Principal Investigator (PI)
The Foundation is providing £242,100 in support.
Agnieszka Latka, Véronique Flamand, Ilaria De Angelis
1. Use of a Galleria mellonella larvae infection model to rapidly screen depolymerases for lead selection while minimizing the number of mice to be used.
2. Expertise and technical assistance in the use of cell lines (epithelial cells for invasion assays and macrophages for phagocytosis assays) for the in vitro clinical evaluation of depolymerases.
3. Intensive interaction with a GSK DMPK expert to evaluate potential in vivo toxicity and hemolysis of the lead depolymerase.
BACKGROUND
Multidrug-resistant Klebsiella pneumoniae is an emerging opportunistic pathogen belonging to the ESKAPE group and is marked by the World Health Organization (WHO) as a critical priority pathogen, resistant to all or nearly all available antibiotics (Calfee 2017, Tacconelli et al. 2018). Since antibiotic treatment options of multidrug-resistant K. pneumoniae infections are limited, new alternative methods of therapy are highly urgent. Apart from drug resistance, K. pneumoniae efficiently escapes the immune system response due to the production of a polysaccharide capsule. This crucial virulence factor forms a protective shield enabling bacteria to avoid phagocytosis or complement-mediated killing (Majkowska-Skrobek et al., 2018). Klebsiella capsules show a large diversity in composition distinguished by at least 78 different capsular serotypes (Ktype) and at least 186 capsular locus types (KL-types) (Lam et al., 2022), though a more limited set of capsular serotypes are prevailing among epidemiological clones. Some studies reported K1 and K2 in the top 5 of most common K-loci (Ssekatawa et al., 2021; Afolayan et al., 2021; Choi et al., 2020). Moreover, K1 and K2 are the most common capsular types associated with hypervirulence (Choby et al., 2020). Hypervirulent Klebsiella pneumoniae (hvKp) are able to increase capsule production, rendering them hypermucoid and explaining their higher virulence than classical strains. They are able to infect healthy individuals and cause communityacquired infections, starting from the carrier in the gastrointestinal tract. HvKp can cause liver abscesses, central nervous system infection, endophthalmitis, infections developed at multiple sites and metastatically spreading (Russo & Marr, 2019). Multidrug-resistant hypervirulent stains emerge by exchange of mobile elements carrying resistance determinants and hypervirulence determinants between classical and hypervirulent Klebsiella pneumoniae strains, further deteriorating the threat posed by these strains (Russo & Marr, 2019).
Bacteriophages are viruses infecting bacteria and part of the ecosystem to keep bacteria under control. Every phage infection is initiated by the recognition of a specific receptor located on the bacterial cell surface by the phage’s receptor-binding protein (RBP). In the case of K. pneumoniae, the capsular polysaccharide often serves as the primary phage receptor and accordingly K. pneumoniae phages have evolved RBPs comprising serotype-specific enzymatic domains that depolymerize the polysaccharide capsule and initiate the infection process (Leiman and Molineux 2008). These RBPs are therefore also called depolymerases and are a highly promising avenue of investigation to develop efficient antivirulence compounds targeting K. pneumoniae.Capsule degradation upon exposure to specific depolymerases renders the otherwise serum-resistant Klebsiella bacteria susceptible to complement-mediated killing. After decapsulation, the bacteria are more easily phagocytized by macrophages (Majkowska-Skrobek et al., 2018). Klebsiella-specific depolymerasesdecrease the resistance to innate defense systems and eliminate the virulence of K. pneumoniae, resulting in the elimination from the infected body. In addition, resistance development against phage-encoded depolymerases is associated with capsule loss, equally resulting in virulence loss (Kaszowska et al., 2021).
Accordingly, mice infected by K. pneumoniae are rescued upon treatment with specific phage-encoded depolymerases (Wu et al., 2023). Another recent study showed that a phage depolymerase is more effective than its parental phage in the treatment of thigh soft tissue K. pneumoniae infections in mice without inducing adverse behavioural affects or toxicity (Volozhantsev et al., 2022). Furthermore, depolymerases were able to protect Galleria mellonella larvae from infection with a multidrug-resistant K. pneumoniae (Majkowska-Skrobek et al., 2018; Gorodnichev et al., 2021).
PROBLEM STATEMENT AND RESEARCH QUESTIONS
Phage-encoded depolymerases adhere to the innovation criteria of the World Health Organization (WHO) for new antibiotics (novel class, new mode-of-action, no cross-resistance with existing resistance mechanisms). In addition, due to their enzymatic mode-of-action with a high turnover rate, only small amounts of depolymerase are required to achieve meaningful and significant rescue in mice infection models (in the microgram range). A simple rule of thumb for protein-based biologicals is that the cost per mg can be reduced to 1 dollar after extensive CMC optimization (depending on the protein). Low- and middle-income countries can therefore benefit from cheap depolymerase-based therapeutics, if high income countries invest a priori in their expensive CMC development. A successful CMC development requires to start from highly stable and efficient depolymerase with the best possible production yield.A current limitation is the high specificity for capsular serotypes. Nevertheless, phages have countered this high capsular diversity by evolving an equally high diversity of depolymerases. This is an ancient, continuous and dynamic process. In addition, capsule diversity is more constrained when focusing on local epidemiological clones. In this project, we will focus on capsular serotypes K1 and K2, which are highly prevalent, circulating serotypes and are associated with hypervirulence. There are a multitude of K1 and K2-specific depolymerases known (Lin et al., 2014; Hoyles et al., 2015; Pan et al., 2017; Solovieva et al., 2018; Blundell-Hunter et al., 2021; Dunstan et al., 2021; Pertics et al., 2021; Lin et al., 2022; Tu et al., 2022; Volozhantsev et al., 2022), but a systematic comparison of their drug potential is missing.
We formulate the following three research questions:
(1) What are the best K1- and K2-specific depolymerases in terms of protein yield, efficacy and protein (thermo)stability? These three metrics are essential parameters for affordable and efficient therapeutics to be used in regions in which keeping a cold chain is not as trivial as in the Western world. We will evaluate individual depolymerases and cocktails thereof.
(2) Can we broaden the specificity of specific depolymerases to cover more serotypes?
(3) Can we prevent neonatal sepsis by gut decolonization of pregnant mothers (vertical transfer) and efficiently treat neonates that are infected through horizontal transfer?
PROJECT VISION/GOAL
We envision to create affordable, efficient and stable therapeutics to reduce the prevalence and mortality rate of neonatal sepsis in low- and middle-income countries.
SCIENTIFIC RATIONALE
• Wild-type depolymerases have a proven track record in terms of in vitro and in vivo activity. Specifically, their biochemical activity of degrading the capsular polysaccharides, requiring microgram amounts only, along with their high thermostability because of their trimeric state (Majkowska-Skrobek et al., 2016 and 2018), has been translated to powerful antivirulence components in in vivo experiments. The antivirulence concept based on decapsulation turning the bacterial cells vulnerable for the innate immune system, has been supported by experimental data (Majkowska-Skrobek et al., 2018; Kaszowska et al., 2021). In this project, we particularly focus on serotypes K1 and K2 and perform an in-depth, systematic comparison for the best performing depolymerases in terms of production yield, activity and (thermo)stability, capitalizing on the natural diversity due to the practically infinite number of phages. Enzymes typically diverge in their enzymatic and physicochemical parameters. The proposed systematic approach will enable to select for the most suitable candidates, fully relying on our established methodological expertise.
• Vertical transfer from the colonized pregnant mother or horizontal transfer in the immediate postnatal environment are recognized as important transmission routes for K. pneumoniae to neonates, resulting in the onset of neonatal gut flora dysbiosis, disturbed immunity maturation and sepsis. Upon evaluation of the efficacy (therapeutic, prophylactic) of the selected depolymerases in an invertebrate Galleria mellonella larvae infection model, we will evaluate the effect of depolymerases on the vertical transmission route (preventing neonatal sepsis) in mice and the treatment efficacy on neonates that were infected horizontally.
STUDY DESIGN AND METHODOLOGY
The study design is split in two in vitro trajectories (WP1 and WP2) that focus on the composition and characterisation of a depolymerase cocktail comprising the K1 and K2 depolymerases with the highest drug potential, and the engineering of depolymerases with enhanced serotype promiscuity and thermostability, respectively. The two trajectories can be considered as a conservative and innovative approach, respectively. Thereafter, the most promising depolymerases will be selected and analyzed for their clinical potential with in vitro (including cell lines and red blood cells) and in vivo (larvae, mice) assays to support their further development as a novel modality.
ETHICAL CONSIDERATIONS
In WP3 we will perform in vivo experiments at the Tres Cantos site and ULB. Prior to the experiments, approval from the responsible ethical committees will be obtained and all diligent efforts will be done to execute the experiments according to the best ethical standards. Prior to the mice experiments, we will use cell lines, red blood cells and the invertebrate Galleria mellonella larval infection model, minimizing the number of mice experiments needed.