"Using Phage Display and Antibodies for Detection of Foreign Molecules and Treatment of Disease"
Dr. Fagan has also provided links to three background readings:
Again, whoever is responsible for a discussion question, please be sure to include one based upon what is sure to be a thoughtful seminar.
Dr. Fagan's seminar lecture, Using Phage Display and Antibodies for Detection of Foreign Molecules and Treatment of Disease, important methods and technology that is utilized in her laboratory and also current research her students and herself are conducting. Monoclonal antibodies are antibodies that are produced by the same immune cells and therefore have a very high binding specificity to a single antigen or molecule, which has become valuable in the field of medicine. Also presented was the technique of phage display, which uses transformation of bacteria by a bacteriophage that displays an antibody fragment on its surface in order to isolate monoclonal antibodies and construct phage display library. This technique has been effective for developing human monoclonal antibodies. Antibiotic-resistant bacteria has been identified as one of the greatest threats to public health around the entire world. How could phage display technology be used to combat the increase in bacteria that have become resistant to current antibiotic treatments and what are the pros and cons of phage display for this application?
ReplyDelete*Correction : . . . Treatment of Disease, DISCUSSED important methods . . .
ReplyDeleteI found an interesting paper highlighting the pros and cons of phage therapy.
ReplyDeletehttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278648/
"In terms of “Pros,” for example, phages can be bactericidal, can increase in number over the course of treatment, tend to only minimally disrupt normal flora, are equally effective against antibiotic-sensitive and antibiotic-resistant bacteria, often are easily discovered, seem to be capable of disrupting bacterial biofilms, and can have low inherent toxicities. In addition to these assets, we consider aspects of phage therapy that can contribute to its safety, economics, or convenience, but in ways that are perhaps less essential to the phage potential to combat bacteria. For example, autonomous phage transfer between animals during veterinary application could provide convenience or economic advantages by decreasing the need for repeated phage application, but is not necessarily crucial to therapeutic success. We also consider possible disadvantages to phage use as antibacterial agents. These “Cons,” however, tend to be relatively minor.
Potential disadvantages are:
-Not all phages make for good therapeutics.
-The problem of narrow host range.
-Phages are not unique pharmaceuticals.
Because phages infect and kill using mechanisms that differ from those of antibiotics, specific antibiotic resistance mechanisms do not translate into mechanisms of phage resistance. Phages consequently can be readily employed to treat antibiotic-resistant infections."
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ReplyDeletePhage display is a molecular technique through which foreign proteins including peptide and antibodies fragments are expressed at the surface of phage particles. The phage therefore become vehicles for expression that not only carry within them the nucleotide sequence encoding expressed proteins, but also have the capacity to replicate. This procedure entails insersion of the nucleotide sequence encoding the protein to be displayed into a phage or phagemid genome as a fusion to a gene encoding a phage coat protein The DNA hybrid and the phage gene are then put inside E. coli bacterial cells for amplification. affinity selection is done through biopanning. Examples of bacterial used include XL1-Blue E. coli, SS320, TG1, and ER2738. The peptide or protein of interest is eventually expressed in either the minor or major coat protein.
ReplyDeleteMany bacteria have evolve over time and can develop a resistance to antibiotics. Because phages are target specific, meaning only a one or very few bacterial strains are targeted upon, it is easier to develop new phages than new antibiotics. A time period of only a few days or weeks is needed to acquire new phages for resistant strains of bacteria, whereas it can take years to obtain new antibiotics. When resisting bacteria evolve, the assigned phages also evolve, so when super bacterium appears, an equivalent super phage fights it as long as the phage is derived from the same environment.
Compared to antibiotics, phages go deeper into the infected area. Antibiotics, concentration on the hand quickly decrease as they go below the surface of the infection. The replication of phages is concentrated on the infected area where they are needed the most, while antibiotics are metabolized and removed from the body. Multiple side effects like allergies, intestinal disorders, and yeast infections have been observed when using antibiotics are overcome through this technology. Some of the disadvantages of phage therapy are that the size of the phage is large compared to the chemical therapeutic agent and may not be able to access all the place in the body and that still some bacteria can still develop resistance to some phages. Also because of the specificity of the phages, it may be hard to use if the offending bacteria is not known
Phage display is new technique that is finding success and versatility in the scientific community. MRSA is a highly drug resistant bacteria which makes infections a serious threat to the host’s health. Since antibiotics are becoming less effective in the treatment of MRSA, different approaches need to be investigated in order to find new treatment plans. Phage display could be the answer to this problem. If capsule specific antibodies could be developed against staph, scientists could attach a unique toxin to kill the staph or possibly introduce a new protein in the form of plasmid, making it more susceptible to host defenses. However, this could also lead to other problems. Phage is difficult to control and kill meaning that it will keep infecting and replicating itself in potential targets. If the antibody isn’t specific enough to differentiate MRSA from normal S. Aureus, the phage will target both. This would remove the normal flora S. Aureus and removing an innate host defense. Another opportunistic pathogen could colonize the area and create more problems.
ReplyDeleteBecause infectious diseases are able to spread rapidly and globally, antimicrobial drug resistance is a serious concern for human health. Phage-display technology is recognized for the ability to bind to a wide range of antigens, meaning it can play an important role to combat the rising concern of these diseases. Phage-display technologies to combat drug resistance include the M13 phage, library construction and biopanning. The M13 phage is ideal for displaying large insertions; however infectivity of the phages may be reduced.
ReplyDeleteBecause mainstream broad-spectrum antibiotics are the most popular option to combat microbial disease, focus has not been on developing a means to combat specific pathogens.
Phage display may provide advantages in facilitating screening of diverse molecular targets to obtain high-potency candidates suitable for therapeutic development within a much faster timescale than that achievable for small molecules. A disadvantage is that not all peptides or proteins can be selected using phage-display. Furthermore, some peptide may only function when it is an integral part of the phage coat protein, rather than in the free solution process.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421897/
Bacteria are able to evolve quickly due to their rapid division rate and facilitated means of passing on genetic information through horizontal gene transfer (from one bacteria to another without sexual and asexual reproduction). This passing of genetic information can actually pass resistance for certain antibiotics between bacteria. Finding something that could disrupt these resistance genes could be beneficial in preventing bacterial resistance from spreading and becoming a serious issue like MRSA. If phage display could find protein-protein interactions that could be useful in the creating of a drug to target the resistance genes in bacteria or find protein interactions that can shutdown essential bacterial protein production, it is very possible to use this technique to combat bacterial diseases that have become resistant to antibiotics. Some pros to the technique include that phages are effective against antibiotic-sensitive and antibiotic-resistant bacteria. Some cons could be that phages are host-specific. They might not be able to infect all types of bacteria and phage modifications/type changes might be needed. Some competing methods being developed and improved to look at protein evolution are yeast display, bacterial display, ribosome display, and mRNA display.
ReplyDeleteSome of the pros of using phages are that they are very specific, they will not harm the bacteria and normally flora around the targeted bacteria. Using phages in animals has not produced many side effects and also due to the specifity of phages, they do other produce bacteria resistance in bacteria of the body. Some more pros of phages are that they have an inexhaustible supply, in that evolution is driving phages to constantly change as bacteria develop resistance.
ReplyDeleteSome of the disadvantages of using phages are that there are no studies on humans that have been published yet, experiments are currently under way. The great specificity of phages can be a disadvantage when the type of bacteria is not known. Bacteria can be specific to phages, they have a mechanism which incorporates part of the genetic material of penetrating phages(transformation). Phages can be excreted from the body by the immune system, and also phages are larger than antibiotics, so the areas they can reach need to be investigated more.
Some of the pros of using phages are that they are very specific, they will not harm the bacteria and normally flora around the targeted bacteria. Using phages in animals has not produced many side effects and also due to the specifity of phages, they do other produce bacteria resistance in bacteria of the body. Some more pros of phages are that they have an inexhaustible supply, in that evolution is driving phages to constantly change as bacteria develop resistance.
ReplyDeleteSome of the disadvantages of using phages are that there are no studies on humans that have been published yet, experiments are currently under way. The great specificity of phages can be a disadvantage when the type of bacteria is not known. Bacteria can be specific to phages, they have a mechanism which incorporates part of the genetic material of penetrating phages(transformation). Phages can be excreted from the body by the immune system, and also phages are larger than antibiotics, so the areas they can reach need to be investigated more.
Dr, Fagan’s presentation was very interesting! I was very intrigued by the final outcome and application of her research. Ideally being able to detect if human blood and determine the exact location of said person could be useful in many aspects. Antibiotic resistance is an increasing problem partly due to the inappropriate use of antibiotics. People often do not complete their course of antibiotic because they just discontinue taking the medication when they feel symptoms have resolved. This poses a risk for the remaining bacteria or virus to continue to replicate and become resistant to that antibiotic. Phage display could be useful in combating this problem by utilizing the phages to produce monoclonal antibodies that can then be given to the person suffering as a form of treatment to prevent spread of infection. This ofcourse is the ideal outcome, however some antibodies may not be compatible with all species or may not have the ability to be given as therapy for diseases.
ReplyDeleteGeorge Smith introduced phage display technology in 1985. Researchers used it as an expression vector that is able to present a foreign amino acid sequence available to binding an antibody. T4 phages are good example of well studied dsDNA T4 phages and have relatives which infect Aeromonas, Vibrio, Acinetobacter and other bacteria. T4 like phages’ genomes has similar homologies with T4. Phages as antibacterial agents have a lot of features to be alternative of chemical antibiotics.
ReplyDeleteAdvantages:
• Unlike chemical antibiotics, to kill a single bacterium, you need only a single phage
• Relatively, being inexpensive
• Phages are easily discovered
• Less disruption of normal flora
Disadvantages:
• Phage selections
• Limitation of phage’s host,
• Individuality
• Being unfamiliar with the phages.
http://www.highveld.com/virology/phage.html
http://www.scielo.br/scielo.php?pid=s1415-47572005000100001&script=sci_arttext
Phage display is a powerful tool for selecting peptides, proteins, or antibodies with specific binding properties from a large number of variants in phage libraries.The starting point is usually a phage library. There are several types of phage display libraries including peptide libraries, protein libraries and antibody libraries. For example, peptides can be selected from peptide libraries, which commonly expressed variants of peptides of 7 to 20 amino acids in fusion with the protein pIII or pVIII of the phages. To select phage display antibodies it is neccesary to have or construct a phage library that display antibody fragment on the surface of the phage. The genomic information coding for antibody variable domains to construct antibody libraries is usually derived from B cells of immunized or non – immunized donors. 3 types of common phages used in phage display are the M13, F1 , FD.
ReplyDeleteAdvantages:
Diversity and abundance
Automated parallel screening.
Rapid optimization.
Phage display can be used for selection of proteins, peptides, or antibodies with affinity and specificity to a molecule or protein of interest from phage libraries. It can be helpful for preservation of unstable hybridoma clones and to identify molecules that can be recognized and internalized by eukaryotic cells
http://phagedisplay.net/
http://www.wikinvest.com/stock/Dyax_%28DYAX%29/Advantages_Phage_Display_Technology_Therapeutic_Drug_Discovery
Speed and cost effectiveness.