Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
2001-12-18
2004-07-06
Leffers, Gerry (Department: 1636)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S069700, C435S069100, C435S320100, C435S091400, C435S252300, C536S023100, C536S023400
Reexamination Certificate
active
06759229
ABSTRACT:
TECHNICAL FIELD
This invention relates to compositions and methods for killing bacteria.
BACKGROUND
Throughout recorded history virulent bacterial infections have been a bane to mankind. Until recently, it was assumed that drug antibiotics had largely eradicated virulent bacteria. It is now apparent, however, that bacteria have circumvented the effects of single-point targeted drug antibiotics. Consequently, there is a need to develop new anti-bacterial agents that can be used to supplement or replace conventional drug antibiotics.
Like animal cells, bacterial cells are subject to infectious agents that are present in their environment. Viruses known as bacteriophage, or phage, specifically infect bacterial cells. Bacteriophage are the natural enemies of bacteria and, over the course of evolution, have developed proteins which enable them to infect a bacterial host cell, replicate their genetic material, usurp host metabolism, and ultimately kill their bacterial host cell.
Research into the use of bacteriophage as therapeutic agents for treatment of bacterial infection began sometime in the late 19th century, predating the development of conventional drug antibiotics. By 1920, Edward Twort and Felix d'Herelle, two noted pioneers in bacteriophage research, were isolating bacteriophage from several bacterial species and using them as anti-bacterial agents. During the early 1940′s, however, antibiotics were introduced to the world as a broad range treatment for bacterial infections, and bacteriophage therapy research went into decline.
Early clinical studies of phage therapy were plagued with poor experimental design, with few controls and little documentation, variable success due to the indiscriminate use of phage to treat a broad range of bacterial infections, and the use of procedures that introduced bacterial toxins into patients and loss of effectiveness of the isolated phage.
The lack of knowledge and scientific expertise needed to understand bacteriophage and their interaction with bacteria also hindered efforts to improve phage therapy. For example, differences between the biological interaction of bacteriophage strains with their species-specific bacterial host in vitro as compared to in vivo have posed considerable difficulty. Although bacteriophage can be selected for their lytic virulence (immediately replicating and then inducing bacterial host cell lysis following infection) in vitro, such selection does not guarantee against the conversion of a seemingly lytic phage to a temperate phage (entering into a state of lysogeny via integration of the bacteriophage genome into the bacterial genome followed by a quiescent period during which lytic proteins are not expressed) in vivo. These conversions result in lysogenic bacteria that are resistant to further bacteriophage infection, thus reducing the effectiveness of phage therapy.
Since the early 1940′s drug antibiotics have become the choice for treating virulent bacterial infections. Several problems associated with this approach are now becoming evident. The misuse and overuse of drug antibiotics has contributed to the rise of antibiotic resistant bacterial strains. Moreover, since drug antibiotics are non-specific with respect to the types of bacteria that they effect, the bacterial flora that naturally occur within the body are killed along with the disease-causing bacterial pathogen. At least 200 identified bacterial species normally inhabit the human body, and many of the these species synthesize and excrete vitamins vital for human health, promote the development of certain tissues, e.g., lymphatic tissue, e.g., Peyer's patches, and stimulate the production of cross-reactive “natural” antibodies that react with pathogenic bacteria. Moreover, natural bacterial flora greatly inhibit colonization by non-indigenous bacteria through normal niche colonization or by producing substances and bacteriocins that can inhibit and kill foreign bacteria. Conventional broad spectrum antibiotics risk killing the non-pathogenic bacteria that are responsible for these beneficial effects.
Bacterial drug resistance was evident at the onset of drug antibiotic therapy, and drug resistant virulent strains of both gram-negative bacteria (including pathogenic strains of
Escheria coli
) and gram-positive bacteria (including pathogenic strains of Staphylococcus and Streptococcus) have become increasingly resistant to drug antibiotics. This increased resistance arises primarily from selection for virulent-resistance strains by the presence of drug antibiotics, resulting in the lateral transfer of resistance genes between different strains and species of bacteria. Epidemic outbreaks have been attributed to a single clone of a benign or virulent progenitor, as well as spontaneous multi-clonal populations within a community setting when drug antibiotic usage is increased. Although decreased usage of antibiotics may improve the odds of generating a population of virulent bacteria that are less resistance towards antibiotics, much contradictory evidence is beginning to surface. For example, a study in Finland found that the incidence of
Streptococcus pyogenes
resistance to macrolide decreased after macrolide treatment was reduced in favor of treatment with erythromycin. However, a follow-up study reported a subsequent 17% increase in
Streptococcus pyogenes
resistance to erythromycin. Another growing concern is the increasing number of multi-resistant bacteria. In 1968 approximately 12,500 people in Guatemala died from an epidemic of Shigella, caused by a bacterial strain that contained a plasmid encoding genes resistant to four different antibiotics (Davies (1996)
Nature
383:219). Population genetics studies of virulent bacteria causing disease outbreaks or increases in frequency and virulence have shown that the distinct clones responsible for the acute outbreaks are often characterized by unique combinations of virulence genes or alleles of those genes.
Increasing drug antibiotic resistance has resulted in increased dosage levels and duration of antibiotic treatment. These practices are associated with hypersensitivity and serious side effects in a growing number of patients (see Cunha (2001)
Med Clin North Am
85:149; Kirjavainen and Gibson (1999)
Ann Med
31:288; Lee et al. (2000)
Arch Intern Med
160:2819; and Martinez et al. (1999)
Medicine
78:361). The increasing hypersensitivity and side effects are not being seriously addressed and have so far been clinically under-evaluated (Demoly et al. (2000)
Bull Acad Natl Med
184:761; and Gruchalla (2000)
Allergy Asthma Proc
21:39). As an example of one serious side effect that is becoming increasingly prevalent, especially in children, the use of antibiotics has been shown to be positively associated with the development of asthma and atopy. The mechanisms underlying these associations remain largely unknown (von Hertzen (2000)
Ann Med
32:397).
Drug antibiotics and their effects are not isolated to individuals under the supervision of a doctor's care, but are a communal health issue. Molecular population studies have identified healthy humans that are VRE (vancomycin-resistant enterococci) carriers. An increase in VRE strains in healthy farm animals is associated with the increased use of the antibiotic avoparcin. There is currently a tentative link between the consumption of farm animals and VRE transference to people (Bates (1998)
J Hosp Infect
27:89). Data on antibiotic resistance profiles of several food born pathogens provides ample evidence that antibiotic resistance traits have entered the microflora of farm animals and the food supply produced from them (Teuber (1999)
Cell Mol Life Sci
56:755).
SUMMARY
The present invention is based, at least in part, on the development of intracellular peptide toxins and peptide-like toxins that are toxic to a cell when inside the cell, but relatively non-toxic to the cell when outside the cell. Such peptide toxins and peptide-like toxins are useful in the production of a recombinant bacteriophage that eff
Fish & Richardson P.C.
Leffers Gerry
President & Fellows of Harvard College
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