Antibody Resistance Mutations

This article has already been stolen by various news outlets as a full guard, and the vaccine will not help, and again we will all die. This is of course complete nonsense, even having nothing to do with what was actually done in this article. And the article is actually very good and interesting. But it will take a fairly long introduction to explain it.

In vitro evolution
Let’s start from afar. Let’s say you have a new antiviral drug and want to understand how a virus can develop resistance to it. How can you find out? The classic approach is in vitro selection . We let the virus multiply in cell culture, while adding this antiviral drug, but in concentrations that do not completely suppress the virus, and so that it can continue to multiply, albeit not very well. As the virus multiplies, it will mutate and eventually a mutation will appear that makes it resistant to this drug. Such a mutated virus will multiply faster than the wild one and will gradually displace it in cell culture. We will see that, firstly, the virus began to multiply faster (our drug no longer holds it back), and secondly, almost all viruses in the culture carry some kind of new mutation.

It sounds easy and simple, but as is often the case, in reality this approach has many problems. I will not list them all, I will only point out a few. Firstly, it is difficult to balance the concentration of the drug so that sufficient selection pressure is exerted on the virus, but at the same time it can still actively multiply. If we add too little, the mutated virus will not have enough advantage over the wild one for it to multiply. Add too much – the virus does not multiply well, which means that mutations appear slowly and it will take a very long time. Secondly, these experiments are very laborious – the virus must be constantly re-inoculated into new cells, and in large volumes, and for quite a long time. Thirdly, just random mutations constantly accumulate in viruses, and at the end of the experiment it happens that you have to dig for a long time to find out which of the mutations really gives resistance, and which one appeared by chance. This is sometimes very difficult to do because resistance has been acquired through two or even three different mutations, each of which individually has no effect or has a weak effect.

Deep Scanning
Jesse Bloom’s group (I know a couple of people from his lab ) takes an interesting approach that gets around some of these problems. They use new technologies for DNA synthesis and quantitative sequencing . First, they create a library of mutant viruses. They order the synthesis of a viral gene, but not an ideal copy of it, but with mutations that change each of the amino acids one by one for all 19 possible alternatives (total amino acids – 20). Something like this: The first three nucleotides (an amino acid is encoded by three nucleotides) are random in all possible combinations, and then a perfect copy. Separately, a gene is synthesized in which the second three nucleotides are random, and the rest are a perfect copy. Then – the third three nucleotides are random, and the rest is a perfect copy. They all mix together and these mutated genes are inserted back into the virus and thus in one fell swoop they create all possible mutants in which each of the amino acids in the protein under study is mutated into all the other nineteen. They call it “deep mutation scanning”.

Then new sequencing technologies come to the rescue , i.e. determination of the nucleotide sequence of genes. They are based on the fact that sekviniruyutsya small gene fragments, which later with the help of computers is going to complete the sequence. But they can be used in a different way. If you have a mixture of different but similar genes (as in the library of mutant viruses obtained above), then the computer can say not to collect one sequence from the obtained fragments, but to calculate the frequency of all the variants available in the mixture. Thus, you can take the resulting library of mutant viruses, and get for each mutant what percentage it is in the total mixture. After that, we can take these viruses and, for example, infect cells with them in the presence of our drug, wait for the appearance of a new generation of viruses, and again analyze them by sequencing . If a mutant reproduces poorly in the presence of a drug, its percentage in the population will go down. If it’s good, then up. Thus, we can quickly analyze a huge number of mutations for resistance to a given drug.

This approach, of course, does not solve all problems, and it has serious drawbacks. One of the main ones is that if mutations in more than one amino acid are required for drug resistance, then this method will not detect them.

All this is quite difficult, if you have any questions – ask in the comments . And I will write about the results of the article a little later.

local_offerevent_note December 28, 2020

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