Developing Host-directed therapy in Cryptococcal Meningitis

BACKGROUND
Cryptococcal meningitis (CM), caused by the environmental yeast Cryptococcus neoformans, is a severe opportunistic infection that occurs in patients with impaired immunity such as in HIV/AIDS. It is the most common cause of adult meningitis in sub-Saharan Africa and despite the role-out of HIV treatment results in up to 165,000 deaths per year. New treatment strategies are urgently needed and boosting the host immune response has been suggested as a potential way to improve patient outcome. However, better understanding of the host immune response is required to identify the specific groups of patients who will benefit and to ensure there are no detrimental consequences of immune therapy.

Small clinical trials have previously demonstrated some benefit from administering interferon gamma to patients with CM, along with anti-fungal drugs. Interferon gamma is produced by the immune system and plays a vital role in the body's defence against Cryptococcus, stimulating white blood cells such as monocytes and macrophages to engulf and kill the fungus. However, this treatment has never been tried in large numbers of patients and exploratory data suggest that the main benefits might be restricted to certain patients who were not able to produce sufficient interferon gamma themselves. In previous studies of CM we have identified a pattern of immune response in the blood that is strongly associated with early mortality, even when other important factors are accounted for. We think that this immune signature may hold the key to identifying patients with CM at high risk of early death and potentially allow targeted immune therapy (such as interferon gamma) or targeted intensification of anti-fungal therapy to improve outcome.

AIMS
We would like to study this immune response further, to understand whether it is an isolated problem or part of a wider deactivation of the host immune response and whether it can be reversed through immune modulatory treatment such as interferon gamma and other molecules. We would also like to examine how it performs as a prognostic test and whether simplified assays that approximate the immune signature could be used to guide therapy.

APPROACH
We propose nesting an immunological sub-study within the phase 3 AMBITION-CM trial, currently enrolling patients with CM at 6 sites across sub-saharan Africa . This will examine the immune response in a variety of ways including measuring gene expression in the blood, along with the expression of activation markers and the cytokine responses of white blood cells in the blood and cerebrospinal fluid. We will use a variety of analysis techniques to determine whether this immune signature, and more simple versions of it, can be used to predict mortality or slow rate of clearance of infection (a proxy for poor outcome). We will also explore whether impaired cellular responses can be returned to normal in the laboratory by adding different immune modulating agents to patients cells.

APPLICATIONS
This study will generate important data to inform and guide future trials of targeted interferon gamma therapy, either alone or in combination with other immune therapies. We also hope that it may provide sufficient data to allow the development of immune assays to predict poor outcome allowing the targeting of treatment. In addition to these direct benefits it will provide important information regarding immune therapy to researchers studying other difficult-to-treat infections.

BACKGROUND
Cryptococcal meningitis (CM) remains a significant cause of death in AIDS patients in sub-Saharan Africa. Modulating the host immune response is a potential method to improve outcome and trials of adjunctive interferon gamma (IFNg) are promising. However, further study is required to understand which patients are likely to benefit and any unintended consequences. We have previously reported an immune phenotype associated with mortality characterised by monocyte and T cell deactivation, raised neutrophils and IL-10. This study aims to explore this phenotype further to determine whether it can be used to better predict poor outcome and guide anti-fungal and immune modulatory therapy.

SPECIFIC AIMS
1. Better understand the immune response in CM, particularly whether monocyte and T cell deactivation is associated with mortality
2. Determine whether immune deactivation can be reversed with immune modulatory agents
2. Establish whether simplified immune assays can be used to predict poor outcome and guide anti-fungal and immune modulatory therapy

APPROACH
We propose a prospective observational study nested at 2 sites of the AMBITION-CM trial (https://blogs.lshtm.ac.uk/ambition/). We will assess the host immune response in cryopreserved samples from patients with CM using a variety of techniques including RNA-sequencing of blood, flow cytometry of PBMCs and cerebrospinal cells, whole blood LPS stimulation assays and phagocytosis assays. We will examine associations between this immune signature and poor outcome. Additional experiments will examine ex vivo reversibility of immune deactivation by incubation with IFNg and other cytokines. We will then examine the performance of simplified immune assays in predicting poor outcome over the whole trial cohort.

IMPACT
This will provide important data to inform future trials of immune modulation in CM and allow the development of prognostic assays/scoring systems to guide intensification of treatment.

This is a translational immunology project and builds on observations from previous cohort studies. It will significantly increase our understanding of the role of the host immune response in the pathogenesis of cryptococcal meningitis.

The most immediate beneficiary of this research will be the scientific community researching the immunology and biology of Cryptococcus neoformans and the host response during infection. These people will be engaged via a number of publications and international conference presentations detailed in the "Communications Plan". Much of what is known about the immune response to Cryptococcus is based on animal research so this study will provide important information regarding the similarities and differences between the human and animal immune response. While murine and in vitro models are critical components of biomedical research in human conditions, relating such observations back to what happens in patients is vital. Furthermore, many basic scientists use Cryptococcus as a model organism to help understand fundamentals of immunology, phagocytosis and intracellular pathogen survival. This study may prove useful in improving the understanding of cellular processes and may be translatable to other intracellular infections.

Ultimately the most significant potential impact in the long term would be to provide vital information to inform future trials of immune modulatory therapy in cryptococcal meningitis. Immune modulatory treatment does hold great potential in the treatment of fungal infections but a clear understanding of the immune response is required before this can be safely undertaken. Data generated may also enable the development of immune assays to help predict poor outcome allowing intensification of anti-fungal therapy or targeted immune modulation. Such benefits may also be translatable to other difficult other invasive mycoses such as Candidiasis and Aspergillosis and similarly difficult-to-treat bacterial infections.

It is also possible that this project may have more direct clinical benefits, most likely through the development of prognostic scoring systems utilising clinical and laboratory tests. Should such a prognostic score/assay be developed we will lobby the relevant departments of health, public health and policy writing to ensure the relevant authorities are aware of the findings. We will also ensure that the data is presented at conferences or educational events attended by doctors treating patients with CM. Should highly significant findings be made we will liaise with the University of Birmingham communications and media relations department to provide information to the media. Should any commercially valuable intellectual property be identified we will liaise with University of Birmingham Enterprise Ltd to ensure intellectual property is protected.