulated blood and liver‐derived MAIT cells. Type I interferons contribute to MAIT cell activation by riboflavin producing bacteria, and act directly on MAIT cells. Together, this suggests type I interferons are important early modulators of the MAIT cell TCR response.
The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant ...adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration. There are twelve serotypes and many capsid variants of rAAV. However, a comparative study to determine which rAAV is most efficient at transducing primary human melanocytes has not been conducted. We therefore sought to determine the optimum rAAV variant for use in the in vitro transduction of primary human melanocytes, which could also be informative to future in vivo studies. We have screened eight variants of rAAV for their ability to transduce primary human melanocytes and identified rAAV6 as the optimal serotype, transducing 7-78% of cells. No increase in transduction was seen with rAAV6 tyrosine capsid mutants. The number of cells expressing the transgene peaked at 6-12 days post-infection, and transduced cells were still detectable at day 28. Therefore rAAV6 should be considered as a non-integrating vector for the transduction of primary human melanocytes.
Mucosal associated invariant T (MAIT) cells are anti‐microbial innate‐like T cells that are abundant in blood and liver. MAIT cells express a semi‐invariant T‐cell receptor (TCR) that recognizes a ...pyrimidine ligand, derived from microbial riboflavin synthesis, bound to MR1. Both blood and liver derived (ld)‐MAIT cells can be robustly stimulated via TCR or by cytokines produced during bacterial or viral infection. In this study, we compared the functional and transcriptomic response of human blood and ld‐MAIT cells to TCR signals (Escherichia coli or the pyrimidine ligand) and cytokines (IL‐12 + IL‐18). While the response of blood and ld‐MAIT cells to TCR signals were comparable, following cytokine stimulation ld‐MAIT cells were more polyfunctional than blood MAIT cells. Transcriptomic analysis demonstrated different effector programmes of ld‐MAIT cells with the two modes of activation, including the enrichment of a tissue repair signature in TCR‐stimulated MAIT cells. Interestingly, we observed enhancement of IL‐12 signaling and fatty acid metabolism in untreated ld‐MAIT cells compared with blood MAIT cells. Additionally, MAIT cells from blood and liver were modulated similarly by TCR and cytokine signals. Therefore, we report that blood and ld‐MAIT cells are fundamentally different but undergo conserved changes following activation via TCR or by cytokines.
We demonstrate that liver‐derived MAIT cells are more responsive to stimulation with proinflammatory cytokines than blood MAIT cells. The transcriptome of unstimulated liver‐derived MAIT cells differs from blood MAIT cells, with significant differences in metabolic pathways and cytokine signaling. Together this suggests tissue specific modulation of MAIT cell function.
PDF
