1 00:00:01 --> 00:00:05 Today, we're talking about immunology as you may recall. 2 00:00:05 --> 00:00:12 Let's roll. Yes, let's roll. 3 00:00:12 --> 00:00:16 And by the way, admire your little finger, 4 00:00:16 --> 00:00:21 because most of the planet isn't as gifted as you are. 5 00:00:21 --> 00:00:26 Not everyone was guaranteed so many brains as you and I, 6 00:00:26 --> 00:00:31 seriously, have been fortunate enough to have been granted. 7 00:00:31 --> 00:00:35 Let me just finish up what we were talking about last time, 8 00:00:35 --> 00:00:44 which was the cell cycle. 9 00:00:44 --> 00:00:48 You may recall that we talked about oncogenes. We talked about Ras and 10 00:00:48 --> 00:00:52 the Ras oncoprotein, which sends out mytogenic signals. 11 00:00:52 --> 00:00:56 And, what it does, just to summarize what we were talking about 12 00:00:56 --> 00:01:00 is it pushes cells from the beginning of the G1 phase of the 13 00:01:00 --> 00:01:05 cell cycle up to a decision point in the life of the cell. 14 00:01:05 --> 00:01:09 It's called the restriction point. And here, just by way of review, 15 00:01:09 --> 00:01:13 the cell has to decide whether or not it's going to commit itself, 16 00:01:13 --> 00:01:17 essentially irreversibly, to go through the rest of the cell cycle, 17 00:01:17 --> 00:01:22 or whether it'll stay in G1, and may even retreat from the cell cycle 18 00:01:22 --> 00:01:26 into G0. And Ras pushes this decision forward. 19 00:01:26 --> 00:01:30 The retinoblastoma protein, which we talked about the last time, 20 00:01:30 --> 00:01:35 stands as the guardian of the gate right here, the RB protein. 21 00:01:35 --> 00:01:38 And, the retinoblastoma protein holds this gate shut unless and 22 00:01:38 --> 00:01:41 until certain preconditions have been satisfied, 23 00:01:41 --> 00:01:45 on which occasion the retinoblastoma protein opens up the restriction 24 00:01:45 --> 00:01:48 point gate, and allows the cell to go through the rest of the cell 25 00:01:48 --> 00:01:52 cycle. And now, interviewed from this perspective, 26 00:01:52 --> 00:01:55 we can begin to understand how hyperactivity of Ras, 27 00:01:55 --> 00:01:59 and how the inactivation of this RB, this retinoblastoma, tumor 28 00:01:59 --> 00:02:02 suppressor protein have such disruptive destabilizing effects on 29 00:02:02 --> 00:02:06 the proliferative controls of the cell. 30 00:02:06 --> 00:02:10 Keep in mind, this is a negative actor on cell proliferation. 31 00:02:10 --> 00:02:14 It's a tumor suppressor gene which must be inactivated in many cancers. 32 00:02:14 --> 00:02:18 This is a proto-oncogene, or an oncogene, which must become 33 00:02:18 --> 00:02:22 hyperactivated. Now, I want to move from that into 34 00:02:22 --> 00:02:26 the topic of today, and that's the whole issue of 35 00:02:26 --> 00:02:31 immunity. And, much of our immunity comes from 36 00:02:31 --> 00:02:35 understanding the way we deal with what viral infections. 37 00:02:35 --> 00:02:39 The fact is, just to cite one arbitrary viral infection, 38 00:02:39 --> 00:02:44 relatively few people die from viral infections these days because we can 39 00:02:44 --> 00:02:48 become immunized against them. The first immunizations already 40 00:02:48 --> 00:02:52 began in the late 18th century, believe it or not, when a physician 41 00:02:52 --> 00:02:57 in England called Edward Jenner first noticed that women who had 42 00:02:57 --> 00:03:01 worked as milkmaids milking cows, and who got a disease called cowpox 43 00:03:01 --> 00:03:06 from milking the cows seemed to be immune to the disease of smallpox 44 00:03:06 --> 00:03:10 which was by that time realized to be spreading in epidemic, 45 00:03:10 --> 00:03:14 so a highly infectious agent, and one which actually killed quite 46 00:03:14 --> 00:03:19 a few people. And Jenner intuited correctly, 47 00:03:19 --> 00:03:24 in retrospect, that the experience of these milkmaids and their 48 00:03:24 --> 00:03:29 exposure to cowpox exposure somehow protected them, 49 00:03:29 --> 00:03:35 gave them indeed lifelong protection from subsequent smallpox infection. 50 00:03:35 --> 00:03:39 Subsequently to that, the sores from the cowpox infection 51 00:03:39 --> 00:03:44 were scraped, and the exidate, the fluid, was scratched into wounds 52 00:03:44 --> 00:03:49 of people in order to immunize them. And so, immunization them. And so, 53 00:03:49 --> 00:03:54 immunization already began in the 1790's, taking a sore from the skin 54 00:03:54 --> 00:03:59 of a cowpox infected patient, injecting that into the skin of 55 00:03:59 --> 00:04:04 somebody who required immunization, and as a consequence, hoping that 56 00:04:04 --> 00:04:09 this would confer in them lifelong protection. 57 00:04:09 --> 00:04:13 In some cases, actually the individuals who were 58 00:04:13 --> 00:04:17 infected in that way actually came down with smallpox or some virulent 59 00:04:17 --> 00:04:21 form of this cowpox, but in most other cases these 60 00:04:21 --> 00:04:25 individuals actually acquired a lifelong immunity. 61 00:04:25 --> 00:04:29 In fact, the very word vaccine, which was used already at the time, 62 00:04:29 --> 00:04:33 comes from the Latin word vaccinus which means a cow. 63 00:04:33 --> 00:04:37 And, in the north side of Cambridge Common there's the Benjamin 64 00:04:37 --> 00:04:42 Waterhouse which is still there. He was the first physician to 65 00:04:42 --> 00:04:47 introduce smallpox vaccination into this country already in the end of 66 00:04:47 --> 00:04:52 the 18th century. If we fast forward to a situation 67 00:04:52 --> 00:04:57 like poliovirus, we have situations in this country 68 00:04:57 --> 00:05:02 where in the 1930's-1940's, there were epidemics of poliovirus. 69 00:05:02 --> 00:05:06 If one began to examine who was susceptible and who wasn't, 70 00:05:06 --> 00:05:10 it was clear that children who were, for example, born and raised in 71 00:05:10 --> 00:05:14 middle and upper-middle class houses were very susceptible for much of 72 00:05:14 --> 00:05:18 their lives. For example, in southern California there were 73 00:05:18 --> 00:05:22 dramatic examples, whereas children who grew up across 74 00:05:22 --> 00:05:26 the border in, let's say, Tijuana, 75 00:05:26 --> 00:05:30 Mexico, rarely came down with paralytic polio. 76 00:05:30 --> 00:05:34 Poliovirus, as one soon learned, was a virus which infects not only 77 00:05:34 --> 00:05:38 the gastrointestinal tract, and creates a form of mild diarrhea, 78 00:05:38 --> 00:05:43 but it may be one out of 100 persons the virus escapes from the GI tract, 79 00:05:43 --> 00:05:47 from the gastrointestinal tract, invades into the central nervous 80 00:05:47 --> 00:05:52 system, and actually creates debilitating paralysis, 81 00:05:52 --> 00:05:56 most of which is not healed. And some people have lifelong 82 00:05:56 --> 00:06:01 paralysis. Other individuals whose paralytic 83 00:06:01 --> 00:06:05 paralysis goes away, actually when they grow older, 84 00:06:05 --> 00:06:09 30, 40, 50 years later, they begin once again to experience the 85 00:06:09 --> 00:06:13 paralytic symptoms that arose as a consequence of their childhood 86 00:06:13 --> 00:06:17 infection. And at this time, one began to try to figure out why 87 00:06:17 --> 00:06:21 children living in Tijuana, Mexico rarely came down with 88 00:06:21 --> 00:06:25 poliovirus infections, whereas those who grew up up north 89 00:06:25 --> 00:06:30 like, say, in southern California did indeed do so. 90 00:06:30 --> 00:06:34 And one came to the conclusion that the children growing up in Tijuana, 91 00:06:34 --> 00:06:38 Mexico were frequently exposed very early in their life to contaminated 92 00:06:38 --> 00:06:42 water, sewage contaminated water, and they as a consequence acquired a 93 00:06:42 --> 00:06:46 lifelong immunity without getting sick, whereas children who grew up 94 00:06:46 --> 00:06:51 in rather sterile conditions further north never had any exposure to the 95 00:06:51 --> 00:06:55 virus. And when it hit them as young adults or as teenagers, 96 00:06:55 --> 00:06:59 it created devastating effects. And this indicates, once again, 97 00:06:59 --> 00:07:03 that somehow one's exposure to an infectious agent, 98 00:07:03 --> 00:07:07 historical exposure has a dramatic effect on one's susceptibility 99 00:07:07 --> 00:07:12 to a virus. And in the case of poliovirus, 100 00:07:12 --> 00:07:16 we're dealing here with an agent which is much simpler than the 101 00:07:16 --> 00:07:20 retroviruses we talked about last time like RSV. 102 00:07:20 --> 00:07:25 Poliovirus also has a single stranded RNA genome, 103 00:07:25 --> 00:07:29 and that's encapsidated in a proteinaceous coat, 104 00:07:29 --> 00:07:33 which is made up only of viral proteins, so it's very simple single 105 00:07:33 --> 00:07:37 stranded RNA proteinaceous coat. The single stranded RNA is actually 106 00:07:37 --> 00:07:40 polyadenylated at the three prime end. So, it can serve as a 107 00:07:40 --> 00:07:44 messenger RNA. It's of the same polarity that has 108 00:07:44 --> 00:07:47 a plus polarity, which means that it can be 109 00:07:47 --> 00:07:50 translated immediately. I'm distinguishing that from the 110 00:07:50 --> 00:07:54 other flavor of single stranded RNA which could be of the complementary 111 00:07:54 --> 00:07:57 strand, which could exist because one could make double stranded RNA 112 00:07:57 --> 00:08:01 which obviously cannot be translated. 113 00:08:01 --> 00:08:05 So, this can serve as a messenger RNA, and just as an aside, 114 00:08:05 --> 00:08:10 the way that poliovirus replicates is totally different from that of 115 00:08:10 --> 00:08:14 retroviruses. What happens is that poliovirus makes its own polymerase. 116 00:08:14 --> 00:08:19 You go from single stranded RNA to double stranded RNA, 117 00:08:19 --> 00:08:24 i.e. the complementary copy is made, and that in turn is used as a 118 00:08:24 --> 00:08:29 template for making more single stranded RNA, progeny RNA. 119 00:08:29 --> 00:08:33 And note here that there's no DNA at all involved. In fact, 120 00:08:33 --> 00:08:37 poliovirus can grow inside a cell that has been deprived of its 121 00:08:37 --> 00:08:41 nucleus. Moreover, if one stops nuclear DNA dependent 122 00:08:41 --> 00:08:45 transcription, that has no effect on this. 123 00:08:45 --> 00:08:49 But you'll notice correctly that these kind of steps here involved 124 00:08:49 --> 00:08:54 RNA dependent RNA polymerases. And in the normal physiology of a 125 00:08:54 --> 00:08:58 cell, that such a polymerase, such an enzyme never operates. 126 00:08:58 --> 00:09:02 And therefore, poliovirus must make among its other proteins an RNA 127 00:09:02 --> 00:09:06 dependent RNA polymerase that can mediate these steps: making a 128 00:09:06 --> 00:09:10 complementary copy of this strand, to get the complementary strand, and 129 00:09:10 --> 00:09:15 then making progeny plus strand RNAs. 130 00:09:15 --> 00:09:19 Again, it has a very simple capsid of several viral proteins which wrap 131 00:09:19 --> 00:09:24 it up. Now, if one wants to assess the potency of poliovirus RNA, 132 00:09:24 --> 00:09:28 one can grow it in a Petri dish. One way of figuring out how much 133 00:09:28 --> 00:09:33 poliovirus RNA is in a solution is to make a monolayer of cells like 134 00:09:33 --> 00:09:38 this, and then take various dilutions of a virus stalk. 135 00:09:38 --> 00:09:41 And when one talks about a virus stalk, one talks about a solution of 136 00:09:41 --> 00:09:44 virus particles. One can't really see them. 137 00:09:44 --> 00:09:47 And even if one could see them under the electron microscope, 138 00:09:47 --> 00:09:51 one wouldn't really know what fraction of the ones you were seeing 139 00:09:51 --> 00:09:54 were actually biologically competent. But most interestingly, 140 00:09:54 --> 00:09:57 you can take a solution of poliovirus particles, 141 00:09:57 --> 00:10:01 place it on a monolayer of cells here, which can be infected 142 00:10:01 --> 00:10:05 by poliovirus. In contrast to what I told you last 143 00:10:05 --> 00:10:09 time about RSV, where an infected cell can tolerate 144 00:10:09 --> 00:10:13 the continued presence of a viral infection without dying, 145 00:10:13 --> 00:10:17 poliovirus is a highly cytopathic virus, and by that I mean it 146 00:10:17 --> 00:10:21 replicates inside cells and it kills them during the course of 147 00:10:21 --> 00:10:25 replicating inside these cells. So, here what one has is just a 148 00:10:25 --> 00:10:29 poliovirus will infect a cell here, and then it will begin to spread 149 00:10:29 --> 00:10:34 from that cell to neighboring cells in the surrounds. 150 00:10:34 --> 00:10:37 And in so doing it will create, it will erode a hole right here in 151 00:10:37 --> 00:10:41 the monolayer whose presence, a so-called plaque, signifies the 152 00:10:41 --> 00:10:45 fact that there was an initially infecting virus particle there that 153 00:10:45 --> 00:10:49 spreads centrifugally, that spread outward, from the 154 00:10:49 --> 00:10:53 initially infected cell. In detail, if you want to do this 155 00:10:53 --> 00:10:57 experiment really nicely, what you do after you infect the 156 00:10:57 --> 00:11:01 initial monolayer of cells, which I show here in section, 157 00:11:01 --> 00:11:05 is you put on a layer of agarose, or something, above the infected 158 00:11:05 --> 00:11:09 monolayer, and that ensures that if there's any viral spread, 159 00:11:09 --> 00:11:13 it will be from cell to cell spread in a certain neighborhood rather 160 00:11:13 --> 00:11:17 than virus particles getting into solution and swimming around all 161 00:11:17 --> 00:11:21 over the plate and affecting cells helter-skelter. 162 00:11:21 --> 00:11:24 So here, we might want to infect an initial cell right here in the 163 00:11:24 --> 00:11:28 monolayer, and now this agarose will confine the subsequent spread of 164 00:11:28 --> 00:11:32 progeny virus to adjacent cells in the area, again eroding, 165 00:11:32 --> 00:11:36 ultimately, a hole here which we call a plaque. 166 00:11:36 --> 00:11:40 And if we count the number of plaques, that will tell us how many 167 00:11:40 --> 00:11:45 biologically active virus particles there were in the initial solution 168 00:11:45 --> 00:11:49 that was previously applied to that monolayer culture of the susceptible 169 00:11:49 --> 00:11:54 cells. One could use such monolayer cultures, in fact, to propagate 170 00:11:54 --> 00:11:58 poliovirus. And one can take the poliovirus 171 00:11:58 --> 00:12:02 coming out of those monolayer cultures, and actually use them to 172 00:12:02 --> 00:12:06 inject into human beings in order to vaccinate them to confer on them 173 00:12:06 --> 00:12:09 immunity. But if you do that with wild type poliovirus, 174 00:12:09 --> 00:12:13 then what will happen is that you may inadvertently give that person 175 00:12:13 --> 00:12:17 whom you're attempting to immunize a nasty poliovirus infection which 176 00:12:17 --> 00:12:21 could paralyze them, might even kill them. 177 00:12:21 --> 00:12:24 When I was growing up, poliovirus infections were much 178 00:12:24 --> 00:12:28 dreaded because we knew of people who were being kept alive in iron 179 00:12:28 --> 00:12:32 lungs that breathe for them because their autonomic nervous system had 180 00:12:32 --> 00:12:36 been destroyed by a poliovirus infection. 181 00:12:36 --> 00:12:40 So, what happens is therefore if one wishes to immunize somebody, 182 00:12:40 --> 00:12:45 if one wishes to vaccinate them against poliovirus, 183 00:12:45 --> 00:12:49 one needs to inject them with an attenuated virus, 184 00:12:49 --> 00:12:54 that is, a virus whose ability to create disease, 185 00:12:54 --> 00:12:58 whose pathogenic abilities, pathogenic, remember, refers to the 186 00:12:58 --> 00:13:03 ability to create disease. You want to use an attenuated virus, 187 00:13:03 --> 00:13:07 which might be able to go into the body. It might be able to evoke 188 00:13:07 --> 00:13:12 immunity, but it will not create a major disease response. 189 00:13:12 --> 00:13:16 And this was dealt with in two different ways. 190 00:13:16 --> 00:13:21 Jonas Salk, one of the pioneers of creating a poliovirus vaccine took 191 00:13:21 --> 00:13:25 the poliovirus particles that have been produced by culturing them on 192 00:13:25 --> 00:13:30 monolayers of monkey kidney cells, and he treated the poliovirus 193 00:13:30 --> 00:13:35 briefly with a little bit of formaldehyde. 194 00:13:35 --> 00:13:39 And formaldehyde, as you may know, reacts with the 195 00:13:39 --> 00:13:43 amine groups of the ribonucleosides, of the purines and pyrimidines, and 196 00:13:43 --> 00:13:47 therefore kills the virus. And that kill virus, having been 197 00:13:47 --> 00:13:51 treated briefly with the formaldehyde, the virus particle is 198 00:13:51 --> 00:13:55 still essentially intact, was then injected into individuals. 199 00:13:55 --> 00:14:00 Alternatively, 200 00:14:00 --> 00:14:04 his deadly rival, because they two hated each other's 201 00:14:04 --> 00:14:08 guts, Sabin decided on another strategy for making a vaccine stalk. 202 00:14:08 --> 00:14:13 And that is, he took poliovirus which had been passage from one cell 203 00:14:13 --> 00:14:17 culture to the next over extended periods of time, 204 00:14:17 --> 00:14:22 over several years. So, it had been passage in vitro. 205 00:14:22 --> 00:14:26 When we say in vitro, in this context we mean poliovirus had been 206 00:14:26 --> 00:14:30 taken from one plate, put into another plate of cells, 207 00:14:30 --> 00:14:35 taken to another plate of cells. In vitro here implies in culture 208 00:14:35 --> 00:14:39 rather than the other alternative. In vivo means that the virus is 209 00:14:39 --> 00:14:43 being passaged in a living organism. And as it turns out, when you 210 00:14:43 --> 00:14:48 passage poliovirus in vitro from passing it just from one culture of 211 00:14:48 --> 00:14:52 cells to the next, infecting one after another in a 212 00:14:52 --> 00:14:56 serial or sequential fashion, then the virus is selected for its 213 00:14:56 --> 00:15:01 ability to proliferate in the cultured cells in vitro. 214 00:15:01 --> 00:15:05 But it gradually loses its ability to create disease in vivo in that 215 00:15:05 --> 00:15:10 there is no Darwinian selection to favor its disease causing ability. 216 00:15:10 --> 00:15:14 And consequently, virus that Saben used wasn't inactivated with 217 00:15:14 --> 00:15:19 formaldehyde. It was a simply attenuated because of extensive in 218 00:15:19 --> 00:15:23 vitro propagation. And when he injected that into 219 00:15:23 --> 00:15:28 young people, very rarely did they ever come down with poliovirus 220 00:15:28 --> 00:15:32 infection, and the virus was able to replicate to some extent in their 221 00:15:32 --> 00:15:37 bodies without causing disease, and to create lifelong immunity. 222 00:15:37 --> 00:15:41 I.e. such an individual was protected from subsequent poliovirus 223 00:15:41 --> 00:15:46 infection ever again. And, as it turned out, 224 00:15:46 --> 00:15:50 this was a very nice way of protecting because it also turned 225 00:15:50 --> 00:15:55 out the monkey kidney cells that were used to propagate the virus 226 00:15:55 --> 00:15:59 also contained certain monkey viruses. For example, 227 00:15:59 --> 00:16:04 it came out after years that there was a DNA tumor virus. 228 00:16:04 --> 00:16:07 We've been talking about RNA tumor viruses until now, 229 00:16:07 --> 00:16:11 but there's a DNA tumor virus called SV40, which is a very potent tumor 230 00:16:11 --> 00:16:15 virus in hamsters, and mice, and rats. 231 00:16:15 --> 00:16:18 And it turns out that SV40 lingered. It lurked in monkey kidney cell 232 00:16:18 --> 00:16:22 cultures. And, nobody knew about it. 233 00:16:22 --> 00:16:26 Whenever you put poliovirus into many of these cultures, 234 00:16:26 --> 00:16:30 not only did poliovirus replicate, but all of a sudden so did SV40. 235 00:16:30 --> 00:16:34 And therefore, many of the stalks of poliovirus 236 00:16:34 --> 00:16:38 that were used to inject people like myself, I lived around the corner in 237 00:16:38 --> 00:16:42 Pittsburgh from Jonas Salk, and the children in the Pittsburgh 238 00:16:42 --> 00:16:46 public schools were his first guinea pigs. Many of us were exposed to 239 00:16:46 --> 00:16:50 poliovirus stalk which actually had more SV40 particles in it than 240 00:16:50 --> 00:16:54 poliovirus particles because of this inadvertent contamination. 241 00:16:54 --> 00:16:58 There probably were between 30 and 40 million people who were immunized 242 00:16:58 --> 00:17:03 with poliovirus and inadvertently with SV40 virus. 243 00:17:03 --> 00:17:07 And one is that epidemiology in the subsequent years to figure out 244 00:17:07 --> 00:17:11 whether that had led to any increased rate of cancer because it 245 00:17:11 --> 00:17:15 could well have. There could have been an epidemic 246 00:17:15 --> 00:17:19 of cancer in this country, millions of people affected because 247 00:17:19 --> 00:17:23 of this unknown, and at the time almost unknowable 248 00:17:23 --> 00:17:27 contaminating virus. As it turns out, people who were 249 00:17:27 --> 00:17:31 infected with SV40 inadvertently have no higher rates of cancer than 250 00:17:31 --> 00:17:35 anybody else even though this virus is a very potently tumorogenic virus 251 00:17:35 --> 00:17:39 like RSV, but doing so in this case in rodent cells and ostensibly 252 00:17:39 --> 00:17:43 not in humans. So, public health measures can 253 00:17:43 --> 00:17:47 sometimes have unforeseen side effects or consequences that nobody 254 00:17:47 --> 00:17:51 anticipates ahead of time. Now, getting back to the whole 255 00:17:51 --> 00:17:56 strategy of poliovirus infection, this raises the issue of what it was 256 00:17:56 --> 00:18:00 in the poliovirus that was able to evoke the subsequent lifelong 257 00:18:00 --> 00:18:05 immunity. And in telling you that, I mentioned the following. 258 00:18:05 --> 00:18:09 You can make antiserum or you can make serum from an individual who's 259 00:18:09 --> 00:18:13 been immunized with poliovirus. And, recollect, we mentioned this 260 00:18:13 --> 00:18:17 before, the way to make serum is to allow blood to clot. 261 00:18:17 --> 00:18:21 The red cells and the platelets aggregate in the bottom, 262 00:18:21 --> 00:18:25 and what remains on top is simply serum. You can spin out all the 263 00:18:25 --> 00:18:30 residual cells and so all you get is sort of a straw colored fluid. 264 00:18:30 --> 00:18:34 And that serum from an individual who's been immunized with poliovirus 265 00:18:34 --> 00:18:38 stalk can actually be used to, you can add it to the poliovirus 266 00:18:38 --> 00:18:42 stalk prior to adding these virus particles to the Petri dish. 267 00:18:42 --> 00:18:46 So, here's the Petri dish as before, and what you find is that if you add 268 00:18:46 --> 00:18:50 serum from an individual who's been immunized to a solution of 269 00:18:50 --> 00:18:54 poliovirus particles, and then you take that mixture and 270 00:18:54 --> 00:18:58 put them on the plate, you no longer get any of these 271 00:18:58 --> 00:19:03 plaques that I referred to before. 272 00:19:03 --> 00:19:07 And that indicates that within the serum, there is some kind of factor 273 00:19:07 --> 00:19:12 which is neutralizing the infectivity of the polio virus 274 00:19:12 --> 00:19:16 particles, i.e. the poliovirus particles are in some 275 00:19:16 --> 00:19:21 way prevented from creating an infection, and their plaque forming 276 00:19:21 --> 00:19:26 ability, their ability corrode these holes in the monolayers of 277 00:19:26 --> 00:19:31 subsequently infected cultures is now compromised. 278 00:19:31 --> 00:19:35 And such activity suggests that such an individual has actually antiserum, 279 00:19:35 --> 00:19:39 i.e. some kind of reactivity which prevents the poliovirus particle 280 00:19:39 --> 00:19:44 from doing its thing. In fact, I can tell you that such 281 00:19:44 --> 00:19:48 antiserum or such neutralizing antiserum can be achieved in a 282 00:19:48 --> 00:19:53 number of ways. One way to do it is just to take 283 00:19:53 --> 00:19:57 the protein capsid, the outer coat of the poliovirus 284 00:19:57 --> 00:20:02 particle that I described before and inject that into an individual. 285 00:20:02 --> 00:20:06 And that will on its own already evoke a measure of antiviral 286 00:20:06 --> 00:20:10 immunity. It will evoke a concentration of antiserum in the 287 00:20:10 --> 00:20:15 blood of a patient. Or, if you want to do it in a very 288 00:20:15 --> 00:20:19 much more modern way, you can sequence the proteins of the 289 00:20:19 --> 00:20:24 poliovirus and determine the amino acid sequence. 290 00:20:24 --> 00:20:28 And when you do that, you can synthesize through organic 291 00:20:28 --> 00:20:33 synthesis oligopeptides of 10 or 20 amino acids long. 292 00:20:33 --> 00:20:36 And these oligopeptides reflect different parts of the poliovirus 293 00:20:36 --> 00:20:39 coat protein. So, if here's the sequence of one of the 294 00:20:39 --> 00:20:43 capsid proteins, recall that capsid refers to the 295 00:20:43 --> 00:20:46 coat that's shielding or protecting in this case poliovirus, 296 00:20:46 --> 00:20:50 the single stranded viral RNA genome. So, if here's one of the capsid 297 00:20:50 --> 00:20:53 proteins, what you can do is to figure out the amino acid sequence 298 00:20:53 --> 00:20:57 of this segment of the capsid protein, and then rather than 299 00:20:57 --> 00:21:00 cutting it out from the capsid protein, you just synthesize it by 300 00:21:00 --> 00:21:04 organic synthesis, ten or twenty amino acid residues 301 00:21:04 --> 00:21:08 long. And when you inject that 302 00:21:08 --> 00:21:12 oligopeptides into an individual, that may also evoke some kind of 303 00:21:12 --> 00:21:16 neutralizing antibody response. In fact, as you might correctly 304 00:21:16 --> 00:21:21 imagine, if we can look in more detail at the capsid structure, 305 00:21:21 --> 00:21:25 I'm just going to draw it in a very primitive way here, 306 00:21:25 --> 00:21:29 it's obviously much more complicated. But if this is the capsid here, 307 00:21:29 --> 00:21:34 it has an inside and an outside. And the antibodies that you make 308 00:21:34 --> 00:21:39 against an oligopeptide that is sticking out here on the outside 309 00:21:39 --> 00:21:43 will be neutralizing, i.e. the antiserum will be able to 310 00:21:43 --> 00:21:48 recognize the outside of the virus particle and somehow inactivate it. 311 00:21:48 --> 00:21:53 But if you were to use an oligopeptide antigen from over here, 312 00:21:53 --> 00:21:57 which might be a part of the capsid protein that is deep inside, 313 00:21:57 --> 00:22:02 well even though you might have a good reactivity with oligopeptide it 314 00:22:02 --> 00:22:07 would not be neutralizing. And that suggests the notion that 315 00:22:07 --> 00:22:11 whatever is present in the antiserum is recognizing some external portion 316 00:22:11 --> 00:22:16 of this protein which is sticking out. And that external portion is 317 00:22:16 --> 00:22:21 called an antigen. So, there's a specific chemical 318 00:22:21 --> 00:22:25 structure which is being recognized by the antiserum, and we 319 00:22:25 --> 00:22:30 call that an antigen. In principal, there are dozens of 320 00:22:30 --> 00:22:34 distinct antigens on the surface of a poliovirus particle, 321 00:22:34 --> 00:22:39 each of which one can, in principal, make an immunizing antibody against. 322 00:22:39 --> 00:22:44 Each of those can therefore be considered to be an antigen. 323 00:22:44 --> 00:22:48 And, how can we imagine this antiserum as actually successfully 324 00:22:48 --> 00:22:53 neutralizing the virus particle? And here, this depended on actually 325 00:22:53 --> 00:22:58 the biochemical characterization of antisera because one soon came to 326 00:22:58 --> 00:23:03 learn that within antisera are what we call antibodies. 327 00:23:03 --> 00:23:07 Or, keep in mind that in biology you never should ever use a simple 328 00:23:07 --> 00:23:11 Anglo-Saxon word if you can use a much more complicated Greek or Latin 329 00:23:11 --> 00:23:16 one. So, we can always call these immunoglobulins. 330 00:23:16 --> 00:23:20 You see the word right there. Immunoglobulin represents an 331 00:23:20 --> 00:23:25 antibody molecule, and it turns out that much of the 332 00:23:25 --> 00:23:29 protein in the soluble fraction of our serum consists of such 333 00:23:29 --> 00:23:34 antibody molecules. And these antibody molecules have a 334 00:23:34 --> 00:23:38 very interesting structure which I'd like to dwell upon momentarily. 335 00:23:38 --> 00:23:42 But before I do that, I'd just want to anticipate what I'm about to say 336 00:23:42 --> 00:23:46 by indicating that we now realize that an antibody molecule can 337 00:23:46 --> 00:23:50 recognize an antigen on the surface of the virus particle, 338 00:23:50 --> 00:23:54 and the antibody molecule can actually physically bind to this 339 00:23:54 --> 00:23:58 antigen. So, we have an antigen antibody complex, 340 00:23:58 --> 00:24:02 and that binding of the antibody molecule to the surface of the virus 341 00:24:02 --> 00:24:06 particle is what results functionally in the neutralization 342 00:24:06 --> 00:24:11 of its infectivity. Or to put it another way, 343 00:24:11 --> 00:24:15 given the fact that this particular capsid protein might be repeated, 344 00:24:15 --> 00:24:19 let's say, 60 times around a spherical surface of a virus 345 00:24:19 --> 00:24:23 particle, there might be, in fact, 60 different antibody 346 00:24:23 --> 00:24:27 molecules recognizing this repeated antigen which occurs on each of 347 00:24:27 --> 00:24:31 these capsid proteins. And therefore, 348 00:24:31 --> 00:24:35 you can imagine in a very approximate way that if you have an 349 00:24:35 --> 00:24:39 effective neutralizing antibody, it's coating the surface of the 350 00:24:39 --> 00:24:42 virus particle of antibody molecules, and that clearly obstructs the 351 00:24:42 --> 00:24:46 ability of the virus particle to initiate a subsequent round of 352 00:24:46 --> 00:24:49 infection. Here's what a single antibody molecule looks like, 353 00:24:49 --> 00:24:53 and it has some critically important features. First of all, 354 00:24:53 --> 00:24:57 there is a specific region of the antibody that recognizes 355 00:24:57 --> 00:25:01 the antigen. In this case, I'm using as the 356 00:25:01 --> 00:25:05 example the antigen an oligopeptide on the surface of the poliovirus 357 00:25:05 --> 00:25:09 particle. And that is located in this part of the antibody molecule. 358 00:25:09 --> 00:25:14 Note, by the way, the antibody molecule is a heterotetramer. 359 00:25:14 --> 00:25:18 It has two light chains, two small chains here on the right, 360 00:25:18 --> 00:25:23 and two heavy chains. Note that the cysteine disulphide bonds are 361 00:25:23 --> 00:25:27 holding the entire assembly together. So here, one is not relying on 362 00:25:27 --> 00:25:32 hydrogen bonds to hold together this heterotetramer. 363 00:25:32 --> 00:25:36 And note the following, that there are portions of the 364 00:25:36 --> 00:25:41 antibody molecule which may be specifically varied from one 365 00:25:41 --> 00:25:46 antibody molecule to another over here, and other portions which are 366 00:25:46 --> 00:25:50 standard operating hardware. And let me elaborate on that for a 367 00:25:50 --> 00:25:55 moment. If you look in the serum of an individual and you try to analyze 368 00:25:55 --> 00:26:00 the chemical structure of the antibody molecules in their serum, 369 00:26:00 --> 00:26:04 you find that for a given type of antibody molecule like this, 370 00:26:04 --> 00:26:09 all the antibody molecules share this constant here: light 371 00:26:09 --> 00:26:13 purple domain. They all have that in common. 372 00:26:13 --> 00:26:17 They all have in common also the light blue domains. 373 00:26:17 --> 00:26:21 But if you were able, and we'll discuss this in a moment 374 00:26:21 --> 00:26:25 how you could do so, if you were able to analyze this 375 00:26:25 --> 00:26:29 other portion, you would find that when you look at 376 00:26:29 --> 00:26:33 one antibody molecule, it has a certain amino acid sequence 377 00:26:33 --> 00:26:37 here, which is involved in antigen recognition, and that this amino 378 00:26:37 --> 00:26:41 acid sequence on the light and heavy chain varies from one heterotetramer 379 00:26:41 --> 00:26:45 to another heterotetramer. And again, I'm going to reinforce 380 00:26:45 --> 00:26:50 that. But I want to indicate that there are, within the human 381 00:26:50 --> 00:26:55 antiserum, millions of structurally distinct antibody molecules. 382 00:26:55 --> 00:27:00 They share in common the structure down here up to this point. 383 00:27:00 --> 00:27:03 And thereafter, each of them has its own 384 00:27:03 --> 00:27:06 idiosyncratic, its own unusual, 385 00:27:06 --> 00:27:09 its own particular private antigen recognition site, 386 00:27:09 --> 00:27:13 which stems from a region or a pocket of the protein which is 387 00:27:13 --> 00:27:16 involved in antigen recognition and has variable amino acid sequences. 388 00:27:16 --> 00:27:19 Here, you see the way that an x-ray crystallographer would actually 389 00:27:19 --> 00:27:23 visualize this molecule, and you begin to realize that the 390 00:27:23 --> 00:27:26 schematic that I showed you before, in fact, is really doing a little 391 00:27:26 --> 00:27:29 bit of violence to the real amino acid sequence, 392 00:27:29 --> 00:27:33 to the real three dimensional structure, excuse me. 393 00:27:33 --> 00:27:36 Now, note something else that is implicit in what I've just said but 394 00:27:36 --> 00:27:40 I haven't said it explicitly, there are actually two antigen 395 00:27:40 --> 00:27:44 recognition sites, one here and one here. 396 00:27:44 --> 00:27:48 So, this antibody is in that sense bivalent. And if there were, 397 00:27:48 --> 00:27:51 for example, two poliovirus particles, one over here and one 398 00:27:51 --> 00:27:55 over here, you could imagine in principal, although I'm not drawing 399 00:27:55 --> 00:27:59 things to scale, that this antibody molecule could 400 00:27:59 --> 00:28:03 actually use one of its antigen recognition sites to bind over here 401 00:28:03 --> 00:28:07 to one poliovirus particle, and this to bind to another 402 00:28:07 --> 00:28:11 poliovirus particle. Now, I've told you that the antigen 403 00:28:11 --> 00:28:15 is recognized by this antibody is an oligopeptide, which constitutes a 404 00:28:15 --> 00:28:20 distinct chemical structure. How many different oligopeptides of 405 00:28:20 --> 00:28:25 ten amino acids long are there in the mathematical universe? 406 00:28:25 --> 00:28:30 How many conceivable ones are there? 407 00:28:30 --> 00:28:34 How many amino acids are there? 20? So what's the number? I heard 408 00:28:34 --> 00:28:38 you all say 1020, right? So, that's an awful lot. 409 00:28:38 --> 00:28:42 That's more than you can shake a stick at. So that means that there 410 00:28:42 --> 00:28:46 are, in principal, essentially an infinite number of 411 00:28:46 --> 00:28:50 oligopeptides of ten amino acids long, and each one of those could, 412 00:28:50 --> 00:28:54 in principal constitute a distinct antigen that could be recognized by 413 00:28:54 --> 00:28:58 the antibody molecule. Note that what's happening here is 414 00:28:58 --> 00:29:02 that a protein antigen such as one of these oligopeptides is being 415 00:29:02 --> 00:29:06 recognized by another protein, which is the antibody molecule. 416 00:29:06 --> 00:29:10 And therefore, we begin to imagine there's some 417 00:29:10 --> 00:29:14 kind of lock and key complementarity where somehow the amino acid 418 00:29:14 --> 00:29:18 sequence here recognizes and binds a complementary fashion to the antigen 419 00:29:18 --> 00:29:22 that is being recognized, and there's a physical association 420 00:29:22 --> 00:29:26 which allows the antibody molecule then to attach tightly to the 421 00:29:26 --> 00:29:30 antigen bearing poliovirus particle. 422 00:29:30 --> 00:29:34 And, by the way, to put it another way, 423 00:29:34 --> 00:29:39 it could be that some of these poliovirus capsid proteins have not 424 00:29:39 --> 00:29:43 yet been assembled in a virus particle. And even if they aren't, 425 00:29:43 --> 00:29:48 in principal a free protein, a capsid protein of poliovirus, 426 00:29:48 --> 00:29:53 could also be recognized by an antibody molecule and be bound by 427 00:29:53 --> 00:29:57 the antibody molecule. Now, this creates several major 428 00:29:57 --> 00:30:02 conceptual problems. How on earth can the body make 429 00:30:02 --> 00:30:06 antibody molecules that are able to recognize a poliovirus particle? 430 00:30:06 --> 00:30:10 Well, you'll say that's easy. Clearly, in our genes there must be 431 00:30:10 --> 00:30:14 some kind of nucleotide sequence which has up here the ability encode 432 00:30:14 --> 00:30:19 an antigen-binding site. Note, by the way, the 433 00:30:19 --> 00:30:23 antigen-binding site's actually composed of two different proteins. 434 00:30:23 --> 00:30:27 Here's part of the antigen-binding site. Here's another part of the 435 00:30:27 --> 00:30:31 antigen-binding site. So, it must be on two different 436 00:30:31 --> 00:30:35 genes. But, these two different genes create an antigen-binding site 437 00:30:35 --> 00:30:39 which can recognize poliovirus. Bind the poliovirus antigen and 438 00:30:39 --> 00:30:43 neutralize it. Well, that's well and good. 439 00:30:43 --> 00:30:47 That's perfectly reasonable, but let me tell you what's 440 00:30:47 --> 00:30:51 unreasonable about that. Each of us during his or her 441 00:30:51 --> 00:30:55 lifetime are going to be infected by literally hundreds of viruses. 442 00:30:55 --> 00:30:59 You get cold viruses almost every two, three, four, five 443 00:30:59 --> 00:31:03 times a year. By the time you get old, 444 00:31:03 --> 00:31:07 you actually have acquired immunity to many of these, 445 00:31:07 --> 00:31:11 but during the course of one's lifetime, the immune system has been 446 00:31:11 --> 00:31:15 able to generate antiviral immunity and eliminate virtually all the 447 00:31:15 --> 00:31:19 viral infections you ever got. How many people do you know who 448 00:31:19 --> 00:31:23 have actually died of a viral infection? And yet each one of 449 00:31:23 --> 00:31:27 these viruses is in principal able to replicate throughout your body 450 00:31:27 --> 00:31:32 and kill you, and they never do? So that means that the immune system 451 00:31:32 --> 00:31:36 is extremely effective in developing an antibody or antibodies which 452 00:31:36 --> 00:31:40 could neutralize infecting virus particles. You might have the cold 453 00:31:40 --> 00:31:44 for a day, a week, or two weeks, but eventually the 454 00:31:44 --> 00:31:48 immune system develops enough antibodies and other strategies to 455 00:31:48 --> 00:31:52 eliminate the viral infections from your body. I remember when my 456 00:31:52 --> 00:31:56 grandfather was 94 years old and he got a cold. And he hadn't had a 457 00:31:56 --> 00:32:00 cold for 20 years because by the age of 75, he had been exposed to almost 458 00:32:00 --> 00:32:04 every virus imaginable. And so, when he got a cold at the 459 00:32:04 --> 00:32:08 age of 94, his brain was still working, he'd forgotten how to blow 460 00:32:08 --> 00:32:12 his nose. It just hadn't happened to him. He didn't know what it was 461 00:32:12 --> 00:32:16 like to have a cold anymore. So, it just goes to show you that 462 00:32:16 --> 00:32:20 the immune system can be very versatile and very successful. 463 00:32:20 --> 00:32:24 But this creates a major conceptual problem. How do our genes know how 464 00:32:24 --> 00:32:28 to create antigen-binding sites in our antibodies that recognize all of 465 00:32:28 --> 00:32:32 the infectious viruses that are going to attack us during 466 00:32:32 --> 00:32:36 a lifetime. Do we have a gene for neutralizing 467 00:32:36 --> 00:32:40 smallpox? Do we have a gene for neutralizing poliovirus and 468 00:32:40 --> 00:32:44 adenovirus, which gives us bad head colds. And, rabies virus and 469 00:32:44 --> 00:32:48 vesicular stomatitis virus, and all other kinds of viruses that 470 00:32:48 --> 00:32:52 we are going to experience in a lifetime. Well, 471 00:32:52 --> 00:32:56 possibly we do have a gene for each one of those. But, 472 00:32:56 --> 00:33:00 then I'm beginning to raise some other questions if you believe that, 473 00:33:00 --> 00:33:04 which you shouldn't. First of all, there are many 474 00:33:04 --> 00:33:08 different distinct oligopeptide antigens on the surface of the 475 00:33:08 --> 00:33:12 poliovirus particle. If you look at the antibodies that 476 00:33:12 --> 00:33:16 had been developed against a poliovirus capsid protein in an 477 00:33:16 --> 00:33:19 immunized individual, they have one antibody that 478 00:33:19 --> 00:33:23 recognizes one part of these capsid protein, and another antibody that 479 00:33:23 --> 00:33:27 recognized another part. So, here's the surface of the 480 00:33:27 --> 00:33:31 capsid protein blown up now, and we can imagine there are 481 00:33:31 --> 00:33:35 different oligopeptide epitopes on different parts of the protein. 482 00:33:35 --> 00:33:39 And they're all ten amino acids long, for example. The fact is, 483 00:33:39 --> 00:33:43 in individuals who have anti-poliovirus antibody, 484 00:33:43 --> 00:33:48 you can find that there's an antibody that recognizes this 485 00:33:48 --> 00:33:52 antigen, and one that recognizes this antigen, and another that 486 00:33:52 --> 00:33:57 recognizes this antigen. So, the number of distinct antigens 487 00:33:57 --> 00:34:01 which the immune system has succeeded in making antibodies 488 00:34:01 --> 00:34:05 against, vastly exceeds the number of infectious agents we're going to 489 00:34:05 --> 00:34:10 experience in a lifetime. And, that begins to undermine your 490 00:34:10 --> 00:34:14 confidence in the notion that when we're born, we already possess the 491 00:34:14 --> 00:34:18 genes to recognize different infectious agents, 492 00:34:18 --> 00:34:22 and to construct neutralizing antibodies against them. 493 00:34:22 --> 00:34:26 There's also another flaw in this whole notion that we inherit a set 494 00:34:26 --> 00:34:30 of genes that enable us from the get-go to make specific 495 00:34:30 --> 00:34:34 antibodies. And that flaw comes from the 496 00:34:34 --> 00:34:38 following notion. Evolution cannot anticipate which 497 00:34:38 --> 00:34:42 infectious agents each one of us is going to experience in a lifetime. 498 00:34:42 --> 00:34:47 Let's say that some of us experience a totally new kind of a 499 00:34:47 --> 00:34:51 viral infection which had never been experienced by our ancestors. 500 00:34:51 --> 00:34:55 If the way of neutralizing that virus, and defending us against that 501 00:34:55 --> 00:35:00 infection depended on using an antibody molecule whose sequence was 502 00:35:00 --> 00:35:04 encoded in our germ line in the genes we inherited at birth, 503 00:35:04 --> 00:35:08 then we might be in bad shape if that virus was a novel virus to 504 00:35:08 --> 00:35:13 which the human race had never been exposed. 505 00:35:13 --> 00:35:17 And in arguing that, I'm telling you that it's impossible 506 00:35:17 --> 00:35:21 to imagine a situation where our germ line, the set of genes we are 507 00:35:21 --> 00:35:26 born with that we start life with, already contains the information for 508 00:35:26 --> 00:35:30 making each one of the different kinds of antibodies that 509 00:35:30 --> 00:35:34 are in our antiserum. How many different kinds of 510 00:35:34 --> 00:35:38 antibodies are in our antiserum? Well now, the whole notion becomes 511 00:35:38 --> 00:35:42 even more stark because there's probably millions, 512 00:35:42 --> 00:35:46 maybe even ten or 100 million distinct antibody molecules floating 513 00:35:46 --> 00:35:50 around in our blood. Each one in this case has the same 514 00:35:50 --> 00:35:54 constant region down here. You see the constant region, 515 00:35:54 --> 00:35:58 and each one of these 100 million distinct antibody species has its 516 00:35:58 --> 00:36:01 own antigen-combining site. Now, if we want to pursue that 517 00:36:01 --> 00:36:05 further, we have to begin to imagine how the immune system can make so 518 00:36:05 --> 00:36:09 many different antibody molecules. This itself is a major conceptual 519 00:36:09 --> 00:36:13 challenge. And now we have to go and know a little bit of cell 520 00:36:13 --> 00:36:16 biology because if we look at the immune system, 521 00:36:16 --> 00:36:20 and there are cells forming in the immune system, 522 00:36:20 --> 00:36:24 we find a set of cells that are called B cells or a subset of B 523 00:36:24 --> 00:36:28 cells that are called plasma cells. And the plasma cells, and there's a 524 00:36:28 --> 00:36:32 whole bunch of different plasma cells. 525 00:36:32 --> 00:36:35 So the plasma cells are floating around in the circulation, 526 00:36:35 --> 00:36:39 and the plasma cells are able to secrete antibody molecules into the 527 00:36:39 --> 00:36:43 serum. And they are abundant. They're around by the billions 528 00:36:43 --> 00:36:47 inside our body. And these plasma cells are 529 00:36:47 --> 00:36:51 constantly secreting antibody molecules into the serum around them 530 00:36:51 --> 00:36:55 into the plasma around them. And these are the antibody 531 00:36:55 --> 00:36:59 molecules I've been talking about here, the antibody molecules that 532 00:36:59 --> 00:37:03 are capable of neutralizing poliovirus particles. 533 00:37:03 --> 00:37:06 So, this now creates another conceptual question. 534 00:37:06 --> 00:37:10 Let's imagine for the sake of argument that in our blood stream 535 00:37:10 --> 00:37:13 there are 100 million distinct antigen species floating around. 536 00:37:13 --> 00:37:17 And I don't mean 100 million molecules. I mean there's a million 537 00:37:17 --> 00:37:21 molecules, and each one of these species has a different 538 00:37:21 --> 00:37:24 antigen-binding site. So, there may be many 539 00:37:24 --> 00:37:28 antigen-binding sites. There may be many antibody 540 00:37:28 --> 00:37:32 molecules. There may be million of this kind of 541 00:37:32 --> 00:37:36 antibody that recognize this antigen. There may be millions of this 542 00:37:36 --> 00:37:40 antibody recognizing this antigen, millions of this antibody 543 00:37:40 --> 00:37:44 recognizing this antigen on the surface of poliovirus. 544 00:37:44 --> 00:37:48 And I'm saying there could be a million distinct species of 545 00:37:48 --> 00:37:52 antibodies, each species being defined by the antigen that 546 00:37:52 --> 00:37:56 recognizes and by the structure of its antigen-recognizing site, 547 00:37:56 --> 00:38:00 its antigen-binding site. In other words, 548 00:38:00 --> 00:38:03 to repeat myself, what distinguishes one species of 549 00:38:03 --> 00:38:06 antibodies from another is the amino acid sequence, 550 00:38:06 --> 00:38:09 the structure of this particular region of the antibody molecule that 551 00:38:09 --> 00:38:12 makes one species of antibody different from the other. 552 00:38:12 --> 00:38:16 And with all that in mind, we have two alternative scenarios. 553 00:38:16 --> 00:38:19 Let's again say there's a million different species of antibodies 554 00:38:19 --> 00:38:22 being secreted into the antiserum at any one point in time. 555 00:38:22 --> 00:38:25 This cell could make all million. This cell could make the whole 556 00:38:25 --> 00:38:28 million species. This cell could, 557 00:38:28 --> 00:38:32 and this cell could. In other words, 558 00:38:32 --> 00:38:38 each one of these B cells could be multitalented, 559 00:38:38 --> 00:38:43 able to simultaneously make a million different kinds of antibody 560 00:38:43 --> 00:38:49 molecules. The opposite model is as follows, and that is that this B 561 00:38:49 --> 00:38:54 cell makes one species of antibody. We'll call it antibody A, and this 562 00:38:54 --> 00:39:00 species makes another species of antibody molecule. 563 00:39:00 --> 00:39:04 So, this B cell makes antibody A. This B cell makes antibody B. This 564 00:39:04 --> 00:39:08 B cell makes antibody C. And keep in mind that when I'm 565 00:39:08 --> 00:39:12 saying antibody A, B, and C, I mean A has a certain 566 00:39:12 --> 00:39:17 antigen-binding site. Be has another antigen binding site. 567 00:39:17 --> 00:39:21 C has yet another antigen binding site. And, we can distinguish 568 00:39:21 --> 00:39:25 between these two alternative mechanistic models by looking at the 569 00:39:25 --> 00:39:29 disease called multiple myeloma. And what you have in multiple 570 00:39:29 --> 00:39:33 myeloma is the following. All of a sudden, 571 00:39:33 --> 00:39:37 the blood stream becomes full of much greatly elevated levels of 572 00:39:37 --> 00:39:41 antibody molecules. They're present in the blood stream. 573 00:39:41 --> 00:39:45 Now, normally, if you look at all the antibody 574 00:39:45 --> 00:39:48 molecules in the blood stream and you were to separate them by some 575 00:39:48 --> 00:39:52 kind of electrophoretic technique which distinguished them on the 576 00:39:52 --> 00:39:56 basis of subtle differences in the amino acid sequence, 577 00:39:56 --> 00:40:00 and let's not worry about exactly how you do that. 578 00:40:00 --> 00:40:03 But we've talked about a million different antibody species. 579 00:40:03 --> 00:40:07 They're chemically slightly different from one another by virtue 580 00:40:07 --> 00:40:11 of the antigen-recognizing site. And if you could separate them by 581 00:40:11 --> 00:40:15 some kind of system that separated them on the basis of their charge, 582 00:40:15 --> 00:40:18 you'd find a whole spectrum of different antibody molecules, 583 00:40:18 --> 00:40:22 a million in this large, heterogeneous mixture of antibody 584 00:40:22 --> 00:40:26 molecules, each species found somewhere or another in this very 585 00:40:26 --> 00:40:30 broad distribution of 100 million distinct species which are all mixed 586 00:40:30 --> 00:40:33 together in the antiserum. If you look at the serum or the 587 00:40:33 --> 00:40:37 plasma of a patient suffering from multiple myeloma, 588 00:40:37 --> 00:40:41 what you find is the following, that one species of antibody 589 00:40:41 --> 00:40:45 molecules dominates over all the other ones. And so now, 590 00:40:45 --> 00:40:49 whereas that single species might only be on average one millionth of 591 00:40:49 --> 00:40:53 the total antibody complement in the blood. In those suffering from 592 00:40:53 --> 00:40:57 myeloma, or sometimes it's called multiple myeloma, 593 00:40:57 --> 00:41:01 now one single antibody species may represent 60, 70, 594 00:41:01 --> 00:41:05 or 80% of all the antibody molecules in the blood. 595 00:41:05 --> 00:41:09 And clearly something has gone very wrong. And if you look at what 596 00:41:09 --> 00:41:14 happened in the case of an individual of multiple myeloma, 597 00:41:14 --> 00:41:18 if we look at that diagram up there, what you see now is that instead of 598 00:41:18 --> 00:41:23 there being many different B cells that are equivalently represented in 599 00:41:23 --> 00:41:28 the blood stream, now one of the B cell types has 600 00:41:28 --> 00:41:32 begun to multiple uncontrollably, and this vasicular B cell type, 601 00:41:32 --> 00:41:37 which I indicate up there, which happens to be specifically 602 00:41:37 --> 00:41:42 able to make antibody C in this model, now this particular species 603 00:41:42 --> 00:41:46 of B cells is now a predominant constituent of the population of B 604 00:41:46 --> 00:41:50 cells in the blood. So just to reiterate, 605 00:41:50 --> 00:41:54 in the normal blood, there are a million different sub-populations of 606 00:41:54 --> 00:41:58 B cells. But in these individuals, one of the sub-populations of B 607 00:41:58 --> 00:42:01 cells has expanded. It's created a monoclonal growth. 608 00:42:01 --> 00:42:05 Keep in mind monoclonal refers to the fact that all the cells in this 609 00:42:05 --> 00:42:09 tumor, and there is a tumor, descend from the same ancestor. 610 00:42:09 --> 00:42:13 So, it's a monoclonal growth. It's a kind of cancer. 611 00:42:13 --> 00:42:17 It's a kind of blood cancer. And all the cells in this 612 00:42:17 --> 00:42:21 population make the identical antibody molecule, 613 00:42:21 --> 00:42:25 which explains this very homogeneous peak here riding above the very 614 00:42:25 --> 00:42:29 heterogeneous mixture of antibodies, the background heterogeneity that 615 00:42:29 --> 00:42:33 normally characterizes normal serum. 616 00:42:33 --> 00:42:36 And what that clearly indicates is that each different B cell or plasma 617 00:42:36 --> 00:42:40 cell as you will like to call it in our normal serum is specialized to 618 00:42:40 --> 00:42:44 make its own particular kind of antibody. It's not that a single B 619 00:42:44 --> 00:42:48 cell can make a million different antibodies. Each B cell makes a 620 00:42:48 --> 00:42:52 very specific kind of antibody. And when I say a specific antibody 621 00:42:52 --> 00:42:56 or a specific antibody species, again I'm referring to an antibody 622 00:42:56 --> 00:43:00 that has a specific antigen-binding site. 623 00:43:00 --> 00:43:04 Or to put it another way, all of the antibody molecules coming 624 00:43:04 --> 00:43:09 out of a single B cell are identical with one another. 625 00:43:09 --> 00:43:14 That, then, raises the question of how the B cell learns how to make 626 00:43:14 --> 00:43:19 that antibody molecule and not other antibody molecules. 627 00:43:19 --> 00:43:23 There's yet another puzzle we have to answer, and that's the following. 628 00:43:23 --> 00:43:28 If you look at the serum response of an individual to a viral 629 00:43:28 --> 00:43:32 infection, it looks like this. And here, let's look at what's 630 00:43:32 --> 00:43:36 present in the ordinate, and it's on a log scale as you can 631 00:43:36 --> 00:43:40 see here, and what's present on the abscissa. If one is initially 632 00:43:40 --> 00:43:44 exposed to an antigen and develops an immunity, then here is the wave 633 00:43:44 --> 00:43:48 of antibody production and the concentration of antibody against, 634 00:43:48 --> 00:43:52 let's say, poliovirus that is present in the blood of a recently 635 00:43:52 --> 00:43:56 infected individual. They're exposed to the antigen, 636 00:43:56 --> 00:44:00 or in this case the poliovirus capsid protein. 637 00:44:00 --> 00:44:04 In the days and weeks that follow, they develop a significant level of 638 00:44:04 --> 00:44:09 antibody which is used to neutralize, in this case the infected poliovirus 639 00:44:09 --> 00:44:13 particles. And then once the poliovirus is cleared from the 640 00:44:13 --> 00:44:18 system, i.e. once all the infectious particles have been neutralized, 641 00:44:18 --> 00:44:23 then the antibody molecules go down almost to zero, 642 00:44:23 --> 00:44:28 and there's virtually no antibody against poliovirus left around. 643 00:44:28 --> 00:44:32 But look what happens when that individual is re-exposed to 644 00:44:32 --> 00:44:37 poliovirus years later. All of a sudden, now he or she 645 00:44:37 --> 00:44:41 mounts a massive antibody response vastly higher than the initial one. 646 00:44:41 --> 00:44:46 Well, you say it's only twofold higher than this. 647 00:44:46 --> 00:44:50 But let's look at the ordinate. This is a logarithmic plot. So, 648 00:44:50 --> 00:44:55 on this plot, the secondary antibody response is maybe 100 or 200 times 649 00:44:55 --> 00:45:00 more vigorous, much higher antibody production. 650 00:45:00 --> 00:45:04 Let's think about this time years later when that person is exposed to 651 00:45:04 --> 00:45:08 a second virus. Let's say that person is exposed 652 00:45:08 --> 00:45:13 subsequently to cold virus, adenovirus. Does the exposure to 653 00:45:13 --> 00:45:17 poliovirus here in the blue curve help that person develop an 654 00:45:17 --> 00:45:21 adenovirus antibody response? The answer is absolutely not. 655 00:45:21 --> 00:45:26 There's no cross-immunity. And so, when that individual years later 656 00:45:26 --> 00:45:30 becomes exposed to adenovirus, there's once again the same kind of 657 00:45:30 --> 00:45:34 initial response that happened as a consequence of previously being 658 00:45:34 --> 00:45:39 exposed to poliovirus. It's a rather weak response. 659 00:45:39 --> 00:45:43 It's effective enough in developing the initial virus eliminating 660 00:45:43 --> 00:45:47 ability, but it's not very strong. What is this telling us? I'm glad 661 00:45:47 --> 00:45:51 I asked that question. It's telling us that that immune 662 00:45:51 --> 00:45:55 system is able to remember over a period of months, 663 00:45:55 --> 00:45:59 years, and decades that a previous exposure to this antigen has 664 00:45:59 --> 00:46:03 occurred because here the immune system in this second red curve 665 00:46:03 --> 00:46:07 knows, somehow remembers, that there was an exposure years 666 00:46:07 --> 00:46:12 earlier. And not only does it remember and 667 00:46:12 --> 00:46:16 respond rapidly, but it responds much more vigorously. 668 00:46:16 --> 00:46:20 And here now, we begin to recognize some of the 669 00:46:20 --> 00:46:24 outlines of immunity because the immune system remembers this earlier 670 00:46:24 --> 00:46:28 exposure, and when it's provoked a second time, through an accidental 671 00:46:28 --> 00:46:32 and inadvertent exposure to poliovirus on a second occasion, 672 00:46:32 --> 00:46:36 now it really goes to town. And now it creates antisera which is 673 00:46:36 --> 00:46:40 much more vigorous than it was during the first exposure. 674 00:46:40 --> 00:46:44 If the first exposure had never occurred, there wouldn't be such a 675 00:46:44 --> 00:46:47 vigorous response. Look at this one over here. 676 00:46:47 --> 00:46:51 So now, we begin to realize two mysteries that we have to explain 677 00:46:51 --> 00:46:55 next time in our further discussion of the immune system. 678 00:46:55 --> 00:46:59 First of all, how do B cells figure out how to make so many different 679 00:46:59 --> 00:47:03 kinds of antibody molecules. And secondly, how does the immune 680 00:47:03 --> 00:47:07 system remember from one decade to the next, that exposure to a 681 00:47:07 --> 00:47:12 previous antigen has occurred which merits a vigorous response. 682 00:47:12 --> 47:17 See you then on Friday.