As mentioned on the prior page, the three "top performing" MALT-targeting sequences, from the Round 2 screening tests (or, more precisely, the top three performers which did not contain cysteine residues, to avoid unwanted complications) were selected for inclusion in a "tandem-triple" MALT-targeting sequence.

          Since we were already accustomed to working with Inovirus phages, and since they are small and easy to engineer, we hired a contract company to assemble a set of "first testable phage constructs" suited for use in "antibody production tests", containing both:

          (i) the "three-in-tandem" MALT targeting sequence, at the outer tips of all 5 copies of the long tentacle-like cp3 proteins on each particle; and,

          (ii) a well-known antigen sequence (the "HA-tag epitope", which first appeared in a troublesome influenza strain about 60 years ago). It is widely used for testing, largely because monoclonal antibodies that will bind to it are readily available, at reasonably low "mass-manufactured" costs.

          The HA-tag antigen sequence was placed in some, but not all, of the small brick-like cp8 proteins that are packed together to assemble the cylindrical capsid which encloses the phage DNA, in Inovirus phages. It was discovered, in the 1970s, that if a peptide sequence longer than about 6 amino acids was added to all of the cp8 proteins, the resulting viruses would have severe difficulty in assembling (or "packaging") themselves. Therefore, scientists began inserting a second engineered cp8 gene into the phage genome, controlled by a relatively weak or inducible gene promoter, so that a longer foreign peptide can be inserted into several hundred copies of the cp8 proteins, randomly distributed among nearly 3000 copies of the unmodified ("wild-type") protein.

          Those constructs were tested in both mice, and pigs, and in both types of animals, testing via both ELISA, and SDS-PAGE/Western, clearly showed that a single nasal infusion of those phage particles, with no adjuvants, and no booster dosages, triggered "robust" formation of both secreted IgA dimers, in saliva, and internal IgG antibodies, in blood serum.

          During the lead-up to those tests, several challenges were encountered with the Inovirus phage constructs, including a severely time-wasting episode of instability. When we looked into that problem, we learned that researchers have known, for decades, that the classic "fd-tet" construct – which contains not just a simple tetracycline resistance gene, but an entire "tetracycline resistance complex" which is self-regulating, and which is not expressed unless tetracycline is present – is inherently unstable, for not just one but two reasons:

          (i) the tetracycline resistance complex was inserted into the "long inter-gene region" of the starting-point fd phages; and, Inovirus phages have a natural ability to spontaneously delete any foreign DNA which has been inserted into that region; and, any phages which happen to delete any foreign DNA from that region can reproduce more rapidly than phages carrying that "unwanted baggage", and will soon overrun any subsequent batches of phages grown from those earlier batches; and,

          (ii) the "tetracycline resistance complex" came from a transposon, and transposons (which often are called "jumping genes") are known to spontaneously jump from one genome, to another.

          Therefore, when the first constructs designed to carry a currently-active and important antigen sequence were being planned, the initial plan was to shift over to a different Inovirus construct, using two important modifications developed by the Jonathan Gershoni group, as described in Enshell-Seijffers et al, "The rational design of a 'type 88' genetically stable peptide display vector ..." Nucleic Acids Res. 29(10): E50 (2001).

          However, questions and concerns began to arise over how quickly, reliably, and consistently a "filamentous" phage can be taken in, by an immune cell. If scaled up to a 1/8-inch thickness (i.e., comparable to a strand of cooked spaghetti), an Inovirus phage would be nearly 2 feet long; and, while any human can happily imagine slurping in a strand of cooked spaghetti that long, if it is coated in butter and a tasty sauce, our ability to accomplish that feat depends heavily on having a tongue, teeth, and a pre-existing, long, generally tubular, and happily-receptive digestive system. However, immune cells have none of those things; instead, they need to form a special pouch, which will become a phagosomal bubble, from the same membrane material which makes up their outer membrane, any time they take in a particle. Therefore, simple logic suggests that a flexible filamentous phage will end up being "wadded up", in some haphazard rather than controlled way, as it gets stuffed into a phagosome that is trying to remain spherical.

          To avoid that problem, it was decided to shift over to using a "lytic" phage (i.e., a phage with a roughly spherical "head" component, which can be grabbed and pulled in quickly and conveniently, by an immune cell, in a manner comparable to a hand grabbing a nugget, or a gem), as the starting point, when plans began to firm up for creating the first phage constructs that would be suited for the first "pathogen challenge tests".

          That work is described on the next page.

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