Session 10: The Immune Response in Health and Disease

00:00:11;20 Well, hello.
00:00:12;20 My name is John Schiller.
00:00:13;27 I work in an intramural program of the National Cancer Institute.
00:00:18;14 And the topic of this presentation is to address the question of why HPV virus-like particles
00:00:26;25 work so well.
00:00:29;12 This is an extension of the previous talk I gave, that described the basic biology of HPV,
00:00:35;09 its association with cancer, and the development of the vaccines.
00:00:41;03 Now, in that talk, I went over some of the evidence of why this vaccine worked so well.
00:00:48;25 First of all, it induces long lasting and virtually complete protection from
00:00:53;25 incident infection and disease by the vaccine-targeted types, and, remarkably, it does this
00:01:00;07 even after a single dose.
00:01:03;01 It's the first subunit vaccine that appears to have these characteristics.
00:01:09;03 It induces sterilizing immunity in most subjects, so that they never get infected.
00:01:15;14 And this makes it the first sexually... first vaccine that's highly effective against
00:01:21;24 a sexually transmitted mucosal infection.
00:01:26;07 So, why is it important to try to understand why the vaccine works so well?
00:01:30;28 Well, as somebody who developed... helped develop this vaccine, it's of basic biological interest.
00:01:38;14 I mean, we want to know just for curiosity.
00:01:41;01 But above that, determining why the vaccine works so well provides biological plausibility
00:01:48;00 for the potential of using a single dose subunit vaccine in the future.
00:01:52;14 And importantly, we hope would inform development of future subunit vaccines, so they could
00:01:58;12 have these same remarkable characteristics as the HPV VLP vaccine.
00:02:04;14 So, as most things in biology, explanations are multifactorial.
00:02:10;20 But in this case, we think that there are three primary reasons why these vaccines
00:02:15;26 work so well.
00:02:17;07 And I'll be going into the first two in some detail.
00:02:20;08 The first is that the vaccines are exceptionally good at inducing neutralizing antibodies,
00:02:26;15 which we think is the main effector mechanism for the activity of this vaccine.
00:02:31;23 Secondly, the unique infection mechanism that vac... that HPVs use make them
00:02:39;10 exceptionally susceptible to neutralizing antibodies.
00:02:42;22 And we didn't know either of these things when we first started developing this vaccine.
00:02:46;18 And finally, just basic molecular biology, is that HPVs have DNA genomes, and so
00:02:55;08 they don't rapidly evolve to evade antibody responses.
00:03:00;10 The mutation rates, since they use the cellular machinery to replicate the DNA,
00:03:05;10 is basically the same as our own genome.
00:03:08;07 So it's much different than RNA viruses such as HIV or hepatitis C, where they replicate
00:03:15;07 as a swarm.
00:03:16;17 And so trying to inhibit infection... you're basically trying to attack a moving target.
00:03:24;06 Now, there are several reasons why we think that antibodies are likely to be
00:03:29;08 the main mediators of immune protection.
00:03:32;26 First of all, high levels of virus-neutralizing antibodies are routinely generated by VLP vaccination.
00:03:39;00 And if you look at the cross-protection seen in the clinical trials against types
00:03:43;28 that are not specifically targeted by the vaccine, it largely mirrors the antibody-mediated cross-protection
00:03:52;04 we see in in vitro assays.
00:03:54;14 And I'll describe those in vitro assays a little bit later.
00:03:57;01 And I think most importantly, that protection from the vaccine can be passively transferred
00:04:05;07 in the serum from a vaccinated animal to a naive indiv... animal in animal challenge models.
00:04:15;19 And again, I'll show you some evidence for that a little bit later.
00:04:18;17 Now, this vaccine is remarkably consistent in inducing antibody responses.
00:04:25;24 So, this shows the sero-conversion rates in women to individual HPV VLP types for the
00:04:34;26 Merck vaccine Gardasil.
00:04:37;11 And what you can see is that 100%... basically 100% of women sero-convert, to a rounding error,
00:04:44;04 for each of the four types in the vaccine.
00:04:48;03 Now, I'm gonna give you a little bit of a primer on what we expect from antibody responses
00:04:54;27 to different types of situations.
00:04:57;20 And so, if you look at the antibody responses to a real virus infection, what you see is that
00:05:03;06 the titers go up very rapidly within a few days to weeks.
00:05:09;00 And then they... the responses start to decay, initially quite rapidly as short-lived
00:05:15;14 plasma cells in the blood die off, but then this is followed over a period of years by
00:05:21;14 the generation of long-lived plasma cells which can then go to the bone marrow and
00:05:26;25 continually pump out antibodies indefinitely.
00:05:29;17 Now, in contrast to this is what is normally seen with a subunit vaccine such as
00:05:36;28 tetanus toxoid or diphtheria toxoid, where, again, you get this early peak in a number of weeks,
00:05:43;18 and then a decay, a rapid decay, and then a slower decay, but the decay keeps going on.
00:05:50;03 We don't reach this plateau phase as we normally see with a real virus infection.
00:05:55;15 Now, the surprising thing is if you look at the antibody response, in this case over ten years,
00:06:02;08 to a VLP vaccine, in this case Cervarix.
00:06:07;05 And what you can see is that, especially in the 15-to-25-year-olds, which are
00:06:11;04 the targets for the immunization programs, that after that initial decay there's a plateau,
00:06:18;05 a stabilization of the antibody responses, that for all the world looks like a real virus infection.
00:06:24;08 But it's important to point out that this is a true subunit vaccine.
00:06:28;17 It's composed of only one protein, 360 copies of the L1 protein that assemble into this
00:06:35;13 virus-like particle.
00:06:37;14 And even more remarkably, we get this stabilization, in this case out to 7 years, the longest we've
00:06:44;01 been able to look, by even a single dose of the HPV vaccine.
00:06:51;05 And if you look at the difference between three doses and one dose,
00:06:54;24 the difference in antibody responses at the plateau stage is only fourfold.
00:07:00;20 And it still remains almost a log higher than what we see after natural infection.
00:07:07;21 Now, this data that I just showed you is from the Costa Rican trial, NCI-sponsored trial,
00:07:15;14 of Cervarix, which contained a more powerful adjuvant -- immune stimulatory molecules --
00:07:23;03 than did Gardasil.
00:07:24;04 So, it was an interesting question to know whether we could get the same type of stabilization
00:07:30;06 of antibody response after a single dose of Gardasil.
00:07:34;28 And what you can see here is that there's emerging evidence, now out to four years
00:07:39;23 in an interrupted Indian trial, that Gardasil also can generate a stable antibody response
00:07:46;14 despite using a simple aluminum salts adjuvant.
00:07:49;13 And the difference between threefold... three doses and one dose is, again, only about fourfold.
00:07:58;26 Now, the data I've shown you so far in terms of the antibody response was data that was
00:08:03;25 mostly done with what's called an ELISA, which is an in vitro assay that measures antibodies
00:08:11;08 that bind to the antigen, in this case the VLPs, and so can measure both antibodies
00:08:17;20 that have the ability to inhibit infection and also those that can't inhibit infection,
00:08:22;10 that may be low... low binding activity or just not of the right type.
00:08:27;17 And so it's also important to look at the ability of the antibodies induced
00:08:31;24 in these trials to prevent infection.
00:08:34;24 And to do this we use what's called an in vitro neutralization assay, which then
00:08:41;01 measures only antibodies that can prevent infection.
00:08:44;00 Now, HPV, real HPVs, you really can't use them in a neutralization assay for two reasons.
00:08:52;08 One, there isn't a good source of the real HPVs.
00:08:54;27 And secondly, if HPVs infect a cell in culture, it doesn't do anything that's easily scored
00:09:01;22 as an infectious event.
00:09:03;25 And so we needed to develop a technology of high titer what we call pseudoviruses,
00:09:10;11 that rather than transmit the real genome they transmit a marker plasmid, which,
00:09:15;23 when the gene is ex... the marker gene is expressed is easily scored as an infectious event.
00:09:21;08 And several years ago, we developed a technology for generating these high titer pseudoviruses
00:09:28;11 for use in neutralization assays.
00:09:30;14 I'm just gonna go over really quickly how this occurs, because there's a few little tricks involved.
00:09:36;18 So, what we do is we transfect with the marker plasmid, which is less than 8 kb,
00:09:43;00 which is the maximum size that can be packaged by the HPV variants.
00:09:48;24 We co-transfect that with a plasmid that contains the genes for the two virion proteins,
00:09:55;22 L1 and L2, and we need both in order to generate infectious particles.
00:09:59;28 The VLPs for vaccination, incidentally, to remind you, are only L1.
00:10:06;09 And the trick is, one, we codon modified the genes so that they can be more highly expressed,
00:10:13;08 because we get rid of negative regulatory elements in the genes that normally
00:10:17;17 restrict expression to terminally differentiated epithelium, and we put it on a plasmid that's too big
00:10:22;13 to package.
00:10:24;12 The other trick is that the plasmid we want to have packaged, we want to make
00:10:28;26 lots of copies so we can generate a lot of virus.
00:10:32;00 And so we put on is the SV40, which is a polyomavirus... the origin of replication from that virus,
00:10:38;11 and transfect cells that express SV40 T antigen.
00:10:43;02 And so it's able to induce replication of this plasmid.
00:10:47;17 We get a lot of copies of the plasmid replicated, packaged by the virus-like particle, and generate,
00:10:53;20 as you can see, very high titer stocks, 10^10 per mL.
00:10:57;07 So, one of the interesting things, then, was to look at the relationship between binding
00:11:03;24 and antibodies in the neutralization... in an ELISA assay, shown on the x-axis,
00:11:09;24 with neutralization titers that presumably correlate better for protection.
00:11:16;13 And what you can see here is that with the 3-dose data that there's a very strong correlation
00:11:22;02 between neutralizing titers and ELISA titers.
00:11:24;22 They're virtually one-to-one.
00:11:27;03 But even more interesting, after a single dose there's the same relationship.
00:11:31;08 So, what we conclude is that the quality of the antibody responses doesn't increase
00:11:38;00 with boosting.
00:11:39;19 You get just as good a quality of the antibodies at one dose as with three doses.
00:11:46;14 Another interesting observation we made in these trials is if you look at the antibody
00:11:50;17 avidity, which is the strength of... which is basically a measure of the strength
00:11:55;12 at which the antibodies, the serum antibodies, bind their target antigen, in this case the virus-like particles.
00:12:01;04 And the way you measure this is that you do an ELISA under more and more stringent conditions
00:12:07;16 by washing it with molecules that are called chaotropes, such as guanidine hydrochloride.
00:12:14;03 And we made the surprising observation that, over time, if you look at the three-dose...
00:12:20;28 over the first four years, there was a continual increase in the affinity of the antibodies
00:12:27;05 for the antigen.
00:12:29;06 But even after a single dose, we get the same type of affinity maturation, because at 48 months
00:12:34;15 there's almost no difference between the affinity measurements for three versus one dose.
00:12:39;23 But then we get a stabilization, so that between year four and year seven there's
00:12:44;24 essentially no difference.
00:12:46;07 But again, part of the reason we think one dose will be able to work well to
00:12:50;15 prevent infection is, again, the quality of the antibody responses doesn't seem to go up with boosting.
00:12:56;23 So, from a biological point of view, from an immunological point of view, this is
00:13:01;02 a very interesting observation, because we wouldn't expect affinity maturation to continue for a long time.
00:13:07;28 So, it raises the question, where is this occurring and how is this occurring?
00:13:12;07 And so, normally what happens is when you have a naive B cell it sees the antigen
00:13:16;28 in the prime.
00:13:18;01 It gets activated, goes to the lymph nodes, and goes through what's called
00:13:23;06 a germinal center reaction, which increases, by inducing [somatic hypermutation], increases the avidity
00:13:29;14 of the antibodies overall.
00:13:32;02 And so if we're getting continued avidity maturation, does that mean that we're getting
00:13:37;04 the VLPs to be maintained on the antigen-presenting cells to B cells in the lymph node,
00:13:44;03 the follicular dendritic cells, for four years?
00:13:46;17 It's possible, but we think that's kind of unlikely.
00:13:51;08 But what happens after the germinal center reaction is that you generate three types
00:13:55;15 of cells.
00:13:56;18 You generate long-term... short-term plasmablasts in the blood that pump out antibodies
00:14:01;19 for a while and then die.
00:14:03;18 You generate memory B cells, which are the reserve component, so they don't make antibodies
00:14:10;26 until they're re-exposed to antigen.
00:14:13;04 And then you generate, as I mentioned, the long-lived plasma cells, which mostly reside
00:14:18;08 in the bone marrow.
00:14:19;08 And our guess is that what we're looking at here is that we're looking at preferential survival
00:14:23;12 of the plasma cells that have gotten strong signals through their B cell receptors
00:14:30;02 for survival in the bone marrow niche.
00:14:36;08 Now, the central question that we're addressing is, why do the virus-like particle vaccines
00:14:44;17 act so differently than your typical subunit vaccines like tetanus or diphtheria toxin?
00:14:50;20 And we believe that the most important feature is that the immune system, especially the
00:14:56;16 B cell compartment, has evolved to recognize the dense, repetitive protein array
00:15:04;08 that's displayed on these virus-like particles as dangerous microbial structures.
00:15:09;13 Such that, when the B cell receptors, which are just the antibodies that are on the surface
00:15:15;04 of a particular B cell, when they engage monomeric proteins you generate weak activation signals,
00:15:24;07 generally low-level antibody responses that don't last very long.
00:15:28;15 In contrast, when you oligomerize the B cell receptors by their interaction with
00:15:35;17 highly repetitive antigens like the virus-like particles, this leads to strong survival and proliferation signals,
00:15:44;11 high levels of antibodies, and long durations.
00:15:47;21 So, we believe that this oligomerization of the B cell receptor by the antigen is key
00:15:54;23 for inducing long-lived plasma cells.
00:15:58;13 And there was... the hypothesis was actually made, interestingly enough, by Bachmann and Zinkernagel
00:16:07;19 basically about the same time, the same year that we developed the virus-like particles,
00:16:11;18 that B cells specifically recognize particulate antigens with epitope spacing
00:16:17;11 of 50-100 Angstroms, because this spacing is common on microbial surfaces, for instance
00:16:23;21 the viral... virus shell, like our VLPs, or on bacterial components like bacterial protein.
00:16:30;16 But protein complexes with this spacing rarely if ever occur in vertebrate animals,
00:16:37;12 at least on surfaces that are exposed to the systemic immune system.
00:16:41;03 And so you can sort of think of the B cell receptors as having been evolved as antigen-specific
00:16:46;07 pattern recognition receptors.
00:16:48;17 Now, VLPs also do other good things in addition to sending strong signals through the B cell receptor.
00:16:56;02 They have just the right size for efficient trafficking to lymph nodes, where they can
00:17:00;04 engage B cells and T cells to start an immune response.
00:17:04;07 They're preferentially and readily phagocytized by antigen-presenting cells because of their size,
00:17:10;20 so that they induce strong T helper responses.
00:17:15;10 And lastly, their poly-valency leads to stable binding of natural low-avidity IgM and also complement.
00:17:23;04 And that... normally, if you had a monomeric antigen, this low-avidity natural IgM would just fall off.
00:17:30;18 But because you have five surfaces that now can be engaged, it's going to have a semi-stable complex,
00:17:35;09 which... which has been shown to promote their acquisition by follicular dendritic cells
00:17:41;00 and prime an antibody response.
00:17:44;23 Now, it's important to point out that not all virus-like particle vaccines are
00:17:52;02 created equally with regard to the immune response.
00:17:55;15 So, a good example of this is the hepatitis B vaccine, which is a very good vaccine,
00:18:01;17 but if you look at the response in humans or in animals it's not as immunogenic as
00:18:07;24 the HPV VLP vaccine.
00:18:09;00 For instance, one dose induces a poor antibody response in many individuals.
00:18:13;28 And even if you give three doses, the titers continue to wane over time.
00:18:19;09 But the reason this vaccine works well is that it induces a good memory response.
00:18:24;05 And so, if you do get exposed to hepatitis B, it may induce a little bit of an infection,
00:18:29;01 because you don't have enough onboard antibodies from long-lived plasma cells, but these
00:18:34;02 memory B cells kick in, generate a whole bunch of plasmablasts that generate new antibodies
00:18:39;26 that control infections, so it protects from disease if not initial infection.
00:18:44;28 And so it's interesting to think about why the HPV structure works really well and
00:18:50;23 the hepatitis B vaccine structure doesn't work so well.
00:18:53;17 And the structure is shown here.
00:18:55;18 And there are quite a few differences between the two structures.
00:19:02;08 For instance, the hepatitis B particles have far fewer repeats: 24 spikes as opposed to
00:19:09;04 360 copies of an element.
00:19:11;26 So, there may be too few repeats.
00:19:15;20 It's floating in a lipid membrane, rather than being very rigid and structural.
00:19:20;11 And so that... this may inhibit the cross-linking of the B cell receptors.
00:19:25;05 It may simply be too small.
00:19:26;20 It's 22 nanometers versus 55 nanometers for an HPV.
00:19:31;06 And also the key antigenic determinants may not all be folded correctly when it's
00:19:35;21 made in the hetero... heterologous systems, for instance in yeast, where most of
00:19:40;01 the hepatitis B vaccines are made.
00:19:42;00 So, to conclude, the HPV immunogenicity... you know, we think that the VLP... the HPV vaccine
00:19:51;00 is so immunogenic because of the VLP structure.
00:19:55;28 And perhaps we've had too low of expectations of VLP vaccines in the future, because these
00:20:01;18 expectations were based on simple subunit vaccines that take multiple doses to protect
00:20:07;27 and don't protect long term without boosts.
00:20:10;26 But maybe the VLPs should be compared to acute virus infections and the effect of live virus vaccines
00:20:18;13 that also have the high-density virus-like display of their surface elements,
00:20:24;15 and in some cases can protect even after a single dose.
00:20:29;16 So now, turning to the virology of the system.
00:20:32;27 As I described in my last talk, HPV viruses have a very unusual life cycle, in which
00:20:41;17 they can only produce their virions, complete their life cycle, in a stratified squamous epithelium.
00:20:48;14 And so, again, the reason they do this, presumably, is because these virus structures are very immunogenic.
00:20:55;02 And by delaying expression and assembly until the terminal differentiation of the epithelium,
00:21:01;24 where they are not under close immune surveillance, they're able to avoid induction of a
00:21:07;25 potent neutralizing antibody response and also T cell responses to the particles.
00:21:13;11 And so because of this, the virions are seldom exposed to the systemic immunity.
00:21:20;09 They... their escape mechanism is essentially ignorance.
00:21:23;18 And so, now, this becomes the... the heel... the Achilles' heel of the virus, because the
00:21:30;01 virus has not evolved defenses against systemic exposure of their virion components.
00:21:38;05 For instance... which we can readily do by taking these virus-like particles and injecting them
00:21:42;17 parenterally, intramuscularly.
00:21:44;04 So, one of the questions that came up when we were developing this vaccine is,
00:21:51;04 how could the antibodies we generate by this vaccine, which are systemic, they're mostly IgG floating around
00:21:57;24 in the blood, protect against a local mucosal infection, for instance of the cervical genital tract?
00:22:06;13 As many of you know, the protection at most mucosal sites is mainly mediated by IgA.
00:22:13;09 And systemic immunization is very poor at inducing IgA.
00:22:16;26 Now, we think that this vaccine can protect from local mucosal infection by two basic mechanisms.
00:22:25;11 First of all, the cervical vaginal tract is unusual in that about half the antibodies
00:22:30;05 in cervical vaginal mucus are actually IgG, which are delivered across the epithelium
00:22:38;22 by the neonatal Fc receptor, which is in the epithelial tissue of the cervical vaginal tract.
00:22:45;16 Incidentally, it's the same receptor that functions to pump antibodies
00:22:51;06 from the mother to the fetus across the placenta.
00:22:54;22 And the other mechanism that we think, that actually may be more relevant, is the fact that
00:23:00;27 in order for this virus to infect it has to enter through sites of trauma because
00:23:07;12 it needs to initially bind the basement membrane that divides an intact epithelium from
00:23:14;01 the underlying dermal compartment.
00:23:15;22 In a minute, I'll explain why we think that this needs to be the case.
00:23:21;09 And so, when the virus has to bind the basement membrane, it'll be exposed to direct exudation
00:23:31;02 of interstitial and capillary antibodies at this site of trauma.
00:23:35;18 And we think that that is actually the main reason how the vaccine works, in part
00:23:41;12 because the vaccine is very good at protecting against genital warts that are caused by HPV6 and 11,
00:23:47;11 and many of those occur on cornified skin that isn't covered by mucus.
00:23:53;23 So, we know that this second mechanism must be sufficient for protection.
00:23:58;22 To show how the virus actually infects its target tissue, the cervical vaginal epithelium,
00:24:04;24 we developed a mouse model.
00:24:06;11 And in this mouse model, what we do is that we infect with these HPV pseudoviruses.
00:24:12;19 For instance, we can infect ones with red fluorescent protein so you can see
00:24:17;14 the fluorescent tissue or individual cells microscopically.
00:24:21;17 Or we can infect with pseudoviruses that express luciferase, and by putting in
00:24:26;17 the substrate for luciferase, luciferin, we can see that... whether we have infection or not.
00:24:32;04 And what we found is that in order to get infection, we need to get physical or chemical
00:24:37;13 disruption of the tissues.
00:24:39;13 And so the question was, well, why do we need to disrupt the tissue in order to get infection?
00:24:45;13 And this is one of the more surprising things that I've found in all my studies of papilloma viruses.
00:24:51;23 In that, if you looked at intact tissue, whether they're stratified squamous epithelium
00:24:57;17 or simple columnar epithelia, there's absolutely no binding to intact tissue.
00:25:02;27 Now, in... the virus is shown here in red... in green, because we use a little trick
00:25:08;12 where we can label it with a fluorescent dye that emits green upon excitation.
00:25:15;21 And if we get infection, we can... we... the cells turn red, because we transduce the RFP gene.
00:25:22;15 So, where the [viruses] are is green in the next few slides.
00:25:25;14 Where we get infection, the cells are red.
00:25:28;23 But what we found is that as soon as we disrupted the epithelium, there was very strong binding
00:25:35;17 to the basement membrane that separates the epithelium from the dermis.
00:25:42;06 You can see it lights up like a Christmas tree.
00:25:44;08 And it's... it's really remarkable that even the basolateral surface of the epithelium
00:25:48;13 -- which is just below the word epithelium in the left-hand panel --
00:25:52;11 there's absolutely no binding initially.
00:25:55;03 But after a relatively long period, about a day, you can see that there's now,
00:25:59;12 shown by the arrows in the middle panel... there's now transfer to the cells.
00:26:03;14 And in a period that, again, takes between two and three days, we can start to see
00:26:08;19 infected cells that are shown in red.
00:26:13;28 And so, to make a long story short, we went on and characterized this process and...
00:26:18;10 why does it need to bind the basement membrane?,
00:26:20;25 what happens?,
00:26:22;09 and how does it eventually infect the keratinocytes?
00:26:26;01 And so what happens is that initially the virus can only bind to specifically modified forms of
00:26:31;18 heparan sulfate proteoglycans that are on the basement membrane but not on
00:26:36;28 apical surfaces or basolateral surfaces of epithelial cells.
00:26:41;04 This induces a conformation change in the capsid which exposes the minor capsid protein, L2,
00:26:46;21 which is shown in yellow here, to cleavage by a protease called furin.
00:26:53;17 And this causes the external portion of L2 to flip out of the way, expose a binding site
00:26:59;22 on the major capsid protein, L1, that can now engage a keratinocyte-specific receptor
00:27:06;13 and do infection.
00:27:08;14 But the important point of... in terms of antibody-mediated protection, is that
00:27:12;21 this whole process takes hours.
00:27:15;10 This is by far the slowest infecting virus we've ever come across.
00:27:19;05 Most viruses infect in seconds to minutes.
00:27:22;07 And so it provides an extremely long opportunity for antibodies to interrupt that process.
00:27:31;06 And we actually then went on and looked to see, if you take a vaccinated animal,
00:27:35;05 what happens to the virus if you put it in the... in the female genital tract, in comparison
00:27:39;20 to no antibodies?
00:27:41;19 So, mice incidentally generate about the same titers of antibodies as humans,
00:27:45;17 so it's kind of a good model.
00:27:47;17 And so, shown on the left, you can see that that's what happens in a mock vaccinated animal,
00:27:52;10 where you see strong binding to the basement membrane.
00:27:54;23 In the middle, you see what happens to the particles in a mouse that had been vaccinated.
00:28:01;12 And you can see you get these little funky dots all over, some on the tissue, some not.
00:28:08;14 And we found, by doing co-staining with other markers, that mainly where those viruses are...
00:28:15;24 is that they're attached to neutrophils and monocytes/macrophages.
00:28:20;04 So, we think that what happens is that, in the normal levels of antibodies that
00:28:24;08 would be induced in a woman... that they're enough to coat the virus such that it can't interact
00:28:31;04 with the basement membrane.
00:28:32;24 And in floating around, the antibodies, the constant region of the antibodies, serve as handlebars.
00:28:41;00 And they get gobbled up by these phagocytic cells.
00:28:43;19 Now, one thing we were interested in, and that is, what happens when the antibody titers
00:28:48;05 get lower, at the plateau stage?
00:28:51;13 And will the fourfold difference between the antibody titers between three and one dose
00:28:57;06 influence the long-term protection?
00:29:00;07 And to do this, we did what are called passive transfer experiments.
00:29:03;06 So, we vaccinated a rabbit with the VLPs, took out the sera, and then diluted that sera,
00:29:11;19 transferred it to a naive mouse that hadn't been vaccinated, and then challenged
00:29:16;11 that mouse with the pseudoviruses.
00:29:17;14 And asked the question, how much can the sera from the rabbit be diluted and still protect
00:29:24;17 against high-dose intravaginal challenge with our pseudovirus?
00:29:29;03 And this result was the most surprising result I think I've ever had in my career.
00:29:35;23 And what we found is that when you dilute the sera, even if you dilute the serum 10,000-fold,
00:29:42;07 you can still get protection from high dose challenge.
00:29:44;21 So, that rabbit... the antibody concentration in that rabbit was 10,000 times higher
00:29:50;17 than it needed to protect in that particular animal.
00:29:55;00 So, rabbits produce about 10 times higher responses than mice or humans.
00:30:01;00 And so from this, we would predict that humans are at least a couple orders or three orders
00:30:05;13 of magnitude higher in levels of antibodies than needed to protect it from challenge,
00:30:10;04 if you believe that this model replicates what's happening during natural infection.
00:30:18;06 And so it has also been interesting to look at... well, at these low levels of antibodies,
00:30:21;28 how does the vaccine protect?
00:30:23;09 Is it the same mechanism or something different?
00:30:25;22 And so at... when we transfer high volumes of serum, just a 100-fold dilution, you basically...
00:30:31;06 it looks like the same thing as in a vaccinated animal, where you don't get basement membrane binding,
00:30:36;24 you get this clumpy stuff that's binding to neutrophils, and you don't
00:30:41;06 get any basement membrane binding.
00:30:43;18 But at low levels, there's something different.
00:30:45;28 We actually see binding to basement membrane, albeit maybe at lower levels.
00:30:50;14 We see this conformational change to expose the L2, which is shown in the middle lower panel,
00:30:55;20 but we never see stable acquisition by the keratinocytes.
00:31:02;13 And so there could be two explanations for this.
00:31:04;28 One is that while it's sitting on the... on this basement membrane for hours, the neutrophils
00:31:11;21 are still able to come in and gobble up.
00:31:13;20 And incidentally, since these are points of trauma, these are also areas that are
00:31:17;06 going to attract neutrophils and monocytes/macrophages.
00:31:21;14 Or the levels of antibodies needed to block cell surface association are just less than
00:31:28;17 the number of antibodies needed per virus to block HSPG binding to the basement membrane.
00:31:36;28 But in either way, it looks like there's a different mechanism of protection at low levels
00:31:42;00 of antibodies.
00:31:43;06 To conclude, we then went on and looked at what levels of antibodies were needed
00:31:50;14 for protection in the in vitro neutralization assay, which we think mainly prev...
00:31:56;01 prevents infection by this binding... covering up the cell surface binding sites, and protection in vivo.
00:32:04;17 And what this slide shows is that you can get protection in vivo at over
00:32:11;18 100-fold lower levels of antibodies than what you can get in vitro, despite the fact that in vivo
00:32:19;05 we use more challenge virus than we do in vitro.
00:32:22;09 So, what this says is that, clearly, the in vitro assays are missing some potent mechanisms
00:32:28;24 of infection that are happening in vitro... in vivo.
00:32:32;24 And we think that this may be due to this activity of these phagocytes.
00:32:37;20 But because of this type of data, we basically believe that if you can detect antibody responses
00:32:43;17 in women they're going to be strongly protected from challenge from natural infection.
00:32:51;02 So, to conclude, the VLP vaccines are very effective at preventing virus infection
00:32:58;27 for two main reasons.
00:33:01;07 The VLP structure is exceptionally potent at inducing durable neutralizing antibody responses.
00:33:08;10 And we think that this observation is actually going to be translatable to other future prophylactic vaccines.
00:33:15;07 At this point, if you want to make a vaccine that induces consistent and durable high levels
00:33:20;20 of antibodies, I think you almost have to try a virus-like displayed vaccine.
00:33:26;04 And many, many vaccines now being developed are now using this principle in future-generation vaccines.
00:33:33;06 But the other reason is that the slow virus infection process makes it... and the fact
00:33:38;00 that it has to initially bind sites of trauma, make it very susceptible to inhibition by antibodies.
00:33:45;00 Now, this is going to be much less translatable to other viruses, where they infect much more readily.
00:33:51;15 But the revol... we think that, overall, the results provide a rationale for HPV VLPs
00:33:57;11 the first subunit vaccine that may be able being to induce durable protection after a single dose,
00:34:02;20 and certainly inform development of other subunit vaccines.
00:34:06;10 Thank you very much.

00:00:07.18 My name is Lalita Ramakrishnan and I'm a professor of Immunology and Infectious diseases at the
00:00:12.17 University of Cambridge.
00:00:14.11 I work on tuberculosis, and I'm going to introduce you to this disease and share with you some
00:00:21.00 vignettes about the curious pathogenic personality of its causative agent, Mycobacterium tuberculosis,
00:00:28.18 which we often also call the tubercle bacillus.
00:00:32.17 Now, tuberculosis is mostly a disease of the lungs, and what do you think of when you...
00:00:40.10 when you think of a TB patient?
00:00:41.18 Do you think of someone who's thin, very thin, emaciated, and in fact the disease has been
00:00:48.01 called consumption over the ages.
00:00:51.02 People also tend to have fevers, loss of appetite, they cough a lot, and often, as you can see
00:00:58.06 here, they cough up blood.
00:01:01.09 If you were to look at an X-ray of their chest,
00:01:04.07 you would see that their lung is ravaged by TB.
00:01:08.06 As well, you can see a big cavity in there that is teeming with bacteria that they're
00:01:13.13 coughing up in that sputum, there.
00:01:16.09 Now, while we often think of TB as a lung disease, in fact TB can affect multiple organs.
00:01:23.16 So, on that upper left panel, there, you see a lung that has been ravaged by TB.
00:01:30.08 But you can see here that many, many other organs -- pretty much every organ in the body --
00:01:35.07 can be affected by TB.
00:01:37.03 The only thing is that TB, as I have taught my medical students for decades, now -- is
00:01:45.12 transmitted through the lungs, as you can see here.
00:01:50.00 The bacteria spew out of an infected patient and land in the lungs of an unfortunate individual
00:01:55.22 who happens to be next to this person.
00:01:57.15 So, TB in any other organ is going to be a dead-end disease, and so in that... in terms
00:02:04.23 of the pathogen's survival it doesn't do very much for the pathogen.
00:02:10.20 Now, TB was discovered by these two physicians: Jean-Antoine Villemin was a French physician
00:02:21.10 who first identified TB to be an infectious disease, and then Robert Koch really elaborated
00:02:31.00 on this elegantly in 1882.
00:02:33.21 So, before this for many, many centuries, people had recognized... well, not centuries,
00:02:40.18 even -- millennia... people had recognized that TB was a single disease entity.
00:02:45.19 The ancient Greeks knew this, for example, but it's these two gentlemen who... who identified
00:02:51.22 it as an infectious agent, and then Koch actually figured out that the disease was caused by
00:02:57.09 this particular bacterium.
00:02:59.08 Now, at the time, in the late 1800s, when these umm... when... when Villemin and Koch
00:03:05.17 were... were making these important discoveries, TB was a terrible problem in Europe.
00:03:12.03 So, a seventh of Europe's population was dying of this disease, and a quarter of... of the
00:03:22.17 working adults of Europe were... were dying of TB.
00:03:26.06 Now, imagine how that would be to have a quarter of the workforce decimated
00:03:30.18 by a single infectious disease.
00:03:33.15 And, in fact, we all know that there are many, many famous people who died of TB,
00:03:38.22 and I've shown...
00:03:40.07 I'm showing you some of their pictures, here,
00:03:43.06 and you can see that these are all young European men.
00:03:46.12 Well, one of them, the guy on the right there, Ramanujan, is obviously not a young European
00:03:50.20 man -- he was an Indian man -- but it turns out that he... he was a mathematical genius
00:03:55.20 who was recognized by a mathematician in Cambridge, a guy called GH Hardy, and Hardy invited Ramanujan
00:04:03.23 to come to Cambridge and... and do some math with him, and that's when Ramanujan came down
00:04:10.03 with TB and, as you can see, died some years later of it.
00:04:14.06 Now, we think of TB as a disease of the past, something that opera singers got, and poets
00:04:21.17 and musicians of the past, and maybe something that our grandparents had or if you're older,
00:04:27.19 like me, your parents, so here... so we here in North America and Europe really don't think
00:04:34.01 of TB as something that's... that is... is something to worry about anymore.
00:04:41.07 And, in fact, people will always... often ask me, is TB... oh, you work on TB?
00:04:46.22 Is TB back now?
00:04:48.14 But actually, TB never went away.
00:04:51.03 And, in fact, there's more TB now than there... there ever was before.
00:04:55.18 And, as you can see, it's concentrated in certain parts of the world: in Africa, as
00:05:02.02 you can see; and pretty much all of Asia; in Europe; and we have Russia that's affected
00:05:07.16 by TB; and... and then so is South America.
00:05:11.18 And... and this is very sad and so it's... it's important to stop for a minute and think
00:05:17.10 about why this might be the case.
00:05:19.06 And, in my view, there are two reasons for this.
00:05:22.18 The first is socioeconomic.
00:05:24.22 So, TB...
00:05:26.14 TB is a disease that disproportionately affects the poor, and this is because of the fact
00:05:35.16 that it transmits best in very... in crowded conditions with poor ventilation.
00:05:40.20 So, for example, your... your urban shantytown would be a place where TB would spread a lot.
00:05:48.00 In terms of who gets TB, malnutrition is a major risk factor, cigarette smoking is a
00:05:53.19 risk factor, environmental smoke... so, you know, smoke from... from... from cooking in
00:05:59.06 these crowded, unventilated environments is a risk factor.
00:06:04.08 And then there are more modern risk factors such as diabetes and also HIV.
00:06:10.18 And so about 35% of the... of the TB in the world today is amongst people who have HIV.
00:06:19.16 From a medical standpoint, there are all... there are issues that have made
00:06:24.06 TB difficult to eradicate.
00:06:26.17 So, for one thing, we don't have an effective vaccine against TB.
00:06:31.18 There was... there's a vaccine that was developed in 1921 -- I'll tell you a little more about
00:06:37.02 it later -- but it's basically not that effective.
00:06:40.06 In terms of antibiotics, we've had antibiotics since 19... and since 1950, and in fact the
00:06:48.11 anti... the four antibiotics that we use today were all developed between 1952 and 1962.
00:06:54.23 But the problem is that, to... to... to reliably cure TB, you need to treat a person with three
00:07:03.04 to four antibiotics for six months.
00:07:06.01 Now, anyone with... anyone of you who's tried to take antibiotics even for a week will...
00:07:11.17 will realize how hard that is.
00:07:13.22 When you start to feel better, you stop taking antibiotics.
00:07:17.07 Now, imagine if you had to do this in a place where you didn't have great access to health
00:07:23.07 care and perhaps you had to trek a long way to get your antibiotics, and it meant losing
00:07:28.09 a day's work.
00:07:29.09 So, it's not... it's easy to see why people stop taking medicines when they start to feel
00:07:34.04 better, and then... unfortunately what happens then is that the... the bacteria... the dis...
00:07:40.11 the infection... the disease relapses and they get contagious disease again.
00:07:46.19 And perhaps it's because of this that we now not only have TB persisting in the world,
00:07:53.14 but we also have drug-resistant TB.
00:07:56.09 And you can see that that has affected many parts of the world.
00:08:01.01 And drug-resistant TB comes in many flavors, depending on its extent.
00:08:07.00 So, if the bacterium is... is resistant to just the umm... the frontline drugs that I
00:08:14.10 told you about -- rifampicin and isoniazid -- then it's called multi-drug-resistant TB,
00:08:20.04 and then you have to treat the TB with other drugs that are not as good, for sometimes
00:08:24.03 as long as 18 months.
00:08:26.01 But then you can get what is called extensively drug-resistant TB or even
00:08:30.01 totally drug-resistant TB, which is basically a death sentence.
00:08:35.09 And so... so not only has the problem not gone away, but the problem has been compounded
00:08:43.20 by an alarming rise of resistant TB, and what... what is even more scary is that this multi...
00:08:51.12 extensively drug-resistant TB is perfectly able to transmit from individual to individual.
00:08:56.07 So, it's... it's a very good infectious agent.
00:08:59.20 So, let's have a quick look at the life cycle, the so-called life cycle of TB.
00:09:06.19 So, as I've alluded to, it's transmitted from person to person.
00:09:11.12 So, person coughs it up, it lands in the lung of the unfortunate individual next to them,
00:09:17.18 and then it gets into these cells that are called macrophages, and then tricks the macrophage
00:09:25.09 into taking it in, and forms these big aggregates that we call tubercles.
00:09:33.09 And then it has to break out of the tubercle to get out again and be contagious.
00:09:38.11 So, here is a fundamental difference between TB and some of the other... world's other
00:09:45.00 great bacterial killers: TB is completely dependent on causing... producing active disease
00:09:53.10 in the host in order to transmit.
00:09:56.02 So, it's sort of an obligate pathogen, which... and I use that word in a slightly different
00:10:02.08 sense than other people do at times.
00:10:05.10 Whereas if you think about other great bacterial killers, such as, let's say... let's take
00:10:12.03 the instance of plague... plague is really not a human... it's an accidental human disease.
00:10:17.17 So, this... this... this infection that has decimated humanity over the years is really
00:10:23.23 an accident, and human infection has no relevance to the evolutionary survival of... of the...
00:10:31.11 of the plague bacillus.
00:10:32.22 This is even true for things like the pneumonia bacterium, the pneumococcus, or the... or
00:10:37.18 the meningococcus that causes devastating meningitis, or your life...
00:10:43.07 your strep-eating streptococcus.
00:10:45.20 In all of these cases, these pathogens, these bacteria are just mucosal colonizers.
00:10:50.24 They live in our mucosal tracts and disease is occasional and accidental,
00:10:55.19 devastating as it is.
00:10:57.21 Not so for TB.
00:10:59.08 It needs to produce disease in order to... to transmit and survive, evolutionarily.
00:11:05.15 And, perhaps this explains why there's... there's a certain... there's a curious feature
00:11:12.10 that... that TB has, and that is it lacks what we call these classical virulence factors.
00:11:18.02 It lacks capsules that... that bacteria have to avoid being eaten by a macrophage, flagella
00:11:26.12 that bacteria use for motility, pili, toxins... pilis... pili are used for adhes... adhesion,
00:11:36.04 toxins are used... well, they're basically cell pois... they poison the host cell.
00:11:40.22 And in fact this is all talked about very nicely, both by Stanley Falkow and also by
00:11:46.14 Ralph Isberg in... in prior iBiology talks.
00:11:50.09 So, TB doesn't have any of these.
00:11:53.12 So, how is it so successful?
00:11:56.00 Well, there's... there's... there's a few things and I'm gonna illust...
00:12:01.02 I'm going to tell you about a couple of them.
00:12:03.08 So, one thing it does have is this waxy coat that, as you can see, gives the colonies a
00:12:09.12 characteristic appearance.
00:12:11.06 And then, if you were to stain the bacterium, and that... that stain there is actually taken
00:12:15.12 from a patient sputum, you'll see a... a... that it stains in the characteristic way,
00:12:22.18 where there are very specific stain and we call it... we call them acid-fast bacilli,
00:12:28.00 or some people call them red snappers, and this is because of that complex cell wall.
00:12:33.01 So, let's have a quick look at that cell wall.
00:12:35.22 So, if you look at this cartoon, that... there's a layer just above the cytoplasmic membrane
00:12:42.14 that is the peptidoglycan that... that pretty much all bacteria share.
00:12:47.13 But then you can see that, above it, it's got a really complex lipid... array of lipids.
00:12:54.08 Some of these lipids are complexed to sugars, others are complexed to proteins, and these...
00:13:01.11 this... these lipids seem to contribute a lot to the ability of mycobacteria to evade
00:13:08.09 the host and sort of... and... and... and... and... and be pathogens.
00:13:13.14 And I'm going to illustrate this for you with a discovery that was made in my lab by a PhD
00:13:18.16 student, CJ Cambier.
00:13:20.15 So, CJ wanted to really look at that very early event of TB.
00:13:26.20 So, after it gets into a host, how does it get into a macrophage and survive?
00:13:33.18 And macrophages are primary immune defense cells, and their job is to come to bacteria
00:13:41.05 and eat them and kill them.
00:13:43.20 And here's a video of a macrophage in culture eating bacteria.
00:13:48.15 You'll... you... this is a... umm...
00:13:52.00 I've...
00:13:53.00 I've...
00:13:54.00 I've basically taken this video from Manuel Amieva at Stanford, who also gave it to Stanley
00:14:00.02 Falkow, so you'll see this video in Stanley Falkow's iBiology lecture as well.
00:14:05.10 And you can see there's macrophages reaching out and eating bacteria, and Stanley refers
00:14:11.24 to this as macrophages eating peanuts from a bowl.
00:14:17.09 And now... when bacteria get in to macrophages, your garden-variety bacterium is killed, and
00:14:29.09 Stanley also talked about this in his lecture.
00:14:33.07 The way this happens is that the bacteria have on their surface those... you see those
00:14:38.07 funny little protrusions?
00:14:39.21 Those are called PAMPs, for pathogen-activated molecular patterns.
00:14:44.11 And these PAMPs activate in the host a pathway, a signaling pathway called
00:14:52.17 the Toll-like receptor signaling pathway.
00:14:55.08 And this pathway brings macrophages to the bacteria, and they'll eat them up and then
00:15:01.17 they can kill them using various microbicidal mechanisms.
00:15:04.22 So, you can see how, if a mucosal pathogen is... is in... in the right place for the
00:15:10.15 host, as in on the... on the outside of the mucosa, the host might let it be, but if it
00:15:15.01 tried to get in or get... or... or sort of started to wander, the host would send out
00:15:20.07 these macrophages that would then kill it.
00:15:23.14 Now, I've already told you that TB has to get in, it has to traverse this barrier to
00:15:29.06 get in -- it doesn't want to hang out with these pesky commensals.
00:15:32.11 It wants to get away from them and deal solely with the host.
00:15:37.05 And so, the TB has a... also has a ton of these PAMPs, so, how does it do this?
00:15:44.19 And what CJ found was that... that... that it uses some of these cell surface lipids
00:15:50.20 to coat the PAMPs.
00:15:51.20 So, the blue lipids here are something called PDIM, and PDIM coats these PAMPs, so now it
00:15:59.18 prevents TLR-mediated cell migration and engulfment.
00:16:05.00 But, then, it still needs to get in, so how does it do that?
00:16:09.16 Well, it adds on another lipid, and this lipid is a phenolic glycolipid, and this phenolic
00:16:15.08 glycolipid brings in a new and different kind of macrophage that is not microbicidal -- it...
00:16:24.18 it can engulf the bacteria, but it's permissive to the growth of the... of the bacteria.
00:16:30.09 So, in other words, mycobacteria are telling the host, "Thank you.
00:16:35.01 Don't worry about getting... about coming along with your macrophages.
00:16:38.23 I'll bring in a different kind of macrophage from you that can help me get in and survive".
00:16:44.05 So... so, here... just to reiterate, your bacteria on the left are your garden-variety
00:16:51.11 bacteria that will get killed by microbicidal macrophages that are recruited by the Toll-like
00:16:58.08 receptor pathway.
00:17:01.04 The mycobacteria don't use that pathway, they... they...
00:17:05.15 they hide from it from using these lipids.
00:17:08.22 And then, instead, they turn on... they... they activate the production of the chemokine
00:17:15.14 that recruits these permissive macrophages, that, as you can see, take up the bacteria
00:17:20.17 and then take them inside.
00:17:24.16 Now, this is a very... this... this... this use of the... of these two lipids is a very
00:17:29.08 nice evasion strategy, but if you think about it there's a... there's a problem with this.
00:17:34.15 And the problem is that we inhale TB and... into our nasal... into our nasopharynx, and
00:17:43.17 our nasal... our nasopharynx is full of bacteria that trigger TLR signaling.
00:17:51.15 So, even if TB has as a... the... even if mycobacterium has a way to evade Toll-like
00:17:58.11 signaling, it's... there's going to be a lot of Toll-like signaling going on right there,
00:18:05.21 and so it would be basically caught in the crossfire, in this battle between the host
00:18:11.16 and the bacterium, and... and... and essentially be collateral damage.
00:18:17.10 So, the bug has evolved a trick for this.
00:18:21.05 And that is that, unlike a lot of respiratory pathogens that are transmitted in the upper...
00:18:27.21 through the upper muc... respiratory mucosa, TB goes deep down inside.
00:18:34.05 And that's again illustrated in this cartoon you've seen before.
00:18:37.22 But watch of those red droplets, right?
00:18:40.00 They go deep down into the alveoli of the lung.
00:18:43.01 So, TB is not a disease of the upper respiratory tract.
00:18:46.09 It has to go really... down... deep down in small droplets that cont... that contain maybe
00:18:52.02 one, at the most, ten bacteria.
00:18:55.12 And this... this has been known for quite some time, both through human epidemiological
00:19:00.08 studies of how TB spreads and also through animal models.
00:19:04.22 And this is a very nice experiment that illustrates this.
00:19:07.21 So, these researchers at Johns Hopkins University in 1948 gave rabbits TB and they collaborated
00:19:16.08 with some engineers, and what they did was to devise a way to give rabbits TB so that...
00:19:23.21 in large droplets that contained 10,000 bacteria.
00:19:27.08 And these... these bacteria got stuck in the upper airway -- they could... they would follow
00:19:32.12 them just by killing rabbits and transecting them and seeing where the bacteria landed.
00:19:38.00 They also, then, took bacteria and put them in small droplets, so that one to three bacteria
00:19:42.18 were given to the rabbits, and they showed that they... these droplets went to the bottom
00:19:47.13 of the lung, to those alveoli that I talked about.
00:19:50.15 And then they followed what happens... happened to the rabbits, and you can see that the...
00:19:55.02 the rabbits that got more bacteria didn't get infected, whereas the rabbits that got
00:20:00.15 fewer bacteria got infected.
00:20:03.10 And so TB has known that more is not better -- less is better --
00:20:09.18 and has devised an additional strategy.
00:20:13.02 So, it has to use two lipids and it has to minimize its droplet size... size.
00:20:19.06 And so I illustrate for you, here, how TB escapes from the lower air... from the other
00:20:25.00 commensals and the... from the commensals, sorry, in the upper airway, and goes down
00:20:29.08 to the lower airway where it's all by itself and can do this
00:20:32.19 recruiting trick using its lipids.
00:20:36.01 Now, this has... there's... there's a couple of instructive points here.
00:20:44.05 One is that TB is less infectious because of... because it has to use the small-drop...
00:20:50.04 droplet strategy, it's actually not... not nearly as infectious as a common cold, or
00:20:55.05 measles, or other things that have to transmit in the upper airway.
00:21:00.14 Conversely, we could think about it as the commensal flora being protective against TB,
00:21:08.11 as we're finding more and more that they are against many infections, and TB doesn't seem
00:21:12.20 to be an exception.
00:21:15.11 But finally, TB has managed with this strategy and has been around since before the Neolithic
00:21:25.00 demographic trans... transition.
00:21:27.11 It's been around for something like 70,000 years.
00:21:31.06 So, the strategy is working very well for it.
00:21:36.02 So, these two lipids that I just finished telling you about turn out to be very important
00:21:41.24 determinants in the evolution of pathogenesis.
00:21:47.02 So, pathogenic mycobacteria evolved from soil-dwelling mycobacteria, and the acquisition of these
00:21:53.06 lipids was a major part of that step.
00:21:57.06 But, when we move on to now look at the next phase with... of... of the... of the life
00:22:04.21 cycle of these bacteria, which is living within this macrophage they've recruited and going
00:22:10.23 on to form the granuloma, there's a little bit of a surprise.
00:22:17.10 Because it turns out that the determinants that are used to survive in the macrophage,
00:22:23.07 bona fide virulence determinants that are studied quite a lot, turn out to be also present
00:22:30.18 in the soil-dwelling bacteria and it looks like they've just been repurposed from soil-dwelling
00:22:36.01 bacteria to... to confer virulence.
00:22:41.11 And one such example is the bacterial efflux pump, which... we don't know exactly what
00:22:48.05 it does in the soil, but it's very possible that this... this pump was used, or is used,
00:22:55.00 in the soil-dwelling mycobacteria to pump out antimicrobials or antibiotic-like molecules
00:23:02.19 that other soil-dwelling organisms put out, because it's... it's... it's a warfare out
00:23:08.01 there between these different organisms in the soil, and that's why a lot of antibiotics
00:23:12.12 were discovered in soil-dwelling organisms, to protect themselves against other organisms.
00:23:19.06 And this insight came from a graduate student in the lab, Kristin Adams.
00:23:26.00 And what Kristin found was that these bacterial efflux pumps got induced in the pathogenic
00:23:33.23 mycobacteria when they went into macrophages -- transcriptionally induced -- and they then
00:23:39.10 protected the bacteria against the macrophage and allowed them to survive in the macrophage.
00:23:45.23 So, they were macrophage-induced virulence factors.
00:23:50.08 So, here... but here's the twist: When Kristin looked a little bit more at this, she found
00:23:56.17 that they also mediate a phenomenon called bacterial tolerance.
00:24:01.20 So, bacterial... sorry, antibiotic tolerance.
00:24:06.11 Antibiotic tolerance is a phenomenon where the bacterium doesn't become genetically resistant
00:24:11.09 to an antibiotic, but nevertheless is... is... is phenotypically resistant to the antibiotic
00:24:19.17 in the... in the absence of any genetic resistance.
00:24:23.23 And it's often induced by particular environmental conditions.
00:24:27.19 This phenomenon of tolerance has been known for a long time and has been thought to be
00:24:31.11 the reason that TB takes so long to treat.
00:24:35.13 But the model that has been proposed for this is that when bacteria enter the host and enter...
00:24:42.13 and become part of the host granuloma, the tubercle, they essentially become dormant.
00:24:48.01 They undergo metabolic and a replicative arrest and, as a result, they become resistant to...
00:24:58.16 as resistant as intolerant to the antibiotics that typically tend to cart... target bacterial
00:25:06.10 determinants that are needed by actively growing bacteria, for example, your cell wall or your
00:25:11.02 ribosome or your transcriptional machinery.
00:25:15.13 And so, in this model, the slow...
00:25:18.02 most slowly growing bacteria would be the ones that would be the most tolerant.
00:25:22.18 And a lot of attempts to make new antibiotics to shorten TB treatment
00:25:27.24 are predicated on this model.
00:25:30.02 However, when... when Kristin looked, she found that these same pumps that allow the
00:25:37.01 bacteria to survive in the macrophage also mediate tolerance against frontline antibiotics
00:25:45.13 used for TB, and in fact every antibiotic that she tested was... was... the bacteria
00:25:54.11 underwent tolerance to that antibiotic upon entering a macrophage.
00:25:59.16 So, this has a profound clinical implication, because, now, the most... in her model or
00:26:05.19 in her observations, the most rapidly growing bacteria are the ones that are most tolerant
00:26:11.13 to the antibiotic, presumably because they're pumping it out.
00:26:15.18 And there's a... there's a... there's a... there's a clinical in here with this... with
00:26:22.16 this finding, because there are efflux pump inhibitors available,
00:26:27.21 bacterial efflux pump inhibitors.
00:26:30.00 Many of these just happen to be drugs that are around for other purposes.
00:26:34.02 And the one that we honed in on, or Kristin honed in on, was a drug called verapamil,
00:26:38.18 which is a calcium channel blocker that's used to treat high blood pressure, and cardiac
00:26:43.15 arrhythmias, and migraines.
00:26:46.09 And she found that if she treated the... the macrophages that were infected with verapamil,
00:26:52.09 along with the standard chemotherapy, they now did... they were killed much better with
00:26:57.13 standard chemotherapy.
00:26:59.00 And, based on these results, a clinical trial is... is going to start in India to see if
00:27:05.07 verapamil will shorten the course of treatment.
00:27:08.03 From an evolutionary standpoint, it's kind of interesting, because we're talking about
00:27:13.23 a... a pump that was used... or... and is used in soil-dwelling mycobacteria, presumably
00:27:20.02 to pump out antibiotics.
00:27:21.15 Then, over the course of most of the history of a... a pathogenic mycobacteria, it was
00:27:27.19 repurposed to... to fight macrophage defenses.
00:27:33.00 And then, in the last hundred years or so, since the advent of... or less, actually,
00:27:38.09 fifty years or so... since the advent of chemotherapy, that ancestral function has come back to help
00:27:45.02 the bacterium withstand the... the modern post-antibiotic era.
00:27:52.11 Moving along, we've got the bacteria now living in a... in what is called a granuloma, or
00:27:59.15 a tubercle, and the... while the initial cells that come and form this granuloma are macrophages,
00:28:07.10 the body then brings in, the host... the immune system brings in many more different cells
00:28:11.22 to... to come in and help fight the bacteria, and yet, in many... in... in a proportion
00:28:18.16 of cases, the bacterium is still able to survive in this rather complex structure.
00:28:25.05 And this brings me to... to a point that I want to make, which is that you'll hear many
00:28:32.24 times people say, and you'll read in books,
00:28:36.07 that a third of the world is infected with TB.
00:28:39.21 And the idea there is that they're infected with TB, they've got it under control, and
00:28:44.06 then it's ... but at any given time they can reactivate this latent disease, and... and
00:28:50.23 get transmissible... and get morbid and transmissible TB.
00:28:56.16 But, actually, this is based on... on the... on the TB skin test, which simply tests to
00:29:08.03 see if you've ever been infected with TB.
00:29:11.21 And so the fact that your skin tests positive doesn't mean you have TB infection now; it
00:29:16.15 means you once had TB infection.
00:29:18.13 You could still have it or you could have cleared it.
00:29:21.17 And if you really go and look at old epidemiological studies, old and new, you'll find that most
00:29:28.00 people who get infected with TB actually clear it using this arsenal of immune defenses,
00:29:36.06 and... and a combination thereof that I've listed for you, here.
00:29:41.20 And so, actually, most people eradicate TB, and a few people go on to have primary disease,
00:29:48.24 and latency and reactivation disease are no doubt present, but in my view, based on my
00:29:55.14 reading of the epidemiological studies, are a minority of... of cases.
00:30:02.16 And this is very nicely shown here, in a study done in Amsterdam, where contacts of active
00:30:10.02 TB sufferers were followed meticulously, and if they got TB at all they got it within the
00:30:16.01 first few months of exposure.
00:30:17.21 So, the immune system does a pretty good job of killing TB.
00:30:23.12 And if that's the case, then why don't we have a vaccine for TB?
00:30:27.18 Now, the vaccine for TB has been the Holy Grail.
00:30:30.13 Well, in fact... a vaccine for TB was made and was first administered in 1921.
00:30:36.05 It was a live attenuated vaccine.
00:30:39.19 And it's the... while it's the... we still use it to this day in highly endemic areas,
00:30:45.07 because it does offer a modicum of protection against disseminated TB and meningeal TB in
00:30:50.14 kids, which as you'll see in my third lecture is a terrible disease, but obviously we still
00:30:55.16 have a lot of TB, though it's the world's most widely administered vaccine, and that
00:31:00.21 means it doesn't protect very well at all.
00:31:03.20 And efforts to improve its efficacy, or to make whole new vaccines,
00:31:08.10 have not worked very well.
00:31:10.07 So, why is that?
00:31:11.18 Well, that's a paradox; we don't really understand why, but I'd like to tell you a few things.
00:31:16.06 First of all, most people don't get TB when they're infected -- they're protected naturally.
00:31:21.19 But, if you do get TB, then very... then you're not completely protected against a second
00:31:29.21 infection, which is different from the case of, say, smallpox, where if you get smallpox
00:31:34.18 once then you don't get it again.
00:31:36.15 People can get TB again.
00:31:39.00 On the other hand, there's very clear data that show that if you've... if your skin tests
00:31:45.03 positive but don't... never manifest a disease, you're... you're somewhat protected.
00:31:51.00 And that's nicely shown here in a study of nurses in Norway, who, in the pre-antibiotic
00:31:56.09 era, were found to be either skin test-positive and skin test-negative, and then they were
00:32:01.14 put amongst the TB patients to take care of them, and the ones who were skin test-negative
00:32:07.02 were more... much more likely to get TB.
00:32:09.11 So, these... the people who were skin test-positive but had never manifested TB were somewhat
00:32:14.13 protected against TB.
00:32:15.21 And if we can understand this, look at these data again with new eyes and understand them
00:32:20.20 a bit better, maybe we can try to think about ways to... to... to recapitulate the protection
00:32:29.03 that many people in fact seem to have.
00:32:31.19 So, in closing, we've got a bacterium here that is possibly one of the world's most successful
00:32:39.02 bacteria, that evolved from non-pathogenic environmental mycobacteria, doesn't really
00:32:44.23 have a great many classical virulence factors, but seems to use a sort of stealth mechanism
00:32:52.14 to survive.
00:32:54.19 And, even though it doesn't produce disease in most of the people that it infects, it's...
00:33:02.03 that's good enough for it, because it produces just enough disease to... to have survived
00:33:08.13 over the... over... over the eons.
00:33:12.08 That is not to minimize the impact of TB.
00:33:15.03 TB killed nearly eight... two million people last year, or year... in 2015, and caused
00:33:21.16 disease that... that debilitated them in about 10 million people.
00:33:27.22 And perhaps the most chilling thing to consider is that TB has been with us for more than
00:33:33.13 70,000 years and it's still with us.
00:33:36.23 It predates the transition to an agrarian culture.
00:33:40.21 It... it... it resisted urbanization -- in fact, it thrived with urbanization.
00:33:48.03 It's resisted modern medical technologies, like antibiotics and attempts to make a vaccine.
00:33:57.14 And so it's a... it's a... it's a pretty... it's a pretty tough and wily bacterium.
00:34:06.17 And if you stay tuned for parts two and three, I'll tell you about some more insights we've
00:34:11.16 had about this bacterium using a very interesting and unusual model to derive these insights.
00:34:23.12 Okay, thank you.

00:00:07.21 My name is Lalita Ramakrishnan.
00:00:09.16 I'm a professor of Immunology and Infectious diseases at the University of Cambridge.
00:00:14.08 And, in this lecture, I'm going to continue to tell you a little bit more about tuberculosis,
00:00:20.16 focusing on a structure called the tubercle.
00:00:24.13 Now, just to recapitulate the lifestyle or the life cycle of Mycobacterium tuberculosis,
00:00:33.02 which is the causative agent of tuberculosis, the bacterium that causes it, the bacteria
00:00:39.01 are exhaled by... in coughs by people who are infected, get inhaled by individuals near
00:00:48.22 them, and then they enter cells that are called macrophages.
00:00:52.18 But what happens next is that they... they induce these macrophages to form structures
00:01:01.16 called granulomas, and these granulomas can become quite elaborate.
00:01:09.14 At first, they're comprised just of macrophages, but then many other immune cells come in and
00:01:15.13 they can form of a fairly complex structure.
00:01:19.21 And another thing to note is that the macrophages within this granuloma undergo a specialized
00:01:27.15 differentiation called epithelioid transformation, where they form these finger-like, interdigitated
00:01:34.03 projections between each other to form a very compact, organized structure.
00:01:42.23 Now, this structure... pathologists call these structures granulomas, and granulomas were...
00:01:51.22 are... are associated with many, many, many diseases, both infectious and non-infectious.
00:01:59.19 And they can often be quite pathological.
00:02:02.03 But granulomas... but... but the single biggest cause of granulomas is tuberculosis.
00:02:09.13 And, in fact, granulomas were first discovered in the context of tuberculosis in 1679, some
00:02:18.13 200 years before the bacterium Mycobacterium tuberculosis was... was discovered.
00:02:25.09 And, in fact, the microbe... the... the structure used to be called a tubercle, and obviously
00:02:31.23 that... both the bacterium and the disease are named for the granuloma.
00:02:35.16 So... but it turns out that granulomas are very primitive structures; they've probably
00:02:43.03 evolved... there... they're present even in lower vertebrates in a... in a more primitive
00:02:47.11 form, and they probably evolved to wall off foreign bodies -- you could imagine a thorn
00:02:54.02 being... being circumscribed by that... by such a structure until it's...
00:02:59.03 it's sort of dissolved.
00:03:01.00 But these so-called foreign body... body granulomas have a very low turnover of macrophages; the
00:03:07.14 macrophages just come and sit there and it's not a particularly inflammatory structure,
00:03:11.14 or so it's thought.
00:03:13.19 In contrast, the types of granulomas that form with tuberculosis, and pretty much any
00:03:18.02 medically significant granuloma, tend to be high turnover granulomas where there's a rapid
00:03:25.10 death of macrophages and a repopulation by new ones,
00:03:28.22 and they can be very inflammatory structures.
00:03:31.22 There are many traditional animal models that are used to study TB.
00:03:36.11 The oldest ones are the ones... the rabbit and the guinea pig, which were used by
00:03:42.19 Villemin and Koch, respectively, at the time that they discovered TB.
00:03:48.11 They used these animals to... to pass the bacterium from one animal to another to show
00:03:54.19 that it was associated with... with TB.
00:03:58.18 The most commonly used model now is the... is the mouse.
00:04:01.21 And this makes a lot of sense because mice have a wonderful array of
00:04:08.03 immunological and genetic tools.
00:04:10.06 One issue is that mice don't... that most mice... mouse strains don't develop the...
00:04:18.07 the nice, tight granulomas that are associated with human disease, but there are some recently
00:04:25.06 identified mouse strains that do, and so those could actually be quite good.
00:04:33.07 Another more recently used model is the non-human primate, and this is a good model because
00:04:40.04 it really recapitulates human disease.
00:04:43.03 But the problem is, of course, they're expensive, there are ethical considerations, and obviously
00:04:48.06 cannot be used widely.
00:04:50.19 So, in... on this backdrop, my story and my engagement with TB and the granuloma came
00:05:00.24 when, as a postdoctoral fellow at UCSF -- I was a clinical infectious disease fellow and
00:05:07.08 had to do a postdoctoral fellowship -- I approached Stanley Falkow at Stanford to... to go to
00:05:14.07 his lab and study TB, which he didn't study at the time,
00:05:17.14 but he studied many other bacterial pathogens.
00:05:20.22 And Stanley said to me, forget it, I don't have the specialized containment facilities
00:05:27.00 you need to study TB, which is a human aerosol pathogen and, besides, he said, TB grows so
00:05:33.05 slowly I'll be dead before you get your first result.
00:05:36.18 And that was in 1991, and I'm happy to say he's still alive, and... and...
00:05:42.17 and quite well.
00:05:45.13 And... so... what he told me... he gave me an insight and he said, look, there are other
00:05:53.19 strains of mycobacteria, there are other species of mycobacteria that are pathogenic in other
00:05:59.01 animals, that are natural pathogens of other animals.
00:06:02.20 And he said, I...
00:06:03.20 I'm pretty sure there are these ones of... of marine life of fish, because I've seen
00:06:09.19 people get them who were fishermen.
00:06:13.19 He had worked at Brown and knew that Portuguese fishermen got these... this disease... mycobacterial
00:06:20.15 disease on their digits and on their soft tissues.
00:06:24.22 And so I went off to the UCSF library and looked at this very classic manual.
00:06:31.13 It's called Bergey's Manual of Systemic Bacteriology, and I... and I found what he was talking about.
00:06:37.16 He was talking about... he was probably talking about a bacterium called Mycobacterium marinum
00:06:43.13 that was thought to be a close relative of human TB, of the human TB bacterium, and it
00:06:50.01 gave fish TB.
00:06:52.24 It turns out that it also infects humans, and this has been known since the 50s, and
00:07:00.02 I can personally attest to it.
00:07:02.18 And it gives humans disease on their extremities, as Stanley already knew, and... and many clinicians,
00:07:08.23 particularly dermatologists and infectious disease clinicians, already know this.
00:07:13.12 But if you look inside that lesion, you'll see a classic granuloma and actually in many
00:07:18.20 cases it can be indistinguishable from the granulomas caused by the human TB bacterium.
00:07:27.03 But, of course, we know that this bacterium also infects fish, and it was first identified
00:07:34.03 to do so in the Philadelphia Aquarium, where in 1926 fish were dying of some mysterious
00:07:42.20 wasting disease, very similar to human TB.
00:07:46.12 And when they tried to culture these fish to see what bacterium they had, why were they
00:07:52.23 dying?, they couldn't culture anything, but when they looked at the fish by histology
00:07:57.24 they could see these classic red snapper bacteria that looked very much like TB.
00:08:04.05 And then Aronson had the bright idea to culture the... to try to do the cultures at a low
00:08:09.21 temperature that was commensurate with... with the low body temperature of the fish,
00:08:14.02 and then he was able to cultivate, he was able to culture Mycobacterium marinum.
00:08:19.08 And, since then, we've had Mycobacterium marinum sequenced at the Sanger Center, and it turns
00:08:26.22 out to be the closest genetic relative of the human TB bacterium, so I guess we also
00:08:32.16 got quite lucky.
00:08:34.00 And it turns out that Mycobacterium marinum also infects zebrafish.
00:08:39.14 Zebrafish are a pet develop... a pet organism of developmental biologists and are a natural
00:08:46.09 host to Mycobacterium marinum.
00:08:48.20 So, I've got for you, here, down below in... in the bottom panel, the... a human TB granuloma
00:08:56.14 stained by hematoxylin and eosin, which will only stain the host cells but not the bacteria,
00:09:01.20 and what you can see is that you've got a nice organized structure, which is cellular,
00:09:07.23 as evidenced by blue nuclei on the edges, but in the center where that arrowhead is,
00:09:14.12 you'll see that the structures become acellular, because it's undergone necrosis, just as we
00:09:21.20 know that human TB granulomas do.
00:09:24.03 But here's, now... let's take a look, now, at the... at a zebrafish granuloma, and in
00:09:30.03 this granuloma you can see... which... which I've stained, here, with a stain that also
00:09:36.22 stains the bacteria, you can see that it looks very similar and it's a nice cellular organized
00:09:43.13 structure and you can see that there are a few bacteria within macrophages, but where
00:09:48.12 the macrophages have necrosed there are tons of bacteria,
00:09:51.12 which is exactly what you would expect.
00:09:55.11 But the great feature of the zebrafish that makes them so enticing to developmental biologists
00:10:01.11 is that they have a prolonged larval phase when they're transparent.
00:10:06.09 And so you can actually watch things happen and people watch developmental processes happen,
00:10:12.24 but... so we asked, well, can we put in bacteria and watch infection happen?
00:10:18.10 And so, they have a cavity that's called the hindbrain ventricle, which is the equivalent
00:10:22.24 of something in our brain, close to our brain called the fourth ventricle, and so we put
00:10:27.09 in some bacteria there -- I've shown it to you with an arrowhead, there -- and what we
00:10:32.03 saw very quickly was that macrophages came, and you'll see the macrophages sort of chasing
00:10:40.14 after the bacterium like a cat after a mouse, and eventually you'll see this mac... mac...
00:10:45.17 macrophage gets it.
00:10:47.12 And there you've got an infected macrophage.
00:10:51.20 You can now follow these infected macrophages out of the cavity -- this is a few days later
00:10:57.12 -- and you can see that it's just moseying along.
00:11:02.04 The bacteria have grown in the macrophage and -- because it's a permissive macrophage
00:11:08.19 for the... for the... for the bacterium -- and there it is.
00:11:13.20 But what was really exciting to us was that you could see, within a few days,
00:11:19.02 a granuloma form.
00:11:20.22 And here you can see that we've got a granuloma that's already formed and what you're going
00:11:25.08 to see, where that white... white arrow is, a new uninfected macrophage is going to come
00:11:32.00 and then it's going to enter the structure.
00:11:34.20 So, watch this.
00:11:37.13 See?
00:11:39.11 There it comes, and it's going to squeeze its way in between and get in there.
00:11:50.21 So, the granuloma is a highly chemotactic structure
00:11:54.21 that is recruiting new macrophages to come to it.
00:11:59.19 And then we could show all this by... by engineering fish that were transgenic, so that they had
00:12:07.06 green florescent macrophages and red fluorescent neutrophils, which is another cell type that
00:12:12.11 is somewhat involved in granulomas, but not as much as macrophages.
00:12:16.17 And what you can... and now we've infected the fish with blue fluorescent bacteria and
00:12:20.10 you can see that the... that we've got a nice tight bona fide epithelioid granuloma with
00:12:28.00 infected macrophages.
00:12:29.13 So, this was good.
00:12:31.10 As we were developing this model, my colleague and friend, David Sherman,
00:12:37.11 made a suggestion to us.
00:12:40.16 He... so, it's... people have been searching for virulence determinants in mycobacterium,
00:12:50.03 and one exciting discovery was that a specialized secretion system called the ESX-1 or RD1 locus,
00:12:58.18 which I've shown in white in that top panel, the white genes in the top panel,
00:13:03.24 were involved in virulence.
00:13:05.16 And this was very exciting because these... it turns out that this was the locus that
00:13:10.09 was missing in the attenuated vaccine strain, BCG, that was made by serial passage in...
00:13:17.14 in the 1920s, and now we finally knew the molecular basis of its attenuation.
00:13:24.19 So, Mycobacterium marinum, not surprisingly, has a locus that looks virtually identical.
00:13:31.19 And David kept telling me, look, make a mutation in this and let's see how it really works,
00:13:38.10 because everyone knows it's attenuated, but a lot of these animal models are black boxes
00:13:42.24 because you only get to see the end result, and he could see that we would be able to
00:13:47.01 get some insights about the actual sequence of what was got... what was different.
00:13:52.04 And, when I was a bit slow to do this, he actually had someone in his lab make the mutation...
00:13:59.04 the mutant for me, and he gave it to us and he said, take this.
00:14:02.02 So, at this point, we were sort of shamed into doing this quickly, and we was
00:14:07.03 Hannah Volkman, who had joined my lab at... as a graduate student, and Hannah showed very quickly
00:14:12.08 that, yes, if you put this mutant into zebrafish larva, it was attenuated.
00:14:17.16 The animals didn't die and if you looked at the bacterial counts you could see that the
00:14:23.03 bacteria didn't grow as well.
00:14:24.03 So, this was good because it showed us that it was behaving just like you would expect.
00:14:28.12 But, here came the surprise.
00:14:30.21 And, at this point... by this point, Hannah had recruited some of her colleagues -- Dana
00:14:36.12 Beery, on the left, who was a technician in the lab, and Hilary Clay, a graduate student
00:14:41.01 who was Hannah's very good friend -- and she got them to join in this... in this quest
00:14:46.01 to see what was going on.
00:14:48.15 And what they found was something quite interesting.
00:14:52.13 Because, if you look at the fish on top, they are infected with wild-type bacterium, and,
00:14:58.19 if you look at the close-up on the top right,
00:15:01.17 you can see that a nice big granuloma has formed.
00:15:04.22 But if you look at the mutant, what you can see is that, even if you inject many, many
00:15:09.22 more bacteria, just to compensate, so that you get more... as many bacteria as with the
00:15:16.10 wild-type, the macrophages pack up with the bacteria, as you see on that bottom-right
00:15:21.09 panel, but there's... they don't form granulomas.
00:15:25.13 Now, this seemed opposite of what you'd expect, because if... if granulomas are good for the
00:15:32.09 host, as what everyone in TB... in the field of TB thought... people have thought that
00:15:38.12 the granuloma is a critical host protective structure that walls off the bacteria and,
00:15:46.00 while it's not always successful in eradicating the bacteria, it sure as heck tries to do
00:15:51.14 so, and is... is... is pretty good at it.
00:15:54.12 Now, if that's the case, then we should see more granuloma formation with that mutant,
00:16:00.03 but we saw less.
00:16:02.05 So, Hannah took a close look at this and she was able to observe fish as the granuloma
00:16:08.24 formed by serial imaging, and what she showed was that, when the granuloma formed, the number
00:16:16.19 of infected macrophages went up dramatically, as did the number of bacteria.
00:16:21.19 So, the granuloma was actually promoting growth rather than restricting growth.
00:16:26.18 So, why might this be?
00:16:28.15 Because here we are saying that a... the... you know, this... this immunological structure
00:16:34.07 that really should be killing the bacteria is actually promoting growth.
00:16:40.24 And the answer to this came of... both from Hannah's work and from a new graduate student
00:16:49.20 who joined, an MD/PhD student, Muse Davis, and what we found was happening was that,
00:16:57.02 when there's an infected macrophage, when new macrophages come to it, for some reason,
00:17:04.13 if they have that ESX1 locus, the bacteria are spreading quickly from macrophage to macrophage.
00:17:12.16 You can see, within 48 hours, you've gone from one infected macrophage in that top-left
00:17:17.12 panel to many infected macrophages, whereas if you didn't have that locus, if the bacterium
00:17:23.24 didn't have that locus, then that one mac... macrophage just remains one great big macrophage.
00:17:29.09 And the bacteria are just growing in it, but obviously they're not doing as well as if
00:17:33.09 they can spread to new macrophages.
00:17:37.20 And so it turned out that the bacterium is
00:17:40.07 using this locus to spread from one macrophage to another.
00:17:44.16 Now, how does this happen?
00:17:46.13 What... what Muse found was that, if the initial macrophage was infected with bacteria that
00:17:54.03 contained this locus, then somehow that macrophage was able to exert a rapidly chemotactic effect
00:18:03.18 on... on macrophages around it, or... or even far away, so that they came.
00:18:10.18 And you can see that the macrophages in the top panel have these protrusions that reflect
00:18:16.15 that they're highly chemotactic and are responding to a chemotactic gradient, and our racing
00:18:22.13 into this structure.
00:18:24.07 In contrast, if that initial macrophage didn't have this locus, then the incoming macrophages
00:18:31.03 are coming in very, very slowly, and they clearly are not experiencing a chemotactic
00:18:36.15 gradient; they don't have that big... great big protrusion.
00:18:40.18 And, even once they get into the granuloma, they behave very differently.
00:18:45.06 The wild-type macrophages move far and wide within the granuloma, and rapidly, whereas
00:18:50.13 the mutant macrophages, the few that do come, are just sort of sitting there
00:18:54.23 like bumps on a log.
00:18:58.20 And this is the kind of movie that gave us that insight that I just talked to you about,
00:19:04.10 that macrophages are used... exploited by the bacteria to spread from cell to cell.
00:19:11.01 So, what happens is that, when a given infected macrophage packs up with... packs up with
00:19:17.08 bacteria, because the bacteria can grow within it, it undergoes an apoptotic death, where
00:19:24.10 it's dead but it's preserved its membranes.
00:19:27.19 And now what happens is this dead cell is recognized by the incoming macrophages, that
00:19:34.22 come and eat it.
00:19:36.05 And I'm going to show you an example, here, of where one... one dead macrophage with the
00:19:42.07 white arrow is going to be engulfed by an incoming macrophage.
00:19:46.19 So, watch this happen.
00:19:48.02 It's... it... you're going to watch it, it's going to come from below, it's kind of like
00:19:52.04 that movie Jaws, where the... you know, the shark comes from below.
00:19:56.06 And, look at it, it's eating it bite by bite.
00:19:59.22 And this is why macrophages might be called what they are -- macrophage for "big eater".
00:20:05.12 And now you've got... this macrophage has been eaten by a new macrophage.
00:20:12.12 But you're going to say, wait a minute, why would this spread the infection?
00:20:15.18 You've gone from one macrophage to another macrophage, so all you've done is conserved
00:20:19.12 the bacteria.
00:20:21.04 But it turns out, when Muse looked closely, that, on average, a given macrophage, given
00:20:26.14 infected, dead macrophage, was eaten, on average, by 2.3 macrophages every 24 hours.
00:20:34.03 And so you can imagine... you can see how the bug is using the macrophage to expand
00:20:39.24 its numbers.
00:20:42.06 And not only that, but we showed that the bacterium also induces the death of the macrophages.
00:20:47.21 Here's a TUNEL stain on the left and this... this death... there are probably many bacterial
00:20:53.03 determinants that do this, but one of them is that self... same locus, ESX1, that also
00:20:58.11 induces the macrophages to come.
00:21:00.06 So, it's got a two-pronged effect.
00:21:02.05 It's inducing death of the infected macrophage and, separately, it's recruiting new macrophages
00:21:07.18 to come to it so that they can engulf the dead macrophage, and... and produce infection.
00:21:15.22 So, to summarize this, let's take a look at what happens in the mutant first.
00:21:23.19 Because, in the mutant, the granuloma might actually be functioning as a host-protective
00:21:29.14 structure, as it might be meant to be.
00:21:34.06 It's you've... you've got an infected macrophage, it dies at a slow rate, macro... new macrophages
00:21:42.23 are slowly recruited at a... at a respectable pace.
00:21:46.10 And, now, they can eat the new macrophage one by one on one, and there's some time for
00:21:53.07 the cells to also kill the dying cell, to also kill the bacteria before they're eaten,
00:21:59.10 so you could imagine there's an attrition of bacterial infection.
00:22:02.19 This might be why the BCG vaccine strain is attenuated.
00:22:06.11 But, paradoxically, instead of sort of thwarting these host-protective processes, as you might
00:22:14.19 imagine a pathogenic bacterium might do, the... the pathogen actually accelerates these processes,
00:22:21.05 so it converts them from being a host-protective to a host-detrimental process.
00:22:26.04 So, simply by speeding up the rate of cell death, and speeding up the recruitment of
00:22:30.19 new macrophages, it's... it's just using the macrophage niche to spread in from cell to
00:22:37.12 cell, and therefore expand itself in the granuloma.
00:22:40.20 And this is quite a nifty thing to do, I think.
00:22:45.04 So... okay.
00:22:46.13 But the question now is, how do... how do... how does this ESX1 locus induce the recruitment
00:22:53.04 of new macrophages?
00:22:54.21 And so, for this part, Hannah was joined by Tamara Pozos, who was a pediatric infectious
00:23:00.13 diseases fellow who joined the lab, and together they did a microarray where they looked at
00:23:05.10 fish that were infected with wild-type or mutant bacteria to identify host genes that
00:23:11.03 might be different between the two.
00:23:13.00 And the gene that stuck out was matrix metalloproteinase 9.
00:23:17.18 And this is an extracellular... an... an enzyme of that... that models...
00:23:23.06 remodels the extracellular matrix.
00:23:25.22 And they were even able to show, not only by transcriptional analyses but also by doing
00:23:31.06 a gelatinase assay on the fish for the actual activity of this enzyme, that MMP9 was induced
00:23:39.20 upon wild-type infection but not upon mutant infection.
00:23:44.16 Now, that's fine.
00:23:47.03 But if MMP9 is... induction of MMP9 is responsible for the ESX-mediated acceleration of macrophage
00:23:57.13 recruitment, then, if you make a MMP9 mutant, that mutant should be attenuated even with
00:24:03.05 wild-type infection.
00:24:04.08 And, sure enough, when they looked they saw that the MMP9 mutant, which is shown on the
00:24:09.17 bottom, there, that fish, was attenuated for infection, and it had very few granulomas.
00:24:17.15 So, it was behaving just like the bacterial mutant and therefore it was the partner.
00:24:24.09 So, so this was nice and then... but then we started to look into what MMP9 does and
00:24:30.03 then it turns out that MMP9 is involved in the pathogenesis of arthritis, and cancers,
00:24:37.14 and other inflammatory conditions, and often in those cases the mac... it's the mac...
00:24:43.07 it's a macrophage that is the bad actor, that is making MMP9 and... and causing trouble
00:24:49.06 in these lesions.
00:24:50.06 So, of course, we thought, well, okay a macrophage gets infected with the bacteria it now induces
00:24:56.17 MMP9 in the macrophage, and now that... that is secreted and calls in new macrophages.
00:25:03.11 But when we did in situ analyses there... to look at where the MMP9 was being made,
00:25:10.13 here is a granuloma, and the MMP9 is labeled green and the macrophage... macrophages are
00:25:17.14 labeled red, and what you can see is that the MMP9 is not in the... in the macrophages
00:25:22.15 of the granuloma.
00:25:23.17 But, rather, it's in the epithelial cells surrounding the granuloma.
00:25:29.13 And so what... what seems to be happening is that an infected macrophage secretes something
00:25:37.18 from that secretion system that goes and talks to the epithelial cell
00:25:43.02 and induces it to make MMP9.
00:25:45.14 And the MMP9 now calls in new macrophages, and this... this... this strategy was called
00:25:52.09 "subversion from the sidelines" by my friend and colleague, Bill Bishai at Hopkins, and
00:25:57.20 I rather like how he put it.
00:26:00.11 So, why might that be?
00:26:03.05 You could imagine that the bacterium wants to tamp down immunity in the infect... in
00:26:10.05 the actual... in the macrophage itself, because it has to survive in there.
00:26:13.21 So, it's doing that and, meanwhile, it's inducing an inflammatory program in a neighboring cell,
00:26:19.24 so that it can bring more macrophages, infect, and then subvert them.
00:26:25.03 Okay.
00:26:26.04 So, but... but... so this is so... so this is how the bacterium uses the innate immune
00:26:33.23 phase of the granuloma to promote its... its growth and expansion.
00:26:40.14 Of course, then the bac... the granuloma matures and other things happen and, as I told you,
00:26:47.01 one of the things that happens is epithelioid transformation, these tight interdigitated
00:26:52.09 projections, that too has been known for, oh, a hundred years or so, and that very reasonably
00:27:00.01 was thought to be a host-protective mechanism that would sort of wall off the bacteria and...
00:27:09.08 and perhaps be... somehow help the host, despite all these strategies of the bacterium.
00:27:14.06 Well, very recently, work from David Tobin's lab, also done in the zebrafish, that this
00:27:20.11 too turns out not to be the case.
00:27:24.10 Mark Cronin and David Tobin have shown that epithelioid transformation of the macrophage
00:27:31.09 is also something that the bacterium is benefiting from.
00:27:34.16 If they inhibit it, then they can... they get less infection than if it's there.
00:27:43.00 And, of course, they're working out the details of how this might be, but what it's telling
00:27:47.02 you is that practically every step that we might predict will help the host can be taken
00:27:52.21 advantage of by the bacterium.
00:27:56.04 So, in my first lecture, I told you that most people actually clear infection and they do
00:28:02.11 so after the adaptive phase of the... of the granuloma has kicked in.
00:28:06.22 So, it's very clear that... that at some point the granuloma can fight back and can... can
00:28:14.21 eradicate or at least suppress infection.
00:28:17.14 And many, many people who specialize in the areas of adaptive immunity and TB are... are
00:28:24.17 working on this.
00:28:26.14 But I want to close by saying that, while the adaptive immunologists may not know as
00:28:32.11 much about this as they want to, and they're working hard to figure it out, the bacterium
00:28:37.01 seems to know quite a little... quite a bit about this... these immune mechanisms, because
00:28:42.11 what these people so... who worked on it so far can tell you is that it tries really hard
00:28:48.08 to delay and inhibit adaptive immunity, so that the adaptive immune elements that come
00:28:54.18 into the granuloma do so late.
00:28:58.10 And this gives time for the mechanisms that I've just been telling you about to help bacteria
00:29:04.20 expand in the innate context.
00:29:07.20 I'll close by thanking the many people whose research I've described to you -- they're
00:29:14.04 both students and from my lab, as well as colleagues and collaborators from outside
00:29:19.19 my lab, and, indeed, from across the world.
00:29:23.08 Thank you.