Michael Fischbach
@mfgrp.bsky.social
Liu (Liao) Family Professor of Bioengineering, ChEM-H @Stanford.
Major distinguishing feature: it is *pre-emptive*. The host reaches outside, grabs bacteria (or bacterial fragments), brings them inside, & develops an immune response against them. In a very real sense, the host is *vaccinating itself* against the colonist. 41/55
December 11, 2024 at 4:30 PM
Major distinguishing feature: it is *pre-emptive*. The host reaches outside, grabs bacteria (or bacterial fragments), brings them inside, & develops an immune response against them. In a very real sense, the host is *vaccinating itself* against the colonist. 41/55
OK, so what did we learn? Let's start w/ the initial question: What does the immune system intend to do when it sees a colonist? Don't want to over-generalize, but at least for S. epi, the immune response is much more pathogen-like that we had realized. 40/55
December 11, 2024 at 4:30 PM
OK, so what did we learn? Let's start w/ the initial question: What does the immune system intend to do when it sees a colonist? Don't want to over-generalize, but at least for S. epi, the immune response is much more pathogen-like that we had realized. 40/55
Aap-sc-TTFC & the conventional vaccine both elicit a potent response in the bloodstream. However, when we looked in the pulmonary & nasal mucosa, we found that Aap-sc-TTFC had elicited a strong IgA response; the conventional nanoparticle vaccine had not. 39/55
December 11, 2024 at 4:30 PM
Aap-sc-TTFC & the conventional vaccine both elicit a potent response in the bloodstream. However, when we looked in the pulmonary & nasal mucosa, we found that Aap-sc-TTFC had elicited a strong IgA response; the conventional nanoparticle vaccine had not. 39/55
To our surprise, Aap-sc-TTFC elicited a massive response - even stronger than the original genetically encoded (Aap-TTFC) strain, and almost as potent as the hottest conventional vaccine we could find. Titers from engineered S. epi are stronger than a typical mRNA vaccine. 36/55
December 11, 2024 at 4:30 PM
To our surprise, Aap-sc-TTFC elicited a massive response - even stronger than the original genetically encoded (Aap-TTFC) strain, and almost as potent as the hottest conventional vaccine we could find. Titers from engineered S. epi are stronger than a typical mRNA vaccine. 36/55
Djenet engineered S. epi to express a variant of Aap that displays SpyCatcher. When she incubated these cells with recombinant GFP-SpyTag, not only were the cells labeled but the copy number was very high (we estimate ~40,000-50,000/cell). 32/55
December 11, 2024 at 4:30 PM
Djenet engineered S. epi to express a variant of Aap that displays SpyCatcher. When she incubated these cells with recombinant GFP-SpyTag, not only were the cells labeled but the copy number was very high (we estimate ~40,000-50,000/cell). 32/55
Christopher had a clever idea. He had been making nanoparticle vaccines in which the particle bears a SpyCatcher domain, the immunogen is SpyTagged, and the two 'click' together spontaneously. Why not express SpyCatcher on the surface of S. epi? 31/55
December 11, 2024 at 4:30 PM
Christopher had a clever idea. He had been making nanoparticle vaccines in which the particle bears a SpyCatcher domain, the immunogen is SpyTagged, and the two 'click' together spontaneously. Why not express SpyCatcher on the surface of S. epi? 31/55
Djenet (wisely) ignored my advice and decided to do it anyway. Having received no help from me, she did what everyone in my lab does when they run into trouble: walked downstairs to consult @cobarnes27.bsky.social. 30/55
December 11, 2024 at 4:30 PM
Djenet (wisely) ignored my advice and decided to do it anyway. Having received no help from me, she did what everyone in my lab does when they run into trouble: walked downstairs to consult @cobarnes27.bsky.social. 30/55
She proposed an alternative: make the immunogen in mammalian cells and then attach it (after the fact) to S. epi. I dismissed the idea, arguing that the bacterial cells would divide, the immunogen would dilute, and the antibody response would be weak. Seemed inelegant. 29/55
December 11, 2024 at 4:30 PM
She proposed an alternative: make the immunogen in mammalian cells and then attach it (after the fact) to S. epi. I dismissed the idea, arguing that the bacterial cells would divide, the immunogen would dilute, and the antibody response would be weak. Seemed inelegant. 29/55
...which were (more than) sufficient to protect against a lethal challenge by tetanus toxin. 26/55
December 11, 2024 at 4:30 PM
...which were (more than) sufficient to protect against a lethal challenge by tetanus toxin. 26/55
When she colonized mice with an engineered strain of S. epi displaying TTFC within Aap, the mice developed very high titers of antibody against TTFC... 25/55
December 11, 2024 at 4:30 PM
When she colonized mice with an engineered strain of S. epi displaying TTFC within Aap, the mice developed very high titers of antibody against TTFC... 25/55
She tried a few different strategies. The one that worked best was to replace the parallel beta-helix domain with our model antigen, a C-terminal fragment from tetanus toxin (TTFC) that is non-toxic but can elicit immunity against tetanus. 24/55
December 11, 2024 at 4:30 PM
She tried a few different strategies. The one that worked best was to replace the parallel beta-helix domain with our model antigen, a C-terminal fragment from tetanus toxin (TTFC) that is non-toxic but can elicit immunity against tetanus. 24/55
Having seen that the antibody response targets a giant cell surface protein, Djenet wondered: what if we express a non-native immunogen on the cell surface? Will the mice develop immunity against the new antigen? 23/55
December 11, 2024 at 4:30 PM
Having seen that the antibody response targets a giant cell surface protein, Djenet wondered: what if we express a non-native immunogen on the cell surface? Will the mice develop immunity against the new antigen? 23/55
It consists entirely of repeat sequences. The B domain (& blue N-terminal domain) are direct amino acid repeats, while the beta-solenoid is a structural repeat. All of these presumably fold cooperatively after snaking through the secretion pore. 21/55
December 11, 2024 at 4:30 PM
It consists entirely of repeat sequences. The B domain (& blue N-terminal domain) are direct amino acid repeats, while the beta-solenoid is a structural repeat. All of these presumably fold cooperatively after snaking through the secretion pore. 21/55
Aap is a giant (140 kDa) tree-like protein whose rigid 'trunk' (the B domain) sticks straight up through the peptidoglycan meshwork, displaying a parallel beta-helix domain that extends above the arbor on the bacterial surface. 20/55
December 11, 2024 at 4:30 PM
Aap is a giant (140 kDa) tree-like protein whose rigid 'trunk' (the B domain) sticks straight up through the peptidoglycan meshwork, displaying a parallel beta-helix domain that extends above the arbor on the bacterial surface. 20/55
There are only 9 predicted sortase substrates in the two S. epi isolates we used. Djenet narrowed it down to one that is the predominant (though not exclusive) target of the antibody response: a truly bizarre protein named Aap. 19/55
December 11, 2024 at 4:30 PM
There are only 9 predicted sortase substrates in the two S. epi isolates we used. Djenet narrowed it down to one that is the predominant (though not exclusive) target of the antibody response: a truly bizarre protein named Aap. 19/55
Indeed there was: a mutant missing the gene that encodes sortase, the enzyme responsible for attaching proteins to the cell wall of Gram-positive bacteria. This made things easy - sortase substrates can be ID'd computationally by their C-terminal LPXTG tag. 18/55
December 11, 2024 at 4:30 PM
Indeed there was: a mutant missing the gene that encodes sortase, the enzyme responsible for attaching proteins to the cell wall of Gram-positive bacteria. This made things easy - sortase substrates can be ID'd computationally by their C-terminal LPXTG tag. 18/55
She took a series of S. epi mutants, each one missing a conserved structure on the cell surface. By incubating them with serum from a mouse colonized with the wild-type strain, she asked: is there a mutant to which the antibodies no longer bind? 17/55
December 11, 2024 at 4:30 PM
She took a series of S. epi mutants, each one missing a conserved structure on the cell surface. By incubating them with serum from a mouse colonized with the wild-type strain, she asked: is there a mutant to which the antibodies no longer bind? 17/55
Next, Djenet wanted to figure out: what molecule on the bacterial cell surface is the target of the antibody response? 16/55
December 11, 2024 at 4:30 PM
Next, Djenet wanted to figure out: what molecule on the bacterial cell surface is the target of the antibody response? 16/55
Our paper went in a different direction. First, Djenet asked whether the antibody response to S. epi is conserved in humans. She found that serum samples from healthy adults harbor very high levels of IgG against common strains of S. epi. More on this below. 15/55
December 11, 2024 at 4:30 PM
Our paper went in a different direction. First, Djenet asked whether the antibody response to S. epi is conserved in humans. She found that serum samples from healthy adults harbor very high levels of IgG against common strains of S. epi. More on this below. 15/55
They report that S. epi induces a tertiary lymphoid structure near the site of colonization; antibodies are made in this structure and in conventional germinal centers in the draining lymph node. Langerhans cells are required for all of this to happen... 13/55
December 11, 2024 at 4:29 PM
They report that S. epi induces a tertiary lymphoid structure near the site of colonization; antibodies are made in this structure and in conventional germinal centers in the draining lymph node. Langerhans cells are required for all of this to happen... 13/55
Shortly thereafter, we learned that Inta Gribonika in the Belkaid lab had made a similar observation (neither of us realized we were both working on it!). Yasmine is one of my dearest collaborators; we coordinated our efforts from that point forward. 12/55
December 11, 2024 at 4:29 PM
Shortly thereafter, we learned that Inta Gribonika in the Belkaid lab had made a similar observation (neither of us realized we were both working on it!). Yasmine is one of my dearest collaborators; we coordinated our efforts from that point forward. 12/55
Unlike a normal antibody response following vaccination - which rises after the prime, falls back down, and rises again after the boost - the antibody response to S. epi keeps rising and rising until ~6 weeks... and then stays there indefinitely. 11/55
December 11, 2024 at 4:29 PM
Unlike a normal antibody response following vaccination - which rises after the prime, falls back down, and rises again after the boost - the antibody response to S. epi keeps rising and rising until ~6 weeks... and then stays there indefinitely. 11/55
The experiment was very simple. We didn't prep the mice in any way; Djenet dipped a Q-tip in a bacterial culture and gently swabbed the head of the mouse. The result was striking: not only is there a systemic antibody response, but it is extremely potent. 10/55
December 11, 2024 at 4:29 PM
The experiment was very simple. We didn't prep the mice in any way; Djenet dipped a Q-tip in a bacterial culture and gently swabbed the head of the mouse. The result was striking: not only is there a systemic antibody response, but it is extremely potent. 10/55
So she decided to test whether, in addition to T cells, there was a B cell response as well. 9/55
December 11, 2024 at 4:29 PM
So she decided to test whether, in addition to T cells, there was a B cell response as well. 9/55
Djenet grew suspicious that the immune response against this (generally harmless) commensal might be more aggressive than we had realized. When the adaptive immune system means business, both arms are typically engaged. 8/55
December 11, 2024 at 4:29 PM
Djenet grew suspicious that the immune response against this (generally harmless) commensal might be more aggressive than we had realized. When the adaptive immune system means business, both arms are typically engaged. 8/55