Orally administered P22 phage tailspike protein reduces salmonella colonization in chickens: prospects of a novel therapy against bacterial infections

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans.     

Direct injection of functional single-domain antibodies from E. coli into human cells

Intracellular proteins have a great potential as targets for therapeutic antibodies (Abs) but the plasma membrane prevents access to these antigens. Ab fragments and IgGs are selected and engineered in E. coli and this microorganism may be also an ideal vector for their intracellular delivery. In this work we demonstrate that single-domain Ab (sdAbs) can be engineered to be injected into human cells by E. coli bacteria carrying molecular syringes assembled by a type III protein secretion system (T3SS). The injected sdAbs accumulate in the cytoplasm of HeLa cells at levels ca. 10⁵–10⁶ molecules per cell and their functionality is shown by the isolation of sdAb-antigen complexes. Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. These results are proof-of-principle for the capacity of E. coli bacteria to directly deliver intracellular sdAbs (intrabodies) into human cells for analytical and therapeutic purposes.     

Selection,characterization, and thermal stabilization of llama single domain antibodies towards Ebola virus glycoprotein

Background

A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity.

Results

An immune phage display library containing more than 10⁷ unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15–17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies.

Conclusions

SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.

     

Distribution Dynamics of Recombinant Lactobacillus in the Gastrointestinal Tract of Neonatal Rats

One approach to deliver therapeutic agents, especially proteins, to the gastro-intestinal (GI) tract is to use commensal bacteria as a carrier. Genus Lactobacillus is an attractive candidate for use in this approach. However, a system for expressing exogenous proteins at a high level has been lacking in Lactobacillus. Moreover, it will be necessary to introduce the recombinant Lactobacillus into the GI tract, ideally by oral administration. Whether orally administered Lactobacillus can reach and reside in the GI tract has not been explored in neonates. In this study, we have examined these issues in neonatal rats. To achieve a high level of protein expression in Lactobacillus, we tested the impact of three promoters and two backbones on protein expression levels using mRFP1, a red fluorescent protein, as a reporter. We found that a combination of an L-lactate dehydrogenase (ldhL) promoter of Lactobacillus sakei with a backbone from pLEM415 yielded the highest level of reporter expression. When this construct was used to transform Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus, high levels of mRFP1 were detected in all these species and colonies of transformed Lactobacillus appeared pink under visible light. To test whether orally administered Lactobacillus can be retained in the GI tract of neonates, we fed the recombinant Lactobacillus casei to neonatal rats. We found that about 3% of the bacteria were retained in the GI tract of the rats at 24 h after oral feeding with more recombinant Lactobacillus in the stomach and small intestine than in the cecum and colon. No mortality was observed throughout this study with Lactobacillus. In contrast, all neonatal rats died within 24 hours after fed with transformed E. coli. Taken together, our results indicate that Lactobacillus has the potential to be used as a vehicle for the delivery of therapeutic agents to neonates.     

Mouse intragastric infusion (iG) model

Direct intragastric delivery of a diet, nutrient or test substance can be achieved in rodents (mice and rats) on a long-term (2-3 months) basis using a chronically implanted gastrostomy catheter and a flow-through swivel system. This rodent intragastric infusion (iG) model has broad applications in research on food intake, gastrointestinal (GI) physiology, GI neuroendocrinology, drug metabolism and toxicity, obesity and liver disease. It achieves maximal control over the rate and pattern of delivery and it can be combined with normal ad libitum feeding of solid diet if so desired. It may be adopted to achieve infusion at other sites of the GI system to test the role of a bypassed GI segment in neuroendocrine physiology, and its use in genetic mouse models facilitates the genetic analysis of a central question under investigation.     

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.     

The macaque gut microbiome in health, lentiviral infection, and chronic enterocolitis

The vertebrate gut harbors a vast community of bacterial mutualists, the composition of which is modulated by the host immune system. Many gastrointestinal (GI) diseases are expected to be associated with disruptions of host-bacterial interactions, but relatively few comprehensive studies have been reported. We have used the rhesus macaque model to investigate forces shaping GI bacterial communities. We used DNA bar coding and pyrosequencing to characterize 141,000 sequences of 16S rRNA genes obtained from 100 uncultured GI bacterial samples, allowing quantitative analysis of community composition in health and disease. Microbial communities of macaques were distinct from those of mice and humans in both abundance and types of taxa present. The macaque communities differed among samples from intestinal mucosa, colonic contents, and stool, paralleling studies of humans. Communities also differed among animals, over time within individual animals, and between males and females. To investigate changes associated with disease, samples of colonic contents taken at necropsy were compared between healthy animals and animals with colitis and undergoing antibiotic therapy. Communities from diseased and healthy animals also differed significantly in composition. This work provides comprehensive data and improved methods for studying the role of commensal microbiota in macaque models of GI diseases and provides a model for the large-scale screening of the human gut microbiome.     

Thermal stability of single‐domain antibodies estimated by molecular dynamics simulations

Single‐domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T_m) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High‐temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the T_m values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per‐residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.     

Immunodiagnostic reagents using llama single domain antibody-alkaline phosphatase fusion proteins

Naive libraries of single domain antibodies (sdAbs) enable rapid isolation of binders to nearly any target. These binders, however, lack the benefits bestowed by in vivo affinity maturation and typically have low affinity toward their targets. We expressed five low-affinity toxin binding sdAbs, previously selected from a naive library derived from variable regions of llama heavy chain-only antibodies, as fusions with a hyperactive mutant Escherichia coli alkaline phosphatase (AP) and examined the impact on apparent affinity and utility. AP spontaneously dimerizes in solution, effectively dimerizing the fused sdAbs, imparting avidity in place of the lower affinity monomeric interactions. The sdAb-AP fusion also combines the target recognition domain with a signal transduction domain, commonly used in enzyme-linked immunosorbent assays (ELISAs). The functional affinity of the sdAb-AP fusions, often increased by a factor of 10 over unfused sdAbs, and their utility as tracer reagents in ELISAs was dramatically improved, giving limits of detection of 300 ng/ml or less, whereas parental sdAbs gave no discernible signal at the toxin concentrations tested. The fusion of sdAbs to AP presents a valuable route to facilitate the implementation of sdAb-based immunoreagents rapidly selected from existing naive libraries toward new or emerging threats.     

Single Domain Antibody Multimers Confer Protection against Rabies Infection

Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90–95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection.     

Harmful fungi in both agriculture and medicine

Most fungi are saprophytic and not pathogenic to plants, animals and humans. However, a relative few fungal species are phytopathogenic, cause disease (e.g., infections, allergies) in man, and produce toxins that affect plants, animals and humans. Among such fungi are members of the Aspergillus and Fusarium genera as well as other genera (e.g., Alternaria, Mucor) comprising the emerging pathogen group in humans. These fungi present a common threat to both agricultural production and the health of healthy and immunocompromised individuals. Taken together, these relative few fungi can cause huge economic losses to agriculture, loss of food for consumption, and serious, often fatal diseases in humans and animals. Plants may be a source of antifungal compounds since they have had to develop compounds to resist infections by fungi present in their environment.     

Bloodmeal analysis reveals avian Plasmodium infections and broad host preferences of Culicoides (Diptera: Ceratopogonidae) vectors

Changing environmental conditions and human encroachment on natural habitats bring human populations closer to novel sources of parasites, which might then develop into new emerging diseases. Diseases transmitted by host generalist vectors are of special interest due to their capacity to move pathogens into novel hosts. We hypothesize that humans using forests for recreation are exposed to a broad range of parasites from wild animals and their vectors. A corollary of this is that new vector-host, parasite-host, and vector-parasite associations could eventually develop. Thus, we expect to observe atypical vector-host associations. Using molecular bloodmeal analysis via amplification of the mtDNA COI gene we identified the vertebrate hosts of Culicoides (Diptera: Ceratopogonidae) species in a sub-urban forest of Southwestern Germany. Bloodmeals were also checked for haemosporidian infections by amplifying a fragment of the mtDNA cyt b gene. We identified a total of 20 Culicoides species, thirteen of which fed on humans. From 105 screened bloodmeals we obtained high quality sequences for 77 samples, 73 (94.8%) originated from humans, two from livestock (Bos taurus and Equus caballus), and two from wild birds (Sylvia atricapilla and Turdus merula). We found that four Culicoides species previously assumed to feed exclusively on either birds (C. kibunensis) or domestic mammals (C. chiopterus, C. deltus, C. scoticus) fed also on humans. A total of six Culicoides abdomens were infected with avian haemosporidian parasites (Plasmodium or Haemoproteus), four of those abdomens contained blood derived from humans. Our results suggest that parasites of wild animals may be transferred to humans through infectious bites of Culicoides vectors. Further, we show that Culicoides vectors believed to be a specialist on specific vertebrate groups can have plastic feeding preferences, and that Culicoides are susceptible to infection by Plasmodium parasites, though vector viability must still be experimentally demonstrated.     

Single-domain antibodies: a versatile and rich source of binders for breast cancer diagnostic approaches

Noninvasive early detection of breast cancer through the use of biomarkers is urgently needed since the risk of recurrence, morbidity, and mortality is closely related to disease stage at the time of primary surgery. A crucial issue in this approach is the availability of relevant markers and corresponding monoclonal antibodies suitable for the development of effective immunodiagnostic modalities. The identification of such markers from human pathological lesions and the isolation of specific antibodies using conventional approaches remain major challenges. Camelids produce functional antibodies devoid of light chains in which the single N-terminal domain of the heavy chain is fully capable of antigen binding. When produced as an independent domain, these so-called single-domain antibody fragments (sdAbs) or nanobodies have several advantages for biotechnological applications owing to their unique properties of size (13 kDa), stability, solubility, and expression yield. In this work, we have generated phage display libraries from animals immunized with breast cancer biopsies. These libraries were used to isolate sdAbs against known and relevant antigens such as HER2, or several cancer-specific sdAbs against unknown targets. We describe the identification of one these targets, cytokeratin 19, using affinity purification in combination with mass spectrometry. Some of these sdAbs were used in several straightforward diagnostic applications such as immunohistochemical analysis of tumor samples, multiplexed cytometric bead array analysis of crude samples, or an immune enrichment procedure of rare cells. Here, we demonstrate that phage display-based selection of single-domain antibodies is an efficient and high-throughput compatible approach to generate binders with excellent characteristics for the fast development of diagnostic and prognostic modalities.     

Parasite interactions in natural populations: insights from longitudinal data

The physiological and immunological state of an animal can be influenced by current infections and infection history. Consequently, both ongoing and previous infections can affect host susceptibility to another parasite, the biology of the subsequent infection (e.g. infection length) and the impact of infection on host morbidity (pathology). In natural populations, most animals will be infected by a succession of different parasites throughout the course of their lives, with probably frequent concomitant infections. The relative timing of different infections experienced by a host (i.e. the sequence of infection events), and the effects on factors such as host susceptibility and host survival, can only be derived from longitudinal data on individual hosts. Here we review some of the evidence for the impact of co-infection on host susceptibility, infection biology and pathology focusing on insights obtained from both longitudinal studies in humans and experiments that explicitly consider the sequence of infection. We then consider the challenges posed by longitudinal infection data collected from natural populations of animals. We illustrate their usefulness using our data of microparasite infections associated with field vole (Microtus agrestis) populations to examine impacts on susceptibility and infection length. Our primary aim is to describe an analytical approach that can be used on such data to identify interactions among the parasites. The preliminary analyses presented here indicate both synergistic and antagonistic interactions between microparasites within this community and emphasise that such interactions could have significant impacts on host-parasite fitness and dynamics.     

Detecting emerging transmissibility of avian influenza virus in human households

Accumulating infections of highly pathogenic H5N1 avian influenza in humans underlines the need to track the ability of these viruses to spread among humans. A human-transmissible avian influenza virus is expected to cause clusters of infections in humans living in close contact. Therefore, epidemiological analysis of infection clusters in human households is of key importance. Infection clusters may arise from transmission events from (i) the animal reservoir, (ii) humans who were infected by animals (primary human-to-human transmission), or (iii) humans who were infected by humans (secondary human-to-human transmission). Here we propose a method of analysing household infection data to detect changes in the transmissibility of avian influenza viruses in humans at an early stage. The method is applied to an outbreak of H7N7 avian influenza virus in The Netherlands that was the cause of more than 30 human-to-human transmission events. The analyses indicate that secondary human-to-human transmission is plausible for the Dutch household infection data. Based on the estimates of the within-household transmission parameters, we evaluate the effectiveness of antiviral prophylaxis, and conclude that it is unlikely that all household infections can be prevented with current antiviral drugs. We discuss the applicability of our method for the detection of emerging human-to-human transmission of avian influenza viruses in particular, and for the analysis of within-household infection data in general.     

A natural asymptomatic herpes B virus infection in a colony of laboratory brown capuchin monkeys (Cebus apella)

Herpes B virus (BV) infection of macaques persists in the natural host, but is mainly asymptomatic. However, BV can cause fatal disease in humans and in several non-macaque species such as capuchin monkeys (Cebus apella). The BV infection described here in a colony of capuchin monkeys was persistent but asymptomatic. Initially the infection was detected serologically in five out of seven animals. However, using polymerase chain reaction (PCR) developed specifically for BV, we found the virus in all seven clinically healthy animals. It is probable that the infection was transferred from BV-infected macaques housed in different cages but in the same room for several years. We have no evidence to indicate that similar asymptomatic infections may occur in other New World species but the possibility should not be discounted. We recommend that the housing of capuchin monkeys in close proximity to macaques should be avoided and that greater caution should be used when handling capuchin monkeys and possibly other New World species that have been in contact with macaques. All may act as a source of BV infection in humans, hence routine, repeated testing of all primates is essential.     

Hedgehog signaling in gastrointestinal development and disease

The development of the gastrointestinal (GI) tract and its associated parenchymal organs depends on Hedgehog signals from the endoderm to the surrounding mesoderm. During development, Hedgehog signaling is essential for patterning the GI tract along anterior-posterior (A-P), dorsal-ventral (D-V), and radial axes, as well as in maintenance of stem cells. Our knowledge about these roles for Hedgehog signaling is derived from studies of developmental defects that result from disrupted or activated Hedgehog signaling in model organisms including mouse, chick, and frog. These studies provide evidence for distinct roles of specific Hedgehog ligands in GI development. Studies in model organisms have also elucidated how Hedgehog signaling may function in development and function of the GI tract in humans. Several diseases and congenital syndromes are known to result from genetic defects in Hedgehog signaling components, and this pathway may ultimately prove to be an important target for future diagnostic and therapeutic tools.     

Edible plant vaccines: applications for prophylactic and therapeutic molecular medicine

The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. Genes encoding bacterial and viral antigens are faithfully expressed in edible tissues to form immunogenic proteins. Studies in animals and humans have shown that ingestion of transgenic plants containing vaccine proteins causes production of antigen-specific antibodies in serum and mucosal secretions. In general, the technology is limited by low expression levels for nuclear-integrated transgenes, but recent progress in plant organelle transformation shows promise for enhanced expression. The stability and immunogenicity of orally delivered antigens vary greatly, which necessitates further study on protein engineering to enhance mucosal delivery. These issues are discussed with regard to the further development of plant-based vaccine technology.     

Nitric oxide mediates intestinal pathology but not immune expulsion during Trichinella spiralis infection in mice

The relationship between intestinal pathology and immune expulsion of gastrointestinal (GI) nematodes remains controversial. Although immune expulsion of GI helminth parasites is usually associated with Th2 responses, the effector mechanisms directly responsible for parasite loss have not been identified. We have previously shown that while the intestinal pathology accompanying the expulsion of the GI parasite Trichinella spiralis may be dependent on IL-4 and mediated by TNF, parasite loss is independent of TNF. In contrast, intestinal pathology in other disease models has been attributed to Th1 cytokines, although it closely resembles that seen in helminth infections. Whereas production of inducible NO synthase (iNOS) in the gut is important for both homeostasis of the epithelial layer and in protection against pathogenic microorganisms, overproduction of NO has been implicated in the pathogenesis of a number of inflammatory conditions. We therefore investigated the role of NO in T. spiralis infection using iNOS-deficient mice. iNOS-/- and iNOS-/+ mice were infected with T. spiralis, and parasite expulsion and intestinal pathology were followed. Parasite expulsion proceeded similarly in both groups of animals, but significant intestinal pathology was only observed in the heterozygous mice. Thus it appears that, although the protective effects of Th2 responses in GI helminth infection do not require NO, this mediator contributes substantially to the associated enteropathy. NO may therefore be an important mediator of enteropathy in both Th1- and Th2-inducing conditions.     

Predictors of Hepatitis B Cure Using Gene Therapy to Deliver DNA Cleavage Enzymes: A Mathematical Modeling Approach

Most chronic viral infections are managed with small molecule therapies that inhibit replication but are not curative because non-replicating viral forms can persist despite decades of suppressive treatment. There are therefore numerous strategies in development to eradicate all non-replicating viruses from the body. We are currently engineering DNA cleavage enzymes that specifically target hepatitis B virus covalently closed circular DNA (HBV cccDNA), the episomal form of the virus that persists despite potent antiviral therapies. DNA cleavage enzymes, including homing endonucleases or meganucleases, zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR-associated system 9 (Cas9) proteins, can disrupt specific regions of viral DNA. Because DNA repair is error prone, the virus can be neutralized after repeated cleavage events when a target sequence becomes mutated. DNA cleavage enzymes will be delivered as genes within viral vectors that enter hepatocytes. Here we develop mathematical models that describe the delivery and intracellular activity of DNA cleavage enzymes. Model simulations predict that high vector to target cell ratio, limited removal of delivery vectors by humoral immunity, and avid binding between enzyme and its DNA target will promote the highest level of cccDNA disruption. Development of de novo resistance to cleavage enzymes may occur if DNA cleavage and error prone repair does not render the viral episome replication incompetent: our model predicts that concurrent delivery of multiple enzymes which target different vital cccDNA regions, or sequential delivery of different enzymes, are both potentially useful strategies for avoiding multi-enzyme resistance. The underlying dynamics of cccDNA persistence are unlikely to impact the probability of cure provided that antiviral therapy is given concurrently during eradication trials. We conclude by describing experiments that can be used to validate the model, which will in turn provide vital information for dose selection for potential curative trials in animals and ultimately humans.