Precision-Guided (MALT-Targeting) Mucosal Vaccines

          When the facts and factors described on the previous page are explained in a logical step-by-step manner, focusing on each step that will lead to the desired result, while excluding anything that might distract from a challenging pathway, that pathway might seem “obvious”, as that term is used in patent laws around the world.

          However, the startup company which owns this website – and, the patent rights to this invention – will state without apology that these insights did not simply “arrive”. They could not be assembled, logically, until we figured out which facts were truly important, and then, how we could assemble them, into a logical, step-by-step system. We spent literally years, patiently and persistently trying other approaches, which didn’t work, before we finally noticed several things that were well off to the side of the pathway we originally tried to follow. And, that was when we became ready, and able, to combine multiple different insights, components, and relationships, into a cohesive system that can actually work.

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          When boiled down to a concise summary, the crucial steps on our new screening test include the following:

          STEP 1: Tiny droplets (containing 20 million phages from a phage library, for each mouse) were slowly “infused” (i.e., via a micro-pipette, rather than injected via a needle) into the nostrils of each mouse, in multiple different (sedated) mice;

          STEP 2: We waited 90 minutes, to allow the droplets and phages to slowly travel through the nasal airways in the snout of the mouse, and to give the M cells and dendritic cells enough time to perform all five of the steps listed above, but not enough time for the dendritic cells to begin digesting and breaking apart the phages they had pulled in;

          STEP 3: The mouse was anesthetized, then sacrificed. To slow down cellular activity and prevent activated dendritic cells from breaking apart the phages that had triggered their activation, ice-cold saline was forcibly injected into the aorta, and emerged from the vena cava (as an alternate approach, the head can be severed quickly, then placed inside a refrigerator).

          STEP 4: A “skull section” was created, which exposed the MALT patches in the nasal airways (which are in known locations, in mice and rats). Using a stereo-microscope, a very thin brush was used, with gentle pressure, to harvest surface and near-surface cells from the MALT patch locations.

          STEP 5. The harvested (phage-exposed) cells were loaded into the top chamber of a dual-chamber vertical tube device, which had been custom-assembled from a sawed-off segment of a plastic pipette having a diameter that allows the final to be lowered, vertically, into a well on a 48-well ELISA plate. The bottom chamber (i.e., the bottom of the well on the plate) contained a supply of the CCL19 chemo-attractant. The top and bottom chambers were separated from each other by a filter having pores with 5 micron diameters, which had been glued into place using a rubber ring as a gasket, to render the top chamber watertight.

          Most animal cells have diameters of roughly 10 microns, and dendritic cells are even larger. However, they evolved with an ability to squeeze through the gaps between neighboring cells in soft tissues, in ways similar to the “pseudopod” travel of amoebas, or to how an octopus can squeeze through a small opening, to get to a crab or fish. Therefore, the only cells that were both ABLE . . . AND, MOTIVATED . . . to squeeze through the 5-micron filter, were dendritic cells which had already moved their CCR7 receptor proteins to their outer surfaces, by the time they were loaded into the separation tube.

          STEP 6: The harvested phage-exposed cells were re-warmed to body temperature, and were given two hours, to give any activated/transformed dendritic cells time to squeeze through the filter. The separation tube was then lifted out and discarded, and any cells that had reached the bottom chamber were broken apart, by a detergent that will break apart cell membranes, but not phages enclosed within proteins. Whatever was released, when the cells were broken open, was plated onto a “lawn” of fresh host bacteria cells, on an agar plate.

          STEP 7: Within two days, a few phage colonies began to appear, and “clonal isolates” from each colony was transferred into a fresh batch of host cells, in broth. When enough new “clonal” phages (i.e., all descended from a single ancestor, and having the game genetic content) had reproduced, the phages were purified, and a DNA preparation was made from each clonal isolate. The “foreign insert” sequences (and the “primer sequences” which flanked the foreign inserts) were “amplified”, by polymerase chain reaction (PCR), and those DNA preparations were shipped to a DNA sequencing lab.

          The resulting DNA sequences revealed what the amino acid sequences were, in the “winning” phages that had “won” the competition which was created by the screening test. Those were the phages which were carrying randomly-generated “foreign insert peptides” which had functioned – successfully and effectively – as “MALT-targeting HYPER-pathogen peptides”, which had been able to initiate, and then drive, all the way to completion, not just one or two cellular responses, but an entire series of five different and distinct cellular responses, by two different types of immune cells, as described on the previous page.

          In a grand total of only 90 minutes, phages carrying those short “HYPER-pathogen” signaling/transport peptides were able to complete a process that likely requires an entire day, or even more, when vaccines are injected into muscles, rather than applied topically (and immediately), directly onto the surfaces of surface-mounted lymph nodes, on mucous membranes that are readily and directly accessible to vaccines in nasal sprays, or in lollipops or lozenges.

          A major, major additional factor also should be noted: when “immature” dendritic cells in a MALT patch transform into antigen-presenting cells, and leave the docking sites behind M cells and begin searching for T and B cells that are working together inside a germinal center of a lymph node . . . well, those antigen-presenting cells will already be inside a lymph node. So, they won’t have to look far, or go far, to get where they need to go.

          When that “already inside a lymph node” factor . . . is combined with the additional fact that . . . MALT-targeting mucosal vaccines can convert immature dendritic cells into fully-transformed antigen-presenting cells >> IN ONLY 90 MINUTES << . . . well, people should begin wondering, and asking, whether these types of “MALT-targeting” mucosal vaccines will become the fastest-acting vaccines that ever have been (and, possibly, ever can be) created.

          We hope to learn the answer to that question, as soon as possible.