As described on the previous page, when it came time to move beyond "antibody production" tests and into "pathogen challenge" tests, we made two important changes, to create an entirely new type of engineered phage particle:

          (i) we shifted out of using "filamentous" phages, which get "wadded up" in uncontrollable and unpredictable ways, when they get stuffed into a generally spherical phagosomal bubble, and we chose a type of "lytic" phage (the T7 class of phages), with a generally round "head" component, since that type of component can be easily and rapidly grabbed, and pulled in, by immune cells; and,

          (ii) we stopped using an "easily tested" antigen (the HA-tag epitope, isolated from an influenza strain that was important 60 years ago), and obtained guidance from a specialist in influenza research, who recommended an influenza antigen called FI-6 (that is, capital F, and capital I, sometimes mistakenly called the F-16 antigen, and sometimes mistakenly interpreted as the F-lower-case-L-6 antigen). Its sequence, in single-letter code, is KESTQKAIDGVTNKVNS, and more information on it can be found in the NIH's epitope database, at www.iedb.org/epitope/162644. That antigen sequence was selected, because it is present on the surfaces of a VERY wide variety of different strains of influenza, as described in Corti et al, Science 333: 850-856 (2011).

          That specialist steered us toward influenza, mainly because it can infect such a wide range of different animal types (including some types of mice, which can greatly reduce the costs of the initial tests), and because numerous labs already work with it. However, the choice of influenza, for the first pathogen challenge tests, will bring into the process a number of complicating factors, which require attention.

          One complication arises from the mechanism that influenza viruses use, to infect cells. Most mucosal pathogens can penetrate into cells by binding to only a single specific type of protein, on the surfaces of the cells it targets. As three examples, HIV viruses (which cause AIDS) can only bind to CD4 receptors, which are present only on certain types of immune cells; the spike proteins on COVID viruses bind to a surface protein called "angiotensin-converting enzyme-2" (ACE2); and most "rhinoviruses" (which cause "common colds"; "rhino" is the Greek root for "nose") bind to a cell protein called "intercellular adhesion molecule-1" (ICAM-1). However, influenza viruses use a very different mechanism. Each influenza particle carries several hundred copies of a protein called "hemagglutinin" (HA), and those proteins can grab hold of any "glycosylated protein" (i.e., any protein which has sugar groups attached to its strand of amino acids; roughly half of all animal proteins are in that category) which has a "sialic acid" group (i.e., a specific type of sugar molecule) at the tip of one of the sugar chains that are attached to the protein. Sialic acid groups are commonly used, to terminate the glycosylation process; therefore, they are present on lots of different proteins. That is why influenza viruses can infect so many different body parts, in a specific animal, and why it can infect so many different types of animals.

          And, to make influenza viruses even more difficult and challenging, they are among the fastest-mutating viruses ever discovered.

          And, to make the initial "MALT-targeting pathogen challenge tests" even more of a challenge, the engineered phage particles will be carrying only a single specific influenza antigen (i.e., the FI-6 antigen, as mentioned above). In direct contrast, the flu vaccines that are issued every year (actually, twice a year, for the northern and southern hemispheres), all contain a mixture of multiple different particles, carrying multiple antigens, created by different manufacturing batches.

          All those factors make it extremely difficult to create truly effective vaccines against influenza; so, it may turn out to be a mistake, to choose influenza as the pathogen that will be used in the very first round of pathogen challenge tests, to evaluate the efficacy of MALT-targeting vaccines; so, if it turns out to be a bad choice, subsequent tests will use a different antigen, from a different pathogen which infects animals via a more conventional pathway. However, if the decision to choose influenza for the first pathogen challenge tests turns out to be a good decision, it will open more doors, and lay a better foundation for future work, more quickly, and more convincingly. So, time will tell.

           To provide a bit more information to help readers understand T7 phages, they have a surface protein which appears in two different forms, which are called the 10A form (which appears in about 400 copies/particle), and the 10B form (which appears in about 40 copies/particle). The somewhat longer 10B form is created when the "translation" process (which occurs when a ribosome "reads" the codons on a strand of mRNA, and uses those codons as "instructions" to add another amino acid, as specified by each successive codon) "crashes through" a first stop codon, and continues translating a longer protein (the 10B version), until it reaches a second stop codon. ​Therefore the FI-6 antigen sequence was positioned in the 10A segment (to provide about 400 copies of that antigen, per particle), and the MALT-targeting sequence was positioned in the 10B segment (to provide only about 40 copies/particle).

          Additional details concerning the "genetic cassette" design of those T7 phage constructs will be disclosed in a patent application, rather than in this website. For now, it should be noted that the phrase, "genetic cassette," is used to indicate that a certain type of plasmid or phage construct has been designed and assembled in a certain way, to make it simple and easy to delete a specific DNA sequence, and replace that deleted sequence with a new and different sequence. Therefore, the "cassettes" that are being created, to create MALT-targeting vaccine particles, have been specifically designed to make it easy to "swap out" the antigen sequence, or the MALT-targeting sequence, and to either swap out, or delete, the selectable marker gene.

          As a result, by using those cassettes as a starting reagent, we can provide custom-assembled MALT-targeting T7 phages, carrying any antigen sequence that a qualified requesting company or research group specifies, at a low cost, provided that the requesting company or group makes a firm commitment to actually test those particles, in pathogen challenge tests, in at least one type of animal. More information on that offer is available, HERE.

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