Precision-Guided (MALT-Targeting) Mucosal Vaccines

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          This page describes the screening test that was created and used to isolate those few phage particles, out of a billion candidates, which happened to be carrying foreign insert peptides which can function as potent “MALT-targeting” sequences when added (in small numbers) to mucosal vaccine particles.

          To better understand the challenge, and how it was met, the reader needs to understand that the goal was to identify, and isolate – in still-viable, still-reproducing form – those few phages which were able to trigger – and then drive, all the way to completion – not just one, but an entire series and sequence of four different and distinct steps, involving not just one but two entirely different types of immune cells. Those steps can be summarized as:

          (i) an M cell on the outer surface of a MALT patch needed to pull – into the cell – a phage particle carrying a “winning” foreign insert peptide; and, that phage particle had to be inside a special type of membrane bubble, called a “phagosome”; 

          (ii) the M cell then had to perform a special process (which can be done only by M cells) called “trans-cytosis", which means that the M cell had to rapidly push, pull, and hustle the phagosome containing that phage all the way through the cell, and then mash that phagosomal bubble against the “basal” outer membrane of the cell, so that the phagosome membrane, and the cell membrane, would merge together (which happens, because both membranes are made of exactly the same types of molecules), in a way which would expel the phage particle out of the  M cell, in “naked and exposed” form again, into a special pouch (called a “docking site”) on the basal side of the M cell. And – equally important – that transfer and expulsion process had to be completed before a different type of organelle, called a “lysosome” (containing high acidity, and aggressive digestive enzymes) managed to merge with the phagosome (that's what happens to nearly all phagosomes, as part of how cells break apart and digest any particles they pull in); 

          (iii) the docking site had to already contain an "immature” dendritic cell, which needed to be waiting for a “pathogen delivery” by the M cell which created and which controlled that docking site; and,

          (iv) the immature dendritic cell had to use its surface receptors to analyze that particle, determine that it appeared to be a dangerous and important pathogen, and irrevocably commit to maturation (aka transformation, activation), which required it to move its CCR7 receptors to its outer surface.

​          It also must be emphasized that the concepts and means to achieve these goals didn't simply fall or move into place, quickly, or logically. Instead, the scientist who did this work had spent nearly 4 years as a full-time employee, with this project as his only assignment, before he finally realized how to create this screening test, and then actually used it successfully. 

          So, the steps described below eventually came together, and formed not just one, but two different “rounds” of screening tests. The first round was a big step forward, which could purify (or at least greatly enrich) the numbers and concentrations of phages that had been taken inside dendritic cells, or which at least were clinging to the surfaces of dendritic cells, while being analyzed by those cells. Then, when that set of tests was nearing completion, the scientist realized how he could create an even better screening test, and how he could use the phages he had already isolated, from the first round of tests, in a way that enabled him to evaluate and rank how well, and how potently, the different candidate peptide sequences could perform each and all of the desired tasks.

          That two-cycle process can best be explained, by describing the first round of tests under the heading, “The CD4 Receptor Tests”, and describing the second round under the heading, “The CCR7 Receptor Tests”. 

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ROUND 1:  The CD4 Receptor Tests (isolated any dendritic cells – immature, or activated)

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           STEP 1: We purchased a high-quality “phage display library” from New England Biolabs (catalog number E8210S), 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: Liquid droplets with about 20 million phages/animal were slowly “infused” into the nostrils of a sedated mouse, via a micro-pipette (50 mice were used for that stage, for a total of a billion candidate particles). That allowed the particles to enter the nasal airways 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 break apart and digest the phage particles, the mouse was painlessly euthanized, and ice-cold saline was injected into the aorta, under mild pressure, to slow down any digestion of the phages by cells, without killing the cells. The cold saline emerged from the cut ends of the vena cava (the upper and lower main veins that return oxygen-depleted blood to the heart), pooled in the chest cavity, and was suctioned out. A “transverse skull section” was created, which exposed the MALT patches in the nasal airways. Surface and near-surface cells from the MALT areas were harvested, using a very thin brush with gentle lateral pressure, under a binocular microscope.

​           STEP 4: The harvested cells from the nasal lining were processed, to isolate any cells with CD4 receptors on their surfaces (which includes dendritic cells). This process used tiny metal beads with enough iron content to be attracted to magnets. “Activated complement proteins” were coupled (indirectly but firmly) to the beads, since CD4 receptors bind to those proteins. Cells which became firmly coupled to the beads were purified by using a magnet to pull the beads into a clump, located halfway up a vertical column of liquid, pressing against the inside wall of the tube which held the suspension of beads and cells. All liquid (and any unwanted cell debris and other particles) below “the magnetized clump” 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 a highly enriched set of dendritic cells.

          STEP 5: The membranes of the enriched dendritic cells were broken apart, using a detergent which dissolves cell membranes, but not proteins (which cover and enclose the phages). That released any phages which had been pulled inside those dendritic cells, or which were clinging to the surfaces of the dendritic cells. Those phages were “plated” at low density on “lawns” of fresh host cells, on agar plates. “Clonal colonies” of the “First Round Winner Phages” were selected, and reproduced in fresh batches of host bacteria. 

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ROUND 2:  The CCR7 Receptor Tests (isolated only those dendritic cells that committed to activation/maturation)

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          The first screening round, described above, isolated 145 different “First Round Winner” phages. However, while that work was being done, the scientist doing that work continued to study and learn more about how dendritic cells change, when they shift from “immature” to “antigen-presenting” cells. As a result of that inquiry, he realized there was a way to design and run a better screening test, which would NOT select any and all dendritic cells, and which, instead, could select only those dendritic cells which had been given a phage particle carrying foreign protein sequences which made that phage particle appear to be dangerous enough, and important enough, to merit an antibody-forming response. In other words, he figured out how to create a screening test which could isolate only those dendritic cells which had irreversibly committed to “maturation” into mobile “antigen-presenting cells” that would leave the docking site of an M cell, and begin looking for a gathering of T and B cells in a “germinal center” inside a lymph node. 

          He created that screening test, by figuring out how to use and exploit the following fact: when dendritic cells “commit” to activation/maturation, they move multiple copies of a special receptor, called the CCR7 receptor, from inside the cell, to the outer surfaces of the cell. After those receptors reach the outer surfaces of a dendritic cell, they can be contacted by a chemo-attractant cytokine called CCL19, which is being slowly and constantly released by T cells located inside the “germinal centers” of lymph nodes. Activated dendritic cells will always travel in the direction of the highest number of signals they are receiving, from the CCR7 receptors distributed all around the surface of the cell. By “climbing the gradient” (i.e., always moving in the direction of the highest concentration of CCL19 molecules), activated dendritic cells will eventually find, and enter, the places where those CCL19 molecules are coming from . . . which is where T cells and B cells are gathered together, inside lymph nodes, waiting for a mobile immune cell to “present” an antigen to them, so that they can begin working to create antibodies which will bind to that antigen.

          Rather than starting over and putting the 145 “First Round Winner” phages into a freezer for storage, the scientist realized how he could use those "Winners" to create a “potency ranking” which would indicate the best and most potent performers, from among those 145 phages. That led to the following steps . . . 

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          STEP 6: Two mixed batches of phages were created, with one batch containing 72 of the 145 “First Round Winner” phages, and the other batch containing the other 73 “First Round Winner” phages, all in roughly equal numbers. The concentration of particles in each of the 145 starting batches was measured (by using light absorption at 280 nm wavelengths), and concentrated starting batches were diluted, when necessary to provide roughly equal numbers of each starting batch, in the two large mixtures.

          STEP 7: While that was being done, the scientist also created a set of “dual chamber” separators, by using cement and rubber gasket rings to glue a microporous filter disk into a plastic tube, which was created by sawing off a segment of a plastic pipette. The pipettes that were used had outer diameters which enabled each cut-off segment to fit comfortably, when lowered into one of the wells on a multi-well ELISA plate.  An aliquot of liquid with a supply of the CCL19 chemo-attractant was placed in the bottom of each chamber, along with a small number of medium-sized glass beads, to enable and promote better diffusion of the CCL19 molecules upward through the liquid, and up through the filter as well, into the upper chamber of that separator device

          STEP 8:  Droplets containing 20 million phages/animal were inserted into the nostrils of each mouse, as described above; 90 minutes were allowed to pass, to give the mucosal cells time to begin handling any dangerous-looking phages but not digest or dismantle them; the mouse was painlessly euthanized; ice-cold saline was injected into the aorta, to slow down cell activity and phage digestion; a skull section which exposed the MALT patches in the nasal airways was created; and, a mixture of mucosal surface and near-surface cells was harvested, using a thin brush, as in Round 1.

          STEP 9: A quantity of liquid suspension containing harvested phage-exposed mucosal cells was pipetted into the dual chamber device, on top of the filter disk, so that the cells would settle downward, through the liquid, and come to rest on the top surface of the micropore filter disk.

          The filter disks that were chosen and used, for this separation step, have pore diameters of 5 microns. Most animal cells have diameters of about 10 microns (dendritic cells are even larger, with their multiple petal-like surface extensions). Because they are larger than the pores, most types of animal cells cannot squeeze through a 5-micron pore, in a filter. However, because activated dendritic cells need to be able to travel and permeate through soft tissues (albeit slowly), by using the same type of “pseudopod” motion that amoebas use (octopuses can also do it), activated dendritic cells can squeeze through 5-micron pores. But, they will do so, only if they are motivated to do so. And, they are absolutely driven, and compelled, by their nature and by “what they were born to do”, to do whatever they need to do, to travel in the direction of the highest concentration of CCL19 chemo-attractant molecules that their CCR7 receptors are detecting.

          Therefore, dendritic cells which had already moved their CCR7 receptors to their outer surfaces – in other words, only those dendritic cells which had irreversibly committed to activation, transformation, and maturation – were the only cells that were both able, and motivated, to squeeze downward, through that 5-micron filter, to get to the bottom chamber, where the concentration of CCL19 was highest. So, everything above the filter was discarded, and the cells below the filter were collected, and broken apart, to release the phages which those dendritic cells were carrying.  They were plated on agar, to create clonal colonies, and their foreign DNA inserts were sequenced, using “Polymerase Chain Reaction” (PCR) and Sanger 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” which did not contain any cysteine residues (to avoid possible complications involving “disulfide bond” formation, which will alter the shapes of proteins). 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 “linkers” that increase flexibility and accessibility). Those phages became our “​first testable constructs” carrying MALT-targeting sequences AND a “testable antigen”, as described on the next page.

 

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