Session 10: The Immune Response in Health and Disease
Transcript of Part 1: Why Do HPV Virus-Like Particle Vaccines Work So Well?
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.