FanPost

OT: COVID-19, testing and vaccination: How do they work?


Greetings everyone.

As the Jays prepare to start the season in Texas, a state controlled by science-denying elected officials who reopened everything, to play in a stadium full of fans and what may indeed constitute a super spreader event (what could go wrong...), I thought I could post here some basic things about the biology of the virus, its testing (fast and slow) and the vaccines against it. After all, this is my professional background.

This is a long read. But it might explain some uncertainties some readers might have, especially with regards to the different vaccines out there.

First of all, some molecular biology basics. Molecular biology of coronavirus and vaccines

The mechanism of action of RNA and DNA vaccines, also illustrate the virus life cycle and molecular biology basics.

As you probably all know, our genetic information is stored in a molecule called deoxyribonucleic acid (DNA). There are four basic compounds, or bases, in DNA: tyrosine (T), adenosine (A), cytosine (C) and guanosine (G). All four are triphosphates (three straights PO4- groups attached to them), making DNA negatively charged molecule. Crick and Watson, along with Maurice Wilkins (and Rosalind Franklin who got ignored there) got the Nobel Prize in 1962 for elucidating DNA's structure: a double-helix, so two chains linked together. Those chains are strictly matched: T with A, G with C. DNA stores information by the sequence of its nucleotides. DNA itself does not do much; it is the blueprint for proteins which are the true actors of life.

However, there are no pathways for translation from DNA to protein. An intermediate is needed, which might actually be the oldest molecule of them all: ribonucleic acid (RNA) (it carries an hydroxyl group (OH) that DNA molecules lack, hence the different name). An hybrid, it can store information like DNA does, do some enzymatic activities like proteins do, and crucially can bind directly to aminoacids to put them in sequence and thus, to create proteins. There are a few different RNA molecules: messenger RNA (mRNA), which is the intermediate between DNA and protein, is the interesting one for us (the others are: transfer RNA which bind the amino acids; ribosomal RNA, which are the actual structures translating genetic code to protein, and microRNA (miRNA) that are short sequences that influence mRNA translation.

So, in short, DNA -> mRNA -> protein.

All living organisms on Earth follow this pathway. If RNA came first to store genetic information, it was displaced by DNA...

That is, unless you consider viruses as living or not.

Viruses are true parasites: they cannot live on their own. "Living" here means accomplish their full life cycle. That is actually true for a number of other parasites, such as the paludism amoeba, which have complex life cycles, but those do have their own cells to reproduce their genetic material. Think of viruses like computer viruses: outside the computer, they can't act. They need computers (memory sticks are not sufficient) to reproduce.

Viruses therefore need hosts to reproduce. They carry genetic information and some proteins necessary to deliver that information to the target cell and then use it to reproduce itself.

Viruses, however, are not bound to DNA. Many carry DNA, but others carry their genetic information on mRNA. Examples of the latter include HIV, the influenza viruses and the coronaviruses. All three will be used to explain some stuff here.

Coronaviruses are thus named because of their structure, a protein ball with spikes that can look like a crown when visualized with electron microscopy. They are the most complex RNA viruses. We now know of 7 strains that can infect humans. 4 are relatively harmless and are among the cold-causing viruses. A first deadly one was the SARS (Severe Acute Respiratory Syndrome) of 2003, of which Toronto was an important infection cluster outside China. A second deadly one emerged in the Middle East in 2012 and named MERS. In late 2019, a new strain, similar to the 2002 SARS, was discovered in patients in Wuhan, China. Thus, SARS-CoV-2 was discovered. In contrast to the 2003 SARS outbreak (that virus newly renamed SARS-Cov-1), this one did not disappear by itself. The reason there is no vaccine yet for SARS-CoV-1 is that the virus disappeared before the vaccine could be developed.

Yet a difference between 2003 and 2019 can already appear. It took months to sequence the genetic sequence of SARS-CoV-1. It took a few days for SARS-CoV-2. That technology has improved in a big way.

As mentioned above, coronaviruses are RNA-viruses. That means they have to deliver RNA to the interior of the target cell, but not to its nucleus. Viruses need an entry point to the cell; SARS-CoV-2 uses the angiotensin-converting-enzyme 2 as a binding partner. These entry partners can specific which cells are infected by viruses. Then, the mRNA can be translated directly to proteins, such as a RNA polymerase (for replications) and the proteins necessary for new virus assembly. This typically can overload a cell, which may die and disperse the new virus particles around.

In contrast, DNA viruses need translation to RNA as an added step. Some DNA viruses can also infiltrate the nuclei and act as host genes, if only for a time. This, combined with the advantage of DNA stability (compared to RNA, which is a lot more unstable), creates advantages for viruses like the herpes virus family. A special family, the retroviruses, transcribe their RNA into DNA, which is then inserted directly into the host's genome. The host's cell cannot then get rid of it. Retroviruses are the only know beings known to do reverse transcription. A useful fact for science.

Testing

Once armed with the genetic sequence of the virus, testing for that genome's presence in patients can happen. The technique used, called PCR for polymerase chained reaction, uses two concepts which are not present in animals. The first, key for PCR, is the extremophile bacteria (or archea) which live in high temperature waters close to hot vents, at the bottom of oceans. These organisms possess a DNA polymerase which is very resistant to heat. PCR uses that DNA polymerase: the DNA double-strand is separated by heat (95°C), then primer sequences (which are selected bases on the target gene sequence) attach to the new single strands of DNA at a lower temperature (typically around 55-65°C). Finally, the DNA polymerase can synthesize new double chain DNA on each single chain, effectively doubling the target sequence's number of replicates. With enough of these cycles, which increase your sequence of interest exponentially, you can amplify the SARS-CoV-2 specific sequence you found before enough to be able to detect it. If it is not there, there is no detection. Let's thank Kary Mullis, Nobel Prize in chemistry in 1993, for that invention.

But there is a problem: PCR does not work for RNA. Retroviruses come to the rescue. As mentioned before, they possess a reverse transcriptase to go from RNA to DNA (then called complementary DNA, or cDNA). That enzyme was isolated long ago, and with PCR, makes of a technique called RT-PCR.

Finally, the use of fluorescent probes, which help in quantification of the PCR results with each cycle (in contrast to just the traditional measure of DNA at the end), make it a RT-qPCR (Reverse-transcription-quantitative-PCR).

This test is very specific, and very sensitive. The only thing needed is to know the sequence of SARS-CoV-2, and the selection of a specific sequence in it to test. The drawback is time. To complete the RT and then the PCR, at least 4 hours are needed in the machines, without the time to prepare the samples.

But it's also the reason why vaccine development was so rapid.

VACCINES

Delivery of genes to living organisms (bacteria, yeast, plants, animals) has been done in research for a long time. But gene therapy, which was a big promise some 20 years ago, faltered. It is not because there was no efficiency in gene delivery in target models. But human usage, with its tons of ethical issues to begin with, never got off the ground. Delivery systems also lagged behind.

Traditional vaccine development centers on antigen response. Vaccines were all developed by isolation of the infectious agent, then trying to find a way to render it non-infectious while keeping the immune response it induced, for our adaptive immunity system to remember it and be more efficient in case of a new infection.

The same is true for the testing: rapid testing depends on antigen testing, which requires detectable amounts of the virus itself, or the antibodies against it.

But that can take a lot of time, trials and errors. Antidotes against antigens are usual horse-produced anti-serum: inject the infections agent in a horse, then isolate its serum, which contains antibodies against the virus, for human use. The advantage of the horse here is: it is big and may give a lot of serum without actually dying. The animal used may vary. But that requires successful infection of the target animal, which is not always possible. But although this may provide protection, it is not a vaccine. For that, we need bioreactors to produce the antigen itself. The easiest way, when it works, is to chemically "castrate" the infectious agent, then inject it to the patient. That works remarkably often, but not for everything, and does tend to create too big an inflammatory response. But that is what the diphtheria vaccine is, for instance.

In contrast, the production of mRNA from the gene can be done as soon as you know its sequence. It's not much more difficult to use its cDNA. Those are, in contrast to more traditional pathways, ridiculously simple, and cheap. It's not for nothing that the first vaccine for COVID-19, the Pfizer-BioNTech, was simply made by a couple in Germany (BioNTech's two employees, basically).

The problem was then: does it work to just inject it? And how to do this?

Pfizer-BioNTech, and Moderna, chose the mRNA path, delivered in functionalized lipid particles called liposomes. Astra-Zeneca/Oxford and Johnson & Johnson each used DNA instead, inserting it in adenoviruses and using their infectious properties to deliver the DNA to the target cells. Each included the sequence for the same protein, the famous spike protein giving coronaviruses their names.

Adenoviruses are named after DNA: they use DNA for their genome, and carry it inside the host nucleus. There, it functions as a plasmid: it is transcribed but never integrated into the host genome, and is eventually cleared away. Adenoviruses used in research are also deficient in reproduction genes, and therefore cannot replicate. In their wild form, they mostly cause the common cold in humans.

The companies all use the same strategy: incite our own cells to produce the spike protein to be recognized as an antigen. Specialized cells gobble up the antigen, break it down and express it in different patterns as antigens for B lymphocytes (which produce antibodies) to recognize.

None of these companies were technological pioneers in this development. But there were no vaccines using this strategy before (which is also being developed for the Zika virus). And there was no previous experience of it working in clinical settings. They also had to find proper delivery vehicles, such as the functionalized liposomes.

It's fantastic news that it works. But RNA, as mentioned before, is rather unstable. Hence the need for freezing for the Pfizer and Moderna vaccines. RNA also helps coronaviruses mutate into multiple variantes. The influenza virus strains are living proof of that.

I also don't have any insight into the efficacy of the different vaccines on the market right now.

PREVENTION

Of course, we can't really avoid this one. There are no 100% proof protection measures against COVID-19, which has proved a remarkably infectious agent (measles is the gold standard there, it is so ridiculously infectious). No vaccine is really efficient 100%, and we don't yet know how long immunity against COVID-19 lasts. That's why global reach of the vaccine is important: vaccination greatly reduces the odds of developing an infection, and community immunity greatly reduces the odds of the infection reaching you (or vulnerable persons). Washing hands, wearing face covering is also important. Neither reduce all risk, but all act as an additional layer of protection, and all layers work together. Finally, the more we prevent contamination of people, the less chance the virus has to mutate in a profitable way.

Most mutations are bad for the organism having the mutation. It's probable that less than 0.0001% of mutations help the organism survive. But every once in a while, by chance, one mutation might provide a benefit, and that mutation is then selected through natural selection and comes to dominate. Our work is to reduce the number of occasions for such a beneficial mutation to happen.

Vaccines also don't prevent infections. They mightily increase our body's response to the infection, rending it impotent very quickly and extinguishing contamination chances. With immunization, your body is primed to kick the infectious agent out, that's it. It's not totally foolproof, but it is sure a lot better than no immunization at all.

And there is absolutely no immunization advantage by having the actual virus rather than the vaccine (a trope we see sometimes). None.

Stay Safe !

Don't hesitate to ask questions!

Editor's Note: This is a FanPost written by a reader and member of Bluebird Banter. It was not commissioned by the editors and is not necessarily reflective of the opinions of Bluebird Banter or SB Nation.