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.