Joachim Jakob Bugert

Background: Neurotropic viruses infect the central nervous system (CNS) resulting in severe neurological and long-term complications, including permanent cognitive impairments. However, for many of these neurotropic viruses, like tick-borne encephalitis virus (TBEV), there are no clinically approved antiviral drugs. The blood-brain barrier (BBB) is a key entry point for neurotropic viruses, as well as a barrier for drug delivery to the brain. To aid in the identification of BBB-penetrating antivirals we established a BBB-permeable antiviral screening assay (BASA). Methods: BASA combines antiviral assays with an in vitro BBB transwell model consisting of primary human brain microvascular endothelial cells (HMBEC) and pericytes (HBMVP). The combination of our in vitro BBB transwell model with astrocyte-enriched cell cultures provided a tri-culture model to investigate TBEV infection at the BBB. Results: The suitability of BASA for identifying BBB-permeable antivirals was validated by testing clinically approved anti-human immunodeficiency virus (HIV) drugs with known CNS-penetrating properties. Infection experiments reveal that TBEV crosses the intact BBB to infect astrocytic target cells. Through the application of BASA, we identify a novel BBB-permeable antiviral against TBEV (compound 3d). Conclusions: We establish a modular assay system for the early evaluation of CNS penetration potential of novel antiviral compounds. The development of tools like ours can help promote early-stage preclinical optimization of dedicated therapeutics against neurotropic viruses, while reducing the need for animal experiments.

A validated catalog of 105 recombinant depolymerases from Klebsiella phages covers 58 KL-types. 46 novel enzymes from prophages, jumbo phages, and common phages are linked to any known enzymatic activity against 14 classical serotypes and 12 genome-defined KL-types. Using activity-based profiling, structure prediction, and domain dissection, we developed a function-guided classification and a five-class structural catalog. This framework reveals highly specific enzymes active against up to three capsule types. K47 CPS was degraded by three diverse protein groups. Structurally similar depolymerases degrading particular CPS were found in distinct phage taxa, with highly conserved enzymes in Drulisvirus specific to K1-, K2-types. The exclusive depolymerases were found in siphoviruses targeting K2 and K62 serotypes. A case study of five structurally similar enzymes degrading KL22/KL37/KL111 and KL25/KL119 capsules suggested specificity switching via amino acid changes or C-domain modification. Klebsiella phage depolymerases catalog sheds light on their diversity, evolution, and potential application.

Adjuvant therapy with bacteriophage (phage) cocktails in combination with antibiotics is a therapeutic approach currently considered for treatment of infections with encapsulated, biofilm forming, and multidrug-resistant Klebsiella pneumoniae (Kp). Klebsiella phage are highly selective in targeting a bacterial capsule type. Considering the numerous Kp capsule types and other host restriction factors, phage treatment could be facilitated when generating phages with a broad host range. A modified ‘Appelmans protocol’ was used to create phages with an extended host range via in vitro forced DNA recombination. Three T7-like Kp phages with highly colinear genomes were subjected to successive propagation on their susceptible host strains representing the capsule types K64, K27, and K23, and five Kp isolates of the same capsule types initially unsusceptible for phage lysis. After 30 propagation cycles, five phages were isolated via plaque assay. Four output phages represented the original input phages, while the fifth lysed a previously non-permissible Kp isolate, which was not lysed by any of the input phages. Surprisingly, sequence analysis revealed a novel N15/phiKO2-like phage genome (vB_KpnS_KpLi5) lacking substantial homologies to any of the used T7-like phages. This phage is not a chimeric recombinant of the applied T7-like phages, but represents a temperate phage that was induced from Kp due to the application of the input phages phages (cocktail), but not by any of them individually. Adapted phages with chimeric genomes and extended host range derived from input phages were not observed. Induction of temperate phages may be a stress response caused by using multiple phages simultaneously (i.e., by destabilization of the cell wall due to an unspecific binding of the phages). Successive use of different phages for therapeutic purposes may be preferable over simultaneous application in cocktail formulations to avoid undesired induction of temperate phages.

Adjuvant therapy with bacteriophage (phage) cocktails in combination with antibiotics is a therapeutic approach currently considered for treatment of infections with encapsulated, biofilm forming, and multidrug-resistant Klebsiella pneumoniae (Kp). Klebsiella phage are highly selective in targeting a bacterial capsule type. Considering the numerous Kp capsule types and other Kp host restriction factors, phage treatment could be facilitated when generating phages with a broad host range A modified ‘Appelmans protocol’ was used to create phages with an extended host range via in vitro forced DNA recombination. Three T7-like Kp phages with highly colinear genomes were subjected to successive propagation on their susceptible host strains representing the capsule types K64, K27, and K23, and five Kp isolates of the same capsule types initially unsusceptible for phage lysis. After 30 propagation cycles, five phages were isolated via plaque assay. Four output phages represented the original input phages, while the fifth lysed a previously non-permissible Kp isolate, which was not lysed by any of the input phages. Surprisingly, sequence analysis revealed a novel N15/phiKO2-like phage genome ( vB_KpnS_KpLi5 ) lacking substantial homologies to any of the used T7-like phages. This temperate phage was only induced in the presence of all input phages (cocktail), but not by any of them individually. Induction of temperate phages may be a stress response caused by using multiple phages simultaneously. Successive use of different phages for therapeutic purposes may be preferable over simultaneous application in cocktail formulations to avoid undesired induction of temperate phages. (243)

A bstract: Epidemiological data suggest that clade IIb monkeypox virus (MPXV) is readily transmitted during sexual contact, even before symptom onset. However, presymptomatic shedding of MPXV remains to be demonstrated. Here, we prospectively followed up 25 individuals after high-risk exposure to MPXV. Daily anorectal, genital, and saliva samples and weekly blood and oropharyngeal samples were collected along with clinical information. During follow-up, 12/18 (66.0%) sexual and 1/7 (14.0%) non-sexual contacts showed evidence of MPXV infection by PCR, five of whom had low viral loads and no typical MPXV symptoms. In 5/6 (83.3%) patients with typical symptoms, viral DNA was detected as early as four days before symptom onset and in three of them, replication-competent virus was found. These findings emphasize the high risk of MPXV transmission during sexual contact and confirm the existence of presymptomatic viral shedding of MPXV. Sexual contacts of an MPXV-infected partner should abstain from sex irrespective of symptoms.

In May 2022, human monkeypox infections including human-to-human transmission were reported in a multi-country outbreak in Europe and North America. With the first two German cases, we are providing clinical and virological findings, including the detection of monkeypox virus DNA in blood and semen. It is important to raise awareness among health professionals of the rapid and correct identification and diagnosis of this disease, which is probably still under-reported in Europe. We describe the first two cases of monkeypox (MPX) infections in Germany to highlight the importance of recent developments for health professionals worldwide and to share further observations related to human-to-human transmission in these cases. MPX is an orthopoxvirus infection that, with the exception of a few imported cases in the past, was previously thought to be endemic only in West and Central African countries [1, 2]. Beginning in 2018, the United Kingdom (UK) Health Security Agency has reported several imported cases associated with travellers from these countries [3]. One case reported on May 7, 2022, had a travel history to Nigeria. However, the two other cases within a family reported on May 13, 2022, neither had a travel history nor an epidemiological link to the previous case. On May 17, 2022, four additional UK cases were reported involving individuals who described themselves as belonging to the group of men who have sex with men (MSM). Subsequent testing for monkeypox virus (MPXV) in symptomatic MSM patients reporting to sexual health and sexually transmitted disease (STD) clinics in the UK and elsewhere revealed an increasing number of confirmed MPX cases in Europe [1].

Despite numerous advances in personalized phage therapy, smooth logistics are challenging, particularly for multidrug-resistant Gram-negative bacterial infections requiring high numbers of specific lytic phages. We conducted this study to pave the way for efficient logistics for critically ill patients by (1) closely examining and improving a current pipeline under realistic conditions, (2) offering guidelines for each step, leading to safe and high-quality phage supplies, and (3) providing a tool to evaluate the pipeline’s efficiency. Due to varying stipulations for quality and safety in different countries, we focused the pipeline on all steps up to a required phage product by a cell-free extract system. The first of three study runs included patients with respiratory bacterial infections from four intensive care units, and it revealed a cumulative time of up to 23 days. Ultimately, adjustment of specific set points of the vulnerable components of the pipeline, phage isolation, and titration increased the pipeline’s efficiency by 15% and decreased the maximum required time to 13 days. We present a site-independent practical approach to establish and optimize pipelines for personalized phage delivery, the co-organization of pipeline components between different institutions, non-binding guidelines for every step, and an efficiency check for phage laboratories.

Background: Multidrug-resistant Klebsiella pneumoniae spp. (kp) are emerging agents of severe infections of the respiratory, urinary tract and wounds that can progress to fatal septicemia. The use of bacteriophages is currently being considered as an effective alternative or adjuvant to antibiotic therapy. Results: In this study, we report capsule (K)-typing of 163 carbapenem-resistant Kp (CRKP) isolated 2014–2018 at the Military Hospital of Instruction of Tunis (MHT), Tunisia, by partial amplification and sequencing of the Kp wzi gene. The most prevalent K-type overall was K64 with 50.3% followed by K17 and K27 (22.7 and 11.0%, respectively). K64 Kp strains were most common and associated with increased case/fatality rates, especially at the intensive care unit (ICU). Using a K64 Kp strain we isolated and characterized a lytic Kp phage, vB_KpP_TUN1 (phage TUN1), from wastewater samples of the ICU at the MHT. TUN1 belongs to the Autographiviridae family and specifically digests K64 Kp capsules most probably via a depolymerase encoded by gp47. Furthermore, we successfully assembled phage TUN1 in a non-replicative host (E. coli) raising the possibility of in vitro assembly in the absence of live bacterial hosts. We propose that phage TUN1 is a promising candidate to be used as an adjuvant or an alternative to antibiotic therapy in CRKP infections, facilitating regulatory approval of phage therapy. Conclusions: K64, K17 and K27 are the most common wzi capsule types in this geographical location in Northern Africa. The lytic phage TUN1 efficiently lyses K64 Kp strains associated with increased case/fatality rates at body temperature. Together with its ability to be rescued in a non-replicative host these features enhance the utility of this phage as an antibacterial agent.