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SARS-CoV-2: The COVID-19 Pandemic [Part Two]

Editor: This article is the second of a two-part series. Check out Part One here.


The first article on COVID-19 looked at the biology of SARS-CoV-2 on a phylogenetic, structural and genomic level.


If you haven’t read it yet, be sure to check it out first, because in this article we will be applying the knowledge by looking at the various types of SARS-CoV-2 diagnostic tests and treatments as well as the different categories of vaccines that are currently being developed.


Controlling the virus

In response to any infectious disease outbreak, strategies must be implemented to limit the spread of the pathogenic agent.

For COVID-19, steps should be taken to prevent the transmission of the virus. These include hygiene measures such as handwashing/sanitising and regular cleaning of surfaces, social distancing and utilising PPE (personal protection equipment). Travellers arriving from countries with high infection rates should isolate, as should those displaying COVID-19 symptoms. These measures are particularly important given the high transmissibility of SARS-CoV-2, as well as the fact that asymptomatic people are capable of infecting others.

Furthermore, once low levels of transmission have been reached, it is important that they are maintained. Alongside the above measures to reduce the risk of exposure, functioning public health systems are necessary to quickly detect new suspected cases or clusters, test and isolate confirmed ones, followed by tracing and quarantining close contacts.


Testing

There are three main categories of testing for SARS-CoV-2, each with their own benefits and drawbacks:

1. Testing for RNA

An RT-PCR (reverse transcription-polymerase chain reaction) test was rapidly developed following the moment when SARS-CoV-2 genome was published in early January. The test (shown in Figure 1a) involves amplification of viral nucleic acid (if present) in nasopharyngeal samples, which can then be detected, and a positive test result returned.

This nucleic acid test is highly sensitive (meaning that it can distinguish between other coronaviruses and common viruses responsible for colds) and specific. Sensitivity refers to the test’s ability to correctly identify those with the disease whilst specificity is the ability to correctly identify people without the disease.


The main disadvantages of RT-PCR testing are that it is high cost and relatively time-consuming, although hundreds of samples can potentially be tested in parallel so these issues aren’t very significant. Another problem with testing for the presence of viral RNA is that it has been found that SARS-CoV-2 RNA can persist in individuals for weeks or months after infection, even if they aren’t infectious. Essentially, this assay fails to distinguish between people who can and can’t infect others.


2. Antigen testing

Antigen testing looks for viral proteins (called antigens) in samples, such as the SARS-COV-2 nucleoprotein. These tests are beneficial as they identify individuals with a high viral load (meaning that they have loads of viral particles their body) and therefore are most likely to be infectious. Like the polymerase chain reaction (PCR), these tests can be carried out in labs.

In contrast, rapid diagnostic tests (RDTs) are also being developed to detect viral antigens, such as the spike or membrane protein, outside of the lab. These tests are normally lateral flow immunoassays (like those used for pregnancy tests) which provide results in around 30 minutes. An illustration of this test is shown in Fig.1b.

Whilst these point-of-care tests (i.e. carried out at/near the site of the patient; outside of the lab) would be extremely useful for increasing the frequency of tests that are being carried out to help control the pandemic, a significant issue is the low sensitivity and specificity of these tests. Unlike PCR, there is no amplification step in RDTs, so false negative results will occur at a higher rate (meaning low sensitivity) - this is where someone infected with the virus is incorrectly diagnosed as not having it.


Meanwhile, a relatively high proportion of results will be false positives (i.e. where someone is identified as infected when they aren’t) due to the low specificity of the test. This is because the test may detect antigens from coronaviruses other than SARS-CoV-2, such as seasonal hCOVs (human coronaviruses) responsible for colds.


3. Antibody testing

Detecting the presence of antibodies specific to SARS-CoV-2 indicate whether someone has been infected and could potentially tell us the degree of immunity they have against future infections. Such tests could also help study the true extent of the virus’s spread in a population, as it can identify individuals who were asymptomatically infected. Lateral flow immunoassays can be used when testing for antibodies or antigens.


Antibody testing also has its limitations. Firstly, it is not yet known whether antibodies confer immunity against further infections, or how long antibodies and immunity lasts for. Secondly, we don’t know whether the anti-SARS-CoV-2 antibodies are neutralising antibodies’ (i.e. capable of blocking viral infection) and therefore provide immunity. Finally, following infection, it can take weeks for seroconversion to occur. Seroconversion is the point in viral infection when antibodies against the virus are produced. If someone is in this ‘window period’ - where they are infected but don’t have detectable antibodies - they will receive a false negative result. For this reason, antibody testing is generally used to determine if an individual has previously had COVID-19.

Figure 1: (a) The process of a polymerase chain reaction (PCR) test for SARS-CoV-2. This figure is adapted from American Society For Microbiology (2020)’s COVID-19 Testing FAQs and created using BioRender. (b) Diagram showing a lateral flow immunoassay for detection of the SARS-CoV-2 spike (S) protein. The same concept applies for different antigens, such as the SARS-CoV-2 membrane protein, and for antibody testing. The process first begins with (1) the sample is added to the test device, with buffer to help the sample flow. This is followed by (2) the samples flowing and eventually reaching the conjugation pad. This pad contains antibodies specific to the SARS-CoV-2 S antigen (anti-S antibodies) which are conjugated to gold nanoparticles. Spike proteins in the sample will bind to the antibody-nanoparticle conjugate to form a complex. Moving forward, (3) the complex moves to the nitrocellulose membrane which has the test and control lines. The test line contains immobilised antibodies specific to the S protein, whereby these antibodies can only bind to the S + anti-S antibody + nanoparticle complex to give a coloured line. They won’t bind if the S protein is absent; in this case, no coloured line is produced. (4) The next line is the control line. This contains immobilised antibodies specific to the unbound conjugate (anti-S-antibody + gold nanoparticle). The unbound conjugate will bind to give a coloured control line, regardless of whether the test result is positive or negative. The control line confirms that the test is functioning. The figure was adapted from Generon, SARS-CoV-2 (Covid-19): Diagnosis by IgG/IgM Rapid Test and created using BioRender.


Treatments

There is currently no specific treatment for COVID-19, however schemes such as WHO’s “Solidarity” clinical trial and the UK’s RECOVERY (Randomised Evaluation of COVID-19 Therapy) are investigating the effectiveness of various drugs against the disease.

The main focus has been on drugs which have the potential to reduce viral load and mortality and improve clinical outcomes. Some COVID-19 patients, particularly those with co-morbidities, will suffer from complications and require hospitalisation where these can be managed. These complications include pneumonia, ARDS, kidney and liver injury, secondary bacterial infection, sepsis, and cardiovascular problems.

The three most promising types of treatment are:


1. Antiviral therapies

These will interfere with viral replication and infection, with the aim of reducing viral load and infectivity of COVID-19 patients.

Remdesivir is an anti-viral drug (initially discovered as an anti-Ebola drug) which reduces the recovery time of people infected with SARS-CoV-2 and has been approved for use on severely ill patients in several countries. It is a nucleotide analogue – meaning that it competes with RNA nucleotides for incorporation by the virus’ RNA-dependent RNA polymerase into new RNA copies. However, once incorporated, it acts as a chain terminator and prevents the RNA molecule being extended any further, thus preventing genome replication.


2. Blocking the cytokine storm

We know that excessive inflammation induced by a ‘cytokine storm’ (that is part of the host immune response) is responsible for some COVID-19 symptoms, so preventing the overactivity of the immune system could be a useful therapy for patients.

Dexamethasone, a steroid drug, dampens immune activity to prevent the hyperinflammation. In a large UK trial, COVID-19 patients on ventilators were given a placebo or dexamethasone. 41% of those who received the placebos died, whilst only 29% of those who received the steroid died. Dexamethasone is recommended for severely ill patients and is the first drug capable of reducing the death rate (remdesivir shortens recovery time but doesn’t lower mortality). Because it reduces immune activity, the drug isn’t administered to infected individuals outside of hospital.

Another way to block the cytokine storm is by inhibiting one (or more) of the signalling molecules involved in the hyperinflammation. Studies looking at the pathogenesis of SARS-CoV-2 pneumonia found several pro-inflammatory cytokines which had been upregulated (i.e. their expression has been increased), and therefore are likely to be responsible for the inflammatory cascade. One of these cytokines is IL-6 (interleukin-6).

Monoclonal antibodies (antibodies with a single specificity) which block the receptor for IL-6 could be another therapeutic strategy to improve patient prognosis. Such drugs are currently used to treat some autoimmune diseases and are undergoing clinical trials for use in coronavirus patients.


3. Neutralising antibodies (from convalescent plasma or monoclonal antibodies)

Convalescent plasma is plasma (the liquid component of blood; without cells) from people recovering or have recovered from infection. They are likely to have antibodies specific to the causative pathogen. These antibodies could then be purified and used to provide others with passive immunity (i.e. immunity provided by the transfer of antibodies) by neutralising viral particles.

This therapy was effective in SARS and MERS patients and was used in the 1918 Spanish Flu pandemic, so there is hope that it could be successful in treating COVID-19 patients. This has been supported by small trials which suggest that convalescent plasma can reduce chances of death and recovery times. The US FDA (Food and Drug Administration) authorised it on the 23rd of August for emergency use in hospital patients, however the lack of large, randomised trials means that the efficacy of convalescent plasma therapy cannot yet be confirmed.


Monoclonal antibodies (mAbs) can be produced commercially using cells or by injecting mice with the antigen specific to the desired antibody. Several companies have developed mAbs with the potential for treating COVID-19 patients. When President Donald Trump was infected with SARS-CoV-2, he received an experimental mAb treatment. This particular treatment, known as REGN-COV2, was developed by Regeneron Pharmaceuticals and is a cocktail of two mAbs, both of which bind to the virus’s spike protein to neutralise it. Such mAb treatments are expensive to develop and produce, and would be reserved for high-risk, severely ill patients. The antibody cocktail is currently undergoing clinical trials in the UK.

Vaccines

Despite developed measures to limit the spread of the pandemic as well as improve the treatment of COVID-19 patients, politicians tell us that we will only be able to return to normal when a preventative vaccine is developed.


We have seen with many other deadly viruses, such as smallpox and polio, that global vaccination programmes are essential for controlling epidemics and can even pave the way for disease eradication. As soon as the SARS-CoV-2 pandemic began, the race to find a vaccine commenced. Progress in this field has been made at record speed, particularly with the help of data from SARS-CoV and MERS-CoV vaccine development.

Despite this, there are numerous scientific and logistical numerous barriers to overcome. How long will vaccine-induced immunity last? Will the vaccine protect from infection by the virus or just reduce the severity of the disease? Can enough vaccine be made to meet global demand?

There are over 180 vaccine candidates being developed, based on various mechanisms of action. Some of these mechanisms are more traditional, whilst others have never before been licensed.

Let’s take a look at some of the types of vaccines being explored. The categories are illustrated in Figure 2.


Involves using a whole, killed virus which is no longer capable of infecting cells or replicating. However, it is still a foreign pathogen and is therefore immunogenic (stimulates an immune response). The main advantage of this method is that the immune system can target the S, E, M and N proteins.

This strategy is used successfully in polio and influenza vaccines.


2. Live attenuated vaccines

Based on viruses which have been weakened and therefore don’t replicate efficiently in humans. The virus doesn’t cause disease but still stimulates the immune system, so the resulting immune response is very similar to that of a natural infection.

Live attenuated vaccines have been successful in the past, particularly as replication of the virus means that the immune system is exposed to structural and non-structural viral antigen. A significant disadvantage of this method is that there is a risk of the attenuated virus reverting to the wild type version - this occured with the oral polio vaccine and is responsible for vaccine-derived cases of polio.


3. Recombinant viral-vectored vaccine

Involves a replication-deficient or attenuated viral vector which has been genetically modified to express the SARS-CoV-2 S protein. The virus is very effective at stimulating the immune system, allowing an antibody-mediated and cell-mediated immune response to be mounted against the S antigen. This method is also advantageous as it doesn’t require handling of the live SARS-CoV-2 virus.

The AstraZeneca vaccine at the University of Oxford is based on this principle, whereby they are using the non-replicating ChAdOx1 vector (an attenuated chimpanzee-infecting adenovirus) which expresses the SARS-CoV-2 S protein. At the point of writing, the vaccine has entered Phase III of clinical trials.


Contain viral antigens, which are produced as recombinant proteins or synthetic peptides. A recombinant protein is produced from recombinant DNA - this is foreign DNA which is artificially introduced into a host cell which expresses the genes. Synthetic peptide vaccines are made by identifying the sequence of amino acids which induces immunity and producing artificial (i.e. synthetic) peptides from the sequence to use in a vaccine.

This method is useful if a response against a single antigen can provide immunity. For SARS-CoV-2, this could be one of its structural proteins. Like the recombinant viral-vectored vaccine, this vaccine has the benefit of not needing the live and infectious virus. This means that they are safer to produce and don’t carry the risk of reversion.


Use plasmid DNA or RNA which carries the gene (S gene) encoding a viral antigen (S protein) as well as a mammalian promoter for expression of the gene. This means that when the vaccine is administered, the gene will be transcribed and translated into the S protein, which will induce a cell-mediated and antibody-mediated immune response. The main appeal of this vaccine is its relative safety and ease of production.


The Moderna Therapeutics mRNA vaccine contains mRNA, encoding the SARS-CoV-2 S protein, enclosed in a lipid coat to allow the nucleic acid to enter the recipient’s cell. The mRNA is then translated into the immunogenic viral antigens. The vaccine has entered Phase III clinical trials in the United States at the point of writing.

Meanwhile, Imperial College London is carrying out clinical trials of a self-replicating mRNA vaccine. Self-replicating mRNA, in comparison to the non-replicating mRNA in the Moderna vaccine, contains the S gene as well as additional genes encoding RNA replication machinery. The benefit of self-replication is that it enables increased antigen production, even from only a small vaccine dose.

Figure 2: A summary of the COVID-19 vaccine platforms being developed. This figure was adapted from Calina et al. (2020)'s Towards effective COVID‑19 vaccines: Updates, perspectives and challenges and created using BioRender.


Why is this important?

Learning about SARS-CoV-2 is obviously critical given that it is the causative agent of a global pandemic which doesn’t seem to be ending any time soon. The scientific community needs to study emerging viruses like this one for various reasons; from determining its mechanism of transmission and pathogenesis to unravelling the complex immune response. The knowledge acquired from this research contributes to efforts to find therapies for COVID-19 patients as well as a vaccine to prevent infection.


The COVID-19 infodemic is another issue that we currently face. The infodemic - an excess of information, both accurate and inaccurate - means that misinformation about SARS-CoV-2 is seriously endangering our ability to control the virus and is responsible for preventable deaths. This means that educating the public about the nature of the disease, the threat it poses, and the significance of a vaccine is even more important.


Looking ahead of the present pandemic, each virus outbreak, whether it is HIV/AIDS, Ebola, influenza or SARS-CoV-2, teaches us a huge amount about viruses, epidemiology, public health and the human immune system. Furthermore, lessons can be learnt from the decisions taken, research carried out and mistakes made today which will be invaluable in future pandemics. With climate change, urban expansion into wild habitats, altered land use and changing demographics, novel viruses are becoming increasingly common. In fact, whilst viruses make up only 15% of all human pathogens, they are responsible for 43% of emerging human pathogens. This is just another indication that the COVID-19 pandemic will not be the last, so it is vital that we do everything we can to prepare ourselves for the future.

Author: Ambar Khan, BSc Biological Sciences

Disclaimer: All figures created using BioRender are intended solely for educational purposes and not for profit.

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