Joana Vidigueira

 

Abbreviations

SARS: Severe Acute Respiratory Syndrome, CoV: coronavirus, MERS: Middle Eastern Respiratory Syndrome, DNA: deoxyribonucleic acid, RNA: ribonucleic acid, ACE2: angiotensin-converting enzyme 2, APC: antigen presenting cells, mRNA: messenger RNA, RT-PCR: reverse transcription polymerase chain reaction

Introduction

Viruses are biological agents that are only able to survive and multiply within a living host. When outside of a host cell, viruses exist as viral particles that are made up of genetic material surrounded by a protein coat that can also include lipids. The viral genome encodes the structural proteins that make up the viral particle as well as proteins that the virus needs to be able to replicate. In order to replicate faster and more efficiently, viruses keep the size of their genomes as small as possible and rely on many components of the host cell’s machinery to replicate. Once they enter the cell, most viruses have mechanisms in place that induce the host cell to preferentially produce viral proteins over host proteins. These proteins are packaged into viral particles that are released upon lysis of the host cell, as illustrated in Figure 1. Viruses come in a variety of shapes and forms and are classified according to a range of different properties, including the type of genetic information that they contain, which can be composed of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

 


The virus binds a receptor on the surface of the host cell. The viral particle is then taken into the cell, and can release its genome. The genetic information is replicated, and viral proteins are made, using the protein synthesis machinery of the host cell. The new proteins and the genome are then packaged into a new viral particle which is released from the cell. This is the general process through which some viruses replicate, including the SARS-Cov-2 virus.

How does our immune system respond to infection with SARS-CoV-2 and how do vaccines help us?

Infected cells eventually die and can then be taken up by immune system cells such as dendritic cells (DCs) and macrophages. These cells are called antigen-presenting cells (APCs), as they have the ability to present fragments of the proteins that they engulf on their surface. The APCs move to lymph nodes where they can present the antigen to T cells, activating them. There are two types of T cells and these are the killer T cells and the helper T cells. While killer T cells can directly kill infected cells, the role of the helper T cell is to activate B cells (3, 5). When B cells interact with helper T cells, they become activated by maturing into antibody-producing cells. These cells are responsible for producing the antibodies that will recognize the infectious agent and trigger an immune response against it (3). A pool of memory T cells are maintained long term. This allows the body to rapidly mount a response on encountering the same pathogen (5). Similarly, memory B cells circulate in a dormant state and become activated when the same antigen is detected again, initiating a stronger response than in the first infection. Antibodies are proteins of the immune response that specifically bind an antigen, which is a molecule that has been recognized by the body as being foreign. The binding of an antibody to an antigen can neutralize the pathogen by blocking it, recruit other immune cells that can engulf and destroy the infected cell and the pathogen, or trigger an inflammatory response. As we age, our immune responses become slower and less efficient. This phenomenon is called immune senescence and goes towards explaining why older individuals are more likely to suffer with COVID-19 symptoms and to develop more severe and life-threatening covid infections (6). COVID-19 vaccines focus on inducing this antibody-response (7). Antibodies that bind specific regions of the S1 and S2 subunits of the spike protein have been seen to have a neutralising effect. The vaccines that are currently on the market target the receptor binding domain of the S1 subunit of the spike protein in an attempt to block binding of the virus to the host cells (6). The vaccines artificially induce immune responses against the spike protein so that upon infection with SARS-CoV-2, memory cells can rapidly respond against it. In the UK and EU/EEA, there are two major classes of COVID-19 vaccines available. These are mRNA and DNA vaccines. The differences between these are shown in Figure 3. Different companies have manufactured these vaccines (see Figure 3). In the UK and EU, many people were offered the ‘Comirnaty’ vaccine, produced by Pfizer/ BioNTech (Figure 4). The effectiveness of these vaccines appears to decline over time and as a result, it has been necessary to offer booster vaccinations (7, 8). These booster vaccinations expose the immune system to the antigen for a third time, triggering an immune response and the development of antibodies and improving immune memory. The emerging new variants pose a potential threat to vaccine efficiency and further support the need for boosters  as well as improved vaccine design (8).

 

Two major types of vaccines are being used to tackle the COVID-19 pandemic. Inactivated virus vaccines make use of viruses which are not capable of replicating in humans. These viruses can carry and deliver molecules to our cells. In the case of Covid-19 vaccines, the molecule being delivered is DNA containing the sequence for the spike protein of the SARS-CoV-2 virus. In the nucleus, the DNA is read and copied into a messenger RNA (mRNA) molecule in a process called transcription. The mRNA is transported out of the cell’s nucleus and can then be read to produce the protein, in a process called translation. The virus does not contain the machinery necessary to carry out transcription and translation and therefore, uses the host cell’s machinery (9). The other type of vaccines are mRNA vaccines. For these vaccines, the initial nuclear step is bypassed and the mRNA is directly delivered to the cell to produce the spike protein. The spike protein is then presented on the cell surface, where it can be recognised by other immune cells. As a consequence, an immune response is elicited. Memory immune cells are then capable of recognizing this same protein when the organism is exposed to the virus. This recognition allows the immune system to quickly respond to infection and fight it (9).

How do the different variants of SARS-CoV-2 differ from one another?

For the greater part of 2020, it seemed that the version of SARS-CoV-2 virus that was spreading around the world was the same. However, since late 2020, various mutations have emerged which have generated ‘variants of concern’. Since viruses compact their genetic information as much as possible, their genome is highly conserved and so it is expected that most mutations will inactivate the virus or reduce its ability to infect and cause disease (virulence). A proportion of mutations would be expected to have no effect on the virulence of the virus. However, certain mutations can alter the functional properties of SARS-CoV-2, modifying its infectivity and the severity of disease it can cause, and the way in which the virus interacts with the elements of the host’s immune system. The emergence of such mutations is normally due to the adaptation of the virus to the changing immune profile of the population, as more people become infected and/or are vaccinated (10). The first of these mutations was detected in the UK in September 2020, in a variant of Covid which is now known as the Alpha variant. Subsequently, in October 2020, the Beta variant was detected in South Africa and the Delta variant was detected in India. In December 2020, the Gamma variant surged in Brazil. Most recently, in November 2021, the Omicron variant was detected in South Africa. Although other variants have been detected, these are the ones that have been considered to be variants of concern. While the Alpha and Beta variants have nine spike mutations, the Gamma and Delta variants have eleven, and the Omicron variant has thirty-two (11). These mutations to the spike protein alter the virus’ properties, including its infectiousness and the consequences for infected individuals. The effects of different spike mutations on viral properties and their clinical consequences are summarised in Figure 5. The variants that surged through populations in 2020 have all been determined to be more transmissible, with the Delta variant having the largest increase in transmissibility (97%) (12). The mutations that affect transmissibility and the severity of disease have been found to be those mutations that cause changes in the structure of the spike protein. The spike protein binds to receptors on the cell surface of the patient’s cells. The high number of mutations presented by the Omicron variant has caused great concern amongst scientists worldwide. However, so far it does not seem to cause different symptoms from the remaining variants. Because of the differences in the spike protein of the Omicron variant, it seems that the immune memory created by current vaccines does not allow the body to respond to this variant as effectively. For instance, the antibodies which are rapidly produced might not recognise or bind the spike protein as effectively. However, the Omicron virus cannot completely evade the immune response triggered after vaccination and so, previous immunity from vaccination is still important in fighting infection with this variant (11, 13). It is, however, important to note that studies on the Omicron data are lacking to allow full understanding of this variant.  One important difference which has been found with Omicron is that rates of reinfection appear to be higher than for the other variants of concern identified (14). As will be discussed later, the high number of mutations on this variant could impact
the current protocols for vaccination.

 

Each of the letters above corresponds to a particular amino acid within the amino acid sequence of the Spike protein. Amino acids are encoded by DNA and mutations can cause changes in the amino acids that make up a protein, changing its properties. These are some of the most important mutations labelled according to their effects on the viral properties and predicted clinical consequences of each of them (15). The receptor-binding domain is the part of the Spike protein which binds to the ACE2 protein. Mutations outside of this domain can still cause alterations in the protein structure, which can in turn, affect the behaviour of the virus.

How do PCR and antigen tests identify the presence of the SARS-CoV-2 virus in samples?

There are two main types of tests that can detect the presence of SARS-CoV-2 in sample swabs. These are the RT-PCR and the Antigen tests. RT-PCR stands for reverse transcription polymerase chain reaction. This test is able to detect specific sequences within the RNA molecule of the virus with high specificity and sensitivity. Therefore, it is considered the most effective way to determine whether an individual may be infected with SARS-CoV-2. Figure 6 shows the steps of the procedure that is followed for the detection of the SARS-CoV-2 virus using a fluorescence-based RT-PCR test. This type of test involves amplification of the viral sequences. This means that specific sequences which are known to be part of the RNA of SARS-CoV-2 are duplicated in multiple cycles to obtain a sample which contains a detectable amount of them. If there is no virus present, these sequences cannot be amplified. However, if enough virus is present, the sequences will be amplified and detectable.

 

Fluorescent-based RT-PCR tests involve collection of a sample by nasopharyngeal swab, extraction of nucleic acids (RNA and DNA) from the sample, synthesis of complementary DNA (cDNA) and PCR. RNA is a nucleic acid with a single strand while DNA has two strands. In the reverse transcription step, the enzyme reverse transcriptase can copy the information in the RNA molecule onto another strand to create a cDNA molecule. For PCR, the cDNA is first amplified in a way that incorporates a fluorescent label. Only specific viral RNA sequences are amplified. The fluorescence is detected as the number of amplification cycles increases. If after a low number of cycles, the level of fluorescence is higher than a set limit (threshold), then that means that there was a large enough amount of viral RNA in the sample and therefore, the test result is positive (16, 17).
Antigen tests, also known as rapid tests, rely on antibodies that are raised against specific viral proteins. Figure 7 shows how antigen tests work. These are the most commonly used tests and those which most people will be familiar with. Figure 8 presents an example of an antigen/ lateral flow test you might have used at home.

 

A nasal swab is collected, and the sample is mixed with an extraction buffer solution in a tube. The mixture is then added to the sample well on the lateral flow device. As the sample flows into the chamber, antibodies (green) raised against viral proteins (antigens), like the nucleoprotein, for example, can bind to the viral antigens. These antibodies have properties that make them visible. As the sample continues to move across the lateral-flow device, it encounters antibodies (orange) on the test line (T) that can bind the viral protein-bound antibodies. If the sample contains viral proteins, the antibodies bound to these will accumulate on the test line, and a band will form. On the control line (C), there is another type of antibody (blue) present that detects unbound anti-viral protein antibodies. If the sample has been correctly applied, the antibodies will reach the control line and a red line will become visible (17).

 

The amplification step in PCR tests means that even very small amounts of the virus can be detected using this test. This makes the PCR test the most reliable test for covid. PCR tests can in certain instances, give a positive result even after weeks or months have passed since the initial infection. However, the detection of viral RNA may not always indicate that the individual is still contagious and able to spread the virus to others.

How should testing and vaccination adapt to the new SARS-CoV-2 variants?

So far, both PCR and antigen tests have proven to be effective in detecting the presence of the virus, regardless of the variant (18, 19). This is because the test detects conserved viral proteins, like the nucleoprotein, rather than the spike protein which changes from variant to variant. Therefore, testing appears to remain effective in detecting new cases of infection with SARS-CoV-2, even when variants contain extensive mutations. On the other hand, the current vaccines seem to be less effective against this new variant. Variants with only a few mutations do not change the spike protein enough that it cannot be recognized by the immune system. However, the Omicron variant seems to partially evade vaccines and it is possible that future variants could completely evade the current vaccines. The question now is what this means for the public and how we can prevent this type of immune evasion.

Recent data on the effectiveness of booster doses of the Pfizer/BioNTech vaccine against the Omicron variant seem promising (20). It has been suggested that the booster might help with the process of affinity maturation, which involves the production of antibodies that have higher affinities for the antigen that they bind, in this case, the spike protein (20). However, the booster might not be effective in stopping future variants. A recent trial in Israel showed that administering another booster of Pfizer or Moderna, a fourth dose, does not largely increase protection against any of the variants (21, 22). These results seem to indicate that there is an upper limit, and it is not possible to increase immunity by giving more boosters of the same vaccine. However, this trial had a small sample size and further studies will be needed to validate these results (21). A big issue with Covid-19 vaccines is that it is not known how long immunity lasts, and this also seems to vary according to the variant. Therefore, it will be important to complement the current data on boosters with more long-term studies to determine the effectiveness and the need for boosters. 

Besides boosters, another strategy to improve efficacy of vaccines against new variants is to create variant-specific vaccines. These have been developed for Omicron in an attempt to obtain optimal protection against this variant. However, it is important to note that these will take time to develop once a new variant is identified. In addition, current data seems to indicate that Omicron-specific boosters do not offer an advantage compared to boosters of the current vaccines (23-25). The data available comes from animal studies and has not been peer reviewed, meaning that these results should not be taken as absolute proof of the inefficacy of these boosters. In a study done on eight primates with the Moderna vaccine, the Omicron-specific booster (third dose) showed similar increases in antibody production to the current booster (24). However, it is unclear how long this effect lasts. A study in mice with the Moderna vaccine showed similar results (25). Furthermore, this and another study both showed that giving mice the Omicron-specific vaccine only on all doses impaired the ability of the antibodies to inhibit other variants (23, 25, 26). A different study looked at a ‘replicating RNA’ vaccine which includes an enzyme that amplifies the mRNA sequence in the vaccine. The Omicron-specific version of this vaccine did not produce an elevated immune response against the Omicron variant, compared to vaccine based on the original SARS-CoV-2 variant, in hamsters. However, when they administered one dose of the vaccine targeting the original variant and two doses of the Omicron-specific variant, they saw an increased immune response against the Omicron variant (23, 27). These studies indicate that a single booster of a variant-specific vaccine will not be the solution but maybe having two doses of that vaccine could be effective (23). It will be important to look at these studies in combination with the results from the ongoing trials in humans being performed by Pfizer and Moderna.

 

Conclusion

Overall, there is still much that remains to be understood about the Covid-19 variants. Further epidemiological studies are needed to improve our understanding of how dangerous each variant actually is and whether it will be necessary to develop new vaccines that specifically target the mutated spike protein of these variants in order to offer optimal protection. The sequencing technologies that are currently available, allow us to quickly identify and sequence new variants, enabling us to develop appropriate vaccines in a much shorter time frame than was ever possible previously. These advances make it more attainable to develop strategies to combat new variants in a short period of time. The ability to recognize and target the precise nucleotide sequences that are mutated in each variant allows us to react rapidly and means that we are now in a good position to tackle this problem. It will also be important to carry out more studies on boosters and variant-specific vaccines for each variant that emerges, as mutations give the virus different properties which can change how our immune system reacts to it.

 

Useful Links

For more information on the symptoms of Covid-19 and up-to-date guidance on how to act if you have symptoms, want to get vaccinated or have other Covid-19 related questions, visit https://www.nhs.uk/conditions/coronavirus-covid-19/

For more information about Covid-19 and the SARS-Cov-2 virus, visit: https://www.who.int/health-topics/coronavirus

For up-to-date data on the spread of SARS-CoV-2 in the UK, visit: https://coronavirus.data.gov.uk/

For general information about coronaviruses and SARS-CoV-2:

For more information about Covid-19 tests:

For more information about Covid-19 immunity and vaccines:

For general information about protein synthesis, viruses, and the immune system:

 

References

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