Phage-TB · Mycobacteriophages to treat tuberculosis - Passed station or future promise?

Mycobacteriophages to treat tuberculosis - Passed station or future promise?

Introduction Tuberculosis (TB) has troubled humanity for millennia. Incidence rates and mortality declined with improving living conditions and the discovery of anti-tuberculous treatment. However, TB is still a threat to public health worldwide; it still lies dormant in approximately a quarter of the world’s population and HIV fuels the TB epidemic in areas of high prevalence (Houben 2016). The emergence of drug-resistance to TB drugs poses an increasing challenge to TB control. Over the past two decades, the emergence of multi (MDR) and extensively (XDR) drug resistant strains of M tuberculosis are an increasing threat to public health in endemic areas. Treatment of drug-resistant TB is complex, toxic, poorly tolerated and costly (median cost per person treated for TB in 2019 was US$ 860 for drug-susceptible TB vs. US$ 5,659 for MDR-TB) (WHO TB report 2020). Outcome data in the WHO 2020 TB report show success rates of 85% for susceptible TB, while only 57% for MDR-TB. Novel or repurposed antibiotics are still being introduced, but this pipeline will dry out in the near future. It is high time to start exploring novel therapeutic approaches that synergize with antibiotics and improve the chances of success of tuberculosis treatment. In addition to M tuberculosis, non-tuberculous mycobacteria (NTM) are emerging pathogens of increasing clinical importance (Johansen 2020). Antibiotic resistance is an enormous problem especially with certain NTM like M abscessus (Griffith 2007). Treatment is often toxic and prolonged courses of multiple antibiotics are needed. Therefore, also for these emerging pathogens novel treatment strategies are urgently needed. Bacteriophages (or “phages”) are viruses that kill bacteria. Phages were first described by Frederick Twort and Felix d’Hérelle in the early 20th century. Both independently discovered small infecting agents of unknown nature that D’Hérelle first used to treat dysentery. Without knowing their exact nature or biology, phages with activity against pathogenic bacteria such as Neisseria meningitis, Salmonella typhi, Shigella dysenteriae, Vibrio cholerae and Yersinia pestis were soon discovered and used for treatment of these conditions. It is no surprise that phage treatments worked somewhat unreliably given the poor understanding of their nature and the impossibility to consistently provide high-quality products. The “golden age of phage therapy” lasted from the 1920s until effective antibiotics with a much wider spectrum almost completely replaced phage treatments in the 1950s. Phage technology survived in Eastern Europe where phage-based products remain available to this day (Luong 2020). Phages represent the most abundant biologic entity on earth and can be found in every natural environment including the human body. The number of phage particles on earth has been estimated to be in excess of 1031, and it has been calculated that 1025 phages attack a bacterium every second of the day (Hatfull 2011). Phages have a head containing double-stranded DNA inside a protein shell and a tail of various size and contractility (Figure 1). Phages are sized around 100nm, placing them between large proteins (10nm) and bacteria (2µm). Phages have high genetic diversity Other than antibiotics, they are very selective down to attacking only a single subspecies of bacteria. They can be either lytic or temperate. Lytic phages infect bacterial cells, replicate and lyse the bacterial cell, where temperate or lysogenic phages can integrate their DNA into the bacterial chromosomes and persist dormant until a lytic cycle is triggered (Figure 2). In recent years, with the increase of antimicrobial resistance and the lack of development of novel antibiotic agents, phages have gained new interest in various industries including medical treatments where the regulatory requirements are the most stringent. Although phage treatments are used in some parts of the world since a century, most reports available in English are observational or anecdotal. Incidental case reports or controlled trials show promising results and excellent safety (Hawkins 2010 and Wright 2009), but randomized controlled trials aiming at registration of a phage product are still lacking. Mycobacteriophages were isolated only in 1947 from soil (Gardner 1947) and were thus not used for treatment but in laboratories to differentiate between the types of mycobacteria they can infect. In the new millennium mycobacteriophages made a resurgence as substrates for training courses in which such phages were isolated from natural environments, amplified and their genome sequenced, offering insight in their genetic diversity. To date over 11,000 mycobacteriophages have been isolated and almost 2,000 have been fully sequenced (Hatfull 2017). The first therapeutic use of mycobacteriophages was described in a cystic fibrosis patient, suffering from a disseminated, highly resistant M abscessus infection after lung transplantation (Dedrick 2019). He was treated with a cocktail of three phages by intravenous and topical administration. The phages were genetically modified to ensure lytic activity. Phage therapy was well tolerated and associated with objective improvement of skin lesions, abdominal lymphadenopathy and dynamic lung function tests (Dedrick 2019). However, in a later case of an immunocompetent adult with pulmonary M abscessus infection, success of therapy was impaired, most probably due to the development of neutralizing antibodies; an issue warranting attention in future trials (Dedrick 2021). Reports of therapeutic application of mycobacteriophage treatment for TB in vitro and in guinea pigs have been promising, but reports in humans are still lacking (Azimi 2019). Recently, a cocktail of 5 diverse mycobacteriophages was assembled from phages that double-infect M smegmatis and M tuberculosis and have been engineered to be stricly lytic (Guerrero-Bustamente 2021). This phage cocktail is now available to TASK to start exploring its efficacy in human tuberculosis patients. There are various modes of administration of phages possible (Wienhold 2019, Abedon 2015). Success of phage therapy is dependent on the number of phages reaching the site of disease and infecting extracellular bacteria to start a self-augmenting lytic reaction. Therefore, for pulmonary TB, a combination of intravenous and inhalation administration should be explored first. Importantly, it has been demonstrated that phages can be spray dried to respirable powders which would facilitate administration in the field and thus make such a product practically relevant (VandenHeuvel 2013). Phages are quickly removed from circulation after intravenous administration and concentrate at sites of disease (Luong 2020, Schooley 2017), from where they are eliminated once there are no more targets available for propagation. As treatment success is associated with the levels of phages reached at site of disease relatively high doses will be required. Tolerability and anti-phage antibodies will be important issues to consider. A recent review reported adverse events occurring in 21% of patients treated with phage therapy for various infections, however all events were mild and transient (Luong 2020). In an era of increasing antibiotic resistance, novel therapeutic concepts to treat mycobacterial infections are urgently needed. Because the genetic diversity of M tuberculosis is relatively low individual phage preparations for each patient are not necessary. Mycobacteriophage treatments can be designed that can be used on all TB patients “off the shelf”. Apart from being a novel treatment option for drug-resistant TB, it might as well be of interest for treatment shortening of all forms of TB and NTM infections, alone or combined with antibiotics. More rapid sputum conversion will reduce transmission. All this can impact on global TB control and might provide novel treatment options for infections with atypical mycobacteria in immunocompromised hosts outside TB endemic areas. Project aims The mycobacteriophage project at TASK is run by an international consortium with multiple funders and partners. The aim of the consortium is to develop clinically useful mycobacteriophage treatments for TB as well as NTM infections. I aim to make contributions in the following areas: - Phage discovery in areas of high prevalence of TB (Cape Town) - Best administration methods for the existing anti-TB phage cocktail (Cape Town) - Preparation of first clinical trials with phages in TB (Cape Town) - Selection of promising lytic TB phages from collected phages during the first phase of this project (Nijmegen) This project is a novel and original approach to treating drug resistant TB. It follows calls to action by WHO and other organisations who see an increasing threat to global public health, and with only few new TB drugs in the pipeline alternative therapeutic targets are urgently needed. Advanced technologies allowing characterization, purification and quantification of mycobacteriophages allow for renewed interest in this pre-antibiotic treatment strategy. Phages are relatively easily grown on clinical isolates and due to their specificity to infect their bacterial host, phage treatment is generally well-tolerated and non-toxic, in contrast to several first and second line TB drugs. If our mycobacteriophage hunt for more phages that infect M tuberculosis is successful, and the translation can be made into purified and adequately dosed administration of mycobacteriophages, a virtually inexhaustible addition will be made to the therapeutic armamentarium for drug-resistant as well as drug-sensitive TB. Simplification of isolation, purification and amplification processes might in the future allow therapy tailored to a specific mycobacterial strain that is not M tuberculosis.

Data: CORDIS, © European Union