Science in Action: Drugs and Vaccines for the New Coronavirus Covid-19

05/11/2020

The way science has dealt with the pandemic is an example of its effectiveness and versatility. Within days of the recognition of an outbreak of atypical pneumonia in China caused by a virus, the virus could be isolated and the sequence of its genetic material unraveled, information that was immediately deposited in international databases so that anyone could have access to it. 

With this data, it was possible to establish with certainty that a new coronavirus was responsible for the disease, and in just a few weeks it was determined what its components are and the role they play in its life cycle.

Of some of them, the most relevant ones, it was possible to establish their three-dimensional structure by crystallography techniques and by computational protein modeling techniques. This is essential for the development of drugs and vaccines against the new coronavirus. Also, in record time, it was possible to establish not only how the virus spreads, but also the mechanism by which it replicates inside our cells and the evolutionary course it is taking.

The relatives of the new coronavirus

The genetic material of the new coronavirus (SARS-CoV-2) is a huge RNA strand of nearly 30,000 nucleotides, one of the largest known genomes of RNA viruses. The sequence of the genome itself is invaluable, as it allows us to first recognize the closest relatives of the virus. Secondly, it demolishes all those conspiratorial ideas that this virus had escaped from a laboratory and, finally, it determines where it came from and what its structural components are.

When the genome sequence of this new virus was compared against the sequences of other viruses deposited in databases, it was determined that the cause of COVID-19 (the disease that produces SARS-CoV-2) was a new beta-coronavirus, related to two others of the same family that caused serious epidemics in the recent past: the virus that caused SARS (Severe Acute Respiratory Syndrome) and the MERS virus (Middle East Respiratory Syndrome).

It was also discovered that it is very similar, in more than 96%, to the genome of other beta-coronaviruses that have been isolated from bats, their natural reservoir, and to some beta-coronaviruses isolated from pangolins; mammals that are traded illegally in China. These observations allow us to establish that the origin of SARS-CoV-2 occurred first when a coronavirus originally found in bats acquired the ability to infect other types of animals, possibly pangolins, and then some additional mutations allowed it to infect humans.

Portrait of SARS-CoV-2

The analysis of the genome indicates that the genome of the new coronavirus has the instructions to produce 20 proteins, but only four of them are present in the mature virus (virion) and therefore they are called structural proteins. The other 16 are important in the replication of the viral genome and in the formation of new viral particles.

The first of the structural proteins, the S (for a spike, spicule), is what gives the virus its peculiar crown shape when observed under the electron microscope and through it the virus recognizes which cells can infect and initiate the invasion process. This initial recognition occurs when the S protein interacts with the ACE2 (Angiotensin-converting enzyme 2) protein, a receptor found on the surface of the cells in some of our tissues. ACE2 is particularly abundant in the epithelial cells lining the nose, bronchioles, and pulmonary alveoli.

The location of ACE2 explains the respiratory symptoms that characterize COVID-19 and why we can easily become infected if we come in contact with the droplets and aerosols (smaller droplets) that a sick person produces when sneezing, coughing, talking, or singing. This makes it clear that wearing a mask is very important to protect ourselves and others. I want to emphasize that ACE2 is a protein whose original function is not to capture the coronavirus, but is related to the regulation of blood pressure.

For the S-protein of the virus to interact with ACE2 it has to be processed by an enzyme that is found on the surface of many of our cells and that cuts it into two segments: only in this way can the infection process be initiated. The M protein is the most abundant in the virus and is inserted in the membrane that covers it. It is thought that the M protein contributes to the spherical shape of the virus. On the other hand, the E protein (envelope) is found in low quantities, but it is very important for the virus to assemble correctly and then be released from the infected cells. Finally, the N (nucleocapsid) protein has a section that is embedded in the membrane of the virus and another that "peeks out" from the inside. The N protein makes contact with the viral genome and organizes it to be arranged in an orderly manner within the viral particle. As it is on the surface of the virus, the four structural proteins are the elements that our immune system first detects and indicate that it must produce antibodies against them.

An essential tool for the adequate management of this and other pandemics is an agile and precise diagnosis to identify infected people, to offer them adequate care, and to isolate them the necessary time to avoid that they infect other people. This is especially relevant in phase 1 of an epidemiological contingency, that is, when the first epidemic outbreaks occur in a given country due to people who became infected abroad and return sick (imported cases). 

Properly tracing these first chains of contagion is very useful for containing an epidemic. This strategy was especially useful in efficiently controlling SARS and MERS, diseases that were much more lethal (percentage of infected people who died) than is now the case with COVID-19. Infected people quickly became ill and were stranded in their homes.

In addition, only those who showed symptoms of the disease were contagious. The most serious problem we have now in controlling the pandemic is that there are many infected people who have no symptoms, but are carriers of the virus. This makes it very difficult to trace all the chains of infection. 

And to make things even more difficult, the symptoms that characterize COVID-19, such as fever, headache, dry cough, tiredness, etc. are also characteristic of other viral diseases. So an accurate diagnosis is essential not only to combat the pandemic, but to channel human and economic resources in an appropriate manner.

Tests to detect the virus

The diagnostic systems we have to detect SARS-CoV-2 are all based on the amplification of a small piece of its genetic material, exclusive of this virus, which is not present in other coronaviruses circulating in the human population (alpha-coronavirus) and are responsible for a considerable percentage of the colds we suffer from year after year. 

The diagnosis only needs a sample of nasopharyngeal secretion of the patient suspected of being infected, taken with a swab, which isolates the genetic material of the virus if it is present. In general, the results are obtained within a few hours, but if the laboratories that perform the test are saturated, the results will take a few days.

We can also identify those who were sick, even if they have not presented symptoms, thanks to the fact that cured people, convalescent people, or those who have at least seven days of being infected have high levels of antibodies against the virus in their blood. Rapid tests are based on detecting these antibodies with a device similar to pregnancy tests. This process is so efficient that results can be obtained in a few minutes.

People with high levels of antibodies to the virus can no longer get sick, at least as long as those levels remain high. In the case of SARS and MERS, antibodies could be detected for up to a year after infection. We still don't know what happens with COVID-19, but it could be less time.

People who tested positive on the rapid test and have stopped having symptoms for a few days are no longer contagious; they no longer need to be quarantined. If rapid testing is applied massively (which is not the case in Mexico) health authorities can identify infected people who do not have symptoms and thus cut the chains of transmission. In addition, they can determine with greater certainty who has come into contact with the virus and assess whether or not we are emerging from the pandemic.

Monitoring the course of the epidemic by counting only suspicious cases will likely mean underestimating it, especially where there is hospital overcrowding. Rapid testing could also play a role in determining in a workplace, for example, whether employees can safely enter and resume their duties.

Therapies and medications

One of the problems with COVID-19 is the variety of its symptoms and their intensity. Some people show no or only mild symptoms, while others die in intensive care units. There are factors that help COVID-19 become severe, such as age, sex (men die significantly more than women), and so-called comorbidities, i.e. those who already suffer from other conditions such as diabetes, obesity, hypertension, cancer, or heart problems become more seriously ill.

There is another element to take into account: many of the patients who become seriously ill do so not because the virus is winning the battle, but because their immune system reacts so violently that it attacks their own tissues. This phenomenon is known as a cytokine storm, since these are the molecules that we produce precisely to modulate the immune response; if they are produced in excess they can lead us to the grave.

Before starting to talk about medications, it must be clear that to develop a new molecule with therapeutic value, approximately 10 years of research and development are needed; this is the time it takes to ensure that it really works, that it does not have harmful side effects and that it has been approved by the corresponding health authorities, such as the Federal Commission for Protection against Sanitary Risks in the country.

The cost of developing molecules with medical value has been calculated at about 800 million dollars. So starting from scratch is not the best strategy. What is being done is to test already known and proven drugs for other diseases or viral infections, which could potentially have an effect against the new coronavirus. If a molecule is found that has even a partial action, it can be chemically modified to enhance that action.

Current strategies to medically combat COVID-19 can be roughly classified into two groups. The first is to find drugs that prevent the virus from infecting us or blocking its life cycle. Many of them seek to prevent the virus from sticking and invading our cells, either by blocking the ACE2 receptor or by interrupting the function of the structural S protein of the virus. One of the ways that are being experimented with is to trick the virus with soluble ACE2 protein or incorporated in nanoparticles. If there is an excess of ACE2, the virus will find these molecular lures and thus be prevented from contacting the ACE2 in our cells. Experiments were done on mice and some patients are promising.

Drugs are also being tested against the AIDS virus that inhibits the enzyme that cuts the S-protein into two segments, preventing it from being activated and the virus from entering our cells. Another approach is to test drugs that inhibit the enzymes involved in virus replication that have worked successfully in infections caused by other RNA viruses.

In the same line, there are also drugs that are known as nucleotide analogs, designed to make the replication system of a virus go wrong and generate abnormal and non-functional genomes. However, the therapy that promises to be successful in the short term is to use the blood serum of convalescent patients, with high levels of antibodies against the virus. This serum, given intravenously, would allow the patient's body to use these antibodies to fight the virus without waiting for the immune response itself.

The second group of strategies uses drugs that prevent exacerbated inflammation and cytokine storm. One of the drugs being tested is interferon-beta, a cytokine that is useful in the treatment of multiple sclerosis and also serves to combat the side effects of some infections. Pilot tests show that it may help in coping with COVID-19. There are also antibodies produced in the laboratory, such as Tocilizumab, that block the action of one of the cytokines that is critical for the cytokine storm to occur.

Dexamethasone, a drug commonly used in the treatment of arthritis and certain allergies, has recently received a lot of attention in the press; there is evidence that it significantly helps people with COVID-19 who are severe, but not those with minor symptoms. As for hydroxychloroquine, a cheap and effective medicine against malaria and which was thought to be useful against the new coronavirus, it has been found not to work and also causes higher mortality in patients with COVID-19.

Vaccines

Many governments and some pharmaceutical companies are developing various types of SARS-CoV-2 vaccines. An account of the strategies being followed to achieve this appeared in Nature magazine on April 28th of this year. What a vaccine does is trick the body into mounting an efficient defense response without getting sick. It is easy to say, but complicated to do so, since it must be experimentally determined that the vaccine is capable of inducing antibodies that neutralize the virus and that it does not cause significant side effects.

All new vaccines and medicines have to be clinically evaluated in three phases. The first one is with healthy volunteers to evaluate if the vaccine really awakens the immune response. The second phase is done with more people and the third phase usually includes thousands of individuals; the objective is to determine the effectiveness and safety of the vaccine.

There are already eight vaccines that are in phase III of evaluation. The most advanced is that of the Modern biotechnology company, which is testing it on 30,000 people. It is closely followed by vaccines from the German company Pfizer-BioN- Tech, the Chinese company Sinopharm and the University of Oxford in collaboration with the company AstraZeneca.

On 11 August, Russia announced that it had already approved a vaccine, but it has only been tested in 76 people and has not completed phase III. Unfortunately, it is not enough to have a good vaccine, it must also be manufactured and distributed and this is a titanic undertaking. In the best case, the vaccine will be ready and accessible in several months, so the best way to get rid of COVID-19 is to avoid getting infected by applying the recommended sanitary measures. Take care!

Molecular diagnostic tests

Molecular tests such as RT-PCR tests detect the genetic material of the virus.

Sampling

A swab is inserted into the nose (or throat) to absorb the secretions.

Transfer of the sample

The sample is kept at 2-8 °C for up to 72 hours during transfer to the laboratory.

RNA extraction

Purified RNA is extracted from the deactivated virus.

Transcription and amplification (RT-qPCR)

One or more controlled heating and cooling cycles are performed to convert the purified virus RNA into DNA and then make millions of copies of the DNA.

Result

When DNA binds to specific probes, fluorescence (light) is produced that the machine can see; the test result will be "positive" for SARS-CoV-2 infection if it crosses the threshold within 40 cycles.

Rapid Serological Antibody Test

Sample preparation

A drop of serum or blood is placed in the sample well; a saline phosphate buffer is added.

Antibody-antigen recognition

Antibodies specific to SARS-CoV-2 in the sample will bind to the SARS-CoV-2 antigen conjugates in the pad (which also contains colloidal gold and rabbit gold IgG conjugates) forming an antigen-antibody complex.

Detection of SARS-CoV-2 antibodies

The sample migrates to the test band and the antibody-antigen complexes bind to the anti-human IgG/IgM antibodies.

Control antibody detection

The results appear after 2-10 min. Positive: 2 strips; one on the test strip and one on the control strip. Negative: 1 strip on the control strip.

Interpretation of the result

The test contains an internal control that must display a color stripe regardless of the test band. The gold rabbit antibody conjugate (from the pad) binds to anti-rabbit IgG antibodies.

To develop a new molecule with therapeutic value requires approximately 10 years of research with an estimated cost of $800 million.

In essence, there are 2 strategies:

Drugs that prevent the virus from infecting us or block its life cycle.

Drugs that prevent exacerbated inflammation and cytokine storm.

There are over 176 vaccines in development, several of which are already being tested in humans.

Preclinical Phase 1 vaccine - safety testing and dosing in a small number of people.

Preclinical Phase 2 vaccine - expanded safety testing in hundreds of people of different ages.

Preclinical Phase 3 Vaccine - large scale efficacy testing. Why do we need a good diagnosis?

Approved vaccines.

By Miguel Ángel Cevallos, a frequent collaborator of ¿Cómo ves?, he is a doctor in basic biomedical research and a specialist in bacterial molecular genetics. He works at the Center of Genomic Sciences of the UNAM.