Carl R. Merril, Dean Scholl, and Sankar Adhya

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Selection and Characterization of Therapeutic Phages Factors

Phage are the most abundant entities on the planet (estimated to be more than 1030 phage particles (13). However, only a few phage strains will prove to be effective as therapeutic antibacterial agents. There are a number of factors that can affect the therapeutic efficacy of phage chosen for use as antibacterial therapeutic agents. Studies of phage multiplication in bacteria in vitro may provide information as to bacterial host range and a good first approximation of whether the phage may be appropriate for a particular clinical infection. However, observation of phage multiplication in defined culture media does not take into account the interactions of the phage with the bacteria in the clinical environment. Bacterial gene expression and phenotype may be affected by numerous variables in the clinical milieu ranging from differences in the basic nutrients to altered physiological parameters including pH and ionic strength. In addition, clinical infections have the added complication of interactions between the innate and active immune systems with both the infectious bacteria and the therapeutic phage. While phage therapy, as described in the introduction, has had a relatively long, but checkered history, careful scientific study of factors associated with therapeutic efficacy remain to be explored.

Phage chosen as antibacterial therapeutic agents need to be well characterized, the genome sequenced, and much of the biology of the phage well understood before it is developed for therapeutic use. Phage host range, virulence, stability, interaction with both innate and active immune systems as well as their possible capacity to lysogenize and transduce must be understood to provide the greatest chance of efficacy and safety.

In addition, therapeutic phage strains need to be tested and selected for their ability to function in the milieu of the human physiological systems. Interactions with both the innate and active immune system need to be minimized for phage strains that are employed for the treatment of systemic infections and pharmacokinetic parameters need to be determined.

Host specificity of phage

The host range of phage is generally narrower than that found in antibiotics selected for clinical applications. Most phages are specific for one species of bacteria and many are only able to lyse specific strains within a species. The limited host range of phage can be both an advantage and disadvantage in phage therapy.

The advantage of a narrow phage host range is that the use of such phage in antibacterial therapy results in less harm to the normal body flora and ecology. Antibiotics, in contrast, with their ability to affect a wide range of bacterial strains often disrupt the normal gastrointestinal flora. Such side effects of antibiotic therapy can result in opportunistic secondary infections by organisms such as Clostridium difficile (8). While this type of side effect should not be a problem with phage therapy, the narrow phage host range does require a means of determining the specific phage strain needed for each infection that is to be treated. This requirement for the use of phage as an antibacterial agent presents two major problems in the current clinical setting. The first problem is the need to have battery of well characterized phage available for a broad range of pathogens and second, there must be a timely method available to determine which phage strain will be effective for a given infection (as discussed in: V. Methods for the Rapid Determination of Phage Specific for Infecting Pathogen).

Many different phage strains will need to be identified, characterized, and developed to cover even a portion of the bacterial diseases that could be good candidates for phage therapy. However, using current molecular techniques it may be possible to enhance the host range of some phage, thus reducing the number of phages that need to be developed. For example it has been found that coliphage K1 5 is a "dual" specificity phage that encodes two different tail proteins allowing it to attack and replicate on both K1 and K5 strains of E. coli (62). One tail protein found on phage K1-5 is a lyase protein, similar to that of phage K5 (specific for the K5 polysaccharide capsule) and a second tail protein found on this phage is an endosialidase similar to a tail protein found in phage K1E (specific for the K1 polysaccharide capsule). In addition, the genomic region encoding these proteins is almost identical to the genomic construct found in the salmonella phage SP6 which codes for a protein that binds to the salmonella O-antigen (Figure 48-1) (63).

Figure 48-1

The observation of a similar tail genome motif in both the salmonella phage SP6 and the coliphage K1E, K5, and K1-5 suggests that this genomic construct might serve in the development of a modular phage platform that could operate over a wide bacterial host range. The development and use of such a “modular” phage would also save considerable time and effort over that required for the characterizing completely new phages for each bacterial strain.

Other mechanisms that have been found in nature to expand the bacterial host range of phage. These include the site-specific recombination systems that permit phage to switch between alternative tail fiber proteins (61) or the reverse transcriptase, by which a Bordetella phage provides for variations in its tail fiber proteins (44). It may be possible to adapt these mechanisms to extend the host range of therapeutic phages.

Parameters besides tail fibers and their interaction with cellular receptors can be important for host specificity. Restriction/modification systems may limit the host range of phage in some bacterial strains. It may be possible to address this problem by engineering phages with genomes that do not contain restriction sites recognized by the non-permissive host. Alternatively phages could be produced in bacterial strains that provide DNA modification(s) that allow the phage to escape restriction in the targeted bacteria strain. Another approach that might be employed to address the restriction/modification problem, when engineering a phage, is exemplified by a mechanism used by the phage T7. This phage gene expresses a gene, O.3, early in the infection process which codes for a protein that is a potent inhibitor of type I DNA restriction and modification enzymes (51). A construct containing this gene might be adapted for use in other phage strains or it may be possible to modify T7 phage to expand its bacterial host range for E. coli infections.

In some cases phage may fail to replicate in a particular host because they lack one or two genes essential for the replication of the phage. Such gene(s) can be identified and then incorporated in the phage genome. For example, Lambda phage does not normally replicate in Salmonella. However, when a Lambda phage library containing copies of the E. coli genome were tested it was found that Lambda phage carrying the E.coli nus A gene could replicate in a Salmonella strain, provided that the receptor protein for Lambda attachment is already expressed in the Salmonella strain (S. Adhya, unpublished observations).

In addition to the factors addressed above, bacteria grown with standard laboratory protocols may not behave the same when they are growing in the milieu of an infection. Karakawa noted that Staphylococcus aureus rarely expresses the capsular polysaccharides found in clinical isolates when the bacteria are grown in the laboratory (33). Given such a change in the bacterial capsule, a phage discovered using bacterial grown in the laboratory may not be able to multiply in the same bacterial strain in an infected animal. In the early phage literature there are reports body fluids, (serum, pus, ascetic fluids, cerebrospinal fluid, urine and bile) that inhibits the infectivity of phage against typhoid, colon bacilli and staphylococci (17, 14). More recently, it has been reported that phage that infected certain strains of E. coli, that are not expressing the cell surface protein Ag43, in standard laboratory growth media, may be inhibited by concentrations of bile salts similar to that found in the gastrointestinal tract (28). The Ag43 protein is a phase variable protein whose expression is associated with E. coli biofilm formation (18). Recognition of these problems is important in isolating phage for clinical applications.

Undesirable phage genes

While phage can be used to treat bacterial infections they can also play a major role in bacterial pathogenesis. A number of phage genes have been discovered that encode toxins, or factors that enhance bacterial virulence. They may also contribute, through transduction, to the transmission of antibiotic resistance genes (81). It may be possible to reduce the occurrence of such adverse effects by sequencing the genome of phages of interest for therapeutic applications and using this sequence information to search for homologies with known toxin genes, islands of pathogenicity or genes that foster integration of DNA into the bacterial genome. A table of known phage encoded toxin genes is provided below:

(Table 48-1)

The presence of such toxin genes or genes with similar sequences can be found be found by searching phage genomes against Genbank online using the Basic local alignment search tool, BLAST (2). In addition, this approach can be used to search for drug resistance genes, phage genomic integration factors, or other potential genes that may increase virulence of a bacterial strain. Such BLAST searches take into account only similarities to known genes and it can certainly be assumed that there are other yet to be identified toxin and potentially detrimental genes that do not have sequence similarity to anything in the databases at this point in time. However knowledge of toxin, drug resistance and other potentially troublesome genes is increasing rapidly as is the number of completely sequenced phage and bacterial genomes. For these reasons, such database searches will become increasingly useful and they should help to assure that phage chosen for use as antibacterial therapeutic agents are free of genes that might potentially damage bacterially infected humans, animals or plants being treated with phage therapy.

Pharmacokinetics of phage therapy

Phage in Mammalian Host

Pharmacokinetic data concerning phage therapy is still in a rudimentary state despite the long history of phage use and study. Many early clinical applications of phage therapy employed oral administration of phage preparations with little or no effort to determine phage uptake or distribution. While oral administration may have diminished the possible side-effects from contaminants, including endo- and exotoxins that are often present in filter “purified” phage preparations, it may not have been the most effective route for the treatment of systemic infections. Determination of the most effective therapeutic regime(s) for phage therapy requires pharmacokinetic information.

There were some early practitioners of phage therapy who recognized the need to learn the fate of phage injected into animals. However, these early researchers generally employed qualitative methods and they only reported whether lysis had occurred following the incubation of ground up tissue or drops of blood with the host bacteria in liquid media. Despite these limitations, such efforts led to the observation in 1921 that phage injected into the circulatory system of rabbits could still be found in the spleen long after no trace of phage could be found in other organs or the blood (3). This finding was corroborated twelve years later, in 1933, in an experiment in which, three days after the intravenous injection of a rabbit with phage the animal was sacrificed and the liver, spleen and blood were examined for phage. The liver and spleen, “crushed to a pulp in a mortar,” and a sample of blood were independently incubated in growth media with the host bacteria. At that time phage could no longer be detected in the blood or the liver but it could be found in the spleen (24). One of the first quantitative studies of the fate of phage in animals was performed by Nungester and Watrous (55). They reported that following the intravenous inoculation of 109pfu of a staphylococcus phage into albino rats, the titer in the blood dropped to 105pfu in five minutes and about 4X101 pfu in two hours. This rapid elimination of phage from the circulatory system was attributed the organs of the reticulo-endothelial system, primarily the liver and spleen.

In experiments using T4 phage as a probe of the immune system, Inchley (35) found that the liver phagocytosed more than 99% of the phage within 30 minutes after inoculation and that it removed 12 times as much phage as the spleen, as measured by the uptake of 51Cr-labelled phage. Additional studies demonstrated that the liver inactivated the phage at a higher rate than the spleen, as measured by pfu’s of phage that could be detected in these organs and the rate of loss of 131I-labeled T4 phage in these organs.

A study of the distribution of phage was reported in the previously mentioned study by Geier et al. (30) that employed germ free mice. These mice had no detectable antibody activity to the Lambda phage. Despite the lack of these antibodies the animals also displayed rapid elimination of the phage from the circulatory system and retention of active phage, as measured by pfu, in the spleen (Figure 48-2). As there were no detectable antibody levels for the phage employed in these experiments this initial reaction to phage by the animals must be attributed to the innate immune system. This study also demonstrated only trace amounts of phage in blood and organs of the mice that received phage by oral administration.

Figure 48-2.

Based on the results of these experiments demonstrating the ability of the mammalian host defense systems to remove phage, a serial passage selection method was developed to identify phage variants with a capacity to remain for longer periods in the circulatory system (50). This system was used in mice to select two E. coli phage Lambda variants that demonstrated 16,000 and 13,000 fold greater capacities to remain in the circulation evade the animals host defense systems; similar results were obtained with the Salmonella typhimurium P22 phage. These long circulating mutant phages were demonstrated to be of value in treating animals with bacterial infections. In these experiments there was less morbidity in E. coli infected mice treated with the long-circulating Lambda phage mutants. In similar experiments conducted with Salmonella typhimurium P22 phage, mice infected with Salmonella typhimurium also displayed less morbidity and mortality when they were treated with the long circulating mutant P22 (50).

The experiments above suggested that the loss of phage was due to interactions with the reticulo-endothelial system. In addition, innate immune system blood factors have also been found to be of importance. Sokoloff et al. (70) using a T7 phage peptide display library found a correlation between the peptides displayed and survival of the phage in the rat circulatory system. Phage displaying carboxy-terminal lysine or arginine residues had longer circulating half lives. In addition, in rat serum T7 phage inactivation was associated with complement activation. The T7 phage displaying carboxy-terminal lysine or arginine residues were found to be protected from this complement-medicated inactivation by binding to the C-reactive protein which is normally elevated in rats and mice. However, in human serum phage resistant to inactivation were found to display peptides containing tyrosine residues, not lysine or arginine as in the rat experiments. In human serum the protective protein may be a2-macroglobulin and not C-reactive protein, as found in the rat and mouse serum experiments, because in contrast to the rat, C-reactive protein is not normally elevated in human serum (70). These T7 phage peptide library experimental results may also help to explain the finding that there was a substitution of a lysine for a glutamic acid residue in the E capsid protein in the long circulating mutant Lambda phage, used as an antibacterial therapeutic agent in mouse experiments (50).

In addition to selecting phage that can remain longer in the circulatory system, it has been possible by using phage display libraries, to select phage that display specific peptide sequences that appear to influence the binding or uptake of phage by the vascular endothelium in specific regions of the body (76). This in vivo screening method has also been employed on one patient in an effort to develop a molecular map of the human vasculature (4). The ability to target specific regions of the body may be useful for the treatment of localized infections.

In some infections, the pharmacokinetics of the whole organism may be secondary to the ability to deliver phage intracellularly. This would be particularly important in diseases such as tuberculosis in which the intracellular infection of macrophages can serve as a reservoir for spread of the infection throughout the body. In this regard, Broxmeyer et al. (12) have demonstrated that it is possible to deliver the lytic phage TM4 to intracellular locations in macrophages, by using Mycobacterium smegmatis, an avirulent mycobacterium, as a vector. In their experiments they showed that such treatment could reduce the titers of Mycobacterium avium or Mycobacterium tuberculosis in infected macrophage cultures (12).

Another unique feature concerning the pharmacokinetics of phage, unlike most other therapeutic agents, is that they contain a genome. There is evidence that phage genomes can gain direct entrance to mammalian cells. It has been reported that phage genomic fragments have been found in mammalian cells following oral exposure to phage DNA (21; 65). M13 and Lambda phage DNA was found in the cells of the gastrointestinal tract, peripheral white bloods, and the cells of the liver and spleen using PCR. Phage DNA could be detected for up to 24 hours in the spleen and liver following a single oral dose of phage DNA. However, when phage M13 DNA was fed daily for 1 week, Doerfler et al. (21) were able to isolate clones containing M13DNA from the mouse spleen. One of these clones contained a 1299nt pair fragment of M13 DNA covalently linked to an 80nt DNA segment with 70% homology to the mouse IgE receptor gene (64). In addition, when pregnant mice were regularly fed phage M13 DNA, evidence of M13 DNA could be detected in the fetuses with in situ hybridization methods. In some rare fetal cells this M13 DNA appeared to be associated with the chromosomes (65). There also have been reports of phage induced enzyme activity in mammalian cells, albeit at low levels, following exposure to phage or phage DNA (47; 34). The integration of phage DNA into the genomes of mammals, as the result of phage therapy for an infection, might result in the loss of heterozygosity of tumor suppressor genes. However, the effects from such events are probably minimal given that phages are associated with bacteria in our colon, nose, throat and skin throughout our normal life span. While phage gene delivery and expression in mammalian cells may normally be rare, phage are currently being genetically engineered to enhance these capacities. Such engineered phage may be able to serve as vectors for targeted gene delivery in mammalian cells (40).

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Carl R. Merril, Dean Scholl, and Sankar Adhya iconBebout, G. E., Scholl, D. W., Kirby, S. H., and Platt, J. P., eds., Subduction: Top to bottom, Volume 96: Geophys. Mono.: Washington, dc, agu, p. 223-228

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