This session evaluates two examples of the interaction between pathogens and the human immune system. First, it characterizes human papillomavirus (HPV) pathogenesis and discusses how the molecular mechanisms of viral entry allow the HPV vaccine to work with high efficacy. Second, this session provides an overview of tuberculosis pathogenesis, the life cycle of the M. tuberculosis bacteria, and how M. tuberculosis uses the host immune system to increase infectivity.
Why Do HPV Virus-Like Particle Vaccines Work So Well?
Concepts: Human Papillomavirus (HPV), HPV susceptibility to neutralizing antibodies, B cell receptor/Virus-like particle complex, and importance of viral infection timeline
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.
An Introduction to Tuberculosis: The Pathogenic Personality of the Tubercle Bacillus
Concepts: Overview of tuberculosis (TB), TB prevalence worldwide, life cycle of TB, TB pathogenesis (infection of macrophages & granulomas), efflux pumps, and TB vaccine
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...
00:13:54.00 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.
The Troublesome Tubercle in Tuberculosis
Concepts: TB bacterium's use of granuloma to attract macrophages and increase infectivity, Mycobacterium marina - a model to study TB in zebrafish, granuloma promotes growth of the TB bacteria, and expansion of disease via macrophage apoptosis
Note: This video is optional for this session.
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.
00:11:39.11 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.
00:26:26.04 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.
- John Schiller iBioSeminar: Human Papillomavirus (HPV) Vaccines to Prevent Cancer
- Lalita Ramakrishnan iBioSeminar: Tuberculosis Pathogenesis
Dr. Lalita Ramakrishnan is a professor of immunology and infectious diseases at the University of Cambridge, UK. She received her medical degree from the Baroda Medical College in India, and her PhD in Immunology from Tufts University in Boston. After completing her medical residency at Tufts and a fellowship in Infectious Diseases at the University… Continue Reading
Dr. John Schiller is a National Institutes of Health (NIH) distinguished investigator and a professor at the National Cancer Institute at the NIH. Schiller completed his bachelor’s degree in molecular biology at the University of Wisconsin-Madison (1975), and received a doctorate degree in Microbiology at the University of Washington in Seattle (1982). Schiller continued his… Continue Reading
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