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Human Papillomavirus (HPV) Vaccines to Prevent Cancer

Part 1: Human Papillomavirus (HPV) Vaccines to Prevent Cancer

00:00:11;18 Well, hello.
00:00:12;24 My name is John Schiller.
00:00:14;01 I work in the Intramural Program of the National Institutes of Health.
00:00:18;26 And today I'll be giving a lecture on HPV vaccines to prevent cancer.
00:00:23;15 And what I'll be covering is initially the basic biology of HPV and its association with cancer.
00:00:31;15 And then talk about the development of the vaccines to prevent the cancers that are caused
00:00:36;07 by HPV.
00:00:37;24 And then, in the last third, talk about some of the implementation issues that have arisen
00:00:43;06 with these vaccines, and what we've been doing to try to overcome those implementation issues.
00:00:50;20 So, first of all, a little basic molecular biology.
00:00:55;02 So, HPVs all have 8-kilobase double-stranded genomes.
00:01:01;06 And they are divided into two regions.
00:01:03;14 One is called the early region, which encodes factors involved in the replication and transcription
00:01:10;09 of the viral genome, and setting up the cells for viral replication.
00:01:16;26 There are two genes in the early region, E6 and E7, which are selectively retained
00:01:24;00 and expressed in cancers.
00:01:26;01 E6 binds the tumor suppressor gene p53, and E7 binds the tumor suppressor Rb.
00:01:34;09 The other region of the genome is what's called the late region... region, which I'll mainly
00:01:39;01 be talking about today, which encodes two proteins: the major virion protein, L1,
00:01:45;08 and the minor virion protein, L2.
00:01:48;17 So, the structure of the virion is depicted on this slide.
00:01:52;21 It's a non-enveloped virion, about 55 nanometers with a regular icosahedral array composed of
00:02:00;10 72 pentamers of the L1 major capsid protein and up to 72 copies of the minor capsid protein,
00:02:08;08 L2.
00:02:09;08 And in the middle, not shown here, is the DNA genome, which is histone-bound.
00:02:17;15 Now, the HPVs have a very unusual life cycle.
00:02:22;09 The life cycle takes place entirely within the stratified squamous epithelia.
00:02:28;10 And we believe that this type of life cycle has evolved primarily to escape immune [surveillance].
00:02:34;17 So, this is a tricky virus.
00:02:36;22 What happens is it initially has to bind the basement membrane
00:02:39;19 -- and I'll talk about that in my second lecture --
00:02:42;26 which then initiates the infectious process of basal keratinocytes.
00:02:46;20 Now, once it infects these lower cells, it expresses very low levels of its virul...
00:02:53;03 of its proteins, just the early proteins, and has autonomous replication at
00:02:58;20 relatively low copy number.
00:03:00;19 But, relying on signals involved in terminal differentiation of the epithelium,
00:03:06;28 there is an induction of over-replication of the genome and the late gene expression, leading to
00:03:12;23 virion assembly and release.
00:03:15;08 And so, by making these virions and exposing them only to the external surface, it essentially
00:03:22;27 evades the immune system, keeps the immune system ignorant of these very immunogenic particles,
00:03:29;04 until quite a delayed period.
00:03:32;19 So, the reason we want to develop HPV vaccines is that HPVs cause cancers, a lot of cancers.
00:03:40;12 It's been estimated that approximately 5% of all cancers are caused by HPV infection.
00:03:48;08 And the worldwide burden is shown on this slide.
00:03:50;19 And it's important to note that cervical cancer represents the majority of the HPV-associated
00:03:57;17 cancers worldwide, about 500,000 cases and 250,000 deaths.
00:04:03;26 It's also important to note that of the 12 or 15 types that are considered oncogenic,
00:04:09;21 two types, 16 and 18, account for 70% of cervical cancer.
00:04:14;10 And they actually account for approximately 90% of the other HPV-associated cancers,
00:04:22;26 which are fewer in number and a lower attributable fraction.
00:04:27;15 Essentially 100% percent of cervical cancers are believed to be caused initially by
00:04:32;23 an HPV infection.
00:04:34;19 Now, in the US, of the attributable cancers that are caused by HPV, the proportions
00:04:42;21 vary somewhat differently than worldwide.
00:04:44;17 And as you can see, cervical cancer doesn't dominate as much.
00:04:48;20 And there are more male cancers, shown in in gray, than there are worldwide.
00:04:53;27 And the reason for this is two-fold.
00:04:56;24 One is we have PAP screening, which decreases cancer...
00:05:00;13 cervical cancer by approximately 80% in the United States.
00:05:04;16 And secondly, there's been what's considered an epidemic of oropharyngeal cancer in the
00:05:09;27 last several decades.
00:05:11;12 It's essentially doubled.
00:05:12;28 And many of these cancers -- as shown in gray, again -- are caused in males.
00:05:19;07 Now, HPVs, and the oncogenic HPVs, actually infect many sites in both the male and female
00:05:26;23 genital... genitalia.
00:05:28;11 So for instance, there's many infections in the vaginal and vulvar regions of the women,
00:05:34;02 and the penile skin of men.
00:05:37;12 But the reason we believe that HPV predominates in cervical cancer is because there's a place
00:05:45;03 in the anatomical regions of the cervix called the transformation zone
00:05:50;07 that's particularly susceptible to carcinogenic progression.
00:05:53;26 And this is the region where single columnar epithelia meets stratified squamous epithelia.
00:05:59;20 And quite frankly, we really don't understand why this particular site is so prone to
00:06:05;16 HPV-induced carcinogenesis.
00:06:07;22 But this incidentally is also the site in which most of the cancers in anal cancer arise
00:06:12;26 as well.
00:06:14;11 Now, this shows the timeline of [HPV] infections in relation to cervical cancer.
00:06:20;22 And what you can see is that the rates of cervical HPV infection rise very rapidly
00:06:29;18 in young adults, and the lifetime incidence of genital HPV infection is considered to be over 80%.
00:06:36;13 So, having a lifetime HPV infection is almost synonymous with being sexually active.
00:06:42;18 But most of these infections go away spontaneously and do not really pose risk for cancer.
00:06:50;15 You need a persistent infection to get cancer.
00:06:54;11 But about a decade later, you see a peak in precancerous lesions.
00:06:58;01 And then about another decade later, you can see that you get a peak in cancer incidence.
00:07:05;00 So, really, persistent infection with oncogenic HPV is a critical risk factor for progression
00:07:13;00 to precancer and cancer.
00:07:16;08 And because HPV infections are so common, they're rapidly acquired after sexual debut.
00:07:22;07 And this is just data from the US and UK that shows, quite strikingly, that within two years
00:07:28;07 of initiating sexual activity almost half of the young women have had an HPV infection.
00:07:35;07 Now, it's important to point out that current PAP screening is quite effective,
00:07:40;22 but it's what we call secondary prevention, which targets adult women who already have their infections
00:07:48;06 and have been diagnosed with a cervical abnormality for further follow up
00:07:52;16 and eventually ablative treatment.
00:07:54;26 And what we're talking about with the vaccines, now, is what we call primary prevention,
00:08:00;23 which can target both adolescent girls and boys, and eliminate the initiating event and
00:08:07;14 prevent all this downstream follow-up that's required in treatment for secondary prevention.
00:08:14;20 So, as I mentioned, the real viruses have three components.
00:08:20;08 They've got L1 pentamers.
00:08:21;28 They've got L2 monomers.
00:08:24;21 And then the HPV genome.
00:08:26;26 But through molecular biology tricks, we can express just L1 and L2 alone, and generate
00:08:33;13 what are called virus-like particles.
00:08:35;26 Now, in animal models, we were able to determine that L1 contains most of the determinants
00:08:43;00 to which neutralizing antibodies can bind.
00:08:46;22 And so that the current vaccines are made up of what we call L1-only VLPs, virus-like particles.
00:08:53;14 So, they're essentially made up of 360 copies of a single protein, this L1.
00:08:59;28 And therefore they're considered to be subunit vaccines because they're non-infectious
00:09:04;17 and non-oncogenic, because they're just made up of this single protein.
00:09:09;15 And again, we've shown in animal studies that I won't show that VLPs induce extremely high titers
00:09:14;01 of neutralizing antibodies that are at least a log higher than what is seen after
00:09:20;21 a natural infection.
00:09:22;26 So, based upon the promising preclinical data, three distinct HPV L1 VLP vaccines have been
00:09:31;21 commercialized.
00:09:33;06 And they're made by two manufacturers: GlaxoSmithKline and Merck.
00:09:37;21 And they vary in valency, the number of types included;
00:09:42;09 the adjuvant, which is the immune stimulatory component in the vaccine;
00:09:46;27 and the production system in which the VLPs are made.
00:09:51;07 So, Cervarix, which is made by GlaxoSmithKline, is bivalent.
00:09:55;19 It contains the two types, 16 and 18, which cause 70% of cervical cancer.
00:10:02;18 This immune stimulatory product that is used is a proprietary one.
00:10:06;28 It's called AS04.
00:10:09;01 It consists of a simple aluminum salt, which would be the standard adjuvant, plus what's
00:10:15;00 called monophosphoryl lipid A, which is a TLR agonist that
00:10:21;19 further stimulates the immune response.
00:10:24;23 And it's the first example where the FDA has approved this type of immune stimulator
00:10:31;08 for a prophylactic vaccine.
00:10:34;11 Gardasil is quadrivalent.
00:10:37;13 It contains the same two high-risk HPV types, 16 and 18.
00:10:41;18 In addition, it contains HPV 6 and 11, which cause about 90% of genital warts.
00:10:47;25 It doesn't cause cancers, but is responsible for genital warts.
00:10:53;26 It contains a much more common aluminum salts vaccine.
00:10:59;25 Gardasil-9, which has been more recently developed and is actually the vaccine that's now
00:11:05;22 most available in the United States, is nonavalent.
00:11:09;15 So, in addition to the two types in Cervarix, it contains the five next types that are
00:11:15;25 most often seen in cancer.
00:11:18;06 But other than that, it's very much formulated the same way.
00:11:21;10 Now, the production system also varies.
00:11:24;09 So, Cervarix is made in insect cells, which was the production system we initially
00:11:30;18 used in our preclinical studies in a proof-of-principle evaluation, whereas Gardasil is made in
00:11:38;14 Saccharomyces cerevisiae yeast, common bread yeast.
00:11:41;28 So, the vaccines are administered by intramuscular vaccination,
00:11:48;18 generally at one and either one or two months... at zero, one, or two months, and then six months.
00:11:56;08 But more recently, an abbreviated schedule of one and six months has been adopted
00:12:02;22 in the EU, Canada, and more recently in the United States for individuals less than 15 years old,
00:12:09;17 based upon the finding that those two doses in the younger age groups is equivalent
00:12:16;01 in the antibody responses that are induced in comparison to the three doses in young adults,
00:12:21;26 in which the efficacy was demonstrated in the clinical trials.
00:12:26;24 So, this slide shows a timeline of HPV association with cervical cancer versus vaccine development.
00:12:35;06 And the take-home message here is that basic development to a public health intervention
00:12:43;23 generally takes decades.
00:12:45;18 So, HPV16 was first discovered in 1982 by Harald zur Hausen and his colleagues in Germany,
00:12:54;09 and he later went on to win the Nobel Prize for that discovery.
00:12:57;27 It took us 10 years in the laboratory to develop an attractive HPV vaccine particle that
00:13:05;08 we could show generated high titers of antibodies that could prevent infection.
00:13:09;16 And that was in 1992.
00:13:12;01 Now, during the decades of the '90s and '80s, there was a whole series of studies
00:13:19;05 -- case control and prospective epidemiological studies, and also laboratory studies,
00:13:25;05 some of which my group was involved with --
00:13:27;17 that led to the conclusion that HPV infection was the
00:13:31;24 first necessary cause of a cancer, in this case, cervical cancer.
00:13:37;08 And this correlated with the time in which we conducted the preclinical animal studies,
00:13:43;08 the proof-of-concept studies, and started clinical trials, again in 1999, leading to
00:13:49;17 the first efficacy data in 2001, licensure in females in 2006 and in males in 2009.
00:13:58;01 And you can see that even though in 1992 we had the prototype for what was going to
00:14:04;10 become the commercial vaccine, it still took 15 years in order for it to be widely distributed in people.
00:14:11;12 Now, an important thing to note is that the basic understanding of how HPV causes infec...
00:14:19;17 causes cancer was critical for developing these vaccines.
00:14:25;14 And that's because we wanted a vaccine that could be licensed for preventing cancer,
00:14:30;17 but we could not use cancer as a primary endpoint in the trial.
00:14:35;03 And there are two reasons for this.
00:14:37;15 One, what we're trying to do is prevent initial infection, and it takes decades to go
00:14:44;07 from initial infection to cancer, which would be far too long for a controlled clinical trial.
00:14:49;02 And secondly, because we have PAP screening, we identify premalignant lesions and
00:14:54;11 have to remove them.
00:14:55;20 And so it's unethical in a trial with active follow-up to allow women to go on to cancer.
00:15:01;16 But fortunately, by that time we understood that these premalignant lesions,
00:15:07;19 particularly CIN3 -- cervical intraepithelial neoplasia grade 3 --
00:15:13;11 was a necessary precursor for cervical cancer.
00:15:16;18 And the FDA and other regulatory bodies were convinced that if we could demonstrate protection
00:15:21;22 against intermediate- and high-grade cervical lesions that we would be preventing future cancers,
00:15:28;05 and therefore could license the vaccine for prevention of cancer.
00:15:34;16 And so there were several large-scale trials that were done, then, to demonstrate that
00:15:39;17 these vaccines could work against disease endpoints.
00:15:43;22 And remarkably, the vaccines were extremely effective in these efficacy trials.
00:15:51;15 This is the first vaccine against a sexually transmitted disease that has shown any substantial efficacy.
00:15:58;19 And what you can see here is that for both Cervarix and Gardasil, if you look in women
00:16:08;17 who had no genital HPV infection at entry and restrict the analysis to the types
00:16:15;15 that are targeted by the vaccine, that there was 100% protection against
00:16:20;05 the most cancer-proximal endpoint we could look at, which was CIN3.
00:16:25;04 There was also strong protection against genital warts with Gardasil, which contains HPV6 and 11.
00:16:32;17 Now, the protection in men looks as if it may be slightly less, but it's important
00:16:37;10 to note that we think that we miss more prevalent infection at the time of vaccination in men,
00:16:43;14 and so that's... a lot of these, what look like breakthrough infections,
00:16:47;06 are actually emergence of prevalent infections that pre-exist prior to vaccination.
00:16:53;11 Now, Gardasil-9, which again was approved in late 2017, the clinical trials were a little different.
00:17:03;10 Because at that point there were two vaccines that were already FDA-approved.
00:17:08;06 And so it was considered unethical to test them against the placebo control.
00:17:13;03 So, what the FDA and other regulat... regulators agreed to is to demonstrate that they
00:17:18;14 could protect against high-grade dysplasias against the new types in Gardasil-9, the five oncogenic types,
00:17:26;05 and had immunogenicity equivalents to the four types that were already there.
00:17:32;08 In other words, the antibody levels were no different to the four types in common.
00:17:37;06 And what they found in this trial is that there was 96% efficacy against CIN2/3 of
00:17:43;04 five new types in comparison, direct the comparison, to Gardasil-4,
00:17:47;13 and the antibody types to the common VLPs... the antibody titers to the common VLPs were no different.
00:17:55;20 So, in addition to having demonstrated protection against neoplastic disease against cervical lesions,
00:18:05;22 there is also data that it protects against anal lesions and vulvar/vaginal lesions
00:18:11;14 in clinical trials.
00:18:12;22 But as you can see here, there's no evidence that it protects against lesions on the penis.
00:18:18;08 And the reason for this is that penile intraepithelial neoplasias are actually relatively uncommon
00:18:23;15 in comparison to the ones listed above.
00:18:25;17 And the trials were not particularly large.
00:18:28;10 And secondly, we don't know that it protects against infections... lesions at the oro-pharynx,
00:18:34;25 and the reason for this is the premalignant lesions have not been yet identified.
00:18:39;15 Now, I think we have to start think... stop thinking about the HPV vaccines as a new kid on the block.
00:18:47;24 It's still considered one of the newer vaccines, but it's already been commercially available
00:18:53;06 for 12 years and has been licensed in 82 countries, and over 270 million doses have been given globally.
00:19:00;09 So, I think we have to consider this just one of the standard, routine vaccines to deliver
00:19:04;22 to adolescents.
00:19:06;20 Now, we think that the way this vaccine works primarily is that it protects from initial infection
00:19:12;26 by generating antibodies that bind the virus and prevent them from
00:19:18;14 initially infecting people.
00:19:19;24 And the reason we think that is that most vaccinees never test positive for HPV DNA
00:19:26;13 using sensitive PCR assays.
00:19:28;14 And also the fact is that when we do see breakthrough infections, they happen predominantly early
00:19:36;01 during the trials.
00:19:37;24 So, why would we get more infections early rather than later, when normally immunity
00:19:43;10 is waning?
00:19:44;10 And the reason, again, we think this is because what we're actually measuring is
00:19:49;10 emergence of infections that were present at the time of vaccination,
00:19:53;08 who then emerged during the vaccination period.
00:19:56;08 And so really it could not be prevented by a prophylactic vaccine.
00:20:00;10 So, it's also important, although these vaccines seem like they're very potent at
00:20:06;02 preventing initial infections by the types targeted by the vaccine,
00:20:09;11 there are some things that the vaccines don't do.
00:20:12;06 And they don't prevent infection or disease by most of the other HPV types.
00:20:17;03 There's a few very closely related types that we do see partial protection from it.
00:20:21;23 And lastly, it's important to note that they do not induce clearance of infection or prevent
00:20:28;11 progression of lesions once they've occurred.
00:20:31;08 And this is why it's really very important, to get the biggest bang for the buck, to vaccinate
00:20:36;08 young women and young men before they become sexually active,
00:20:39;07 because once they have the infections the vaccine is not going to do any good for that particular infection.
00:20:46;18 Now, it's important to note that despite what maybe you can read if you google HPV vaccines
00:20:52;26 and safety, the vaccines actually have an excellent safety record.
00:20:56;23 So, low grade and transient infection site reactions are common.
00:21:01;24 And when there are systemic reactions, such as fever, they're generally mild and self-limiting.
00:21:06;23 A certain number of the vaccinees do faint during the vaccination.
00:21:14;01 But we believe that this is primarily due to seeing the needle, and the fainting
00:21:19;17 is no more than other needle injections in this age group.
00:21:24;11 Important to point out is that there's no pattern of serious adverse events,
00:21:28;06 either in the trials or in post-licensure surveillance, that would suggest that there's a causal relationship
00:21:34;17 of the vaccine to any serious adverse event.
00:21:38;18 So, turning to effectiveness, in vaccinology we have two different terms.
00:21:44;27 Efficacy means how well the vaccine works in a clinical trial.
00:21:49;15 And effectiveness is used to describe how well the vaccine uses in general use,
00:21:55;18 in the public, for instance in national immunization programs.
00:21:58;23 And we're starting to accrue, now that the vaccines have been available for more than a decade,
00:22:02;19 substantial evidence that these vaccines are also very effective in general use.
00:22:10;13 And there's data both in the levels of infections, prevention of genital warts,
00:22:14;25 and prevention of cervical lesions.
00:22:17;03 And I'm just gonna give you a little flavor of some of the data that's starting to
00:22:20;17 come out from some of these national immunization programs.
00:22:23;23 So, this slide shows the effect of Cervarix vaccination in England after the beginning
00:22:31;10 of their vaccination program.
00:22:33;19 And what it shows is that, in the younger age group, 16 to 18, in which 50% are vaccinated
00:22:40;16 by year two or three, there was a 50% reduction in the prevalence of HPV16 and 18 in the population.
00:22:48;26 And after three to four years... after four to six years, when over 70% were vaccinated,
00:22:54;28 there was a 76% drop in the prevalence in the population.
00:22:59;10 But it's important to note that in the older young women, who... there was no vaccination,
00:23:07;00 there was no drop in HPV infection rates.
00:23:08;15 And as a matter of fact, it may have even been increasing slightly.
00:23:14;25 There's also evidence, for instance this evidence from Australia, that the rate of intermediate-
00:23:20;00 and high-grade dysplasias is also dropping in the vaccinated population.
00:23:24;00 So, here you see that in under-18-year-olds, the rate of CIN2/3 is declining rapidly since
00:23:31;24 the start of vaccination, which is indicated by the needle; whereas we maybe are starting
00:23:37;17 to see a little bit of a drop in the 18-to-20-year-olds, where there's some vaccination
00:23:43;07 but relatively poor coverage;
00:23:45;12 and no drop in the older women, who are not being vaccinated.
00:23:49;25 Now, this vaccine, then, therefore we think has an enormous potential for decreasing
00:23:57;06 the burden of HPV-associated disease.
00:24:00;19 And this shows what we think the potential is for cervical cancer.
00:24:06;04 So, in a non-vaccinated scenario, the expectations are that, over the next 65 years,
00:24:14;20 worldwide we will see 19 million cases of cervical cancer and 10 million deaths.
00:24:20;26 And you can see that the majority of these cases will occur in lower/middle-income and
00:24:26;02 lower-income countries.
00:24:28;26 Now, in the ten years since we've initiated the vaccine programs worldwide,
00:24:37;10 it's been estimated that we've prevented a little over 350,000 cases and 150,000 deaths.
00:24:45;25 And the majority of these are in upper-income countries or high-income countries.
00:24:54;08 And again, the reason for this is that this is where the vaccine is being delivered.
00:24:59;25 And so, if you look at this number, 150,000, from a preventive point of view, we're disappointed.
00:25:09;07 But this sort of points out the real potential of cancer prevention.
00:25:14;27 Because if we had shown you that 150,000 women were cured of cancer, everybody would be
00:25:22;02 really excited about it.
00:25:23;02 And it points out that for prevention, the individual who's not going to get the cancer
00:25:28;14 doesn't know it.
00:25:29;14 And so it can tend to be underappreciated in relationship to cancer treatments.
00:25:34;26 And it's not only in low-resource countries that this vaccine is being underutilized,
00:25:40;11 but also in countries like the United States.
00:25:42;17 So, this slide shows the uptake over time since the introduction of two other vaccines
00:25:49;20 for adolescents versus the HPV vaccine.
00:25:53;20 And what you can see is that the three-dose coverage is only a little above 30%,
00:25:58;16 and the one-dose coverage is about 50%, which is much less than the other two vaccines.
00:26:04;25 So, what do... what can we do to increase uptake, particularly in low-resource settings?
00:26:10;08 Well, importantly, both [companies] have committed to sell the vaccine to GAVI,
00:26:16;16 which is a global alliance that buys vaccines and distributes it to the 72 lowest-income countries in the world.
00:26:25;13 They decided to buy it for five dollars a dose, which is much, much less than the market price.
00:26:32;01 Another possibility that we're working with is with vaccine manufacturers in
00:26:36;17 emerging countries, for instance in India and China and Brazil, who, again, could potentially
00:26:42;05 manufacture the vaccine and sell it at a lower price.
00:26:45;19 We certainly need to address vaccine hesitancy, particularly in families but also in
00:26:51;13 health care providers who seem to be a major impediment to increasing uptake of the vaccine.
00:26:58;23 And finally, that's something I'll deal with now, is that what happens if we can deliver
00:27:02;28 fewer than three doses.
00:27:05;00 It's problematic trying to set up vaccination programs where adolescent girls,
00:27:10;05 and in some cases boys, have to come in three times in six months.
00:27:15;01 And so we want to determine whether we can deliver fewer than three doses, for instance
00:27:20;11 even a single dose.
00:27:21;18 And the reason we think this might be possible comes from post hoc analysis of trials,
00:27:28;26 for instance this NCI sponsored trial in Costa Rica, which used Cervarix.
00:27:36;10 And what this table shows is that if you look at the end of seven years, the total number
00:27:42;20 of HPV16 and 18 infections in women who got... who got three doses, which is what they were
00:27:50;17 supposed to get, versus the women who for various reasons only got one dose, is no different.
00:27:57;19 The confidence intervals are wider for the one dose, because there's actually
00:28:01;20 relatively few women who didn't get the full three doses, but they're completely overlapped.
00:28:07;00 If you look at the types in which we get partial protection, HPV31, 33, and 35, you can see that,
00:28:13;16 again, there's no difference in the total number of infections by these types
00:28:18;16 at the end of seven years.
00:28:20;02 And if you look at other oncogenic or nononcogenic types, which we know the vaccine
00:28:24;13 does not protect against, again, the accumulated incidence is the same.
00:28:29;05 And this is an important observation because what it says is that the women who get
00:28:34;01 only one dose are not some unusual subset that have lower exposure rates to genital HPV infections.
00:28:41;04 This is just another way of looking at the data, where if we just look cross-sectionally
00:28:46;08 at the end of seven years... who has infections?
00:28:49;20 So, in comparison to the control group that didn't get vaccinated, you can see that
00:28:55;13 there's essentially no difference between the women who got one, two, and three doses for nononcogenic types,
00:29:01;17 for the cross-reactive types shown in red, or for the targeted types, HPV16 and 18, shown in blue.
00:29:10;12 So, there are actually two other trials, again, post hoc analysis, that demonstrated that
00:29:16;22 at the end of four years, for instance in the GlaxoSmithKline, the corporate-sponsored trial,
00:29:22;25 they also showed similar efficacy at one, two, and three doses.
00:29:26;17 And for Gardasil, there was an interrupted Indian cluster randomization trial,
00:29:32;06 where they were supposed to compare two doses to three doses, but for political reasons
00:29:38;02 the vaccine trial was stopped, and so some of the women only got one dose.
00:29:41;22 And again, after seven years, they showed similar protection in women regardless of
00:29:47;10 the number of doses they received.
00:29:49;24 So, the question then is, well, now, is it time?
00:29:53;09 Should we adopt a single dose vaccination program based upon this data?
00:29:57;28 Well, we believe that because these are this post... these... these findings are post hoc,
00:30:03;17 that they provide insufficient evidence to generally promote implementation of
00:30:09;00 single dose HPV vaccination programs.
00:30:11;27 Now, they may possibly induce early adoption, and particularly in low-resource settings
00:30:18;13 where they can't afford to give three doses or even two doses, with the contingency that
00:30:24;20 if one dose doesn't appear to work that the women can then be boosted.
00:30:31;06 That type of implementation issue or program may be justified.
00:30:36;09 But we think that to really put this all on very firm, strong footing, and be able to
00:30:43;05 convince regulators to approve single dose vaccination programs,
00:30:46;23 we need to do a well-controlled randomized controlled clinical trial to compare one and two doses.
00:30:52;26 And the NCI has now undertaken this study in Costa Rica with support from the Gates Foundation.
00:31:00;05 And it'll be a four-armed trial where we'll be comparing one and two doses of Cervarix,
00:31:03;26 and one and two doses of Gardasil-9, with 5,000 12-16-year-old young ladies in each arm.
00:31:12;01 We'll do a survey of HPV prevalence in unvaccinated young women in the same region of the same age
00:31:17;16 so we can get some sort of idea of pseudoefficacy.
00:31:22;24 And we expect the trial to last for four years with long term follow up.
00:31:26;17 So, to conclude, the HPV vaccines are safe and very effective at preventing
00:31:33;06 incident infection and disease by the vaccine-targeted types.
00:31:37;11 The vaccines have great potential for reducing the burden of HPV-induced cervical and
00:31:43;13 other types of cancers worldwide.
00:31:45;21 So the primary challenge now is implementation, to see that the vaccines reach the individuals
00:31:52;00 who are most in need in them.
00:31:53;14 And we believe that the way to maximize implementation is to demonstrate that a single dose is enough
00:32:00;09 in a randomized clinical trial, which could be transformative for implementation of this vaccine.
00:32:07;02 So, I would like to conclude by thanking some of the collaborators that have been working
00:32:13;02 in my lab and in other places around the world.
00:32:16;00 And particularly, Doug Lowy, who's been my co-PI on all the work that happened
00:32:21;04 in our lab related to the HPV vaccines for the last 20 years.
00:32:25;05 So, thank you very much.

Part 2: 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.

Videos in this Talk
  • Part 1: Human Papillomavirus (HPV) Vaccines to Prevent Cancer
    Part 1: Human Papillomavirus (HPV) Vaccines to Prevent Cancer
    Audience:
    • General Public
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 2: Why Do HPV Virus-Like Particle Vaccines Work So Well?
    Part 2: Why Do HPV Virus-Like Particle Vaccines Work So Well?
    Audience:
    • General Public
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
Speaker: John Schiller
Total Duration: 1:07:10
Recorded: April 2018
All Talks in Human Disease

Talk Overview

Human Papillomavirus (HPV) causes 5% of all cancers worldwide, and the first vaccine against HPV was approved in 2006. In this seminar, Dr. John Schiller provides an overview of HPV virus and infection, compares the three FDA approved vaccines against HPV, and explains the endpoints used in the clinical trials to prove vaccine efficacy. After a decade of using the vaccine, retrospective studies now allow us to evaluate the possibility of using single-dose vaccination, which could lead to an increase in the general use of the vaccine (implementation), and improve HPV-related cancer prevention.

In his second seminar, Schiller discusses the high efficacy of HPV vaccine, which is exceptionally good at producing neutralizing antibodies and also benefits from the low mutation rate of HPV. Coming to a better understanding of the efficacy of the HPV vaccine will provide evidence to support single-dose vaccination and aids in the development of new vaccines.

Speaker Bio

John Schiller

John Schiller

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

Playlist: Cancer

Related Resources

Schiller J and Lowy D (2018) Explanations for the high potency of HPV prophylactic vaccines. Vaccine pii: S0264-410X(18)30019-7

Schiller JT and Lowy DR (2012) Understanding and learning from the success of prophylactic human papillomavirus vaccines. Nat Rev Microbiol 10(10):681-92

Reader Interactions

Comments

  1. Becky says

    “Human Papillomavirus (HPV) causes 5% of all cancers worldwide” … Really? I would appreciate the research that showed this. I have heard differently from someone who worked on the HPV vaccine. The marketing strategy included dropping some zeros and shifting a decimal or two to create the illusion of this figure. Also, the HPV causes specific cancers, not all…. and they differ between men and women. “Also, “the high efficacy of HPV vaccine” is a purely projected prognosis and there is as yet no appreciable data to prove that statement fully. In fact there is more data cropping up each month, suggesting that the vaccine has inadvertently caused some ovarian, colon and prostate cancers and rendered many women barren as well has having had a side effect of paralysis in varying degrees to young women the world over. While I understand how it is meant to work, that the theory and rationale is perfect, we have seen too many other variables, and in the light of the sheer magnitude of highly qualified whistleblowers who have specifically condemned this vaccine, it would be pertinent for parents to do a lot more research on how it has behaved in reality. I have some serious reservations about the air of marketing in this ‘biology’ lecture…. I would appreciate some bone fida clarity on this. Thanks 🙂

    Reply

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