Welcome to week two, COVID-19: Health Systems andPandemics. Once again, I’m Dr. Michael Sulzinski, professor of microbiology and immunology atGeisinger Commonwealth School of Medicine. Welcome again to our MBS graduate students,medical students, members of the Geisinger community and any others who may be joiningus to learn about SARS-CoV and COVID-19. We’ll start off with a look back andlook ahead. What we covered last week, last week we covered COVID-19 as a new disease.We introduced the topic of a zoonotic infection, that is an infection from animals to humans. Andwe talked about how with the SARS-CoV-2 likely had its origins originally inin the bat population, underwent some genetic change, that we call antigenic shift, to moveto some yet unidentified intermediate host which again underwent another antigenic shiftso that the virus is now transmissible to humans as part of its host range. We talkedabout how the first incidence of disease, pulmonary disease, was described on the verylast day of 2019. And we talked about this as a new disease that had been characterized quicklyover the coming weeks and months. Next we talked about how the virus spreads.We talked about person-to-person transmission, and the public health, their recommendations tominimize the risks of infection.We talked about the consequences of infection and said thatthey varied widely, on one extreme asymptomatic infection with no symptoms at all and on theother extreme severe sometimes fatal infections. We talked about the nature of viruses. We saidthat all viruses are made up of two components, a genome and a capsid. Genome consistsof either DNA or RNA but never both. We said that SARS-CoV-2 wasan RNA virus and as an RNA virus we said that there was a special tendency ofviruses with RNA genomes to mutate and to evolve quickly. We talked about the virus replicationcycle. We listed the six steps that each virus takes to replicate and make more of itself. Andwe said that the first step, the attachment step, was arguably the most important because ifattachment takes place infection takes place. Attachment is a very specific interactionbetween something on the surface of the virus with something on the surface of the cell. We talked about the specificproperties of SARS-CoV-2.We talked about its shape and its name. Talkedabout the fact that it has an envelope and as an enveloped virus what that meant in terms ofits ability to survive outside the body. Focusing further on the envelope, we talked a lot about aparticular protein known as the spike glycoprotein projecting through and extending from theenvelope giving the virus its characteristic crown appearance. We said that the spike glycoprotein wasimportant because it is the molecule that will be recognized by the host receptor. We said that thespike glycoprotein is important because it is the molecule that will be recognized by our immunesystem as something foreign to the body and as such, we’ll see today, that that spike glycoproteinis going to be a key protein for vaccine design.We ended up talking last week about thekey discoveries over the past nine months. Those key discoveries including the determinationand publication of the complete genome of SARS-CoV-2, 30,000 bases of ribonucleicacid in a particular order, and I will remind you today of how important that breakthrough hasbeen in our ability to do diagnostics as well as vaccines. We talked about the fact that onlyless than 10 major strains of the virus have been detected circulating through the populationand we said this was remarkable for an RNA virus which at least has the potential to producehundreds if not thousands of different strains. Last time we said that a key discovery was theability to cultivate or replicate the virus in tissue culture and living cells in the laboratory.And we talked about the fact that there was an animal model, that is to say within the hostrange of this virus there are specific animals that could be inoculated with purified virus and after inoculation those animals would developclinical disease.Today working ahead with week two, we’re going to talk about testing therapy andvaccine strategies. Testing will include both diagnostics and screening. PCR tests willdetect the SARS-CoV RNA genome. Serological tests to detect antibodies throughoutthe body made against the virus. We’ll talk about drug therapy and convalescent serum usinga procedure known as passive immunization. And we’ll spend a lot of timetalking about vaccine strategies and the plans for a safe and reliablevaccine for global distribution. For those of you who may be enrolledas a student in class, these are the learning objectives for week two: To explain howPCR is applied as a diagnostic test to detect SARS-CoV in nasopharyngeal samples. Todescribe how antibodies produced against the virus are being applied to determine present and pastinfection, how those antibodies might be produced for vaccination purposes. To summarize the evidenceto date on antiviral drug therapies.Outline the current strategies for global development ofvaccines using criteria of safety and efficacy. And to have an understanding of the technical and practical challenges ofproduction and distribution, summarize ethical considerations associatedwith vaccine testing, delivery and administration.Let’s begin with an overview ofdiagnostics specifically for COVID-19. By diagnostics our goal is to detect some markerof infection, such that infected persons have that marker that is detectable and uninfectedpersons do not. Well what is the marker? The marker could be the virus itself. Marker couldbe parts of the virus, viral RNA, viral proteins. Markers could also be antibodies made by thebody in response to the virus or its parts. So the goal for COVID-19diagnostics is to identify those persons that are infected who have that marker to medicallytreat them if appropriate, to isolate them from others who are not infected and to follow howthe disease moves throughout the population. We’ll begin with a discussionon polymerase chain reaction, PCR. PCR is a method widely usedto make millions of copies of a very small quantity of nucleic acid.So itfollows the rule of the proverbial needle in a haystack, so that if a person has a small amountof that particular nucleic acid in their body, PCR test is an amplification strategy wheresmall quantities of that target become amplified such that there are millions of copies of it whichare much easier to detect. In order to design a PCR test, requires the knowledge of the genome. Youneed to know what the sequence of the genome is, at least partially, and again that highlightswhat we talked about in week one, the importance of establishing what that genome was in terms of thesequence. PCR is considered to be the gold standard of diagnostic tests, the most reliabletest in terms of clinical diagnosis. If you look at the illustrationon the left you see the sample being taken as a nasopharyngeal swab.Second part, which we’ll go into detail on the next slide, shows how the DNA is amplified in thetest tube in the laboratory. And the third part talks about detection and determiningwhether the sample is positive or negative. So polymerase chain reaction is described as a DNAamplification test.Well we know that the genome of SARS-CoV is RNA so the very first stepin this assay is going to be to take the viral RNA and switch it over to a DNA. After thathappens we do the procedure of amplification which involves heating and cooling the samples inthe presence of small pieces of DNA called primers. The primers are specific for the nucleocapsidgene of the SARS-CoV. So this brings tremendous specificity for the amplification. Soit’s not going to amplify just any nucleic acid it’s going to amplify specifically the nucleicacid of the virus that we’re interested in. So every time the sample is heated and cooled,heated and cooled we call that a cycle of PCR. We typically do the procedure for 20 or 30 cyclesand with each cycle the amount of amplified DNA doubles so we get a quick logarithmic increase inthe number of copies of a specific nucleic acid.The details then for SARS-CoV-2 PCR, it’sa nasopharyngeal swab obtained from the patient. Swab is then placed in a tubewith preservatives, sent to the lab. Sample is partially purified, treated torelease the viral RNA from the sample. Sample’s put in a test tube, and again as I justmentioned the very first step is to take the viral RNA and convert it to DNA, that happensusing an enzyme called reverse transcriptase. So when we see RT-PCR.RT-PCRcan refer to reverse transcriptase where the target is actually RNAthat needs to be converted to DNA. Once that conversion takes place as I saidthe DNA is amplified over many cycles of PCR such that there is an amplification many millionfold. Positive sample is then detected by the shape of a curve caused by fluorescence that is detected,determined and observed on a computer monitor. A little bit of details for the PCR test,there are actually three different sets of these primers that are specific. Two of the primerpairs target the viral N gene, or nucleocapsid gene, as we described last week as being associatedwith the viral RNA inside the virus. There is a single primer pair that’s an additionalprimer pair that confirms that the sample is amplification competent, in other words we aredetecting a human gene in the sample to verify that the sample is amplifiable.This is reallyimportant in validating a negative result. We also have negative controlsand positive controls. Positive controls typically used for a non-infectiouspositive control DNA version of the viral RNA. A flow chart showing you how the test is done.Again starting with our nasopharyngeal swab, sample is collected, kept at the refrigerated temperatureuntil it makes it to the lab. RNA extraction is step number three to partially purify the RNA.Step number four our real-time PCR. The little q in front of the PCR says quantitative, so ourtest is actually going to be able to tell us how much viral RNA is present in a sample. Ourtest results, number five there, I’ve actually made this a little bit easier to see on thisslide, showing you positive and negative results. Again we are looking indirectly at amplificationby looking at increases in fluorescence because the more amplification that takes place the morefluorescence that is determined by a meter inside the instrument.So for a positive test seenon the left we see a typical sigmoid curve. Sample is exceeding some threshold limit telling us thatit’s a positive sample, whereas the negative sample shown on the right does not have that fluorescenceand therefore is interpreted as a negative. So the PCR test detects a part of the N gene.Remember the N gene is a relatively stable gene in the genome, that’s why the nucleocapsidgene was chosen as the target here. There are some challenges with PCR as reportednationwide. And the challenges are these the procedure is labor-intensive and expensivewith specialized equipment and personnel. Because of the cost and availability ofPCR, universal testing is not practical. Sampling is difficult, sometimes missespositive samples such that symptomatic persons may test negativeone day, a retest 24 or 48 hours later may test positive. It could well mean that thesample taken was taken too early such that there was not enough virus or viral RNA in this,in the specimen to come up as a positive.An issue particularly early on in the pandemicwas that there was a delay of getting specimens to the lab and the longer the delay the greaterthe chance of sample degradation on the way to the lab. And then finally another challenge isthat this PCR test only detects active infection so it may miss very early infection ifthere’s not enough virus and it is not capable of detecting cleared infection in whichthe virus and the viral RNA is gone from the body. Using PCR as a diagnostictest for SARS-CoV, just a nice summary, using an infected, newlyinfected patient during the incubation period, very early in the incubation period, it couldwell be that the PCR test is going to be negative, not enough virus or viral RNA to come up as apositive. A symptomatic patient will likely test positive. However a recovered patient for whichthe virus and viral RNA has been cleared, this test is going to be negative. There are going tobe some people for which the PCR test is probably not done, and that would be an asymptomaticinfected patient, people with mild or uncharacteristic symptoms that we described last week probablywould not present themselves for a test.And then we have examples of low-risk symptomatic patients,that would be for example young people for which severe disease is not likely, the recommendationmay simply be to stay at home so that you’re not presenting a risk to others and in which case PCRtests probably won’t be done. So the bottom line of this, based on our PCR testing alone we are missingsome unknown number of people that are infected.Let’s switch our discussion to talk about the detection of antibodiesagainst SARS-CoV-2. Serological testing, testing for antibodies orantigens, is based on this particular theory. Persons who have never been infected withSARS-CoV-2 will not have antibodies against the virus in their blood and we saysuch persons are seronegative. Persons who have been infected either with symptoms orwith an asymptomatic infection, either way, will have antibodies against the virus in theirblood and those persons will be zero positive. There are two classes of antibodies that cangive us information whether the infection was a past infection or whether it is an active or veryrecent infection. So for example the presence of IgG antibodies would indicate a past infectionas opposed to IgM antibodies in the blood indicating an infection that is still active orvery recent. For most diseases and most pathogens you can exploit these observations to study howthe virus moves throughout the population and how many people had been exposed or infected.Theoretically antibody testing should be able to determine the presence of antibodies in thepopulation which we call antibody prevalence.The theory is that IgG antibodies in a person’sblood will protect them from future infection if they are exposed to the virus and it shouldn’tmatter whether those antibodies are there in response to a symptomatic infection with illness,asymptomatic infection without illness or symptoms or vaccination, which we don’t havetoday but the hope is hopefully soon. Nonetheless the presence of antibodies, weare expecting a protection from future infection. So the presence of antibodies therefore not onlyindicates past exposure to the virus or some viral protein but at least in the historicalstance, view it also suggests future protection. With antibody testing there are a number ofimportant challenges as reported nationwide and those challenges are issues both withtest sensitivity and test specificity. For the antibody tests, sensitivity is the abilityto detect the presence of antibodies such that an infected person should be testing aspositive, that’s the test with high sensitivity. Specificity is the ability to confirm theabsence of antibodies when they are truly absent, that means an uninfected person should betesting as negative with a good specificity.Does being seropositive protecta person from future infection? Truthfully the answer is we aren’t sure atthis point in time. There have been a few rare documented cases where persons havebeen re-infected with SARS-CoV-2. Why is this an important question? It’san important question because being seropositive and protecting a person is really thebasis of vaccine design. We are intentionally creating a vaccine so that people will seroconvert to be seropositive under the assumption that being seropositive, having those antibodieswould protect you from future infection. In addition to PCR and the antibody tests, thereare a few other tests that are worth mentioning. There are COVID-19 antigen tests. This issomething that you may not have heard about because it is not in the public press. These arelaboratory-based and rapid point-of-care tests, as shown on the right, that detects SARS-CoVviral proteins or antigens in the patient samples. So the antigen test is for diagnosis ofactive infections but not past infection, similar to the way we view the PCR test. However the COVID-19 antigen tests are notnearly as sensitive or as specific as RT-PCR. If you look at the picture to the right, theseare point-of-care tests for COVID-19 antigen. Patient specimen is put into the bottom wherethe specimen block is.There is a capillary action that draws the specimen to the top andwe see a positive test on the left, negative test on the right. This is similar to a pregnancytest, with the same kind of technology where the presence of two bars on the left, thecontrol and the test indicates a positive sample, and on the right a negativesample. If there were no bars, nothing for either C or T, thatwould indicate an invalid test. There are also tests for the management of COVID-19 patients.So beyond the tests that diagnose or detect the virus or its antibodies, there areother tests that are used in the management of patients with COVID-19 and these are teststo detect biomarkers related to inflammation. And again I refer to the last class wherewe talked about the cytokine storm and the MAS. So once patients are diagnosed with the COVID-19disease, these additional tests can be used to inform patient management decisions, particularlyin patients that have severe infections.Look for an influenza SARS PCR test called theFlu SC2 multiplex assay coming in the next week. This is an RT-PCR test that both detectsand differentiates SARS-CoV-2, influenza virus A and influenza B virusfrom upper and lower respiratory specimens. We call this a multiplex assay because witha single patient specimen you can test for SARS-CoV and two influenza viruses. Testwill tell you whether the samples are positive and it will differentiate each of thosethree different viruses from each other. Expect this to be very usefulin the coming flu season. Let’s spend a few minutestalking about anti-viral drugs. Today there are no antiviral drugs that arecurrently approved for patients with COVID-19. This is not particularly unusual for avirus infection. Traditionally most viruses do not have effective antiviral therapy. Thereare some exceptions. Some of the exceptions are hepatitis C, hepatitis B, HIV and influenzavirus. But the fact that we haven’t had a a very good antiviral therapy yet is really notall that unusual.As you are aware over the past several months a number of medications were underinvestigation as therapies for SARS-CoV-2 infection including chloroquine, hydroxychloroquineeither in the presence or absence of the antibiotic azithromycin. Those drugs have notfared very well and are not considered to be useful in therapies for patients with SARS-CoV-2.Still under investigation: antiviral drug Remdesivir is an adenosine nucleotide analogue and is still being examined with clinical trials. There is one investigational therapy that isshowing some signs of promise, that is the drug dexamethasone. In the United Kingdom with arandomized clinical trial showed that this steroid drug could reduce COVID-19 relateddeaths by a third in patients on ventilators. Also those who were receiving oxygen therapy, noton ventilators, had a 20 percent less risk of COVID-19 related death.However for patients withoutserious symptoms dexamethasone did not appear to provide any particular benefit. So for thosemost severe cases dexamethasone has been shown in clinical trials to be efficacious. There isanother experimental treatment using what we call convalescent serum. Convalescent serum meansthe taking a serum from patients that have been infected with SARS-CoV-2 and have survivedthe infection such that the virus is no longer in their blood. So the model is these people who haveconvalescent serum have antibodies in their blood, transfer those antibodies to another person tomitigate their infection. This is a process that we call passive immunization. Early evidenceresults are mixed for passive immunization using convalescent serum and it appears thatnot all antibodies are protective antibodies and passive immunization works for some patientsbut not all. Under development for the next few months expect to see the creationof laboratory-derived monoclonal antibodies. These are highly purified, highly specificantibodies produced in the laboratory and show at least some potential for the treatmentof high risk patients to mitigate their infection.All right let’s talk about vaccines. And of course the number onequestion: When will we have a vaccine? In order to license the new vaccine in the U.S.two requirements must be met. The vaccine must be proven to be safe and efficacious. Safety of coursesays that the vaccine can be administered without serious adverse drug reactions, side effects.Efficacy means, is the vaccine effective? Is it efficacious? Does it do what it was designedto do and that is to prevent infection in people that receive the vaccine? So while lots of vaccinesmay look good on paper, on the drawing board vaccine design is very easy, but in the real worldtake neither safety nor efficacy for granted. Here’s a slide that shows from the FDA’s perspective how vaccine isdeveloped, approved and manufactured. It’s three phases in clinical trials. The firstphase involves about 20 to 100 volunteers. This is a very simple phase one clinical trialasking is the vaccine safe? Are there any serious side effects? And also to determine what might bethe most efficient dose or the safest dose that will still achieve the desired end.Phase two of aclinical trial involves several hundred volunteers, asking again what are the most common short-termside effects? This phase two clinical trial, it does not take a long time. It will then ask how arethe volunteers immune systems responding to the vaccine measured by the amount of antibodiesproduced by people who receive that vaccine A phase three clinical trial involves hundreds orthousands of volunteers asked the question how do people who get the vaccine comparewith people who do not get the vaccine? Still ask the question is the vaccine safe inthis clinical trial? Is the vaccine effective? And we’ll follow a particular vaccine candidatein phase three and talk a little bit more about how phase three clinical trialsare used to evaluate both safety and efficacy. Let’s talk about the main strategiesfor designing vaccines against COVID-19. One of the main new strategies is shown asnumber one on this slide, messenger RNA or mRNA, encoding a SARS-CoV-2 gene.That SARS-CoVgene of course is going to be the spike glycoprotein because that is going to be theimportant antigen. So in making this vaccine design once again you need to have the informationfrom the genome. So that genome sequence comes into play again and how important that is. Soknowing that part of the genome that encodes a spike glycoprotein you can then create andmanufacture in the laboratory a messenger RNA that codes that gene which ultimately willproduce the viral protein after it’s received in the body. We’ll talk about this with this twospecific vaccine candidates in just a minute. The second strategy for vaccine candidate designis to make recombinant SARS-CoV-2 surface protein, particularly ourglycoprotein, a spike glycoprotein in a vector. That vector would be then expressedsuch that you get large quantities of spike glycoprotein.This technique has been used forother kinds of vaccines successfully in the past. The third approach is to use a viral vectorwhich is packaging the SARS-CoV gene for the spike glycoprotein, and I’llgive you a specific example of this, such that the this recombinant virus as a vectoris then going to deliver the viral protein such that the body will recognizethat protein and make antibodies. Number one on our list was mRNA vaccine design.This is a quick graphic that shows how that works. The messenger RNA for the spike protein that thecoronavirus is injected into the muscle, vaccine triggers production of the protein itself andthen antibodies specific against it and therefore we get the production of antibodies againstthe spike glycoprotein. This approach is said to be very safe. Manufacturers say thatthis is a safe vaccine because it is cell free, no infectivity, no virus, not possibleto become infected by virus at any step. They say it’s easy to design based on thenucleotide sequence of the virus.So we have a synthetic viral messenger RNA, synthetic meansit’s actually made by a machine in the laboratory. By taking those, that particular sequence ofnucleic acid and then put together as a messenger RNA. Once that is injectedinto the body it finds its way into cells. In the cytoplasm messenger RNA istranslated on ribosomes to give protein so that messenger RNA information becomestranslated to produce the SARS-CoV spike protein which is then presentedto the immune system to make antibodies.Let’s talk about the first specific vaccinecandidate that is currently under clinical trial. The vaccine is called mRNA-1273. Sothis is a messenger RNA vaccine for spike hypoprotein. It is dispersed andpackaged in liquid lipid nano particles. In general messenger RNAs are labile insidethe cell so you need something to deliver the messenger RNA such that it doesn’t get brokendown. It is difficult to get solid clinical data on how these vaccine candidates areare performing in clinical trial. So a lot of the information really comesfrom public press releases from the companies. This report says that the vaccine candidatemRNA-1273 induced antibody levels that exceed those in human convalescent phase serum. So theobservation here is that by vaccinating people they actually produced more antibodiesthan people that were naturally infected. Expected release of this candidate vaccine wouldbe late 2020.It’s expected that this might be one of the first ones to be approved. U.S. governmenthas already purchased at least one million doses pending FDA approval. It is interesting to notethat the name of the company is Moderna and the end of the company’s name is RNA so that tells youhow they are basing their technology for vaccines. Looking at the, a second leading vaccinecandidate, this one produced by the pharmaceutical company Pfizer.And Pfizer of course has had along history of producing vaccines. They produce a vaccine which is based on mRNA they call it BNT162.Messenger RNA also for the spike glycoprotein. Pfizer actually set up a system wherethey had four different messenger RNAs and internally pitted them one against the otherand the top candidate was this one here BNT162. Again difficult to get hard data on how thePfizer vaccine is working in clinical trials but the reports that are available say that inanimal studies there have been favorable results. That is that BNT162 is able to produceantibodies and able to also produce a CD4+ and CD8+ response. We saythis is a cell mediated response which may be an a very importantcomponent of immune protection. For animal studies the Pfizer vaccine has alsobeen reported to protect vaccinated animals from disease after viral challenge.That’s worth aminute or two of of a little bit of background.So for the laboratory animals last time we talkedabout the host range and we talked about the ability to do challenge infections in animals. Sofor vaccine candidates this becomes an important consideration. In this case you take your vaccinecandidate, healthy animals of course, you will inoculate your healthy animals with the vaccineand then wait a while perhaps a couple weeks after that time, come back and you will intentionallytry to infect those animals with purified virus. We call this a viral challenge. If the vaccineworks it will not only produce antibodies within that animal but it will protect the vaccinatedanimals from developing disease after they have been injected with the virus.And the Pfizervaccine candidate appears to to be able to do that. Another vaccine candidate is AstraZeneca. AstraZeneca has their vaccine called AZD1222.This is a vaccine that is taking a different approach. Does not use a messenger RNA, instead itis using a non-replicating chimpanzee virus to deliver the spike protein. This is anadenovirus that is not infectious to humans. It is theoretically a very safe vaccine. And theidea of course is to target the SARS-CoV spike protein such that once injectedinto vaccine recipients the vaccine recipients then recognize the spike proteinas foreign and produce antibodies against it. This also seems to be a leading contender in terms of the ability to get a vaccine licensed.both the U.S. and European Union governments have already agreed to buy millions of dosesof the AstraZeneca vaccine pending approval. Again difficult to get hard data on theclinical trials, however company does reveal that to date they have receivedfavorable results with animal studies producing high quantities of antibodies, producingboth CD4 and CD8 mediated response.We call this a cell-mediated response and again that mightbe very important in making a good vaccine. And AstraZeneca also reports that vaccinatedanimals with AZD1222 also protects, so vaccinated animals are also protectedfrom disease after a viral challenge. Give you a little bit of detail about thisvaccine. Currently in phase three clinical trials. There are adult volunteers at 80 sites across theUnited States to evaluate whether this vaccine candidate can prevent COVID-19. There are 30,000 volunteers in this very large clinical trial. The 30,000 volunteers are randomly assigned to oneof two groups. One of them is the investigational vaccine group and the other group is aplacebo group. Participants will receive either two injections of the investigationalvaccine or two injections of a saline placebo approximately four weeks apart. So the studyis designed such that there are two-thirds of the study, in people that are enrolled in thestudy, two-thirds of them received the vaccine and about ten thousand people receive a placebo.It is a blind study such that the persons who are receiving the vaccine and the personsthat are administering the vaccine are blind and that they do not know whether the personis getting the actual vaccine or the placebo.So in this phase three clinical trial what you dois administer the vaccine or the placebo and then you follow all the participants, all 30,000 ofthem, and you are going to compare the presence of antibodies against SARS-COV-2. Ifthe vaccine works you would expect that those who have received the investigational vaccine asa group would produce antibodies against the virus whereas those in the placebo group wouldtypically not, unless they were otherwise infected. You would also determine if there is a decreasedincident of disease in those volunteers that receive the vaccine. So this is a little bitdifferent, doing a human component to the clinical trial, because for ethical reasons you can’t doa challenge inoculation.We can’t have people volunteering for the vaccine candidate and thenintentionally try to infect them with the virus. So that is why the animal models becomeinfected for the human clinical trials. We simply follow them over time and ask thequestion as they are naturally exposed to the virus in their environment willthey be protected against disease if they are exposed. So you follow theparticipants looking for antibodies, you are determining whether there is a decreasedincidence in those who receive the vaccine and of course in terms of safety youare also comparing the two groups to see whether there is an increased incidenceof adverse drug reactions or side effects.So I’ve given you just a few samples ofwhat are considered to be the top contenders in this race to get a vaccine licensed. There aremany others, well over a hundred other entities, that are involved in making vaccine candidatesworldwide. In addition to what we described there are other approaches that are alsobeing taken. For example inactivated or killed vaccines where the virus is treatedwith formalin to kill it. This is a standard technique that is proven technology that we usefor many of our vaccines currently. There are other approaches that include recombinant vaccines,using either an adenovirus or baculovirus vectors. The local company Sanofi Pasteur is using abaculovirus vector for its vaccine candidate. There is another approach using what iscalled self amplifying RNA vaccines that is another unproven technology.And many other new approaches. Let’s talk about vaccines and the important challenges that we face for any vaccines in termsof their safety and efficacy.First of all challenges to vaccine safety, andlet’s frame this in the context of present day views on vaccines in general, particularly in theU.S. For mostly unfounded reasons there is a fear or aversion to vaccines. So that is rightoff the bat a challenge to vaccine safety, that we need to have a vaccinethat is going to be proven safe to convince people that this is somethingthat they want to get. So in the U.S. we have 328 million people we are envisioninga vaccine for hundreds of millions of them. COVID vaccine will be tested first in animalmodels and then in human clinical trials to demonstrate vaccine safety but the concernis that in a hurry to get the vaccine out, in a hurry to get the vaccine licensed,we will have limited clinical trials in which limited numbers of persons areenrolled over a limited period of time. The problem is that adverse drug reactions orside effects may be rare and may not be seen until large numbers of persons receive the vaccine.So 30,000 people in a clinical trial sounds like a large number but it might not be large enoughto be able to detect rare adverse drug reactions. If you have a serious adverse drug reactionfor example at a rate of one in a thousand if you administer the vaccine to two hundredmillion people you will expect at a rate of one in a thousand that two hundred thousandpersons will have that adverse drug reaction. So very important that the vaccinesafety be examined very carefully. So there is a valid concern that a rushed,poor vaccine would erode confidence in other vaccines and erode confidence inimproved second- or third-generation vaccines. Remember the rule for vaccines is the rulefor medicine in general, first do no harm.Challenges to vaccine efficacy, many of the vaccine candidates we just describedrely on unproven technological approaches. That doesn’t mean that they won’t work. It doesmean that their success is more unpredictable. For example we don’t have any licensed vaccinesthat use messenger RNA technology right now. Will the antibodies that are inducedas a result of vaccination protect humans? Will it protect them enough againstinfection such that they will not have a serious clinical disease. Will the antibodies thatare produced at the time of vaccination persist or will they start to wane afterweeks, months, years? We would like those antibodies to persist for thelifetime of the vaccine recipient. Are antibodies enough? Many experts predict that anysuccessful vaccine in addition to antibodies must involve T cell responses, CD4 and CD8 Tcells, not just the production of antibodies.We know that tests in animal models donot always predict effectiveness in humans. Inducing antibodies against the virusdoesn’t guarantee protection. Persons most in need of protection, that is the elderly or oftendifficult vaccine recipients because they simply can’t respond the same way that youngerpersons can with a more robust immune system. Finally sometimes vaccines can actuallymake a natural infection worse. We call this antibody dependent enhancement ofsymptoms. It is rare but it can happen. Then there are logistical challenges for vaccines.And probably the major logistical challenge is what we call scale up, that is the manufacturingprocess such that you’re now going from hundreds or thousands of vaccines to now tens of thousandshundreds of thousands and millions of doses. That is a big consideration. Another logisticalchallenge is refrigeration, getting the vaccine under refrigeration from the pointof manufacture to the point of administration. You need to have an effective network distribution,not just for the vaccine but also for associated supplies, needles and syringes.Two doses means thatyou need to track people and get them back for a second dose. If there are multiple doses, if thereare multiple vaccines, what happens if people don’t get two doses of the same vaccine? We are not ableto predict adverse drug reactions when we combine two different vaccines because that willnot have been examined in clinical trials. What about record keeping? Tracing of vaccinerecipients and adverse drug reactions, this may be important. It was important certainly in 1976 whenwe had an outbreak of swine flu in the U.S. and the adverse drug reaction for that vaccine wasan increased risk for Guillain-Barre syndrome. What about informed consent? Who willbe liable for adverse drug reactions? What about the cost of vaccine? Who will pay forit? The cost of supplies and cost of administration and for all of those logistical challengesmultiply everything by two because almost all the vaccine candidates that we described so farhave two dose schedules for complete protection. How do you handle multiple vaccines that might beapproved by the FDA? Which one might you recommend? Who gets the vaccine first?Who gets the vaccine last? Do you require people to get it? Will there bea government requirement? Employer requirements? School district requirementsfor for children and teachers? What about significantly better vaccines in thepipeline? Should you wait or get a vaccine now? If you are already vaccinated and a much bettervaccine now becomes available what do you do? So I’m trying to give you a nice flavor of whatthose questions might be and those questions of course will come into greater focus over thecoming months.But there are lots of challenges in terms of getting a vaccine licensedand distribute it to where it needs to be. So that’s it for week two. Here’s our summary.We started today with a nice diagnostic overview, talking about the principle of diagnostics.We talked about the technical application of polymerase chain reaction, defined RT-PCRas reverse transcriptase and real time PCR. We talked about serological testing for antibodiesand the challenges of specificity and sensitivity. We talked about antiviral drugs. We spent a lot oftime talking about vaccine design and some of the new innovative approaches for the current leadersin vaccine design at least in terms of progress in clinical trials. And then we have we ended uptalking about challenges to vaccine safety and efficacy as well as practical challenges fordistribution. So now we’re looking forward to week number three by Dr.Reema Persad-Clem, facultyat Geisinger Commonwealth School of Medicine. We’ll be talking about pandemic preparation and response,mitigation versus quarantine and other strategies. It’s been my pleasure to be with youfor the first two classes of COVID-19: Health Systems and Pandemics.Whether you are a graduate student, medical student, health professionalin the Geisinger system or part of our wider community, I hope you foundthe first two classes informative and useful. I am looking forward to joining you as a participantin the class as we progress in the weeks ahead..