What are coronaviruses, What is Covid-19?

Coronaviruses get their name from their crown-shaped appearance and can cause a variety of diseases, from a mild cold to severe respiratory syndrome (1).

Researchers assume that the coronavirus was transferred from a bat to humans via an intermediate host.
Photo: Pixabay.

Individuals infected with this coronavirus may develop no symptoms or only mild symptoms typical of influenza or colds (fever, headache, chills, cough, cold, diarrhea, sore throat). However, severe courses of pneumonia up to acute lung failure with fatal consequences are also possible (1, 3). The virus can also cause neurological symptoms such as a loss of sense of smell and taste, headaches, fatigue, nausea, vomiting or a disturbance of consciousness (4).

Since the virus is highly contagious, scientists and politicians are currently making every effort to slow down the increase in new infections as much as possible by setting targets for contact reduction (5).

Recent findings suggest that the virus can be transmitted via surfaces (6), but above all via aerosols in the air, i.e. the air we breathe (7,8). The aim is to be able to provide sufficient medical treatment capacity for serious cases. However, the key is to develop a vaccine.

It is known that the coronavirus enters the cells via a cell receptor called ACE2 (receptor angiotensin-converting enzymes II)  during an infection (9,10). In principle, the receptor-angiotensin-converting enzyme II catalyzes the cleavage of peptide bindings but is also a transmembrane protein and in this function, it is a target for coronaviruses. The enzyme is not only found in lung cells, but also in the heart, kidney, endothelium and gastrointestinal tract (11,12). It plays an important role in the cardiovascular and immune systems and is involved, among other things, in heart function and in the development of high blood pressure as well as diabetes mellitus (13). There might be genetic differences between people in the construction of this receptor, which means that even younger people can be affected by serious lung complications. Studies are currently underway (14).

There is currently no vaccine available and many researchers around the world are working to develop such one. At least 68 vaccine projects have already started (2) and a total of more than 80 clinical trials (15) are currently in progress.

What is the meaning of "shortened development time"?

However, a vaccine for the majority of the population will take some time and, according to experts, be available in 12-18 months earliest. The development process is already being shortened through the use of genetically modified virus particles (vaccine vectors, 16) and vaccine carrier systems, of which compatibility studies are already available.

The first step in vaccine design is to identify the antigens to be used in a vaccine. These antigens contained in the vaccine are intended to activate the adaptive immune system, i.e. the antibody-producing B cells, T helper cells and also the cytotoxic T cells (17). Through these antibodies, the virus can then be eliminated by the cells of the innate immune system, the so-called monocytes by phagocytosis. Depending on the strategy, the scientists will concentrate on an active or a passive vaccination, the development of which takes different time periods.

There are no simple solutions: According to Prof. Christian Drosten, a virologist at the Charité University Hospital of Berlin (18), the coronavirus requires special attention. The antigenic domain of the virus, which can be attacked (neutralized by antibodies), only appears in certain developmental stages of the virus infection.
The worst-case scenario is, that there would be not the wanted antibodies available after vaccination against antigens of other virus stages, but non-specific antibodies. These would not allow the monocytes to eliminate the infection completely. Non-specific antibodies would form a connection with the virus allowing the monocytes to phagocytose, however, the virus could remain active within the monocytes. The result could be an "unbalanced" immune response with a deterioration of the disease.

Problems due to species differences

An "animal model" that reflects the infection of humans is currently urgently searched for all over the world. According to the initial results of the Friedrich-Loeffler-Institute, ferrets might be shortlisted as model animals for human infection with the novel coronavirus and for testing vaccines or drugs (19). In recent years, almost two-thirds of all experiments on ferrets have been carried out to study human infectious diseases. The ferret is considered worldwide as an alternative test system for non-human primates (20). But many researchers apparently also want to study the coronavirus with genetically manipulated "animal models".

The Jackson Laboratory already provides genetically manipulated mice. By nature, the mouse is not suitable because of its differences in the ACEII protein. However, based on earlier research on the ACEII protein, the Jackson Laboratory has developed the so-called transgenic mouse K18-hACE2. The genetic information of the human ACEII protein was incorporated into the mouse's own genetic information. Coronavirus infection is very painful for the mouse: Among other things, it causes an upregulation of inflammatory messengers in the lungs and brain. Three to five days after infection, breathing becomes difficult and lethargic. The animals die within seven days (21).

The immune system is a sensitive "organ": Even different strains of mice and rats show variability between the extent of infarction or stroke, which could be due to differences in the immune system (22). Gender-specific differences in the immune system in both mammals and humans should not be neglected. In mice, for example, gender differences were found in untreated and interferon-induced immune cell types (23), which cannot simply be eliminated by genetic modification. Established mouse inbred strains vary in their immune response pattern as a result of genetic mutations and polymorphisms resulting from intentional selection for research (24).

Animal experiments conducted in parallel with clinical studies

The fact that many researchers have started their first clinical trials without animal experiments is presumably related to the fact that safety information is already available for the drugs in which great hopes are placed. This is because they were developed for a different purpose and, in this context, have already been extensively tested for their efficacy, tolerability respectively safety, e.g. Resochin (25), a drug developed in the 1930s for malaria prophylaxis, the antibody-drug Trimodulin from Biotest (26, 27) or Remdesivir (28), which was originally developed against Ebola. The procedure is not uncontroversial and its effectiveness is not always clearly proven (29, 30). For example, Remdesivir is only approved in certain serious cases when patients already need artificial respiration (31).
Moreover, this does not mean that animal experiments are not carried out in parallel or are postponed to a later stage. Animal experiments cannot yet be completely replaced, but they also show differences in species that should not be underestimated, which is why more and more researchers are turning to new, human-specific methods.

New human-specific techniques provide a major contribution to the clarification of infection mechanisms

Here, the achievements of in vitro methods and their great importance in the elucidation of infection mechanisms should be emphasized. Some examples are:

Cell lines and organoids
A German research team involving infection biologists from the German Primate Centre, scientists from the Charité University Hospital Berlin, the Hannover Veterinary University Foundation, the Berufsgenossenschaftliche Unfallklinik Murnau, the Ludwig Maximilian University of Munich, the Robert Koch Institute and the German Centre for Infection Research has used cell cultures to find an important protein that could offer a possible point of attack against the penetration of the virus into the lung cells (9). For their investigations, the team used a large number of cell lines and primary cells and infected them with the virus. Afterward, they examined, among other things, cell viability, the absorption mechanism of the viruses into the cells and coronavirus expression. They identified a cellular protein called TMPRSS2, which is important for the penetration of the coronavirus into cells. It is a protease that has an important function in binding the virus to the ACEII receptor (see above), through which the virus can enter the cell and multiply. Their findings have provided scientists with a starting point for combating the virus.

Scientists at the Max Planck Institute for Infection Biology in Berlin, led by Director Thomas F. Meyer, have developed a model of the human lung epithelium, with which they can investigate active substances against the corona virus Sars-CoV-2 (32). The organ-like cultures (organoids) developed from human epithelial cells of the lower respiratory tract are developed into surface cells of the alveoli, (alveolar epithelial cells). The so-called type 2 pneumocytes produce vital proteins that reduce the surface tension of the alveoli preventing them from collapsing. Numerous pathogens, including the Sars-CoV-2 virus, attack type 2 pneumocytes. With the lung organoids, the researchers are able to test the effect of antiviral drugs and vaccines against the new virus (32).

Pulmonary alveoli-on-a-Chip
The Jena-based start-up company Dynamic42 GmbH, together with other partners of the InfectoGnostics research cluster - the Center for Sepsis Care and Control (CSCC), the Leibniz Institute for Natural Product Research and Infection Biology Hans Knöll Institute (HKI), the University Hospital Jena (UKJ) and the University of Jena - has developed an artificial model of a pulmonary alveolus ("Alveolus-on-a-Chip"), which enables direct work with human cells. The artificial pulmonary alveolus on a microfluidic chip provides results that are closer to the human situation than animal experiments. Using their model, the researchers were able to show that the simultaneous infection of viruses and bacteria (influenza with bacterial superinfection by staphylococci) damages the protective inner layer of blood vessels (endothelium). As a result, pathogens and their toxic metabolites spread faster in the lungs and lead to sometimes severe pneumonia (33, 34).

An international team of researchers involving the Institute of Molecular Biotechnology (IMBA) in Vienna uses organoids to show how SARS-Cov-2 affects blood vessels and kidneys and how the viral attack can be slowed down by the active substance hrACE2.  It was shown that the virus can directly infect the organoids and multiply in these tissues. This provides important information on the development of the disease and the fact that severe cases of COVID-19 are associated with multi-organ failure and signs of cardiovascular damage (35).

There are also considerations to use digital models in the future that work with patient data and dispense with the intermediate step of animal testing. These models have the potential to avoid at least part of the experiments using animals (36).

Human relevant research must be the future

The selection of examples shows the potential of new, human-specific methods, which are increasingly available for important questions producing reliable results.  As a member state of the European Union, the National Committee of the Netherlands (NCad) was already convinced in its exit scenario in 2016 that the use of laboratory animals for the production and release of biological products, such as vaccines, can be phased out gradually by 2025 - while maintaining the safety level. However, this does not apply to regulatory pre-clinical research, which will take longer. Vaccines that have already been tested for market acceptance for registration and for which batches are already produced in a uniform manner would not need to be retested in animal experiments for release (37).

Literature:

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