Precision-Guided (MALT-Targeting) Mucosal Vaccines

          This topic (and the next page) are included, to reassure any vaccine companies – or researchers who might be interested in MALT-targeting mucosal vaccines – that we did the work described herein, and that this isn't some effort to defraud or swindle anyone. It requires, first, a brief summary of phages, and what they are (and, that requires a bit of history). That is followed by a summary of what “phage display libraries” are, and of how “screening tests” can be thought-up, and used, to identify which particular phage particles, out of millions of “candidate” or “contestant” particles, happen to be carrying a foreign insert peptide which will cause those particular particles to be treated and processed in some particular way, by some particular type of cell, or tissue type, or animal.

          That is “starting-point" information, to get someone ready to understand what is on the next page. That page describes the specific screening test we created, and used, to identify those particular phages (from among a billion candidates/contestants) which happened to be carrying foreign insert peptides which triggered and drove both M cells, and “immature dendritic cells”, in MALT patches, to “determine” that those particular phages were so dangerous, and so important, that they would cause a full-scale antibody-forming response  to start up and move into action, as quickly as possible.

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WHAT ARE “BACTERIOPHAGES” (WHICH ARE NOW CALLED JUST “PHAGES”)?

 

          As a very brief introduction to “phages”:

          1. People had been experimenting for hundreds of years with various types of lenses, including “magnifying lenses”, when the Dutch fabric merchant Van Leeuwenhoek became interested in trying to make them better, in the 1670s, so that he could more closely examine the thinnest, tiniest threads in the fabrics he handled. Once he got started, he kept refining and improving his magnifying lenses, until he could clearly see (in samples of water, rather than fabrics) microbes that actively moved, which people initially called “animalcules”.

          2. Within a few decades, after seeing and categorizing numerous types of bacteria and other microbial cells, scientists realized that there was an entire category of microbes that were infective, somehow, but which were too small to be seen by even the best light microscopes of that era. Those came to be called “viruses”, after the Greek root word for “virulent”. Until the 1930s, when electron microscopes were invented and scientists could actually “see” and begin to seriously study viruses, no one knew what viruses were, or how they could reproduce.

          3. In the 1890s, scientists realized that there was some type of “virus”, in some of the rivers in India, which could kill and inactivate the bacteria which caused cholera; and not long afterward, a different scientist discovered a similar “virus” that could kill and inactivate the bacteria which caused dysentery. When World War I began, the French armies were the first to develop liquid drinks carrying those viruses, which they fed to their troops, to “immunize” those troops against cholera and dysentery.

          4. As scientists began looking for and finding other viruses that could attack and destroy other types of bacteria that caused other diseases, they realized that each such virus could attack only a very specific and limited class of bacteria. So, they named that entire category of viruses “bacteriophages”, from the Greek root “phage”, which translates into "aggressive eating", comparable to “devour” (as opposed to just “nibbling”). Later, “bacteriophages” was shortened to just “phages”.

          5. The noun “phage” has come to refer to any virus which: (i) can infect only some limited group, type, or class of bacterial cells; and, (ii) is classified as “non-pathogenic”, and not dangerous, since phages cannot infect plants or animals, in any way. The search for new and additional types of phages became active and motivated in the early 1900s, because “phage therapy” grew into a major and crucial branch of medicine, before the advent of sulfa drugs and then penicillin. If someone was infected by a bacterial pathogen, the scientists and physicians of that era could usually figure out what type of bacteria it was, and they would administer, directly to the infected site, a batch of phages which could kill that type of bacteria. That approach has recently come back into favor, to help fight certain types of antibiotic-resistant bacteria.

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PHAGE LIBRARIES (aka PHAGE DISPLAY LIBRARIES)

 

          In the 1970s and 1980s, a group of scientists (led by Prof. George Smith, at the University of Missouri, who later won a Nobel Prize for that work) began developing new and clever ways to work with a specific class of phages called “Inoviridae” (aka Inoviruses). They are phages with extremely small genomes, which infect E. coli cells, and which have “filamentous” shapes that enable them to wriggle out of pores they create in E. coli cell membranes, without killing their host cells (which greatly reduces the number of fragments from dead cells, and makes Inovirus phages easy to purify and use).  They also go through a double-stranded "replicative phase”, which enables them to be handled and treated just like plasmids. And, they form the “capsids” (or shells, sleeves, etc) which enclose their single-stranded DNA, by simply continuing to add more and more of the small brick-like “coat protein 8” (cp8) proteins to a capsid that is being formed, until the “tail end" of the DNA strand reaches the assembly site; that is also highly useful, since it allows exceptionally long foreign inserts to be added to Inovirus phages.

          All of those useful and helpful factors were combined, in Inovirus phages,  and that is why Smith and his research teeam selected Inovirus phages as their starting point, and raw material, when they began trying to figure out how to create “phage display libraries” (which now are also called simply “phage libraries”).

          Summarized briefly, Smith and his team (and other research groups that later became involved, and contributed additional useful methods) figured out how to insert a short DNA segment, created by “random chemical synthesis,” into a single specific site, in the genomes of Inovirus phages, in a way that would cause the altered DNA strand to encode and create engineered proteins which are carrying a small additional segment of also-random amino acids. The most popular and widely used phage libraries that are being sold today carry a set of 12 amino acids, as a “foreign insert”, added to the outer tips of the long cp3 proteins, which function like the tentacles of squids that are hunting for food in the deep ocean, where there is no light. Each phage particle has 5 copies of the cp3 proteins, but all five copies are encoded by a single cp3 gene, so all five tentacle-like cp3 proteins, on any specific phage particle, will have identical copies of the foreign insert peptide which happens to be carried by that particular phage particle.

          The work required to create really good phage libraries took decades; but, now that that work has been completed, and now that companies can make them quickly and efficiently, using computer-controlled machines, anyone can buy a phage library with about a trillion different “candidate” particles, all in a single small tube, for less than $800 (e.g., www.neb.com, catalog number E8210S, which is a “kit” that also includes monoclonal antibodies and magnetic beads, all for $719 as this is being written).

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WHAT ARE "SCREENING TESTS", AND HOW ARE THEY USED?

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          The “trick” to using any phage display library comes in thinking up some new and useful type of “screening test”, which will somehow identify which particular phages – out of thousands or millions of candidates/contestants, in a small “aliquot” of a liquid suspension of phage particles (i.e., a quantity of liquid having a known and specific volume, which will contain some known number or portion of the molecules or particles, taken from a larger batch of that liquid), will be taken in and processed, in some particular way that is of interest, when all of the particles in that aliquot are treated in a certain way.  Almost all “screening tests” will create some type of “fair competition” between the particles, such as by contacting all of the candidate/contestant particles with a specific type of cell or tissue, and seeing which particles are pulled inside those cells (or, as alternate examples, by passing an aliquot of particles through an “affinity column” or other device; or, by infusing or injecting them into a lab animal, and then looking to see which ones reach some particular targeted cell or tissue type).

          The basic rule of “screening tests” is that no one can predict, in advance, which particular particles will be able to do “the XYZ trick”. So, if a scientist hopes to isolate and identify those few particles which can perform “the XYZ trick,” s/he will need to figure out two things: (i) how to pit millions of phages against each other, as “candidates” or “contestants” in a fair competition; and, (ii) what type of isolation or purification process the scientist can use, to identify (and, usually, to isolate, preferably in a still viable and reproductive form) those specific phages which happened to be carrying a foreign insert which enabled them to become “the winners” in that competition.

          So . . . now that that starting-point information has been explained, the next page will describe the specific steps, and the specific screening tests, that were created and used to identify those few phages which happened to be carrying randomly-generated foreign peptide inserts that can function as potent “MALT-targeting” peptide sequences.

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