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II INTERNATIONAL CONGRESS of Biosanitary Research for Young Researchers Plenary Conference The next lecture will be given by Doctor Enjuanes. The connection is via the online platform. So we must shortly be picking up the signal. Let us wait a few seconds for him to connect And I will introduce him to you. Hello. Can you hear me? Yes… Well, I will now introduce… About the echo… It cannot be fixed. I would like to introduce Doctor Luis Enjuanes. Who… Good morning! How nice to see you! We have been on tenterhooks over the connection hoping to see you. Here you are at last. Well, I believe little needs to be said to introduce this personage, this highly respected Spanish scientist, whom we have been following during the pandemic. For those younger listeners who may not know him yet, he is a virologist who has been working for over 40 years. Thirty-five of those in coronavirus. He is a virologist professor at the Universidad Autónoma de Madrid. and at the Pasteur Institute at Paris. He was named Distinguished Senior Virologist by the Spanish Society of Virology. He is member of the American Academy of Microbiology and International member of the National Academy of Sciences of USA. He was awarded the Medal to Merit in Research and University Education by the Spanish Ministry of Science and Innovation. National Biotechnology Award 2022. He is Expert Consultant at NIH and for the World Health Organisation… and I have a long list of merits here. I think the key is to be grateful that we all have the privilege of hearing you live telling us about the latest news on how current research is going. Thank you very much for being here. Okay. I am going to try and share my screen. And… Let me open this file… I do not know if you can see the presentation. Can you see the presentation? Can you see it? -Yes, they can. They do. Okay, I am going to go full screen and try to activate a laser pointer. -There it is. Okay. Finally, it was not clear to me, shall I speak in Spanish or in English? -Spanish, as… Okay. Right, probably for the better. I am going to talk to you (I am aware of the time) about emerging human coronaviruses. and the pathogenesis they produce, as well as how we can protect ourselves against them. Well, I am speaking in Spanish, but the slides are going to be written in English. As you can see. Viruses are great generators of genetic variability. In fact, due to the exchange of genetic material between the viruses and their host viruses seem to be a major driving force in the evolution of higher organisms. In fact, in humans, viruses are continually reinventing themselves thanks to a highly creative process never seen before in nature. Thus, for instance, virus families as the coronaviruses have evolved to such an extent that they can infect different families of animals or even people. These are categorised into different genres: Alpha, Beta Genre Delta, Epsilon and Gamma. These infect wild animals and people; domestic animals, mice and people; wild animals… they infect bats, and also birds. But the key point of this slide is that all these viruses, all these coronaviruses, come from the bats that are flying at the moment on all continents. That is, here at the European continent and in Murcia at the present moment. They transport all those viruses. To date, we have discovered seven human coronaviruses. The first four ones, here in black, were first discovered in 1960 and are now attenuated. If you get infected, they only cause the common winter cold. The last three, however, here in red, were first discovered in 2002, in 2012, and the last one in 2019. And they are deadly to humans. To give you an overview of these viruses, what they have done so far: In 2002, SARS-CoV 1 infected 8000 people in six months, killed 10% of them and spread to 29 countries. MERS-CoV came from the Middle East. It appeared ten years later. From then until now, in ten years, it has infected only 2650 people but, mind you, it has killed 37% of them. That is, one out of every three. And it has spread to 27 countries. However, SARS-CoV-2, the latest human coronavirus, emerged in 2019 and, attention, has infected 600 million people and killed 6 million. Its mortality rate is less than 2%. But it has spread to 253 countries. Hence, it has caused a pandemic. Nowadays, with these viruses we are already in a phase (it has taken almost three years) in which we were able to ascertain that apart from the everyday infections, there are those patients who have a long-term disease due to an infection with SARS-CoV-2. These viruses result in several pathologies, giving rise to long-term problems: cardiovascular, neurological, psychological, haematological, pulmonary, etc. If we were to create a human body diagram and place the pathologies they cause, as you can see, from the brain to any other organ, it affects all major systems. To break one down, the one that affects blood circulation, the cardiovascular, leads to Myocarditis, Heart Failure, Arrhythmias, etc. This is a problem that society will have to face from now on. As some people did apparently recovered from the viral infection, but are still suffering from some very serious after-effects. The first coronavirus deadly to humans appeared in south-east China, near Hong Kong, in the Guangdong province. There, the World Health Organisation requested that all moving animals be hunted down. It was found that, out of 150 species, there were three, civet cats, racoons and visons, that harboured the virus. Especially in the civet cats, where it replicated at the highest speed. They carried a virus that was virtually identical, according to the genome sequence. The bad news was that there these civet cats are considered a delicacy and were served in restaurants. And that is how it passed on to us. The second coronavirus deadly to humans, which appeared in 2012 in the Middle East, in Saudi Arabia, spread to the neighbouring countries. From there it went to South Korea. It infected 180 people and killed 80. It was terrible. The carriers -keep in mind that the origin was Saudi Arabia- were the camels, which are widely used there, as cargo animals, as racing animals… Just as horses here. The problem was also that people were in the habit of drinking the hot urine and milk freshly drawn from the camels, and so people got infected. And where does the current coronavirus come from? The SARS-CoV-2. Until recently we did not know the origin but now, as of a few weeks ago, we do. The natural reservoir, as for all coronaviruses, is bats. Suspicions were raised, as they had been before, that the intermediate hosts were small mammals closer to humans than bats. And that one of them had passed it on to humans. But this had never been proven. These coronaviruses had been shown to be differentiating and making several evolutionary branches. But the human coronaviruses which first appeared in the city of Wuhan in central China, essentially created two evolutionary branches: called the Line A and the Line B. Then it became clear that Line B was the first, but both had a recent common predecessor. These viruses, the first 200 or 300 viruses, emerged in the city of Wuhan, in this dark grey area in the centre of China. North of the Yangtze River, which crosses the city (the third largest river in the world), there was, and still is, a flea market where all sorts of things were sold, but also live animals for human consumption. To the south there was, and still is, the Wuhan Institute of Virology, which our friend Trump accused of having released the virus, by mistake or by design, and that this caused the current pandemic. However, reliable scientific data, recently published in two papers in Science, have proved… (This is the Yangtze River, which flows through the city of Wuhan.) that the pandemic epicentre was here. (This was checked several times.) and that the virus was not initially seen south of the Yangtze River. This is the Huanan market structure, quite a large flea market. Each grid represents a market stall, but the positive cases were mainly found in this area. In fact, the pandemic epicentre was here, when looking at all the market stalls. This area is where they were selling the raccoons, which actually are the very source of the virus. It is important to note that, when Trump accused Wuhan Virology Laboratory that they were responsible for the outbreak of the virus, the virus that was isolated, the virus that was spreading, had a four-amino acid insert that changed its behaviour. However, the virus that was being studied at the Wuhan Institute of Virology did not have such an insertion. This insertion has made the virus tremendously dangerous for reasons that I will now briefly summarise. The most direct explanation for its origin is a zoonotic event. That it spread from a lower mammal to humans. There is currently no evidence that SARS-CoV-2 originated in a laboratory. And, more importantly in practical terms, biological material was found in the racoons faeces. In the iron cages where the raccoons were kept there in the Huanan market, they found viruses with a sequence 99.993% identical to the virus that had caused the pandemic. Raccoons are members of the dog family. and these little animals are the ones that transferred the virus to us. On the left, you can see a virus particle. This is the S protein on the virus spicules that identifies the receptor, that there is a cell, displayed on the right. However, when the identification occurs, there is binding but the virus is not internalised. This is the cut-off point I mentioned, which is cleaved by furin. (Here is the molecular structure.) If we extend the protein S towards the middle, this is the proteolytic cleavage zone. Why is this important? This proteolytic cleavage is caused by furin, a protease that is present in all the tissues of the organism and that, therefore, facilitates the entry of the virus into any of our tissues, any important organs, which can lead to many pathologies. In fact, the previous virus, SARS-CoV-1, which emerged in 2002, infected only the lungs and enteric tract, while the current virus infects all the major organs of the body and can cause more than 50 pathologies. What history has proven, as you all already know, is that the virus evolves quite fast and has given rise to many strains: Alpha, Beta, Gamma, Delta, and recently, the Omicron. In this evolution, however, the virus has been attenuated. Fortunately, the latest variation, the Omicron, when tested in mice, when mice get infected, if we monitor the weight loss caused by the pathology that the virus produces in these mice, if the virus is one of the Omicron strains (shown in the upper lines) being attenuated, no apparent weight loss occurs. However, when infected with an earlier strain, It causes a rapid weight loss that soon leads to death, right? But the Omicron does not, implying that the current virus is attenuated. Let us see… It was also possible to observe… Okay, something happened here, this is not working… Right. When studied in hamsters, the same thing happened. If the lung of a hamster is infected by the Delta strain, at days 3 and 6 after the infection, when looking at the pulmonary alveoli, it is clear that the Delta strain has infected a lot and an infiltration of immune response cells has occurred by day 6. By day 3, these red areas show the presence of the virus, which increases on day 6. However, when these hamsters are infected with the Omicron variant, at days 3 and 6, the pulmonary alveoli are clear, there is oxygen exchange, and they do not die. And when we check for the virus, by day 3 there is no trace of it, and by day 6, as you can see, there is just one cell infecting it. The same has been observed in humans. When you look at the human bronchi, at the epithelial cells, red denotes infection, whereas if we look at the ciliated cells of the bronchi, viral particles can be seen by electron microscopy, even viral factories within vesicles. Interestingly, when the respiratory tract is analysed in humans, either the bronchi or the lungs, and we observe where the Omicron variant has grown the most, (the blue columns) these are the highest in the bronchi, the upper part of the lung. This facilitates the spread of the virus. Yet, when you look at the bottom of the lung, the Omicron columns are the lowest, indicating less presence of viruses and also less pathology, as this is where these respiratory viruses cause the pathology. And what has happened over time and what is on the horizon? The first prediction is that this virus will not be eradicated. that it will become a seasonal virus due to its easy transmission. And its evolution is constrained by two key factors. The first one: this is a virus that has evolved, has optimised itself and has increased its infectivity, by better joining to the cell's receptor. And another important point: these coronaviruses have an RNA genome which is constantly evolving, and what it does is to change its envelope so that the immune response we may have, induced by a vaccine or an infection, does not recognise it. This makes it evade the immune response. There is always a persecution of the virus by the immune system that responds to it. What happens today? Fortunately, there are companies that have quickly developed vaccines. There are many types of vaccines. The inactivated vaccines, based on a purified protein, induce very little immunity. It is a very short-term immunity. There are live-attenuated vaccines, which means virus-based, which can replicate. This makes these vaccines very efficient. They mimic the natural route of infection, they replicate and amplify, inducing very high immunity, and a long-lasting immunological memory. But, beware, there are safety concerns, because they are based on virus mutants attenuated but replicable, they might evolve and revert to virulent. There are other vaccines, such as the AstraZeneca's vaccine, that are based on viral vectors. In AstraZeneca, as is well known, they used an adenovirus in which they incorporated the S protein from the virus spicules, the major inducer of neutralising antibodies. These are well-studied vectors, they are very safe and very efficient. Then, as you know, in a record time, a year and a bit, some vaccines based on mRNA that encodes the virus's spicule protein, which is the major inducer of neutralising antibodies. And Moderna and AstraZeneca's vaccines were developed rather quickly. However, these vaccines, which were extremely useful to society and saved many lives, are based on a non-amplifying mRNA. You give a microgram, you get a microgram. We have been working with coronaviruses for a long time, and have developed what are known as RNAs replicons, self-amplifying mRNAs that can self-amplify up to 1000 or 5000 times. Being self-amplifying, they produce a very strong immune response As you will see, the fact that they cannot be propagated makes them safe, so that they are no longer a virus. What were our goals when the pandemic struck? We have been working with coronaviruses for 35 years and one of our most recent objectives was to study the molecular basis of virulence. A virus is not virulent because it grows a lot. A virus is virulent when it carries virulence genes. In general, virulence genes are those which inhibit the innate immune response which takes effect in two or three days. Excuse me, in two or three hours. and which is very important for our organism. If we identify the genes that are responsible for the immune response inhibition and we remove them, the virus becomes attenuated. An attenuated virus is a potential vaccine. Our laboratory had the good fortune to be the first one in the world who designed a cDNA clone of the genome. These viruses have a large RNA genome. Genetic engineering is not performed on RNA, it has to be done on cDNA. We were able to make a copy and put genes in and take genes out to study them and see if the virus was attenuated or not. But how to study if the virus was attenuated or not? Using an animal model that was developed in collaboration with North American colleagues, from the Iowa and Chicago Universities. Creating mice susceptible to the human coronavirus. Coronaviruses are species-specific. The ones that infect dogs, mice, cats or humans are different variants. Therefore, we had to create, by transgenesis, mice that were susceptible to the virus. We began removing the genes one by one using genetic engineering techniques. We were able to remove the envelope gene, which is very low in copy number but produces a large amount of E protein, which constitutes a virulence factor. By removing the E gene, we found that the virus was attenuated. But not if it carried the E gene. To test this, we conducted an experiment: We infected a group of mice with the virus minus the E gene, and another group with the gene. Four and six days post-infection we analysed the alveoli. When the virus was missing the E gene, the alveoli were clean, they were able to exchange oxygen and so much so that the mice did not die. However, if the virus carried the E gene, within 4 days of infection there was a terrible infiltration of immune response cells, and by day 6 the lungs were waterlogged. Indicating pulmonary oedema and death for the infected animal or person. Thus, we wondered if we could use this attenuated virus to make a vaccine. Two groups of mice were studied, one infected with the Delta-E virus and another uninfected, the control group. We monitored the weight loss. Three weeks post-infection, post-immunisation, (Three weeks to let the immune response develop.) we analysed whether the unvaccinated mice lost weight or not. And they did. When challenged with a lethal dose of the virus, they lost weight rapidly. Then we tracked the live mice, the survivability, and found that four days post-infection, there was not one mouse left. However, those immunised did not lose weight and survival was 100%. We had to check whether this deletion mutant of the E gene that we had created, if it could be replicated. And yes, we could, more than the original (the red columns). And if it could make messengers, too. These are crucial, as they code the immunising proteins. Then we looked at whether this deletion mutant without E could disseminate or not. We tested it 3 and 6 days post-infection and found no virus. What does this mean? That this virus cannot spread. If it cannot spread it is no longer a virus, it is an RNA replicon derived from the virus genome. This makes the vaccine we developed safe to use, since we are not injecting a virus, but a replicon that cannot spread. Actually, I said we removed one gene, but we removed first one, then two, four, five to improve the safety of the vaccine. We observed that the mutant in which we had targeted five genes could be grown successfully after 3 and 6 days (orange columns). We also looked at whether it made mRNAs, which encode the proteins of the virus. And it did. Here, the orange columns. And we thought: "This is a potential vaccine". We studied it by immunising mice and waiting three weeks for the mice to develop immunity. There were two groups: those we had immunised and those we had not (in grey). Three weeks after immunisation we challenged it with 100 000 infective doses and noticed that the unimmunised lost weight rapidly and that there were no survivors among the unimmunised. However, those who had been immunised with different altered viruses did not lose weight and the survival rate was 100%. Now, a very interesting observation is that when we immunised the mice and then challenged them with a large dose of virus, if we looked at days 2, 4 and 6 after immunisation how much virus was in those immunised mice (the orange columns), we found none of them had any virus. As opposed to the current vaccines, Moderna, Pfizer, etc., which you know that even after three doses you can still be infected by the virus and spread it. This is called sterilising immunity, since the immune mouse does not develop the virus. We have developed one intranasal vaccine prototype which is what we call the Virus Like Particles formation, formation of VLP. Well, these particles… If we infect a cell with the complete virus, and analyse the cytoplasm by electron microscopy, we see vesicles filled with virus. If we infect it with the RNA replicon we have generated, which is imprisoned inside them, VLP are formed in which the RNA replicon is introduced and cannot spread. And our vaccine is an RNA replicon encapsulated in a VLP, something we had achieved by creating so called packaging cells that express the gene that we had taken away from the virus and made it unable to spread. Seeing how this worked, when the SARS-CoV-2 appeared, (as we had done all this with the MERS) we tried to apply the same principles. This took us a year, due to the different genetic structures of the two viruses. To make sure that the vaccine we were making was safe, the whole team (this is a laboratory-wide effort) worked to alter different areas of the genome that were almost 30 000 nucleotides apart. This means that a recombination event of this vaccine with another of the coronaviruses that infect us can repair one of these errors. However, it does not seem logical that it repaired something that is 30 000 nucleotides away. We intervened in these areas by modifying this nsp1 protein, seeing different areas of it that are highly conserved. If they are conserved, it is because that is important. Then we modified the domain where the four amino acids were inserted (those I mentioned earlier), which made it possible for the enzyme furin, (which is in all organs and helps the virus to infect anything) so that it cannot spread, making this a safe vaccine. Then came the question of which S protein to put in the vaccine. The logical thing to do seemed to be to put the S of the Omicron variant, the one that has spread all worldwide. In the United States accounts for 94% of current infections. But what we saw is that Omicron, which is a variant that has evolved a lot, induces immunity against Omicron, but not against the earlier ones (Alpha, Beta, Gamma, Delta). Therefore, it was unwise to immunise against Omicron alone, instead we had to immunise with a variant carrying the Omicron S protein, and one of the old ones, because any of the old viruses induces antibodies against all the strains Alpha, Beta, Gamma and Delta, and also against Omicron. However, since it has evolved so much, it has lost strength against this variant. So now vaccinations have to be double. We developed two replicons of SARS-CoV-2, one carrying the Omicron variant and one carrying the delta. We recovered them with protein expression systems. (We removed them to amplify them.) Proteins are very well expressed on their own or in combination. And we have found that these variants, when we infect the mice (which is where we did the evaluation), show great levels of S protein antibodies with one immunisation and saturation with two immunisations. And also that these antibodies target the S protein domain which attaches to the receptor, thereby blocking the entry of the virus. We have also found that these vaccines induce T responses, which are important to prevent the evolution of the virus. By immunising mice with these replicons, we found that three weeks post immunisation (to allow the immune response to develop), if they were not immunised, they rapidly lost weight. And at day 8 after injecting them with a lethal dose survival was zero. But if they had been immunised, they did not lose weight, they actually gained weight, and the survival rate was 100% for the five mutants made. In summary, we have developed a vaccine with a number of properties. (So far we have only tested it in mice.) This is based on an RNA, not a virus, that is deficient in propagation, which makes it safe. This vaccine, unlike others, expresses several viral proteins, not just one. It induces neutralizing antibodies and T cell responses. Immunisation is done intranasally. This is essential because these are respiratory viruses. Immunity in respiratory mucous membranes is only effectively induced in them. These have showed very good results. Finally, as I mentioned, this vaccine produces sterilising immunity in the humanised mouse model. We are on to other challenges, trying to make a vaccine based on chemically defined things, which is what companies like. Only two components. One is the replicon RNA transcribed in vitro, and the other is a polymer that protects the former from degradation and facilitates cell entry. We found that these replicons perform well, for instance expressing the S protein in the cell, yet technically they are very large, (the first one we evaluated). So we are making small, medium and larger replicons to make the production process more economical. I will leave it here in case you want to ask me any questions. or if you would like me to clarify something. I would like to thank my colleagues. Our team has 16 members: Ten of them are doctors, and the rest will become so very soon. I run the coronavirus laboratory of the National Biotechnology Centre with the help of co-directors Isabel Sola and Sonia Zúñiga. Here are the people from the lab who contributed to develop the vaccines. And I would like to mention our collaboration with US teams, particularly the group of S. Perlman from the University of Iowa, which essentially developed the transgenic mice, the ones I mentioned earlier. That is all. […] There is too much echo now… Congratulations on the hard work. I think that for our students this is great example of didactic lecture, research of the highest quality and impeccable presentation. This is what you should be aiming for. Now let us see if anybody in the room has any questions. They must have plenty. I work in immunology so I have a lot of questions to ask. Does anyone in the room have questions? Could you hand him the microphone? As you will not hear it on the platform, he will ask the question and I will repeat it from here, from the computer. Okay? -Perfect. Whenever you want. He is one of the students who did an oral presentation this morning. Hello, good morning. Can you hear me? -Yes. First, I would like to thank Dr Enjuanes. This really has been… -You will repeat the question, right? -What? -Yes, there is a delay because the talk is being watched live and here it is streaming. -Right. First, I would like to thank Dr Enjuanes for the talk. It has been quite enlightening and, above all, encouraging, seeing the research potential we can perform in Spain and the technology we have available. Somewhat related to what was said earlier in the round table. I have recently graduated and this question may be asked out of ignorance, but how long, more or less, does it take to sequence all the genomics of the virus? After all, it is true that we have… -Could you repeat that? […] -How long does it take to sequence the complete virus genome? Well, nowadays this is done at lightning speed. When I was an intern, many years ago, these sequences were our task. But now, to be honest, that is not competitive, and we get it done elsewhere, either the sequencing department of the university campus, or the samples are sent to Korea, which is a bit far away but their prices are very competitive. They can sequence it and within a week, two weeks at most, we get the results back. Thank you very much. Any more questions? There seem to be no questions online… Well, I wan to ask you some questions. The first one is, when will we have this vaccine? when will we be able to take it? We are looking forward to it. The million-dollar question. We cannot give dates. We have just performed the so-called first trial test, which is to see that it works in an experimental animal mouse model, now we are with hamsters, and next with macaque monkeys. And we are already in contact with companies to make a… […] (-Why is it overlapping?) -Please, go on, we can still hear you… We are still optimising the performance and we have to test it on macaque monkeys first. If everything goes well and a multinational company shows interest in the work, we will go ahead. If not, it will not be possible. But we will not stop working on developing and improving this vaccine… […] -We are struggling to hear you, I wonder if the problem is here or there. Well, let us continue. Maybe by lowering the sound a little it will not reverberate in the room. Well, I do not know… Regarding your comment, Are not the multinationals fighting to get your vaccine on the market? They should. Well, I regret to inform you that, as expected, the multinationals are bringing their vaccine to the market. and trying to stop others from doing so. Because these multinationals' decisions are not taken by scientists, but by finance executives. And they, logically, are looking after their company. They… […] …Laboratories need access to infrastructures to do the experiments on mice and all of this. We have them, fortunately, but even though they are of high quality, they are still limited, which also slows us down a bit. But also because this is a rather original vaccine, and it is a bit complex. Another question, about respiratory tract infections. Mucosal immunity, that protective immunity, why are not more groups looking for mucosal immunity vaccines? Why has such vaccine not been available so far? Well, that is something that only two million people know. Approval of a vaccine is much easier for intramuscular administration than approval for intranasal administration. Out of fear that something that enters through the nose, so close to the brain, might jump the blood-brain barrier. First of all. And because there were some cases before, when they were given intranasally, that produced facial paralysis. A vaccine for the influenza virus. Fortunately, this has now changed dramatically, and since last year the influenza vaccine can be given intranasally, since those problems have been solved, and this will also facilitate the approval of vaccines, such as Pfizer's or Moderna's, which are administered intranasally. This is essential. Because mucosal immunity is only clearly induced when the antigen, the vaccine, is present both in the respiratory tract and lungs. So there is going to be a big turnaround. We know that all the multinationals are now concerned about updating their vaccine to be intranasal, and (this may take a bit longer to come) so that it is a dual vaccine. So that it can combat the Omicron variant and one of the previous variants, so that individuals are protected against any of the variants. My next question is regarding the decision on a new booster dose for the population of the existing vaccines. I do not know if you would rather answer it or pass. What are your thoughts on that? I say yes, we should get the booster vaccine, as soon as possible. In Spain, it seems that immunisation, initially for older people, is planned to start in October. This is very important. But I would recommend that people get vaccinated only if it is a dual vaccine. If it accounts for 2 strains of the virus, the Omicron and one of the other strains. Otherwise it is almost better to wait. But I think that will be the case, because these multinationals already have the dual vaccine ready. Perfect. For those not used to the concept of immunity, this is what is done yearly with the influenza virus vaccine. New strains are introduced to protect against new outbreaks. In this regard, you also mentioned the speed at which the new strains change. The forecast of new strains appearing and the launching into the market, how did you assess that? The new vaccines to be introduced already include some new strains, or else it makes little sense. Usually, for the vaccine manufacturers and for us, in the annual update of the vaccine, the scientific-technical problem is of no great relevance. The control check is harder, checking that the new vaccine, with the new variant, does not cause any side-effects. But this has been the case for many years, for instance with the influenza vaccine. The influenza virus is a highly evolving RNA virus. Every year we can get a different strain and therefore vaccines are updated annually. And that is already happening with coronaviruses as well. An annual update will remain, for the virus has come to stay. And above all, its reappearance intensifies seasonally, with the arrival of autumn and winter. […] One last question. Your experience, your professional career, is an impressive example for them. But I would like to ask… The life of a scientist has changed a lot since you started. I would like you to tell us a bit about what drives a person of your age to keep working and fighting so hard. What drives your passion to keep doing what you do? And, on the other hand, What difficulties do you think young people face when doing research in a country like Spain, with our characteristics? The first thing to say to young people is that research work is fascinating. You need to have a strong vocation because it is hard work. When you are doing research, most of the time you are not successful. Then you have a few months of glory when you get results when you develop something that actually works. But most of the time is spent in the process of finding it. And when you find something it is just stage one, but then you have to move on to the next stage back to perfecting the vaccine or whatever you are working on. It is fascinating. If you take genes out of a virus, you can attenuate it and make vaccines. But you can also do the opposite, you can become a terrorist. Because there are viruses that are not so dangerous, but if you put these virulence genes in them, they kill people. Anyway, people normally make vaccines and things like that, or they look for antivirals. Students need to consider this seriously, they need to set a life project, and follow it. Obviously, adapting it according to the circumstances. But if they lack a project and have not made a decision beforehand nor have they set goals for themselves, I think they will not do the crazy things that some of us older ones have done, such as moving to the United States, leaving to further your studies… That supposes a major financial and personal sacrifice So they must think it through, it is best to know that it is exciting, but certainly not easy. No job is easy, but some are harder than others. There is a question in the room. Yes, I have two questions. Thank you very much for the presentation. I do not know if he can hear me, I think he is talking right now. […] Okay. My first question is: Autogenous vaccines are becoming more and more common nowadays, in the urological field, for example, for urinary tract infections, and these are administered in the mucous membranes of the mouth, not in the respiratory tract. Are there any studies supporting that the effect is the same administered via the oral mucosa and it is also valid? Perhaps that would make people less apprehensive than intranasally. -Could you hear the question? -No, not at all. […] Let me try to relay it to you. Is there an autovaccine, for mucosal immunity that might give rise to less concerns about using it? Is that it? -Yes, just as there are now autovaccines for the oral mucosa, which are tailored to each patient… -Usually, vaccines against viruses… There may of course be some exceptions, but they have to be virus-specific. They are made ad hoc. You have to insert some antigens to induce a specific immune response. So one vaccine works for one virus and not for others. What we have are some substances so-called immunologic adjuvants, that help to promote the immune response and activate it in a generalised way. But this is only relatively effective unless you combine it with the capacity to promote such response against a particular pathogen. That is why all vaccines have, to simplify, two components. The antigen that comes from the virus and an adjuvant that enhances the immune response it produces. Today's vaccines, in general, 99.9% of them, have these two components. -Right, well, the question was, basically, if instead of nasal administration, whether administration in the oral mucosa has been seen to be effective as well. As now there are, for example, vaccines made for urinary tract infections that target the bladder mucosa, but are delivered through the oral mucosa. I seem to be missing the point. Since they are all mucous membranes, is the mouth one equally valid for mucosal immunity? Maybe people would better tolerate that approach than the intranasal one. On account of what you explained earlier, the blood-brain barrier problem. One of the participants is asking if the vaccine administration could be oral instead of intranasal. The vaccine administration. If you think that oral administration would be less problematic than intranasal because of the blood-brain barrier issue. Most likely, yes. There are also oral vaccines being developed for coronaviruses. But bear in mind one of the principles I mentioned earlier, which is old and correct, which is that mucosal immunity is compartmentalised. It is induced in that part of the body in which the mucosa is located. If I immunise my right eye, my right eye is immunised, not the left. The antigen must then be delivered to the respiratory mucosa, where the virus enters. If you ingest something there is always a part of the antigen (especially with sprays) that can reach the enteric tract, but some part reaches the respiratory tract too. It is a combined effort. An exclusively intragastric vaccination would effectively immunise the gastrointestinal tract. But in the vast majority of cases, the virus infects our respiratory tract. So it would be rather a waste of the antigen. To immunise where the virus enters, the respiratory mucosa, we have to administer it there. Alright. My last question is, based on what you said about multinationals being interested What is the way forward if, after so much work, no multinational is interested? What would happen? Is there another way for it to go ahead? Because otherwise it would be such a pity. Well, this is the million-dollar question. If after so much work and success, without a multinational company that could provide the financial support to get to a powerful clinical trial… If that did not work, what would happen to everything you discovered? Well, then we would have created a coronavirus-based vaccine model and we would have developed a technology that would make a very good vaccine that induces sterilising immunity. Time will select the vaccines that will survive. We are in contact with companies facilitated by the Spanish National Research Council. It is true that companies are not foolish, if they see a vaccine that could be very interesting… For the moment, those who already have one have their financial plans, but we are not making any assumptions. We are still working on the vaccine, working on the performance, and we are convinced (this is a very important principle) that the project we are involved in is worthwhile, it is interesting, and it will certainly be implemented in the future. That is all. Do we have any more questions in the room? There are not on the platform either. We are nearing the end. I wanted to ask… Just an estimate. I know it is difficult to say for sure how long it will take. We have seen during the pandemic that vaccines were in the market within months. That was a very exceptional thing. But, more or less, on average, how long does the pre-clinical phase take to get to the clinical phase? Now that we are experimenting with animals, about to test macaques, -How long will it take? -If all goes well, (this said with no strings attached, as we know these things get delayed) we could have it by the end of next year. But everything has to go smoothly. Our vaccine is brand new. New technology, new processes. We have to identify genes, we have to optimise it and we know that this takes work. Nor do we want to create expectations that will not be fulfilled. What we can say is that we are working on something that has already been done, and that is very promising. We believe this is one of the most effective out there. It might be late, but we will have a very solid vaccine. All right. Thank you very much. It was a pleasure to have you with us. We send you lots of energy and support. Good luck and may everything go well. And may you soon be nominated for a Nobel Prize, so that we have another Spanish Nobel. Okay. Thank you all very much. This concludes the session until this afternoon. -Best regards, it has been a pleasure. -Thank you. See you later.