TWiV 1367: Turning Off Hepatitis B Virus Without Cutting its DNA

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0:00This Week in Virology, the podcast about viruses, the kind that make you sick.

0:10From Microbe TV, this is TWIV, This Week in Virology, episode 1367, recorded on October 16th, 2026. I'm Vincent Draconello, and you're listening to the podcast all about viruses. Joining me today from Austin, Texas, Rich Condit. Hi there. It's been a while. It has been. It's been so long that I am slow in getting up the weather report, but I can tell you that

0:42we got a cold front coming through, so it's like, here it is, 67 degrees, which is probably today's high, and it's going down to something like 54 overnight. So we're finally, I think, out of the worktop. And it's really windy. And tonight, my grandson, who plays in the high school band, I'm going to go to their performance, and they have a color guard that Harper used to be in, she's now off to college, that they, their big deal, right, is waving flags and tossing

1:21them in the air and that kind of stuff. And they got like 25 mile an hour winds tonight. So they're going to have a challenge. Oh yeah, that's going to be hard. Yeah. That's going to be hard. They'll have to be picking them up. Yeah. I'm sure they got contingency plans. Yes. We'll modify. Here it's 22 and cloudy. It's been pretty mild lately after a little bit of a cold spell. Also joining me today from Madison, New Jersey, Brian Barker. Hi. It's great to be here. It is also 22 Celsius, 72 Fahrenheit here, which may be the only times

1:55ever that it's been warmer in Madison than in Austin. Very cloudy, but a good day to do a podcast. And from Ann Arbor, Michigan, I was going to say Madison again, from Ann Arbor, Michigan, Kathy Spendler. Hi, everybody. Here it's 56 Fahrenheit, which is 13 Celsius. That's a little bit windy, drizzly right now, and so the sky is definitely gray. Actually, all three of you I haven't seen in a bit. Yeah. I was just thinking it's great to be back and see all you guys again.

2:28I have seen Alan. I have seen Angela. And then a couple of weeks ago, Brianne was the last one. Kathy, you're still in your office. Yes. Rich and Kathy have been AWOL. Yeah, I've been AWOL. It's true. Good to see you back, Rich. Absolutely. Good to be back. Actually, it's not true. You can do whatever you want. There's no such AWOL on TWIV. That's why we have like six people, right? So eventually we'll get two or three. It all works out. It all works out. And now, you know, on TWIN, we're now down to two people.

2:58Is that right? So last year, Jason Shepard left, and now Vivian decided to leave after five and a half years. How often does TWIN drop? It's once a month. But it's, I mean, I want to get, I always wanted to get another senior person to replace Jason, and I have someone in mind. So I will pursue that. But in the meantime, it's, Tim is very good. He knows what he's talking about, and he likes explaining

3:30things. He's very clear. So we had Jason back actually to visit a couple of weeks ago. He had a very interesting new paper in, I forgot what journal, but it's about his favorite protein, ARC, which is a retroviral gag protein that forms capsids. And he thinks it's involved in neurotransmission. And it looks like it's ferrying tau from neuron to neuron now, right? So phosphorylated tau moves around, right? And that's bad. That's what you see in Alzheimer's and so forth. So

Remembering Helen Branswell

4:02that was interesting to have him back. Okay. The news this week is Helen Branswell has passed away, 69 years old. She's most recently was writing for Stat, and she was always a good writer, clarity, and got her facts right and so forth. And I always wondered what she started out as. And as I'm reading her column here, and yeah, she was a literature major and then started doing journalism and then got interested in science. It appealed to her and became a science writer. But she was very good. She understood things and wrote clearly. So we're going to lose a

4:41good science writer. Yeah, the testimonies were good because they all commented on how they looked forward to the questions that she had. She was well prepared and she asked the key questions. Yes. Yes. And there's a line here that says she could be thorny or something. What word did they use? Something like that. She could be tough. Okay. And I felt that a couple of times when I emailed her. She could be really tough, but that's okay.

5:11I confess I wasn't really familiar with her. I probably read some of her stuff, but it wasn't something that stuck in my head, but this article is great and really paints a fine portrait of this person. I always felt like her articles stood out. Yeah, they were good. Where is this quote about her? All right. It doesn't really matter. She had a big heart. No, it was just before that. Oh, by her own admission, she was a bit gruff, right? Yes. Okay. All right. Now we have some papers for

Influenza A and pregnancy study

5:49you. Two papers. And the first is in Science Advances. And the title is Influenza A virus infection induces immune dysregulation in the placenta and fetus without vertical transmission in non-human primates. We have the first author is Orlando Cervantes. And there are two co-second authors, Sydney Sun and Hazel Huang. And the corresponding author is Christina Adams Waldorf. And they are at

6:27the University of Washington Seattle Children's Hospital, the Washington National Biomedical Research Center and Seattle Children's Research Institute. And Kathy has a summary. Oh, also, at least somebody with a subscript to a couple of them are from, oh, wait, no, no, three, Case Western Reserve School of Medicine. I missed that. Yeah, there it is. Case Western Reserve. And then somebody from Portland. So Pacific Northwest plus Cleveland, Ohio. Okay. Yeah. So I have a summary. So influenza,

7:02influenza is a particular threat for pregnant people, both to the mother and to the child, including things like maternal mortality and stillbirth, and in the child, sometimes neuropsychiatric disease. And so it's important to understand the effects of influenza A, or IAV, as they call it here, on the placenta and the developing fetus. And how systemic disease in the mother coordinates immune changes, immune changes in the placenta and the fetus, and whether there are correlations of

7:35those immune changes with disease severity and fetal development are the focus of this work. So they really want to try and look at those two sort of intermediate things between the mother, well, the placenta and then the fetus itself. And so mice and rats have been used a lot as models, but here they're going to use a non-human primate model, pigtail macaques. And the reason is that it more carefully or better mirrors the gestation in humans and larger mammals. So they have a gestation that's

8:13longer, almost six months long than rodents, which are, you know, three to four weeks. And they have more chances for exposure to flu because of that. And their placenta and labor physiology are more similar to humans than the rodent ones are. So in brief, what they did in this study was they inoculated 11 of these pigtail macaques in the third trimester. And for controls, they had what they call historical controls that had been used as uninfected animals in prior studies. So they

8:46monitored these for up to five days after infection, whether they had sniffles or cough or things like that. And they euthanized them at five days. And then they did a lot of studies of the various tissues. They compare the infected animals in their overall results to the controls. But they do make a point that unexpectedly, two of the infected macaques were stillborn. And that, as I said, was

9:17unexpected. So then they report other interesting findings. There was not vertical transmission, in other words, no transmission of the virus from the mother to the child. So I interpret that as saying that the function of protecting the fetus from infection by the placenta worked in the case of the placenta. And in two cases, however, they did find viral RNA in the placenta, but they didn't get virus into the infant or fetus. And then maternal infection generated a type 1 interferon response in

9:57the placenta. And then they had altered fetal immune cell populations in the fetus, both innate immune cell population responses and adaptive immune cell responses. The maternal lung viral load and interferon alpha were rarely associated with the placental immune perturbations. So they weren't so much linked to the placental immune perturbations. But the maternal viral load and interferon alpha levels

10:30were linked to fetal immune cell populations. So the conclusion is that the maternal influenza A disease severity is associated with dysregulated fetal immunity. And in other words, just like the title says, influenza A virus infection induces immune dysregulation in the placenta and fetus without vertical transmission in non-human primates.

10:57Okay. Now, as you heard, influenza A infections during pregnancy have a risk of maternal mortality and also problems for the baby, like stillbirth, a neuropsychiatric disease. And that's been reported in multiple populations. And this has mostly been studied in mice. And you can see mice and rats, you can see a range of outcomes, preterm birth, low birth weight, stillbirth.

11:28So in these models, you see very good activation of immunity in the maternal compartment. Right. Well, and I think the one thing that comes up here that's really important is that some of the the things that they see in terms of the neuropsychiatric disease in children or some of the other infections are with infections that are not thought to be vertically transmitted. You know, we don't usually think about influenza as something that is vertically transmitted.

11:58And so actually doing the investigation here is important. But some of these previous studies that they cite kind of have this idea or support the idea that perhaps some details of what's going on with mom's immune response during pregnancy is influencing fetal development. And that's going to, when I think about some of the big picture take home messages of this paper,

12:31that becomes really important.

12:35So it's been previously shown that you get vascular inflammation in the mothers, you have placental growth restriction, and fetal brain hypoxia. But very few studies have looked at immune perturbations in the fetus following exposure to maternal influenza A virus. And so in other words, the extent to which systemic maternal disease coordinates the immune changes in the placenta and the fetus are not really well

13:09studied. And so that's what they, yeah. No, I was just gonna say, and the thing I really like here is they're talking about those changes in the placenta and in the fetus, because you could imagine maybe there are changes in the placenta and that's sort of buffering a change and keeping it from getting to the fetus versus the change actually happening in the fetus. And so it's nice to actually say, okay, is the change happening to the placenta, but that is there also a change happening further downstream of the fetus or is the placenta sort of taking the hit?

13:40So what they're looking at here is they want to see the impact of maternal infection. They're using the H1N1 2009 pandemic influenza virus, the impact of maternal infection on the placenta and the fetus. They're using pigtail macaques, macaquea nemestrina, because the gestational period is closer to humans. You know, mice have a really short gestational period, so it's not a long time for the viral

14:12effects to propagate to the placenta and the fetus. So these non-human pionomates have longer gestation. They have similar placental structure and labor physiology as humans. So they had 11 pregnant pigtail macaques. They inoculate them with H1N1 2009 in the third trimester using a combination of intranasal, intratracheal, and intraocular and intraoral roots all at once. They want to make sure

14:43they're infected. And I've seen that before in primates, non-human primates. And the results are compared with a historical control. So they don't actually, they don't have controls in this experiment. They have, but they have a lot of these pigtail macaque studies going on at the institution. So they have other uninfected pregnant pigtail macaques and they use to study. And they have been inoculated with various things or with saline. So that's the control

15:20cohort. And then in the flu cohort, they do a C-section and they necropsy the dam and the fetus at five days after inoculation. So that's the whole experiment, five days, right? They look at clinical signs of illness, of course, during the period of five days. They see coughing, wheezing, increased respiratory effort, nasal discharge, decreased appetite in seven of the 11 animals, 64%. And the four others didn't show any clinical symptoms. It's pretty amazing,

15:53right? They hammered them in all these different ways with virus. And 10 to the 7th PFU, that's a lot. And they still did not, four of them did not develop symptoms. Two animals had the most severe disease and one of them, Blue 9, experienced a stillbirth. All of the mothers showed lung histopathology in the infected group and not in the control group, of course.

16:24Stillbirths were diagnosed at the endpoint in two of 11 animals. So the gestation period is 134 days for, well, for these two stillbirths, 134 and 121 days. It's about 74% of the full-term macaque pregnancy, which is 172 days. And in their six years of data at this Washington National Biomedical Research Center, stillbirth occurred in 2.6% of pregnancies. So having two here is high.

17:05Okay. So they look at the placental histopathology in the two stillbirth cases. Basically, it looks the same as other influenza A virus-infected animals and also some control, some of their control animals. None of the placentas show active inflammation of the chorioamniotic membranes. And basically, they say this is in line with high background rates of marginal placental infarctions and necrotizing villitis in macaque placentas. Even if you don't infect them with influenza A virus,

17:42you see this at a reasonable rate, at some rate. Okay. Virus. So they do RT-PCR to measure influenza viral RNA in the maternal lungs, placenta, and fetal organs. So at five days post-infection, all the flu animals, that's the infected ones, had high levels of viral RNA in their lungs. Maternal plasma is negative for influenza RNA. And in placental chorionic villus tissue,

18:15influenza RNA was detected in only two of the cases, both with low copy numbers. It took fetal tissues from brain, brainstem, spinal cord, meninges, heart lungs, liver, spleen, thymus, all negative for influenza virus RNA in all of the macaques.

18:36So overall, maternal lung, wide distribution of influenza virus, but infrequent detection of viral RNA in placental tissues. So no evidence for congenital infection. So how wide a tissue distribution would you expect in the mom other than lungs? I mean, it's going to be, flu is going to be pretty much confined to the lungs, right? Yeah. That's where it should be. Yeah. Yeah. I remember the 1918 flu paper in cinemologous macaques

19:12had both seasonal flu and 1918 flu, and they saw some broader tissue distribution with 1918, but the seasonal was pretty restricted. And they looked at a lot of tissues in that paper. Now, avian flu would be different. There you'd see some other, and that's been done in many studies. But yeah, and seasonal influenza is pretty much a lung infection. And so people may be asking, why do we find it in wastewater? Because you swallow mucus that has the virus in it,

19:44passes through. I was just thinking that, you know, the absence of RNA in the placenta doesn't necessarily mean that the placenta is doing something special relative to keeping the virus infection away, because the virus is pretty much confined to lungs anyway. Right. Although when it makes it to the placenta, it doesn't seem to make it across. Right. Yes. Yeah. So it's sort of, the placenta seems to be doing its job that way. Right.

20:15Yeah. The placenta did its job. Although, how would it get to the placenta?

20:21Fetal tissues are all negative. I mean, somehow it's, something's happening, but it's not in the blood. So I don't know how else it would get to the placenta. Right? Yeah. I don't know. I mean, this is one infection where you would not expect transplacental transmission, right? Right. It's not viremic, but, you know, Zika, you would, and so forth. Right. It's not Torch. Yeah. It's not Torch. Still. But something's happening in the placenta. We don't know what it is, do we, Mr. Jones? Right. And the whole point of the study is that, you know, in humans, the infection can, in pregnant

20:53women can cause problems. So this is not to take away from the question. So something's happening and you're going to see something's happening in the placenta, but we don't know how it happens. Okay. So then they look at cytokine responses in the placental disc and the amniotic fluid. Chronic villus tissue from the placenta disc of flu animals had greater concentrations of some inflammatory and antiviral cytokines than the controls like

21:23IL-1 beta, IL-18 interferon beta, and others. In the amniotic fluid, many cytokine concentrations were elevated as well. So basically what the result here is that you see perturbations in cytokines associated with maternal influenza infection. And these are compartment specific. And the elevated cytokines in the chorionic villus tissue were not always transported into the amniotic fluid

21:55compartment. So the placenta knows something's going on. Yes, exactly. That's a good way to put it. It knows something's going on. The placenta is responding. Well, we don't know how it's sensing that. Right. It doesn't share its secrets yet with us. We will not learn at the end of this paper either. But something must be circulating that's not virus, right? Potentially, yeah. So, Breanne, what would be a candidate for that? Would it be a cytokine? I mean, my first thought is a cytokine, but, you know, maybe a cytokine, maybe a lipid mediator,

22:31since those would be harder to study and wouldn't show up in this Luminex. I don't know. Yeah, yeah.

22:37Okay. Then they look at immune cell frequencies in the placenta. They do flow cytometry-based immunophenotyping. The classical monocyte was decreased in the placental disc of the flu versus control cohort, and they don't find any relationship between stillbirth and the frequency of immune cell populations. So that doesn't explain the stillbirths. So basically, all right, so that's what they find, a reduction in maternal IV infection.

23:10You get a reduction in classical monocyte frequency there in the placenta. So, then they do a transcriptional profile on the placenta to look at what's altered, right? RNA-seq on the chorionic villus tissues.

23:26I love this terminology. Listen to this, folks. We reported total RNA sequencing. Okay, that's fine, RNA-seq. And validated our results by direct gene counting using a nanostring N-counter assay.

23:40Oh, my gosh. Technology. I don't know what a nanostring N-counter. Do you know what that is, anyone? Nope. A really tiny string that you know. Really tiny string. We're doing physics. I think nanostring is the company's name. We used it in my lab. So, how do you gene count? So, does it go through the data or what? What is it? I'm not sure what they're doing here. We used it for a completely different way. Well, the point is that they're validating the RNA-seq. So, in the placental chorionic villus tissue, 185 genes are upregulated and 178 were downregulated, right?

24:15That's compared to the control group. And what do you see? Upregulated interferon-stimulated genes, like IFIT3, MX1, OAS2. This is in the placentas. Now, these are going up. You also see enrichment of pathways related to the type B1 interferon response. Negative regulation of viral processes like genome replication and viral entry. Okay. So, there are gene changes. And so, again, something's going on, but we don't know.

24:45We don't know why or how.

24:49They also looked at these RNA-seq results to identify pathways differentially expressed by influenza A virus. They got two different clusters of differentially expressed genes. Cluster 1 are downregulated in flu versus control, and cluster 2 are upregulated in flu versus control. And let me just tell you the bottom line here. We don't need to go through all of it. This shows that you get influenza infection of the mom gives you upregulation of antiviral innate immune genes, downregulation of critical cell functions like cell cycle, metabolism, and ubiquitination, which is consistent with an antiviral response.

25:34And it doesn't matter how severe the disease was in the maternal lung. You get this in all of the placentas. Yeah. So, no matter how sick mom is, if she's making an immune response, her placenta changes. Yeah. Yeah. They ask whether placental gene expression correlated with clinical score, lung virus load, or some immunologic correlate of lung disease, weak correlation between maternal lung disease indicators and placental gene expression, weak.

26:10So, the change in the gene expression profile in the placenta is basically turning on an immune response. Yeah, exactly. So, the placenta knows that there's an infection going on. Put it that way. The placenta knows there's an infection going on. And so, given that we don't see vertical transmission, perhaps that is helping to block transmission.

26:41We don't exactly know that. But the placenta seems to be doing something that might be useful. Well, the placenta doesn't necessarily know whether this is an infection that could be systemic and a real threat. Right, exactly. It just knows there's something going on, right? So, it's covering all the bases. Yep. Rich, this is Ballad of a Thin Man. Ballad of a Thin Man? Do you know the Bob Dylan song?

27:07Ooh, boy. I'm going to have to look that up. Okay, I'll tell you the line that I'm thinking about. So, there's something going on, but you don't know what it is, do you, Mr. Jones? Yes, right. Very good. And that's what's happening in the placenta. Right.

27:20Good show title. Ballad of a Thin Man. Ballad of a Thin Man. Fetal immune cell frequencies in the lung, lymph node, and blood. By flow cytometry, they look at fetal lung, lymph nodes, and blood. They find a perturbation of innate immune cells and T cells in the infected versus control cohorts. In the lung of the fetus, the monocyte frequencies, non-classical monocytes, plasmacytoid dendritic

27:52cells, and CD4 T cells are all higher in the flu cohort. Similar pattern in the fetal lymph node. And there are some other immune populations that went higher in the lymph nodes, like natural killer T cells and others, but they say they were not significant. In the whole blood, there is a higher frequency of certain cells as well. Again, not significant. Overall, so basically, the maternal infection perturbs fetal innate and adaptive immune cells

28:26in organs, lymphoid compartments, and blood, even where the virus is not crossing the placenta. Okay. In the absence of vertical transmission. So yes, mom making an immune response changes what's going on in the baby's immune system. Mm-hmm.

28:44Okay. Then they wanted to try and make correlations between the maternal lung disease and fetal immune cell populations. So in the placenta, there weren't many correlations, but there were many correlations between lung disease in the mother and fetal immune cell populations across lungs, lymph nodes, and blood. And so there are positive correlations between maternal disease severity and higher frequencies of fetal T cell populations, dendritic cells, which make a lot of interferon, and maternal lung

29:20type 1 interferon concentrations were positively correlated with CD8 T cell frequencies in the fetal blood and lungs. So it suggests that maternal viral influenza disease is associated with perturbations in these immune cells across multiple compartments. So maybe it's interferon, Brianne. It could be interferon. I mean, what I see when I get to this is the more severe mom's infection is, and it very well could be interferon, but the more severe the infection is, the more change is happening

29:56in the baby's immune response. And to me, this is some of the most important data or one of the most important take-home messages because when I see this paper, honestly, my first thought was sort of like, oh, no, because I can imagine the ways that some people would take this paper in a really bad direction. And I certainly know people who, whenever they see something like this, will say, oh, see, these immune

30:36responses during pregnancy, they're real bad. And somehow they'll take it to a vaccines during pregnancy, real bad type of thing. That would be too much. That's totally different, right? Right. But what I think is really key here is that you can notice that when mom has more severe disease, the baby has a larger impact on immune responses. And one really great way to make sure mom doesn't have severe disease is to vaccinate

31:07her, especially to vaccinate her early. But to vaccinate her at all is going to prevent all of these sort of severe infection outcomes. And so if your biggest issue here is, oh, see, changes in mom's immune responses are so bad. This, I think, says to you, yeah, making sure that mom doesn't have severe infection. That vaccine that can prevent severe infection is going to prevent some of this change in the fetus immune response. Yeah. Yeah. All right.

31:40So those are the data. Basically, maternal influenza infection induces a cascade of immune defenses, including interferons. And this somehow makes the placenta transition into a defensive state. So you shield the fetus from infection, but you may get some immune injury to the placenta and fecesis and even stillbirth. When the mom is infected. When the mom's infected, yeah. So they say here, we don't find vertical transmission.

32:12So our data suggests that immunologic rather than virologic signals are responsible for profound changes in the fetal immune landscape, which is what we've been talking about, right? Yeah. I highlighted one. Maybe you did the same thing. Yeah. Vertical. Yeah. I highlighted the same phrase you did. So this paper makes me think of Carolyn Coyne. Yes. And I would love to hear from her on this because she could tell us all about placental biology

32:46and the barrier and pathogen response. But, I mean, the fetal blood system and the maternal blood system are separate, right? But there is diffusion, at least of small molecules, across from maternal to fetal blood and the other way around, waste products. So there is an opportunity to exchange materials, but I can't say any more than that because I

33:20don't know, from mom to fetus and otherwise. But in this case, we don't know what sorts of molecules are signaling the placenta that something is awry. And that, to me, is where this goes next. It would be very interesting. Yes. Yeah, absolutely. And they do, in the very end of the paper, go on a little. Vaccines are important. Vaccines really help. Vaccines prevent morbidity, mortality, and stillbirth. Yeah, they say, despite recommendations that pregnant women get seasonal flu vaccines,

33:53only half of pregnant women in the U.S. typically receive it each year. That's not good, folks. Not good. You need to do that. But then the last sentence is very interesting. Whether mitigation of maternal influenza virus disease through vaccination preserves the normal trajectory of fetal immune development is an important question. So in other words, you're vaccinated. You're not going to get severe disease, but you will get infected, right? Is that going to still induce these changes in the fetus?

34:23That's a really neat question, right? Right. We need to know that. I think it must mitigate it because we saw some relationship here between severity of disease. That's my thought, too. Yeah. Yeah. So, yeah. In the meantime, until those studies are done, you should get vaccinated. Anyway, I thought that was cool. I already did.

34:44Me, too. I got vaccinated flu and COVID. Yep. I was too early to get the mRNA flu vaccine, though. I got it. Oh, SIVA. You got it? Yep. Cool. Yep. Do you? So in the clinical update the other day, Daniel did a paper. You know, some people get vaccines and they get fever and some people get vaccines and they don't get any fever. I heard about this paper. So this paper says the ones who get fever have a gut microbiome, which has got a lot of bacteria

35:17with flagella and the flagellin is activating inflammation, right? And so they think maybe it's what they eat that predisposes towards flagellin-containing bacteria. Interesting, right? All right.

Hepatitis B epigenetic therapy

35:33So now we're going to move to the liver. And I thought that Rich Condit would enjoy this paper particularly. Everybody will enjoy it. It's pretty cool. I think it's something different. And this is in Nature Biomedical Engineering. I don't think we ever did a paper from that journal before. Development of an Investigational Epigenetic Silencer Therapy to Transcriptionally Inactivate Viral DNA in Chronic Hepatitis B.

36:03Yeseina Angelero Rodriguez and Shang Zhang are the two first, co-first authors. And Jennifer Marlowe is the last. And this is from Enchroma Bio, a company in Boston, Massachusetts. She chooses the San Raffaele Scientific Institute in Milan, the Instituto Nazionale Genetica Moleculare Romeo e Enrica Invernizzi in Milan.

36:33Wow. At the University of Milan as well. And Kathy has a summary. Yes, let me find it here. Yeah. So hepatitis B is serious. It's associated with over a million deaths every year from cirrhosis and liver cancer. And this paper proposes a therapy to, quote, cure those people who have chronic infection with hepatitis B virus or HBV, as I'll probably say. So there is an effective vaccine.

37:05We've talked about it a lot. It's best if people get it at birth, as we have talked about a lot. But if people aren't vaccinated and they get the virus infection and it becomes chronic, the treatments that are available don't completely clear the virus. They don't completely, quote, cure the virus. And so this paper is about the development of a therapy and the pharmacology of that developing therapy as a prelude to using it, putting it through clinical trials and development.

37:40So the therapies for this chronic hepatitis B infection must, one, deal with two forms of the viral DNA in infected cells, covalently closed circular DNA and integrated DNA. And then HPV gene expression is regulated by chemical modifications, which is the addition of methyl groups to CPG-rich regions in the viral genome. So there are these Cg dinucleotides, and they can cluster into what are called islands.

38:17And those regions, when methylated, affect the levels of viral DNA replication. And the serum antigen levels that come from the virus in the infected patients. So the therapy has to, if possible, deal with this methylation. So it has to deal with the two forms of DNA and deal with this methylation. And methylation correlates with lower DNA transcription and reduced levels of these viral DNAs and the

38:51viral antigens and reduced DNA replication. I said that about the antigens. I think it's true. Yeah. Okay. So, and then there is an existing first-line therapy now that they call NUCs, N-U-C, all capital letters, S. And we'll hear a little bit more about this later, because they use it as a combination with their therapy that they're proposing here.

39:18And so these NUCs are known to suppress DNA replication and reduce progression to cirrhosis and cancer. But treatment with NUCs is not good enough to clear all the viral antigens and HPV DNA completely. So their therapy uses two components. One is a guide RNA to direct the therapy to the viral genome. And the second is an effector molecule that they designate EE for epigenetic effector.

39:50So I'll tell you a little more about each of those two molecules. The guide RNA, they design to correspond to all the open reading frames across the HPV genome for five different genotypes of HPV, types A through E. And they also design it to have special features. They prepared and screened 121 of these. And then through their analyses, they reduced it to the four best ones. And then they chose the very best one, which is the one we're going to focus on, the guide RNA2,

40:24gRNA2. This guide RNA2 would target their epigenetic effector, EE, to the highest variety of genomes and have the best results based on their preliminary examination of it. Now, features of the EE molecule, it's an mRNA that encodes a fusion protein that has three parts to it. One is it encodes a catechly inactive Cas9.

40:55So this is a molecule that can bind to DNA but doesn't cut it. The second domain is a CRAB domain, K-R-A-B, and that represses transcription. And they really don't talk very much more about that. And the third thing is their DNA methylation effector domain. And so that's where they get the E of the double E. So this is the part, sort of the business end of it. So this first double E that they're going to study, they call double E4000.

41:29And so they test the gRNA2 and double E4000 in cells. And what they're looking for is silencing, that is, reduced transcription of the viral antigen genes due to this methylation. So they see that. And so the result is they see a correlation of methylation of the dinucleotide CPG islands and the silencing. So I wrote in my notes, yay, that's what they're looking for.

42:00Okay, so that was in cells. And then they go on and they test it in mice that have the HBV genome in the mice. So it's a reagent that's useful for these kinds of studies. And they see the reduction of the viral gene expression. And they're going to continue to optimize this. And then instead of using double E4000, they try double E6000 and 7000. And 7000 works out to be the best.

42:32So going forward, the rest of their studies are going to use this guide RNA2 to direct the double E7000 to the site in the viral genome that you want to stop transcription from. And they call this combined therapy CRMA-1001. And that's what's in the title of the paper. And I'm just going to call it CREMA-1001 because it's faster than saying CRMA. And that sounds a little confusing. And based on most of them being in Italy, I think of it as like CREMA-1001.

43:07Okay. So they test this combination of the GRNA2 and the double E7000, aka CREMA-1001, in cells and mice. They try different doses. They combine it with those NUCs, that existing therapy. And that makes it even better, more efficacious. And then they do, for the, I would say, the last half of the paper, large part of the paper, what I call pharmacology. They do pharmacokinetics, biodistribution, that is where the drug goes in the body, and

43:43they do safety and toxicity. They assayed whether it hits unintended targets, and they found no verified unintended targets, which they call VUTs, verified unintended targets. So the grand summary is that CRMA-1001 works. It doesn't rely on the induction of DNA breaks, which makes it safer than other kinds of CRISPR-Cas9-type methods, because it preserves the host genome integrity, which isn't getting

44:15cut up. And they have a lot of good data here for further clinical development for use against chronic HBB. So I just have two comments. One is that they abbreviate a lot of things. And so I could just imagine, I could hear Dixon throughout the whole thing saying, what is that? What does that, what does that, what do those letters stand for? And some of them, I mean, they pretty much define all of them. When I went back and did my PDF search for one that I missed earlier, it was, they were there. But so, and they even define messenger RNA as mRNA.

44:49I didn't need that one spelled out for me, but okay. So anyway, so that was good. And then there was one thing, just while I have the floor, I will say in the, I was reading along in the abstract and one thing really hit me as odd because they're talking about all the good things of it. And then suddenly they say, CRMA-1001, induced transient liver transaminase elevations only at the highest dose tested. And I thought, well, that sounds, that sounds weird. That sounds like a kind of a bad thing.

45:20So what, so in fact, if they had reversed that sentence with the subsequent sentence and said, expression and DNA inhalation profiling revealed no detectable unintended targets in the human genome and CRMA-1001, induced transient liver transaminase elevations only at the very highest dose tested. So it's pretty safe. So those are my two little beefs were those abbreviations. Safe in cells and culture and mice and so forth.

45:51No, I think it's really cool because, you know, the problem with HPV, and we can think of all sorts of chronic viral infections, one that is, you know, I think about more often where the problem is that you have viral genomes still in the cells. And so, yes, it would be cool if you could cut it out, but cutting is a little dangerous. And so here they've come up with this really cool way to take advantage of other things

46:21we know about transcriptional silencing. And that's safer. Obviously, if you silenced all transcription, that'd be bad. But if you can just specifically silence the viral genome transcription, that's cool. And so they actually came up with a cool reagent that lets them do that. And so I can see lots of interesting applications for these reagents that they've described.

46:52And I really also liked the way that they talked about sort of how long the treatment lasted, because that's sort of the would be the downside. If you don't cut out the viral genome, if you're just silencing it, then do you have to take this silencing drug every day for the rest of your life? How long does your silencing work? And they address that nicely here as well. Yes. So this is very interesting. Hepatitis B, 250 million people infected, a million deaths a year, and nobody talks about

47:26it, right? Do you ever hear about it in the press? Only when RFK wants to stop vaccinating babies at birth, right? So this is a serious problem and needs to be dealt with. And as Kathy said, there are two DNA forms. There's the covantly closed circular DNA floating around in the nucleus, and that's the source of proteins and genomes for new virus particles. But then there's an integrated... That's really the active, the infection active form of the virus. Right.

47:56And then the integrated DNA doesn't give you infectious particles, but it does give you mRNAs and protein, including the hepatitis B surface antigen. Yeah, and the integrated form is relevant because of the antigen production and other things as well, is relevant to the persistence of the infection and the ultimate frequent progression to cirrhosis and cervical carcinoma. So the drugs, the nukes, will prevent the production of infectious particles, but they don't get rid

48:31of these DNAs and they don't affect the antigen production. So we need something better than that. And in fact, the drugs, I think, knock back the closed circular DNA, but they don't eliminate it, I don't think. They don't eliminate it. Okay, so you're stuck with that. The drug treatments, it's like HIV. You're stuck with the drugs for the rest of your life, okay? Right. And we should note that there's a highly effective vaccine. So if everybody took the vaccine, we wouldn't have to be talking about this.

49:02Yes. So these people who are infected are not vaccinated, yeah. I was going to say, if some people have their way, maybe people won't be as vaccinated, and then I will be angry.

49:15Okay, so as you also heard, the transcription of the viral genome can be regulated by what we call epigenetic modifications, like DNA methylation at the CPG dinucleotide, and the genome has three regions that can be methylated, and that will influence replication and antigen levels.

49:39So here in this, and we have nukes, as I said, and Kathy said, but they are not great. And also, you have to take them a long time, and that's always a problem. So this approach is to try and do epigenetic silencing of the genome using things that are delivered to the patient. So just a little background. DNA methylation at these CPGs will recruit methyl binding proteins, which in turn recruit

50:11histone deacetylases and chromatin remodelers. And the result is a compaction of the DNA, which transcription factors and RNA polymerase have a lot of trouble getting to when the DNA is compacted around histones. And these methylation can also block the binding of transcription factors, because many of them can't bind a methylated motif. So what they want to do is they've developed this crema, crema miluno, crema 1001, which

50:44is a therapeutic candidate. So you have a guide RNA and an mRNA co-encapsulated in a lipid nanoparticle. And the mRNA is interesting. It sounds like an mRNA vaccine. mRNA encodes an epigenetic effector protein, which is a fusion containing a catalytically inactive Cas9 protein. So remember, this is taken from CRISPR-Cas systems. So the Cas9 protein can be directed to the DNA by a guide RNA.

51:18And in this case, and then the Cas9 will clip the DNA. But they've catalytically enacted it. So it will just bind to the DNA target and not cut it. And it will bring along with it the other part of the effector, which is a transcriptional repression domain and a DNA methylation domain. So the Cas9 binds the guide RNA, right? Right. And so that's what, that goes together with the Cas9 together with the guide RNA is what

51:51provides the targeting. So this is a totally cooked up protein, right? Totally cooked up. This does not exist otherwise. We said, oh, well, we need a targeting domain and we need a methylation and we need to methylate at the target. And while we're at it, why don't we do some transcriptional repression as well? My mind is boggled. It's amazing. Yay, for basic research. Yeah, it's great. Well, you know. Just get creative, right?

52:21Yes. None of this epigenetic stuff existed when I was a child. Okay. And I, you know, I've been a skeptic all the way along. To me, in some ways, the ability to actually use all this stuff to affect a change systemically by God in a mature animal. It's just mind boggling. And it also, you know, proves that all this stuff is true in my mind.

52:52I mean, I'm a skeptic, so everybody else all knew this, but.

52:57So Rich is, as he calls it, totally cooked up protein. That rang bells for me. Is that going to generate an immune response? Oh, actually, that's interesting. It would depend on kind of where the domains of the protein come from, right? Yeah, that's what I was just thinking. Well, Cas9 is from strep pyogenes, so yeah. Right, right, right. So potentially. Yes, but this, as you'll see, the effect occurs very quickly, probably before.

53:32And these things are cleared quickly. And the interesting thing, I'm getting ahead of the game here, the interesting thing is that once you've made the modification, it persists, even in where you have new cells growing in the liver. So the modifications must be propagated, right? Right. Once they're set down. And because they kind of show some of the information about dosing, even if you were to make a big immune response to an early dose, because you don't necessarily have to dose again, it might

54:06be okay that you're making a response to Cas9. Yeah. As long as you don't have to dose again. Right, exactly. Because in people, we don't know. Right. This is all done in mice here. But it may be in people you have to dose once a year, and then you may have to do something different. Right. Well, and Rich, I'll just tell you, a lot of these chromatin modification things were sort of very hot when I was in graduate school. Yeah, for HIV, right? Well, just in general, like when I was in our basic cell biology classes, this was a big

54:36part of it, to the point where my friends and I were sort of thinking, you know, whenever there was something that was happening where you couldn't quite explain the cause, the answer it was always in chromatin. Right. Except for genetic. Right. And so I still kind of think of the answer, if I don't know the answer, is it's just chromatin.

54:55That's very funny. So my pick relates to that in a way. Just remind me to tell you. You remind me. This is really a sideline. But Mike Merschlinski was a vaccinia virus DNA replication type. DNA replication can be complicated, okay? And any time somebody backed him into a corner with a bunch of questions, he would say, oh, it's recombination. And that's so complicated that they'd just shut up. Quit asking him questions.

55:26So basically you get an mRNA containing nanoparticle, right?

55:33So it's got a guide RNA and it's got an mRNA encoding this EE protein, which is going to, the guide RNA is going to bring it to the DNA, and then the EE is going to use the cell's methylation machinery to silence that gene, okay? I'm sorry. In other studies, it's been shown that these changes persist and they can last over a year in mice and in non-human primates. It's very cool. So the other thing that interests me about this, fascinates me about this, is that the

56:06thing that you're administering is just mRNA. Or it's a guide RNA and a messenger RNA that encodes this thing, this fusion protein. So that means that the fusion protein has to be made from the messenger RNA and it has to bind the guide RNA. So in my mind, they have to be in proximity somewhere. So it's as if a given cell takes up one of these particles with both of these RNAs in

56:39them, translates this protein in the same cell that the guide RNA is there, and they manage to find each other in that cell and bind. That, to me, is remarkable as well. Yep. And in the nucleus, right? So the protein is made in the cytoplasm and then it must have an NNLS and it gets in the nucleus and the guide RNA has to go in with it, which I guess is going to happen because Cas is going to be made in the cytosol as well and it'll take the whole thing in.

57:11But yes, there's a lot of Hail Mary type stuff here. But it worked. But it worked. It just worked. Amaze, amaze, amaze. Exactly. So let's tell you how they did this. So they have a panel of 121 HPV-targeted guide RNAs. And this is spanning all the open reading frames across the genome. And they're designed to hit all the different genotypes of HPV.

57:41And they've looked at 7,000 genome sequences, genotypes A through E from the databases. So they screen these 121 guide RNAs using HEP2 cells. So these are HEPG2 NTCP cells. These are human liver cell line that is producing the receptor for hepatitis B virus, which is NTCP. And they infect these with HPV genotype D. And then they put in the lipid nanoparticles with our EEM RNA and the guide RNAs.

58:12And they quantify HEPB antigen in the supernatants to see if any of these have knocked it down. So 51 guide RNAs showed over 50% silencing of either of the two antigens that they looked at. And 25 silenced both biomarkers. And these guide RNAs in these 50 were throughout the genome. So no preferential hotspot for silencing. They picked four of these guide RNAs for additional investigation.

58:44They looked at silencing efficiency in HEPG2 cells that express full-length HBV genomes, now a little bit different. And all of these four showed dose-dependent suppression of viral antigens. With number two, HEPHBVG RNA2 had the most pronounced and durable silencing. So this is working, folks. It's silencing the viral genome. So now they address this experimentally later. But part of the issue here is that you want guide RNAs that are specific for the HBV genome.

59:19And was that part of their screening protocol to start with, is sequences that will hit HBV and not parts of the human genome? So first they looked for HBV sequences. They found ones that worked. And then later they do, they say, is it going to be off target at all? Yeah. I mean, I assume if it was super off target, the cells wouldn't live. Good point. Maybe. Good point. All right. So then, so we got now HBVG2.

59:51By the way, there's a correlation. They look at methylation, actually. They do a method that they can measure the average methylation and they can correlate it with silencing efficiency. And so of GB2, GRNA2 had the highest level of de novo methylation, which had also the highest level of silencing. So then they have a couple of models for further studying this. The first is they deliver the viral genome to cells in an adeno-associated virus, an AAV

1:00:23vector. So they have an AAV vector with the whole HBV genome in it, and they can infect cells with that. And you get episomal HBV DNA in the nucleus. And so they can use their gRNA to assess antigen suppression. So they put their lipid nanoparticles into these cells. They actually can, they put the AAV into mice, and the mice get AAV episomally, and then they can look at this silencing in vivo.

1:00:54And so again, gRNA2 gives you the highest antigen reduction in the AAV-HBV mice. And so now it's the problem that Rich mentioned, even more magnified, because it's trying to get all of those components into the same part of the cell. That's easy enough, perhaps, in cell culture. But now we're actually getting it to go all to the right places in an organism and find the infected cells and get it to the infected cells all correctly in an organism.

1:01:25It's awesome. I had another thought here, in case anybody's wondering. Some people who are listening closely out there may be thinking, wait a minute, why all of these sort of phony substrates for the virus? Why not just infect the animals with the virus, find out what's happened? And the answer to that is you can't. The virus is very specific for humans. So, and that's been a problem with hepatitis B all the way along, is appropriate animal

1:01:55models. We'll come to more of that later. Yeah. And so this AAV-HBV mouse is not ideal because it only has episomal. It doesn't have the integrated, right? Right. But they tested in that. Yeah. And that's good because there's more copies to it. More closely mimics the closed circular DNA phenomenon. So next they want to optimize this EE protein, right, for the best methylation and silencing. But they can't really optimize it in an animal model. So what they do is they find a hepatocyte gene.

1:02:29It's called PCSK9. It's expressed in hepatocytes. They're going to use this as a surrogate to optimize their EE protein, right? Because it can't infect animals with HPV, as Rich said. So they're using guide RNAs against this PCSK locus. They find three that are working, and they can use this to optimize the whole system, okay? And they get very good. They get—so they have one called EE7000, which gives you saturating reductions in PCSK9.

1:03:05And this is now in non-human primates at a single dose of mg per kilogram with durability of two years. So this is showing the optimization of this. So then they take this EE7000 optimized cassette now, and they're going to use that with G2, gRNA2, and that's the final development candidate, which is CREMA1001. So it was optimized on this cellular gene, and then they take the optimized protein and go with it, okay?

1:03:38So now they're going to use another mouse model to further test CRM1001. They have a transgenic HPV mouse model where the entire viral genome is integrated into the mouse. Genome has two copies of the genome, and they're going to see if their drug can silence integrated DNA. And so they do this experiment. They inject the mice with it. They get a single dose—well, a dose-dependent reduction in plasma biomarkers in these transgenic HPV mice following a single dose.

1:04:17They get maximal reductions of 2.7 logs of HP surface antigen and 2.9 logs of HPV DNA. And that suppression is made throughout the seven-month observation period before they sacrificed the animals. All the animals that got over a half milligram per kilogram of this, this is now EE4000. This is the first step. They gave a progressive decline in both antigen and viral DNA about a month, starting at about a month after treatment.

1:04:48And at the end of the study, you get comparable levels of suppression across all doses. And 11 of the 15 mice had undetectable antigen levels.

1:05:01The suppression of antigen then coincides with the emergence of antibodies. Interesting thing. So the antigens are immunosuppressive themselves. As soon as you get rid of those, you can make antibodies to the viral proteins. Let's see. So then at the end of the study, they see a reduction in antigen staining, hepatitis, core antigen staining in the liver. 98% of hepatocytes are negative for core antigen in the highest dose group, even though there's still integrated DNA in all of these cells, right?

1:05:37But it's silenced. They do methylation sequencing. They confirm dose-dependent methylation of the integrated genome, which correlates with reduced viral transcript. And the interesting thing is, so these gRNAs are targeting the specific region. The methylation extends a plus or minus one and a half kilobases on either side of the methylation site. Genome-wide silencing of HPV promoters. This epigenetic stuff is amazing. Yeah. I'm wondering about the role of the induction of the immune response in this whole thing.

1:06:20I'm wondering if that isn't really key to, well, we don't really know, we don't really have a virus model on this yet. But if that won't ultimately be key in clearing the infection. Because after all, in a normal hepatitis B infection, there's a certain percentage of the population that get an acute infection, mount an immune response, clear the infection, and that's that. So it makes me wonder whether induction of the immune response or suppression isn't as important as anything else, put it that way.

1:07:01Yeah, could be. Okay, so this is done with 4,000, which is not optimized. So now they do, they use CREMA-1001, which has the same guide RNA, but it's got EE-7000, which was optimized on the cellular gene. They then evaluate those in transgenic mice. They get greater reduction than the other one so that they say, you know, the optimization worked. The peak levels of the mRNA, the effector mRNA, and the effector protein are found 24 hours post-treatment, and by seven days, you can't find any of them.

1:07:38They're all gone. Seven days after you inject this in, all these components that you put in are gone. The methylation of the viral DNA and the silencing, it peaks at three days post-treatment and persisted through the 14 days of this experiment. They confirm methylation by methylation sequencing, and they also show that in another, a patient-derived hepatoma cell line with integrated hep B, they also see silencing as well.

1:08:14The idea that mRNAs are gone within seven days after delivery is not surprising. Right. I made an emphasis on that because people are always saying that mRNA vaccines last forever, but they don't. Exactly.

1:08:33Okay. So, all right. So that's the conclusion. The methylation works. It's one and a half KB on either side of the target site, and it suppresses antigen production. And so this enhanced E7000 works well.

1:08:51Okay. So now they have these hep G2 with hep B receptor and primary human hepatocytes as models of HPV infection. They support the whole viral replication cycle. They have CCC DNA and integrated DNA in the same cell. So this is the best you can do. It's just a cell, but it's the best you can do. So they use that now to test, and they treat these with their CREMA-1001. They infect them with hep B.

1:09:23The viral antigen, the two antigens, are reduced by 98% at day seven post-treatment. Treated cells show methylation deposition ranging from 0.29 to 66.42%. Okay. So it really works well in those authentic infection models. And then they go back to the AAV-HBV model, and they try their new CREMA-1001.

1:09:54And as Rich said, these deliver a higher DNA load. So these mice get an intravenous dose or three monthly doses of CREMA-1001. They're monitored for six months. You get decreases, over three logs of decrease of the antigen and of the DNA. You get decreases well over a six-month period. And three doses enhances the effect of the CREMA-1001. You get a further decrease in antigen production.

1:10:24And they see that reduction for like 180 days. Yes, yes. They also see an increase in the proportion of animals with antigen and DNA below the assay limit. So the assay can only go down to so far, and they see more and more animals getting to the point where you can't even detect it anymore, which they say is remarkable.

1:10:55So I guess they like that result, the lower limit of detection of the antigen and the DNA. Okay, so next, they want to know if you can combine this with antiviral, a nuke therapy. So they take these AAV, HPV mice. They treat them with Antecavir, which is one of these antivirals, for two weeks to suppress viral replication. Then they give CREMA-1001 an additional two weeks.

1:11:25So the drug alone reduces DNA by about two logs. It has no effect on the antigens, though, as we know from previous studies. And withdrawal of the drug results in a rebound of DNA to baseline levels. But if you give the CREMA and ETV the drug together, you get reductions similar to CREMA-1 alone, but it led to additive DNA suppression. And when the drug effects go away, which is about four months post-treatment, the DNA levels are comparable to those in the CREMA-1001 treated animals.

1:12:01And so then they also look at biomarkers in the liver, and in addition to HPV, mRNA, and total DNA, no changes in episomal DNA levels, which is consistent with the mode of action. It doesn't cut the DNA. It's just silencing it, right? So basically, the CREMA-1001 targets episomal DNA. It's compatible with nuke therapy, which a lot of people are on, right? And it shows additive antiviral activity when combined.

1:12:34All right. So a little bit of pharmacokinetics and pharmacodynamics. A little. A lot. It is quite a bit. Well, this is important stuff, right? They want to know the distribution. For this company and for, yeah, getting this forward. But it's not what we usually get into. No, not at all. But look how quickly the mRNA goes away. Yes. Yeah, so these are given to non-human primates, and these are cinemologous monkeys. They get a single IV infusion of two milligrams per kilogram, and they look at plasma samples for the lipid, the RNA, and they look at multiple tissues.

1:13:13The maximum plasma concentrations you see right at the end of infusion, and then the concentrations decline in the next hour by more than 60%. And then you can barely detect them after 24 hours. This is going away very quickly. The highest tissue concentrations in selected tissues were observed an hour after the end of the infusion in the liver and the spleen. That's the highest levels that they find, and then they decrease over time.

1:13:45By day 25, they're less than 0.1% of the one-hour liver concentration. These were administered how? Intravenously? Intravenously, yes. They do in-situ hybridization to look at the mRNA at one hour post-treatment. They see this in hepatocytes and other cells in the liver. They see it in the spleen. They look at the testes because they're concerned that this might be in germline cells, right? We don't want to do that.

1:14:16And so testes had very low levels of mRNA staining, also ovaries as well. So the primary site of distribution is the liver and the spleen, very little distribution in germline cells. They also wanted to look at the toxicity. So they give a lot of the drug to synomalgus monkeys. And as Kathy already read to you, liver enzymes are elevated, but after four weeks, they go back down. And they don't see any clinical or pathology correlates, so they consider these non-adverse.

1:14:49Okay, so mostly distributed to the liver and spleen and nothing in germline cells. For the folks out there, I questioned the route of inoculation because of the fact that it shows up in the liver and the spleen. I think of the liver as sort of the traffic cop for your circulatory system. The blood all goes through the liver, and the liver sort of checks it out, makes sure everything's okay. If there's bad stuff, it takes it out. If there's not enough good stuff, it puts it in.

1:15:19Your liver can turn a Twinkie into a balanced meal.

1:15:25That sounds like a really good quote.

1:15:29All right, so the last set of experiments, they want to know if these guide RNAs are hitting anything in the genome, our genome, right? So they do whole genome transcriptomic and DNA methylation analyses. This is complicated, so I wrote up a summary here that I'm going to use to guide myself here, if I could get to it. Yeah, here we go. So they do whole genome transcriptomics and methylation sequencing in primary human hepatocytes, spleen endothelial cells, and adrenal cortical cells from nine different people.

1:16:06And they use in silica prediction of guide RNA, gRNA binding sites, allowing up to four mismatches in this guide RNA. That gives you 1,591 potential places in these cells where the guide RNA could stick. Then they do a capture panel to retest those regions, and only four hypermethylated regions are confirmed, all in hepatocytes and all near six genes. None of those six genes show the expression change that would make them a verified unintended target, a VUT.

1:16:41So the authors say, there are no verified unintended targets. That, to me, is really pretty remarkable. It is good. But there are some things that would be discussed at a regulatory level, right? So you do a single donor per cell type, right? So it might vary in more donors. They say if you get methylation, but there's no expression change, that's okay. But you could imagine that methylation alone might be, should get some attention, right?

1:17:15The cell types were picked based on where the drug is mostly found, right, the liver and the spleen. But they didn't look at germline in the same way, so might do that. And then the mismatch tolerance, four mismatches, it's pretty good, but Cas9 rules are a little more forgiving than that. So, you know, more will be done before this gets approved. But it's a good way to say that it doesn't seem to be any major targets in the human genome.

1:17:49So this is the story. So now they say this is probably ready for clinical development, right, to get into people with a phase one first and so forth. And I think this is pretty exciting. This is a totally new. I mean, I look to see if there are any – there are no FDA-approved drugs that silence genes yet, okay? And this one isn't even a gene. This is a viral gene, right? It's not a human gene. There are some under development to silence human genes for some diseases and so forth, but this would be the first viral gene.

1:18:23This to me is a much more sort of nuanced twist on genetic engineering, okay? Yeah, it's very cool. That's where instead of just going in and knocking stuff out, you're tweaking it. You know, we learned a little about epigenetic control for many years, but the key here is the guide RNA, right? So CRISPR-Cas was an important part of this. You couldn't do this – I mean, you could use zinc finger binding proteins, right? But this is better. This is better. Zinc fingers are finished because these are better.

1:18:54By the way, zinc finger proteins you can engineer to bind specific sequences, right? And they were hot just before CRISPR got discovered, and now they're kind of ignored. So one thing I thought of was if the genome could mutate to avoid silencing, right? So maybe – they didn't look at this at all. I said the paper is silent on this, and Kathy picked that up.

1:19:21They didn't say anything about it, but maybe they would want to use two guides targeting different sites, right? Like we used two or three antivirals, right? And they do have a lot of candidates, so they could combine them. So I don't know – you know, this virus is – the DNA is reproducing. So the possibility of mutations arising that would prevent the guide RNA from binding is a theoretical possibility. So I have to say that there was an animal model for hepatitis B, right?

1:19:58Chimpanzees. Yes, right. But we are no longer allowed to do this with – to experiment with chimpanzees. And I'm not advocating that we should. But I always run through my mind, you know, how do we prioritize this sort of research and the animals that are used and that kind of stuff? It's – I don't want to say more other than I think about it, you know?

1:20:30Look, I worry about mice, Rich. Absolutely. I mean, they're sentient. They know what's happening. Yeah, exactly. Thank you. So it would be nice to eliminate them entirely. But we're not there yet. Right. I mean, we're making a value judgment that human life is more valuable than animals. And somehow chimpanzees are more valuable than mice, right? Because they look like us and they have behaviors that are closer to us. But like you say, mice are sentient, right? So – They know when you're sacking them.

1:21:01The littermates know when you – I mean, yeah, for sure. Before reading into this paper at all, when I first saw that the experiments had to do with infecting and sacrificing pregnant macaques, I got – my first reaction was, there better be a pretty good reason to do this. And then, you know, they quickly – it was – provided a justification that was good enough for me. Okay? But I still – I couldn't do it.

1:21:32Personally, I couldn't do it. No, I did a lot of mouse in my career. I'm happy not to do any more. Yeah. Right. I really am. Anyway, let's do it random. So we look forward to – put it this way. We look forward to a day when we don't have to do animal studies anymore. Anyway, this is – as they say, this is CRMA1001 is positioned as a promising, potentially curative approach for individuals living with chronic hep B. So it's very exciting. And I could – you know, if you've done all the preclinical studies in these sorts of models to show that it's efficacious and apparently safe, I can easily see going ahead with human clinical trials, starting off with safety studies, right?

1:22:21I can imagine many of the people who I met at that hepatitis B meeting raising their hands and saying, sure, I'll try it. Yeah. Right.

1:22:33Yeah, I would do a phase one. I think it's – this looks really good. Yeah. So I mean, phase one, you just need people. But you could also imagine, you know, as you get further along, there might be some people who have chronic hepatitis B who might be willing to try. Right. I wonder if in a phase one you would use chronically infected people. Uh-uh. You wouldn't, right? You need healthy people. Yeah. Yeah. Hmm. Okay.

1:23:03All right. Let's do one round of email.

Listener mail on rabies and vaccines

1:23:08Let me take this first one from Carol. Okay. So this is a paper we did now so long ago. Okay. This is the Tau Herpes paper. Who was here for that? Were you here, Brianne? I was not. Oh, nobody was here except me. This is bad.

1:23:23Okay. So there was a question we had. So this paper was all about how Tau is antiviral against herpes simplex virus in neuronal cultures. Oh, wait. I think I was there. Yeah, I think you were.

1:23:39And so here's Carol's email. The anti-interferon gamma antibody may prevent the induction of tetherin, an important cell autonomous antiviral pathway that interferes with egress of virus from the plasma membrane. Thus, in the presence of the anti-interferon gamma, herpes virus may be able to leave the neurons despite the P-Tau.

1:24:04Okay. So we had some question about what these anti—and so Carol has a suggestion, but I cannot put it in context now because I forgot everything.

1:24:13Because I probably have phosphorylated tau. Who knows?

1:24:18Okay.

1:24:22Brianne, can you take the next one? Sure. Tony writes, Vincent, listening to TWIV 1363, and there is a really important correction about rabies. If you wake up with a bat on you, you must get to an emergency room. There are instances where rabies has been transmitted from bats to humans through scratches, so the post-exposure prophylaxis vaccination is usually done. Also, all the rabies studies we've done in unvaccinated bats lead to death. I think most people in this area will argue that rabies kills bats eventually.

1:24:52This is probably true for all mammals, with the rare exceptions of individuals within a species. And that's from Tony Schouts. Who says, oh, we did a—I don't remember how this came up. Okay. But it was stated on TWIV that if you wake up with a bat on you and you're not scratched, you're not bitten, you'll be okay. But that's not correct. I've heard that if there's a bat in your room, you should go get vaccinated, even if you have no idea if you've been bitten or scratched. Okay.

1:25:23Right. And so the person who said that, I did not contradict at the time because they are a veterinarian and I don't, you know, it's their expertise, not mine. But thank you, Tony. Okay.

1:25:35Kathy. Kellen writes, hi, Vincent. The papers you have shared about various vaccines having additional benefits beyond what was originally intended are really exciting. Not sure if you have seen this, but I came across this article in JAMA—oh, my page isn't open for now—JAMA DERM, showing that HPV vaccine can treat precancerous actinic keratoses. And then there's a link that I haven't looked at, and the title is Human Papillomavirus Vaccination

1:26:09and Actinic Keratosis Burden, the VAX Act Randomized Clinical Trial. And so the main outcomes—okay, no, the measure—conclusions and relevance. In this randomized clinical trial, standard alpha papillomavirus vaccination was found to reduce AK burden in immunocompetent individuals with multiple lesions. HPV-targeted vaccines may be useful for management of AK, a chronic relapsing disease, and the

1:26:39most common precancer in fair-skinned populations. So this AK is not ordinarily associated with human papillomavirus? Is that the gist of it? Yeah, this is—I don't know. Okay. Yeah. Pretty weird. Yeah. It's usually caused by sunlight or UV. Okay. Interesting. Huh. Yeah. So another side benefit. Yeah. As opposed to a side effect.

1:27:10Nice. Yeah. Thank you.

1:27:14Rich. John writes, Vincent Daniel Allen. Thought you might be interested in the summary of this in Wheat, W-E, small i-t, which, as you probably know, is run by Jerry Coyne. So this is a podcast? It's whyevolutionistrue.com. Yeah. It's like a blog, right? Yeah. Right. So just word search the post for perspectives to get the start of the discussion together

1:27:47with a number of links. Alan, I got an old girlfriend, also a W&M classmate in Williamsburg, Massachusetts, who has a fair amount of land and some apple trees. She told me that a wolverine had gotten all the apples on one tree while a bear got another. I had no idea. There were wolverines in central Massachusetts. If you have them, too, please mention sometime when it fits in with something. Here in Greater Braddock, all we have is damp groundhogs.

1:28:20Oh, it also crossed my mind, also crossed my window that at least three rabid beavers turned up in two lakes in Maryland, where they have bit, at least bitten, at least one swimmer. The lakes were closed. Best regards, John. This is all referring to discussions that I was not present for. So what's this? So this is a, he sent the link to why evolution is true.

1:28:58What did he say to look for? It might be interesting to summary in the why. Perspectives. To get to the start of the discussion.

1:29:10Perspectives in biology and medicine. The politicization of science. The latest issue is entirely about the politicization of science. Oh, that's, most articles are critical of the trend. Oh, that's cool. Thank you. We should look at that. That's good. I'd be interested in reading that. Yeah. I'm mostly worried about these rabid beavers. Yes. Yeah, bummer. No lake swimming.

1:29:39Yeah. Okay, and I don't remember this business with apple trees and Alan. I have no idea what. I thought he might be here to know this, but he's not. Okay, let me do one more here. Scott writes, you usually discuss Nobel Prizes in medicine, chemistry, and sometimes physics on Twiv. This time of year, I was looking through the works of Ann Carson, who has received the Nobel Prize in literature, and found a poem that rings truth about taxonomy.

1:30:10Made me think about how a large swath of the enterovirus clade aren't enteric. So here's the poem, Between Us and, this is by Ann Carson, Between Us and animals is a namelessness. We flail around generically, camelopardalis is what the Romans came up with, or giraffe. It looked to them like a camel crossed with a leopard, or get the category wrong. A musk ox isn't an ox at all, but more closely cognate with the goat, and when choosing to name individual animals,

1:30:44we pretend they are objects, spot, or virtues, beauty, or just other selves. Bob. It's really good. Reminds me of one of the Feynman books. What was Feynman's first name? Richard. Richard. Richard, where he got a lot, you know, a lot of his sort of informal education was from his father.

1:31:14He talks about going on walks with his father, outdoors and stuff. And he would be asking his father, oh, there's a bird. What's the name of that bird? What's the name of this flower? What's the name of this? And his father would tell him, who cares?

1:31:31It's a flower. Isn't it pretty? It's a bird. It's different than the other birds. This isn't that nice. Okay. Pretty good. We all get hung up on names. But it's interesting to know what something is, isn't it? Well, I, why? Does it help this? I mean, if you see it again, oh, that's a such and such, right? Exactly. I mean, that's what taxonomy is about, is that you can, the name is an identifier for other people, I suppose, and for yourself to group things and stuff.

1:32:01Say, oh, that's the same thing as this thing. I mean, the vast majority of stuff we see in nature, we don't know what it is. And we just look at it and say, oh, that's cool, right? Right. Or not, maybe. But now you have the app on your phone, you could identify everything. Well, not quite everything. Don't believe it. My app's a little lame. You shouldn't believe it, no? Don't always believe it, no. I always have to. Well, it's not AI, it was before AI, right? It doesn't matter. It's based on AI, some of it, yeah. Some of it is. One of my friend's daughters, who is in elementary school, thinks it's funny to use that app on her mom's phone to identify people.

1:32:38Yeah. And so, apparently, I am sick ginkgo. Oh, that's interesting. I always do that. When my app is not giving me information, I always do the control and shine it on a person. And if it comes up human, I think everything's okay. It usually does. Speaking of AI, in the last six months, so my email, so there are TWIV emails, right? There's TWIV at Microbe TV, and there's all the others, completely flooded with AI emails.

1:33:12Would you like to interview blah, blah, blah? And then sometimes they'll say, oh, on your episode where you discussed AI synthesis of a viral genome. So, they'll summarize an episode as if they'd listened to it, and they say, would you like to interview this and such? I mean, they're just sicking AI on us now to say, do you want to interview? So, these are PR firms that are just churning out these invites. They're probably having the AI send the email, too. And they're just cold emailing you to try and get you to interview people.

1:33:44And some of them, I read them, and I'll go, oh, it sounds like they listen. I say, no, it's just an AI that knows the summary of the thing. It's just kind of insidious, you know?

1:33:54Yeah. Wow. I mean, I'm not saying it's all bad, but it's just there are parts of everything that are not as nice, right? Yeah. And this is one of them. And so, now I have hundreds of emails that I have to go through, and I can sort of tell what they are. But you never know if I'm going to – I just boom, boom, boom, delete them quickly. And maybe I'm going to delete a real one by mistake, you know? That's okay. They'll come back. Yeah. If they're supporting, they'll come back.

Picks of the week and sign-off

1:34:20Let's do some pics of the week. Brianne, what do you have for us? So, I have a really cool story about coral and coral bleaching that I read in the New York Times. And so, here are some scientists. We're trying to – we're working with bleached coral, trying to help that bleached coral. And by accident, they had something happen in their lab.

1:34:51So, basically, I believe, as he points out somewhere, a piece of healthy coral got dropped into the sick coral by accident. And they found that if bleached corals are actually in physical contact with a healthy coral of the exact same species, then the algae could transfer and the bleached coral could be healed. And so, it took some time of direct transfer.

1:35:22It took, like, seven days of direct transfer. And it does have to be a genetically identical coral that's in contact with the bleached coral. But it was a really cool way of showing that coral can be healed in this way. I got really excited when I read this because the donor coral had to be genetically identical to the bleached coral that was being healed, which sort of indicated to me that this might be related to some interesting immune-related molecules, since I know people have looked at MHC matching in corals before.

1:35:57So, I wondered if this was somehow related to that knowledge of which coral is genetically identical to the other. But genetically identical corals seem to be able to have their algae recolonize bleached coral and heal them. It looks like they're fusing. It looks a little bit like that, yeah. The pictures are awesome. To me, like, they're fusing. It makes sense, right? So, hold the phone.

1:36:26This means that I know nothing about coral. I mean, I know that it's a creature, right? But that's about it. So, it's a creature that builds a structure, right? To house itself. What sort of a creature is this? So, coral are invertebrates that are building a structure, but that structure houses other organisms. And so, the reason why coral is all sorts of bright colors is because the coral is housing algae and other types of organisms that have photopigments.

1:37:05And so, they do photosynthesis and actually make nutrients for the coral while the coral is providing a nice, healthy home. Okay. So, the coral organism is the landlord, basically. Yeah. Okay. But there's all sorts of other tenants. Yes. So, you get the polyp and the algae, right? Yes, exactly. So, what's the structure made of?

1:37:36I think it's like a calcium. I mean, calcium carbonate is what comes to mind, like a shell of some sort. Yep, exactly. So, to me, it's pretty remarkable. Right. And I double-checked. That there's a species specificity here. Yeah. As you would think, bleached coral, if it had nothing else going on, would be a perfectly suitable habitat unless the structure has something to do with it. Well, so, it seems like part of the reason why the bleached coral is having problems is because there's an environmental change. So, that the environment, maybe if with global, like, climate change, global warming, now the water can no longer support the algae.

1:38:17So, it leaves the coral. And the coral is now unhealthy because it doesn't have its symbiotic partner. Yeah, but that doesn't explain the species specificity. Right. Right. That's what's getting me.

1:38:31Fascinating. Great stuff. So, I thought the species specificity and I started to want to know about, you know, coral MHC because, like I said, I know this. That's where you were going. There's some literature on coral MHC. Okay. So. Go for it. Yeah. So, can you repair coral, bleached corals this way then? So, it looks like in their experiments they said yes, but they didn't have – they weren't really sure how it would work in the wild where you had a really large piece of bleached coral to then somehow find a genetically matching healthy coral, bring it to the bleached coral, and sort of hold it touching the bleached coral for long enough.

1:39:18Not only that, but in many of the circumstances we're talking about, if not most of them, right, the bleaching is an environmental phenomenon. Right, exactly. So, you're – Yeah, it'll just bleach again. Yeah. Yeah. You got to fix the – once we get our act together and cool the planet down again, all right, we can do this to repopulate the coral, all the bleached coral. Yeah. But it's pretty cool to know that it works and I also like that, you know, it happened because the healthy coral fell into the bleached coral.

1:39:51Right. In the lab. Serendipity. Yeah. Very good.

1:39:57Kathy, what do you have for us? I picked something from Saturday Morning Physics, which I picked before when we were talking about the photograph 51 and what all the different lines in the x-ray image tell you about DNA structure. And this one is particularly – was interesting to me. It's entitled The Science of Survey Research by a person who does survey research. And as she says right at the beginning, I'm not a physicist, so why she's at Saturday Morning Physics is what she's trying to explain.

1:40:32But Saturday Morning Physics always has some leeway in terms of who they have and what topics they cover, kind of like our, quote, science picks of the week. But anyway, so this was a really cool – I think it was the first one of this fall season. And she has graphs and really good explanations of what makes a good survey, what makes a good survey question, and when to believe surveys and when not to, and just a number of things that I hadn't really thought about before.

1:41:04And I think it's worth checking out. I had a – I came up with a couple of questions after the episode was over, and I might try and figure out how to get in touch with her about that. Cool. But, yeah, it's good. Nice. That's neat. Rich, what do you have for us? I have a book that was actually given to me by my granddaughter Harper. Remember Harper? Mm-hmm. I do. Little Harper who used to be on the show occasionally. Well, she's now a first year at Oregon State University.

1:41:34And her summer reading for her curriculum there, I think this is the honors curriculum, was a book called Everything is Tuberculosis by an author named John Green, who I was not familiar with, but he is a best-selling author. Oh, he's pretty famous. Yeah. And his brother is also very famous. Right. They're YouTube wizards, right? Yep. And he used to write mostly children's fiction, but he's sort of transitioning into more health-related stuff.

1:42:09And he got – because of a partnership, I think, through their podcast with Partners in Health, he wound up visiting Sierra Leone to a hospital there and met this kid who had tuberculosis, who I think really focused his attention on TB. So, this book actually sort of follows this kid's history, but also does the whole history of tuberculosis from, you know, way back until nowadays and from all sorts of aspects.

1:42:47And it's very nicely done. I was also – I always had in the back of my mind, so what's with the title? Everything is tuberculosis. And to me – and he never answers that question – but to me, it's like in studying tuberculosis, it's a window on human culture, in particular inequities. Yes. Because this is a – because this is a – like several others that are big killers.

1:43:23We just talked about one, hepatitis B. This is a curable disease. And the fact that it still is killing a lot of people is mostly a cultural and economic thing. And he concludes, basically, that this is a – in the modern system, this is not a disease that's caused by a pathogen. It's a disease that's caused by people, okay? Because they aren't taking care of each other. Right. So, it's a very interesting read.

1:43:53Yeah, Daniel's a big fan of this book. Oh, okay. He always talks about it. Good. But he also wrote the Anthropocene Review. That could be interesting. Did you read that? My understanding is that's kind of a memoir that transitions from his ute fiction days to his more recent days. But that apparently – that book apparently was a huge hit. I know nothing more. Okay, my book is another book by Neil deGrasse Tyson.

1:44:28I really like his writing. So, I searched for one. And this one is called Death by Black Hole and Other Cosmic Quandaries. It's – he used to write for Natural History Magazine, I think, up until 2007. So, this is a collection of his essays. But they are just really good. And they cover so many cool things. I was telling Brianne on Immune, this is where I learned that all the hydrogen and helium in the universe was made by the Big Bang.

1:44:59And then everything else, every other element was made in stars. Stars make them and then they blow up and it gets distributed all over. And that's one of the things. So, he goes into all this stuff about astrophysics. This is one I love. This is the nuclear reactions going on in our sun. So, because I read this, the other day I took a picture of the sun. It was a beautiful blue sky. It's just – you can't look at it. But if you take a picture of it, right, then you can see how big it is. It's a star. And you know what it's doing?

1:45:31It is taking hydrogen, which came from the Big Bang, and it's fusing it in the core to helium. It's doing really hot fusion. There's no cold fusion, folks. And the temperature is like, gosh, 70 million Kelvin or something. It's really hot. Anyway, what happens is – this is the blow of my mind part. So, the hydrogen consumed per second, 600 million metric tons per second is being consumed.

1:46:08Wow. Per second. How can this last – it's going to last another 5 billion years. How can it be so big? It's huge, right? Wow. So, that's amazing. And they make helium. You make 600 million metric tons per second. So, there's 4 to 5 million tons that's missing. And that is made into the photons, into the energy that lights up our world, right? Because E equals MC squared, right? You're going to destroy some matter. You've got to make energy out of it. But here's the cool part.

1:46:40So, these photons are made in the core of the sun. It takes them almost 200,000 years to get to the surface before they can fly off and come to Earth. Because there's such a concentration of particles, it just bumps into something and gets destroyed or becomes something else. It has to do this over and over again until it finally – oh, my gosh. This is amazing. So, that's one of the things you can learn in that. But anyway, and then he'll tell you what happens when the sun exhausts the hydrogen fuel.

1:47:12It's just going to happen in about 5 billion years. And then it will – then the whole star is going to start coming apart. And basically, the solar system will be gone. Earth will be fried.

1:47:24And, you know, 5 billion years is a long time. I don't know if any humans are going to be around in 5 billion years. But they better get out off of it. Anyway, this is really good. Plus, there's a whole section on science communication. Okay, where he's always been into that, obviously. But he critiques lots of popular culture stuff based on space, right? And what is one of them? He said, this particular movie, they have a sunrise at the beginning and a sunset at the end.

1:47:55He said, all they did was reverse the video. And he said, that's not how it works. It goes in opposite directions, right? It curves one way and curves – Yeah, that's pretty good. And he said, they're so lazy. All they had to do was consult an astrophysicist and they would know that. And there's lots and lots of examples of that where he gets really upset by people doing the wrong things. It's kind of like us when we see things that's wrong about viruses. We get a little bit upset. Anyway, I really recommend it. It's very fun to read and he really writes very well.

1:48:28Whether you like him or not is another thing. I encountered a relevant joke recently.

1:48:38Running through Austin, we have a major interstate highway, I-35. Everybody's got one of these. It's under construction all the time. And it's a big nuisance because of that. And the local joke is, the sun's going to burn out in 5 billion years. And that's really too bad because they're going to have to finish I-35 in the dark.

1:49:00Right. No, no, no. That's not happening. You're not going to be able to finish anything.

1:49:07Well, the water's going to evaporate. All life is going to be fried. Even stuff that's really deep in the earth, forget it. It's just because it's going to expand. And look, there's a quote here I want to read about scientific communications. Let's see if I can find it. I finished it. I wonder where your atoms are going to be when all that happens, Vincent.

1:49:29Scattered pretty wide, I think. All of us, right? Yeah.

1:49:34Anyway, I can't find the quote. But it's just basically about how lazy people are. Oh, this is actually another aspect of science communication and people not wanting to believe evidence-based information. You know, and he says basically, you know, people have these views that go in the face of evidence-based information, which tells me they don't want to look at evidence and evaluate it. Right. Which is true. Because it's hard. It's hard to do. It's difficult. And we encounter that all the time, of course, in our science, right?

1:50:10And it's a challenge to science communication because we're all trained to do that. And so we kind of make the, it turns out, unwarranted assumption that if you just explain the facts to people and how you put the facts together, that they'll understand and believe you, but no, they zone out after two seconds, you need a pretty picture and a story that's appealing, okay? And that's why the conspiracy theories stick so hard.

1:50:42All right. That is TWIV 1367. Cool. Such a funny number. You can find the show notes at microbe.tv slash TWIV. You can send us questions, comments, picks of the week to TWIV at microbe.tv. And if you enjoy these programs, we'd love your support, microbe.tv slash contribute. Kathy Spindler is Professor Emerita at the University of Michigan in Ann Arbor. Thank you, Kathy. Thanks. This is a lot of fun. And I forgot to say about my pick that the actual lecture starts about eight minutes in.

1:51:14There's a bunch of introductory stuff because it's the first one of the season and stuff. So if you want to fast forward to that, you can. Rich Condit is Emeritus Professor, University of Florida, Gainesville. He's currently in Austin, Texas. Thank you, Rich. Thank you. And always a good time. I want to point out that Dixon is with us. Dixon's there. We mentioned him earlier. And you said that 1367 is a weird number. Yes, indeed. It's a prime number. It is the 219th prime number.

1:51:45AI just told me that. Wow. No, I wouldn't have known. Are you sure it's right? Oh, yeah. It could be fake news.

1:51:55It could be wrong. They make mistakes.

1:51:58Brianne Barker is at Drew University Bioprof Barker on the Blue Sky. Thank you, Brianne. Thanks. I learned a lot. I'm Vincent Racaniello. You can find me at microbe.tv. I'd like to thank the American Society for Virology and the American Society for Microbiology for their support of TWIV Ronald Jenkes for the music and Jolene Ramsey for the timestamps. You've been listening to This Week in Virology. Thanks for joining us. We'll be back next week. Another TWIV is viral.

1:52:29We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week. We'll be back next week.