Carl R. Merril, Dean Scholl, and Sankar Adhya

Скачать 120.99 Kb.
НазваниеCarl R. Merril, Dean Scholl, and Sankar Adhya
Размер120.99 Kb.
1   2   3   4

Phage in mammalian host infected with bacteria

For many pharmacological agents, information on drug distribution and clearance would be sufficient. However, phages are not passive pharmacological agents, they are capable of exponential growth as is the infectious agent, the bacteria. A full knowledge of the pharmacokinetics of phage antibacterial therapy requires knowledge of three dynamic components and their complex interactions: the infected human, the infecting bacteria and the phage. Of these three dynamic components two of them, the bacteria and the phage, are capable of exponential growth during the course of the infection and its treatment. One of the first researchers to recognize the need for quantitative data to determine whether phage can sustain exponential growth in vivo, Rene Dubos, made use of an animal infectious diseases model employing intracerebral injections of Shigella dysenteriae (22). The data obtained from these experiments indicted multiplication of phage, in infected animals treated with phage, at the site of infection, the brain. They also demonstrated that phage treatment was capable of rescuing infected animals (survival of untreated animals was 3.6% while survival of phage treated animals was 72%). These researchers also showed that heat inactivated phage provided no protective effects unless the heat inactivated phage preparations were given days before the bacterial infection. They suggested that this protective effect may have been due to the activation of antibacterial immunity by bacterial products present in the phage lysate.

Figure 48-3

Thirty nine years after the publication by Dubos et al. (22), Smith and Huggins reported results in similar experiments (67). In one of their experiments mice were infected intracerebrally with E. coli K1 and in another they were infected intramuscularly. In both experiments phage were administered intramuscularly. The results of one of these studies in which the mice were infected intracerebrally is illustrated graphically in Figure 48-4. As in the Dubos et al. (22) study the phage levels were highest in the infected tissue, the brain. The phage levels fell as the bacterial levels in the brain decreased. Unfortunately all of the data in Smith and Huggins’ studies was gathered by sacrificing only 3 animals at each time point, so no meaningful statistics could be performed. In addition, the graph does not reflect the fact that animals were dying during the course of this experiment. For example it is not possible by looking at the graph or the data in the tables in the Smith and Huggins (67) paper, to recognize that 50% of the untreated animals died by 72 hours and 75% died by 96 hours. Despite these problems this work has stimulated considerable interest and analysis.

Figure 48-4

Levin and Bull (43) developed a formal mathematical model based on data from the Smith and Huggins (67) study. Analysis based on this model resulted in their suggestion that that the following four elements are critical for successful phage therapy:

I. How "aggressively" phage can lyse bacterial cultures, as determined by adsorption rate, burst size, and latent period is a critical factor. The particular phage that Smith and Huggins found to be most effective therapeutically was both specific against the K1 capsule and was known to lyse bacteria more rapidly in culture than a non-K1 specific phage.

II. The limited efficacy of antibiotics may be due to the fact that antibiotics decay in the animal whereas the phage (if a sufficient population of bacteria is present) will multiply and increase.

III. Even though phage are capable of multiplication, the initial dose of phage must be sufficient to control the bacteria population before it reaches a lethal threshold.

IV. The virulence of the bacteria (how rapidly it can multiply in a host) is also relevant. A more virulent bacteria strain will require either a higher phage dose of phage or a more virulent phage if the infection is to be countered. The fact that the phage resistance E.coli that arose following phage therapy were E.coli K1- and therefore less virulent may or may not have played a role in the Smith and Huggins investigations but it clearly was not favorable for the bacteria. In contrast antibiotic resistant bacterial mutants are usually not less virulent.

The importance of modeling the therapeutic use of phage was also stressed by Payne and Jansen (58). Their model includes terms for the loss of phage due to interaction with the mammalian systems, such as the RES of the innate immune system. Studies using their model suggest that the use of antibiotics may at times interfere with phage therapy.

While these models and the data of Smith and Huggins provide some insights into the pharmacokinetics of phage therapy, it is imperative that statistically correct experiments be performed so that more accurate modeling and pharmacological planning can be developed for the therapeutic use phage. Recently developed methods of visualizing bacterial infections in live animals using bioluminescent strains of bacteria may help in this endeavor (27). In this method bacteria containing a lux transposon cassette provides for bioluminescent bacteria which can be followed in live mice by the use of a high sensitivity CCD camera. The technique has been used to follow pneumococcal infections in the lungs of live mice. By incorporating an appropriate cassette into a phage it should also be possible to follow phage interactions with bacterial infections in vivo.

Immunogenicity of phage

Phage and the innate immune system

The immune response is dependent on two components, the innate and the adaptive immune system (46). The adaptive immune system relies on somatic mutations and clonal expansion of T and B cells in response to an infection. Such clonal expansion requires at least 3 to 5 days to generate a sufficient number of cells to provide an effective level of antibodies. In contrast the innate system is dependent on evolution for the development of its functions and it is inherited in a Mendelian fashion. It includes antimicrobial peptides, the alternative complement pathway and phagocytes, including those of the organs of the reticulo-endothelial system, primarily the liver and spleen. It is the innate immune system that first interacts with a foreign body such as a phage when the animal or person has had no prior exposure to this agent. The same mechanism resulted in the rapid loss of Lambda phage injected into the circulatory system of germ free mice, in the Geier et al. experiments as these mice had no detectable antibody response to Lambda phage (30). Likewise in experiments using T4 phage as a probe of the innate immune system, it was found that the liver phagocytosed more than 99% of that 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 (35). To study the role of the blood components of the innate immune system Sokoloff et al. preinjected rats with GdCl, to inhibit phagocytosis by macrophages, prior to the administration of T7 phage (70). In these experiments the pfu of phage in the circulatory system decreased by 95-99% within 5 minutes. As only 10% of the pfu’s could be detected in the liver, 1% in the spleen and less than 1% in the kidneys, lungs, heart and skeletal muscles it was concluded that most of the phage was inactivated in the blood. This finding was supported by the fact that the half life of the phage incubated in rat serum at 37oC for 30 minutes was determined to be less than 3 minutes. Complement was shown to play a major role in this phage inactivation by experiments in which complement activity was inhibited by cobra venom factor (CFV). When CVF was injected i.p. 20 hours before phage injection the loss of phage from the rat circulatory system was significantly reduced and in in vitro experiments the recovery of phage after a 30 minute incubation in rat serum containing CVF was 50% as contrasted to less than 1% when CVF was not present. As previously mentioned, by selecting T7 phage from a T7 phage peptide library for their ability to remain for longer periods of time in the rat circulatory system it was found that the long circulating trait was peptide specific (70). These long circulating T7 phage were found display peptides with either a carboxy-terminal lysine or arginine residue. Such peptides appear to protect the phage from complement-medicated inactivation in rat or mouse serum by binding to a serum protein. In their rats this “protection” protein is C-reactive protein which is normally elevated in rats and mice (70). This study is consistent with the prior study by Merril et al. (50) in which a Lambda phage mutant with a capacity for long circulating times in the mouse was found to have a substitution of a lysine for a glutamic acid in a major capsid protein. It should be noted that Sokoloff et al. found that the protective protein in human serum may be a2-macroglobulin, rather than C-reactive protein which has this function in rats and mice. This may explain why phage with tyrosine residues in the displayed peptides were protected in human sera rather than those displaying carboxy-terminal lysine or arginine residues as in the rat sera experiments (70). Experiments such as these suggest that it may be possible to select and/or engineer phage for phage therapy with resistance to inactivation by components in the innate immune system.

Phage and the adaptive immune system

Many phage are also potent activators (antigens) of the adaptive immune system. For the past 3 decades Ochs et al. (56, 57) have made use of this capability of phage jX174 to probe the human immune system. In normal individuals injected phage jX174 is cleared within three days and a primary IgM response can be observed that peaks 2 weeks after immunization. If a second injection is made 6 weeks after the primary immunization a IgM and IgG antibody titers increase and peak within one week and subsequent phage injections result in further increases in the IgG titers (57). Patients with severe combined immune deficiency, characterized by absence of both B and T cell functions, display a prolonged clearance of phage, with phage present up to 4 to 6 weeks after the initial injection. In addition, these severe combined immune deficiency patients do not develop a detectable antibody response to phage. They also noted that while jX174 phage is a potent antigen it causes no recognized toxic effects in man (56; 16).

Similar activation of the adaptive immune system was observed in mice inoculated with an anti-vancomycin resistant enterococcus phage, ENB6 (10). After the third in a series of five monthly injections of phage ENB6, titers of IgG and IgM increased above background 3,800-fold and 5-fold, respectively and IgG levels did not change substantially after the third injection. No anaphylactic reactions, changes in core body temperature, or other adverse events were observed in the mice over the course of these multiple injections of phage. It may be possible to developing phage that are less antigenic by using phage peptide libraries or affinity matrixes made up of antibodies from human serum. This type of approach has already been initiated to attempt to modulate the immunogenicity of therapeutically important enzymes (36).

Preparation of Phage for Therapeutic Usage

Early phage therapy applications used phages that were purified by filter sterilization. This method has proven to be insufficient because bacterial debris, including bacterial exo- or endotoxins that might be present, can pass through such filters. These contaminants can result in increased morbidity or in some cases mortality. For example, in a recently published study inoculation of mice, i.p., with of 109 pfu of filter-sterilized Lambda phage lysate, grown on E. coli produced a mild reaction (ruffled fur). However, all of the mice were injected in a similar manner with P22 phage lysate, grown on Salmonella typhimurium, died within 12 hr after inoculation. The endotoxin levels in these preparations was 5 X104 and 5 X105 endotoxin units (EU)/ml, respectively, as determined by limulus amebocyte lysate assay (50). Additional purification by techniques such as equilibrium density centrifugation with cesium chloride can separate phage particles from debris including toxins that do not have the same specific buoyant density as the phage particles. Such centrifugation was able to reduce the endotoxin levels in the phage preparations discussed above to 0.3 X101 and 1X103, respectively. In contrast to the problems noted with the filter purified phage preparations, no adverse effects were noted in mice inoculated i.p with the CsCl equilibrium density centrifugation purified phage preparations (50). Phage have also been purified by precipitation with ammonium sulfate followed by separation through anion exchange columns (78). Phage prepared in this manner were administered to animals without any noticeable ill effects. In addition, Ochs et al. (56) used this phage purification method in a number of his human protocols.

Testing for adverse effects associated with phage preparations should not be limited to observation with healthy animals. Animals that are stressed may have a lower tolerance to endo- and exotoxins. In a recent study a lower survival rate was observed in bacteremic mice treated with a phage strain (known to have no in vitro activity against the bacteria associated with the bacteremia) (10). In this study while the highest doses of the phage preparation produced no reported adverse effects in healthy animals an increased mortality was observed in bacteremic stressed mice. This effect was shown to be phage dose dependent suggesting that stressed animals may be more sensitive to the phage itself or trace amounts of endo- and exotoxins present in the phage preparations than normal animals.

These experiments suggest that the presence of toxins in early phage preparations may explain some of the catastrophic results reported in early attempts to use phage to treat bacterial infections in humans. In one such example, reported in 1932, a phage strain was found that could lyse broth cultures of plague (Yersinia pestis) in less than two hours. However, when a filter sterilized lysate containing this phage stain was injected into rabbits experimentally infected with plague the mortality increased to levels above that found in infected rabbits that were not treated with phage. Furthermore when this phage preparation was then used to treat 33 human patients they all died (mortality from plague is normally 60-90%) (52).

While the omission of purification processes may result in increased levels of contamination, including toxins, the overzealous addition of agents to assure that there is no active bacteria present in phage preparations can also be detrimental. The association of a “weak” phage preparation and the presence of organomercury compounds was made in a 1932 study of commercial phage preparations from a major U.S. pharmaceutical company (74).

The problems associated with the production of phage for clinical use are not insurmountable as evidenced by over three decades of phage use in humans (56,57). In addition, animal experiments have provided evidence that relatively simple phage purification processes, such as CsCl equilibrium density centrifugation, can significantly reduce animal morbidity and mortality.

Methods for the Rapid Determination of Phage Specific for Infecting Pathogen

When a clinician is confronted with a patient with an infectious disease the prudent course of action requires the determination of the identity of the infectious agent. This task can often be time consuming and laborious, involving isolation and identification of the causative agent. Often, given the time needed to make such a determination, physicians use their best judgment to chose and administer a relatively broad range antibiotic that is effective for the suspected bacterial strain while they wait for the culture and antibiotic sensitivity results. In contrast, if phages are to be used in place of antibiotics it is critical to actually determine the strain of phage to be used, given the generally narrow host range displayed by most phage. Such determinations using current technology could easily take days to perform. If phage are to be used as therapeutic antibacterial agents a rapid and inexpensive method to determine the nature of the infectious bacteria and its phage susceptibility is needed.

One approach that can be taken to this problem is based on the use of modified phage containing reporter genes. In this method phage are first isolated and identified as being potential therapeutic agents for a particular species or strain of bacteria. These phage are genetically engineered to encode a reporter gene such that a characteristic color or marker will be produced when the phage infects the specific strain of bacteria that is susceptible to that phage. For example, if different strains of phage, carrying the luciferase reporter gene, were placed in a multiwell plate along with an aliquot of a clinical isolate the emission of light from any of the wells would serve to identify the bacterial strain in the clinical isolate as well as the phage strain that may be used to treat the bacterial infection. Such testing could be performed in hours, instead of the days that traditional culture methods require. This approach has been used with phage carrying the luciferase reporter gene to detect Listeria contamination in foods (45). A similar approach has also been developed for a rapid and relatively inexpensive diagnostic test for Mycobacteria infections in patients suspected to have tuberculosis (15). Alternatively, by placing luciferin/luciferase in the incubation mixture, lysis of bacterial strains by phage will result in the discharge of adenylate kinase which can convert ADP in the reaction mix to ATP. As luciferin/luciferase can utilize the ATP for light emission, the lysis will serve to identify the organism and the appropriate phage without the need to genetically endow the phage with a luciferase reporter gene (66).

Recent advances in mass spectrometry may also provide fast methods for the identification of bacterial strains. It is now possible to rapidly identify and type bacterial strains based on their lipid, protein, and nucleic acid mass fragment “fingerprints” (79). While mass spectrometry is currently being developed to identify bacteria strains it might also be possible to use this approach to determine whether the bacteria are susceptible to a given phage. However, such information is not currently available and it may be impractical to gain sufficient knowledge of bacterial mass “fingerprints” to accurately determine when a particular phage could be useful. Alternatively, one could use phage gene product expression for the development of markers for both bacterial identification and as an indicator of phage susceptibility. In this approach one could use mass spectrometry by placing a clinical sample in growth media to amplify the bacteria followed by infection with selected “therapeutic” phage strains. If the infecting bacteria were susceptible to the phage, mass spectrometry would then detect signature fragments of proteins that are expressed only when infection of the bacteria occurs by a specific phage. Signature fragments, that are not part of the phage virion, would be generated from phage specific proteins such as RNA polymerase, regulatory protein, or lytic enzyme. This approach could be used so that the generation and detection of phage specific products serves the same role as “reporter” gene products in a manner analogous to the detection of marker gene products described above.

DNA microarrays, in conjunction with PCR amplification, are also being developed for the rapid diagnosis of bacterial strains. This technology can be used to determine the susceptibility of bacterial strains to certain antibiotics (31). In principle it may be possible to also develop such a method for the determination of bacterial strains and their phage susceptibility.

1   2   3   4


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

Carl R. Merril, Dean Scholl, and Sankar Adhya iconKick ass carl Hiaasen

Carl R. Merril, Dean Scholl, and Sankar Adhya iconDr Carl Kirstein en prof fj potgieter

Carl R. Merril, Dean Scholl, and Sankar Adhya iconSifakis, Carl, ed. The Encyclopedia of American Crime. 2nd ed. Vol. Ny: Facts

Carl R. Merril, Dean Scholl, and Sankar Adhya iconDean Jaimie L. Hebert, Ph. D

Carl R. Merril, Dean Scholl, and Sankar Adhya iconDescendants of Elizabeth Dean

Carl R. Merril, Dean Scholl, and Sankar Adhya iconDean, Boonshoft School of Medicine

Carl R. Merril, Dean Scholl, and Sankar Adhya iconExcerpted from Dean Lueck, “First Possession”

Carl R. Merril, Dean Scholl, and Sankar Adhya iconLetter-of-intent to executive dean(S)

Carl R. Merril, Dean Scholl, and Sankar Adhya iconDean & Professor Office: ed II, 5/104

Разместите кнопку на своём сайте:

База данных защищена авторским правом © 2014
обратиться к администрации
Главная страница