The world has been hit by a new disease caused by the SARS-CoV-2 virus, which spreads from person to person via droplets and can thus be transmitted both through the air and through direct physical contact with infected secretions. The global economic and healthcare systems are under huge strain, and the crisis is threatening the social interactions of billions of people. By Ali Salanti, professor, PhD, Department of Immunology and Microbiology, University of Copenhagen & Jørgen Kjems, professor, PhD, iNANO, Department of Molecular Biology and Genetics, Aarhus University We quickly learned from China that currently available drugs for viral infections have no significant clinical effect on COVID-19. Consequently, there arose an urgent and critical need to develop new forms of therapeutic treatment as well as a prophylactic (protective) vaccine. The Carlsberg Foundation was the first Danish foundation to respond immediately, providing rapid funding for a project to develop both a COVID-19 vaccine and new diagnostic and therapeutic options. There are all manner of challenges connected with making a globally distributed and effective vaccine for COVID-19. Firstly, the vaccine has to induce effective immunity after a maximum of two immunisations, especially in the COVID-19 target group, primarily older people who generally react less efficiently to vaccines. And then it has to be a vaccine that can be mass-produced and mass-distributed, including to parts of the world where vaccine storage at very low temperature cannot be expected. This is a challenge of hitherto unseen dimensions, and it is not particularly encouraging that, since the introduction of the HPV vaccine almost 15 years ago, no new protective vaccines have emerged on the market. STAY CURIOUS: In search of the “Achilles heel” of the Spike-protein The need for new vaccines The recognition that an effective vaccine is needed before society can be fully reopened has globally led to the launch of a large number of vaccine projects based on widely differing technologies. Under normal circumstances, it takes at least 15 years to develop a vaccine, and the University of Copenhagen, by way of example, has been working to develop a malaria vaccine for almost 30 years. So one might wonder whether there is any basis for the global optimism that a COVID-19 vaccine can be developed in just 18 months. The first important point is that, from experience of previous coronavirus epidemics (MERS, SARS), we know precisely where on the virus we can target a vaccine. On the surface of the virus particle, there is a protein complex, called Spike, which binds a receptor protein on lung tissue cells called ACE-2. It is via this protein–protein interaction that coronavirus enters a cell in order to take over the cell’s machinery for protein synthesis and thereby multiply. Experience from SARS clearly shows that if we can block this interaction, we can also block infection – and, in so doing, the disease and its spread. A second point is that, in order to test whether a vaccine works, we need a relatively large number of individuals at high risk of becoming infected. This is obviously difficult for diseases such as HPV-induced cervical cancer or Ebola, where thankfully the frequency of the disease is relatively low, whereas it seems more realistic to recruit 100,000 people for a COVID-19 vaccine trial and expect that a few thousand of them will become infected. Three possible vaccine technologies The first and fastest COVID-19 vaccines currently being tested in humans are based on injecting genetic material from SARSCoV- 2 in the form of either mRNA or DNA. The principle is the same: to get the body itself to produce elements of SARSCoV- 2 Spike protein and thereby induce a virus-blocking antibody response. This principle has shown promising results in animal models, but an effective vaccine for humans has never been produced using this principle. Our vaccine technology stems from discussions about the central characteristics of the few vaccines developed and tested over the past 30 years that have been sufficiently potent and protective to make it onto the market The challenge is that when you inject genetic material and let the body itself produce the vaccine, you lose the opportunity to mix in other ingredients (adjuvants), making the resulting immune response weak and short-lasting. The next wave of vaccines, which are now also entering the first clinical trials in China and the UK, are adenovirus-based vaccines. In this case, you get a virus that is unable to divide to express elements of the SARS-CoV-2 antigen, but previous trials have not shown a particularly sustained response. And finally, we have a third wave of vaccines, the protein-based vaccines, which include the vaccine being developed at the University of Copenhagen. The research team’s vaccine strategy The research team at the University of Copenhagen, led by Professor Ali Salanti, Professor Thor Theander and Associate Professors Morten Nielsen and Adam Sander, has just developed a new and promising vaccine platform that could prove ideal for delivering a COVID-19 vaccine. Our vaccine technology stems from discussions about the central characteristics of the few vaccines developed and tested over the past 30 years that have been sufficiently potent and protective to make it onto the market. The HPV vaccine, which protects against cervical cancer, is a remarkable exception. This vaccine is based on the empty non-infectious shell of HPV virus, and two vaccinations give lifelong protection. The underlying reason why the HPV vaccine is so extremely effective at inducing a sustained and effective immune response can be found in the fact that the vaccine mimics the normal HPV virus, but with the important exception that it does not contain infectious DNA. Professor Ali Salanti in the lab at the University of Copenhagen. From an evolutionary perspective, mammals have evolved to respond quickly and effectively to viral infections, which would otherwise have the potential to quickly wipe out a species. The body recognises patterns of proteins on the surface of a virus, and also how densely packed those proteins are. These are patterns and densities that we never find on our own cells, and the body can therefore quickly muster a specific antiviral response. The empty HPV vaccine mimics these protein patterns and densities, thereby producing a successful vaccine. Inspired by the principle for this vaccine, we came up with a ”superglue” technology that allows us to affix any sort of virus protein to the surface of an empty virus shell, and what is more with the same density and in the same pattern as the virus’s own proteins. The body recognises patterns of proteins on the surface of a virus, and also how densely packed those proteins are The principle has proven highly effective, and among other things we have shown that we can effectively protect mice against influenza. Our technology is thus optimal for responding to a threat such as COVID-19, where the vaccine component is known and published, and where the aim is a vaccine that is able, highly effectively and after just a few immunisations, to induce a powerful immune response even in people with a weakened immune system and older people, who notoriously have difficulty responding to vaccines. The project is thus focused on producing the Spike protein, or elements of the Spike protein, that SARS-CoV-2 uses to penetrate cells. These proteins are then affixed to an empty, harmless virus particle, and here we decided to use a particle that is similar to HPV but easier to produce in bacteria on a large scale. Fig. 1 The virus particle vaccine principle. In ordinary E. coli bacteria, we can produce the empty virus shell in large quantities. Similarly, we can produce SARS-CoV-2 antigens in insect cell factories. Subsequently, using our superglue technology we can affix the SARS-CoV-2 proteins to the surface of the empty virus shell and the result is a virus particle that displays densely packed and well-organized SARS-CoV-2 antigens similar to the natural SARS-CoV-2 virus. The aim is to create an artificial virus particle that displays the SARS-CoV-2 proteins on its surface in a similar manner as the natural virus and, following injection in humans, will be recognised as a very harmful virus and thus trigger a rapid and powerful response – without, of course, major risk of side effects (see fig. 1). So far, we have succeeded in making the two vaccine components – the artificial virus shell and the Spike protein – at a quality and in a quantity that make it plausible to scale up to billions of doses Clinical testing of the new vaccine The vaccine project at the University of Copenhagen is performed in close collaboration with researchers from Statens Serum Institut under Professor Michael Theisen, Expres2ion Biotechnologies, who are working to supply SARSCoV- 2 proteins, Professor Søren Paludan’s team at Aarhus University, who will test the efficacy of the vaccine against SARS-CoV-2 in vitro, and Professor Peter Garred’s team at Copenhagen University Hospital (Rigshospitalet), who will use the proteins that are produced to develop diagnostic assays. So far, we have succeeded in making the two vaccine components – the artificial virus shell and the Spike protein – at a quality and in a quantity that make it plausible to scale up to billions of doses. At Professor Paludans Laboratories at Århus University we have shown that animals immunized with this virus like particle vaccine induces extremely potent SARS-CoV-2 neutralizing antibodies; a response that appears up to 100x more potent than what we have seen with other vaccines in clinical development. University of Copenhagen’s spin-out company AdaptVac are now transferring the method of production to an approved factory The first clinical trials are expected to commence at the end of 2020, where it will be investigated whether the vaccine has any side effects and whether it also induces the right response in humans. Only in the subsequent outcome trials, in which we vaccinate tens of thousands of volunteers over a period of time, will we find out whether the vaccine also protects against COVID-19 infection. The timeframe for these trials naturally depends on the infection pressure during the period, but we expect to be able to complete them in six months. University of Copenhagen and AdaptVac have teamed up with the Danish Vaccine company Bavarian Nordic to support the clinical development beyond the first clinical trials. We will certainly not be the first team to deliver a vaccine to market, but our overall ambition and hope are to be able to develop the best preventive COVID-19 vaccine that will effectively protect immune-suppressed and older people without side effects, and that can be mass-produced and distributed to parts of the world where the cold-storage capacity is uncertain. The need for a diagnostic test that can show whether a person has been infected with SARS-CoV-2 is also growing, but the quality of available commercial kits varies and the supply reliability is low. It is thus obvious that the highquality proteins we produce to develop the vaccine will also be useful in validating an assay of this kind. This work is well underway at Copenhagen University Hospital, in Peter Garred’s research team, and the prototype test has already shown very high specificity and robustness. The hope is to develop a cost-effective test that can also be used longer term in the general population. Developing RNA drugs against SARS-CoV-2 As stated above, developing a vaccine can be like playing roulette. It may take years for us to find the right strategy, and there is a risk that we might never find a vaccine. It therefore makes sense to open up a number of fronts in the fight against the disease and start developing antiviral drugs, and this is precisely what the research team at Aarhus University, led by Jørgen Kjems, has done. Here too it is history that gives hope of success; for example, although many years of intensive research have not delivered an effective vaccine against HIV-1, today there is an effective antiviral treatment that can keep the virus under control and extend the patient’s life considerably (decades). Unlike vaccines, which are administered to protect against contracting a virus, antiviral drugs are typically used to inhibit the virus’s reproduction in the body once a person has been infected. Right now, a number of clinical trials are ongoing for drugs that have previously shown antiviral effects, but so far only marginal effects have been observed in COVID-19 patients. Fig. 2 The principle for developing anti-SARS-CoV-2 drugs. RNA molecules that bind to the Spike protein are selected from a large library of random sequences and then tested for their ability to block the virus’s binding to ACE-2 or prevent protease cleavage of the Spike protein, which is necessary for the virus to enter the cell. In our project, we will attempt to synthesise biomolecules that can prevent the virus from entering our cells by binding to Spike proteins and blocking the interaction with the ACE-2 protein on the surface of human cells. (See fig. 2). The drugs we will use to target the Spike protein consist of nature’s own building blocks, namely RNA molecules. RNA is largely similar to our genetic material, DNA, but whereas DNA has two strands, RNA normally has just one. This means that the base-pairing we know from the double helix in DNA causes the single strand in RNA to fold together in a complex but well-defined structure. Antiviral bodies fighting Corona virus From a very large library of different RNA sequences (typically 1016), we can select those that best are able to bind the Spike protein, thereby isolating the few molecules in the pool that can potentially disrupt the virus’s entry into the cell. These molecules are isolated, propagated on a large scale and tested individually for antiviral activity. In the first instance, we test whether they prevent cells from taking in harmless SARS-CoV-2 mimics, which are designed to expose the Spike protein on their surface in the same way as the real virus. The most effective RNA molecules will then be tested on real SARSCoV- 2 virus particles under controlled laboratory conditions. In an approach that is completely unique to this project, we have developed a method for incorporating other substances, such as amino acids and sugar groups, in the RNA molecules. These hybrid RNA molecules have been proven to bind to proteins with great strength, often more effectively than naturally occurring antibodies. We hope in this way to find candidates that can effectively block the Spike protein’s binding to ACE-2 and thereby prevent the virus’s passage into the cells. What are the benefits of using RNA molecules to combat viral diseases? Vira have a tendency to alter themselves to avoid nature’s protein-based antibodies, but they have never ”seen” our unnatural RNA hybrid molecules. We therefore expect our strategy to give rise to more broad-spectred antiviral medicine that also will have effect against new variants of the virus, which constantly emerge during a pandemic, and against new types of coronavirus that may attack in the future. Another important consideration is that, unlike vaccines, which can take years to produce, the selection of RNA drugs takes place in vitro without the use of animals in just a few weeks. We therefore hope that this method of producing antiviral drugs can potentially be used as a first line of defence, including in the event of future virus attacks. Finally, our RNA molecules can easily be produced in scalable quantities using both chemical and enzymatic methods.