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George: Some old some new. Can we get the lights? I'm going to tell you eventually about transmitted/founder viruses, but the story begins earlier than that. I was a young buck coming out of University of Michigan as a physician-scientist, and I wanted to study molecular biology. I interviewed with Phil Leder (1934–2020), who said he'd be happy to have me but I'd have to wait a year. I interviewed with [00:00:30] Bob Weinberg (b. 1942) who was not at all interested, and I interviewed with Geoff Cooper who told me he'd be happy to have me except that, I'd have to work with his girlfriend.

I went to Bob Gallo's lab, I walked into his office at Building 37, he looked up at me he said, "Sure, I'll take you," and I will be forever grateful for that. A little known secret at the time though was that the only person at the NIH who had larger lab than Gallo was Flossie [Wong-Staal]. [laughs] [00:01:00] It's shown pretty well here. Here I am in the background, there's Beatrice. These were the two key people, Flossie taught me everything I know about molecular biology at the time, except what Beatrice [Hahn] taught me. This is back in the olden days and as many of you know, me and B, we have rarely been further apart than this in the last 32 years, those were the major players.

George M. Shaw is an immunologist, molecular biologist, and professor of medicine at University of Pennsylvania, Perelman School of Medicine. He is the husband of Beatrice Hahn.

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Now, I'm going to give you two slides, the bookends of my career. This is my first major paper out of the Gallo lab. (1) Here are BH-10 [00:01:30], Beatrice named her clones after herself, Beatrice Hahn. I was more magnanimous, my clones were full length integrated copies of HIV-1, and they're termed HXB for a human retrovirus. Xba was the cloning site and these were the clones, there you go, it doesn't change.

The other side of the bookend is this, and this is a true story. From that early work in 1984.[00:02:00] Those clones were submitted in August 22, 1984, just before the first cloning work came out of Gallo's lab. It took 32 years for that patent to be approved and awarded. Of course, Bob, fortunately—we've had a large patent portfolio in between that supported this, but there are the clones that came out of 1984. That's a pretty good date, I remind you to have now.

What can you do with those clones? What did we do with [00:02:30] those clones? First thing is we did, we detected HIV-1 in brains of babies and adults with AIDS dementia complex, that was pretty cool. (3) The next thing we did is we discovered the virus quasispecies, [00:02:40] extensive variation in vivo. (4) You could do more, though. Jeff Lifson approached me one day and said, "I've got a new assay [00:02:50] for viral RNA in the plasma." John Mellors mentioned that the B DNA and so forth essays by Chiron and so forth. This is the first accurate quantification [00:03:00] of HIV-1 in human plasma, and it's a great collaboration with Jeff and the late Mike Piatak. (5) Finally, David [Ho] just talked about viral dynamics, I don't need to say anything more about that. (6) But with viral dynamics we could begin to apply that to important questions, and here in 1997 Shirley, Bruce and others had shown that CTLs (CD8 cytotoxic T cells) recognized HIV-1 peptides and proteins. But the first [00:03:30] clear demonstration that, in acute and early infection, that CTLs actively engage the virus, recognized peptides, and let the virus turn over and escape was this paper with Seph Borrow. (7) If we could do that with CTL recognition and escape, we decided we would try it for neutralizing antibodies. (8) People forget that in 2002 John Moore was writing review, after review, after review, after review saying, it's not even clear that [00:04:00] neutralising antibodies are doing anything in HIV-1 infection. This was a pretty indication that they were, and this I've done with Peter Kwong.

That's the past, and the past was good, but the future hopefully is better. Now, I'm going to tell you about the future. We specifically asked ourselves, how could we take these kinds of earlier findings forward and make them relevant to HIV cure and vaccine research? Enter Brandon Keele, if I was a young buck in Gallo's lab, [00:04:30] this guy has horns coming out the top up here. Brandon was such a gift to our lab, he came and did, I think, a three-year post-doc with B and then a two-year postdoc with me, and our labs have never been the same since.

We thought about what Ashley [Haase] had written, and we thought about the concept of HIV transmission. (9) That prevention research—we needed to understand the infection process in humans. As we all know, that's very difficult to do because—here is a vagina, [00:05:00], or could be a rectum, a quasispecies of virus damped into the vagina. All we know is that we have to wait two weeks later to get virus out of the plasma, to try to infer what might be going on in here, what might be the relevant viruses? Brandon and I thought about that and in a paper in 2008—and before the paper came out, of course, we considered—We are simple-minded folks and so, we tried to break this down into a concept or a model. [00:05:30] If you imagine here, the diverse virus quasispecies in semen, blood, wherever, it's got to get across a mucosal barrier generally to infect a new person. And one could imagine a variety of scenarios, you could have replication to fit viruses that are poorly efficient, so their R_0's are just barely greater than one. You could have defective viruses, it's not going to go anywhere, but you could have other viruses. All we know is that exposure leads [00:06:00] to transmission infrequently. We imagined—and again, the only thing we can get our hands around are samples from the blood at day 14 to 28 post-infection. If we could get our hands on those samples, what could we do with them?

The punch line, of course, is that it turned out to be a simple concept. By studying the genetic makeup of these viruses, we can infer the exact 10 kilobase nucleotides sequence, and that's [00:06:30] the inferred proteome of that virus. We can do it in the first patient and we can do it in 100 consecutive patients, and that is a powerful technique. (10)

It's based on coalescent theory, here, you could imagine an infection leading to genetic drift and a wide diversification. But the coalescent process brings it back near to the transmission event or at the transmission event. The theory is good, but we needed a technological breakthrough, because the old fashion way of looking with just peak bulk [00:07:00] PCR just basically led to misinformation in the literature.

#credit, priority

#molecular cloning

#cell culture, tissue culture, immortalized cell line

#patents and intellectual property (IP)

Enter John Coffin, thank you, John. John actually wrote a seminal paper with Sarah Palmer in 2005, I happened to be at an ICAAC (Interscience Conference on Antimicrobial Agents and Chemotherapy) meeting—I guess it was ICAAC, one of the two—and wandered by his poster. Where he was using single genome sequencing, something that has been described by [Peter] Simmonds and Andy Leigh Brown in 1990, and by [James] Mullins. (11) [00:07:30] but for a decade and a half, they didn't quite know what to do with it. And John said, "You can use this approach, to eliminate PCR misincorporation, eliminate Taq-mediated recombination and preserve genetic linkage across genes and in fact whole genomes. He wanted to do that, in chronic infection in order to characterize mutations across pol and protease. (12)

Well, we got the idea we could apply that to acute infection and so here we go. (10) Here is about [00:08:00] two or three weeks after acute infection in a human, we sample the plasma, we use single genome sequencing, these are full-length env genes. We were surprised, stunned but in retrospect shouldn't have been but no one ever did this and these are the env sequence. They're all identical to themselves or different by one of two nucleotides. The inference, the coalescent of these is the virus that crossed the vaginal or rectal [00:08:30] mucosa, end of discussion.

If the person's infected with two viruses, what would you might expect? A collection of viruses here and a collection of viruses here is a highlighter plot. These are all identical or nearly so to themselves, these are all identical to themselves, they happen to be different from these by 3%, but it tells you that two viruses were transmitted. That has enormous opportunities for studying SIV viruses with vaccines [00:09:00] and that's what—There are hundreds of papers being written now about that. If this was a concept that was fun. That led to this paper that Bob sponsored in PNAS, and it launched Brandon on his current career.

Now, I'm going to switch from the past to the future, transmitteed/founded HIV-1 genomes, that's what we just described, what they teach us, and I'm going to describe two very recent discoveries from our laboratory that I think are important for vaccine design, and potentially cure research.

#Taq-DNA polymerase

The first [00:09:30] is designer SHIVs for vaccine and cure research. This work was done by the research associate in my lab, that's been with me for sometime Hui Li, done a great job. This paper just came out a couple of months ago in PNAS that describes this designer SHIV work. (13) In essence, what we did that was different from the last 25 years in SHIV research is that, we took transmitted founder env's, [00:10:00] env's that knew were perfect for transmission in humans. They had every single amino acid of 865, highly evolved so as to work, and other people have taken those transmitted/founder viruses, or primary env's, put them into SHIVs and they generally don't work. They don't work.

We hypothesized because they don't bind rhesus CD4 efficiently, and we came upon the idea of substituting a single amino acid at position 375. That idea came out of Finzi, Kwong, Sodroski's [00:10:30] earlier work, multiple papers, in which they showed that at position 375, which is in the Phe43 cavity, it is under strong evolutionary pressure over the vast expanse of primate lentiviruses. (14) That one amino acid is doing something and there's a whole story about that, about what and why, which I won't go into, it's in that paper I just showed you. But we've said, well, we're going to take [00:11:00] HIV-1 env's and we're going to substitute examples of these amino acids. They're all bulky hydrophobic amino acids to replace the wild type serine, which is small, at position 375.

And this was the voilà result. When we did that here, here's HIV 1 subtype D env and A env—this is the famous BG505 from Rogier Sanders and John Moore and Dennis Burton. (15) [00:11:30] Here's Bart Haynes's CHAVI 505 and CHAVI 83. These are four primary HIV-1 env's that we made into SHIVs. And here's viral load, here's days, up to a couple of years of infection. They all replicate, efficiently and well. All of a sudden, we go overnight from a poor model to a good model.

Here is an example of CHAVI 505 HIV-1 env, constructed into a SHIV infecting three rhesus macaques in red, green, and [00:12:00] blue. We're now looking at the elicitation of autologous tier 2 neuts, neutralizing antibodies, compared with the human, who was infected with essentially the identical env. The human developed over a period of 30 to 40 weeks, titers of autologous neuts of about one to a thousand. The three rhesus animals developed higher neuts faster. It's recapitulating the neutralization that one sees in humans.

And now the really [00:12:30] exciting result. Okay? And I have to walk you through this. I showed you before our transmitted/founder viruses. We infer a transmitted founder/virus envelope in the human subject CH505. We asked the question over a period of a year and a half—that's weeks there—does the virus evolve changes? And are the changes random? Are they sprinkled everywhere, or are they highly non-random selected—These are all amino acid mutations in red tick marks, [00:13:00] and the black is in-dels insertions deletions.

You can see that early in infection, the first month of infection, you have random variation. But very soon you get highly selected changes. Here is a rhesus macaque infected with a SHIV, with the homologous envelope, the identical envelope, except for a single amino acid substitution at 375. Well, it doesn't take statistics or a rocket scientist, to realize that all of these highly selected changes in the human are being recapitulated [00:13:30] in the rhesus macaque. We really dug into that.

Bart Haynes has a Nature paper (16) and two Cell papers (17, 18) all on this one subject, characterizing the types of monoclonal antibodies that developed in the humans, and the antibody specificities of the neuts that led to the development and neutralization breadth. There was one set of antibodies called CH235 and CH103, that each are broadly neutralizing monoclonals from this patient. These are the [00:14:00] critical sites that these monoclonal antibodies target. There were some additional strain-specific antibodies in the subject. They're attacking the base of V3, very common site, V3 glycan antibodies. There were additional V4 here, those sites. Then there were glycans, the glycan SHIV, remember from my earlier paper. These are all key residues or key sites where the autologous response in the human is [00:14:30] attacking the virus, and the virus is escaping. And eventually this kind of evolution driving to bnAb production. Well, what happened in the rhesus macaque?

Well, first of all: nearly identical. And I won't show you the data that not only are the positions identical, the actual amino acids are the same. It's saying that in HIV-1 env evolution in the human is closely recapitulated in the rhesus macaque. [00:15:00] That suggests there's something unique about the envelopes of each primary HIV-1 strain, and in retrospect not surprising. They are all genetically, widely diverse, HIV-1 envelopes are. But they must evolutionarily do two things. They must be able to efficiently engage receptors and at the same time, they must be able to avoid neutralizing antibodies. Every primary HIV-1 env has to find an evolutionary solution to that and they do or they don't exist, [00:15:30] and you can recapitulate that in the rhesus macaque.

Well, that's only one animal. What if we go to two more animals? This is rhesus macaque 6070169 infected with the same SHIV. The human data up here is the same. Here are two more rhesus macaque. Now we're up to three rhesus macaques infected with the same SHIV and what is the result? Well, not hard to figure out. Almost the same. This is a real theme. And I won't show you the data [00:16:00] now. We've done the same studies on BG505. The important vaccine strain that Dennis [Burton] works on, the same story, just a different envelope. The patterns are all different but in the human and the rhesus macaque almost indistinguishable. We did it with a third virus, CHAVI 844 that also made bNabs. In the human, in the rhesus almost indistinguishable. This is a real theme and we think this has importance in the future as a molecular guide for [00:16:30] HIV-1 vaccine design and development.

I think I have five more minutes. I'm going to—how many more minutes? It's three?

Alan Perelson (Moderator): Three.

George: Okay. What's the second story I want to tell you about, that's new. This new story is unpublished now and it's really exciting, because I think it challenges paradigms of retroviral gene expression. There are a couple of Nobel laureates here, I think people who have been studying retroviral gene expression for [00:17:00] 50 years. I'm going to tell you, there's a major part of it that hasn't been discovered before. And this is it, that's kind of exciting, I think. With applications to vaccine, research and to cure research.

The 5' leader sequences of SIV and HIV and other retroviruses, but these two are ones we're studying, otherwise referred to as the 5' untranslated region, encodes in its primary and secondary RNA sequence key regulatory elements [00:17:30] required for virus replication. But it is not known, of course, to encode polypeptide products.

We studied, just like we studied humans over time—this is a macaque, rhesus macaque infected four macaques infected with SIVmac239, et cetera, or 766, the same thing. We looked for—This is the transmitted founder viruses at the top. Again, just like in humans, we're looking for evidence of selection, and we see CTL escape mutants, for example.

But then we looked in the 5' [00:18:00] leader region of the four animals. And what in the world is this? This green thing here and with red interspersed?

These are highly selected changes in the five-prime leader sequence of four animals. Well, where is this? Well, it is in the five-prime leader, in this particular case it is in-- This is the dimerization signal. That's the kissing loop, and this is the STEM loop 1 of the five-prime leader sequence. Here you go from five prime to three prime. There's tar, [00:18:30] for example, there's polyadenylation site, there's the primary binding signal, all the things you recognize as regulatory elements. But this highly selected change there is, in this case, in this 5' leader sequence and it's covered a span of about 30 or 40 nucleotides which is just the right size for a CTL epitope.

We went to a bunch more animals, one, two, three, four, five, six, seven, eight animals, all Mamu-B29 positive [00:19:00] animals. In every case, acute infection, striking selection. This is one of the most striking selections in HIV, SIV literature. If you don't have B29, you don't get the change.

Here's the STEM loop blown up.

There's the kissing palindrome. Here's the STEM loop leading up to it. Just 5' of that. These are one-off AUGs. It's not AUG, it is ACG, and [00:19:30] it repeats, ACG. Those are two, one-off AUGs. Secondly, this is a favorable Kozak [consensus sequence] mutant. It's plus one, two, three, four, so at plus four, you have a G (guanine), and at minus one, minus two, minus three, you have an A. That's a very favorable Kozak.

And to make a long story short, we hypothesized that you have a ribosomal scanning. You're expressing viral peptide from this favorable Kozak [00:20:00]. And a peptide is made, and it just so happens that if you infer in the plus one reading frame here, what would be the amino acids up to here? They are K, G, A, G, R, Y, Q, T, A. That is almost a perfect match to a predicted B29 epitope, and these animals are B29.

Next, we did ribosomal scanning—that ACG ACG sitting right here it's the [00:20:30] strongest positive signal of protected fragments in the 5' leader of this animal, of these the sequence in this animal. If we site-directed mutagenize these two ACG-ACG's to UCG's, so it's now it's a two-off, this is a poor potential start site. This red signal right here, goes to there, so it looks really good.

To quickly move forward, we then use ELISpots to ask the question, can we find [00:21:00] ELISpot reactivity to this particular peptide? It's shown right there, that's the KA9 epitope. This is slightly extended on either side here, some positive controls in the plus two and plus three reading frame, and the minus one minus two minus three—so essentially, we made peptides across the entire 5' prime leader in the plus one, plus two, plus three reading frame, minus one, minus two minus three. The only place you get a signal is in the plus one, and it is this KA9, [00:21:30] same in a couple more animals.

Then we make a tetramer of this inferred KA9 epitope, and we say for tetramer staining of the CD4 and CD8 cells, and you have a hugely positive signal right here a few weeks after infection. The animals are making T cells to bind to this thing directly, granzyme positive, perforin positive cells.

Conclusion, 5' prime leader peptides are similarly ex—no, not conclusion. What about HIV-1? 5' leader peptides [00:22:00] are similarly expressed in HIV-1, and similarly recognizes immunodominant CTL epitopes in humans. Here is the 5' leader sequence essentially of HIV-1, and the conclusion I'm not going to show you the data for lack of time. The HIV-1 5' leader expressed the short polypeptides, many of them 9 to 20 amino acids in length across its full length, from TAR all the way through to gag matrix, and encoded, [00:22:30] by all three plus-sense reading frames. These peptides are recognized by CTL's in an MHC class one restricted manner. That's in humans, I'm not going to show you the data.

Can 5' leader polypeptide serve as a novel vaccine target? Now I've finished my talk here. This is a novel part of the genome now. One thing to keep in mind, the 5' leader sequence, illustrated right here, is part of every RNA transcript, whether it's spliced or [00:23:00] unspliced. Whenever HIV is expressed or SIV is expressed, you've got the 5' leader on it.

We took the 5' prime leader of SIV macaque—now it has it in the inferred reading frame, plus one plus two plus three, it has stop codons in there, just by chance, because it's not supposed—it didn't make a real functional protein. These are, I'm going to tell you how DRiPs (makes defective ribosomal products). We got rid of the stop codons and we make a polyprotein construct from plus-strand one [00:23:30] to reading frame one, two and three. We put them into Louis Picker's rhesus CMV, so we don't have to worry about the mamu restriction of the animals and we immunize animals. In this case, we immunize five animals either with, reading frame one construct, reading frame two construct, reading frame three, or all three.

I'll just show you the CD8 T cell response, now. This is [00:24:00] our ICS studies, intracellular cytokine staining. You can see if the animal was vaccinated with a reading frame 1 insert, it makes a strong CTL response to—or a strong cellular response to reading frame one peptides. To reading frame two peptide, look at that 2% of the circulating cells in the vaccinated animal. To reading frame three 4% of the cells. A robust [00:24:30] CTL response. If in fact you vaccinate with all three you do all three.

But the key experiment is this, and this is my last slide. If we now take rhesus CD4 T cells, infect them with SIV, the question is can a productive infection of the target cell express naturally the 5' leader peptides from a natural infection, and can they be recognized and killed by the effective cells [00:25:00] I just showed you in the previous slide?

The answer is: most definitely and most robustly. Here are animals that were vaccinated again with reading from one, two, three, or all three, and they are now reacting with SIV uninfected homologous targets, CD4 T cells, or with homologous HLA matched SIV infected cells. And so the vaccinated animals are recognizing and killing, in a robust fashion—[00:25:30] 5% of its effective CD8 cells, are recognizing these targets.

I'll end right there. Conclusions. Cryptic peptide expression from 5' leader sequences represents a novel DRiP (Defective Ribosomal Product). We don't think these are actually making a functional protein. This is Jon Udell, over the last 20 years looking at DRiPs, making studies on DRiPs. As a new paradigm in SIV, HIV gene expression with implications for viral immunopathogenesis [00:26:00] and vaccine design. My acknowledgements are many funders and collaborators, but I'll leave it with this, this is my major partner.

[applause]

Anchor

	qa
	qa

Alan Perelson (Moderator): Thank you George. I guess we have time for a couple of questions or just eating into our coffee break time. Back there.

Andrew Rice: Why do you think the peptides have no function?

George: I just think [00:26:30] that for several reasons. First of all in the SIV system we have knocked out the reading frame in multiple different ways and we can't see a discernible effect on virus replication of that particular short 14 amino acid peptide that's expressed. Now—[crosstalk]

Andrew: Allocation in vitro?

George: Pardon. In vitro. Then the other thing is if you take any 10 HIV-1 viruses and look at the 5' leader sequence, they're all different. They've all [00:27:00] got stop codons in the inferred reading frame one, two, and three at different places. There are some shared open reading frames, but mainly they're just multiple open reading frames in all three events in plus one, plus two, plus three. It's much more likely that this is a consequence of the fact, that on the one hand, the ribosome is using the scanning mechanism probably in this case [00:27:30] more than the iris approach.

It's scanning this 5' prime leader but the 5' prime leader has evolved obviously to perform all these regulatory functions. My guess at this point is that these peptides that are expressed are an epiphenomenon. They're DRiP's, defective ribosomal peptides, that's what my data suggest right now. Of course, I'm open to look for function.

Alan: Paul.

Paul Bieniasz: [00:28:00] Does the existence of these open reading frames affect the level of expression of the authentic reading frames whose AUG's are 3' primes?

George: Yes, so there's a—the guy from Burke has actually done—you didn't know about this—he did, he asked that question in different ways. If you look evolutionarily at the 5000 HIV-1 sequences in the database, there are only three or four that have a single AUG [00:28:30] in the 5' leader sequence. And we're not even sure the sequences are correct. It's telling you that there's a strong strong evolutionary pressure not to have any AUG's in the 5' leader sequence, because of just the thing you're saying. They would impair ribosomal processing to get to the real genes that's got to be expressed. Burke went in and selectively put in multiple AUG's very carefully, thoughtfully and showed that when he [00:29:00] puts in AUG's at these different spots, instead of the one-off AUG's, that it actually impairs gag expression for example.

Alan: One last question from Bob Siliciano.

Bob Siliciano: Was the CMB experiment done with the vector that give non-classical restricted responses.

George: Yes, this is Louis [Picker's] thing. We actually have a pre-clinical trial underway right now. Which instead of looking at five animals were looking at 24 animals, in which they have been vaccinated [00:29:30] with just the constructs I've talked to you about there. Which are the identical constructs that he uses for all of his other work. The question is, will a rhesus CMV vectored 5' leader only—can we protect animals? If we protect animals with this 5' prime leader, it is potentially a game-changer, because as everyone knows, half of Louis's animals with his canonical inserts are protected half not. He doesn't know why, and he speculates well maybe it's a kinetic thing. We think the [00:30:00] 5' leader, because it's expressed in every cell, in every RNA, that it may give a kinetic advantage. If it works, we may put them together.

Bob: Was a leader present with a HLAE though or was that--?

George: Pardon?

Bob: Was the leader peptide presented with HLAE or classical class one molecules?

George: Well, HLAE, and HLE-2. It's definitely not classical, it's doing exactly the same thing that is others.

Alan: Let's take a 15 [00:30:30] minute coffee break, and try to be back by 11:00. Thank you, George.

[applause]

[00:30:37] [END OF AUDIO]

#structural biology

Citations

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