Structural and functional characterization of two group 2 H3 HA stem antibodies, 2F02 and AG2-G02, shows distinct non-overlapping epitopes, protection in mice, and antigenic changes driven by HA2 position 32 that limit AG2-G02 binding
0:00Welcome to Base by Base, the papercast that brings genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. You know, if there's one thing that unites almost everyone in frustration year after year.
0:13It has to be the flu shot. Oh, absolutely. We all line up, we get the jab. And then a few months later, you hear about a new variant and realize we're already behind. Feels like we're just constantly chasing a ghost.
0:24We are. And that chase really stems from the fundamental nature of the influenza virus itself. I mean, it is constantly mutating its primary target, the hemoglutenin head domain or HA head. And that's the part that our vaccines are designed to hit.
0:40Exactly. That head is what latches onto our cells and because it changes so fast. Our seasonal vaccines are always, you know, playing catch up. It's an immune arms race we're kind of set up to lose. But the big dream, the holy grail, has always been the universal flu vaccine, right?
0:56A single shot that could protect you for decades. Right. And that search has, by necessity, shifted its focus away from that super variable head and onto the structural heart of the virus, the HA stem, the stalk.
1:10Because the stem is supposed to be stable. It's essential for the virus to actually get into ourselves. So the thinking was it couldn't change much. It was the fixed target, the Achilles heel. It was supposed to be.
1:20And that's what this gets really interesting because new research suggests that even this highly conserved stem is, in fact, subtly evolving in humans. And this evolution is quiet, it's sneaky, but it's potentially creating these dangerous blind spots in our collective immunity.
1:37And it could fundamentally change the entire search for that universal vaccine. So today we're going to unpack how the flu is quietly escaping our defenses based by a base. And what that really means for our readiness against the next big threat.
1:48Okay, let's get into it. This deep dive is centered on the paper. Characterization of 2 non-competing antibodies to influenza, H3 and 2, HMAG, gluten and scem reveals its evolving antigenicity. And today, we're celebrating the work of a really collaborative international team.
2:05We're talking research for from the University of Illinois, you're about a Champagne, the Chinese University of Hong Kong, and Guangdong Women and Children Hospital. A huge effort. A huge effort. And they've really pushed our understanding forward on this idea of HA STEM antigenicity, especially for the group 2 influenza strains.
2:23So let's ground this in, you know, what this means for global health. Seasonal flu, especially H1N1 and the infamous H3N2. They cause 1000000s of severe cases every year. And H3 and 2 is a real troublemaker.
2:36It evolves so quickly, you get new variants popping up every 3 to 5 years, and that's what leads to those dreaded vaccine mismatches. Right. And beyond just the seasonal flu, there's always the threat of a zoonotic spillover, a bird flu jumping to humans.
2:49That's the big one. Human populations just don't have strong pre-existing immunity to most of those avian H3 strains. And we've seen them causing severe infections, even deaths in people. So understanding our defenses against those is, well, it's paramount.
3:04So let's look at the virus' architecture. It's all about that hemogluten and protein, HA. Remind us why the stem part, the HA2, is so much more stable than the head, the HA1. Well, the structure itself really dictates its stability.
3:18You see, HA is a trimer, but it works in 2 parts. You've got the HA1, which forms this big exposed head that binds to ourselves. And that's the part that's under constant attacks from our immune system.
3:28Exactly. So it has to mutate constantly to survive. But the HA2, the stem is tucked down closer to the virus's membrane. It holds all the fusion machinery. The tools it uses to physically break into ourselves.
3:41The very tools. And because that job is so fundamental, its sequence can't change very much or the whole virus just stops working. And that's what makes it the ideal target for what we call broadly neutralizing antibodies or beanabs.
3:54Okay, so the stem is the target. But I understand, we split these HA subtypes into group one and group two. We do. And antibodies against the group one stem are pretty well studied, but group two, which includes that problematic H3, is much less explored.
4:09And that's where this study really zoned in. And within that group 2 stem, it's not just one target. There are actually two key spots for antibodies to hit, right? That's right. You have the central stem epitope, which is pretty well conserved across both groups.
4:22And then a bit lower down. You have the lower stem epitope closer to the membrane, which is usually hit by group 2 specific antibodies. So the thinking is, if you can hit both of those spots at the same time.
4:33You'd get an immune response with incredible breadth and power. So the goal here was to characterize 2 brand new group 2 specific antibodies, AG2, G02, and 2 FO2, to see if they could be that perfect non-competing duo for a future vaccine.
4:50A one, two, punch. A one-two punch. But 1st they had to prove they weren't, you know, tripping over each other. That sounds like some serious molecular detective work. How did they even begin to confirm what these antibodies were doing at the atomic level?
5:02Well, it started with a massive search. High throughput screening and machine learning to even find AG2, G02, and 2FO2 in the 1st place from vaccinated people. Then comes the 1st big test. The biolayer interferometry competition assay, a BLI essay.
5:17And that basically tells you if they're fighting for the same parking spot. That's a great way to put it. It tells you if one binds, can the other one still get on? And the initial results were, well, they were incredibly promising.
5:29It suggested they had nearby but non-overlapping epitapes. They weren't fighting. So to actually see this, to get a picture of it. They had to bring out the big guns. Oh, yeah. They turn to cryogenic electromicroscopy, cryo EM.
5:44This technique lets you freeze the molecules in place and take these incredibly high resolution pictures of exactly how the antibodies are physically docking onto the HA stem. And what about proving it works in a real living system, not just in a dish?
5:59Right, the crucial step. So they ran the standard binding and neutralization assays, but the real test was the Invivo challenge. They gave the antibodies to mice and then hit them with a lethal dose of influenza.
6:10And if the antibodies work, the mice live. Simple as that. But perhaps the most insightful part of all this was the cerological analysis. Looking at actual human plasma. From different time periods. Exactly.
6:21They took samples from adults, pre 2003, adults post 2011, and even infants post 2011. And this let them map how our population's immune memory might have silently shifted over decades. It's like taking an immune census.
6:35Okay, so let's get into the results. We know they don't compete, but what did that CryoEM picture actually show? Where do they stick? It was the definitive blueprint. 2FO2 targets that central stem epitope.
6:47The classic well-known spot. The classic spot. Meanwhile, AG2G02 targets the lower stem epitoop, that region down near the membrane. It confirmed they had completely distinct territories. And here's the aha moment for the vaccine strategy, right?
7:00This is it. The low resolution cryo EM showed that you could literally stick 3 copies of AG2G02 and 3 copies of 2FO2 onto a single HA trimer at the same time. Six antibodies on one target. That is maximum coverage.
7:14It is. And their binding mechanics were just, you know, perfectly optimized for this. They both used a specific part of the antibody, the CDR H3 region, but in totally different ways. One might use it for stacking, the other uses it for a sort of chemical grip, same tool, different jobs.
7:29So structurally, they're a dream team. And functionally against the older flu screens, they worked. They protected the mice. Yeah. But this is where the story takes a turn. A very dramatic turn. Did they work against the flu that's circulating right now?
7:42And that's the critical failure point. AG2, GO2, the one that hits the lower stem. It failed. It completely failed to bind or neutralize recent human H3 and 2 strains, specifically the 8 or winds 62021 strain.
7:57So one half of the dream team was suddenly locked out. Locked out completely. The other antibody, 2FO2, still bound to everything, but that lower stem target had just vanished from the modern virus. I can imagine the alarm bells going off.
8:09You have this supposedly conserved region and your key weapon for it is useless. What was the culprit? What did the virus do? So they traced it back. And it was an incredibly subtle change. One single amino acid swap at position 32 on the HA2 protein.
8:24Position 32. Right in the middle of where AG2 GO2 was supposed to bind. And they tracked its history. The old H3 strains from 1968 had a small amino acid there, 309. T for 309. Okay. Right. Around 2004, that got replaced by Isolucine, AG2, G02 could still handle that.
8:43But then around 2008, the isolucino was replaced by a much, much larger and positively charged amino acid. Arginine, R 32 HA2. And that arginine is what's in all the recent strains. It is. So the virus essentially bolted a massive brick onto the side of the protein, right where the antibody needed to talk.
9:01It's a physical block. It's a perfect metaphor, the structural model and confirmed it. That bulky arginine creates what we call a severe steric clash. The antibody physically cannot bind anymore. It proves the lower stem is evolving.
9:12It's not perfectly conserved. But wait, hold on. If that stem is so critical for the virus to function, How could it just stick this huge arginine molecule there without, you know, breaking itself? Not a major fitness cost.
9:23And that is the really key, and frankly, scary question. The virus found a way to accommodate it, which means the pressure from our STEM antibodies isn't strong enough to stop evolution at that site. The stem is more flexible, more adaptable than we ever gave it credit for. Before we get to the big implications, there was another detail about how these antibodies protect, right?
9:45It wasn't just about blocking the virus. That's crucial. The study showed that for real protection in the mice, you need what are called FC mediated effector functions. Meaning the antibody does more than just neutralize.
9:56Right. It's not enough to just block the virus from getting in. The tail of the antibody, the FSC region also has to act like a flag to the rest of the immune system. It has to signal killer cells to come in and clear out the infected cells.
10:09So it's the difference between putting up a roadblock and calling in an air strike? Exactly. When the engineered the antibodies to disable that signaling function, the mice did much worse. Survival was down, viral loads were up.
10:21It tells us that for a truly great universal vaccine, you need antibodies that can do both, neutralize and signal. Okay, so connecting this all to the big picture. The implications here for universal vaccine are huge.
10:35They are. On one hand, we now know that combining antibodies that hit the central stem, like 2FO2 and the lower stem, like AG2G02, is the right strategy. It gives you breath and power. And the fact that these 2 don't compete is the design win.
10:50You can target both sites at once without them interfering with each other. Precisely. This study gives us the atomic blueprint for that, but the shadow hanging over it is that evolution at position 32.
11:01It proves the lower stem is not the fixed target we thought it was. The virus found a loophole. It did, and that subtle molecular change has massive real world consequences, which they saw in that population immunity analysis, the results from the human plasma were genuinely startling.
11:16So tell us what they found when they compared the older generation's immunity to the younger generations. So the pre 2003 adults? The ones exposed to that original T 32 HA2 strain. They had great binding against that old virus, but their immunity was much weaker against the modern R 32 HA 2 variant.
11:33Their immune system was imprinted on the old version, and the infants. The post 2011 infants, who have only ever seen the modern R 32 HA2 version, showed the exact opposite. They have strong binding to the modern virus, but the trade-off is, they've lost that robust immunity that older adults have against the ancestral form.
11:52That sounds like a ticking time bomb for public health. We are collectively forgetting how to fight the original versions of the virus. And that's the long-term risk because that ancestral T32 HA 2 variant is still carried by most avian H3 strains.
12:07Bird flu. Bird flu. So as the modern R 32 HA 2 version keeps circulating in humans, our population level immunity against the form found in birds is just eroding generation by generation. So the older generations have this baseline defense against bird flute, just from their life experience.
12:24But today's kids are completely unprimed for that threat. They're vulnerable if one of those avian viruses makes a jump. And that could dramatically increase the risk from the next zoonotic H3 spillover.
12:34The virus didn't have to change its head to do this. It just made one quiet little change to its supposedly conserved stem, and slowly silently eroded our foundational immunity. So let's wrap this up with the core takeaways.
12:48What are the key things to remember from this deep dive? I think there are 2 major things. First, the study characterized 2 critical non-competing antibotors, giving us a definitive roadmap that proves a good universal vaccine should target both the central and the lower stem at the same time.
13:03But just as critically, it exposed a huge vulnerability in that dream. Natural evolution at position HA 232 has changed the lower stem, weakening our protection against recent strains. And maybe more importantly, wiping out our populations immune memory of the ancestral avian forms of the virus.
13:21So going, given that the younger generations are losing this crucial immunity, what strategies should global public health bodies be looking at right now to preemptively boost our defenses against the inevitable next H3 zoonotic spillover?
13:35I think that's a question we need to answer long before the next pandemic arrives. This episode was based on an open access article under the CCBY 4.0 license. You can find a direct link to the paper and the license in our episode description.
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