James (Jim) J Bull

Bacteriophages provide one of the few hopes for treating infections with drug-resistant bacteria. Rather than replacing antibiotics, however, phages will likely complement treatment for patients already on drugs. A challenge with dual treatment is that antibiotics may inhibit the growth of phages by inhibiting growth of their bacterial hosts, possibly limiting phage efficacy. But might phages be adapted to grow in the drugs? Here, a wildtype T7 was separately adapted to grow on an E. coli K12 in partially-inhibitory concentrations of chloramphenicol (Cm, 2ug/ml) or kanamycin (Kn, 8ug/ml) as well as in a no-drug control. Of the three phage lines separately adapted to each environment: growth rate was elevated over ancestor for all lines, albeit to different degrees, suggesting that pre-adaptation of phages might translate to enhanced treatment success. Yet, all evolved phages improved fitness in both the drug and no-drug environments regardless of their selective environment of evolution, suggesting that a substantial component of selection was merely for better growth. Generalizing this result, a T7 previously adapted to a different E. coli K12 strain without antibiotics also had a significantly higher fitness than the ancestor in all three cellular environments. These data reinforce other work showing that antibiotics inhibit phage growth, but they offer the possibility that phage adaptation for better growth in perhaps any environment – even one lacking drugs – may improve fitness in the presence of drugs.

The phages used to treat bacterial infections in phage therapy are commonly chosen based on their abilities to form plaques on the infecting bacterium-on host range. In practice, phage therapy is not always successful, leaving room for improvement. Here, we use computational models to investigate whether some standard phage properties (burst size, lysis rate, adsorption rate constant, intrinsic decay rate, and growth rate) might serve as predictors of treatment success. As our measure of treatment success, we deviate from many other approaches by calculating the number of phages needed to suppress bacterial densities 100-fold in the short term, given that the patient's immune system is expected to regain control once bacterial numbers are reduced. Numerical analysis of single-phage trials across 2400 combinations of phage phenotypes reveals that, on average, adsorption rate constant and growth rate provide the most useful predictive values, decay rate provides some value, whereas burst size and lysis time offer essentially little or no value. Bacterial density is especially informative of the number of phages required for treatment. There is nonetheless often considerable variation around average behavior for a single phenotype. These results raise the possibility that the adsorption rate constant and growth rate may be especially important in phage therapy performance for both high and low bacterial densities. Given that therapeutic phages are often evolved in vitro for broad host ranges rather than for individual hosts, it should be considered that selection for broad host range may have a downside of compromising adsorption to and growth rate on individual bacterial hosts.

The larval tobacco hornworm, Manduca sexta, has been used in a laboratory setting for physiological studies and for pathogen virulence studies. This moth offers a much larger size than the commonly used wax moth (Galleria mellonella), and it can thus be used for a greater variety of assays, such as repeated sampling of the same individual, growth measurements, and tissue sampling. Yet their occasional use in research has led to a minimally documented set of rearing methods. To facilitate further adoption of this insect model, we expanded on previously reported protocols and developed our own rearing methods, which we report here. Our protocol requires little specialized equipment, with a cost less than $100/month for the feeding and maintenance of a laboratory colony of about five hundred larvae of differing instar phases. The low cost generalized equipment and supplies, and the simplification of the standardized protocols allows for an easy entry point for rearing tobacco hornworm populations. We also describe a few methods that are relevant to the uses of these organisms as infection models.

Viruses differ in the number and types of host tissues in which they replicate. For example, systemically replicating viruses such as measles infect cells and tissues throughout the body, whereas respiratory viruses such as influenza viruses and coronaviruses replicate only in the respiratory tract. Reinfections with respiratory viruses are thought to be driven by ongoing antigenic immune escape in the viral population. However, this does not explain why antigenic variation is frequently observed in respiratory viruses and not systemically replicating viruses. Here, we argue that the rapid rate of waning immunity in the respiratory tract is a key driver of antigenic evolution in respiratory viruses. Waning immunity results in hosts with immunity levels that protect against homologous reinfection but are insufficient to protect against infection with an antigenically different (heterologous) strain. Thus, when partially immune hosts are present at a high enough density, an immune escape variant can invade the viral population even though that variant cannot infect solidly immune hosts. Invasion can occur even when the variant’s immune escape mutation incurs a fitness cost, although any such cost is likely to be short-lived from compensatory evolution. Thus the mutant lineage may replace the wild-type, and as immunity to it builds, the process will repeat. Our model provides a new explanation for the pattern of successive emergence and replacement of antigenic variants that has been observed in many respiratory viruses. We discuss our model relative to others for understanding the drivers of antigenic evolution in respiratory viruses.

Interest in phage therapy – the use of bacterial viruses to treat infections – has increased recently because of the rise of infections with antibiotic resistant bacteria and the failure to develop new antibiotics to treat those infections. Phages have shown therapeutic promise in recent work, and successful treatment minimally requires giving the patient a phage that will grow on their infecting bacterium. Although nature offers a bountiful and diverse supply of phages, there has been a surprising number of patient infections that could not be treated with phages because no suitable phage was found to kill the patient’s bacterium. Here we develop computational models to analyze an alternative approach to obtaining phages with new host ranges – directed evolution via laboratory propagation of phages to select mutants that can grow on a new host. The models separately explore alternative directed evolution protocols for phage variants that overcome three types of bacterial blocks to phage growth: a block in adsorption, temperate phage immunity to superinfection, and abortive infection. Protocols assume serial transfer to amplify pre-existing, small-effect mutants that are initially rare. Best protocols are sensitive to the nature of the block, and the models provide several insights for enhancing success specific to each case. A common result is that low dilution rates between transfers is beneficial in reducing the mutant growth rate needed to ascend. Selection to overcome an adsorption block is insensitive to many protocol variations but benefits from long selection times between transfer. A temperate phage selected to grow on its lysogens can evolve in any of three phenotypes, but a common protocol favors the desired changes in all three. Abortive infection appears to be the least amenable to evolving phage growth because it is prone to select phages that avoid infection.

Many emerging and reemerging pathogens originate from wildlife, but nearly all wild species are unreachable using conventional vaccination, which requires capture of and vaccine administration to individual animals. By enabling immunization at scales sufficient to interrupt pathogen transmission, transmissible vaccines (TVs) that spread themselves through wildlife populations by infectious processes could potentially transform the management of otherwise intractable challenges to public health, wildlife conservation, and animal welfare. However, generating TVs likely requires modifying viruses that would be intended to spread in nature, which raises concerns ranging from technical feasibility, to safety and security risks, to regulatory uncertainties (1, 2). We propose a series of commitments and strategies for vaccine development—beginning with a priori decisions on vaccine design and continuing through to stakeholder codevelopment [see supplementary materials (SM)]—that we believe increase the likelihood that the potential risks of vaccine transmission are outweighed by benefits to conservation, animal welfare, and zoonosis prevention.

Recombination among different phages sometimes facilitates their ability to grow on new hosts. Protocols to direct the evolution of phage host range, as might be used in the application of phage therapy, would then benefit from including steps to enable recombination. Applying mathematical and computational models, in addition to experiments using phages T3 and T7, we consider ways that a protocol may influence recombination levels. We first address coinfection, which is the first step to enabling recombination. The multiplicity of infection (MOI, the ratio of phage to cell concentration) is insufficient for predicting (co)infection levels. The force of infection (the rate at which cells are infected) is also critical but is more challenging to measure. Using both a high force of infection and high MOI (>1) for the different phages ensures high levels of coinfection. We also apply a four-genetic-locus model to study protocol effects on recombinant levels. Recombinants accumulate over multiple generations of phage growth, less so if one phage outgrows the other. Supplementing the phage pool with the low-fitness phage recovers some of this 'lost' recombination. Overall, fine tuning of phage recombination rates will not be practical with wild phages, but qualitative enhancement can be attained with some basic procedures.

Transmissible vaccines offer a novel approach to suppressing viruses in wildlife populations, with possible applications against viruses that infect humans as zoonoses - Lassa, Ebola, rabies. To ensure safety, current designs propose a recombinant vector platform in which the vector is isolated from the target wildlife population. Because using an endemic vector creates the potential for preexisting immunity to block vaccine transmission, these designs focus on vector viruses capable of superinfection, spreading throughout the host population following vaccination of few individuals. We present original theoretical arguments that, regardless of its R value, a recombinant vaccine using a superinfecting vector is not expected to expand its active infection coverage when released into a wildlife population that already carries the vector. However, if superinfection occurs at a high rate such that individuals are repeatedly infected throughout their lives, the immunity footprint in the population can be high despite a low incidence of active vaccine infections. Yet we provide reasons that the above expectation is optimistic. High vaccine coverage will typically require repeated releases or release into a population lacking the vector, but careful attention to vector choice and vaccine engineering should also help improve transmissible vaccine utility.

Commercially available cellulases and amylases can disperse the pathogenic bacteria embedded in biofilms. This suggests that polysaccharide-degrading enzymes would be useful as antibacterial therapies to aid the treatment of biofilm-associated bacteria, e.g., in chronic wounds. Using a published enzyme library, we explored the capacity of 76 diverse recombinant glycoside hydrolases to disperse Staphylococcus aureus biofilms. Four of the 76 recombinant glycoside hydrolases digested purified cellulose, amylose, or pectin. However, these enzymes did not disperse biofilms, indicating that anti-biofilm activity is not general to all glycoside hydrolases and that biofilm activity cannot be predicted from the activity on pure substrates. Only one of the 76 recombinant enzymes was detectably active in biofilm dispersion, an alpha-xylosidase from Aspergillus nidulans. An alpha-xylosidase cloned subsequently from Aspergillus thermomutatus likewise demonstrated antibiofilm activity, suggesting that alpha-xylosidases, in general, can disperse Staphylococcus biofilms. Surprisingly, neither of the two beta-xylosidases in the library degraded biofilms. Commercial preparations of amylase and cellulase that are known to be effective in the dispersion of Staphylococcus biofilms were also analyzed. The commercial cellulase contained contaminating proteins with multiple enzymes exhibiting biofilm-dispersing activity. Successfully prospecting for additional antibiofilm enzymes may thus require large libraries and may benefit from purified enzymes. The complexity of biofilms and the diversity of glycoside hydrolases continue to make it difficult to predict or understand the enzymes that could have future therapeutic applications.