Precision-Guided (MALT-Targeting) Mucosal Vaccines

          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 is a research professor at a veterinary college. He 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 was 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). However, recent analyses suggest that a different sequence, which starts with KSTQ rather than KESTQ, may now predominate over the KESTQ sequence, especially among the H1 and H2 groups of influenza viruses. So, that has become one point of concern, among several.

          Another 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 receptor proteins; the spike proteins on COVID viruses bind to a surface protein called “angiotensin-converting enzyme-2” (ACE2); and most “rhinoviruses” (which cause “common colds”) bind to a cell protein called “intercellular adhesion molecule-1” (ICAM-1).

          However, influenza viruses use a very different mechanism. Each influenza particle carries hundreds of 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 and organs, in any specific animal, and why it can infect so many different types of animals.

          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 different antigens, created by mixing together an assortment of different vaccine particles, created by separate manufacturing batches.

          And, as yet another factor which makes influenza viruses even more difficult and challenging, they are among the fastest-mutating viruses ever discovered.

          Those and other factors make it extremely difficult to create truly effective vaccines against influenza; so, we may have made a mistake, by choosing influenza as the pathogen that will be used in the very first round of pathogen challenge tests, and 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 specific-receptor 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, only time will tell.

           To provide 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 longer 10B form is created roughly 10% of the time, when  “protein translation” (i.e., when a ribosome “reads” the codons on a strand of mRNA, and uses each codon as the instruction to select a single specific amino acid, and add it to the growing strand of protein) “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).

          Another advantage of using T7 phages is that their 10A capsid proteins can carry large and long add-on peptides, with optimal exposure and accessibility. Whenever proteins are created, the end with the “N-terminus” is created first, and in T7 phages, that terminus is the end which fits into the capsid, in a manner which causes each 10A protein to “fit in” with, and adhere to, its surrounding proteins (this is comparable to pressing LEGO blocks together, to form a capsid shell structure that will hold together without requiring any additional adhesive to hold the proteins together). That allows relatively long “add-on” sequences (such as an antigen sequence derived from a pathogen) to be added to the carboxy ends of engineered/modified 10A capsid proteins, since those “carboxy-end add-ons” will protrude in an outward direction, and will not interfere with “packaging” of the capsid shell. As a result, engineered T7 phages can carry antigen add-ons having more than 100 amino acids, and lengths of 200 AA's appear to be achievable (however, extra-long lengths will slow down the replication of the phage particles).

          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 replace either one with any other selected sequence, and to either swap out, or delete, the selectable marker gene. It's also worth noting that the selectable marker gene does not involve any form of antibiotic resistance, which would raise an additional set of concerns and objections. Instead, the selection gene encodes an enzyme called "thioredoxin" (abbreviated as trx), which de-toxifies a class of metabolites that become toxic, if they accumulate to unhealthy quantities. Therefore, colonies of trx-deficient cells can begin growing, but they will not grow larger than pinhead sizes, unless they contain a functioning plasmid or phage which can supply functioning trx enzymes.  

          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;  and, we hereby commit to a $4000 price, for each of the first ten T7 phage constructs we create and sell to qualified research teams. We will see how that goes, and adjust the price, if necessary, after we reach that milestone.

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