TOPIC 7. WHAT ARE PHAGES, AND “PHAGE LIBRARIES”? AND, HOW DID WE “SCREEN” LITERALLY A BILLION DIFFERENT PHAGES, TO IDENTIFY AND ISOLATE LESS THAN 100 WHICH WERE CARRYING POTENT MALT-TARGETING SEQUENCES?
This topic is included, to reassure any vaccine companies or researchers who might be interested in MALT-targeting mucosal vaccines that we did indeed do the work we claim to have done, 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 brief summary of what “phage display libraries” are, and of how “screening tests” can be thought-up, and then 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.
Then, we will describe the specific type of screening test we created, and used, to identify those particular phages, from a phage library, which happened to be carrying foreign insert peptides which triggered and drove both M cells, and “immature dendritic cells”, in MALT patches in the nasal airways of mice, to “determine” that those particular phages were dangerous and important pathogens . . . which, therefore, needed to be pulled in and processed, as quickly as possible, so that an antibody-forming response – which would help fight off those “apparently dangerous and important pathogens” – could be commenced, as quickly as possible.
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 “eating”, and which implies an aggressive form of eating (rather than just “nibbling”), comparable to the English work “devour”. Later, the name “bacteriophages” was shortened to just “phages”.
5. Accordingly, 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 very active, and motivated, 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 some particular type of 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 microbes.
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), which are “filamentous” phages” that have extremely small genomes, and which infect E. coli cells. They created new mixtures called “phage display libraries” (now also called “phage libraries”). Summarized briefly, all of the billion or more phage particles, in a “phage display library”, are exactly the same, EXCEPT FOR a short “foreign insert” DNA sequence, at a known and specific location, in a specific gene of that phage. Today, those short inserts are created by “almost entirely random” chemical synthesis (i.e., the chemical methods are entirely random, except for some non-random steps used to avoid unwanted “stop codons”). The foreign DNA inserts are inserted into a precise location in a gene called the “coat protein 3” (cp3) gene, so that the foreign peptide sequence will appear at the outer tips of all five copies of the long tentacle-like cp3 proteins, which Inovirus phages use to latch onto (and infect) new E. coli cells.
The work required to create really good phage libraries took decades; but, now that that work has been completed, anyone can buy a really good 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).
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 a few million candidates in a small “aliquot” of a liquid suspension of phage particles (in this context, “aliquot” refers to a small quantity of liquid having a known and specific volume, which will contain some known number or portion of the molecules or particles from a larger batch of that liquid suspension), will be taken in and/or 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, a scientist who hopes to isolate those particles which can, indeed, perform “the XYZ trick”, 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 then 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.
THE SCREENING TESTS FOR “MALT-TARGETING” PHAGES
This summary lists several of the major steps that were used, to screen a phage library in a way that isolated only those phage particles which happened to be carrying foreign insert peptides which were able to trigger and drive each and all of the following steps, by M cells and then dendritic cells, in MALT patches:
(i) intake (inside a phagosome) of a phage particle carrying a “winning” foreign insert peptide, into an M cell on the surface of a MALT patch;
(ii) rapid trans-cytosis of the phagosome, through the M cell;
(iii) ejection of the particle, in exposed and naked form again, into a docking site holding an immature dendritic cell, on the “basal” side of the M cell; and,
(iv) analysis of the particle, by the immature dendritic cell, leading to a “determination” that the foreign peptide insert sequence, on that particular particle, showed that that particle was a truly dangerous and important pathogen, to a level and extent which triggered an irreversible commitment, by the dendritic cell, to “activation” (aka “maturation”), which would turn that dendritic cell into an “antigen-presenting cell” which would leave that docking site, and begin searching for a “germinal center” of a lymph node, so that it could present a set of semi-digested “chunks” of surface proteins, from that particle, to the B and T cells in that lymph node.
With the goal of creating and using a new type of screening test to isolate and identify those phages which were carrying foreign insert peptides which could potently drive all four of those steps, all the way to completion, here are the steps we used:
STEP 1: We purchased a high-quality “phage display library” from New England Biolabs, having roughly a trillion total phages from the filamentous Inovirus class, with randomly-generated foreign inserts (12 amino acids long) at the outer tips of their long tentacle-like CP3 proteins;
STEP 2: A small “aliquot” of liquid, carrying about 20 million phages, was “infused” into the nostrils of a sedated mouse (using 50 mice, in that stage, for a total of a billion candidate particles), via a micro-pipette. That allowed the particles, in a liquid suspension, to enter the nasal passages, and contact the MALT patches (which are in a well-known location, in those nasal airways).
STEP 3: After giving the mucosal cells enough time to take in and process any particles they chose to take in, but not enough time for the dendritic cells to begin breaking apart and digesting the phage particles, the mouse was painlessly euthanized, and ice-cold saline was infused into its vasculature, to slow down any digestion of the phages by the cells, but without killing the cells. A “transverse skull section” was created, which exposed the nasal airways in the location where the MALT patches are known to be, in mice. Surface cells from those MALT surface areas were harvested, using very gentle pressure and a very thin brush tool.
STEP 4: The harvested cells from the nasal lining were treated by using a “first screening method”, which selected for any and all dendritic cells, regardless of whether they contained any phages. That first screening method selected for cells which had a specific known receptor protein on their surfaces (dendritic cells have that receptor, on their surfaces). The cellular selection and purification process involved using tiny magnetic beads, with molecules which bind to the dendritic cell receptors, coupled to the magnetic beads. Cells which became coupled to the magnetic beads (because their surface receptors became bound to the molecules that were coupled to the magnetic beads) were purified, by using a small but strong (neodymium) magnet to pull the beads into a cluster, located halfway up a vertical column of liquid and pressing against the inside wall of the tube holding the liquid. All of the liquid below that clump (and any unwanted cell debris and other particles) were suctioned out of the tube, and the magnet was then pulled away, to release the beads, which were then resuspended in a fresh batch of liquid cell medium. That “washing” process was repeated three more times, to obtain purified dendritic cells. Their membranes were then broken apart, using a special detergent which will attack cell membranes, but not proteins, which cover and enclose the phages. That released any phages which had been pulled inside of any dendritic cells, or which were clinging to the surfaces of the dendritic cells. The selected phages were then “plated” at low density, on a “lawn” of fresh host cells, on top of a semi-solid gel nutrient called agar, in a shallow dish. That allowed “clonal colonies” of the “First Round Winner Phages” to be isolated, and reproduced in fresh batches of host bacteria.
STEP 5. The work described above ended up isolating 145 different “First Round Winner” phages. However, while that work was being done, the scientist who created that screening process continued to study and learn more about what actually happens to dendritic cells, and how they change, when they shift from “immature” to “antigen-presenting” status; and, as a result of that work, he realized there was a way to design and run a better screening test, which would not select any and all dendritic cells, but which, instead, could select only those particular dendritic cells which had already irreversibly committed to the transition, from “immature” to “antigen-presenting”.
We are not going to disclose, in this website, the specific steps and methods the inventor used, to isolate and select those particular cells which had taken in the phages we wanted to isolate and identify, since those steps sit at the heart of an invention which has been described and claimed in a patent application which will be published before long. The details will be in there, and once that application has been published, this website will be updated, to provide a complete downloadable copy, and a “summary guide” to help non-experts understand it.
However, in a spirit of playfulness, and in the hope of triggering some curiosity, and original thought, here is a hint, which should be regarded as a riddle, a tease, and a challenge. The method the inventor created, and used, involved two crucial numbers: 7, and 19.
We hope anyone who already knows what those two numbers refer to, will be thinking, “Well, if they knew about THOSE two things, they might have actually done what they say they did.”
And, anyone who does not know what those two numbers refer to, is invited and encouraged to send copies of this text, to any experts who might be able to provide that information. And, if any experts are able to answer THAT question, they should be asked how THEY would try to design a screening test, to identify phages which can drive dendritic cells all the way through an irrevocable commitment to activate and mature into an “antigen-presenting” mode.
STEP 6: Rather than abandoning the results of the “First Round” screening test, and moving those 145 phages into deep storage in a freezer, the scientist who designed the tests realized that, if he handled them in a certain way, he could use those 145 “First Round Winner” phages to create a “potency ranking” which would indicate the best and most potent performers, from among those 145 phages. Therefore, he created two large mixed batches of phages, with one batch containing 72 of the 145 “First Round Winner” phages, and the other batch contining the other 73 “First Round Winner” phages, all in roughly equal numbers. The concentration of particles in each of the 145 starting batches were measured, and adjusted, to provide roughly equal numbers of each competing phage, by using a spectro-photometer to measure “light absorption” by each starting batch, at 280 nanometer wavelengths (a standard wavelength used to measure “total protein content” in liquid suspensions).
STEP 7: Using the same methods described above, aliquots of about 20 million phages, from either of the two mixed batches, were infused into the nostrils of sedated mice; after a controlled delay, the mice were sacrificed, and ice-cold water was infused into their vasculature; skull sections were created; surface cells were harvested from the nasal airways where MALT patches are located; and, the harvested cells were processed, using the “7 and 19” method.
The resulting “activated, maturing, antigen-presenting” dendritic cells were isolated, their cell membranes were dissolved, and the phages released from those cells were plated, at low density, to create clonal colonies. The phage DNA was processed, using “Polymerase Chain Reaction” (PCR) to create large numbers of copies of only the foreign DNA inserts, and those DNA preparations were sent to an outside lab, for sequencing.
The sequence listings were then sorted, using a computer, to determine which sequences appeared most frequently, among the “Second Round Winners”.
To give the initial tests the best possible chance of success, we selected not just one, but the three “top performers” (or, more precisely, the top performers which did not contain any cysteine residues, to avoid possible complications involving “disulfide bonds” created by cysteine residues, which can seriously disrupt the three-dimensional shape of a protein). Since there is enough room, in the cp3 proteins of Inovirus phages, to add foreign inserts up to roughly 100 amino acids long, and since the total amino acid number in all three MALT-targeting sequences was less than 50, all three were placed together, in tandem, in a “triple” MALT-targeting sequence (with at least two glycine residues between each sequence, to create a “linker” which would allow more flexibility and accessibility). Those phages became our “first testable constructs”, carrying both MALT-targeting sequences and a “testable antigen”, as described on the next page.